key: cord-002689-qakbp4dz authors: brisse, morgan; ly, hinh title: viral inhibitions of pact-induced rig-i activation date: 2017-07-03 journal: oncotarget doi: 10.18632/oncotarget.18928 sha: doc_id: 2689 cord_uid: qakbp4dz nan viral infections are usually detected by toll-like receptors (e.g., tlr3, tlr7/8) and cytosolic rig-i-like receptors (rlrs), such as rig-i and mda5. rig-i and mda-5 sense 5' triphosphorylated dsrna and higher order rna structures generated during virus replication. activated tlr and rig-i/mda-5 signaling pathways initiate effective antiviral innate immune responses, in particular by inducing type 1 interferon (ifn1) production. in uninfected cells, rig-i is kept inactive by a closed conformation, in which its n-terminal card domains and c-terminal (repression) domain (ctd) are kept close to the centrally located helicase domain [1] . upon viral infection, rig-i binds to rna ligand to facilitate its conformational changes, dimerization and multimerization. atp hydrolysis via the atpase function of rig-i has been shown to be critical for rig-i activation. patel and colleagues [2] have provided strong experimental evidence for the rig-i-mediated atp hydrolysis-dependent enhancement of its active oligomerization state, which correlates well with the degree of ifn1 activation in cells. it has recently been reported that another cellular protein, called pact, a 313-aa cellular protein that contains 3 conserved dsrna binding motifs (dsrbms) with dsrbm1 and 2 binding to dsrna and dsrbm3 mediating activation of the dsrna-dependent kinase pkr, can potently enhance rig-i activation to induce ifn1 production [3] . pact potentiates rig-i function via interaction with the c-terminal repression domain of rig-i and activates the rig-i's atpase function to enhance ifn1 production. the same study shows that the interaction between rig-i and pact can be detected endogenously in cells (upon sendai virus infection) and that enforced expression of pact alone has no effect on the ifnβ promoter activity, but when co-expressed with rig-i with a functional helicase activity, pact can significantly augment rig-i activation. pact does not potentiate rig-i-induced activation of nfκb, but rather through activation of irf3 (i.e., its phopshorylation and dimerization in the nucleus). it is noteworthy that the innate immune stimulatory effect of pact has also been observed on mda5-mediated activation of ifnβ promoter but not on mda5-induced activation of nfκb, the exact molecular mechanism of which is not known. influenza virus ns1, mers-cov 4a, herpesvirus hsv1 us11, and ebola virus vp35 proteins have all been shown to directly disrupt the interaction between rig-i and pact, and hence blocks the ability of pact to activate rig-i (4-7). all these viral proteins have rna binding capabilities, yet it isn't clear from the published reports whether dsrna is absolutely required to activate rig-i via pact induction. pact was first identified as a novel cellular partner of influenza ns1 protein via maldi-tof mass-spectrometry analysis of cell lysates of pulldown experiment using anti-flag antibody to the flag-tagged ns1 and in stable-isotope-labeling silac cell culture [4] . ns1 was confirmed to interact with pact by pulldown assay with pact-specific antibodies. targeted mutagenesis of ns1 revealed that integrity of the rna-binding domain of ns1 was necessary for interaction with pact. however, it is unclear whether rna is required in the process of rig-i activation through pact in influenza virus-infected cells. it has also previously been shown that 4a, 4b and m proteins of coronaviruses can inhibit ifn1 production, with 4a having the capacity to bind to dsrna and inhibit mda5. the dsrna binding region of 4a has a high degree of sequence homology to other coronaviral dsrna binding proteins. however, 4a from bcov-hku4 (another beta coronavirus) does not bind dsrna. when hek293t cells were transfected with 4a from dsrna binding mers-cov and bcov-hku5 and non-dsrna binding bcov-hku4, the ifnβ promoter activity was suppressed in the dsrna binding 4a expressing cells but was not affected in the non-dsrna 4a expressing cells, indicating that dsrna binding is necessary for inhibition of ifn1 production [5] . furthermore, mers-cov 4a inhibited ifn1 production when stimulated by pact. mers-cov 4a and bcov-hku5 4a were confirmed to interact with pact through co-immunoprecipitation (co-ip), but the interaction was ablated by rnase treatment, indicating that rna was necessary for the interactions. like influenza ns1 and mers-cov 4a proteins, us11 protein of hsv1 is a dsrna binding protein and has been shown to associate with pact, pkr, mda5 and rig-i in addition to 2′,5′-oligoadenylate synthetase (oas). interaction between us11 and pact was confirmed by co-ip and was not affected by rnase digestion, indicating that their interaction is likely rna-independent [6] . like the other dsrna binding viral proteins, the dsrna binding c-terminal region on the ebola virus vp35 protein was found to be essential for pact binding and that several charged or polar amino acids were necessary for interaction. both dsrna and pact can activate editorial www.impactjournals.com/oncotarget rig-i while vp35 inhibits rig-i in a dose-dependent manner in both activation scenarios, which the authors have suggested that vp35 blocks rig-i both by shielding dsrna from detection and by preventing pact binding to rig-i [7] . like us11 of hsv1, vp35 binding to pact was not affected by the addition of ssrna-specific rnase a or dsrna-specific rnase iii. in summary, it appears that several human viral proteins share a common strategy to evade innate immune activation by interacting with and thus inhibiting pact from activating rig-i. these known viral proteins have rna-binding properties, yet it still isn't entirely clear whether rna binding is an absolute requirement to inhibit pact-induced rig-i activation. additional investigations into the potential role of dsrna in pact-induced augmentation of rig-i activation are therefore warranted. those studies may lead to development of broad-spectrum antiviral or immune-modulatory therapeutics for diverse families of viruses that can inhibit pact's immune modulatory function. this is an open-access article distributed under the terms of the creative commons attribution license 3.0 (cc by 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited key: cord-285339-pwy1ry4n authors: tarigan, ronald; shimoda, hiroshi; doysabas, karla cristine c.; ken, maeda; iida, atsuo; hondo, eiichi title: role of pattern recognition receptors and interferon-beta in protecting bat cell lines from encephalomyocarditis virus and japanese encephalitis virus infection date: 2020-06-18 journal: biochem biophys res commun doi: 10.1016/j.bbrc.2020.04.060 sha: doc_id: 285339 cord_uid: pwy1ry4n bats are potential natural hosts of encephalomyocarditis virus (emcv) and japanese encephalitis virus (jev). bats appear to have some unique features in their innate immune system that inhibit viral replication causing limited clinical symptoms, and thus, contributing to the virus spill over to humans. here, kidney epithelial cell lines derived from four bat species (pteropus dasymallus, rousettus leschenaultii, rhinolophus ferrumequinum, and miniopterus fuliginosus) and two non-bat species (homo sapiens and mesocricetus auratus) were infected with emcv and jev. the replication of emcv and jev was lower in the bat cell lines derived from r. leschenaultii, r. ferrumequinum, and m. fuliginosus with a higher expression level of pattern recognition receptors (prrs) (tlr3, rig-i, and mda5) and interferon-beta (ifn-β) than that in the non-bat cell lines and a bat cell line derived from p. dasymallus. the knockdown of tlr3, rig-i, and mda5 in rhinolophus bat cell line using antisense rna oligonucleotide led to decrease ifn-β expression and increased viral replication. these results suggest that tlr3, rig-i, and mda5 are important for antiviral response against emcv and jev in rhinolophus bats. role of pattern recognition receptors and interferon-beta in protecting bat cell lines from encephalomyocarditis virus and japanese encephalitis virus infection 1 bats are known as natural reservoirs of some deadly zoondotic viruses, which have a high impact on human health and include filoviruses (marburg viruses), lyssaviruses (rabies and ablv), coronaviruses (sars and mers-cov), and henipaviruses (hendra and nipah viruses) [1] . emcv, a potential omnipresent zoonotic agent, has a broad host range with rodents as its natural reservoir [2] . as yet, there has been no report of emcv isolation from bats; however, miniopterus fuliginosus is supposed to be one of the natural hosts of emcv due to the detection of a genome fragment of the virus in the bat's fecal guano [3] . jev is a mosquito-transmitted flavivirus with humans as the definitive hosts and pigs as the amplification hosts [4] . jev have been isolated from multiple species of fruits and insectivorous bats in several asian countries [5] . experimental infection of jev in some insectivorous and fruit bats showed persistent viremia and viral replication in multiple organs without pathological symptoms or signs of encephalitis [6] . bats remain asymptomatic after viral infection partly due to suppression of viral replication at an early stage of innate immune response in them [7] . some bat species have "always on" interferon system because of higher and constitutive expression of interferonalpha, persistent activity of isgs, constitutive stat1 phosphorylation, and broad tissue distribution of irf7 in unstimulated bat primary cells derived from pteropus alecto, and artibeus jamaicensis [8] . moreover, some bat species have a special mechanism to suppress excessive inflammation during mers-cov infection as identified in a bat cell line derived from eptesicus fuscus and bat primary immune cells derived from p. alecto [9, 10] . innate immune response is initiated through recognition of viral pathogens by the prrs and will converges at irf3, which is followed by phosphorylation of irf3 leading to initiation of antiviral responses such as activation of type i ifn, isgs, and proinflammatory cytokines [11] . previous studies have showed that tlr3, rig-i, and mda5 signaling is critical for ifn-b production and susceptibility of cells against emcv and jev infection in humans and mice [12e16] . only a few studies are available on prrs and type i ifn signaling in bats during viral infection. activation of tlr3, rig-i, and mda5 after poly(i:c) stimulation have been described in p. alecto kidney cell line, desmodus rotundus fetal lung cell line, and e. fuscus kidney cell line; however, their role in stimulating type i ifn production has been clarified only in the e. fuscus kidney cell line [17e19]. here, the influence of emcv and jev on prrseifneb signaling in bat cell lines derived from four bat species (pteropus dasymallus, rousettus leschenaultii, rhinolophus ferrumequinum, and miniopterus fuliginosus) was examined. bhk-21 (syrian hamster, kidney), hek293t (human, kidney), fbkt1 (ryukyu flying fox, pteropus dasymallus, kidney), demkt1 (leschenault's rousette, rousettus leschenaultii, kidney), bkt1 (greater horseshoe bat, rhinolophus ferrumequinum, kidney), and yubfkt1 (eastern bent-wing bats, miniopterus fuliginosus, kidney) cell lines were maintained in dmem supplemented with 10% fbs, 2% l-glutamine, 0.14% nahco 3 , and 1% penicillin-streptomycin. the bat kidney cell lines were established as previously described [20, 21] and were derived from proximal epithelial tubules cells based on the presence of aqp1 and muc-1, specific markers for proximal and distal epithelial cells, respectively (supplementary fig. 1 ). partial dna sequence of aqp1 and muc-1 from all cell lines can be found in supplementary fig. 2 the emcv strain used in this study, niid-nu1, was provided by dr. kazuya shirato (national institute of infectious diseases, japan). the genome sequence of niid-nu1 strain of emcv was determined (genbank accession number lc508268). the emcv strain niid-nu1 was clustered into group 1a and most closely related to emcv gs01 and pv21 strains, which were isolated from pigs (supplementary fig. 4 ; and supplementary table 2 ). the jev strain, jev/sw/chiba/88/2002, was isolated from swine serum [22] . both emcv and jev were propagated in bhk-21 cells and the virus titer was determined by plaque assay. all kidney cell lines were infected with emcv and jev at moi of 1.0 and 0.01 in dmem supplemented with 2% fbs medium inside biosafety level 2 laboratory. the cells were seeded at a concentration of 5 â 10 4 cells/well in a 24-well plate and were infected with emcv and jev at moi of 1.0 and 0.01. both mock and emcv-jev-infected cells were washed with pbs and were harvested using 0.25% trypsin-edta at 1, 2, 3, 4, 5, 6, and 7 days post infection (dpi). assessment of cell growth (cell viability and total live cell numbers) was carried out by trypan blue dye exclusion test. unstained or live cells were counted using automated cell counter. total rna was collected from mock and emcv and jev-infected cells with isogen ii (nippon gene) and rna cleanup were performed using rneasy mini kit (qiagen). the cdna was synthesized using superscript iv first-strand synthesis system (invitrogen). qrt-pcr was performed using roche lightcycler 96 in conjunction with thunderbird sybr qpcr mix (toyobo) as per the manufacturer's instructions. relative expression level of the target genes (tlr3, rig-i, mda5, and ifn-b) were normalized against gapdh and is expressed as reciprocal of dct. fold change is represented as fold increase of expression level in infected cells over mockinfected cells. viral genome copies was calculated using the emcv and jev standard curves that were created by serial dilution of a known number of emcv and jev pcr-amplified fragments. phosphorothioate antisense rna oligonucleotide (s-oligo) were synthesized by fasmac (japan) using the consensus sequences of tlr3, rig-i, and mda5 genes of three bat species (r. ferrumequinum, m. fuliginosus, and r. leschenaultii) (supplementary figs. 5, 6, and 7). bkt1 cells were transfected with the 120 pmol antisense rna oligonucleotides against tlr3, rig-i, and mda5 (supplementary table 3 ) using pei [23] . the knockdown was verified by measuring the expression level of tlr3, rig-i, and mda5 by qrt-pcr. the bat and non-bat cell lines showed different replication levels of emcv and jev. emcv and jev replicated at a significantly lower level in three bat cell lines (demkt1, bkt1, and yubfkt1) than in non-bat cell lines (bhk-21 and hek293t cells) and fbkt1 cells ( fig. 1a and b) . higher replication level of emcv and jev in non-bat cell lines and fbkt1 cells resulted in massive cell death within 1 dpi for emcv and 3 dpi for jev ( fig. 1c and d) . the cpe in non-bat cell lines and fbkt1 cells after emcv and jev infection with moi of 1.0 and 0.01 appeared earlier than in three bat cell lines (demkt1, bkt1, and yubfkt1). the cpe was observed in non-bat cell lines and fbkt1 cells within 1e2 dpi and 3e5 dpi after emcv and jev infection, respectively ( fig. 1c and d) toll-like receptors bkt1, yubfkt1, and demkt1 cells, the cpe was delayed to 7 dpi for moi of 1.0 and 0.01 without complete lysis ( fig. 1c and d) , but the total live cell numbers were still higher than other cells until 7 dpi (supplementary figs. 8e, 8f , 9d, 9e, and 9f). almost all bat cell lines had a higher basal expression level of tlr3, rig-i, and mda5 than non-bat cell lines ( fig. 2a) . among the emcv and jev infection resulted in a higher expression level of prrs in bat cell lines with a lower viral replication level (demkt1, bkt1, and yubfkt1) (fig. 2b, c, and 2d ). up-regulation of tlr3, rig-i, and mda5 after emcv infection was observed in those cell lines, with a high up-regulation of rig-i and mda5 in demkt1 cells (fig. 2ced) . rig-i and mda5 were also up-regulated in hek293t cells but their expression level was still lower than that in the bat cell lines. down-regulation of all prrs and rig-i was observed in bhk-21 cells and fbkt1 cells, respectively (fig. 2b, c, and 2d) . among jev-infected cells, rig-i and mda5 were up-regulated in all cell lines, except fbkt1 cells (fig. 2c) . down-regulation of tlr3 was observed only in fbkt1 cells (fig. 2b) . ifn-b was highly upregulated in all bat cell lines that showed resistance against emcv and jev infection, except in yubfkt1 cells after emcv infection (fig. 2e ). hek293t and fbkt1 cells also showed upregulation of ifn-b with a lower fold change (fig. 2e) . the knockdown of tlr3, rig-i, and mda5 was confirmed by qrt-pcr in which the only the expression level of prrs was significantly reduced in bkt1 cells (fig. 3a) . the expression level of ifn-b was also decreased in bkt1 cells but it was not as intensive as that of prrs (fig. 3b) . knockdown of prrs in other bat cell lines (demkt1 and yubfkt1 cells) was not successful, as the expression level of prrs was not decreased in those cell lines. knockdown of tlr3, rig-i, and mda5 led to reduced expression level of ifn-b after emcv and jev infection (fig. 3ced) , dramatic cpe at 2 and 5 dpi, respectively (fig. 4c) , and a significantly lower number of live cells than uninfected knocked down cells since days 2 and 3 of emcv and jev infection, respectively ( supplementary fig. 10 ). after emcv infection, the tlr3 knocked down cells showed the lowest expression of ifn-b (fig. 3c) ; however, unpredictably, they did not demonstrate the highest emcv replication level (fig. 4a) . the cells knockdown for mda5 exhibited the highest emcv replication level (fig. 4a) . after jev infection, all cells knocked down for either of the three prrs showed a comparable ifn-b expression level (fig. 3d) , while the cells knockdown for mda5 exhibited a comparatively higher jev replication level (fig. 4d ). in this study, the bat cell lines derived from r. leschenaultii (demkt1), r. ferrumequinum (bkt1), and m. fuliginosus (yubfkt1) showed resistance against emcv and jev infection while massive cell death was observed in bat cell line derived from p. dasymallus (fbkt1). this is the first report on resistance against emcv in bat cells since the lung cells from tadarida brasilensis (tb1.lu cells) did not show resistance against emcv infection [25] . on the other hand, the resistance of bat cell lines against jev infection has been reported in a lung cell line derived from t. brasilensis and primary kidney cells derived from r. leschenaultii, which did not develop cpe until 7 dpi [26] . in this study, the demkt1, yubfkt1, and bkt1 cell lines showed limited cpe, lower replication level of emcv and jev, and higher basal expression level of prrs than other cell lines. the high basal expression level of prrs (tlr3, rig-i, and mda5) has been observed in several bat species, including r. leschenaultii, rhinolophus affinis, and desmodus rotundus [19, 27, 28 ]. this high basal expression level of prrs in the above three species of bats enables quick elimination of emcv and jev through stimulation of type i ifn production. conversely, the fbkt1 cells derived from p. dasymallus showed a low basal expression of prrs. this is consistent with previous study wherein the expression level of tlr3, rig-i, and mda5 in p. alecto kidney was very low compared to that in other tissues; however, the kidneys of r. leschenaultii and r. affinis had an expression level of tlr3, rig-i and mda5 comparable to other tissues [18,28e30] . there seems to be high species dependency of bats in innate immune system [19] . after emcv and jev infection, rig-i and mda5 were highly upregulated in demkt1 and bkt1 cells (fig. 2ced) , which possibly led to up-regulation of ifn-b to reach antiviral state (fig. 2e) , and consequently resulted in limited emcv and jev replication ( fig. 1a and b). inability of fbkt1 and hek293t cells to limit emcv replication might be due to their inability to increase the expression of prrs and ifn-b as high as that in demkt1 and bkt1 cells (fig. 2b , c, 2d, and 2e). similar to other flaviviruses, jev also can antagonizes type i ifn production to evade antiviral immunity and benefit viral replication through suppression of rig-i and mda5 to inhibit type i ifn production [31, 32] . the suppression of rig-i and tlr3 observed in fbkt1 after jev infection should have caused limited increase in ifn-b production (fig. 2b, c, and 2e ). in contrast, jev failed to suppress the prrs in bkt1, demkt1, and yubfkt1 cells. knockdown of tlr3, rig-i, and mda5 resulted in decreased expression level of ifn-b, suggesting that these prrs are important for stimulating ifn-b production in rhinolophus bat cells (bkt1 cells). increased replication level of emcv and jev after knockdown of prrs indicates that tlr3, rig-i, and mda5 are responsible for suppressing emcv and jev replication in bkt1 cells through ifn-b production. ifn-b is predominantly induced by tlr3 after emcv infection but comparable ifn-b expression level was shown in all cells knocked down for prrs after jev infection. in a previous study, knockdown of prrs in e. fuscus kidney cells also showed that ifn-b is predominantly induced by tlr3 than rig-i and mda5 after poly(i:c) transfection [17] . even though the tlr3 knockdown cells had the lowest ifn-b expression level, knockdown of tlr3 did not result in the highest emcv replication level. knockdown of mda5 resulted in the highest emcv and jev replication level among all cells knocked down for prrs ( fig. 4a and 4b ). previous studies have showed that mda5 is the dominant mediator of type i ifn and cytokine response during emcv infection in mice because of rig i degradation by emcv 3c protease [12, 16] . mda5 seemed to have a greater role in stimulating antiviral pathway in rhinolophus bat cells because it was highly up-regulated as compared to other prrs after emcv infection of bkt1 cells and was the only up-regulated prrs after jev infection (fig. 3b, c, and 3d) . it is possible that mda5 stimulates antiviral pathways other than ifn-b during emcv and jev infection of bkt1 cells. in conclusion, tlr3, rig-i, and mda5 play an important role in antiviral response against emcv and jev infections, especially in rhinolophus bats. based on the results of this study and considering the wide diversity of bats worldwide (over 1100 species), the importance of prrs in eliciting antiviral response might be variable among bat species [33] . studying immunity to zoonotic diseases in the natural hostdkeeping it real the encephalomyocarditis virus encephalomyocarditis virus is potentially derived from eastern bent-wing bats living in east asian countries can bats serve as reservoirs for arboviruses? transmission of japanese encephalitis virus from the black flying fox, pteropus alecto, to culex annulirostris mosquitoes, despite the absence of detectable viremia antiviral immune responses of bats: a review immunological control of viral infections in bats and the emergence of viruses highly pathogenic to humans interferon regulatory factor 3-mediated signaling limits middle-east respiratory syndrome (mers) coronavirus propagation in cells from an insectivorous bat dampened nlrp3-mediated inflammation in bats and implications for a special viral reservoir host toll-like receptors and rna helicases: two parallel ways to trigger antiviral responses essential role of mda5 in type i ifn responses to polyriboinosinic: polyribocytidylic acid and encephalomyocarditis picornavirus toll-like receptor 3 is an essential component of the innate stress response in virus-induced cardiac injury roles of tlr3 and rig-i in mediating the inflammatory response in mouse microglia following japanese encephalitis virus infection rig-i mediates innate immune response in mouse neurons following japanese encephalitis virus infection the viral rna recognition sensor rig-i is degraded during encephalomyocarditis virus (emcv) infection lack of inflammatory gene expression in bats: a unique role for a transcription repressor molecular characterisation of rig-i-like helicases in the black flying fox emcv (a) and jev (b) viral copy numbers in tlr3, rig-i, and mda5 knockdown cells 1 dpi with moi of 1.0. (c) cpe observed in tlr3, rig-i, and mda5 knockdown cells 2 dpi for emcv and 5 dpi for jev. statistical significance was calculated using one-way anova, followed by tukey's test development of molecular and cellular tools to decipher the type i ifn pathway of the common vampire bat isolation of novel adenovirus from fruit bat characterization of the envelope glycoprotein of a novel filovirus, lloviu virus molecular epidemiological analyses of japanese encephalitis virus isolates from swine in japan from polyethylenimine (pei)/sirna-mediated gene knockdown in vitro and in vivo a single amino acid substitution in the west nile virus nonstructural protein ns2a disables its ability to inhibit alpha/beta interferon induction and attenuates virus virulence in mice transient high level mammalian reovirus replication in a bat epithelial cell line occurs without cytopathic effect biological characters of bats in relation to natural reservoir of emerging viruses molecular cloning and expression analysis of bat toll-like receptors 3, 7 and 9 molecular characterization of rig-i, stat-1 and ifn-beta in the horseshoe bat molecular characterisation of toll-like receptors in the black flying fox pteropus alecto molecular cloning and expression analysis of bat toll-like receptors 3, 7 and 9 japanese encephalitis virus ns1 0 protein antagonizes interferon beta production west nile virus ns1 antagonizes interferon beta production by targeting rig-i and mda5 a molecular phylogeny for bats illuminates biogeography and the fossil record the authors have no financial or commercial conflicts of interest. supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2020.04.060. key: cord-328549-r56lih8j authors: okamoto, masaaki; kouwaki, takahisa; fukushima, yoshimi; oshiumi, hiroyuki title: regulation of rig-i activation by k63-linked polyubiquitination date: 2018-01-05 journal: front immunol doi: 10.3389/fimmu.2017.01942 sha: doc_id: 328549 cord_uid: r56lih8j rig-i is a pattern recognition receptor and recognizes cytoplasmic viral double-stranded rna (dsrna). influenza a virus, hepatitis c virus, and several other pathogenic viruses are mainly recognized by rig-i, resulting in the activation of the innate immune responses. the protein comprises n-terminal two caspase activation and recruitment domains (2cards), an rna helicase domain, and the c-terminal domain (ctd). the ctd recognizes 5′-triphosphate viral dsrna. after recognition of viral dsrna, the protein harbors k63-linked polyubiquitination essential for rig-i activation. first, it was reported that trim25 ubiquitin ligase delivered k63-linked polyubiquitin moiety to the 2cards. the polyubiquitin chain stabilizes a structure called the 2card tetramer, in which four 2cards assemble and make a core that promotes the aggregation of the mitochondrial antiviral-signaling (mavs) protein on mitochondria. mavs aggregation then triggers the signal to induce the innate immune responses. however, subsequent studies have reported that riplet, mex3c, and trim4 ubiquitin ligases are also involved in k63-linked polyubiquitination and the activation of rig-i. mex3c and trim4 mediate polyubiquitination of the 2cards. by contrast, riplet ubiquitinates the ctd. the physiological significance of each ubiquitin ligases has been shown by knockout and knockdown studies, but there appears to be contradictory to evidence reported in the literature. in this review, we summarize recent findings related to k63-linked polyubiquitination and propose a model that could reconcile current contradictory theories. we also discuss the physiological significance of the ubiquitin ligases in the immune system against viral infection. rig-i is a pattern recognition receptor and recognizes cytoplasmic viral double-stranded rna (dsrna). influenza a virus, hepatitis c virus, and several other pathogenic viruses are mainly recognized by rig-i, resulting in the activation of the innate immune responses. the protein comprises n-terminal two caspase activation and recruitment domains (2cards), an rna helicase domain, and the c-terminal domain (ctd). the ctd recognizes 5′-triphosphate viral dsrna. after recognition of viral dsrna, the protein harbors k63-linked polyubiquitination essential for rig-i activation. first, it was reported that trim25 ubiquitin ligase delivered k63-linked polyubiquitin moiety to the 2cards. the polyubiquitin chain stabilizes a structure called the 2card tetramer, in which four 2cards assemble and make a core that promotes the aggregation of the mitochondrial antiviral-signaling (mavs) protein on mitochondria. mavs aggregation then triggers the signal to induce the innate immune responses. however, subsequent studies have reported that riplet, mex3c, and trim4 ubiquitin ligases are also involved in k63-linked polyubiquitination and the activation of rig-i. mex3c and trim4 mediate polyubiquitination of the 2cards. by contrast, riplet ubiquitinates the ctd. the physiological significance of each ubiquitin ligases has been shown by knockout and knockdown studies, but there appears to be contradictory to evidence reported in the literature. in this review, we summarize recent findings related to k63-linked polyubiquitination and propose a model that could reconcile current contradictory theories. we also discuss the physiological significance of the ubiquitin ligases in the immune system against viral infection. keywords: rig-i, ubiquitin, innate immunity, virus, signaling pathway introduction pattern recognition receptors (prrs) recognize viral nucleic acids and trigger a signal to induce the innate immune responses during viral infection (1, 2) . rig-i is a cytoplasmic rna helicase and a prr that recognizes cytoplasmic 5′ tri-or diphosphate double-stranded rna (dsrna) (3) (4) (5) . rig-i binds relatively short dsrna (<1 kbp) and is involved in the recognition of various viral infections, such as influenza a and b viruses, japanese encephalitis virus, hepatitis c virus (hcv), dengue virus, and west nile virus (6) (7) (8) . after recognition of viral rna, rig-i associates with an adaptor protein, mitochondrial antiviral-signaling (mavs) protein, also called ips-1, cardif, and visa (9) (10) (11) (12) , resulting in the aggregation of mavs on the outer membrane of mitochondria. this ubiquitination of rig-i frontiers in immunology | www.frontiersin.org january 2018 | volume 8 | article 1942 aggregation triggers a signal to induce the expression of type i interferon (ifn) and other inflammatory cytokines (13) . the rig-i protein comprises two caspase-activation and recruitment domains (2cards) at the n-terminal region, an rna helicase domain, and a c-terminal domain (ctd) (14) (15) (16) . viral dsrna binds to the rna helicase domain and the ctd, and 5′ tri-and diphosphate are recognized by the ctd (16, 17) . the n-terminal 2cards are responsible for the association with mavs and, therefore, are required for triggering downstream signaling (5) . in resting cells, the c-terminal region, which includes the ctd and the linker region between the ctd and the helicase domain, suppresses the n-terminal 2cards (14, 18) . binding of the ctd to dsrna induces the conformational change of the rig-i protein, resulting in the release of the 2cards (14) . subsequently, the proteins assemble along viral dsrna and form a nucleoprotein filament (19) . the released 2cards also assemble and form a 2card tetramer structure (20) . the structure functions as a core for mavs aggregation on mitochondria (21) . the rig-i protein harbors lys 63-linked (k63-linked) polyubiquitination required for its activation (22) . trim25 is a ubiquitin ligase and delivers k63-linked polyubiquitin moiety to the rig-i 2cards (22, 23) . the polyubiquitin chains stabilize the 2card tetramer structure (21) . the physiological significance of trim25 in rig-i activation has been shown by several studies (22) (23) (24) (25) (26) (27) . however, recent studies have reported three other ubiquitin ligases, ring finger protein leading to rig-i activation (riplet), mex-3 rna-binding family member c (mex3c), and trim4, which are required for the polyubiquitination and activation of rig-i (28-30). ubiquitin ligases add a ubiquitin chain at k but not r residues of the target protein. there are 18 k residues in the rig-i 2cards, and mass spectrometry analysis revealed that the 2cards fragment carried k63-linked polyubiquitin chains at k99, k169, k172, k181, k190, and k193 (22) . knockdown of trim25 abrogated polyubiquitination of the 2cards fragment, suggesting that trim25 mediates k63-linked polyubiquitination at the 6 k residues of the 2cards (22) . the 2cards fragment has an ability to bind to mavs, and overexpression of the 2cards fragment leads to auto-activation of signaling (5, (9) (10) (11) (12) . an amino acid substitution assay revealed that the substitution of k172, but not of other k residues, with r abrogated the signaling induced by the 2cards fragment (22) . knockout of trim25 severely reduced rig-i-mediated type i ifn production during viral infection. these observations indicate the importance of trim25-mediated k172 ubiquitination (22) . evidence also suggests that trim25 produces unanchored k63-linked polyubiquitin chains in response to viral infection and delivered them to rig-i (23) . the same study also showed that the k172 residue of rig-i was important for non-covalent binding of rig-i with unanchored polyubiquitin chains (23) . considering that mass spectrometry analysis revealed the covalent binding of rig-i with k63-linked polyubiquitin chains, these observations indicate that either covalent or non-covalent binding with polyubiquitin chains is sufficient for rig-i 2cards activation (23, 26) . a structural study of rig-i 2cards tetramer provided evidence that both covalent and non-covalent binding of polyubiquitin chains promotes the formation of the 2card tetramer structure (21, 26) . the trim25 activity itself is regulated by the physical interaction between the trim25 spry domain and rig-i 2cards (31) . the cooperative assembly of trim25 and rig-i facilitates the dimerization of the trim25 ring domain, which is required for trim25 to make polyubiquitin chain (31) . an accumulating body of evidence has shown that trim25 delivers k63-linked polyubiquitin moiety to rig-i 2cards for rig-i activation and that the k172 residue is important for the binding of rig-i to polyubiquitin chains (26, 27, 32) . however, subsequent studies revealed that not only k172 but also other k residues are also important for the binding of rig-i to k63linked polyubiquitin chains. first, shigemoto et al. reported that the expression of the rig-i k172r full-length protein could compensate for a defect in rig-i knockout mouse embryonic fibroblasts (mefs) after sendai virus infection (33) . second, two other ubiquitin ligases, mex3c and trim4, were reported to mediate polyubiquitination of the rig-i 2cards at other k residues (29, 30) . kuniyoshi (30) . mass spectrometry analysis has also revealed the ubiquitination at k48, k96, k170, as well as k172 and k190 of the 2cards (34) . they reported that simultaneous amino acids substitutions at k48, k96, and k172 substantially reduced the polyubiquitination of rig-i (34). these observations suggest that k63-linked polyubiquitination at these k residues can compensate for the loss of k172 binding to the ubiquitin chain under certain conditions (figure 1 ). riplet, another ubiquitin ligase, is also involved in k63-linked polyubiquitination and activation of rig-i. riplet was first isolated by our yeast two-hybrid screening using the rig-i ctd fragment as a bait (28) . an immunoprecipitation assay then confirmed that the protein bound to the ctd fragment, and our studies also indicated that riplet mediates k63-linked polyubiquitination of the rig-i ctd (28) . our mutation analysis indicated that k849 and k851 of the ctd were important for riplet-mediated polyubiquitination, and riplet also targeted other k residues, including k888, k907, and k909 of the ctd and k788 in the linker region between the ctd and the helicase domain (24, 28) (figure 1) . to assess the physiological significance of the protein, we generated riplet knockout mice. knockout of riplet severely impaired the type i ifn and il-6 production in mef, macrophages, and conventional dendritic cells following influenza a virus and an rig-i c-terminal fragment (735-925 aa region), which includes the linker region and the ctd, suppresses the 2card activation (14) , and kageyama et al. reported that the linker region (746-801 aa) was responsible for the auto-suppression (18) . riplet targets the k788 in the linker region. therefore, it is possible that the ubiquitination of the linker region disrupts the auto-suppression. the structure analysis revealed that k849, k851, k858, and k888 of the ctd bind to 5′ triphosphate of dsrna ends (36) . the k849, k851, and k888 of the ctd are targeted by riplet. binding of the ctd to 5′ triphosphate of dsrna was reported to induce conformational change of the rig-i protein (16) . although several rig-i molecules assemble along dsrna, only one rig-i molecule binds the 5′ triphosphate at the dsrna end (19) (figure 2a) . therefore, riplet could access the k849, k851, and k888 of the ctds of the rig-i molecules associating with dsrna (but not the end of dsrna) to induce conformational change of rig-i. these k residues are located at the edge of the ctd basic cleft, which is an rna-binding site (16) . further studies are required to reveal underlying mechanism of riplet-mediated rig-i activation. despite the identification of four ubiquitin ligases, we found that knockout of riplet alone could abolish the polyubiquitination of the endogenous rig-i protein (24, 35) . recently, shi et al. also reported that knockout of riplet is sufficient to abolish the polyubiquitination of rig-i and, therefore, claimed that riplet is a primary ubiquitin ligase and mediates k63-linked polyubiquitination of the 2cards (34) . however, their model appears to be contradict to previous papers showing that trim25 plays a crucial role in rig-i activation. previously, we have postulated a sequential ubiquitination model that riplet-mediated polyubiquitination of rig-i c-terminal region is a prerequisite for the polyubiquitination of the 2cards (figure 2) (24) . this model could explain the apparent discrepancy in the literature, because due to the initial failure to polyubiquitinate the c-terminal region, this would obstruct the subsequent polyubiquitination of the 2cards by other ubiquitin ligases. this indicates that knockout of riplet alone is sufficient to abolish the polyubiquitination of the ctd, the linker region, and the 2cards. in a previous study, we have shown that riplet promotes the binding of trim25 to rig-i (24). this observation supports the sequential model. it is expected that riplet-mediated polyubiquitination leads to the release of auto-suppression and/or conformational change of rig-i, which would allow the access of trim25 to the 2cards and/or promote rig-i assembly along dsrna (figure 2b) . considering that higher-order oligomerization of trim25 with the 2cards is required to induce trim25-mediated polyubiquitination (31), it is not surprising that riplet-mediated c-terminal ubiquitination is a prerequisite for the second ubiquitination by trim25 (figure 2b) . mex3c or trim4 might compensate for the defect of trim25 under certain experimental conditions, because these two ubiquitin ligases target the 2cards in a similar way (figure 1) . although we failed to detect an interaction between riplet and the 2cards fragment, other groups have reported that riplet bound to the 2cards fragment and was involved in the k63-linked polyubiquitination of the 2cards (34, 37) . these observations do not conflict with the sequential ubiquitination model because several ubiquitin ligases can compensate for the loss of trim25 in some conditions. we do not exclude the possibility that riplet is not only involved in the primary ubiquitination of the ctd and the linker region but also the secondary ubiquitination of the 2cards (figure 2 ). type i ifn exhibits a strong antiviral effect, and hence several viruses have evolved to suppress the type i ifn production. hcv is a major cause of hepatocellular carcinoma and persistently infects hepatocytes over several decades without exclusion by the host immune system. a viral ns3-4a protease is required to cleave viral polypeptides and produce mature viral proteins; however, it is also important to suppress the host innate immune response. ns3-4a of hcv cleaves mavs, which results in the release of mavs from mitochondria (12) . several reports have shown that released mavs protein fails to trigger signaling to induce type i ifn production (9) . accordingly, ns3-4a-mediated cleavage of mavs abrogates rig-i-mediated type i ifn production. ns3-4a protein also targets the riplet protein. the ring finger domain is a catalytic domain of the riplet protein, and viral ns3-4a protease cleaves the domain and destabilizes the protein (24) . ns1 protein of influenza a virus also has the ability to suppress type i ifn production (38) . although several mechanisms have been postulated, gack et al. reported that viral ns1 bound to trim25 and inhibited trim25-mediated polyubiquitination of rig-i (39) . in further study, they reported that ns1 protein also targeted the riplet protein and inhibited rig-i polyubiquitination (40) . severe acute respiratory syndrome coronavirus (sars-cov) also interferes trim25 function (41) . the nucleocapsid protein of sars-cov physically interacted with trim25 and inhibited the binding of trim25 to rig-i, resulting in the attenuation of rig-i-signaling (41) . these data imply that viruses obtained the ability to suppress the ubiquitin ligases to escape innate immune responses. conversely, these data indicate the importance of the two ubiquitin ligases, trim25 and riplet, for the antiviral innate immune response. trim25 is also called efp, and it has been shown that trim25/ efp mediates the polyubiquitination of 14-3-3σ and promotes its proteolysis to suppress the growth of breast tumor cells (42) . although other targets of riplet have not been reported, it was shown that mutations on human riplet genes (also called rnf135) are linked to learning disabilities and several neuropsychiatric disorders (43, 44) . thus, it is expected that riplet targets the proteins involved in these conditions. there are several reports that riplet and trim25 are involved in tumorigenesis (42, 45, 46) . as several viruses have the ability to abrogate the described ubiquitin ligases, it is expected that viral protein-mediated inhibition of the ubiquitin ligases affects both innate immunity and other phenomena, such as virus-induced tumorigenesis and neuropsychiatric disorders. there are two protein families related to rig-i called rig-ilike receptors (rlrs). lgp2 is an rlr, and the ctd structure of the protein is similar to that of rig-i (14) . initial studies reported that lgp2 is a negative regulator for rig-i signaling (15, 47) . however, knockout and biochemical studies have revealed that lgp2 functions as a positive regulator of the rig-i pathway (48, 49) . lgp2 is also expressed in cd8 + t cells and is required for cd8 + t cell proliferation (50) . it remains unclear whether lgp2 carries a ubiquitin chain. considering the conservation of the ctd between rig-i and lgp2, it is possible that riplet also targets the ctd of lgp2 and affects lgp2-mediated rig-i activation and cd8 + t cell proliferation. further studies are required to fully elucidate the role of the ubiquitin ligases in the antiviral immune response. ho and mo wrote the manuscript. tk and yf helped the discussion. immune signaling by rig-i-like receptors toll-like receptors and their crosstalk with other innate receptors in infection and immunity antiviral immunity via rig-i-mediated recognition of rna bearing 5'-diphosphates 5'-triphosphate rna is the ligand for rig-i the rna helicase rig-i has an essential function in double-stranded rna-induced innate antiviral responses length-dependent recognition of double-stranded ribonucleic acids by retinoic acid-inducible gene-i and melanoma differentiation-associated gene distinct rig-i and mda5 signaling by rna viruses in innate immunity differential roles of mda5 and rig-i helicases in the recognition of rna viruses identification and characterization of mavs, a mitochondrial antiviral signaling protein that activates nf-kappab and irf 3 ips-1, an adaptor triggering rig-i-and mda5-mediated type i interferon induction visa is an adapter protein required for virus-triggered ifn-beta signaling cardif is an adaptor protein in the rig-i antiviral pathway and is targeted by hepatitis c virus mavs forms functional prion-like aggregates to activate and propagate antiviral innate immune response regulation of innate antiviral defenses through a shared repressor domain in rig-i and lgp2 shared and unique functions of the dexd/h-box helicases rig-i mda5, and lgp2 in antiviral innate immunity nonself rna-sensing mechanism of rig-i helicase and activation of antiviral immune responses the c-terminal regulatory domain is the rna 5'-triphosphate sensor of rig-i 55 amino acid linker between helicase and carboxyl terminal domains of rig-i functions as a critical repression domain and determines inter-domain conformation rig-i forms signaling-competent filaments in an atp-dependent, ubiquitin-independent manner structural basis for dsrna recognition, filament formation, and antiviral signal activation by mda5 structural basis for ubiquitin-mediated antiviral signal activation by rig-i trim25 ringfinger e3 ubiquitin ligase is essential for rig-i-mediated antiviral activity reconstitution of the rig-i pathway reveals a signaling role of unanchored polyubiquitin chains in innate immunity a distinct role of ripletmediated k63-linked polyubiquitination of the rig-i repressor domain in human antiviral innate immune responses hepatitis c virus reveals a novel early control in acute immune response post-translational control of intracellular pathogen sensing pathways viral evasion of intracellular dna and rna sensing riplet/rnf135, a ring finger protein, ubiquitinates rig-i to promote interferon-beta induction during the early phase of viral infection pivotal role of rna-binding e3 ubiquitin ligase mex3c in rig-i-mediated antiviral innate immunity trim4 modulates type i interferon induction and cellular antiviral response by targeting rig-i for k63-linked ubiquitination mechanism of trim25 catalytic activation in the antiviral rig-i pathway ubiquitin-mediated modulation of the cytoplasmic viral rna sensor rig-i identification of loss of function mutations in human genes encoding rig-i and mda5: implications for resistance to type i diabetes ube2d3 and ube2n are essential for rig-i-mediated mavs aggregation in antiviral innate immunity the ubiquitin ligase riplet is essential for rig-i-dependent innate immune responses to rna virus infection visualizing the determinants of viral rna recognition by innate immune sensor rig-i reul is a novel e3 ubiquitin ligase and stimulator of retinoic-acid-inducible gene-i viral infection switches non-plasmacytoid dendritic cells into high interferon producers influenza a virus ns1 targets the ubiquitin ligase trim25 to evade recognition by the host viral rna sensor rig-i species-specific inhibition of rig-i ubiquitination and ifn induction by the influenza a virus ns1 protein the severe acute respiratory syndrome coronavirus nucleocapsid inhibits type i interferon production by interfering with trim25-mediated rig-i ubiquitination efp targets 14-3-3 sigma for proteolysis and promotes breast tumour growth mutations in rnf135, a gene within the nf1 microdeletion region, cause phenotypic abnormalities including overgrowth mutation screening of the ubiquitin ligase gene rnf135 in french patients with autism ring finger protein, promotes the proliferation of human glioblastoma cells in vivo and in vitro via the erk pathway the e3 ubiquitin ligase rnf135 regulates the tumorigenesis activity of tongue cancer scc25 cells loss of dexd/h box rna helicase lgp2 manifests disparate antiviral responses lgp2 is a positive regulator of rig-i-and mda5-mediated antiviral responses the innate immune sensor lgp2 activates antiviral signaling by regulating mda5-rna interaction and filament assembly the rig-i-like receptor lgp2 controls cd8(+) t cell survival and fitness the authors thank dr. t. seya and dr. m. matsumoto for their helpful discussion. this work was supported in part by grantsin-aid from ministry of education, science, and culture, and presto jst. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-257052-cik2wmlk authors: ban, junsu; lee, na-rae; lee, noh-jin; lee, jong kil; quan, fu-shi; inn, kyung-soo title: human respiratory syncytial virus ns 1 targets trim25 to suppress rig-i ubiquitination and subsequent rig-i-mediated antiviral signaling date: 2018-12-14 journal: viruses doi: 10.3390/v10120716 sha: doc_id: 257052 cord_uid: cik2wmlk respiratory syncytial virus (rsv) causes severe acute lower respiratory tract disease. retinoic acid-inducible gene-i (rig-i) serves as an innate immune sensor and triggers antiviral responses upon recognizing viral infections including rsv. since tripartite motif-containing protein 25 (trim25)-mediated k63-polyubiquitination is crucial for rig-i activation, several viruses target initial rig-i activation through ubiquitination. rsv ns1 and ns2 have been shown to interfere with rig-i-mediated antiviral signaling. in this study, we explored the possibility that ns1 suppresses rig-i-mediated antiviral signaling by targeting trim25. ubiquitination of ectopically expressed rig-i-2cards domain was decreased by rsv infection, indicating that rsv possesses ability to inhibit trim25-mediated rig-i ubiquitination. similarly, ectopic expression of ns1 sufficiently suppressed trim25-mediated rig-i ubiquitination. furthermore, interaction between ns1 and trim25 was detected by a co-immunoprecipitation assay. further biochemical assays showed that the spry domain of trim25, which is responsible for interaction with rig-i, interacted sufficiently with ns1. suppression of rig-i ubiquitination by ns1 resulted in decreased interaction between rig-i and its downstream molecule, mavs. the suppressive effect of ns1 on rig-i signaling could be abrogated by overexpression of trim25. collectively, this study suggests that rsv ns1 interacts with trim25 and interferes with rig-i ubiquitination to suppress type-i interferon signaling. respiratory syncytial virus (rsv) belongs to the family pneumoviridae and contains a negative-sense single-stranded rna genome. rsv infection is a leading cause of severe acute lower respiratory tract disease and related hospitalization in children and the elderly [1, 2] . despite the global burden from rsv infection, there are no available rsv-specific vaccines or effective therapeutic agents at present. the cellular innate immune system utilizes various sensors including retinoic acid inducible gene-i (rig-i) and toll-like receptors (tlrs) to detect viral infection and activate antiviral immune signaling pathways [3, 4] . among these, rig-i and toll-like receptor 3 (tlr3) have been implicated in early antiviral immune responses against rsv infection in airway epithelial cells [5] . deficiency cells were collected and lysed in triton x-100 cell lysis buffer (50 mm tris-hcl, 150 mm nacl, 5mm edta, 0.1% triton x-100) containing protease and phosphatase inhibitor cocktail (thermo scientific, waltham, ma, usa). lysates were incubated overnight at 4 • c with the corresponding antibody. after the binding reaction, protein a/g resin (sigma, st. louis, mo, usa) were added to samples and further incubated for 2 h at room temperature. the resin was then washed four to five times with cell lysis buffer. anti-flag antibody conjugated resin (sigma) and anti-v5 antibody conjugated resin (sigma) were also used for co-ip in a similar manner. precipitated proteins were eluted with sds sample buffer (250 mm tris-hcl, 10% sds, 30% glycerol, 25% mercaptoethanol, 0.05% bromophenol blue) and subjected to sds-page and immunoblotting. to precipitate gst-tagged fusion proteins, the cell lysates were incubated with glutathioneconjugated beads (sigma) at rt for 1-2 h. after incubation, the beads were washed four to five times with cell lysis buffer, followed by sds-page and immunoblotting. after polyacrylamide gel electrophoresis, target proteins on the gel were transferred to a pvdf membrane (millipore, st. charles, mo, usa). the membrane was incubated overnight at 4 • c with the indicated antibody in 5% bovine serum albumin (bsa, rmbio, missoula, mt, usa) solution for binding. after extensive washing with pbst, the membrane was then incubated with the horseradish peroxidase (hrp) conjugated secondary antibody at rt for 2 h. luminata forte (millipore) was used as the hrp substrate. anti-v5 rabbit antibody (cell signaling, denvers, ma, usa, #13202), anti-v5 mouse antibody (e-bioscience, san diego, ca, usa, #14-6796-80), anti-ha mouse antibody (santacruz, santa cruz, ca, usa, #sc-7392), anti-flag mouse antibody (sigma, #f7425), anti-ubiquitin (p4d1) mouse antibody (cell signaling, #3936), anti-gst antibody (abcam, cambridge, uk, #19256) and hrp conjugated antibodies (cell signaling, #7074, #7076) were used. hek293t cells in 24-well plates were transfected with interferon-β firefly luciferase (0.25 µg/well) and thymidine kinase (tk) renilla reporter plasmids (0.13 µg/well) were transfected along with other constructs as indicated [21] . after 24 h, transfected hek293t cells were lysed in passive lysis buffer (promega, madison, wi, usa) at rt for 15 min. cell lysates were then analyzed by a dual-luciferase assay according to the instruction (promega). all assays were performed in triplicate and repeated at least three times. total rnas were extracted using trizol (thermo scientific, #15596018) according to the instruction. cdnas were generated from rnas (1 µg) using superscript iii reverse transcriptase (thermo scientific, #18080093) and oligo 20 (dt) primers. real-time pcr was conducted using synthesized cdna (2 µl). the mrna levels of interferon-β and interferon-stimulated gene 15 (isg15) were determined using the following primers and normalized to those of β-actin: interferon-β-forward; 5 -aagagttacactgcctttgccatc-3 , interferon-β-reverse; 5 -cactgtctgctggtggagttcatc-3 , isg15-forward; 5 -cctctgagcatcctggt-3 , isg15-reverse; 5 -aggccgtactcccccag-3 , β-actin-forward; 5 -tggaatcctgtggcatccatgaaac-3 , β-actin-reverse; 5 -taaaacgcagctcagtaacagtccg-3 . hek293t cells were seeded onto fibronectin coated coverslips in a 24-well plate. after 24 h, cells were transfected with flag-tagged rsv ns1 and v5-tagged trim25 and incubated for 24 h. then, cells were fixed with 4% paraformaldehyde followed by blocking and permeabilization in viruses 2018, 10, 716 4 of 12 permeabilization buffer (0.5% bsa, 0.2% triton x-100 in pbs) at rt in 5 min. the cells were incubated with anti-flag mouse and anti-v5 rabbit antibodies in 0.5% bsa solution at 4 • c overnight. next day, the cells were extensively washed and incubated with fitc-and pe-labeled secondary antibodies at rt for 2 h. the co-localization between rsv ns1-flag and trim25-v5 was analyzed by nanoscope k1-fluo confocal microscope. data were presented as the mean ± sem. statistical comparisons between the control and treated groups were performed using the student's t-test. a value of p ≤ 0.05 was considered to be significant. to confirm the suppression of rig-i-mediated signaling by ns1, rsv ns1 was transfected along with constitutively active rig-i-2cards (rig-in). ectopically expressed ns1 inhibited interferon-β promoter activity that was induced by rig-in as determined by the luciferase assays in hek293t cells, confirming that ns1 itself is capable of inhibiting rig-i-mediated antiviral signaling ( figure 1a ). consistently, rig-in-mediated induction of interferon-β and isg15 mrna synthesis was significantly hampered by ectopic expression of ns1 ( figure 1b ). similar results were obtained from experimental settings using a549 and hep-2 cells, suggesting that ns1 is able to suppress rig-i-mediated type-i interferon production in various cells including airway epithelial cells ( figure 1c ,d). incubated with anti-flag mouse and anti-v5 rabbit antibodies in 0.5% bsa solution at 4·°c overnight. next day, the cells were extensively washed and incubated with fitc-and pe-labeled secondary antibodies at rt for 2 h. the co-localization between rsv ns1-flag and trim25-v5 was analyzed by nanoscope k1-fluo confocal microscope. data were presented as the mean ± sem. statistical comparisons between the control and treated groups were performed using the student's t-test. a value of p ≤ 0.05 was considered to be significant. to confirm the suppression of rig-i-mediated signaling by ns1, rsv ns1 was transfected along with constitutively active rig-i-2cards (rig-in). ectopically expressed ns1 inhibited interferon-β promoter activity that was induced by rig-in as determined by the luciferase assays in hek293t cells, confirming that ns1 itself is capable of inhibiting rig-i-mediated antiviral signaling ( figure 1a ). consistently, rig-in-mediated induction of interferon-β and isg15 mrna synthesis was significantly hampered by ectopic expression of ns1 ( figure 1b ). similar results were obtained from experimental settings using a549 and hep-2 cells, suggesting that ns1 is able to suppress rig-imediated type-i interferon production in various cells including airway epithelial cells (figs. 1c and 1d). because trim25-mediated rig-i card ubiquitination is an essential step for the successful induction of rig-i-mediated antiviral responses, we explored the possibility that rsv suppresses rig-i activation by inhibiting the trim25-mediated rig-ubiquitination. first, we have tested whether rig-i ubiquitination is suppressed by rsv infection. as seen in figure 2a , ubiquitination of ectopically expressed gst-rig-in is decreased by the presence of rsv (multiplicity of infection = 4), suggesting that rsv is capable of suppressing rig-i ubiquitination to modulate rig-i signaling. next, we have tested whether rig-i ubiquitination is affected by the presence of ns1. as shown in figure 2b , both ns1 and ns2 suppressed the trim25-mediated ubiquitination of rig-in. increasing amounts of ns1 resulted in an augmented effect on reducing the ubiquitinated form of rig-in ( figure 2c ). the results clearly showed that ns1 is capable of interfering with trim25-mediated rig-i ubiquitination to suppress rig-i-mediated interferon production. were repeated at least three times. the results show the most representative data from a single experiment conducted in triplicate. because trim25-mediated rig-i card ubiquitination is an essential step for the successful induction of rig-i-mediated antiviral responses, we explored the possibility that rsv suppresses rig-i activation by inhibiting the trim25-mediated rig-ubiquitination. first, we have tested whether rig-i ubiquitination is suppressed by rsv infection. as seen in figure 2a , ubiquitination of ectopically expressed gst-rig-in is decreased by the presence of rsv (multiplicity of infection = 4), suggesting that rsv is capable of suppressing rig-i ubiquitination to modulate rig-i signaling. next, we have tested whether rig-i ubiquitination is affected by the presence of ns1. as shown in figure 2b , both ns1 and ns2 suppressed the trim25-mediated ubiquitination of rig-in. increasing amounts of ns1 resulted in an augmented effect on reducing the ubiquitinated form of rig-in ( figure 2c ). the results clearly showed that ns1 is capable of interfering with trim25-mediated rig-i ubiquitination to suppress rig-i-mediated interferon production. interaction between rig-in and ns1 was examined using a gst-pulldown assay to test whether ns1 interacts with rig-in similar to ns2. however, we could not detect any interaction (data not interaction between rig-in and ns1 was examined using a gst-pulldown assay to test whether ns1 interacts with rig-in similar to ns2. however, we could not detect any interaction (data not shown). therefore, interaction between trim25 and ns1 was investigated to determine whether ns1 targets trim25 to suppress rig-i ubiquitination. obvious interaction between ectopically expressed viruses 2018, 10, 716 6 of 12 trim25 and ns1 was detected by the co-immunoprecipitation assay, whereas interaction between ns2 and trim25 was not detected ( figure 3a) . furthermore, endogenous trim25 was also co-precipitated with ns1, supporting that rsv ns1 targets trim25 ( figure 3b ). co-localization of ns1 and trim25 in the cytoplasm was detected by confocal microscopic observation ( figure 3c ). to further dissect the interaction between ns1 and trim25, we determined the domain of trim25 that is responsible for this interaction using truncated trim25 domains. flag-tagged ns1 was expressed along with the ring domain, b-box/ccd domain, or spry domain of trim25 and co-immunoprecipitation assays were performed. as depicted in figure 3d , the spry domain sufficiently interacts with ns1, whereas the ring or b-box/ccd domains do not show any interaction. in addition, the spry deleted trim25 mutant failed to interact with ns1, indicating that the spry domain is required for interaction with ns1 ( figure 3e ). figure 4a , figure 4a ). to further confirm the effect of ns1 on rig-i activation, the effect of ns1 ectopic expression on rig-in interaction with mavs was examined. flag-tagged mavs-card-prd and gst-tagged rig-in was co-expressed with increasing amounts of v5-tagged ns1 followed by a co-immunoprecipitation assay using an anti-flag antibody. as seen in figure 4b ,c, the clear interaction between rig-in and mavs-card-prd was decreased by ns1 expression in a dose-dependent manner. these results suggest that rsv ns1 expression diminishes the interaction between rig-i and mavs by interfering with trim25-mediated rig-i ubiquitination. since trim25 oligomerization is crucial for its e3-ligase activity, it has been tested whether ns1 suppresses trim25 e3-ligase activity by interfering the oligomerization. as seen in figure 4d , expression of ns1 did not affect the interaction between interaction between ha-trim25 and v5-trim25 was analyzed by co-ip and immunoblotting using indicated antibodies. all experiments were conducted at least three times with similar results. to confirm that interaction between ns1 and trim25 contributes to the interferon-suppressive effect of ns1, the effect of trim25 overexpression on the suppression of rig-i signaling by ns1 was investigated using interferon-β luciferase promoter assays. as shown in figure 5a , activation of interferon-β promoter activity by polyi:c transfection was suppressed by ns1 expression. the effect of ns1 was abrogated by the overexpression of trim25 ( figure 5a) . similarly, suppression of rig-in-induced interferon-β promoter activity by ns1 was also reversed by the ectopic expression of trim25 ( figure 5b ). these results indicate that ns1 interaction with trim25 contributes to its rig-i suppressive activity. co-ip and immunoblotting using indicated antibodies. all experiments were conducted at least three times with similar results. to confirm that interaction between ns1 and trim25 contributes to the interferon-suppressive effect of ns1, the effect of trim25 overexpression on the suppression of rig-i signaling by ns1 was investigated using interferon-β luciferase promoter assays. as shown in figure 5a , activation of interferon-β promoter activity by polyi:c transfection was suppressed by ns1 expression. the effect of ns1 was abrogated by the overexpression of trim25 ( figure 5a) . similarly, suppression of rig-in-induced interferon-β promoter activity by ns1 was also reversed by the ectopic expression of trim25 ( figure 5b ). these results indicate that ns1 interaction with trim25 contributes to its rig-i suppressive activity. many viruses possess defensive mechanisms against the host immune system for their effective replication. the ns1 and ns2 proteins of rsv have been shown to effectively inhibit the host immune system. ns proteins target diverse proteins related to type-i interferon induction and signal transduction. for example, rsv ns1 upregulates socs1 and socs3 and triggers stat2 degradation [12] . rsv ns1 also inhibits the interferon alpha response by targeting the interferon alpha receptor [13] . ns2 also degrades stat2 to downregulate interferon-mediated jak-stat signaling responses [14] . in addition, ns1 and ns2 exert suppressive activities on the rig-i-mediated antiviral signaling pathway, which is a crucial response against rsv infection. previous studies suggest that ns1 and ns2 inhibit rig-i-mediated signaling by inducing degradation of key molecules such as irf3/7 [21, 22] . in this study, we explored the possibility that these proteins interfere with rig-i signaling by directly inhibiting rig-i activation. indeed, we could demonstrate that ns1 and ns2 suppressed trim25-mediated ubiquitination of rig-i, which is a crucial step for rig-i activation. previously, ns2 has been shown to interact with rig-i, whereas the interaction between ns1 and rig-i could not be detected [15] . thus, suppression of rig-i ubiquitination by ns2 may be due to its interaction with many viruses possess defensive mechanisms against the host immune system for their effective replication. the ns1 and ns2 proteins of rsv have been shown to effectively inhibit the host immune system. ns proteins target diverse proteins related to type-i interferon induction and signal transduction. for example, rsv ns1 upregulates socs1 and socs3 and triggers stat2 degradation [12] . rsv ns1 also inhibits the interferon alpha response by targeting the interferon alpha receptor [13] . ns2 also degrades stat2 to downregulate interferon-mediated jak-stat signaling responses [14] . in addition, ns1 and ns2 exert suppressive activities on the rig-i-mediated antiviral signaling pathway, which is a crucial response against rsv infection. previous studies suggest that ns1 and ns2 inhibit rig-i-mediated signaling by inducing degradation of key molecules such as irf3/7 [21, 22] . in this study, we explored the possibility that these proteins interfere with rig-i signaling by directly inhibiting rig-i activation. indeed, we could demonstrate that ns1 and ns2 suppressed trim25-mediated ubiquitination of rig-i, which is a crucial step for rig-i activation. previously, ns2 has been shown to interact with rig-i, whereas the interaction between ns1 and rig-i could not be detected [15] . thus, suppression of rig-i ubiquitination by ns2 may be due to its interaction with rig-i. we also could not detect interaction between ns1 and rig-i, indicating that ns1 may utilize a separate molecular mechanism to suppress rig-i ubiquitination. in addition, a previous study showed that ns1 co-localizes with mavs and inhibits rig-i interaction with mavs [16] . considering that rig-i ubiquitination is required for its oligomerization and interaction with mavs, it is conceivable that ns1 suppresses rig-i interaction with mavs by interfering with rig-i ubiquitination, as shown in the current study. thus, it was tempting to test the hypothesis that ns1 targets trim25, an e3-ubiquitin ligase responsible for rig-i ubiquitination. moreover, several viruses utilize their proteins to interfere with trim25 activation and subsequent rig-i activation. for instance, the influenza ns1 binds to trim25 and inhibits trim25 multimerization [10] . the sars coronavirus nucleocapsid protein binds to trim25 spry and disrupts rig-i ubiquitination [23] . in this study, the interaction between ns1 and trim25 was demonstrated. since several studies have shown that trim25 migrates to the mitochondria upon viral infection to interact with mavs [24] , the interaction between mavs and ns1 might be a result of the interaction between ns1 and trim25. as expected, ns1 inhibited the interaction between rig-i card and mavs. these results clearly indicate that ns1 is capable of suppressing rig-i activation by targeting trim25 and inhibiting rig-i ubiquitination through trim25. we have shown that ns1 could suppress the interferon-β promoter activity induced by rig-in and mavs, indicating that ns1 is also capable of suppressing downstream signaling. this can be explained by previous studies showing that ns1 can trigger the degradation of critical downstream molecules such as irf3. nonetheless, we have shown that ns1 can suppress trim25-mediated ubiquitination of rig-in without affecting its amount and that its effect on rig-in-mediated interferon-β promoter activity can be reversed by an excessive amount of trim25. these results indicate that inhibition of trim25-mediated rig-i ubiquitination by ns1 contributes to the suppression of rig-i signaling, at least in part. interestingly, the biochemical domain mapping study revealed that the spry domain of trim25 is responsible for interaction with ns1. given that the spry domain is responsible for interaction with rig-i, a possible molecular mechanism is that ns1 binds to trim25 and sequesters it to prevent its interaction with rig-i. however, we could not observe a significant reduction in interaction between rig-i and trim25 by ns1 (data not shown). further studies are needed to identify the detailed molecular mechanism of rsv ns1-mediated trim25 dysfunction. rsv ns2 is known to bind with rig-i card and disrupts its activation [15] and we showed that rsv ns1 interacts with trim25 to decrease card ubiquitination. in a previous study, rsv ns1 and ns2 have been shown to exist as homo-or heterodimers in mammalian cells. the rsv ns1-ns2 dimer was detected in mitochondria, suggesting that ns1 and ns2 can cooperate to suppress rig-i signaling [18] . it is also possible that the dimer of rsv ns1 and ns2 interacts with trim25 and rig-i card through each ns protein interacting with trim25 or rig-i card. although we could not observe a significant synergistic effect of ns1 and ns2 co-expression in terms of suppression of rig-i ubiquitination ( figure 1c) , the ns1 and ns2 interaction may have additional functions in rig-i signaling. for instance, it is possible that the ns1/ns2 complex induces rig-i degradation followed by suppression of rig-i k63-ubiquitination for complete inhibition of the rig-i signaling pathway. collectively, this study demonstrates that rsv ns1 interacts with trim25 and suppresses trim25-mediated rig-i activation to evade rig-i-mediated antiviral responses. further studies including structural analysis of protein interaction and analysis of the interaction modes between rig-i, trim25, ns1 and ns2 need to be conducted. the burden of respiratory syncytial virus infection in young children global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis pathogen recognition and innate immunity intracellular pattern recognition receptors in the host response retinoic acid-inducible gene i mediates early antiviral response and toll-like receptor 3 expression in respiratory syncytial virus-infected airway epithelial cells mavs and myd88 are essential for innate immunity but not cytotoxic t lymphocyte response against respiratory syncytial virus the rna helicase rig-i has an essential function in double-stranded rna-induced innate antiviral responses trim25 ring-finger e3 ubiquitin ligase is essential for rig-i-mediated antiviral activity martinez, i. trim25 in the regulation of the antiviral innate immunity influenza a virus ns1 targets the ubiquitin ligase trim25 to evade recognition by the host viral rna sensor rig-i modulation of host immunity by human respiratory syncytial virus virulence factors: a synergic inhibition of both innate and adaptive immunity respiratory syncytial virus ns1 protein degrades stat2 by inducing socs1 expression respiratory syncytial virus non-structural protein 1 facilitates virus replication through mir-29a-mediated inhibition of interferon-alpha receptor respiratory syncytial virus nonstructural proteins upregulate socs1 and socs3 in the different manner from endogenous ifn signaling human respiratory syncytial virus nonstructural protein ns2 antagonizes the activation of beta interferon transcription by interacting with rig-i respiratory syncytial virus ns1 protein colocalizes with mitochondrial antiviral signaling protein mavs following infection viral degradasome hijacks mitochondria to suppress innate immunity multiple functional domains and complexes of the two nonstructural proteins of human respiratory syncytial virus contribute to interferon suppression and cellular location a novel p38 mitogen activated protein kinase (mapk) specific inhibitor suppresses respiratory syncytial virus and influenza a virus replication by inhibiting virus-induced p38 mapk activation roles of rig-i n-terminal tandem card and splice variant in trim25-mediated antiviral signal transduction a novel mechanism for the inhibition of interferon regulatory factor-3-dependent gene expression by human respiratory syncytial virus ns1 protein effects of nonstructural proteins ns1 and ns2 of human respiratory syncytial virus on interferon regulatory factor 3, nf-kappab, and proinflammatory cytokines the severe acute respiratory syndrome coronavirus nucleocapsid inhibits type i interferon production by interfering with trim25-mediated rig-i ubiquitination mavs ubiquitination by the e3 ligase trim25 and degradation by the proteasome is involved in type i interferon production after activation of the antiviral rig-i-like receptors this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors declare no conflict of interest.viruses 2018, 10, 716 key: cord-299964-sn5o3ugb authors: xue, qiao; liu, huisheng; zhu, zixiang; yang, fan; xue, qinghong; cai, xuepeng; liu, xiangtao; zheng, haixue title: seneca valley virus 3c protease negatively regulates the type i interferon pathway by acting as a viral deubiquitinase date: 2018-11-05 journal: antiviral res doi: 10.1016/j.antiviral.2018.10.028 sha: doc_id: 299964 cord_uid: sn5o3ugb the mechanisms that enable seneca valley virus (svv) to escape the host innate immune response are not well known. previous studies demonstrated that svv 3c(pro) suppresses innate immune responses by cleavage of host proteins and degradation of irf3 and irf7 protein expression. here, we showed that svv 3c protease (3c(pro)) has deubiquitinating activity. overexpressed 3c(pro) inhibits the ubiquitination of cellular substrates, acting on both lysine-48and lysine-63-linked polyubiquitin chains. svv infection also possessed deubiquitinating activity. the ubiquitin-proteasome system was significantly involved in svv replication. furthermore, 3c(pro) inhibited the ubiquitination of retinoic acid-inducible gene i (rig-i), tank-binding kinase 1 (tbk1), and tnf receptor-associated factor 3 (traf3), thereby blocking the expression of interferon (ifn)-β and ifn stimulated gene 54 (isg54) mrnas. a detailed analysis revealed that mutations (h48a, c160a, or h48a/c160a) that ablate the cys and his residues of 3c(pro) abrogated its deubiquitinating activity and the ability of 3c(pro) to block ifn-β induction. together, our results demonstrate a novel mechanism developed by svv 3c(pro) to promote viral replication, and may also provide a novel strategy for improving ubiquitination-based therapy. seneca valley virus (svv), belonging to the picornaviridae, is a positive-sense, single-stranded rna virus that is most closely related to cardiovirus (hales et al., 2008) . svv was first isolated in the united states in 2002 as a contaminant in the cell culture of human fetal retinoblasts (segales et al., 2017) . afterward, a large number of svv infections, which were characterized by porcine idiopathic vesicular disease, were observed in the united states, canada, brazil, and china (xue et al., 2018) . in china, the first case of svv infection was identified in guangdong province in 2015 . subsequently, new svv isolates were identified in guangdong and hubei provinces (qian et al., 2017; zhao et al., 2017) . in 2017, we also identified a novel svv strain in fujian province in china (zhu et al., 2017) . many viruses have evolved strategies to evade innate immune response by inhibiting the host ubiquitination to promote their survival. for instance, human immunodeficiency virus-1 inhibits the antiviral response by the ub-mediated degradation of irf3 (okumura et al., 2008) , porcine reproductive and respiratory syndrome (prrs) virus inhibits nuclear factor kappa-light-chain-enhancer of activated b cells (nf-ĸb) activation by inhibition of the polyubiquitination process of iĸbα . to date, svv 3c pro has evolved mechanism to cleave or degrade innate immune adaptors to escape the host antiviral innate immune response (qian et al., 2017; xue et al., 2018) . however, other mechanisms that enable svv to escape the host innate immune response remain unclear. to determine whether svv can evade innate immune response by inhibiting the host ubiquitination, hek293t cells were transfected with flag-tagged vp1, vp2, 2ab, 2b, 2c, 3d, 3c plasmids along with ha-ub plasmid. at 24 h post-transfection (hpt), the ubiquitinated cellular proteins was assessed by western blotting. the svv 2a, 2c, and 3c pro inhibited the level of ubiquitinated cellular proteins, and 3c pro most significantly inhibited this process ( supplementary fig. 1 ). human dubs are classified into five subfamilies based on their catalytic domains structures. they have a high degree of homology in the two regions known as cys and his boxes that surround the catalytic cys and his residues (nijman et al., 2005) . similar to other picornaviruses, svv 3c pro also possesses a conserved catalytic box with cys and his residues (qian et al., 2017) . therefore, the svv 3c pro was selected for further studies. to determine whether 3c pro functions as a dub, hek293t cells were transfected with increasing amounts of plasmid encoding 3c pro along with ha-ub or empty vector. at 24 hpt, the effect of 3c pro on all https://doi.org/10.1016/j.antiviral.2018.10.028 received 13 july 2018; received in revised form 26 september 2018; accepted 31 october 2018 ubiquitinated cellular proteins was assessed by western blotting. as shown in fig. 1a , expression of 3c pro resulted in a dose-dependent reduction of the level of ubiquitinated cellular proteins compared with that in the empty vector-transfected cells. to further determine which ub linkage type is targeted by 3c pro , hek293t cells were transfected with ha-k63-ub or flag-k48-ub in lieu of ha-ub. at 24 hpt, the cells were collected for western blotting. both k48-and k63-linked ub chains were also processed by 3c pro in a dose-dependent manner ( fig. 1 b and c) . we also analyzed dub activity during svv infection. hek293t cells were mock infected or infected with svv at a multiplicities of infection (moi) of 3 for 12 h. as shown in fig. 2 a, the levels of endogenous ub, k48, and k63 ubiquitinated cellular proteins were reduced in svv-infected cells compared to those in uninfected cells. taken together, these results confirm that svv exerts dub activity during viral infection and 3c pro is a potent viral dub that inhibits ub conjugates formed through k48 or k63 linkages in cellular substrates. svv 3c pro contains cys and his residues. therefore, the three mutants, namely the single-site mutants h48a or c160a, and the doublesites mutants h48a/c160a (3cdm) (xue et al., 2018) , were used to confirm whether the cys and his residues are involved in the dub activity of svv 3c pro . hek293t cells were co-transfected with ha-ub-, ha-k63-ub-, and flag-k48-ub-expressing plasmids along with flag-3c-expressing plasmid or flag-3c mutants expressing plasmids. at 24 hpt, the cells were collected for western blotting. all the 3c pro proteins without cys and his residues lost the ability to inhibit ub conjugates compared with the overexpression of wild-type svv 3c pro (3cwt) (fig. 3 a-c) . to further confirm the dub activity of svv 3c pro , hek293t cells were transfected with flag-3c-expressing plasmid, flag-3cdm expressing plasmid, or empty vector. at 24 hpt, the cells were collected for western blotting. it showed that the 3c pro proteins without cys and his residues could no longer reduce levels of endogenous ubiquitinated ub, k48, and k63 (fig. 2 b) . taken together, these results indicate that the catalytic cys and his residues of svv 3c pro are required for its dub activity. we also investigated whether 3c pro is associated with the deubiquitination of rig-i, tbk1, and traf3. to do so, hek293t cells cultured in 10-cm dishes were transfected with various plasmids. at 32 hpt, cell lysates were immunoprecipitated with anti-flag or anti-myc antibody and analyzed by western blotting. overexpression of 3cwt significantly inhibited the ubiquitination of rig-i ( overexpression of 3cdm, which lacks dub activity, had no such effects. in addition, our results also demonstrated an interaction between 3c pro and either tbk1, or traf3 ( fig. 4b and c). to investigate the interaction between 3c pro and rig-i, hek293t cells were transfected with flag-3c-expressing plasmid or empty vector. at 32 hpt, cell lysates were immunoprecipitated with anti-rig-i antibody and analyzed by western blotting. rig-i pulled down flag-3c (fig. 4d ), which confirmed that 3c pro interacted with rig-i. the interaction of 3c pro and rig-i, tbk1, or traf3 in context of viral infection was further confirmed. hek293t cells were mock-infected or infected with svv (moi 1) for 12 h. the cell lysates were immunoprecipitated with anti-3c antibody and subjected to immunoblotting analysis. 3c pro pulled down rig-i, tbk1, and traf3 in svv-infected cells (fig. 4e) . a reverse immunoprecipitation experiment was subsequently performed using anti-rig-i, tbk1, or traf3 antibody, which showed that rig-i, tbk1, or traf3 also immunoprecipitated 3c pro (fig. 4f ). these results indicate that the interaction of 3c pro with rig-i, tbk1, and traf3 in the context of viral infection is involved in the suppression of ubiquitination levels. endogenous ubiquitination levels of rig-i, tbk1, and traf3 in svv-infected cells were further assessed. hek293t cells were mock infected or infected with svv (moi 3) for 12 h. cell lysates were immunoprecipitated with anti-rig-i, tbk1, or traf3 antibody and analyzed by western blotting. the results showed that the ubiquitination levels of endogenous k48 for tbk1, and k63 for rig-i and traf3 were reduced during svv infection (fig. 4g ). taken together, these results indicate that svv and 3c pro inhibit the ubiquitination of rig-i, tbk1, and traf3 in a dub-dependent manner. ubiquitination and deubiquitination are important mechanisms that are involved in regulating type i ifn signaling pathways (peisley et al., 2014) . to date, many cellular ub ligase enzymes can regulate these processes. for example, the e3 ubiquitin ligase rnf128 or nrdp1 directly enhance ubiquitination of tbk1, which facilitates the activation of tbk1 (song et al., 2016; wang et al., 2009) . meanwhile, many cellular dubs negatively regulate type i ifn signaling pathways. for example, usp3, usp38, and usp19 target rig-i, tbk1, and traf3 for deubiquitination, respectively, thereby blocking the activation of type i ifn signaling pathways (cui et al., 2014; gu et al., 2017; lin et al., 2016) . dub activity also has been demonstrated in many bacteria and viruses, such as salmonella enterica serovar typhimurium, fmdv, prrsv, herpesviruses, coronaviruses, and bunyaviruses, and dub fig. 3 . svv 3c pro inhibits the ubiquitination of cellular proteins in a manner that is dependent on its dub activity. (a) hek-293t cells were seeded in six-well plates, and the monolayer cells were transfected with 1 μg ha-ub-expressing plasmid along with 1 μg flag-3cwt-expressing plasmid, 1 μg flag-3c h48a-expressing plasmid, 1 μg flag-3c c160a-expressing plasmid, or 1 μg flag-3cdm-expressing plasmid. the empty flag vector was used in the transfection process to ensure that the same number of cells received the same amount of total plasmids. at 24 hpt, the cells were collected for western blotting. similar transfection and analysis were performed for k63-ub (b) and k48-ub (c) as described above. (caption on next page) q. xue et al. antiviral research 160 (2018) 183-189 enzymes play multiple roles in regulating bacterial or viral infections (fiskin et al., 2016; sun et al., 2010; van wijk et al., 2017; wang et al., 2011) . here, svv 3c pro also has dub activity. to investigate whether svv 3c pro can block the type i ifn signaling pathway, quantitative polymerase chain reaction (qpcr) analysis was performed to determine the transcript levels of the ifn-β, isg54, and isg56 genes during viral infection. relative expression of mrna was calculated based on the comparative cycle threshold (ct) (2 −δδct ) method (schmittgen and livak, 2008) . the qpcr primers used in this study are listed in table 1 . it showed that only flag-3cwt was able to inhibit sev-induced ifn-β, isg54, and isg56 mrna expression (fig. 5a ). to determine whether 3c pro affected the replication of svv, hek293t cells were seeded in 6-well plates, and the monolayer cells were transfected with flag-3c-expressing plasmid or empty vector. at 24 hpt, the cells were infected with equal amounts of svv (moi 1). at 12 h post-infection (hpi), viral rna and protein levels were examined. overexpression of 3c pro significantly enhanced replication of svv (fig. 5b) . the ubiquitin-proteasome system (ups) plays important roles in the degradation of proteins, the immune response, and signal transduction (casorla-perez et al., 2017) . the ups is a double-edged sword in viral pathogenesis: the ups is necessary for many viruses replication by maintaining the proper functions of viral proteins (barrado-gil et al., 2017; luo, 2016; wang et al., 2016) ; the ups can constitute host immune system to reduce viral replication (luo, 2016) . for example, the ups was essential for coronavirus replication (raaben et al., 2010) , whereas coronavirus papain-like proteases can act as dubs that block type i ifn production (clementz et al., 2010) . proteasome inhibitors mg132 can inhibit the ups. to determine the impact of the ups on the replication of svv, hek293t cells were infected with an equal amount of svv (moi 1). at 4 hpi, the cells were incubated with or without mg132 for 8 h. viral rna and protein levels were examined. mg132 significantly inhibited replication of svv (fig. 5c ), which indicated that the ups was also essential for svv replication. therefore, we speculated that the ups plays an important role in maintaining functions of svv proteins. the abundance of rig-i, tbk1, and traf3 during svv infection remained unclear. hek293t cells cultured in 6-well plates were mock infected or infected with svv. the abundance of rig-i, tbk1, and traf3 were compared at 0, 6, and 12 h after svv infection. the results showed that svv infection had no impact on the abundance of rig-i and traf3, but did inhibit expression of tbk1 (fig. 5d) . it is well known that reduction of the k48-linked polyubiquitin can stabilize tbk1. however, our results indicated that svv or 3c pro reduced expression of tbk1. to clarify this unexpected result, a broad-range deubiquitinase inhibitor, pr-619 (sigma-aldrich), was selected for further study (altun et al., 2011) . the ubiquitination levels of endogenous k48 for tbk1 was enhanced after pr-619 incubation ( supplementary fig.2 ). hek293t cells were transfected with flag-3cexpressing plasmid or infected with svv (moi 1). then, the cells were treated with or without pr-619. cell lysates were analyzed by western blotting. the expression of tbk1 in the 3c pro -transfected and svv-infected cells was enhanced comparing with that in the pr-619-treated cells (fig. 5e ), which indicated that 3c-induced reduction of the k48linked polyubiquitin of tbk1 enhanced the expression of tbk1. however, as a whole, 3c pro and svv still reduced the expression of tbk1. svv 3c pro possesses protease activity. therefore, we speculated that 3c pro reduced the expression of tbk1 by its protease activity, and 3c pro partly stabilized tbk1 by its dub activity. to further investigate whether svv 3c pro can block rig-i-and tbk1-induced type i ifn signaling pathway, we performed a luciferase reporter assay. hek293t cells grown in 24-well plates were co-transfected with 0.1 μg/well of the plasmid ifn-β-luciferase (ifn-β-luc) along with 0.01 μg/well of plasmid prl-tk and plasmids encoding flag-rig-i, myc-tbk1, flag-3cwt, flag-3cdm, or empty vector. at 24 hpt, the cells were lysed and analyzed with a dual-specific luciferase assay kit. as shown in fig. 5f , rig-i-, and tbk1-mediated ifn-β promoter activity was reduced in the presence of flag-3cwt but not flag-3cdm. to further confirm this effect, we conducted a qpcr analysis to determine the levels of the rig-i-and tbk1-mediated endogenous transcription of the ifn-β and isg54 genes. the results showed that flag-3cwt also inhibits rig-i-and tbk1-induced ifn-β and isg54 mrna expression (fig. 5g) , which is in accordance with previous findings showing that svv 3c pro significantly inhibited the endogenous transcription of the ifn-β and isg56 genes that is mediated by rig-i and tbk1 (qian et al., 2017) . taken together, these results indicate that svv 3c pro promotes replication of svv and suppresses rig-i-and tbk1induced type i ifn production in a manner that is dependent on its dub activity. in the present study, we provide direct evidence that svv 3c pro is a novel viral deubiquitinating enzyme. however, more work will be required to determine if other picornavirus 3c pro , such as encephalomyocarditis virus, enterovirus 71, coxsackievirus a16, or fmdv, have dub activity. our data also uncover a novel mechanism by which svv 3c pro antagonizes type i ifn induction, i.e., by deubiquitinating the critical signaling moleculars rig-i, tbk1, and traf3. our results comparing various 3c pro mutants suggest that the dub activity of 3c pro enables it to block induction of the ifn-β promoter and the fig. 4 . svv 3c pro inhibits the ubiquitination of rig-i, tbk1, and traf3. hek-293t cells were seeded in 100-mm dishes, and the monolayer cells were cotransfected with 2 μg ha-ub-expressing plasmid, 1 μg flag-3cwt-expressing plasmid, 1 μg flag-3cdm-expressing plasmid, and the rig-i (a), tbk1 (b), or traf3 (c) expression plasmids (4 μg). mg132 (20 nm) was added at 30 hpt. cell lysates were prepared at 2 h after treatment and immunoprecipitated with anti-flag or anti-myc antibody, and the ub conjugation of the proteins was detected by western blotting with anti-ha antibody. the input tagged proteins were detected with the indicated antibodies. (d) hek-293t cells were seeded in 100-mm dishes, and the monolayer cells were transfected with 5 μg flag-3c-expressing plasmid or empty vector. at 32 hpt, cell lysates were immunoprecipitated with anti-rig-i antibody and analyzed by western blotting. the whole-cell lysates and ip antibody-antigen complexes were analyzed by ib using anti-flag and anti-rig-i antibodies. (e, f) hek-293t cells were seeded in 100-mm dishes, and the monolayer cells were mockinfected or infected with svv (moi 1) for 12 h. cell lysates were immunoprecipitated with anti-3c antibody and analyzed by western blotting (e). similar infection and ip experiments were carried out as described above. however, the lysates were immunoprecipitated with anti-rig-i, tbk1, or traf3 antibody and subjected to western blotting (f). (g) hek293t cells were cultured in 10-cm dishes, and the monolayer cells were mock infected or infected with svv (moi 3) for 12 h. cell lysates were immunoprecipitated with anti-tbk1, rig-i, or traf3 antibody and analyzed by western blotting. the whole-cell lysates and ip antibody-antigen complexes were analyzed by ib using anti-k48, k63, tbk1, rig-i, traf3, or vp1 antibodies. hc represents heavy chain. lc represents light chain. the qpcr primers used in this study. sequences (5′-3′) target gene xue et al. antiviral research 160 (2018) 183-189 endogenous transcription of the ifn-β and isg54 genes. studies have indicated that svv 3c pro mutants that abrogate cys and his residues lost protease activity (qian et al., 2017; xue et al., 2018) . here, our results indicated that svv 3c pro mutants that abrogate cys and his residues also lost dub activity. therefore, we speculate that the cys and his residues of svv 3c pro may contribute to both protease and dub activities. 3c pro might suppress type i ifn response by different mechanisms in host cells. svv has been identified as a novel oncolytic virus against several human cancers (burke, 2016) . ubiquitination and deubiquitination are mechanisms that also play important roles in regulation of cancer (kaushal et al., 2018) . whether svv can inhibit cancer progression through 3c pro dub activity is still unclear and will need to be investigated in future studies. it may provide a novel method for cancer therapy. (caption on next page) q. xue et al. antiviral research 160 (2018) 183-189 fig. 5. svv 3c pro inhibits rig-i-and tbk1-induced type i ifn production. (a) hek293t cells were seeded in 12-well plates, and the monolayer cells were transfected with 0.5 μg flag-3cwt-expressing plasmid, flag-3cdm-expressing plasmid, or empty vector. at 24 hpt, the cells were mock infected or infected with sev (100 hemagglutinating activity units) for 12 h. the expression of ifn-β, isg54, and isg56 mrnas was determined with qpcr assay. (b) hek293t cells were seeded in 6-well plates, and the monolayer cells were transfected with 1 μg flag-3c-expressing plasmid or empty vector. at 24 hpt, the cells were infected with an equal amount of svv (moi 1) for 12 h. expression of viral rna was determined by qpcr assay. expression of viral vp1 protein was detected by western blotting. (c) hek293t cells were seeded in 6-well plates, and the monolayer cells were infected with svv (moi 1). at 4 hpi, the cells were incubated with or without 20 μm mg132 for 8 h. expression of viral rna was determined by qpcr assay. expression of viral vp1 protein was detected by western blotting. (d) hek293t cells were seeded in 6-well plates, and the monolayer cells were mock infected or infected with svv (moi 1) for 0, 6 and 12 h. expression of tbk1, rig-i, traf3, and viral vp1 proteins were detected by western blotting. (e) hek293t cells were seeded in 6-well plates, and the monolayer cells were transfected with 1 μg flag-3c-expressing plasmid or infected with svv (moi 1). then, the cells were treated with or without 25 μm pr-619 for 6 h. cell lysates were analyzed by western blotting. (f) hek293t cells grown in 24-well plates were co-transfected with 0.1 μg/well of ifn-β-luc along with 0.01 μg/well of prl-tk plasmid and 0.1 μg/well of plasmids encoding flag-rig-i, myc-tbk1, flag-3cwt, flag-3cdm, or empty vector. at 24 hpt, the cells were lysed. the dual-specific luciferase assay kit was used to analyze the luciferase activities of firefly and renilla. the data represent the means and standard deviations from three independent experiments. (g) hek-293t cells were seeded in six-well plates, and the monolayer cells were co-transfected with 1 μg flag-rig-i-expressing plasmid, 1 μg flag-3cwt-expressing plasmid, 1 μg flag-3cdm-expressing plasmid, or 1 μg empty vector. the empty vector was used in the transfection process to ensure that the same number of cells received the same amount of total plasmids. similar transfection were performed for myc-tbk1 as described above. at 24 hpt, the expression of ifn-β and isg54 mrnas was determined with qpcr assay. activity-based chemical proteomics accelerates inhibitor development for deubiquitylating enzymes the ubiquitin-proteasome system is required for african swine fever replication oncolytic seneca valley virus: past perspectives and future directions the ubiquitin-proteasome system is necessary for the efficient replication of human astrovirus deubiquitinating and interferon antagonism activities of coronavirus papain-like proteases usp3 inhibits type i interferon signaling by deubiquitinating rig-i-like receptors global analysis of host and bacterial ubiquitinome in response to salmonella typhimurium infection usp19 suppresses cellular type i interferon signaling by targeting traf3 for deubiquitination complete genome sequence analysis of seneca valley virus-001, a novel oncolytic picornavirus deubiquitinating enzymes in cancer stem cells: functions and targeted inhibition for cancer therapy usp38 inhibits type i interferon signaling by editing tbk1 ubiquitination through nlrp4 signalosome interplay between the virus and the ubiquitin-proteasome system: molecular mechanism of viral pathogenesis a genomic and functional inventory of deubiquitinating enzymes hiv-1 accessory proteins vpr and vif modulate antiviral response by targeting irf-3 for degradation structural basis for ubiquitinmediated antiviral signal activation by rig-i seneca valley virus suppresses host type i interferon production by targeting adaptor proteins mavs, trif, and tank for cleavage the ubiquitin-proteasome system plays an important role during various stages of the coronavirus infection cycle analyzing real-time pcr data by the comparative c(t) method e3 ubiquitin ligase rnf128 promotes innate antiviral immunity through k63-linked ubiquitination of tbk1 the cysteine protease domain of porcine reproductive and respiratory syndrome virus nonstructural protein 2 possesses deubiquitinating and interferon antagonism functions linear ubiquitination of cytosolic salmonella typhimurium activates nf-kappab and restricts bacterial proliferation the e3 ubiquitin ligase nrdp1 'preferentially' promotes tlr-mediated production of type i interferon the leader proteinase of foot-and-mouth disease virus negatively regulates the type i interferon pathway by acting as a viral deubiquitinase the ubiquitin-proteasome system is essential for the productive entry of japanese encephalitis virus complete genome sequence of seneca valley virus ch-01-2015 identified in china seneca valley virus 3c(pro) abrogates the irf3-and irf7-mediated innate immune response by degrading irf3 and irf7 emergence of novel seneca valley virus strains in china this work was supported by grants from the national natural sciences foundation of china (no. u1501213 and 31672585), the national key research and development program of china (2016yfd0500901), the gansu science foundation for distinguished young scholars (no. 1606rjda313) and the gansu science foundation for young scholars (1606rjya280). the authors declare no competing financial interest. supplementary data to this article can be found online at https:// doi.org/10.1016/j.antiviral.2018.10.028. key: cord-305737-bnzd7b25 authors: rehwinkel, jan; reis e sousa, caetano title: targeting the viral achilles’ heel: recognition of 5′-triphosphate rna in innate anti-viral defence date: 2013-05-23 journal: curr opin microbiol doi: 10.1016/j.mib.2013.04.009 sha: doc_id: 305737 cord_uid: bnzd7b25 some rna virus genomes bear 5′-triphosphates, which can be recognized in the cytoplasm of infected cells by host proteins that mediate anti-viral immunity. both the innate sensor rig-i and the interferon-induced ifit proteins bind to 5′-triphosphate viral rnas. rig-i signals for induction of interferons during rna virus infection while ifits sequester viral rnas to exert an anti-viral effect. notably, the structures of these proteins reveal both similarities and differences, which are suggestive of independent evolution towards ligand binding. 5′-triphosphates, which are absent from most rnas in the cytosol of uninfected cells, are thus a marker of virus infection that is targeted by the innate immune system for both induction and execution of the anti-viral response. targeting the viral achilles' heel: recognition of 5 0 -triphosphate rna in innate anti-viral defence jan rehwinkel 1 and caetano reis e sousa 2 some rna virus genomes bear 5 0 -triphosphates, which can be recognized in the cytoplasm of infected cells by host proteins that mediate anti-viral immunity. both the innate sensor rig-i and the interferon-induced ifit proteins bind to 5 0 -triphosphate viral rnas. rig-i signals for induction of interferons during rna virus infection while ifits sequester viral rnas to exert an antiviral effect. notably, the structures of these proteins reveal both similarities and differences, which are suggestive of independent evolution towards ligand binding. 5 0triphosphates, which are absent from most rnas in the cytosol of uninfected cells, are thus a marker of virus infection that is targeted by the innate immune system for both induction and execution of the anti-viral response. the interferon system is one of the cornerstones of innate immunity in mammals ( figure 1 ). interferons were discovered as soluble factors secreted by virally infected cells that protected uninfected cells from subsequent viral challenge [1] . the type i interferons (referred to as ifn in this review) include interferon-b and multiple interferon-a subtypes, all of which signal through a common receptor ifnar [2] (figure 1 ). all nucleated cells can produce ifn in response to nucleic acids generated during virus infection. detection of these nucleic acids is carried out within the infected cell by host proteins residing in the cytosol, such as rig-i-like receptors (rlrs) and other virus-sensing receptors [3] ( figure 1 ). in addition, some cells possess the ability to detect viruses in the extracellular milieu using members of the toll-like receptor (tlr) family. this generally involves endocytic uptake of viruses or remnants of virally infected cells into vesicular compartments surveyed by tlr3, 7, 8 and 9, all of which bind nucleic acids ( figure 1 ). either rlr or tlr signalling then induces expression of the ifn genes ( figure 1 ). given the abundance of nucleic acids in uninfected healthy cells and the potential of ifn to contribute to autoinflammatory and autoimmune diseases [4] , it is not surprising that sophisticated mechanisms ensure that activation of rlrs and tlrs occurs only during infection. one of these mechanisms is the recognition of viral rna genomes bearing a 5 0 -triphosphate (5ppp) moiety by rig-i, a member of the rlr family. 5ppp groups are present on the genomes of many rna viruses (table 1) , but are not found on most cellular rnas in the cytosol. as such, the presence of 5ppp rna in the cytoplasm is a molecular signature of virus infection. secreted ifn acts in autocrine and paracrine fashion to turn on the transcription of several hundred ifn-stimulated genes (isgs) (figure 1 ) [5] . examples include the genes encoding (i) rig-i and other virus sensing receptors (providing a positive-feedback loop), (ii) proteins involved in cell-intrinsic anti-viral defence such as rnasel, mxa or tetherin and (iii) molecules facilitating adaptive immune responses such as cd80 and cd86. one family of isgs encodes the ifit proteins that are characterized by tetratricopeptide repeats [6, 7] . ifits control translation initiation, cell proliferation and cell migration, and exert anti-viral effects against a variety of rna viruses and human papillomavirus [6, 7] . interestingly, it has recently been found that some ifits bind to 5ppp rnas and antagonize viruses by sequestering viral rna [8 ,9 ] . here, we review recent progress on the recognition of 5ppp rna by rig-i and ifits and its subsequent impact on cell-intrinsic immunity to virus infection. studies on the types of rna that can activate rig-i have attracted much attention but have mostly been based on transfection of synthetic rnas into reporter cells. such experiments revealed that the most potent rig-i agonists are rnas that contain a triphosphate moiety and are base-paired at the 5 0 -end. rna sequence is largely irrelevant so long as it does not impact on the base-pairing. a blunt end without overhangs is most effective and can be provided in trans by hybridization between two rna molecules or in cis by complementarity between the 5 0end and 3 0 -end of a single rna molecule (figure 2a ). these findings have been reviewed in detail elsewhere [10, 11] and are well supported by structural data (see below). less is known about the types of rna that activate rig-i during virus infection. however, work from us and others showed that rig-i recognizes the 5ppp rna genome of influenza a virus (iav) and sendai virus in infected cells, and that both the 5ppp and the secondary structure are required for rig-i-dependent ifn induction [12, 13] . thus, these two studies in virus infection models nicely validate the predictions made by analysis of the rig-i-mediated response to synthetic rnas [12, 13] . recent work shows that rig-i is recruited to and activated by bunyavirus nucleocapsids, native complexes containing a 5ppp rna genome and viral proteins [14 ] . this demonstrates that rig-i can indeed gain access to viral rnas under physiological conditions. like viral rna, bacterial rnas can possess 5ppp termini and secondary structures that are predicted to make them rig-i agonists. consistent with this notion, rig-i can act as a sensor of bacterial rna and may help maintain homeostasis to gut microbiota [15, 16] . interestingly, some rnas lacking 5ppps also trigger rig-i upon transfection into cells. these rnas include short double-stranded rnas made by chemical synthesis 486 host-microbe interactions: viruses [52] a ifit1 inhibits viral mrna translation of parainfluenza virus type 5, another member of the paramyxovirus family [54] . the mechanism of inhibition is unknown but is unlikely to involve 5ppp rna binding as viral mrnas are capped. [17, 18] , certain forms of the enzymatically prepared rna poly i:c [19] and dephosphorylated double-stranded rnas made by in vitro transcription [20] . further supporting the notion that non-5ppp rnas can activate rig-i is the observation that poly i:c triggers rig-i signalling in a cell free reconstitution assay [21] . however, it remains unclear whether 5ppp-independent rig-i signalling occurs in infected cells. it will be important to identify natural rnas that activate rig-i in cells infected with different viruses, including positive sense rna viruses, to validate the extent to which rig-i can be activated independently of 5ppps. magnetic resonance and x-ray crystallography elucidated the structure of the ctd in the absence [17, 24] and presence of rna [25, 26, 27] . they showed that the ctd contains a positively charged pocket that can accommodate 5ppp ends of rna molecules and thus provided a structural explanation for detection of this type of rna [25, 26] . much progress has been made in the last two years determining the structure of the other rig-i domains and of the full-length protein [22, 23] . without an rna ligand, full-length rig-i adopts an autorepressed state. the helicase domain is in an open conformation, flexibly linked to the ctd and bound to the second card [28 ,29,30] (figure 2c ). for downstream signalling to occur, the cards need to be free (see below). however, the card2-helicase interaction observed in rna-free rig-i constrains this freedom and thus explains autorepression [28 ,30] (figure 2c ). crystal structures of rig-i bound to short doublestranded rnas [28 ,31 ,32 ] or to a 5ppp hairpin single-stranded rna [33 ] (figure 3b ) reveal how the autorepressed state is dismantled by structural rearrangement upon rna agonist binding. the helicase adopts a closed conformation and completely surrounds the basepaired rna along its length, while the ctd caps the 5 0end by directly interacting with the 5ppp and the first few nucleotides [28 ,31 ,32 ,33 ] (figures 2d and 3b) . the binding site for card2 on the helicase domain is now involved in rna binding, thus allowing the cards to be released [28 ,31 ,32 ,33 ] . furthermore, in the rnabound conformation, a pincer domain (also called bridging helices) places the ctd in a position overlapping with that previously occupied by card2 [28 ,31 ,32 ,33 ] (figure 2d ). rna binding is therefore predicted to push away the cards. this is likely to happen in two steps: first, the ctd recognizes the 5ppp group and, second, the helicase domain binds to the basepaired region (figure 2c ,d) [22, 23] . the hydrolysis of atp is thought to tighten the helicase domain around the rna, further favouring card release [33 ] . mda5 is another rlr. a recent crystal structure of the mda5 helicase domain and ctd in complex with double-stranded rna shows that these domains form a ring around the rna, leaving both ends free to extend [34 ] . this is in contrast to the equivalent domains in rig-i that cap one end of 5ppp base-paired rna, due to tilting of the rig-i ctd towards the rna [34 ] . this structural difference, together with the absence of a 5ppp-binding pocket in the ctd of mda5 [25,34 ,35,36] , may explain why the two rlrs recognize different types of rna and mediate ifn responses to different viruses [37, 38] . upon rna agonist binding and card exposure, rig-i initiates a signal transduction cascade that ultimately results in ifn gene transcription [3] . mavs is the rig-i and mda5 proximal adaptor protein. it mediates activation of kinases such as tbk1, which phosphorylate transcription factors including irf3 that then translocate to the cell nucleus to trigger transcription of ifn genes (figure 1 ). mavs has a c-terminal transmembrane domain and a single n-terminal card, which engages in card-card interactions with rig-i. this interaction requires binding to k63-linked polyubiquitin chains generated by the ubiquitin ligase trim25 (figure 2e) [21, 39, 40 ] . one study suggests that trim25 mediates covalent ubiquitylation of rig-i at lysine 172 in the second rig-i card [39] . a different study indicates that the same lysine binds non-covalently to trim25generated free ubiquitin chains [21] . card-associated ubiquitin chains are key for signalling as they trigger formation of rig-i tetramers (figure 2f ) [40 ] . the resulting card clusters recruit multiple mavs molecules [40 ,41 ] . surprisingly, the stoichiometry of this reaction reveals signal amplification through self-propagation (figure 2f,g) . mavs forms fibrils that convert additional mavs molecules into the fibrillar conformation, in a self-amplification mode reminiscent of prions [41 ] . the mavs fibrils in turn mediate activation of tbk1 [41 ] (figure 2g) . a subsequent study confirmed the prion-like behaviour of mavs and showed that it can also be triggered by the cards of mda5 [34 ] . selfpropagation allows for sensitive detection of minute amounts of viral rna: it has been estimated that 20 molecules of 5ppp rna are sufficient to activate irf3 via the rig-i/mavs pathway [21] . an important question for future studies is how signalling is terminated and how the self-propagating prion conformation of mavs is cleared. another area for future research is the spatiotemporal regulation of rlr signalling and how it is governed by the localization of mavs [42] . interestingly, mavs has been found on mitochondria, peroxisomes and so-called mams (mitochondrial-associated endoplasmic reticulum membranes) [43, 44] . much remains to be learned about how mavs localization is regulated and how this impacts on rlr signalling during infection with different viruses. the human genome encodes four ifits (ifit1-3, ifit5) whereas mice have three (ifit1-3) [6, 7] . the expression of these proteins is potently induced by ifn and their anti-viral function has been reviewed elsewhere [6, 7] . recent functional and structural data indicate that the anti-viral effect of ifit1 and ifit5 is due to their ability to recognize 5ppp rna. a proteomics study identified all four human ifits in a pull-down with synthetic 5ppp rna as bait [9 ] . ifit1 and ifit5 directly bound to the rna, whereas ifit2 and ifit3 did so indirectly, through association with ifit1 [9 ] . three viruses that produce 5ppp rna (vesicular stomatitis virus (vsv), rift valley fever virus and iav) showed enhanced replication in human or mouse cells depleted of recognition of 5ppp rna in anti-viral defence rehwinkel and reis e sousa 489 current opinion in microbiology rig-i and ifits recognize 5ppp rna. (a) several rna viruses generate 5ppp rnas, such as viral genomic rnas. these rnas are recognized by rig-i that induces ifn production (green). ifns then induce the expression of ifits that bind and sequester 5ppp viral rnas, preventing their translation, replication and/or packaging (red). note that ifits can also be induced directly by rig-i/mavs (dashed arrow) [43] . (b) the top row shows overall structures of rig-i and ifit5 in complex with 5ppp rna; the bottom row zooms in on the 5ppp binding sites. the rig-i structure is the rig-i dcard plus 5ppp rna hairpin complex described in [33 ] (pdb code 4ay2). ifit5 is shown in complex with 5ppp-oligo-c [8 ] (pdb code 4hor). amino acid residues contacting the 5ppp are shown in stick format and dashed lines indicate hydrogen-bonding interactions. ifit1 whereas replication of encephalomyocarditis virus (emcv) that lacks 5ppps was normal [9 ] . what is the molecular basis of ifit1 mediated inhibition of virus replication? in vitro assays showed that recombinant ifit1 blocks translation of 5ppp containing reporter rnas, while having little effect on translation of the corresponding dephosphorylated rna [9 ] . ifit1 also precipitated viral rnas from cells infected with vsv or iav [9 ] . together, these observations suggest that ifit1 recognizes and sequesters viral 5ppp rnas and thereby prevents their replication, packaging and/or translation. structural data demonstrate that human ifit5 has a positively charged cavity accommodating 5ppp rna [8 ] (figure 3b ). structure-guided mutation of residues involved in 5ppp rna binding diminishes the anti-viral effects of ifit5 and ifit1 against vsv and iav [8 ,9 ] . similar to 5ppp rna recognition by rig-i, the interaction between ifit5 and rna is not sequence specific [8 ,22,23] . however, while rig-i is most strongly activated by blunt ended 5ppp base-paired rna [11] , a 5 0 -overhang is required for rna to be accommodated in ifit5 [8 ] . whether this reflects binding of different types of rna to ifits and rig-i or whether base-paired rig-i agonists are unwound for ifit recognition remains to be determined. genetically modified mice provide further evidence for a role of ifits in host defence against viruses [9 ,45,46,47] . ifit2-deficient animals are highly susceptible to intranasal vsv challenge [46] , and similar data have been reported for ifit1 à/à mice [9 ] . it is unclear why another study using ifit1-deficient mice independently derived from the same es cell line failed to reproduce these results [9 ,46] . interestingly, ifit1 à/à mice and cells are more susceptible to infection with viruses lacking 2 0 -o-methyltransferases, including mutants of west nile virus, coronavirus and poxvirus [45, 47, 48] . the 2 0 -o-methyltransferases encoded by the wild-type counterparts of these mutant strains modify the cap structure of viral messenger rna by 2 0 -o-methylation. cellular messenger rnas are methylated at this position by a host-encoded methyltransferase, suggesting that some viruses mimic this to circumvent recognition by ifit1 and, possibly, mda5 [45, 47, 48] . this indicates that ifit1 may interact not only with 5ppp rna, but also with capped rna lacking 2 0 -omethyl groups, although the latter interaction could be indirect, perhaps via other ifits. indeed, a large spectrum of different rnas may be targeted by ifits: ifit5 not only binds to 5ppp rna but also to transfer rnas that bear a single 5 0 -phosphate [49 ] and ifit2 interacts with au-rich rnas independently of 5 0 -phosphorylation [50] . it will be important to identify and characterize rnas bound by ifits in virally infected cells to further understand the role of these proteins in immune responses to virus infection. the ifn system operates in two phases: first, virus presence is detected, resulting in the expression of ifn; second, ifn induces isgs that exert anti-viral effects (figure 1 ). recent work shows that the ifn system targets 5ppp rnas during both phases: both rig-i, a virus sensor that induces ifn expression, and ifits, effector molecules that execute anti-viral activities, can specifically recognize 5ppp rnas. as such, 5ppp rnas appear to be achilles' heel of many rna viruses in their interaction with the innate immune system (figure 3a ). why is this the case? many rna viruses use primer-independent means of replication. 5ppp moieties on viral rna genomes, antigenomes and some viral transcripts are an inevitable consequence of primer-independent initiation of rna synthesis by a single ribonucleoside-triphosphate [51] . the fundamental importance of rna replication to the life cycle of rna viruses may explain why viruses that use primer-independent strategies to initiate rna synthesis have been forced to maintain 5ppp groups during evolution -despite the selective pressure exerted by the ifn system that detects these moieties. it is noteworthy that other mechanisms to initiate rna synthesis and the removal of 5ppp groups have evolved in some virus families. for example, emcv uses as primer for rna replication a protein known as vpg, which becomes covalently attached to the 5 0 -end of the viral genome [51] . hantaan virus, crimean-congo haemorrhagic fever virus and borna disease virus posttranscriptionally process the 5 0 -end of their genomes to leave a mono-phosphate [52] . consistent with these facts, none of these viruses trigger ifn induction via rig-i and emcv is not restricted by ifit1 [9 ,38,52] (table 1) . notably, viruses that maintain 5ppp rna and are recognized by rig-i often encode proteins that specifically target and inhibit rlr signalling [53] . studies of the sensors that detect rna viruses and the identification of isgs that restrict them remain challenges for the future. papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest virus interference. i. the interferon interferons, interferon-like cytokines, and their receptors innate recognition of viruses type i interferonopathies: a novel set of inborn errors of immunity a diverse range of gene products are effectors of the type i interferon antiviral response the isg56/ifit1 gene family the broad-spectrum antiviral functions of ifit and ifitm proteins structural basis for viral 5 0 -ppp-rna recognition by human ifit proteins this study reveals the structural basis of how ifits interact with 5ppp rna ifit1 is an antiviral protein that recognizes 5 0 -triphosphate rna rigorous detection: exposing virus through rna sensing the chase for the rig-i ligand -recent advances preference of rig-i for short viral rna molecules in infected cells revealed by next-generation sequencing rig-i detects viral genomic rna during negative-strand rna virus infection incoming rna virus nucleocapsids containing a 5 0 -triphosphorylated genome activate rig-i and antiviral signaling this study reports that rig-i is activated by bunyavirus 5ppp rna genomes contained in nucleocapsids, which demonstrates that this innate receptor can sense viral rna in its native complex with viral proteins mitochondrial antiviral signaling protein (mavs) monitors commensal bacteria and induces an immune response that prevents experimental colitis rig-i detects infection with live listeria by sensing secreted bacterial nucleic acids nonself rna-sensing mechanism of rig-i helicase and activation of antiviral immune responses a structural basis for discriminating between self and nonself double-stranded rnas in mammalian cells lengthdependent recognition of double-stranded ribonucleic acids by retinoic acid-inducible gene-i and melanoma differentiationassociated gene 5 molecular mechanism of signal perception and integration by the innate immune sensor retinoic acid-inducible gene-i (rig-i) reconstitution of the rig-i pathway reveals a signaling role of unanchored polyubiquitin chains in innate immunity a structure-based model of rig-i activation structural insights into the activation of rig-i, a nanosensor for viral rnas the cterminal regulatory domain is the rna 5 0 -triphosphate sensor of rig-i structural and functional insights into 5 0 -ppp rna pattern recognition by the innate immune receptor rig-i the structural basis of 5 0 triphosphate double-stranded rna recognition by rig-i c-terminal domain crystal structure of rig-i c-terminal domain bound to blunt-ended double-strand rna without 5 0 triphosphate structural basis for the activation of innate immune pattern-recognition receptor rig-i by viral rna this paper describes the structure of full-length rig-i and reveals an important interaction between card2 and the helicase domain that explains rig-i autorepression the rig-i atpase domain structure reveals insights into atp-dependent antiviral signalling structure and dynamics of the second card of human rig-i provide mechanistic insights into regulation of rig-i activation structural basis of rna recognition and activation by innate immune receptor rig-i these studies report the crystal structure of the rig-i helicase domain and ctd in complex with rna and provide insights into the conformation of the activated receptor structural insights into rna recognition by rig-i see annotation to ref visualizing the determinants of viral rna recognition by innate immune sensor rig-i see annotation to ref structural basis for dsrna recognition, filament formation, and antiviral signal activation by mda5 this work on the structure of the mda5 helicase domain and ctd highlights similarities and differences between rig-i and mda5 that may explain why these rlrs recognize different types of rna structural basis of double-stranded rna recognition by the rig-i like receptor mda5 solution structures of cytosolic rna sensor mda5 and lgp2 cterminal domains: identification of the rna recognition loop in rig-i-like receptors exposing viruses: rna patterns sensed by rig-ilike receptors differential roles of mda5 and rig-i helicases in the recognition of rna viruses trim25 ring-finger e3 ubiquitin ligase is essential for rig-i-mediated antiviral activity ubiquitin-induced oligomerization of the rna sensors rig-i and mda5 activates antiviral innate immune response this paper shows that activated rig-i forms tetramers and that noncovalent binding of rig-i and mda5 to k63-linked ubiquitin chains is required for signaling to mavs mavs forms functional prion-like aggregates to activate and propagate antiviral innate immune response this study shows that mavs forms prion-like aggregates that activate tbk1 and thus reveals an unexpected mechanism of signal amplification in the rig-i pathway defining the subcellular sites of innate immune signal transduction peroxisomes are signaling platforms for antiviral innate immunity mitochondrialassociated endoplasmic reticulum membranes (mam) form innate immune synapses and are targeted by hepatitis c virus 0 -o methylation of the viral mrna cap evades host restriction by ifit family members interferoninduced ifit2/isg54 protects mice from lethal vsv neuropathogenesis 0 -o methylation of the viral mrna cap by west nile virus evades ifit1-dependent and -independent mechanisms of host restriction in vivo ribose 2 0 -o-methylation provides a molecular signature for the distinction of self and non-self mrna dependent on the rna sensor mda5 using biochemical and structural analysis, this work shows that ifit5 also binds transfer rnas bearing 5 0 -monophosphates crystal structure of isg54 reveals a novel rna binding structure and potential functional mechanisms fields virology processing of genome 5 0 termini as a strategy of negative-strand rna viruses to avoid rig-idependent interferon induction viral tricks to grid-lock the type i interferon system isg56/ifit1 is primarily responsible for interferoninduced changes to patterns of parainfluenza virus type 5 transcription and protein synthesis we thank andreas pichlmair, delphine goubau and safia deddouche for their helpful comments, and martin jinek for his help in preparing figure 3 . we apologize to our colleagues whose work could not be cited due to space limitations. crs is funded by cancer research uk, erc and foundation bettencourt-schueller. jr is funded by the uk medical research council. key: cord-288390-p1q3v1ie authors: habjan, matthias; pichlmair, andreas title: cytoplasmic sensing of viral nucleic acids date: 2015-02-07 journal: curr opin virol doi: 10.1016/j.coviro.2015.01.012 sha: doc_id: 288390 cord_uid: p1q3v1ie viruses are the most abundant pathogens on earth. a fine-tuned framework of intervening pathways is in place in mammalian cells to orchestrate the cellular defence against these pathogens. key for this system is sensor proteins that recognise specific features associated with nucleic acids of incoming viruses. here we review the current knowledge on cytoplasmic sensors for viral nucleic acids. these sensors induce expression of cytokines, affect cellular functions required for virus replication and directly target viral nucleic acids through degradation or sequestration. their ability to respond to a given nucleic acid is based on both the differential specificity of the individual proteins and the downstream signalling or adaptor proteins. the cooperation of these multiple proteins and pathways plays a key role in inducing successful immunity against virus infections. viruses are the most abundant pathogens on earth. a finetuned framework of intervening pathways is in place in mammalian cells to orchestrate the cellular defence against these pathogens. key for this system is sensor proteins that recognise specific features associated with nucleic acids of incoming viruses. here we review the current knowledge on cytoplasmic sensors for viral nucleic acids. these sensors induce expression of cytokines, affect cellular functions required for virus replication and directly target viral nucleic acids through degradation or sequestration. their ability to respond to a given nucleic acid is based on both the differential specificity of the individual proteins and the downstream signalling or adaptor proteins. the cooperation of these multiple proteins and pathways plays a key role in inducing successful immunity against virus infections. almost all cells express germ-line encoded sensors with the ability to recognise virus infections and to initiate defence systems necessary to limit virus spread and pathogenicity. in technical terms, a sensor is 'a device that detects events or changes in quantities and provides a corresponding output without affecting the original trigger'. sensors follow certain rules that include selective sensitivity to a specific measured property and insensitivity to other properties likely to be encountered. in analogy to technical terms, virus sensors convert a signal (virus infection) to an output that instructs the cell to take further actions. the magnitude of its activation is characterised by properties related to the exact nature and the quantity of the trigger. the targets of these sensors can be incoming virus particles [1] , particular viral proteins [2] as well as general integrity of the cell [3] . however, the yet best understood sensors involved in antiviral defence are activated by viral nucleic acids [4] . endosomal toll-like receptors sample the extracellular milieu or cytoplasmic contents that are delivered into endosomes through autophagy. in this review we concentrate on intracellular nucleic acid sensors and effector proteins that evolved to mediate specialised tasks including, firstly, expression of cytokines such as type i interferons (ifn-a/b); secondly, modulation of cellular machineries required for virus replication and thirdly, direct inhibition of virus growth ( figure 1 ). induction of cytokines utilises at least two distinct pathways either involving the adaptor proteins mitochondrial antiviral-signalling protein (mavs) or stimulator of interferon genes (sting). activation of either pathway regulates transcription of cytokines, which are key signals to shape adaptive immunity to induce an intracellular 'antiviral state' characterised by expression of antiviral defence proteins. some of the latter proteins are activated by viral nucleic acids and in turn re-wire cellular machineries to limit virus spread. other proteins directly bind viral nucleic acid and impair functionality through steric hindrance or degradation. to understand how viral nucleic acids are sensed by the innate immune system it is important to consider the different types of nucleic acids generated after virus infection. viruses are intracellular pathogens that require cellular translation and host metabolism, but provide their own replication machinery. independence of the host for multiplication of viral genomes allows high replication rates, which is often associated with pathogenicity [5] . 24-48 hours after infection approximately 25% of rna can be of viral origin (p. hubel and a. meiler, unpublished). viral nucleic acids accumulate in compartments typically devoid of cellular nucleic acids and often possess or lack modifications or physical properties that are not normally associated with cellular rna or dna ( figure 2 ). rna polymerases commonly generate rna with a 5 0 triphosphate group (ppp-rna). cellular rna polymerases cotranscriptionally modify newly synthesised rna at the 5 0 terminus. in case of mrna an inverted guanine nucleotide cap is added and methylated at the n7-position as well as the 2 0 o position of the first ribose of the rna strand (cap1 mrna) ( figure 2 ) [6] . these modifications are necessary to mark mrna for further processing and export into the cytoplasm, where translation takes place. other cellular rnas are cleaved and have a 5 0 monophosphate in case of transfer (t)rna, most ribosomal (r)rnas and small nucleolar (sno) rnas [7] . some small rnas bear a terminally methylated 5 0 triphosphate (u6 snrna, 7sk rna) or are further processed to a hypermethylated 2,2,7-trimethylguanosine cap (tmg) cap (snrnas). in addition, more than 100 modifications on internal nucleotides of cellular rnas have been described, some of which are critical to tame activation of the innate immune system. total cellular rna isolated from cells and transfected into indicator cells does not activate the innate immune system, whereas the products of most viral rna polymerases are strong stimuli of antiviral responses [8] . negative strand rna viruses such as orthomyxo-viruses, paramyxo-viruses and bunyaviruses commonly generate full-length genomic ppp-rna and short 5 0 ppp subgenomic rna, which have strong immunostimulatory potential [4] . to avoid the cellular defence system many viruses mimic cellular mrna-like cap structures by encoding capping enzymes (e.g. flaviviruses, coronaviruses, poxviruses, and reoviruses), 'steal' cap structures from cellular mrnas for their transcripts (e.g. orthomyxoviruses, bunayviruses) or trim their genomic rna to display only monophosphorylated termini (bunyaviruses, bornaviruses) [9, 10] . picornaviruses and caliciviruses mask their rna with a covalently 5 0 genome-linked viral protein (vpg). in addition to the cap itself, 2 0 o methylation of the first ribose of mrnas is an additional modification that is highly conserved between viruses and their hosts, evidenced by the presence of dedicated viral proteins that catalyse this reaction [10] . lack of 2 0 o methylation renders viruses highly vulnerable to the antiviral activity of the interferon system [11, 12] . a type of rna often associated with viral replication is double-stranded rna (dsrna). dsrna could be either the result of replication intermediates (for rna viruses), generation of genomic rna (for dsrna viruses), convergent transcription (for dna viruses), or of the presence of secondary structures found in viral rnas (e.g. the ires structure of ssrna viruses) [5] . however, the definition and exact nature of dsrna still remains enigmatic. using an antibody raised against dsrna, it was found that such rna is produced in cells infected with dna viruses as well as some rna viruses, such as flavi and picornaviruses [8] . however, although double-strandedness is an important feature recognised by virus sensors, it does not seem to be the only important determinant to stimulate fulminant antiviral responses since different double-stranded homopolymers vary considerably in their ability to induce ifna/b. furthermore, dsrna is commonly generated by convergent transcription of cellular rna polymerases and is involved in transcriptional and post-transcriptional gene silencing [13 ,14] . despite the presence of cell-generated dsrna no spontaneous synthesis of ifn-a/b is apparent nor is transfection of total cellular rna containing detectable dsrna molecules or plasmid-based convergent transcription capable to induce significant levels of ifn-a/b [13 ] . a possible explanation for the lack of stimulatory activity of cellular rna may be insufficient concentration of dsrna as proposed by a recent study showing that nuclear dsrna is digested by the endonuclease dicer [15] . it may be that the latter function is used by orthomyxoviruses that replicate in the nucleus to reduce the abundance of viral dsrna in the cytoplasm. since most virus sensors and signalling molecules are localised in the cytoplasm, the cellular nucleus is considered not to promote sensing and signalling of virus infection. indeed, cellular dna, present in the nucleus does not elicit ifn-a/b whereas double-stranded dna (dsdna) introduced into the cytoplasm through transfection or virus infection induces an innate immune response [4] . however, simple compartmentalisation is insufficient to explain the ability of the innate immune system to recognise dna viruses that replicate the cytoplasmic sensing of viral nucleic acids habjan and pichlmair 33 differences between cellular and viral nucleic acids. synthesis of host rna (red) from nuclear dsdna (blue) is achieved by three cellular rna polymerases. rna polymerase ii synthesises mrna, ncrna and some snrnas, whereas rna polymerase iii generates trna and 5s rrna. rrnas are produced by rna polymerase i. virus-derived dna and rna are present in the cell either as genomes, transcripts or replication byproducts. indicated are particular differences at the rna 5 0 and 3 0 end, such as cap structures and methylations (e.g. cellular mrna harbouring an n7-methylated guanine cap structure and 2 0 o-methylation at the first and/or second ribose). *5s rrna harbours a 5 0 triphosphate group; **u6 and 7sk rna both have a 5 0 gamma-monomethyl phosphate, and srp rna has a 5 0 triphosphate. abbreviations: ds, double-stranded; mrna, messenger rna; rrna, ribosomal rna; trna, transfer rna; snrna, small nuclear rna; ncrna, non-coding rna; m7g, n7-methylated guanine cap; m, 2 0 o-methylation; p, phosphate group; tmg, hypermethylated 2,2,7-trimethylguanosine cap; vpg, viral protein genome-linked; a(n), poly(a) tail. nucleus [16 ] . it is therefore likely that additional yet unknown features of viral dna can be sensed by the innate immune system. among the best characterised cytoplasmic proteins involved in virus sensing are rig-i-like receptors (rlrs), a family of dexd/h-box helicases which specifically identify viral rnas and have the ability to stimulate expression of ifn-a/b and other cytokines (figure 3 ) [4, 17] . the founding member of this family, retinoic acid inducible gene-i (rig-i) bears two n-terminal caspase activation and recruitment domains (cards) required for signalling, a central helicase domain that mediates binding to dsrna and a c-terminal repressor domain, which binds 5 0 tri-phosphorylated, di-phosphorylated or dephosphorylated rna ends [18] [19] [20] . rig-i forms oligomers along the bound rna in an atp-dependent manner, the cards oligomerize and allow signalling through card-card interactions with mavs [21, 22] . activation in addition requires dephosphorylation and ubiquitination of rig-i [23] . optimal rig-i ligands are consisting of blunt dsrna formed by two complementary rnas (e.g. reovirus) or generated by intramolecular base pairing as is proposed for the sensing of influenza a virus ribonucleoprotein complexes [20] . other proteins belonging to rlr helicases are mda5 and lgp2. lgp2 lacks functional card domains and therefore cannot induce signalling. however, lgp2 appears to be an important co-factor to facilitate sensing of some viruses [24] . recently, l-antisense rna expressed by encephalomyocarditis virus (emcv) has been identified to associate with lgp2 [25] . l-antisense rna activates mda5, raising the possibility that lgp2 prepares ligands for sensing through other rlrs. mda5 appears to be activated by structural properties of viral rnas, but there is no unifying feature known that could generally explain mda5 activation. instead, a number of different rnas are proposed to activate mda5. firstly, long synthetic dephosphorylated dsrna stimulates mda5 whereas shorter dsrna loses this ability [26, 27] . secondly, replication intermediates consisting of dsrna and generated by picornaviruses [28, 29] . thirdly, high molecular weight rna generated during replication and likely bearing branched rna molecules activates mda5 [8] . the most commonly used synthetic mda5 stimulus, poly-i:c would most likely form such structures. fourthly, a specific sequence in the l-region present on the antisense single-stranded genomic rna of emcv appears to stimulate mda5 [25] . fifthly, for measles virus a sequence bias towards au-rich regions was proposed to be associated with mda5 activating activity [30] . sixthly, mutant coronaviruses that generate rna lacking 2 0 o methylation on the first ribose are a stronger mda5 agonists than corresponding wild-type viruses highlighting the possibility that mda5 may sense a chemical modification on the rna 5 0 end [31] . similarly to rig-i, n-terminal cards of mda5 are required for downstream signalling but unlike rig-i, mda5 oligomerizes along dsrna in a head to tail manner and positions the cards in an elongated structure that activates signalling through mavs [32] . in addition to cytoplasmic rna sensors, cells are equipped with sensors of cytoplasmic dna. dna sensing shows considerable cell-type specificity but in general two different concepts of dna sensing seem to emerge: firstly, direct activation of ifn-a/b and secondly, generation of second messengers that are activating other proteins to induce ifn-a/b. proteins directly activating ifn-a/b via the sting pathway are dna-dependent activator of irfs (dai) [33 ] and the more recently identified interferon-inducible protein 16 (ifi16) [34] . ifi16 belongs to the family of pyhin proteins and contains a pyrin domain and two dna-binding hin domains. ifi16 is able to induce ifn-a/b after infection with herpes simplex virus 1 (hsv-1) and human immunodeficiency virus 1 (hiv-1) as well as transfected dna [34, 35] . proteins generating a second messenger include rna polymerase-iii (rnapiii) and cyclic gmp-amp synthase (cgas). rnapiii binds at-rich regions in viral dna genomes to produce ppp-rna, serving as ligand for rig-i [36, 37] . cgas belongs to the nucleotidyltransferase family and upon dsdna-binding generates cyclic 2 0 -5 0 gmp-amp (cgamp) from atp and gtp [38,39 ,40-42] . cgamp binds and directly activates sting and can also cross cell barriers to activate innate immune responses in adjacent cells [43] . although the exact viral ligand has not yet been defined, lack of cgas in human or mouse cells impairs interferon responses to dna viruses and transfected dna [44 ] . another set of nucleic acid sensors that activate transcription another subset of sensors directly affects cellular machineries to impair virus growth. these proteins include 2 0 5 0 oligoadenylate synthetase (oas), dsrna-dependent protein kinase r (pkr) and absent in melanoma 2 (aim2). dsrna binding to oas catalyses the conversion of atp to 2 0 5 0 -linked oligoadenylates, which activate the latent ribonuclease rnasel to degrade cellular and viral rnas [42] . rnasel cleavage products have been demonstrated to stimulate the mavs pathway but the exact mechanism is not known. pkr is a serine/threonine kinase that is activated either by dsrna of at least 30 bp in length or by ppp-rna and suppresses general translation by phosphorylating eukaryotic initiation factor 2 alpha (eif2-a) [45, 46] . in addition pkr induces apoptosis and regulates cytokine expression, most likely by modulating mrna stability. some nucleic acid binding proteins, such as the pyhin family member aim2, regulate post-translational processing and cell death [47] . aim2 binds dna and triggers the activation of the inflammasome, a molecular platform responsible for the maturation of interleukin 1b (il-1b) and il18 as well as triggering cell death. the rna-binding helicases rig-i [48] and dhx33 [49] have also been implicated in inflammasome activation, but the precise molecular details remain to be determined. innate sensing leads to expression of effector proteins with the ability to sequester, modify or degrade viral nucleic acid (figure 1 ). sequestration of viral rnas can be achieved by interferon-induced proteins with tetratricopeptide repeats (ifits). although combinations of ifits are expressed in a species-specific manner most ifits are highly induced in expression after virus infection. ifits bind viral rna through a deep binding cleft formed by a complex arrangement of tetratricopeptide repeats [50, 51] . ifit1 preferentially binds single-stranded capped non-2 0 o-methylated (cap0) or 5 0 triphosphorylated (ppp) rna, ifit5 exclusively binds ppp-rna [12, 52 ] . ifits compete with the function of other rnabinding proteins, such as cellular translation initiation factors and/or viral proteins. since rna-binding by ifits is highly specific, translation or localisation of cellular mrnas is not affected by ifit proteins [12] . an alternative strategy to directly target viral nucleic acids is to modify or degrade them. rna-specific adenosine deaminase 1 (adar1) or members of the dna-specific apolipoprotein b mrna-editing enzyme, catalytic polypeptide-like (apobec) family deaminate nucleotides to introduce mutations, which potentially impacts rna secondary structure, stability and protein-coding capacity [45, 53] . apobec3a and 3b recognise the hepatitis b virus core protein and target core-associated dna to impair virus growth [54] . viral nucleic acids are directly targeted for degradation by 2 0 5 0 oligoadenylate-activated ribonuclease rnasel [45] and by zinc-finger antiviral protein (zap), which specifically targets viral mrnas for degradation through recruitment of the cellular exosome machinery [55 ] . dna degradation through three prime repair exonuclease 1 (trex1) is required to restrict endogenous retroviruses [56] . mutations in trex1 have been linked to autoimmune diseases, clearly highlighting the importance of nucleic acid metabolising enzymes to reduce the abundance of stimulatory nucleic acids. more recently it has also been shown that the skivl2 exosome is important to reduce stimulatory rna [57 ] . virus infection activates a restricted set of sensor and effector proteins that modulate cellular pathways and directly target viral nucleic acid, thereby shaping the innate immune response. despite remarkable progress in the last few years to uncover modifications that are sensed by the innate immune system, many questions still remain to be answered. the natural ligand of cytoplasmic sensors, for instance, is often not well understood, nor do we know the exact localisation of virus sensing in the cytoplasm. furthermore, numerous cellular pathways and second messengers contribute to innate immunity to viral pathogens and cell biological processes are similarly prominent in contributing to virus defence. we thus anticipate that even more entangled relationships between viruses and hosts are likely to be uncovered in the future. papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest novel paradigms of innate immune sensing of viral infections viruses and toll-like receptors sensing of cell death by myeloid ctype lectin receptors cytosolic sensing of viruses the influenza virus rna synthesis machine: advances in its structure and function cap and capbinding proteins in the control of gene expression molecular biology of the cell reis e sousa c: activation of mda5 requires higher-order rna structures generated during virus infection processing of genome 5 0 termini as a strategy of negative-strand rna viruses to avoid rig-idependent interferon induction conventional and unconventional mechanisms for capping viral mrna 0 -o methylation of the viral mrna cap evades host restriction by ifit family members sequestration by ifit1 impairs translation of 2 0 o-unmethylated capped rna convergent transcription induces transcriptional gene silencing in fission yeast and mammalian cells ifit1 specifically binds viral rna and thereby provides a mechanism allowing very specific targeting of viral nucleic acids without harming cellular functions inverted alu dsrna structures do not affect localization but can alter translation efficiency of human mrnas independent of rna editing human nuclear dicer restricts the deleterious accumulation of endogenous double-stranded rna nuclear ifi16 induction of irf-3 signaling during herpesviral infection and degradation of ifi16 by the viral icp0 protein dicer degrades dsrna in the nucleus to avoid activation of cytoplasmic dsrna receptors that activate the innate immune system rig-i-like receptors: cytoplasmic sensors for non-self rna the cterminal regulatory domain is the rna 5 0 -triphosphate sensor of rig-i structural and functional insights into 5 0 -ppp rna pattern recognition by the innate immune receptor rig-i antiviral immunity via rig-i-mediated recognition of rna bearing 5 0 -diphosphates rig-i forms signalingcompetent filaments in an atp-dependent, ubiquitinindependent manner molecular imprinting as a signal-activation mechanism of the viral rna sensor rig-i dephosphorylation of the rna sensors rig-i and mda5 by the phosphatase pp1 is essential for innate immune signaling lgp2 is a positive regulator of rig-i-and mda5-mediated antiviral responses identification of an lgp2-associated mda5 agonist in picornavirus-infected cells lengthdependent recognition of double-stranded ribonucleic acids by retinoic acid-inducible gene-i and melanoma differentiation-associated gene 5 molecular mechanism of signal perception and integration by the innate immune sensor retinoic acid-inducible gene-i (rig-i) visualisation of direct interaction of mda5 and the dsrna replicative intermediate form of positive strand rna viruses mda5 detects the double-stranded rna replicative form in picornavirusinfected cells in vivo ligands of mda5 and rig-i in measles virus-infected cells ribose 2 0 -o-methylation provides a molecular signature for the distinction of self and non-self mrna dependent on the rna sensor mda5 structural basis for dsrna recognition, filament formation, and antiviral signal activation by mda5 dai (dlm-1/zbp1) is a cytosolic dna sensor and an activator of innate immune response multiple mda5 molecules assembles along dsrna to arrange card domains for downstream signalling ifi16 is an innate immune sensor for intracellular dna ifi16 senses dna forms of the lentiviral replication cycle and controls hiv-1 replication rna polymerase iii detects cytosolic dna and induces type i interferons through the rig-i pathway rig-i-dependent sensing of poly(da:dt) through the induction of an rna polymerase iii-transcribed rna intermediate cyclic gmp-amp synthase is a cytosolic dna sensor that activates the type i interferon pathway structural mechanism of cytosolic dna sensing by cgas in this landmark paper the authors discovered cgas as dna sensor that generates the cyclic dinucleotide cgamp as signalling molecule cgas produces a 2 0 -5 0 -linked cyclic dinucleotide second messenger that activates sting ] is the metazoan second messenger produced by dna-activated cyclic gmp-amp synthase oas proteins and cgas: unifying concepts in sensing and responding to cytosolic nucleic acids cell intrinsic immunity spreads to bystander cells via the intercellular transfer of cgamp pivotal roles of cgas-cgamp signaling in antiviral defense and immune adjuvant effects this paper describes a novel type of signal distribution utilising cgamp, a small dinucleotide, which can cross cell barriers through gap junctions. the use of cgamp as signalling molecule allows immediate response to virus infections in bystander cells, bypassing type i interferon signalling protein kinase pkr and rna adenosine deaminase adar1: new roles for old players as modulators of the interferon response bevilacqua pc: 5'-triphosphate-dependent activation of pkr by rnas with short stem-loops aim2 activates the inflammasome and cell death in response to cytoplasmic dna recognition of rna virus by rig-i results in activation of card9 and inflammasome signaling for interleukin 1 beta production the dhx33 rna helicase senses cytosolic rna and activates the nlrp3 inflammasome ifit1 is an antiviral protein that recognizes 5 0 -triphosphate rna structural basis for viral 5 0 -ppp-rna recognition by human ifit proteins inhibition of translation by ifit family members is determined by their ability to interact selectively with the 5 0 -terminal regions of cap0-, cap1-and 5 0 ppp-mrnas the authors demonstrate that viral rna is bound in a groove formed by structural elements of ifit proteins. this binding mechanism is potentially flexible to accommodate additional ligands with high specificity in a similar manner apobec proteins and intrinsic resistance to hiv-1 infection specific and nonhepatotoxic degradation of nuclear hepatitis b virus cccdna the zinc-finger antiviral protein recruits the rna processing exosome to degrade the target mrna a long-sought issue on apobec-mediated virus targeting was how these proteins detect viral nucleic acid. it appears that apobec3a and apo-bec3b sense the hbv core protein associated to viral dna and this is required for specific targeting trex1 prevents cell-intrinsic initiation of autoimmunity the skiv2l rna exosome limits activation of the rig-i-like receptors the authors want to thank friedemann weber, gareth brady and the members of the innate immunity laboratory for critical input and suggestions. mh is funded by an alexander von humboldt fellowship. ap is funded by the max-planck free floater programme, an erc starting grant (ivip), infect-era (erase) and the german research foundation (pi 1084/2-1). key: cord-254549-ev0oesu0 authors: kutikhin, anton g; yuzhalin, arseniy e title: c-type lectin receptors and rig-i-like receptors: new points on the oncogenomics map date: 2012-02-24 journal: cancer manag res doi: 10.2147/cmar.s28983 sha: doc_id: 254549 cord_uid: ev0oesu0 the group of pattern recognition receptors includes families of toll-like receptors, nod-like receptors, c-type lectin receptors, and rig-i-like receptors. they are key sensors for a number of infectious agents, some of which are oncogenic, and they launch an immune response against them, normally promoting their eradication. inherited variations in genes encoding these receptors and proteins and their signaling pathways may affect their function, possibly modulating cancer risk and features of cancer progression. there are numerous studies investigating the association of single nucleotide polymorphisms within or near genes encoding toll-like receptors and nod-like receptors, cancer risk, and features of cancer progression. however, there is an almost total absence of articles analyzing the correlation between polymorphisms of genes encoding c-type lectin receptors and rig-i-like receptors and cancer risk or progression. nevertheless, there is some evidence supporting the hypothesis that inherited c-type lectin receptor and rig-i-like receptor variants can be associated with increased cancer risk. certain c-type lectin receptors and rig-i-like receptors recognize pathogen-associated molecular patterns of potentially oncogenic infectious agents, and certain polymorphisms of genes encoding c-type lectin receptors and rig-i-like receptors may have functional consequences at the molecular level that can lead to association of such single nucleotide polymorphisms with risk or progression of some diseases that may modulate cancer risk, so these gene polymorphisms may affect cancer risk indirectly. polymorphisms of genes encoding c-type lectin receptors and rig-i-like receptors thereby may be correlated with a risk of lung, oral, esophageal, gastric, colorectal, and liver cancer, as well as nasopharyngeal carcinoma, glioblastoma, multiple myeloma, and lymphoma. the list of the most promising polymorphisms for oncogenomic investigations may include rs1926736, rs2478577, rs2437257, rs691005, rs2287886, rs735239, rs4804803, rs16910526, rs36055726, rs11795404, and rs10813831. of reactive oxygen species, pyroptosis, angiogenesis, and, consequently, tissue remodeling and repair. [1] [2] [3] [4] there are four main groups of pattern recognition receptors, ie, toll-like receptors, nod-like receptors, c-type lectin receptors, and rig-i-like receptors, and genes encoding them are broadly expressed, eg, in epithelial cells, endothelial cells, keratinocytes, lymphocytes, granulocytes, fibroblasts, and neurons. [1] [2] [3] [4] a summary of the most modern conceptual data about members of these groups and about their structure and function can be obtained from recent comprehensive reviews by kawai and akira, 1 elinav et al, 2 osorio et al, 3 and loo and gale. 4 the completion of the human genome project and widespread distribution of genotyping technologies have led to an enormous number of studies devoted to associating inherited gene polymorphisms with various diseases. single nucleotide polymorphisms may result in amino acid substitutions altering protein function or splicing, and they can also change the structure of enhancer sequences during splicing 5 and affect mrna stability. 6 single nucleotide polymorphisms may alter transcription factor binding motifs, change the efficacy of enhancer or repressor elements, 7 and alter the structure of translation initiation codons that may lead to downregulation of wild-type transcripts. 8 gene polymorphisms located in leucine-rich repeats constituting ectodomains of many pattern recognition receptors may affect the ability of these receptors to bind pathogens they normally recognize, 9 single nucleotide polymorphisms in transmembrane domains can lead to defects of intracellular receptor transport that prevent receptors localizing to the cell membrane, 10 and, finally, polymorphisms in the cytosolic domains may result in altered interactions with adaptor proteins or in disrupted receptor dimerization. therefore, there are many avenues by which single nucleotide polymorphisms may alter pattern recognition receptor expression and activity. because pattern recognition receptors recognize a number of oncogenic infectious agents and launch an immune response against them, inherited variation in their structure may modulate cancer risk and, possibly, influence cancer progression. in addition, pattern recognition receptors bind a lot of endogenous ligands, 1-4 so polymorphisms of genes encoding them can affect risk and/or progression of some autoimmune disorders and, consequently, cancer risk and/or progression, given that there is a fundamental and epidemiological association between many autoimmune diseases and cancer risk. although there are a lot of studies investigating the association between single nucleotide polymorphisms in genes encoding toll-like receptors and nod-like receptors and the risk and features of cancer progression, there is an almost complete absence of articles analyzing the correlation between polymorphisms of genes encoding c-type lectin receptors and rig-i-like receptors and cancer risk or progression. this can be explained by the fact that the first wave of studies devoted to the association of polymorphisms of genes encoding toll-like receptors and nod-like receptors with cancer risk appeared only in 2004, and the number of such papers was relatively small until 2008. in addition, known hypotheses about the infectious agents causing human cancer and their recognition by pattern recognition receptors suggested that toll-like receptors and nod-like receptors should play a major role in the immune response against biological carcinogens. however, more recent findings concerning specific potentially carcinogenic ligands of c-type lectin receptors and rig-i-like receptors were only obtained in the last few years, 3,4 so there has not been enough time as yet to conduct comprehensive investigations between single nucleotide polymorphisms of genes encoding c-type lectin receptors and rig-i-like receptors and cancer risk. however, there is some evidence supporting the hypothesis that inherited features of c-type lectin receptor and rig-i-like receptor structure can be associated with increased cancer risk. certain c-type lectin receptors and rig-i-like receptors recognize pamps of oncogenic infectious agents. 3, 4, 11, 12 c-type lectin receptors: on the basis of known associations between inherited structural variations in toll-like receptors and nod-like receptors and cancer risk, 1,2 and according to data about cancer types caused by carcinogenic infectious agents, 11, 12 it is possible to suggest that risk of lung cancer may be modulated by polymorphisms of the mrc1, cd209, clec7a, clec6a, and clec4e genes, oral cancer risk by single nucleotide polymorphisms of the mrc1, cd207, cd209, clec6a, and clec4e genes, risk of glioblastoma and colorectal cancer by polymorphisms of the cd209 gene, hepatocellular carcinoma risk by polymorphisms of the cd209 and rig-i genes, and risk of lymphoma, multiple myeloma, nasopharyngeal carcinoma, and esophageal and gastric cancer by single nucleotide polymorphisms of the rig-i gene. in addition, single nucleotide polymorphisms of mrc1, cd207, ly75, cd209, clec1b, and clec4a genes may correlate with cancer types associated with hiv-1 infection. certain polymorphisms of genes indicated above may have functional consequences on the molecular level that can lead to association of such single nucleotide polymorphisms with risk or progression of some diseases that may modulate cancer risk, so these gene polymorphisms may affect cancer risk indirectly. in addition, polymorphisms of these genes correlating with diseases that are not related to cancer risk may also be useful in oncogenomics because they may have functional consequences at the molecular level as well, although they have not been investigated in relation to association with cancer risk or progression. for instance, it was suggested that variant alleles of mrc1 rs2477637, rs2253120, rs2477664, rs692527, rs1926736, and rs691005 gene polymorphisms are associated with development of asthma 13 (eg, variant a allele of rs1926736 was connected with decreased asthma risk). in addition, alter et al 14 found that the variant a allele (s396) of rs1926736 (g396s) polymorphism is associated with a lower leprosy risk and, conversely, g allele (g396) correlates with increased risk of this disease. interestingly, g396 did not influence leprosy risk in combination with t399 and l407 (amino acids resulting from variant alleles of rs2478577 and rs2437257, respectively). 14 the authors noted that all three of these mrc1 gene single nucleotide polymorphisms map to the second c-type lectin domain (ctld2) of the mrc1 protein, with their in vitro results suggesting that a direct interaction between ctld2 and an accessory receptor molecule is necessary in order for microbial ligand recognition to occur. 14 it is logical to propose that such interaction would be sensitive to g396 only in the context of the a399-f407 haplotype, and not in the context of the t399-l407 haplotype. 14 thus, rs1926736 may have substantial functional consequences at the molecular level, but this depends on its relationship with other single nucleotide polymorphisms in the same exon. finally, hattori et al 15 showed that a variant allele of rs691005 polymorphism, located within the 3′ untranslated region of the mrc1 gene, is associated with a higher risk of sarcoidosis. because of its location, it is feasible that this single nucleotide polymorphism may alter the regulatory binding sequence and influence mrna expression. 15 the only study investigating the association of polymorphisms of genes encoding c-type lectin receptors and rig-i-like receptors with cancer risk is a study by xu et al. 16 they investigated single nucleotide polymorphisms of the cd209 gene and found that the gg genotype of the rs2287886, aa genotype of the −939 promoter polymorphism, and the g allele of the rs735239 single nucleotide polymorphism were connected with higher nasopharyngeal carcinoma risk. 16 polymorphisms in the promoter of the cd209 gene and in the cd209 gene were also associated with hemorrhage in patients with dengue fever (g allele of rs4804803), 17,18 modulated tuberculosis risk (g allele of rs4804803, a allele of rs735239), [19] [20] [21] higher celiac disease risk in hla-dq2-negative cases (g allele of rs4804803), 22 increased ulcerative colitis risk in hla-dr3-positive patients (g allele of rs4804803), 23 higher susceptibility to cytomegalovirus infection (g allele of rs735240 and c allele of rs2287886), 24 protection from lung cavitation 20 and fever during tuberculosis 25 (gg genotype and g allele of rs4804803), decreased hiv-1 infection risk (gg genotype of rs4804803), 21 accelerated progression to acquired immune deficiency syndrome in hiv-1-infected hemophiliacs (c allele of rs2287886), 26 decreased human t-lymphotropic virus type i infection risk (g allele of rs4804803, a allele of rs2287886), 27 increased severity of liver disease during hepatitis c virus infection (g allele of rs4804803), 28 and better prognosis following severe acute respiratory syndrome (g allele of rs4804803). 29, 30 it was shown that the a allele of the rs4804803 single nucleotide polymorphism may increase gene expression in vitro, 17 expression in subjects with the g allele may result in an impaired immune response against hepatitis c virus, 28 m. tuberculosis, 19, 21 and bacteria potentially causing celiac disease 22 and ulcerative colitis, 23 that elevates the risk of diseases caused by these infectious agents. such a decreased immune response may protect from hemorrhage during dengue fever, 17 from lung cavitation, 20 from fever during tuberculosis, 25 and from lung injury during severe acute respiratory syndrome 29,30 as a result of less cytokine production and diminished activation of immune cells. however, from the point of view of vannberg et al, 20 conversely, lower cd209 gene expression as a consequence of g allele of rs4804803 polymorphism may protect against tuberculosis because of decreased production of proinflammatory cytokines such as interleukin-4. further fundamental, translational, and clinical studies are necessary to clarify these discrepancies. nevertheless, although there are a number of reasons for the discrepancies between studies devoted to the association between cd209 single nucleotide polymorphisms and development of tuberculosis, but confounding host, bacterial, and submit your manuscript | www.dovepress.com dovepress dovepress environmental factors between different study populations should be taken into account. in addition, mezger et al 24 demonstrated that alleles of rs735240 and rs2287886 polymorphisms may also influence cd209 gene expression and thus affect transcription factor binding. in relation to the clec7a (dectin-1) gene, it was also found that a variant allele of rs16910526 polymorphism is associated with impaired cytokine production by macrophages 31, 32 and with a defective response to aspergillus and candida invasion. 33, 34 the variant s form of i223s polymorphism was characterized by a lower capacity of the receptor to bind zymosan. 35 among polymorphisms of genes encoding rig-ilike receptors, rig-i single nucleotide polymorphisms are the most investigated. pothlichet et al 36 conducted a comprehensive study investigating the functional consequences of rs36055726 (p229fs) and rs11795404 (s183i) polymorphisms. they found that the variant allele of rs36055726 results in a truncated constitutively active rig-i (that leads to permanent production of proinflammatory mediators, particularly antiviral), and, conversely, the variant allele of rs11795404 induces an abortive conformation of rig-i, causing formation of unintended stable complexes between card modules of rig-i and between rig-i and its downstream adapter protein, mavs, rendering rig-i incapable of downstream signaling and further cytokine synthesis. 36 moreover, shigemoto et al identified a variant of rs11795404 as a loss-of-function allele. 37 ovsyannikova et al 38, 39 showed that a minor allele of rs10813831 polymorphism is associated with a decrease in the rubella virus-specific granulocyte-macrophage colony-stimulating factor/interleukin-6/igg response, whilst a variant allele of rs3824456 is connected with an increase in the rubella virusspecific tumor necrosis factor alpha response, and a variant allele of rs669260 correlates with an increase in the rubellaspecific antibody level. hu et al 40 discovered that a variant allele of rs10813831 polymorphism leads to increased gene expression and, consequently, cytokine production due to an amino acid substitution in the card domain of rig-i that results in functional alteration of this rig-i-like receptor. there are also a lot of studies investigating the role of ifih1/mda5 (the gene encoding mda5 protein that is also a rig-i-like receptor) single nucleotide polymorphisms in the etiology of autoimmune diseases, but almost all of them are devoted to type 1 diabetes and multiple sclerosis, and data about the association of these diseases with cancer risk are conflicting, in that some studies showed an increased risk in patients with type 1 diabetes and multiple sclerosis, 41, 42 and in other investigations no connection or decreased risk of cancer has been observed. [43] [44] [45] [46] [47] [48] [49] taking into account that there are no carcinogenic infectious agents recognizing mda5, it does not seem to be prudent to investigate ifih1/mda5 gene polymorphisms from the oncogenomic point of view. in addition, polymorphisms of genes coding for components of the toll-like receptor signaling pathway may modulate cancer risk as single nucleotide polymorphisms of the tlr gene family. 1 the same statement can be true for c-type lectin receptor and rig-i-like receptor signaling pathways. for instance, a variant allele of rs11905552, encoding mavs/ visa/ips-1, a key downstream signaling molecule of rig-i and mda5, was associated with a particular systemic lupus erythematosus phenotype. 50 it was found that this single nucleotide polymorphism leads to reduced production of type i interferon and other proinflammatory mediators, and also to the absence of anti-rna-binding protein autoantibodies. 50 in addition, variant alleles of rs17857295 and rs2326369 polymorphisms of the mavs/visa/ips-1 gene were associated with nephritis and arthritis in patients suffering from systemic lupus erythematosus. 51 a variant allele of another single nucleotide polymorphism of this gene, rs7269320, showed associations with different clinical characteristics of this autoimmune disease. 51 all the population case-control studies mentioned above are summarized in table 1 . all polymorphisms of genes encoding c-type lectin receptors, rig-i-like receptors, and proteins of their specific signaling pathways that have known functional consequences and may be relevant to oncogenomics are summarized in table 2 . the fundamental basis for the association of the inherited coding variation in genes encoding c-type lectin receptors and rig-i-like receptors with cancer is represented by the defects in the immune response (that are caused by various single nucleotide polymorphisms) against specific carcinogenic infectious agents. some polymorphisms may be valued as the most promising for further oncogenomic investigations on the basis of their association with cancer risk or because of their substantial functional consequences on the molecular level according to the following concept: gene polymorphism may be included on the short list for further oncogenomic studies if: • the single nucleotide polymorphism leads to substantial functional consequences at the molecular level (for instance, it strongly affects transcription, splicing, translation, stability and transport of pre-mrna, mrna, noncoding rna, or protein encoding by the gene, or it noticeably influences signaling of synthesized protein) • it is associated with risk of cancer in population studies • it has functional consequences at the molecular level and it is strongly associated with a condition that significantly increases the risk of cancer (threshold may vary for each cancer type) the gene polymorphism can be also included on the extended list if: • it is characterized by more subtle functional alterations in a gene that, nonetheless, result in qualitative or quantitative alterations of the encoding protein (or noncoding rna) • it is associated with a condition that substantially increases the risk of cancer but has not specifically been identified to increase the risk of cancer. according to this concept, the indicated short list of polymorphisms includes rs1926736, rs2478577, rs2437257, rs691005 (all located in the mrc1 gene), rs2287886, -939 promoter polymorphism, rs735239, rs735240, rs4804803 (all located in the cd209 gene), rs16910526 (clec7a gene), and rs36055726, rs11795404, rs10813831 (all located in the rig-i gene). other polymorphisms mentioned in this article may be added to the extended list for further investigations. polymorphisms with known functional effects (rs1926736, rs2437257, rs691005, rs2287886, rs735240, rs4804803, rs16910526) were associated with relatively significant modulation of risk of diseases (as shown in table 1 ) which is logical and demonstrates the correctness of the studies in which functional consequences of such single nucleotide polymorphisms were analyzed. there are still no comprehensive functional investigations for other single nucleotide polymorphisms correlated with risk of disease, so it is difficult to conclude which of them have independent significance, and which of them are just in linkage disequilibrium with truly functional variants. in addition, pamps of specific infectious agents recognized by each c-type lectin receptor or rig-i-like receptor define cancer types which can be primarily associated with inherited structural variation in the receptors discussed earlier. furthermore, if a single nucleotide polymorphism of a gene encoding a specific c-type lectin receptor or rig-i-like receptor is associated with risk or progression features of certain malignancies, polymorphisms in genes encoding specific signaling molecules constituting pathways of these receptors should correlate with similar neoplasms, if they have substantial functional consequences at the molecular level. the issue of an association of single nucleotide polymorphisms of genes encoding c-type lectin receptors, rig-i-like receptors, and proteins of pattern recognition receptor pathways with various features of cancer progression is open, and only further population studies would be likely to give a definite answer. submit your manuscript | www.dovepress.com reasons for discrepancies in different investigations analyzing the association of polymorphisms in genes encoding c-type lectin receptors, rig-i-like receptors, and the proteins of their signaling pathways with various aspects of cancer development may include confounding host, bacterial, or environmental factors in different ethnicities modulating penetrance of variant alleles and affecting the risk of conditions increasing cancer risk (such as autoimmune diseases, precancerous gastric lesions, tuberculosis, recurrent pneumonia), different bacterial impact on the etiology of such conditions in different populations (that will be reflected in different features of c-type lectin receptor/ rig-i-like receptor-mediated immune response because of specific c-type lectin receptor/rig-i-like receptor-ligand interaction), differences in sample size, in clinicopathological characteristics between study samples, in prevalence of infectious agents in case and control groups, diagnostics, stratification, genotyping methods, and chance. another interesting issue is that associations between single nucleotide polymorphisms of genes encoding c-type lectin receptors and rig-i-like receptors and cancer risk can be skewed by differences between cohorts in various immune responses and infections that may not influence cancer development. the problem is that the design in an epidemiological study having a large sample is very seldom ideal. stratification by status of chronic infection is rather difficult because of their extreme diversity and because of the very high cost of such testing. stratification by an immune response is even more complex because of innumerable peculiarities in functioning of the immune system. therefore, if the study has a perfect funding source, stratification by infection status can be possible, but stratification by immune response status will be far from ideal. unfortunately, to the best of the authors' knowledge, no genome-wide association studies of the connection between polymorphisms of genes encoding the c-type lectin receptor and rig-i-like receptors and cancer risk or progression have been performed, and this can be explained by the relative newness of the problem or perhaps by another unknown reason. summing up, polymorphisms of genes encoding c-type lectin receptors, rig-i-like receptors, and proteins of their signaling pathways may be promising targets for oncogenomics and possibly could be used in programs of cancer prevention and early cancer diagnostics in the future. population and further fundamental studies devoted to their association with cancer risk of progression should shed light on this issue. the authors report no conflicts of interest in this work. toll-like receptors and their crosstalk with other innate receptors in infection and immunity regulation of the antimicrobial response by nlr proteins myeloid c-type lectin receptors in pathogen recognition and host defense immune signaling by rig-i-like receptors hepatic cyp2b6 expression: gender and ethnic differences and relationship to cyp2b6 genotype and car (constitutive androstane receptor) expression plasminogen activator inhibitor type 2 contains mrna instability elements within exon 4 of the coding region. sequence homology to coding region instability determinants in other mrnas single nucleotide polymorphism in 5′-flanking region reduces transcription of surfactant protein b gene in h441 cells c/t polymorphism in the 5′ untranslated region of the apolipoprotein(a) gene introduces an upstream atg and reduces in vitro translation leucine-rich repeats and pathogen recognition in toll-like receptors cutting edge: a common polymorphism impairs cell surface trafficking and functional responses of tlr1 but protects against leprosy role of bacteria in oncogenesis infections and cancer: established associations and new hypotheses genetic variants in the mannose receptor gene (mrc1) are associated with asthma in two independent populations genetic and functional analysis of common mrc1 exon 7 polymorphisms in leprosy susceptibility genetic variants in mannose receptor gene (mrc1) confer susceptibility to increased risk of sarcoidosis sequencing of dc-sign promoter indicates an association between promoter variation and risk of nasopharyngeal carcinoma in cantonese a variant in the cd209 promoter is associated with severity of dengue disease dc-sign (cd209) promoter -336 a/g polymorphism is associated with dengue hemorrhagic fever and correlated to dc-sign expression and immune augmentation promoter variation in the dc-sign-encoding gene cd209 is associated with tuberculosis cd209 genetic polymorphism and tuberculosis disease cd209 gene polymorphisms in south indian hiv and hiv-tb patients a functional variant in the cd209 promoter is associated with dq2-negative celiac disease in the spanish population cd209 in inflammatory bowel disease: a case-control study in the spanish population investigation of promoter variations in dendritic cell-specific icam3-grabbing non-integrin (dc-sign) (cd209) and their relevance for human cytomegalovirus reactivation and disease after allogeneic stem-cell transplantation relationship between polymorphism of dc-sign (cd209) gene and the susceptibility to pulmonary tuberculosis in an eastern chinese population rantes -28g delays and dc-sign -139c enhances aids progression in hiv type 1-infected japanese hemophiliacs dc-sign (cd209) gene promoter polymorphisms in a brazilian population and their association with human t-cell lymphotropic virus type 1 infection variant in cd209 promoter is associated with severity of liver disease in chronic hepatitis c virus infection dc-sign) -336 a . g promoter polymorphism and severe acute respiratory syndrome in hong kong chinese association of a single nucleotide polymorphism in the cd209 (dc-sign) promoter with sars severity functional consequences of dectin-1 early stop codon polymorphism y238x in rheumatoid arthritis dectin-1 y238x polymorphism associates with susceptibility to invasive aspergillosis in hematopoietic transplantation through impairment of both recipient-and donordependent mechanisms of antifungal immunity the y238x stop codon polymorphism in the human β-glucan receptor dectin-1 and susceptibility to invasive aspergillosis early stop polymorphism in human dectin-1 is associated with increased candida colonization in hematopoietic stem cell transplant recipients genetic variation of innate immune genes in hiv-infected african patients with or without oropharyngeal candidiasis study of human rig-i polymorphisms identifies two variants with an opposite impact on the antiviral immune response identification of loss of function mutations in human genes encoding rig-i and mda5: implications for resistance to type i diabetes rubella vaccineinduced cellular immunity: evidence of associations with polymorphisms in the toll-like, vitamin a and d receptors, and innate immune response genes polymorphisms in the vitamin a receptor and innate immunity genes influence the antibody response to rubella vaccination a common polymorphism in the caspase recruitment domain of rig-i modifies the innate immune response of human dendritic cells cancer risk among patients hospitalized for type 1 diabetes mellitus: a populationbased cohort study in sweden cancer incidence in patients with type 1 diabetes mellitus: a population-based cohort study in sweden cancer incidence and mortality in patients with insulin-treated diabetes: a uk cohort study cancer and diabetes -a follow-up study of two population-based cohorts of diabetic patients cancer risk among patients with multiple sclerosis and their parents cancer risk among patients with multiple sclerosis: a population-based register study cancer incidence in multiple sclerosis: a 35-year follow-up multiple sclerosis and cancer in norway. a retrospective cohort study cancer in patients with motor neuron disease, multiple sclerosis and parkinson's disease: record linkage studies a loss-of-function variant of the antiviral molecule mavs is associated with a subset of systemic lupus patients possible association of visa gene polymorphisms with susceptibility to systemic lupus erythematosus in chinese population publish your work in this journal submit your manuscript here: http://www.dovepress.com/cancer-management-and-research-journal cancer management and research is an international, peer-reviewed open access journal focusing on cancer research and the optimal use of preventative and integrated treatment interventions to achieve improved outcomes, enhanced survival and quality of life for the cancer patient. the journal welcomes original research, clinical & epidemiological studies, reviews & evaluations, guidelines, expert opinion & commentary, case reports & extended reports. the manuscript management system is completely online and includes a very quick and fair peerreview system, which is all easy to use. visit http://www.dovepress.com/ testimonials.php to read real quotes from published authors. key: cord-287855-jfrg9soy authors: gaur, pratibha; munjal, ashok; lal, sunil k. title: influenza virus and cell signaling pathways date: 2011-06-01 journal: med sci monit doi: 10.12659/msm.881801 sha: doc_id: 287855 cord_uid: jfrg9soy influenza viruses comprise a major class of human respiratory pathogens, responsible for causing morbidity and mortality worldwide. influenza a virus, due to its segmented rna genome, is highly subject to mutation, resulting in rapid formation of variants. during influenza infection, viral proteins interact with host proteins and exploit a variety of cellular pathways for their own benefit. influenza virus inhibits the synthesis of these cellular proteins and facilitates expression of its own proteins for viral transcription and replication. infected cell pathways are hijacked by an array of intracellular signaling cascades such as nf-κb signaling, pi3k/akt pathway, mapk pathway, pkc/pkr signaling and tlr/rig-i signaling cascades. this review presents a research update on the subject and discusses the impact of influenza viral infection on these cell signaling pathways. infectious diseases such as aids, tuberculosis, malaria, meningitis and influenza remain the most important cause of death and disabilities in human populations worldwide. influenza virus causes epidemics almost every year and occasionally pandemics, due to antigenic drift or antigenic shift [1] . each year influenza virus causes 65 million illnesses and 25,000 deaths in the usa alone. globally, the world health organization (who) estimates the burden of influenza at ~3-5 million cases of severe illness and >300,000 deaths annually. most recently the who declared a phase 6 global pandemic on 11 th june 2009, and more than 209 countries and overseas communities have reported laboratory confirmed cases of pandemic influenza h1n1 2009, including at least 14,142 deaths [2] . influenza virus belongs to the orthomyxoviridae family. orthomyxoviridae are enveloped viruses with negative sense ssrna genome, split into 8 segments, encoding 11 proteins. hemagglutinin (ha) and neuraminidase (na) are 2 important proteins present on the surface of the viral envelope; mutation in these 2 proteins gives rise to the 16 ha and 9 na, different subtypes of influenza virus [3] . the viral rna polymerase complex, composed of the 3 largest gene segments -pb1, pb2, pa and nucleoprotein (np) -is associated with viral rna ribonucleoprotein complex. the 2 matrix proteins, m1 and m2, are the smallest rna proteins and m2 forms ion channels in the viral membrane. the nonstructural gene (ns1) is a multifunctional protein, and ns2 protein is involved in the nuclear export of the viral rnps to the cytoplasm. pb1-f2 protein is a product of the second gene product of pb1. to date it has been documented that influenza viral proteins interact with 1023 host proteins [4] . like other viruses, influenza virus also takes advantage of the host cellular machinery for their efficient replication. according to recent research reports, the following cellular signaling pathways are altered following influenza infection: nf-kb signaling, pi3k/akt pathway, mapk pathway, pkc/pkr signaling, and tlr/rig-i signaling cascades [5] [6] [7] [8] [9] [10] [11] [12] [13] . these pathways are important for viral entry, viral replication, viral propagation and apoptosis, and are involved in antagonizing the host antiviral response. influenza virus manipulates the molecular function of signaling molecules for efficient viral pathogenesis. this review addresses the impact of influenza infection on cellular signaling events. the nfkb/ikb pathway plays a vital role in mediating inflammation, immune response, proliferation and apoptosis [14, 15] . nf-kb is a nuclear factor kb, a transcriptional factor which plays a central role in promoting the expression of more than 150 genes, which in turn governs the cellular status of genes encoding cytokine/chemokines, adhesion molecules and anti/pro-apoptotic genes [6, 16] . nf-kb is present as a complex with its inhibitor ikb. for release from this complex, activation of ikk is required [17] . the ikk complex is composed of active ikk1/ikka, ikk2/ikkb and scaffold protein nemo (nf-kb essential modulator)/ikkg [15] . upon activation, ikk phosphorylates and degrades the ikb protein [18] , which leads to the release of transcriptionally active nf-kb subunits p65/p50 from the inhibitory complex which translocate into the nucleus, where they can 'turn on' the expression of specific genes that have dna-binding sites for nf-kb in their promoters [18, 19] (figure 1 (2)). ikk2 degrades the ikb and activates nf-kb, while ikk1 primarily phosphorylates the other factors of the nf-kb family, such as p100/p52 [15] . it is well established that nf-kb is a major target of most viral pathogens [20, 21] . during viral infection there is activation of the nf-kb signaling pathway and an increase in the gene expression levels of ifn-b/tnfa/il8 [22, 23] , which suggests that ikk-mediated nf-kb signaling is essential for the host innate immune response [24] . for instance, vaccinia virus expresses the tir domain to suppress the tlr/il-1 receptor-induced nf-kb activation [25, 26] . oncogenic viruses such as epstein barr virus (ebv) and kaposi's sarcoma-associated herpes virus (kshv), activate nf-kb during latent infection, which subsequently causes tumorogenesis [27] . thus, viruses have evolved mechanisms to manipulate and thereby evade/modulate host immune responses [28] . several lines of evidence suggest that activation of the nf-kb pathway is a primary requirement for influenza virus infection and its efficient replication [6, [29] [30] [31] . nf-kb activation is biphasic after the influenza infection. early activation of nf-kb has been observed 1 hour post-infection and the later activation was associated with viral replication [32] . some studies also indicate that viral protein overexpression, specifically ha, np and m1 (figure 1 (2)), may be responsible for activation of the nf-kb pathway [5, 20, 29, 31, 33] . these viral proteins are sufficient to transcriptionally activate nf-kb by involving the generation of oxidative radicals which activates ikk as a signal transduction intermediate, a kinase which phosphorylate ikb, thereby regulating nf-kb activity [5] . as a result of activation of this pathway, the host immune response is elicited, to combat which, non-structural protein ns1 of influenza virus comes to its rescue by serving as an antagonist of host ifn response [34] . influenza a virus infection also activates the pi3k/akt pathway. it has been recently reported that the pi3k/akt signaling pathway is induced by the viral ns1 protein to support its efficient replication [7, 35, 36] . pi3k is a family of enzymes that play a pivotal role in regulation of essential cellular functions (cell survival, proliferation, differentiation, etc.) [37, 38] . pi3k family is divided into 3 classes: class i, ii and iii. class i pi3k are heterodimeric enzymes composed of a catalytic subunit, p110, and a regulatory subunit, p85 [39, 40] . they catalyze the generation of phosphatidylinositol-3-phosphate (pip), phosphatidylinositol-3,4-bisphosphate (pip2) and phosphatidylinositol-3,4,5-triphosphate (pip3) [8] . the regulatory subunit of pi3k p85 contains 2 src homology domains, sh2 and sh3. through its sh2 domain, the p85 subunit binds to autophosphorylated receptor tyrosine kinase, in the process activating pi3k. following activation, the p110 subunit converts pip2 to pip3 [41] , which in turn leads to phosphorylation and activation of a number of kinases, including akt/pkb. activation of akt through phosphorylation at thr 308 and ser 473 residues [42] plays a major role in modulating diverse downstream signaling pathways, including cell survival, proliferation, migration, differentiation and inhibition of proapoptotic factors such as bad and caspase-9 by their phosphorylation (figure 1(1) ). the class ii pi3k is composed of 3 catalytic subunits (c2a, c2b, and c2g) but no regulatory subunit. this is involved in the production of pip and pip2 from pi. class iii consists of catalytic (vps34) and regulatory (vps15/p150) subunits that catalyze the generation of pip from pi. [39] . the ns5a protein of hcv directly binds to the sh3 domain of p85 and induces pi3k/akt-mtor signaling to control cell survival [44] . additionally, viruses which cause acute infection such as respiratory syncytial virus (rsv) manipulate pi3k activity for efficient viral replication. other viruses such as sars coronavirus, poliovirus, dengue virus and influenza virus also utilize the pi3k signaling pathway to their advantage [8, [45] [46] [47] . influenza a virus has devised diverse mechanisms to activate pi3k, which in turn leads to activation of several other downstream kinases at different time points during viral replication. though early activation of pi3k has also been reported in influenza b virus infection, late activation only occurs in type a virus [48] . during the later stages of replication, the ns1 protein binds to the sh2 domain of the p85 subunit and activates pi3k, which leads to suppression of cell death [37, 38] . a recently proposed model for activation of pi3k by ns1 suggests formation of an active heterotrimeric complex of p110/p85-ns1 wherein ns1 disrupts the inhibitory interaction interface between p110 and p85 [49, 50] . the activated pi3k/akt pathway is further involved in efficient virus replication and propagation [35, 36] and may also regulate antiviral function, although these processes remain largely unclear. mitogen activated protein kinase (mapk) cascades are involved in the conversion of various extracellular signals into cellular responses as diverse as proliferation, differentiation, immune response and cell death [51, 52] . four distinct subgroups of the mapk family have been well studied [9, 51] : (a) extracellular signal-regulated kinases (erks), (figure 1 (3 c)), (b) the p38 mapk, (figure 1(3 b) ) (c) c-jun n-terminal or stress-activated protein kinases (jnk/ sapk) (figure 1(3 a) ), and (d) erk5/big map kinase 1(bmk1) (20) (figure 1(3 d) ). in mammals 3 major pathways have been identified: mapk/erk, sapk/jnk and p38 mapk. mapk signaling promotes cell survival by a dual phosphorylation event on threonine and tyrosine residues [10] . the upstream mapkk regulates these 4 enzyme activities. the 2 mapkk (mkk3/6, mkk4/7) are responsible for activation of p38 and jnk, respectively. these enzymes are involved in apoptosis and cytokine expression [53] , and can be activated by environmental stress conditions. the upstream raf controls the phosphorylation of mapk/erk kinase (mek) ½, which regulates the activation of erk ½, which plays a regulatory role in cell proliferation and differentiation. lastly, but importantly, enzyme erk5 is activated by mek5 [10] (figure 1(3 d) ). been shown to promote vrnp (viral ribonucleoprotein capsids) traffic and virus production. in the previous study, using specific kinase inhibitors, p38 and jnk have been linked to virus-induced expression of rantes (a chemokine involved in the attraction of eosinophils during an inflammatory response) [54] .another study suggests that erk and jnk are involved in the expression of inflammatory mediator cytooxygenase and phosphorylation of cytosolic phospholipase a2 in bronchial epithelial cells [57] . it has been shown that there is activation of the activator protein-1 (ap-1) during early stages of infection. jnk activation is induced by accumulation of rna produced by viral polymerase. ap-1 is a transcription factor that includes c-jun and activating-transcription-factor-2 (atf-2), whose transcriptional activity is enhanced by jnks [53, 58] , a part of the mapk signalling pathway [23] . ap-1 is also crucial for the expression of interferon-b (ifn-b) and antiviral cytokines [59] . furthermore, inhibition of jnk by dominant negative mutants of mkk7/jnk/c-jun results in impaired transcription from ifn-b promoter during influenza virus infection, thereby increasing virus production [53] . thus, this pathway is important as a mediator of inflammatory response to an influenza infection by co-regulating ifn-b expression ( figure 1(3 a) ). p38 mapk activation regulates the expression of rantes production [47, 60] and chemokines by influenza virus infection [54] . in highly pathogenic h5n1-infected cells, p38 induces tumor necrosis factor (tnf) cytokine [47] . according to a recently published report, il-1b stimulates activation of p38 mapk with prostaglandin e2 production. the p38 inhibitor decreases the release of prostaglandin e2 [57] and also reduces the virus titer, which suggests that p38 mapk activation is essential for inflammatory responses and contributes to the viral replication process. influenza virus infection induces tnf-a in a p38-dependent manner [47] (figure 1(3 b) ). raf/mek/erk pathway is the best proven mapk signaling pathway [51, 61] . many rna viruses induce cellular signaling through mapk cascades. the mechanism of this pathway is initiated by g protein-coupled receptors, which leads to the phosphorylation of downstream molecules and activates the serine threonine kinase raf (dual specificity kinase mek and mapk/erk). erk phosphorylates various substrates, transforms the signals and followes different functions in cells [62] . influenza infection also upregulates this signaling, which is important for efficient export of nuclear rnps. the inhibition of mek blocks this pathway [9] , shown to impair the growth of viruses and decrease the nuclear rnp export [9] . interestingly, it does not affect viral rna or protein synthesis. this suggests that the nuclear rnp export appears in the inducible phase and correlates well with the hypothesis that erk activation occurs in the late phase of infection. the erk ½ are vital for the expression of pro-inflammatory il1b, il-6 and il-8 [63] . erk can regulate the expression of tnf-a, il-12, il-1b, and inhibition of erk by u0126 inhibitor [64] can also reduce the rate of influenza replication by nuclear retention of vrnps ( figure 1(3 c) ). a recent study suggests that raf/mek/erk signaling is activated by proper accumulation of ha/lipid-raft association within the cellular membrane [65] . if there is inadequate transport of ha from cytoplasm to cell surface, this could be a possible reason for the low activation of erk [65] . the viral polymerase complex is responsible for ha accumulation in connection with mapk signaling, supporting the idea that more erk activation follows more efficient nuclear rnp export and increased formation of infectious progeny virions [4] . influenza viral infection induces antiviral responses in the host cell [66] , which include increase in the levels of interferons, primarily of 3 types: ifn a, ifn b and ifn g [67] . ifn activates a number of cellular genes, one of the most prominent being pkr encoding double-stranded rna activated protein kinase [68] . following interaction with dsrna [69] , pkr gets activated and undergoes autophosphorylation. this activated form of pkr phosphorylates the alpha subunit of eukaryotic initiation factor 2 (eif2a), which in turn leads to translational arrest. indeed, reports have suggested a critical role for pkr in mediating ds-rna-induced apoptosis in cells [70] . therefore, in order to counteract the effects of host ifn response and pkr activation, viruses have developed multiple mechanisms to suppress pkr activation [71] . several lines of evidence support the fact that viral genes (vaccinia virus, adenovirus and hepatitis c virus) encode proteins that inhibit the ifn pathway by targeting pkr [72, 73] . for instance, non-structural 5a protein of hepatitis c virus (hcv) causes repression of pkr activation, eventually leading to suppression of host ifn response. during influenza infection, pkr activation is inhibited by 2 processes: (1) iav facilitates binding of p58ipk to pkr, causing inhibition of kinase activity [11, 74] ; and (2) non-structural 1 protein (ns1) of influenza virus blocks activation of pkr. studies carried out in vitro using reticulocyte lysates have suggested that ns1 binds to dsrna causing inhibition of pkr activity and phosphorylation of eif2a, thus inhibiting pkr-induced translational arrest [75] (figure 1(4) ). however, direct interaction of pkr and ns1 has not yet been described. the protein kinase c (pkc) is an upstream molecule of raf, which transmits signals to the downstream molecules for the activation of the raf/mek/erk pathway [65] . the pkc superfamily consists of 12 different isoforms which plays various roles in cells by activating several downstream signaling pathways. pkc is known to play a role in virus entry of enveloped viruses [76] . the viral ha acts as a signaling activator, both inside the cells and at the cell surface. binding of influenza virus ha protein to host cell surface receptor activates pkc [12, 77, 78] , and overexpression of ha inside the cells induces erk signaling. use of a pkc inhibitor, bisindolylmalimide i, demonstrated the inhibition of influenza virus entry, which shows that pkc plays a crucial role in influenza virus entry. it is likely that the pkcbii (pkc isoform) acts in this function. furthermore, there is evidence suggesting that pkc phosphorylates the viral m1 protein and helps in viral replication [20] . the mechanism of this process remains unknown. tlr/rig-i signaling viral infection elicits antiviral response via activation of a variety of pattern recognition receptors (prrs) such as toll-like-receptors (tlr) and rig-i like receptors (rlrs) [17, 79] . while ssrna viruses are known to recognize by toll-like receptor (tlr) 7/8 [80] , dsrna viruses recognize tlr3 and retinoic-acid-inducible protein (rig-i), and a cytoplasmic rna helicase plays a crucial role in detecting ssr-na during influenza a virus infection [81] . rig-i can also recognize dsrna generated during viral replication. during viral infection, rig-i and mda5 play an essential role in initiating antiviral response [82] . rig-i recognizes viral rna in a 5'-triphosphate-dependent manner [83] , following which its n-terminal caspase recruitment domain (card) interacts with a downstream partner, mavs (visa/ips-i/cardif), and activates the antiviral signaling [84, 85] . it has recently been shown that the trim25 (tripartite motif) protein interacts with card of rig-i, which is important for initiating the antiviral cascade [86] . like other viruses, influenza virus also has evolved strategies to antagonize host antiviral responses. trim25 is an ubiquitin ligase required for rig-i activation. rig-i activation leads to the association with the ips-i, which phosphorylates irf3 and follows the activation of ifn-b [13, 87] . the ns1 protein of influenza virus is known to interfere with ifn production by binding to trim25. this process suppresses the rig-i signaling and ifnb production in infected cells [13, 88, 89] . this inhibitory activity was shown to depend on the ns1 rna binding domain. it is believed that ns1 sequesters intracellular dsrna like nucleic acids produced during viral replication, thereby keeping these molecules away from cellular dsrnasensor proteins, as tlr3/7 or rig-i [88] (figure 1(5) ). conclusions influenza virus infection alters many cell signaling pathways involved in important physiological functions of the cell. the virus takes over the host cell machinery to manipulate it for its own benefit. influenza virus affects nf-kb signaling, pi3k/akt pathway, mapk pathway, pkc/pkr signaling and tlr/rig-i signaling cascades by using various mechanisms. most importantly, ns1 protein reduces the antiviral response by activating nf-kb signaling and also activates pi3k/akt pathway for efficient viral replication. the virus misuses these (mapk pathway, pkc/pkr signaling and tlr/rig-i signaling) signaling pathways for inhibition of antiviral effects of cytokines and increasing formation of infectious progeny virions. references: the epidemiology of influenza an epidemiological study of 1348 cases of pandemic h1n1 influenza admitted to singapore hospitals from impact of embryonic passaging of h9n2 virus on pathogenicity and stability of ha1-amino acid sequence cleavage site human host factors required for influenza virus replication expression of influenza virus hemagglutinin activates transcription factor nf-kappa b influenza virus-induced nf-kappabdependent gene expression is mediated by overexpression of viral proteins and involves oxidative radicals and activation of ikappab kinase influenza a virus ns1 protein binds p85beta and activates phosphatidylinositol-3-kinase signaling a new player in a deadly game: influenza viruses and the pi3k/akt signalling pathway influenza virus propagation is impaired by inhibition of the raf/mek/erk signalling cascade ringing the alarm bells: signalling and apoptosis in influenza virus infected cells the cellular inhibitor of the pkr protein kinase, p58 (ipk), is an influenza virus-activated co-chaperone that modulates heat shock protein 70 activity entry of influenza viruses into cells is inhibited by a highly specific protein kinase c inhibitor influenza a virus ns1 targets the ubiquitin ligase trim25 to evade recognition by the host viral rna sensor rig-i nf-kappab signaling differentially regulates influenza virus rna synthesis the two nf-kappab activation pathways and their role in innate and adaptive immunity signaling to nf-kappab influenza a virus lacking the ns1 gene replicates in interferon-deficient systems phosphorylation meets ubiquitination: the control of nf-[kappa]b activity regulation of inducible gene expression by the transcription factor nf-kappab influenza-virus-induced signaling cascades: targets for antiviral therapy? a fatal relationship -influenza virus interactions with the host cell activators and target genes of rel/nf-kappab transcription factors interleukin-1alpha enhances il-8 secretion through p38 mitogen-activated protein kinase and reactive oxygen species signaling in human pancreatic cancer cells nf-kappab and the immune response vaccinia virus protein a46r targets multiple toll-like-interleukin-1 receptor adaptors and contributes to virulence a46r and a52r from vaccinia virus are antagonists of host il-1 and toll-like receptor signaling manipulation of the nuclear factor-kappab pathway and the innate immune response by viruses hostile takeovers: viral appropriation of the nf-kappab pathway active nf-kappab signalling is a prerequisite for influenza virus infection viral induction of the human beta interferon promoter: modulation of transcription by nf-kappa b/rel proteins and interferon regulatory factors regulation of ifn-alpha/beta, mxa, 2',5'-oligoadenylate synthetase, and hla gene expression in influenza a-infected human lung epithelial cells molecular pathogenesis of influenza a virus infection and virus-induced regulation of cytokine gene expression regulation of intracellular signalling by the terminal membrane proteins of members of the gammaherpesvirinae nf-kappab-dependent induction of tumor necrosis factor-related apoptosis-inducing ligand (trail) and fas/fasl is crucial for efficient influenza virus propagation bivalent role of the phosphatidylinositol-3-kinase (pi3k) during influenza virus infection and host cell defence control of apoptosis in influenza virus-infected cells by up-regulation of akt and p53 signaling the nuclear phosphoinositide 3-kinase/akt pathway: a new second messenger system signalling by pi3k isoforms: insights from gene-targeted mice the hepatitis c virus ns5a protein activates a phosphoinositide 3-kinase-dependent survival signaling cascade cloning of pi3 kinase-associated p85 utilizing a novel method for expression/cloning of target proteins for receptor tyrosine kinases signalling through the lipid products of phosphoinositide-3-oh kinase mechanism of activation of protein kinase b by insulin and igf-1 the pivotal role of phosphatidylinositol 3-kinase-akt signal transduction in virus survival activation of the n-ras-pi3k-akt-mtor pathway by hepatitis c virus: control of cell survival and viral replication importance of akt signaling pathway for apoptosis in sars-cov-infected vero e6 cells early phosphatidylinositol 3-kinase/akt pathway activation limits poliovirus-induced jnk-mediated cell death flavivirus activates phosphatidylinositol 3-kinase signaling to block caspase-dependent apoptotic cell death at the early stage of virus infection activation of phosphatidylinositol 3-kinase signaling by the nonstructural ns1 protein is not conserved among type a and b influenza viruses binding of influenza a virus ns1 protein to the inter-sh2 domain of p85 suggests a novel mechanism for phosphoinositide 3-kinase activation mechanism of influenza a virus ns1 protein interaction with the p85beta, but not the p85alpha, subunit of phosphatidylinositol 3-kinase (pi3k) and up-regulation of pi3k activity mitogen-activated protein (map) kinase pathways: regulation and physiological functions map kinases in the immune response influenza virus-induced ap-1-dependent gene expression requires activation of the jnk signaling pathway mitogen-activated protein kinase and c-jun-nh2-terminal kinase regulate rantes production by influenza virus-infected human bronchial epithelial cells mek-specific inhibitor u0126 blocks spread of borna disease virus in cultured cells visna virus-induced activation of mapk is required for virus replication and correlates with virus-induced neuropathology role of mitogen-activated protein kinases in influenza virus induction of prostaglandin e2 from arachidonic acid in bronchial epithelial cells ap-1 function and regulation how cells respond to interferons differential activation of the c-jun n-terminal kinase/stress-activated protein kinase and p38 mitogen-activated protein kinase signal transduction pathways in the mouse brain upon infection with neurovirulent influenza a virus map kinases mitogen-activated protein kinase: conservation of a three-kinase module from yeast to human roles of the erk mapk in the regulation of proinflammatory and apoptotic responses in chicken macrophages infected with h9n2 avian influenza virus sh3gl2 gene participates in mek-erk signal pathway partly by regulating egfr in the laryngeal carcinoma cell line hep2 membrane accumulation of influenza a virus hemagglutinin triggers nuclear export of the viral genome via protein kinase calpha-mediated activation of erk signaling antiviral actions of interferon. interferon-regulated cellular proteins and their surprisingly selective antiviral activities alpha/beta interferons potentiate virus-induced apoptosis through activation of the fadd/ caspase-8 death signaling pathway molecular cloning and characterization of the human double-stranded rna-activated protein kinase induced by interferon possible involvement of doublestranded rna-activated protein kinase in cell death by influenza virus infection molecular mechanisms of interferon resistance mediated by viral-directed inhibition of pkr, the interferon-induced protein kinase effects of protein kinase c inhibitors on viral entry and infectivity the e3l and k3l vaccinia virus gene products stimulate translation through inhibition of the double-stranded rna-dependent protein kinase by different mechanisms evidence that hepatitis c virus resistance to interferon is mediated through repression of the pkr protein kinase by the nonstructural 5a protein the molecular chaperone hsp40 regulates the activity of p58ipk, the cellular inhibitor of pkr binding of the influenza virus ns1 protein to double-stranded rna inhibits the activation of the protein kinase that phosphorylates the elf-2 translation initiation factor signaling through protein kinase c b cell superstimulatory influenza virus (h2-subtype) induces b cell proliferation by a pkc-activating, ca(2+)-independent mechanism influenza virus hemagglutinin stimulates the protein kinase c activity of human polymorphonuclear leucocytes pathogen recognition and innate immunity species-specific recognition of single-stranded rna via toll-like receptor 7 and 8 recognition of double-stranded rna and activation of nf-kappab by toll-like receptor 3 distinct rig-i and mda5 signaling by rna viruses in innate immunity the c-terminal regulatory domain is the rna 5'-triphosphate sensor of rig-i cardif is an adaptor protein in the rig-i antiviral pathway and is targeted by hepatitis c virus the rna helicase rig-i has an essential function in double-stranded rna-induced innate antiviral responses trim25 ring-finger e3 ubiquitin ligase is essential for rig-i-mediated antiviral activity inhibition of retinoic acid-inducible gene i-mediated induction of beta interferon by the ns1 protein of influenza a virus a: identification and characterization of viral antagonists of type i interferon in negative-strand rna viruses intracellular warfare between human influenza viruses and human cells: the roles of the viral ns1 protein key: cord-257886-ytlnhyxr authors: zhao, kuan; li, li-wei; jiang, yi-feng; gao, fei; zhang, yu-jiao; zhao, wen-ying; li, guo-xin; yu, ling-xue; zhou, yan-jun; tong, guang-zhi title: nucleocapsid protein of porcine reproductive and respiratory syndrome virus antagonizes the antiviral activity of trim25 by interfering with trim25-mediated rig-i ubiquitination date: 2019-05-03 journal: vet microbiol doi: 10.1016/j.vetmic.2019.05.003 sha: doc_id: 257886 cord_uid: ytlnhyxr porcine reproductive and respiratory syndrome (prrs) is caused by prrs virus (prrsv), and is characterized by respiratory diseases in piglet and reproductive disorders in sow. identification of sustainable and effective measures to mitigate prrsv transmission is a pressing problem. the nucleocapsid (n) protein of prrsv plays a crucial role in inhibiting host innate immunity during prrsv infection. in the current study, a new host-restricted factor, tripartite motif protein 25 (trim25), was identified as an inhibitor of prrsv replication. co-immunoprecipitation assay indicated that the prrsv n protein interferes with trim25–rig-i interactions by competitively interacting with trim25. furthermore, n protein inhibits the expression of trim25 and trim25-mediated rig-i ubiquitination to suppress interferon β production. furthermore, with increasing trim25 expression, the inhibitory effect of n protein on the ubiquitination of rig-i diminished. these results indicate for the first time that trim25 inhibits prrsv replication and that the n protein antagonizes the antiviral activity by interfering with trim25-mediated rig-i ubiquitination. this not only provides a theoretical basis for the development of drugs to control prrsv replication, but also better explains the mechanism through which the prrsv n protein inhibits innate immune responses of the host. the tripartite motif protein 25 (trim25) is an e3 ubiquitin ligase involved in various cellular processes, including regulating the innate immune response against viruses (heikel et al., 2016) . immediately after a viral infection, pathogen-associated molecular patterns are sensed by host pattern recognition receptors (prrs), thereby allowing cells to distinguish self from non-self and activate an immune response (sparrer and gack, 2015) . for rna viruses, the main cytoplasmic prr is retinoic acid-inducible gene i (rig-i), which can directly recognize and bind viral 5′-ppp rna and short double-stranded rna (chan and gack, 2016) . after recognition, the n-terminal caspase recruitment domains (cards) of rig-i are modified by ubiquitin which is mediated by trim25. such modification is essential for activating a signaling cascade, ultimately resulting in the transcriptional activation of type i and iii interferons (ifns), mediating viral clearance, and inhibiting viral replication and spread (gack et al., 2009; martin-vicente et al., 2017) . various viruses are inhibited by trim25, e.g., coxsackie b virus and poliovirus (schoggins et al., 2014) . porcine reproductive and respiratory syndrome (prrs) is endemic in most pig-producing countries (han and yoo, 2014) , and is caused by prrs virus (prrsv). in 2006, a highly pathogenic prrsv (hp-prrsv) emerged in china, causing high fever, high morbidity, and high mortality . prrsv is an enveloped, single-stranded positive-sense rna virus. its genome is approximately 15.4 kb and encodes at least 10 open reading frames (orfs) (han and yoo, 2014) . orf1a and orf1b encode two large nonstructural polyproteins, ppla and pplb, which are processed into at least 14 nonstructural proteins (snijder and meulenberg, 1998; ziebuhr et al., 2000) . orf2a-orf7 encode structural proteins, including four membrane-associated glycoproteins (gp2a, gp3, gp4, and gp5), three unglycosylated membrane proteins (e, orf5a, and m), and a nucleocapsid protein (n) (dea et al., 2000; snijder and meulenberg, 1998) . among these, nsp1, nsp2, nsp4, nsp11, and been identified and characterized as ifn antagonists (lunney et al., 2016) . further, the n protein is the most abundant protein during infection, accounting for approximately 40% of virion proteins (snijder and meulenberg, 1998) . it plays essential roles in the virus life cycle, including encapsidation of viral rna (spilman et al., 2009) . currently, it is only known that n protein suppresses ifn-β induction by antagonizing irf3 activation (sagong and lee, 2011) . however, other mechanisms through which n protein inhibits ifn-β production are not clear. the nucleocapsid protein of severe acute respiratory syndrome (sars) inhibits type i ifn production by interfering with trim25mediated rig-i ubiquitination (hu et al., 2017) . whereas prrsv and sars both belong to the nidovirales order, whether prrsv n protein inhibits the host innate immune response by interfering with trim25mediated rig-i ubiquitination is not clear. herein, we show that trim25 plays an important role in inhibiting prrsv replication. the n protein interacts with trim25 and suppresses the ubiquitination of rig-i. further, trim25 expression is reduced upon co-transfection with the n protein or prrsv infection. together, these findings suggest a novel mechanism through which prrsv n protein inhibits host innate immunity, and provide improved understanding of the mutual regulatory mechanism between prrsv and the host. human embryonic kidney (hek293t) cells and african green monkey kidney (marc-145) cells were cultured in dulbecco's modified eagle's medium containing 10% fetal bovine serum (gibco, thermo fisher scientific, waltham, ma). cells were maintained at 37°c with 5% co 2 . the highly pathogenic prrsv strain hun4 (genbank no. ef635006) was used for all the experiments (tong et al., 2007) . total rna was extracted from porcine alveolar macrophages (pams) using rneasy mini kit (qiagen, hilden, germany), following the manufacturer's instructions. reverse transcription reactions were performed at 25°c for 5 min and 42°c for 1 h using the m-mlv reverse transcription polymerase system (takara, dalian, china). full-length trim25-and rig-i-encoding sequences were amplified with specific indicated primers. the sequences were then cloned into pcaggs vector to generate pcaggs-trim25-ha, pcaggs-trim25-flag, pcaggs-trim25-myc, pcaggs-rig-i-ha, pcaggs-rig-i-flag, or pcaggs -2 card-flag. orf7 of prrsv hun4 was cloned into pcaggs to generate pcaggs-n-ha, pcaggs-n-flag, or pcaggs-n-myc. all plasmids were constructed by homologous recombination using the nebuilder® hifi dna assembly master mix (new england biolabs; ipswich, ma) according to the manufacturer's instructions. sequences of all primers used for gene amplification will be made available upon request. to investigate the effect of trim25 on prrsv replication, marc-145 cells cultured in 6-well plates were transfected with 2 μg of pcaggs-trim25-flag using x-treme gene hp dna reagent (roche applied science, penzberg, germany). next, 36 h post-transfection (hpt), the cells were infected with prrsv hun4 (multiplicity of infection, moi, of 0.1). after inoculation for 1 h at 37°c, the supernatants were discarded, and cells were washed three times with phosphate-buffered saline (pbs). the supernatant was harvested 12, 24, 36, and 48 h post-infection (hpi), and the cells were lysed using ripa lysis buffer (thermo fisher scientific). viral titers in the supernatants were determined using a microtitration assay, according to the method of reed and muench (reed and muench, 1938) . the amount of the n protein was then detected in cell lysates by western blotting (wb) using a mouse anti-n polyclonal antibody (1:1000) produced by the authors. small interfering rnas (sirnas) against macaque trim25 (genbank no. xm_015119181.1) were synthesized by genepharma (shanghai, china). the sirna molecules used were ccu gga gua uua cgu uaa att (sirna-trim25-1356) and gca ucu acc aua gca ccu utt (sirna-trim25-1007). the control sirna (nc) sequence was uuc ucc gaa cgu guc acg utt. marc-145 cells were seeded in 6well plates and transfected with 50 pm sitrim25 or nc using lipofectamine™ rnaimax transfection reagent (cat. no. 13, 778, 075; thermo fisher scientific) . the cells were lysed in ripa lysis buffer after 36 h of transfection and the effects of sirnas were analyzed by wb using an anti-trim25 monoclonal antibody (cat. no. 13,773; cell signaling technology; danvers, ma; 1:1000). the knockdown efficiency of sirna was analyzed by grayscale scanning with image j software. next, efficient sirna and nc were selected for transfection; 36 hpt, the cells were infected with prrsv hun4 at an moi of 0.1. the supernatant and cells were harvested 12, 24, 36, and 48 hpi, and analyzed based on virus titers and by wb. cell lysates were prepared by harvesting virus-infected or plasmidtransfected cells in ripa or ip-lysate buffer(25 mm tris•hcl ph 7.4, 150 mm nacl, 1% np-40, 1 mm edta, 5% glycerol) at 4°c for 15 min containing 1 mm phenylmethylsulfonyl fluoride and 1 mg/ml of protease inhibitor cocktail (roche). after centrifuging at 12,000 × g for 10 min, the supernatants of cell lysates were mixed with 5 × sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) sample loading buffer (beyotime), and placed in boiling water for 5 min. the proteins were then separated by sds-page and transfected onto a nitrocellulose membrane. the membrane was blocked in 5% skim milk at room temperature for 2 h and then incubated with the indicated primary antibody at 4°c overnight. after washing three times with trisbuffered saline with 0.1% tween 20, the membrane was incubated with horseradish peroxidase-conjugated goat anti-mouse igg(h + l) and/or goat anti-rabbit igg(h + l) secondary antibody (1:5000) for 1 h at room temperature. the target proteins were visualized by treating the membrane with pierce ecl wb substrate (thermo fisher scientific). for the quantification of target proteins, their levels were normalized to the levels of β-actin. to detect the interaction between trim25 and prrsv n protein, hek293 t cells grown in 6-well plates were co-transfected with pcaggs-trim25-flag and pcaggs-n-ha (2 μg each) using x-tremegene dna transfection reagent. at 24 hpt, the cells were lysed with ice-cold ip lysis buffer containing 1 mm phenylmethylsulfonyl fluoride and 1 mg/ml of protease inhibitor cocktail (roche) at 4°c for 15 min. approximately 10% of the lysate supernatant was used as an input control and the remaining lysates were incubated with anti-ha agarose beads (cat. no. a2095; sigma-aldrich; st. louis, mo) or ezview™ red anti-flag ® m2 affinity gel (cat.no. m2426; sigma-aldrich) for 6 h at 4°c. the beads were washed five times with the ip lysis buffer and then analyzed by wb using rabbit anti-flag monoclonal antibody (cat. no. f7425; sigma-aldrich; 1:5000) or mouse anti-ha monoclonal antibody (cat. no. h9658; sigma-aldrich; 1:5000). the interaction between trim25 and rig-i was tested using the same methods with anti-ha agarose beads and wb using mouse anti-ha monoclonal antibody (cat. no. h9658; sigma-aldrich; 1:5000) and mouse anti-myc monoclonal antibody (cat. no. 2276; cell signaling technology; 1:1000). in order to investigate whether the n protein k. zhao, et al. veterinary microbiology 233 (2019) 140-146 competitively affects the interaction between rig-i and trim25, hek293 t cells grown in 6-well palate were co-transfected with pcaggs-rig-i-ha (1 μg) and pcaggs-trim25-myc (1 μg) with or without pcaggs-n-flag (1 μg) for immunoprecipitation. further, to detect the interaction between endogenous (host) trim25 and n protein in prrsv-infected cells, pam cells were grown in 5-cm-diameter dishes and infected with prrsv at an moi of 0.5 for 24 h. the cells were lysed in 500 μl of ip lysis buffer and the supernatants were precleared with protein g-agarose beads (cat. no. 11243233001; roche). the supernatant was incubated with 2 μg of anti-n polyclonal antibody for 8 h. next, 20 μl of protein g-agarose beads was added, followed by an additional 3 h incubation. ip pellets were washed five times with 500 μl of ip lysis buffer, boiled in sds-page sample loading buffer, and analyzed by wb with rabbit anti-trim25 monoclonal antibody (cat. no. ab167154; abcam; 1:2000) and anti-n polyclonal antibody. hek293 t cells were co-transfected with pcaggs-n-ha and pcaggs-trim25-flag, or pcaggs-rig-i-ha and pcaggs-trim25-flag. at 24 hpt, the cells were fixed in 4% paraformaldehyde for 30 min, blocked with 3% bovine serum albumin for 1 h, and permeabilized with 0.1% triton x-100 for 15 min. the transfected cells were incubated with mouse anti-ha monoclonal antibody (cat. no. h9658; sigma-aldrich; 1:2000) and rabbit anti-flag monoclonal antibody (cat. no. f7425; sigma-aldrich; 1:2000) for 1 h at 37°c, and then washed three times with pbs. the cells were then incubated at 37°c for 1 h with donkey anti-mouse igg(h + l) antibody conjugated with alexa fluor 596 (cat. no. 1692912; life technologies; 1:1000) and goat anti-rabbit igg(h + l) antibody labeled with alexa fluor 488 (cat. no. 1674651; life technologies; 1:1000). samples were then stained with 1 μg/ml of 4′,6′-diamidino-2-phenylindole (dapi) for 10 min and examined using a zeiss confocal system. besides, in order to investigate the colocalization of endogenous (host) trim25 and n protein in prrsv-infected cells, the pam cells infected with prrsv were send to colocalization detection with anti-trim25 and anti-n protein antibody. hek293 t cells seeded in a 24-well plate were co-transfected with control plasmids or the indicated expression plasmids (0.25 μg per well) together with 100 ng of a luciferase reporter plasmid; prl-tk plasmid (10 ng) was used as a control of transfection efficiency (huang et al., 2014) . next, 24 hpt, cells lysates were harvested and luciferase activity in the lysates was analyzed using a dual-luciferase reporter assay kit (promega, madison, wi), following the manufacturer's instructions. the results are expressed as the means ± standard deviation (sd) from three independent experiments. statistical analysis was performed using graphpad prism 6 (graphpad, la jolla, ca). the measured values are expressed as the mean with sd. the statistical significance was assessed using student's t-test, with p < 0.05 considered statistically significant. to examine the effects of trim25 on prrsv replication, specific sirna molecules were synthesized to knockdown trim25 expression in marc-145 cells and efficiently reduce trim25 expression. using sirna-1356, the knockdown efficiency was approximately 65% (fig. 1a) . this sirna molecule was used in the subsequent interference experiments. as shown in fig. 1b , n protein levels increased upon transfection with sirna-1356, especially 36 and 48 hpi, compared with those in nctransfected cells. virus titers in the culture supernatants of cells transfected with sirna-1356 were also increased, which was consistent with the expression levels of the n protein, with a significant difference 36 hpi (p < 0.05; fig. 1c ). by contrast, when trim25 was overexpressed, the n protein levels of hun4 were lower than those in the control cells (fig. 1d) . furthermore, there was a significant difference in virus titers between cells transfected with pcaggs-trim25-flag or pcaggs, with an approximate 1.0 log decrease in virus titers from 24 to 36 hpi (p < 0.05; fig. 1e ). these observations suggested that trim25 is a cellular antiviral factor that represses prrsv infection. as shown in fig. 1d , the expression of trim25-flag decreased gradually upon prrsv infection. therefore, we speculated that the virus might inhibit the expression of trim25. to verify this hypothesis, marc-145 cells infected with prrsv or uninfected cells were lysed 12, 24, 36, and 48 hpi, and analyzed by wb. the analysis revealed that the expression of trim25 decreased in prrsv-infected cells at every time point, as compared with the uninfected cells ( fig. 2a) . furthermore, trim25 expression was significantly suppressed when pcaggs-trim25-myc and pcaggs-n-ha were co-transfected into hek293 t cells. these findings indicated that the n protein inhibits the expression of trim25. furthermore, the n protein inhibited trim25 expression in a dose-dependent manner (fig. 2b) . since we demonstrated that prrsv infection and n protein accumulation suppress the expression of trim25, we next investigated whether the n protein of prrsv exerts an inhibitory effect on trim25 expression by interacting with it. to test this, the interaction between trim25 and n protein was investigated by co-ip. based on precipitation with anti-flag agarose, flag-tagged trim25 interacted with hatagged n protein (fig. 3a) . furthermore, n protein was efficiently coimmunoprecipitated with trim25 using anti-ha agarose (fig. 3b) . we also investigated whether trim25 co-localizes with the n protein in hek293 t cells co-transfected with pcaggs-trim25-flag and pcaggs-n-ha. trim25 was mainly located in the cytoplasm ( fig. 3c-a) , whereas the n protein localized in the cytoplasm and nucleus ( fig. 3c-b) . as shown in fig. 3c -d, trim25 and n protein colocalized in the cytoplasm. to examine the interaction of n protein and endogenous trim25 in the context of prrsv infection, virus-infected pam cells were stained with anti-n polyclonal antibody and anti-trim25 monoclonal antibody or immunoprecipitated using anti-n polyclonal antibody and the pellets were detected with anti-trim25 and n antibody. as shown in fig. 3d and 3e, endogenous trim25 was co-localized with n protein and coimmunoprecipitated with the n protein. these observations confirmed that the endogenous trim25 indeed interacts with the n protein of prrsv in prrsv-infected cells. in the current study, we demonstrated that the n protein of prrsv could interact with trim25. further, it has been reported that trim25 interacts with rig-i n-terminal cards and e2 ubiquitin-conjugating enzymes (gack et al., 2007) . whether the n protein can competitively regulate the interaction between trim25 and rig-i was unknown. we first detected the interaction between trim25 and rig-i by co-ip, confirming that trim25 could be immunoprecipitated with rig-i (fig. 4a ). in addition, co-localization analysis demonstrated that rig-i and trim25 co-localize in the cytoplasm (fig. 4b) . further, we k. zhao, et al. veterinary microbiology 233 (2019) 140-146 validated the effect of n protein on the interaction between rig-i and trim25. when rig-i-ha and trim25-myc were co-transfected with n-flag, the interaction between trim25 and rig-i was markedly diminished. meanwhile, when trim25-myc and rig-i-ha were co-transfected without n-flag, the interaction between trim25 and rig-i was not affected (fig. 4c) . these results suggested that the n protein inhibits the interaction between trim25 and rig-i via competitive binding to trim25. to investigate whether trim25-mediated rig-i ubiquitination is regulated by the prrsv n protein, hek293t cells grown in 6-well plates were co-transfected with pcaggs-flag-rig-i (0.5 μg per well) and ha-ubiquitin (0.5 μg per well), and the indicated amounts of the myc-n expression plasmids. cells lysates were immunoprecipitated using mouse anti-ha monoclonal antibody or rabbit anti-flag monoclonal antibody. the experiment revealed that trim25-mediated rig-i ubiquitination was potentiated by sendai virus (sev) infection but was substantially suppressed by increasing the prrsv n protein expression, in a dose-dependent manner (fig. 5) . when viral rna is recognized by rig-i, this protein is modified by ubiquitin, which is mediated by the e3 ligase trim25. hence, this enzyme is essential for activating a signaling cascade that ultimately results in the transcriptional activation of type i and iii ifns. to examine regulation of rig-i activity by the prrsv n protein, a luciferase reporter under the control of the ifn-β promoter (ifn-β-luc) was used to quantify promoter activation. consistent with the inhibition of rig-i ubiquitination by the n protein, ifn-β promoter activation induced by rig-i or rig-i card domain overexpression was significantly inhibited by prrsv n expression, in a dose-dependent manner (fig. 6a, b) . however, co-expression of trim25 with prrsv n significantly counteracted this inhibitory effect mediated by the n protein (p < 0.05) (fig. 6c ). in addition, suppression of rig-i ubiquitination by the prrsv n protein was partially rescued by trim25 overexpression (fig. 6d) . these observations indicated that the n protein inhibits rig-i ubiquitination and that ifn production can be restored by trim25 overexpression. prrsv has been a major threat to global industrial pig farming ever since its emergence in the late 1980s (shi et al., 2010) , and especially or pcaggs (2 μg) were inoculated with prrsv at an moi of 0.1. the cells and supernatant were harvested at the indicated times, and the replication of prrsv was evaluated by wb with an anti-n polyclonal antibody (d) and tcid 50 assay (e). the data are presented as the mean ± sd from three experiments. the statistical significance of differences was determined using student's t-test (*p < 0.05). since the outbreak of hp-prrsv in 2006. to date, although there is some understanding of the mechanisms through which prrsv escapes innate immunity, very little is known about how viral proteins of prrsv antagonize the host innate immune response. therefore, understanding the interactions between prrsv and host proteins will be beneficial for the development of effective therapies to control outbreaks of this disease in the future. in the current study, we demonstrated that trim25 functions by restricting prrsv replication, and that it could act as a host-derived inhibitor of the virus. previously, many host factors that result in cellular resistance to prrsv via different mechanisms were identified song et al., 2017; wang et al., 2016; zhao et al., 2018 zhao et al., , 2017 . for example, cholesterol 25-hydroxylase protects against prrsv infection by converting cholesterol to 25-hydroxycholesterol, which can be used as a natural antiviral agent to combat prrsv infection (song et al., 2017) . further, galectin-3 inhibits replication prrsv by interacting with viral nsp12 in vitro . in the current study, using marc-145 cells, we demonstrated that the overexpression of trim25 restricts the replication of prrsv, whereas knocking down this protein promotes the replication of prrsv (fig. 1) . therefore, trim25 acts as a novel host factor that inhibits the replication of prrsv. trim25 is an e3 ubiquitin ligase enzyme that can regulate the innate immune response against viruses (martin-vicente et al., 2017) . trim25-mediated ubiquitination of the cytosolic prr rig-i is an essential step for the initiation of an intracellular antiviral response (gack et al., 2007) . data presented herein revealed that upon trim25 overexpression, the n protein-mediated inhibition of rig-i ubiquitination and ifn-β promoter activity were diminished (fig. 6) . the host innate immunity was hence activated, leading to a series of signaling cascades and thereby inhibiting prrsv replication. trim25 can activate the host innate immune system and simultaneously induce a series of antiviral responses by promoting the ubiquitination of rig-i and activation of ifn-β promoter activity. however, in the course of natural infection, prrsv can complete the replication cycle and efficiently spread. hence, prrsv has evolved several general strategies to evade the innate immune response. it has been reported that some viral proteins interact with trim25 and inhibit rig-i activation. for example, the non-structural protein 1 (ns1) of influenza a virus interacts with the cc domain of trim25 preventing its dimerization and the k63-linked ubiquitination of rig-i cards, thereby suppressing rig-i signal transduction (gack et al., 2009) . further, trim25 interacts with the n protein of sars-cov, thereby inhibiting the activation of rig-i (hu et al., 2017) . in the current study, we found that the n protein of prrsv inhibits the ubiquitination of rig-i by competitively interfering with the interaction between rig-i and trim25. this might be the mechanism through which prrsv inhibits the antiviral effect of trim25. furthermore, trim25 levels decreased when the cells were infected with prrsv. in addition, when plasmids expressing trim25 and the n protein of prrsv were cotransfected into cells, the expression of trim25 was significantly suppressed. based on this, it would be difficult for trim25 to exert an antiviral effect upon prrsv infection. this might represent another mechanism through which prrsv antagonizes the antiviral response of trim25. besides, the n protein of pedv, another coronavirus, is also able to antagonize ifn-β production (ding et al., 2014) . since prrsv, sars, and pedv all belong to nidovirales, we speculate that the respective n proteins may exert a similar effect of inhibiting trim25mediated ubiquitination of rig-i. however, the effect of pedv n protein on the inhibition of rig-i ubiquitination requires further research. in the present study, we confirmed that trim25 inhibits prrsv replication. further, prrsv can antagonize the antiviral activity of this protein by decreasing its expression and modulating the trim25mediated ubiquitination of rig-i. in addition, the n protein of prrsv inhibits ifn-β production. all these mechanisms improve the understanding of the effect of trim25 on prrsv replication and will further co-ip experiment was performed using agarose beads conjugated with anti-flag monoclonal antibody or (b) agarose beads conjugated with anti-ha monoclonal antibody. the precipitated proteins were analyzed by wb using antibodies against the flag and ha tags. (c) co-localization of the n protein with trim25. hek293 cells were transfected with plasmids expressing ha-n protein and flag-trim25. after 24 h, the cells were fixed and analyzed by ifa to detect tagged n protein and trim25, using mouse anti-ha and rabbit anti-flag monoclonal antibodies, respectively. the nucleus is stained with dapi (blue) in the merged images. (d and e) colocalization and interaction of prrsv n and endogenous trim25. pam cells infected with prrsv were fixed 24 h after infection, and then subjected to indirect immunofluorescence to detect n protein and trim25. in addition, cell lysates from prrsv-infected or mock-infected pam cells were co-ip with mouse anti-n polyclonal antibody, and the precipitates were immunoblotted with the indicated antibodies (for interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article). k. zhao, et al. veterinary microbiology 233 (2019) 140-146 help to understand how prrsv evades the trim25-mediated innate immune response via the n protein. hence, the current study not only offers a new target for the development of drugs to control prrsv spread but also provides an explanation of the mechanism through which prrsv n protein modulates host innate immune responses. in the collection, analysis, and interpretation of data; in the writing of the report; and in the decision to submit the article for publication. 5 . the prrsv n protein suppresses the trim25-mediated rig-i ubiquitination. hek293 t cells were transfected with the indicated plasmids for 36 h, and were infected (with or without sev) for 12 h. anti-flag immunoprecipitates prepared from the cell extracts were analyzed by wb using the indicated antibodies. k. zhao, et al. veterinary microbiology 233 (2019) 140-146 viral evasion of intracellular dna and rna sensing current knowledge on the structural proteins of porcine reproductive and respiratory syndrome (prrs) virus: comparison of the north american and european isolates porcine epidemic diarrhea virus nucleocapsid protein antagonizes beta interferon production by sequestering the interaction between irf3 and tbk1 trim25 ring-finger e3 ubiquitin ligase is essential for rig-i-mediated antiviral activity influenza a virus ns1 targets the ubiquitin ligase trim25 to evade recognition by the host viral rna sensor rig-i engineering the prrs virus genome: updates and perspectives the role of trim25 in development, disease and rna metabolism the severe acute respiratory syndrome coronavirus nucleocapsid inhibits type i interferon production by interfering with trim25-mediated rig-i ubiquitination porcine reproductive and respiratory syndrome virus nonstructural protein 4 antagonizes beta interferon expression by targeting the nf-κb essential modulator galectin-3 inhibits replication of porcine reproductive and respiratory syndrome virus by interacting with viral nsp12 in vitro porcine reproductive and respiratory syndrome virus (prrsv): pathogenesis and interaction with the immune system trim25 in the regulation of the antiviral innate immunity. front. immunol. 8, 1187. reed porcine reproductive and respiratory syndrome virus nucleocapsid protein modulates interferon-beta production by inhibiting irf3 activation in immortalized porcine alveolar macrophages pan-viral specificity of ifn-induced genes reveals new roles for cgas in innate immunity molecular epidemiology of prrsv: a phylogenetic perspective the molecular biology of arteriviruses cholesterol 25-hydroxylase is an interferon-inducible factor that protects against porcine reproductive and respiratory syndrome virus infection intracellular detection of viral nucleic acids cryo-electron tomography of porcine reproductive and respiratory syndrome virus: organization of the nucleocapsid emergence of fatal prrsv variants: unparalleled outbreaks of atypical prrs in china and molecular dissection of the unique hallmark highly pathogenic porcine reproductive and respiratory syndrome the interferon-induced mx2 inhibits porcine reproductive and respiratory syndrome virus replication porcine 2', 5'-oligoadenylate synthetase 2 inhibits porcine reproductive and respiratory syndrome virus replication in vitro mov10 inhibits replication of porcine reproductive and respiratory syndrome virus by retaining viral nucleocapsid protein in the cytoplasm of marc-145 cells virus-encoded proteinases and proteolytic processing in the nidovirales a and b) hek293 t cells seeded in 24-well plates were co-transfected using the firefly luciferase reporter plasmid ifn-β-luc and the renilla luciferase control reporter plasmid prl-tk. for the experiment, pcaggs-rig-i-flag (0.25 μg), or pcaggs -2 card (0.25 μg), pcaggs-n-ha were co-transfected. (c) pcaggs -2 card-flag (0.25 μg), pcaggs-n-falg (0.25 μg) and pcaggs-trim25-myc (0.5 μg) plasmids were cotransfected for the experiment, 24 hpt, the cells were infected with sev, and 16 hpi, whole-cell lysates were analyzed by immunoprecipitation using the indicated antibodies to detect the ubiquitination of rig-i-card. the data are presented as the mean ± sd from three experiments. the statistical significance of differences was determined using key: cord-268438-bjs5oliw authors: jin, yilin; jia, kuntong; zhang, wanwan; xiang, yangxi; jia, peng; liu, wei; yi, meisheng title: zebrafish trim25 promotes innate immune response to rgnnv infection by targeting 2card and rd regions of rig-i for k63-linked ubiquitination date: 2019-12-03 journal: front immunol doi: 10.3389/fimmu.2019.02805 sha: doc_id: 268438 cord_uid: bjs5oliw rig-i-like receptors (rlrs) play important roles in response to virus infection by regulating host innate immune signaling pathways. meanwhile, the rlr signaling pathway is also tightly regulated by host and virus to achieve the immune homeostasis between antiviral responses and virus survival. here, we found that zebrafish trim25 (zbtrim25) functioned as a positive regulator of rlr signaling pathway during red spotted grouper nervous necrosis virus (rgnnv) infection. post-rgnnv infection, zbtrim25 expression was obviously inhibited and ectopic expression of zbtrim25 led to enhanced expression of rlr signaling pathway-related genes. overexpression and knockdown analysis revealed that zbtrim25 promoted zebrafish rig-i (zbrig-i)-mediated ifn signaling and inhibited rgnnv replication. mechanistically, zbtrim25 bound to zbrig-i; in particular, the spry domain of zbtrim25 interacted with the tandem caspase activation and recruitment domains (2card) and repressor domain (rd) regions of zbrig-i. zbtrim25 promoted the k63 polyubiquitination of 2card and rd regions of zbrig-i. furthermore, zbtrim25-mediated zbrig-i activation of ifn production was enhanced by k63-linked ubiquitin, indicating that zbtrim25-mediated zbrig-i polyubiquitination was essential for rig-i-triggered ifn induction. in conclusion, these findings reveal a novel mechanism that zbtrim25 positively regulates the innate immune response by targeting and promoting the k63-linked polyubiquitination of zbrig-i. the innate immune system recognizes pathogen-associated molecular patterns (pamps) by pattern recognition receptors (prrs) as against microbial pathogen invasion (1). retinoic acid inducible gene-i (rig-i)-like receptors (rlrs), as intracellular prrs, composed of rig-i, mda5, and lgp2, recognize non-self signatures of viral rnas in the cytosol of cells. after activated by viral rna, rig-i and mda5 recruited the downstream adaptor molecule, mavs, to their n-terminal caspase-recruitment domains (cards). then, tumor necrosis factor receptor-associated factors (traf) and tank-binding kinase 1/iκ-b kinase ε interacted with mavs, which in turn leads to the phosphorylation and cytoplasm-to-nucleus translocation of interferon (ifn) regulatory factor 3 (irf3), and the activation of type i ifn. subsequent ifns activated a variety of ifn-stimulated genes (isgs) to limit the virus replication (2) . nervous necrosis virus (nnv) is a non-enveloped, singlestranded rna virus belonging to the family nodaviridae. increasing evidence has shown that nnv can infect more than 120 fish species and causes mass mortalities of infected fish worldwide (3) . it has been revealed that rlrs respond in vivo or in vitro to the stimulation of nnv and possess capacities in the induction of ifns and isgs in a variety of fish species. for example, in zf4 cells, expression of rlrs was significantly enhanced post-nnv infection and rig-i knockdown significantly restrained group ii type i ifn activation (4) . our previous studies also suggested that rlr signaling pathway was activated during red spotted grouper nervous necrosis virus (rgnnv) infection in sea perch and its key components possessed anti-rgnnv activities (5, 6) . however, regulation mechanisms of rlr signaling pathway during rgnnv infection is still unclear. rlr-mediated antiviral signaling pathway is tightly regulated at multiple steps in the signaling cascade. several studies demonstrated that posttranslational modifications, including ubiquitination, isgylation, and phosphorylation, were important mechanisms that regulated the rlr signaling pathway, of which ubiquitination was a key regulatory mechanism for rlr pathway (2) . for instance, rnf122 negatively regulated rlr signaling pathway by targeting rig-i (7) . mda5 and mavs were targeted for k48-linked ubiquitination by trim13 and rnf5, respectively, which induced mda5 and mavs degradation and rlrs signal termination (8, 9) . trim25 e3 ubiquitin ligase induced the k63linked ubiquitination of rig-i, which activated rlr signaling pathway to elicit host antiviral innate immunity (10) . trim25, an ifn-inducible e3 ligase, is associated with all kinds of cellular processes, such as the immune response, cancer, and so on (11) . it is becoming evident that trim25 has a dual role in rig-i regulation, since trim25 not only induces k63-linked ubiquitination of rig-i to positive regulate rlr signaling activation but also negatively regulates rig-i activation through inhibiting hla-f adjacent transcription 10 degradation, a negative regulator of rig-i-mediated inflammatory response (12) . multiple fish trim25 homologs have been reported, including rhodeus uyekii (13), epinephelus coioides (14) , and larimichthys crocea (15) . increasing evidence showed that fish trim25 was involved in antiviral immunity and played a pivotal role in rlr antiviral signaling pathway (14) . however, the mechanism by which fish trim25 regulates rlr signaling pathway has not been explored. in the present study, zebrafish trim25 (zbtrim25) was involved in rgnnv infection and was identified as a positive mediator of rlr signaling pathway by binding to and ubiquitinating the caspase activation and recruitment domain (2card) and repressor domain (rd) regions of rig-i, which is different with the findings in mammals. our findings reveal a novel mechanism of trim25 to activate rlr signaling pathway and will help to develop new treatments for viral nervous necrosis disease. all procedures with zebrafish were approved by the ethics committee of sun yat-sen university and the methods were carried out following the approved guidelines. zebrafish wild-type ab line was purchased from china zebrafish resource center. fish were raised with 10 h darkness and 14 h light at 28 • c and were fed with commercial pellets twice a day. all embryos were obtained by natural spawning and staged as previously reported (16) . zbe3 cells derived from zebrafish embryos were cultured at 28 • c as previously described (17) . hek 293t cells were cultured in dmem (invitrogen) enriched with 10% fbs (invitrogen) at 37 • c under a humidified atmosphere of air containing 5% co 2 . rgnnv was propagated in zbe3 cells and stored at −80 • c until use. anti-flag (m20008), anti-myc (m20002), anti-his (m20001l), and anti-ha antibodies (m20013) were purchased from abmart. anti-α-tubulin (ab15246) and anti-gfp antibodies (g1544) were purchased from abcam and sigma, respectively. goat anti-rabbit igg-hrp, goat anti-mouse igg-hrp, alexa fluor 488-labeled goat anti-mouse igg, and alexa fluor 555-labeled goat anti-rabbit igg secondary antibodies were purchased from invitrogen. for in vitro infection, zbe3 cells were challenged with rgnnv [multiplicity of infection (moi) = 1] for 6, 12, and 24 h, respectively. subsequently, rna from cells was extracted to detect the expression of zbtrim25 mrna by quantitative realtime pcr (qrt-pcr). for in vivo infection, rgnnvs (10 8 tcid 50 /ml) were injected into the egg yolk of 50 embryos at the single-cell stage in the experimental group. in the mock group, 50 embryos were injected with dmem. a total of 1 nl of solution was microinjected into each embryo using a microinjector. rna from zebrafish embryo was extracted to detect the expression of zbtrim25 mrna by qrt-pcr at 24 h post-injection. knockdown of zbtrim25 by sirna zbtrim25 sirna (5 ′ -gaatccagttgaagagaaa-3 ′ ) and control sirna were synthesized by ribobio company (guangzhou, china). zbe3 cells were transfected with zbtrim25 sirna or control sirna according to the manufacturer's protocol using lipofectamine 3000 as previously described (18) . twenty-four hours after transfection, zbe3 cells were infected with rgnnv (moi = 1) for 24 h and total rnas were extracted for qrt-pcr analysis. the orf of zbtrim25 (genbank accession no. nm200175.1) was sub-cloned into pcmv-flag or pcmv-myc vectors (invitrogen) to generate recombinant plasmid pcmv-flag-zbtrim25 or pcmv-myc-zbtrim25, respectively. full-length zbrig-i and zbrig-i deletion mutant cdnas encoding amino acids 1-187 (zbrig-i-2card), 188-937 (zbrig-i-2card), 812-927 (zbrig-i-rd), and 188-811 [zbrig-i-(2card+rd)] were inserted into the pegfp-n3 vectors. full-length zbrig-i was inserted into the pet-32a(+) (clontech) vector to generate recombinant plasmid pet-32a(+)-zbrig-i. zbtrim25 deletion mutant zbtrim25-spry and zbtrim25-spry were generated using the pcmv-flag-zbtrim25 plasmid as a template. primers used for amplifying these genes are listed in table s1 . ha-k63ub plasmid was purchased from rebio (shanghai, china). rna extraction and cdna synthesis were performed using trizol (invitrogen) and primescript tm 1st strand cdna synthesis kit (takara) according to the manufacturer's instructions. qrt-pcr analyses of zbtrim25, zbrig-i, rna dependent rna polymerase (rdrp), rlr signaling pathway related genes (mavs, traf3, irf3, and ifn 1), and isg15 were performed as previously described (19) . relative expression levels of target genes were normalized to 18s rrna using 2 − ct methods. data represent the mean ± sd from three independent experiments, each performed in triplicate. primers sequences used for qrt-pcr are listed in table s1 . hek 293t cells, pre-seeded in 24-well plates, were transfected with 250 ng of pgl3-drifn 1-pro-luc plasmid or pgl3-basic empty vector with 25 ng of prl-tk vector (promega) together with pcmv-myc-zbrig-i or pcmv-myc and pcmv-flag or pcmv-flag-zbtrim25 (250 ng per well) for 24 h. then, cells were incubated with poly i:c for 48 h and lysed. luciferase activities were measured using the dual-luciferase reporter assay system (promega). relative luciferase activities were expressed as the ratio of firefly to renilla luciferase activity. the results were the representative of three independent experiments in triplicate. hek 293t cells, pre-seeded in 24-well plates, were transfected with 250 ng of pgl3-drifn1-pro-luc plasmid or pgl3-basic empty vector with 25 ng of prl-tk vector (promega). meanwhile, pcmv-myc-zbtrim25, mutant zbrig-i or empty control plasmids were co-transfected. after being incubated with poly i:c for 48 h, cells were lysed for luciferase assay as described above. at least three independent experiments were performed. hek 293t cells, seeded on glass cover slips, were transfected with pcmv-myc-zbtrim25 and pcmv-flag-zbrig-i plasmids. twenty-four hours post-transfection, cells were washed with pbs three times and fixed with prechilled methanol and then permeabilized using 1% triton x-100 in pbs for 10 min and blocked with 5% normal goat serum for 30 min at room temperature (rt). cells were incubated with anti-myc and anti-flag antibodies for 60 min at rt. finally, cells were washed with pbs and incubated with the appropriate alexa fluor 488 or 555 conjugated secondary antibodies for 1 h. after cell nucleus was stained with hoechst 33342, cells were observed by a confocal microscope (zeiss, germany). co-ip and western blotting experiments were performed as described previously (18) . hek 293t cells in 75-cm 2 flasks were co-transfected with 10 µg of different plasmid combinations for 48 h. then, the cells were lysed on ice with lysis buffer for 15 min and were immunoprecipitated with the indicated antibodies. the precipitated samples and whole-cell lysates (input) were analyzed by immunoblotting with the indicated antibodies. escherichia coli bl21(de3) cells were transformed with pet-32a(+)-zbrig-i or pet-32a(+) plasmids, respectively. then, cells were grown in 50 ml of lb medium (beyotime) containing 0.5 mm isopropyl-1-thio-β-d-galactopyranoside (iptg) (sigma) at 18 • c overnight with shaking at 120 rpm. cells were pelleted by centrifugation at 4,500 rpm for 30 min and lysed in 10 ml of lysis buffer (100 mm sodium phosphate, ph 8.0, 600 mm nacl, and 0.02% tween-20) (beyotime) via sonication on ice. the sonicated mixture was centrifuged at 15,000 rpm at 4 • c for 20 min, and then the supernatant was affinity-purified with dynabead his-tag magnetic beads (invitrogen) according to the manufacturer's instruction. his pull-down assays were performed as described previously with some modifications (20) . his-zbrig-i-magnetic beads were incubated with the lysates of hek 293t cells transfected with pcmv-flag-zbtrim25 or pcmv-flag empty vectors on a roller, respectively. after incubation at 4 • c overnight, the magnetic beads were washed three times with lysis buffer to remove unbound his-zbrig-i and then analyzed via western blotting using anti-flag or anti-his antibodies. his tag protein alone was served as a negative control. ubiquitination assays were performed as described previously with some modifications (21) . hek 293t cells, pre-seeded in 75-cm 2 flasks overnight, were co-transfected with 10 µg of different plasmid combinations. cells were lysed at 24 h after transfection, and then gfp-zbrig-i mutants were immunoprecipitated with anti-gfp antibodies as described above. immunoprecipitates or input were analyzed by immunoblotting with the indicated antibodies. all statistics were calculated using spss version 20. differences between control and treatment groups were assessed by one-way anova. p < 0.05 is considered statistically significantly different. p < 0.01 was considered highly significant. as shown in figure 1a , mrna level of zbtrim25 was downregulated within 24 h after rgnnv infection. meanwhile, we also investigated the expression of zbtrim25 in rgnnvinfected zebrafish embryos at 24 h, and the results were concordant with zbe3 cells (figure 1b) . these data indicated a potential role of zbtrim25 in innate immune response to rgnnv infection. in mammals, trim25 has been suggested to promote ifnβ production by functioning as a key upstream activator of rig-i to activate the rlr signaling pathway (10) . to investigate whether zbtrim25 regulated rlr signaling pathway in zebrafish, the expression of several rlr signaling pathwayrelated genes was measured in zbtrim25 overexpressing zbe3 cells. as shown in figure 2a , overexpression of zbtrim25 markedly enhanced the expression of rig-i, mavs, traf3, irf3, ifn 1, and isg15 during rgnnv infection. similar results were detected in zbtrim25 overexpressing zbe3 cells treated with poly i:c ( figure 2b) . furthermore, coexpression of zbtrim25 with zbrig-i induced a dosedependent increase in ifn activation compared with the zbrig-i overexpression alone (figure 2c) . overexpression of zbtrim25 dose-dependently inhibited rgnnv replication ( figure 2d) . on the contrary, knockdown of zbtrim25 using sirna increased the level of rdrp in rgnnv-infected zbe3 cells (figures 2e,f) . all these results demonstrate that zbtrim25 is a positive regulator of rlr signaling pathway and functions as an antiviral factor during rgnnv infection in zebrafish. to elucidate the mechanism by which zbtrim25 participates in rlr signaling pathway in zebrafish, the interaction of zbtrim25 and zbrig-i was investigated. we co-expressed myc-zbtrim25 and flag-zbrig-i plasmids in hek 293t cells, and immunofluorescence imaging showed zbtrim25 and zbrig-i colocalized in the cytoplasm of hek293t cells (figure 3a) . co-ip against the myc tag revealed that zbtrim25 could interact with full-length zbrig-i but not with flag-vector ( figure 3b) . his pull-down analysis showed that zbtrim25 was directly bound to zbrig-i ( figure 3c ). all these data suggest that zbtrim25 interacts with zbrig-i. to identify the region involved in the zbrig-i/zbtrim25 interaction, firstly, zbrig-i deletion mutants [pegfp-zbrig-i-2card, pegfp-zbrig-i-2card, pegfp-rig-i-rd, and pegfp-rig-i-(2card+rd)] were constructed and cotransfected with flag-zbtrim25 in hek 293t cells ( figure 4a) . zbrig-i-2card, zbrig-i-2card, and zbrig-i-rd could bind to zbtrim25 individually (figures 4b-d) ; however, zbrig-i-(2card+rd) failed to co-precipitate with zbtrim25 ( figure 4e) . these results indicate that zbrig-i binds to zbtrim25 through its n-terminal 2card region and the c-terminal rd region. furthermore, we constructed two truncations of zbtrim25 (zbtrim25-spry and zbtrim25-spry) co-transfected with zbrig-i-2card or zbrig-i-rd in hek 293t cells, respectively ( figure 4f) . we found that the spry domain of zbtrim25 interacted with 2card and rd regions of zbrig-i (figures 4g-j) . collectively, these results indicate that the spry domain of zbtrim25 is responsible for its interaction with 2card and rd regions of zbrig-i. to investigate whether the e3 ligase activity of zbtrim25 is involved in the regulation of zbrig-i, the ubiquitination of zbrig-i was tested in zbtrim25 overexpressing cells. we found that zbtrim25 markedly promoted the k63 polyubiquitination of zbrig-i ( figure 5a) . furthermore, hek 293t cells were transfected with flag-tagged zbtrim25, zbrig-i deletion mutants [gfp-zbrig-i-2card, gfp-zbrig-i-(2card+rd), and gfp-zbrig-i-rd], and ha-tagged k63 ubiquitin, and our results showed that zbtrim25 obviously enhanced the ubiquitination of zbrig-i-2card and zbrig-i-rd (figures 5b,c) , but not zbrig-i-(2card+rd) (figure 5d) . these data suggest that zbtrim25 ubiquitinates both n-terminal 2card and c-terminal rd regions of zbrig-i. it has been reported that ubiquitination of rig-i by trim25 is vital for ifn signaling. thus, the effect of zbtrim25-mediated zbrig-i ubiquitination on zbrig-i's ifn-inducing activities was assessed. our results showed that ectopic expression of zbrig-i-2card and zbrig-i-rd could enhance ifn promoter activity (figure 6a) , and this activation was markedly enhanced by zbtrim25 overexpression (figures 6b,c) . furthermore, overexpression of k63-linked ubiquitin dose-dependently increased the promotion effect of zbtrim25 on zbrig-i-2card and rd mediated ifn 1 promoter activation (figures 6b,c) . these data confirm the importance of zbtrim25-mediated k63 ubiquitination in the n-terminal 2card region and c-terminal rd region of zbrig-i for zbrig-i-mediated ifn induction. rlr signaling pathway plays crucial roles in recognizing viral infections and initiating the antiviral immune response. rig-i, as an important component of rlr signaling pathway, can detect viral dsrnas in the cytoplasm and induce type i ifn production and the secretion of pro-inflammatory cytokines to suppress virus spread during virus infection (22) . multiple studies have demonstrated that the ubiquitination of rig-i plays an important role in the rig-i-mediated antiviral signaling pathway. for instance, trim25, trim4, and mex3c positively regulate rig-i-mediated signaling by targeting rig-i for k63linked polyubiquitination (23, 24) . trim25, well-known as an ubiquitin e3 ligase and an isg15 e3 ligase, is widely involved in the regulation of innate immunity (10, 25) . in mammals, previous reports showed that trim25 enhanced rlrs antiviral pathway by binding viral rna-activated rig-i to induce its k63-linked polyubiquitination and subsequent ifns and isgs production (26) . in teleost fish, several trim25 homologs were reported to play a pivotal role in innate immunity (14, 15) ; however, the mechanisms by which fish trim25 modulates the innate immune response against viruses remain elusive. here, we found that zbtrim25 positively regulated rlr signaling pathway and facilitated zbrig-i-mediated ifn 1 promoter activation, and overexpression of zbtrim25 inhibited rgnnv infection, indicating the conservative antiviral properties of trim25 in fish and mammals. several reports showed that trim25 was involved in the regulation of antiviral innate immunity by targeting rig-i (10, 27) . the mammal rig-i protein contains two n-terminal cardlike domains, a c-terminal rd region and an rna helicase region (28) . in zebrafish, rig-ia (an insertion variant of rig-i) and rig-ib (the typical rig-i) were identified as two transcripts of rig-i, and overexpression of rig-ib in cultured fish cells, but not rig-ia, activated zebrafish type i ifn and induced antiviral response (29) . thus, in this report, we investigated the interaction of zbtrim25 and zbrig-i (rig-ib), and our results showed that zbtrim25 was directly associated with zbrig-i and especially the 2card or rd region of zbrig-i was sufficient for its interaction with zbtrim25. trim25 is characterized by an n-terminal region containing a catalytic ring domain, one or two b-box domains, a coiled-coil dimerization domain, and a c-terminal spry domain (30) . among these domains, spry was associated with protein-protein interactions and/or rna binding (31) . gack et al. reported that the c-terminal spry domain of trim25 interacted with the first card of rig-i, but not the helicase region and rd of rig-i, and this interaction delivered the k63-linked ubiquitin moieties to the second card of rig-i, which facilitated the dimerization of rig-i and subsequent interaction with mavs to induce antiviral signal transduction (10) . we further investigate whether the spry domain of zbtrim25 was responsible for its interaction with zbrig-i. unlike previous studies, we found that the spry domain of zbtrim25 interacted not only with 2card but also with rd regions of zbrig-i. in non-infected cells, rd covered the rna-binding and helicase domains and cards folded over one another, which made rig-i to exist in an auto-repressed conformation. upon virus infection, viral rnas interacted with the rd and the helicase domain of rig-i, which in turn exposed the cards for mavs interaction, thereby triggering antiviral responses (32, 33) . considering the interaction between rd of rig-i and viral rnas, we speculated that the interaction of zbtrim25 and zbrig-i rd might inhibit zbrig-i sensing of viral rnas. meanwhile, it has been known that card domains of rig-i are widely involved in its interaction with other proteins, such as mavs, trim40, and virus proteins (27, 34, 35) . thus, the interaction of zbtrim25 and zbrig-i rd might also make room for other proteins to bind to 2card of zbrig-i, zbtrim25, and other proteins and will work cooperatively in regulation of rlr signaling pathway. the differences between the findings for zbtrim25 and trim25 in mammals indicate that zbtrim25 may regulate rlr signaling pathway in various ways. ubiquitination is a vital post-translational modification for the modulation of rig-i activity. several e3 ubiquitin ligases that mediate k63-linked ubiquitination of rig-i for its activation have been identified. for instance, mex3c overexpression caused the k63-linked ubiquitination of rig-i-2card but not rig-i-2card, and lysines 48, 99, and 169 of rig-i were required for rig-i ubiquitination by mex3c (23) . rnf135 mediated the k63-linked polyubiquitination of rig-i-rd, and lysines 849 and 851 residues of rig-i were crucial for rnf135-mediated ubiquitination (36) . in contrast to rnf135, trim25 mediated the k63-linked polyubiquitination of rig-i-2card, but not rig-i-2card, and the lysine 172 residue of rig-i was critical for efficient trim25-mediated ubiquitination and the ability of rig-i to activate antiviral signal transduction (10) . our results indicated that zbtrim25 mediated k63-linked polyubiquitination of both 2card and rd regions of zbrig-i, which is distinct from the findings in mammals that trim25 only targeted and promoted the k63-linked polyubiquitination of rig-i 2card. in addition, our reporter analysis showed that overexpression of zbrig-i-2card led to the activation of ifn 1 promoter, which is similar with other reports (37) . overexpression of zbrig-i-rd also resulted in the activation of ifn 1 promoter. furthermore, k63-linked ubiquitin is essential for the zbtrim25-mediated enhancement of zbrig-i 2card and rd-dependent ifn 1 promoter activation. zbrig-i possessed capacities in the induction of ifns and isgs to enhance the antiviral response (38) . taken together, these findings suggest that zbtrim25mediated ubiquitination of 2card and rd regions of zbrig-i is crucial for its antiviral innate immune response. however, due to the lack of trim25 or rig-i-knockout zebrafish, we cannot assess the impact of the zebrafish trim25/rig-i pathway at the in vivo level. a recent study demonstrated that zebrafish rnf135 also interacted with and ubiquitinated zbrig-i (39) . further studies will be performed to determine the precise architecture of the zebrafish trim25/rnf135/rig-i protein complex and the mechanism by which zbtrim25 and zbrnf135 worked together to regulate ubiquitination of zbrig-i. it was known that several virus proteins could positively or negatively regulate rlr signaling pathway by targeting its key components or regulatory proteins (22) . for instance, paramyxovirus v proteins interacted with the rig-i/trim25 regulatory complex and inhibited rig-i signaling (27) . influenza a virus ns1 protein bound to trim25 to block ubiquitination of the rig-i (40) . severe acute respiratory syndrome nucleocapsid inhibited trim25-mediated rig-i ubiquitination, causing the inhibition of ifn production (41) . the rgnnv genome encodes a structural (capsid protein, cp) and a nonstructural (rna-dependent rna polymerase, rdrp) protein (3). huang et al. reported that rdrp from ognnv induced ifn by activating irf3, the key regulatory component of rlrs-ifn signaling (42) , indicating that rdrp might be a positive rlr signaling pathway. whether rdrp targets the key components of rlr signaling pathway to exert its positive regulation role is a question that deserves further research. in addition, some mirnas could target critical regulatory proteins of rlr pathway for immune evasion (43, 44) ; whether rgnnv infection-related mirna was also involved in the regulation of rlr signaling pathway needs to be further investigated. in summary, zbtrim25 is identified as a positive regulator of rlr signaling pathway by targeting zbrig-i. the spry domain of zbtrim25 is required for its interaction with 2card and rd regions of zbrig-i. zbtrim25 promotes k63 polyubiquitination of both zbrig-i 2card and rd regions, which subsequently induces the activation of downstream signaling event via mavs and thereby inhibits viral infection (figure 7) . these findings represent a new mechanism underlying the regulation of rlr signaling pathway. all datasets generated for this study are included in the article/supplementary material. the animal study was reviewed and approved by the ethics committee of sun yat-sen university. shaping the landscape of host immunity regulation of rlr-mediated innate immune signaling -it is all about keeping the balance understanding the interaction between betanodavirus and its host for the development of prophylactic measures for viral encephalopathy and retinopathy rig-i specifically mediates group ii type i ifn activation in nervous necrosis virus infected zebrafish cells identification and characterization of the melanoma differentiation -associated gene 5 in sea perch, lateolabrax japonicus characterization and expression analysis of laboratory of genetics and physiology 2 gene in sea perch rnf122 suppresses antiviral type i interferon production by targeting rig-i cards to mediate rig-i degradation trim13 is a negative regulator of mda5-mediated type i interferon production the e3 ubiquitin ligase rnf5 targets virus-induced signaling adaptor for ubiquitination and degradation trim25 ringfinger e3 ubiquitin ligase is essential for rig-i-mediated antiviral activity trim25 in the regulation of the antiviral innate immunity. front immunol ubiquitin-like modifier fat10 attenuates rig-i mediated antiviral signaling by segregating activated rig-i from its signaling platform molecular characterization of tripartite motif protein 25 (trim25) involved in erα-mediated transcription in the korean rose bitterling rhodeus uyekii ring domain is essential for the antiviral activity of trim25 from orange spotted grouper the two trim25 isoforms were differentially induced in larimichthys crocea post poly (i:c) stimulation. fish shellfish immun stages of embryonic development of the zebrafish establishment of a cell line with high transfection efficiency from zebrafish danio rerio embryos and its susceptibility to fish viruses mandarin fish caveolin 1 interaction with major capsid protein of infectious spleen and kidney necrosis virus and its role in early stages of infection interferon regulatory factor 3 from sea perch (lateolabrax japonicus) exerts antiviral function against nervous necrosis virus infection the eukaryotic translation initiation factor 3 subunit e binds to classical swine fever virus ns5a and facilitates viral replication ubiquitination is essential for avibirnavirus replication by supporting vp1 polymerase activity rig-i-like receptor regulation in virus infection and immunity pivotal role of rna-binding e3 ubiquitin ligase mex3c in rig-i-mediated antiviral innate immunity trim4 modulates type i interferon induction and cellular antiviral response by targeting rig-i for k63-linked ubiquitination the ubiquitin-specific protease usp15 promotes rig-i-mediated antiviral signaling by deubiquitylating trim25 mechanism of trim25 catalytic activation in the antiviral rig-i pathway paramyxovirus v proteins interact with the rig-i/trim25 regulatory complex and inhibit rig-i signaling structural features of influenza a virus panhandle rna enabling the activation of rig-i independently of 5'-triphosphate higher antiviral response of rig-i through enhancing rig-i/mavs-mediated signaling by its long insertion variant in zebrafish trim/rbcc, a novel class of 'single protein ring finger' e3 ubiquitin ligases structure and function of the spry/b30.2 domain proteins involved in innate immunity structural insights into rna recognition and activation of rig-i-like receptors the structural basis of 5 ' triphosphate double-stranded rna recognition by rig-i c-terminal domain the e3 ubiquitin ligase trim40 attenuates antiviral immune responses by targeting mda5 and rig-i. cell rep identification and characterization of mavs, a mitochondrial antiviral signaling protein that activates nf-kappab and irf 3 riplet/rnf135, a ring finger protein, ubiquitinates rig-i to promote interferon-beta induction during the early phase of viral infection involvement of zebrafish rig-i in nf-kappab and ifn signaling pathways: insights into functional conservation of rig-i in antiviral innate immunity retinoic acid-inducible gene i (rig-i)-like receptors (rlrs) in fish: current knowledge and future perspectives rnf135 is a positive regulator of ifn expression and involved in rig-i signaling pathway by targeting rig-i. fish shellfish immun influenza a virus ns1 targets the ubiquitin ligase trim25 to evade recognition by the host viral rna sensor rig-i the severe acute respiratory syndrome coronavirus nucleocapsid inhibits type i interferon production by interfering with trim25-mediated rig-i ubiquitination protein a from orange-spotted grouper nervous necrosis virus triggers type i interferon production in fish cell downregulation of microrna mir-526a by enterovirus inhibits rig-i-dependent innate immune response microrna-146a feedback inhibits rig-i-dependent type i ifn production in macrophages by targeting traf6, irak1, and irak2 yj and kj performed all experiments with assistance from yx, wz, pj, and wl analyzed data. kj and my conceived the study and designed experiments. kj and my wrote the manuscript. all authors read and approved the final manuscript. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu. 2019.02805/full#supplementary-material conflict of interest: the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2019 jin, jia, zhang, xiang, jia, liu and yi. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-312886-o3ipzn05 authors: onomoto, koji; yoneyama, mitsutoshi; fung, gabriel; kato, hiroki; fujita, takashi title: antiviral innate immunity and stress granule responses date: 2014-08-19 journal: trends immunol doi: 10.1016/j.it.2014.07.006 sha: doc_id: 312886 cord_uid: o3ipzn05 viral infection triggers the activation of antiviral innate immune responses in mammalian cells. viral rna in the cytoplasm activates signaling pathways that result in the production of interferons (ifns) and ifn-stimulated genes. some viral infections have been shown to induce cytoplasmic granular aggregates similar to the dynamic ribonucleoprotein aggregates termed stress granules (sgs), suggesting that these viruses may utilize this stress response for their own benefit. by contrast, some viruses actively inhibit sg formation, suggesting an antiviral function for these structures. we review here the relationship between different viral infections and sg formation. we examine the evidence for antiviral functions for sgs and highlight important areas of inquiry towards understanding cellular stress responses to viral infection. viral infection triggers the activation of antiviral innate immune responses in mammalian cells. viral rna in the cytoplasm activates signaling pathways that result in the production of interferons (ifns) and ifn-stimulated genes. some viral infections have been shown to induce cytoplasmic granular aggregates similar to the dynamic ribonucleoprotein aggregates termed stress granules (sgs), suggesting that these viruses may utilize this stress response for their own benefit. by contrast, some viruses actively inhibit sg formation, suggesting an antiviral function for these structures. we review here the relationship between different viral infections and sg formation. we examine the evidence for antiviral functions for sgs and highlight important areas of inquiry towards understanding cellular stress responses to viral infection. viral infection and stress granules viral invasion and replication are detected by innate immune sensors in cells, triggering downstream signaling pathways that can ultimately result in the activation of systemic immune responses. several innate immune sensors recognize cytoplasmic viral rna [1] , and lead to the production of ifns which in turn trigger various antiviral pathways aimed at halting viral replication and spread. these antiviral effects include double-stranded (ds) rnadependent protein kinase (pkr)-dependent inhibition of mrna translation, and 2 0 ,5 0 -oligoadenylate synthetase (oas)/rnase l-mediated rna degradation [2] . innate immune responses also trigger the activation of adaptive immunity in the form of t and b cell activation and proliferation, and modulate the phenotype and function of these adaptive responses [3, 4] . in some cases, viral infection also induces the formation of cytoplasmic granules similar to those induced by cellular stresses such as heat, oxidation, hypoxia, and osmotic pressure, which are referred to as stress granules (sgs). sgs are ribonucleoprotein (rnp) aggregates that contain translationally stalled mrnas, 40s ribosomes, and various rna-binding proteins [5] [6] [7] (box 1). activation of the rlr signaling pathways by viral rna innate immune responses are triggered upon recognition of pathogen-associated molecular patterns (pamps) which in the case of viruses are often nucleic acid-based, either rna or dna. viral nucleic acid can be detected by sensors including toll-like receptors (tlr)3, 7/8, and 9, retinoic acid inducible gene i (rig-i)-like receptors (rlrs), and cytoplasmic dna sensors such as dna-dependent activator of ifn-regulator factors (dai), stimulator of ifn genes (sting, also known as mita/eris/mpys), dead box polypeptide 41 (ddx41), and cgmp/camp synthase (cgas) [8] [9] [10] [11] [12] . rlrs, rig-i, melanoma differentiationassociated protein 5 (mda5), and dhx58 [dexh (asp-glu-x-his) box polypeptide 58, also known as lgp2] are all rna helicases and contain the signature dexd/h motif that characterized the dexd/h box family. these proteins are crucial for the detection of cytoplasmic rna [13, 14] . rlrs discriminate self from viral transcripts by recognizing specific biochemical signatures such as ds structure and the presence of a 5 0 -ppp moiety [15] [16] [17] ; self-transcipts lack these viral signatures. the signaling pathways downstream of the founding member of the rlr family, rig-i, are among the best understood. upon viral rna recognition by rig-i, the signal is relayed to the adaptor protein ifn-b promoter stimulator 1 (ips-1, also known as mavs, visa, or cardif), which predominantly localizes to the mitochondrial outer membrane [18, 19] . when viral rna binds at the helicase and the c-terminal domain (ctd) of rig-i, its n-terminal caspase recruitment domains (cards) are covalently modified with k63-linked polyubiquitin chains by the e3 ligase, tripartite motif-containing protein 25 (trim25) [20] , and oligomerize [21] . the ubiquitinated and oligomerized cards bind to the card domain on ips-1 on mitochondria, peroxisomes, and/ or mitochondrion-associated membrane (mam) regions in the endoplasmic reticulum (er) [22] . the translocation of rig-i to these locales is facilitated by the chaperone protein 14-3-3e [23] . the requirement for k63-linked polyubiquitin chains is complex because several reports have demonstrated the importance of non-covalent interaction between rig-i cards and the unanchored k63-ubiquitin chains [21, 24] . box 1. an overview of sg biology cells respond to various insults including heat, oxidative stress, nutrient starvation, and proteotoxic stress by forming cytoplasmic nucleoprotein aggregates termed sgs [5, [68] [69] [70] . multiple rnabinding proteins (rbps) localize to sgs, and some have been used as markers for these cytoplasmic bodies (table i) . although the formation of sgs in live cells can be detected by monitoring fluorescence-tagged sg marker proteins, biochemical isolation of sgs is notoriously challenging because these are not membranesequestered compartments. sg formation has been interpreted as a response aimed at preventing the generation of abnormal proteins by transient stalling of translation in times of cellular stress. stalled transcripts undergo translation upon recovery from stress, or alternatively they are degraded in another granular compartment termed the processing body (p-body, pb) [71, 72] . unlike sgs, pbs are present in the unstressed cell, and contain enzymes for mrna degradation such as decapping enzymes and 5 0 -3 0 exonucleases (table i ). it is thought that transcripts and proteins can move from sgs to pbs (and vice versa), and these aggregates share some of their components (table i) [60] ; however, the mechanisms underlying the proposed exchange of contents are unclear. sg proteins, as defined by studies using proteins characteristic to sgs as markers [73] , are either diffusely distributed in the cytoplasm or localized in the nucleus in normal conditions; stress triggers their aggregation in the cytoplasm [5, 74] . a common event downstream from the aforementioned cellular stresses is phosphorylation of eif2a at serine 51, which is considered to be an initial trigger for sg formation. four eif2a kinases, pkr, gcn2, pkr-like endoplasmic reticulum kinase (perk), and heme-regulated eif2a kinase (hri), can phosphorylate eif2a in mammals (table ii) . the mechanisms that connect eif2a phosphorylation to sg formation remain to be elucidated. some proteins have been shown to be crucial for the formation and or stability of sgs. these include g3bp1, a phosphorylationdependent endonuclease [30, 75] , and t cell restricted intracellular antigen-1 (tia1) and tia-related protein (tiar) that are collectively termed tia1/tiar [74, 76] . removal of these regulators by genomic deletion or rnai blocks sg formation by sodium arsenite, and a mutant form of g3bp1 (s149e) acted as a dominant inhibitor of sg formation [75] . however, because of analytical constraints, the molecular machinery underlying the formation of sgs remains unclear. furthermore, it has been reported that rig-i forms a signaling-competent filament on substrate dsrna independently of ubiquitins [25] . although a recent structural report suggests that the conformation of the active tetramer of rig-i cards can be stabilized by covalent conjugation with ubiquitin chains, and that filament formation may partially compensate for ubiquitin-dependent rig-i activation [26] , further analysis will be necessary to clarify the molecular role of ubiquitin chains in rig-i signaling. the rig-i/ips-1 interaction recruits signaling molecules including tumor necrosis factor (tnf) receptor-associated factors (trafs). subsequent activation of tank-binding kinase 1(tbk1)/inducible ikb kinase (ikki), and ikka/ikkb induces downstream signaling via the ifn regulatory factor (irf) and nuclear factor-kb (nf-kb) pathways, respectively. these pathways ultimately culminate in the activation of the transcription factors irf-3, irf-7, and nf-kb, which activate the transcription of ifn and proinflammatory cytokine genes [27] . these pathways are summarized in figure 1 . moreover, secreted ifn amplifies the expression of isgs, such as rlrs, pkr, and oas, as a host strategy to amplify antiviral signaling. viruses, particularly rna viruses, have been shown to induce the formation of sg-like cytoplasmic bodies (table 1 trends in immunology september 2014, vol. 35, no. 9 and references therein). in some cases these bodies have been given different names in an attempt to distinguish them from sgs; in this review, however, we refer to virusinduced sg-like granules collectively as sgs. many viruses induce sgs through the activation of the eukaryotic translation initiation factor (eif)2a kinases pkr and, in some cases, general control non-depressible 2 (gcn2), which are both triggered by detection of rna in the cytoplasm [28] ( figure 2 ). depending on both the virus and the host cell, different patterns of sg formation have been observed upon infection: stable sg formation, no sg formation, transient sg formation, or alternating (oscillating) sg formation in which sgs form, disperse, and reform during the assays (table 1) . transient sg formation results from dissociation of key components in sgs by viral proteins [29] [30] [31] [32] [33] . for instance, in the case of infection of several picornaviruses, such as poliovirus, coxsackievirus and encephalomyocarditis virus (emcv), transient formation of sgs is associated with the cleavage of ras-gap sh3 domain binding protein-1 (g3bp1) by the viral 3c protease [29] [30] [31] . this was confirmed by the observation that ectopic expression of cleavage-resistant g3bp1 leads to stable sg formation. on the other hand, recent studies have demonstrated that infection with hepatitis c virus (hcv) produces oscillating sgs [34] . hcv strongly activates pkr via the 5 0 -untranslated region (utr) of its genome [35] , thereby inducing sgs [34, 36] , but stress-inducible expression of growth arrest dna-damageinducible 34 (gadd34), a regulatory component of host protein phosphatase 1 (pp1), leads to dephosphorylation of eif2a and terminates sg formation. in the stress-recovered condition, gadd34 protein is rapidly downregulated by an unknown mechanism and the phosphorylated form of eif2a reaccumulates in the cells, resulting in an oscillating pattern of sgs. in cases where viral infection appears to not induce sgs, accumulating evidence suggest that these viruses inhibit sg formation. cells infected with mengovirus or theiler's murine encephalomyelitis virus (tmev), which belong to the cardiovirus genus within the family of picornaviridae, do not exhibit sgs, and this has been proposed to be due to complete inhibition of sg formation by the viral nonstructural protein, leader (l) protein [37] . although l protein is known to block ifn production via inhibition of irf-3 activation [38] , it remains unknown how l limits sg formation. similarly, sgs do not form upon infection of cells with measles virus. in this case the mechanism proposed involves the viral c protein because c-deficient virus, but not the wild type, strikingly induces sgs [39] . although measles c protein has diverse functions during infection, including modulation of viral polymerase activity and inhibition of ifn production [40] , the molecular machinery for sg inhibition remains to be determined. in virus-infected cells, viral rnas activate pkr (or gcn2, in the case of sindbis virus) to initiate assembly of sg through eif2a phosphorylation. eif2a phosphorylation blocks translation of cellular mrnas. translation-stalled mrnas may be transferred to a distinct cellular compartment, the p-body, to be degraded. viral rnas are also recognized by rlrs, which are recruited to sgs with several signaling molecules including antiviral proteins and ubiquitin ligases. the oas-rnase l pathway cleaves viral rnas, and the cleaved rnas may act as ligands for rlrs. ips-1, which is localized on mitochondria and/or mam, forms prion-like aggregates, interacts with rlrs on sgs, and activates ifn-inducing signaling. areas that require further investigation are highlighted with question marks. trends in immunology september 2014, vol. 35, no. 9 in the case of influenza a virus (iav), the viral nonstructural protein 1(ns1) has been shown to inhibit eif2a phosphorylation by blocking pkr activation via viral rna sequestration and physical interaction [41] [42] [43] . during iav infection, the viral nucleocapsid initially accumulates in the nucleus and is then transported to the cytoplasm in the late phase of infection. however, in a mutant iav lacking ns1, the viral nucleocapsid was reported to coincide with sgs in the cytoplasm, to which rig-i is colocalized [42] . these findings suggest that sgs function as a platform for the detection of iav genomic rna by rig-i. as in the cases of mengovirus, tmev, and measles virus, there appears to be a clear benefit, from the standpoint of the virus, to interfering with sg formation. an implication of these findings is that sgs have an antiviral role and that, accordingly, viruses have developed strategies to suppress their formation ( figure 2) . indeed, there are multiple examples of viruses actively inhibiting the formation of sgs through varied mechanisms [44] [45] [46] [47] [48] . an inverse correlation between sg formation and viral propagation has been reported in multiple viral replication systems. in the case of japanese encephalitis virus (jev) infection, sg formation is inhibited by the viral core protein, which directly interacts with sg component, caprin 1 [44] . in cells infected with a mutant virus whose core protein fails to interact with caprin 1, inhibition of sg formation is abrogated and viral propagation is significantly impaired both in vitro and in vivo, suggesting that sgs impact negatively on viral replication. this notion is further supported by studies showing that rlrs localize to virus-induced sgs, suggesting that sgs may act as a platform for viral rna sensing and the activation of downstream signaling pathways [42] . there is a strong correlation between pkr-dependent sg formation and ifn production in some viral infections [29, 42, 49] (figure 2 ). however, a recent study has demonstrated that pkrdependent accumulation of mda5 in sgs is dispensable for triggering ifn responses [50] . further investigations will be necessary to address this discrepancy. whether pkr is required for virus-induced ifn production is controversial. some reports indicate that ifn production is significantly impaired in pkr-deficient cells [29, 42, 49, [51] [52] [53] [54] [55] , whereas others show that deficiency of pkr has no effect [54, [56] [57] [58] . one possible explanation for these observations is that there are differences in the viruses and cell types used in each study. in the early 2000s, several studies demonstrated that virus-induced activation of ifn is independent of pkr in plasmacytoid dendritic cells (pdcs), which are known to be 'ifn-producing cells'. however, subsequent reports revealed that robust ifn induction by pdcs is exclusively induced by tlrs, suggesting a dispensable role for pkr in tlrmediated signaling. by contrast, the pkr-dependency of rlr-mediated signaling is more complicated. because viruses are extraordinary diverse in their genome structures and life cycles, different viruses are likely to produce different rna species during viral replication at distinct locations in the infected cells. thus, the ability of these viral rnas to activate pkr and/or rlrs could be divergent. moreover, as mentioned above, viruses employ a variety of strategies to terminate antiviral responses. for instance, it has been demonstrated that sendai virus (sev)-induced ifn production is independent of pkr [53, 54] . indeed, infection of sev can activate ifn without sg formation [29] ; however, infection by a mutant virus in which accessory protein c is deleted leads to significant activation of pkr and eif2a phosphorylation, with concomitant upregulation of ifn [45, 59] , suggesting that pkr is dispensable for sev-induced ifn activation, but is responsible for the enhancement of ifn production. thus, the pkr-dependency of ifn production might vary depending on the ability of each virus to modulate host responses. this notion is supported by reports in which ifn production following stimulation with a virus-mimetic synthetic dsrna such as poly(i:c) showed significant pkr-dependency [29, 42, 49, 53, 55] . understanding the relationship between mechanisms of viral detection and sgs during viral infection, viral rna, either incoming or produced as a replication intermediate, triggers a series of events. dsrna activates pkr (or gcn2) to initiate assembly of sg via eif2a phosphorylation, and this in turn blocks translation and leads to the recruitment of stalled transcripts into sgs (box 1 and figure 2 ). it has been proposed that, if the stress stimulus is not resolved, the stalled transcripts are transferred to processing bodies (p-bodies, pbs) for degradation [60] . this view requires reexamination because sg formation does not necessarily result in translational shut-off. indeed, it is unclear whether sg formation results in total host cell translational shut-off or translational arrest at limited areas in the cytoplasm. many viruses hijack host cellular compartments to form replication complexes for viral transcription and translation [61, 62] . the fact that ifn is efficiently translated in virus-infected and sg-containing cells [29, 42, 49] suggests that sg formation does not necessarily correlate with total host translational shut-off. viral dsrna contained in sg potentially activates oas to catalyze the synthesis of 2 0 -5 0 oligo a, which activates cytoplasmic endoribonuclease rnase l [63] . rnase l is also detected in sgs of virus-infected cells [42] . activated rnase l may cleave viral rna in sg to block viral transcription and translation, and some cleavage products may act as ligands for rlr [64] . several ubiquitin ligases including trim25, ring finger protein leading to rig-i activation (riplet), and mex-3 rna binding family member (mex3c), that are known to regulate rig-i activation, are also colocalized in virus-induced sg ( figure 2) [65, 66] . although several studies suggest that rlr might be activated in virus-induced sgs [29, 42, 49] , direct evidence to this effect is lacking. rlr-mediated signaling is transmitted via homotypic interaction with ips-1, which is localized on mitochondria, peroxisomes, and mams. it is unclear how rlr-containing sgs can communicate with these organelles and activate antiviral signaling ( figure 2) . a major challenge for sg research is that sgs are difficult entities to isolate for biochemical analyses. the composition of sgs induced by different viruses and in different host cells may vary [49, 67] . the development of novel biochemical isolation approaches and molecular probes for cell biological review trends in immunology september 2014, vol. 35, no. 9 analyses will be crucial in investigating the molecular events taking place in sgs during viral infection. stress responses are known to be crucial for maintaining the homeostasis of living organisms. in response to various stresses, eukaryotic cells initiate stress responses, including the formation of sgs, in which cytoplasmic mrnas are compartmentalized to escape dysregulation. accumulated lines of evidence show that viral infection can also induce stress responses including sg formation concurrently with the initiation of innate antiviral responses via pattern recognition receptors (prrs). the observations that (i) there is a strong correlation between sg formation and ifn production, (ii) there is a reverse correlation between sg formation and viral propagation, and (iii) rlrs are localized in viral-induced sgs together with viral non-self rnas, together strongly suggest that sgs have an antiviral role and possibly function as platform to initiate innate responses. interestingly, this notion clearly indicates that the quality control machinery for 'self rna' and the host defense mechanism against invasion of 'non-self rna' are closely coordinated with each other. moreover, it is interesting to note that these observations may help us to develop a novel therapeutic or preventive strategy for virus-induced infectious diseases. innate immune detection of microbial nucleic acids interferon-inducible antiviral effectors regulation of adaptive immunity by the innate immune system innate receptors for adaptive immunity evidence that ternary complex (eif2-gtp-trna(i)(met))-deficient preinitiation complexes are core constituents of mammalian stress granules stress granules: sites of mrna triage that regulate mrna stability and translatability rna granules: posttranscriptional and epigenetic modulators of gene expression the role of pattern-recognition receptors in innate immunity: update on toll-like receptors rig-i like receptors and their signaling crosstalk in the regulation of antiviral immunity innate recognition of viruses innate immune responses to dna viruses cgas produces a 2 0 -5 0 -linked cyclic dinucleotide second messenger that activates sting the rna helicase rig-i has an essential function in double-stranded rna-induced innate antiviral responses shared and unique functions of the dexd/ h-box helicases rig-i, mda5, and lgp2 in antiviral innate immunity recognition of 5 0 triphosphate by rig-i helicase requires short blunt double-stranded rna as contained in panhandle of negative-strand virus 0 -triphosphate rna requires base-paired structures to activate antiviral signaling via rig-i 0 -triphosphate rna is the ligand for rig-i ips-1, an adaptor triggering rig-i-and mda5-mediated type i interferon induction orchestrating the interferon antiviral response through the mitochondrial antiviral signaling (mavs) adapter trim25 ring-finger e3 ubiquitin ligase is essential for rig-i-mediated antiviral activity ubiquitin-induced oligomerization of the rna sensors rig-i and mda5 activates antiviral innate immune response mitochondrial-associated endoplasmic reticulum membranes (mam) form innate immune synapses and are targeted by hepatitis c virus the mitochondrial targeting chaperone 14-3-3epsilon regulates a rig-i translocon that mediates membrane association and innate antiviral immunity reconstitution of the rig-i pathway reveals a signaling role of unanchored polyubiquitin chains in innate immunity rig-i forms signaling-competent filaments in an atp-dependent, ubiquitin-independent manner structural basis for ubiquitin-mediated antiviral signal activation by rig-i type i interferon gene induction by the interferon regulatory factor family of transcription factors antiviral effect of the mammalian translation initiation factor 2alpha kinase gcn2 against rna viruses encephalomyocarditis virus disrupts stress granules, the critical platform for triggering antiviral innate immune responses inhibition of cytoplasmic mrna stress granule formation by a viral proteinase production of a dominant-negative fragment due to g3bp1 cleavage contributes to the disruption of mitochondriaassociated protective stress granules during cvb3 infection mammalian orthoreovirus particles induce and are recruited into stress granules at early times postinfection importance of eif2alpha phosphorylation and stress granule assembly in alphavirus translation regulation dynamic oscillation of translation and stress granule formation mark the cellular response to virus infection regulation of pkr by hcv ires rna: importance of domain ii and ns5a hepatitis c virus (hcv) induces formation of stress granules whose proteins regulate hcv rna replication and virus assembly and egress the leader protein of cardioviruses inhibits stress granule assembly the mengovirus leader protein blocks interferon-alpha/beta gene transcription and inhibits activation of interferon regulatory factor 3 stress granule formation induced by measles virus is protein kinase pkr dependent and impaired by rna adenosine deaminase adar1 measles virus nonstructural c protein modulates viral rna polymerase activity by interacting with host protein shcbp1 influenza a virus inhibits cytoplasmic stress granule formation critical role of an antiviral stress granule containing rig-i and pkr in viral detection and innate immunity binding of the influenza virus ns1 protein to double-stranded rna inhibits the activation of the protein kinase that phosphorylates the elf-2 translation initiation factor japanese encephalitis virus core protein inhibits stress granule formation through an interaction with caprin-1 and facilitates viral propagation sendai virus c protein plays a role in restricting pkr activation by limiting the generation of intracellular double-stranded rna sendai virus trailer rna binds tiar, a cellular protein involved in virus-induced apoptosis rotavirus infection induces the phosphorylation of eif2alpha but prevents the formation of stress granules interaction of tia-1/tiar with west nile and dengue virus products in infected cells interferes with stress granule formation and processing body assembly dhx36 enhances rig-i signaling by facilitating pkr-mediated antiviral stress granule formation mda5 localizes to stress granules, but this localization is not required for the induction of type i interferon viral infection switches non-plasmacytoid dendritic cells into high interferon producers pkr's protective role in viral myocarditis west nile virus-induced interferon production is mediated by the double-stranded rnadependent protein kinase pkr protein kinase r contributes to immunity against specific viruses by regulating interferon mrna integrity the rna-activated protein kinase enhances the induction of interferon-beta and apoptosis mediated by cytoplasmic rna sensors selective contribution of ifn-alpha/beta signaling to the maturation of dendritic cells induced by doublestranded rna or viral infection irf3 and irf7 phosphorylation in virusinfected cells does not require double-stranded rna-dependent protein kinase r or ikappa b kinase but is blocked by vaccinia virus e3l protein replication-dependent potent ifn-alpha induction in human plasmacytoid dendritic cells by a single-stranded rna virus c and v proteins of sendai virus target signaling pathways leading to irf-3 activation for the negative regulation of interferon-beta production stress granules and processing bodies are dynamically linked sites of mrnp remodeling the dependence of viral rna replication on co-opted host factors virus-host interactomes and global models of virus-infected cells new insights into the role of rnase l in innate immunity small self-rna generated by rnase l amplifies antiviral innate immunity a distinct role of riplet-mediated k63-linked polyubiquitination of the rig-i repressor domain in human antiviral innate immune responses pivotal role of rna-binding e3 ubiquitin ligase mex3c in rig-i-mediated antiviral innate immunity stable formation of compositionally unique stress granules in virus-infected cells cytoplasmic heat shock granules are formed from precursor particles and are associated with a specific set of mrnas the dynamic state of heat shock proteins in chicken embryo fibroblasts stressful initiations p bodies and the control of mrna translation and degradation p bodies: at the crossroads of posttranscriptional pathways mammalian stress granules and processing bodies rna-binding proteins tia-1 and tiar link the phosphorylation of eif-2 alpha to the assembly of mammalian stress granules the rasgap-associated endoribonuclease g3bp assembles stress granules stress granule assembly is mediated by prionlike aggregation of tia-1 tudor-sn and adar1 are components of cytoplasmic stress granules distinct structural features of caprin-1 mediate its interaction with g3bp-1 and its induction of phosphorylation of eukaryotic translation initiation factor 2alpha, entry to cytoplasmic stress granules, and selective interaction with a subset of mrnas mammalian stress granules represent sites of accumulation of stalled translation initiation complexes the deacetylase hdac6 is a novel critical component of stress granules involved in the stress response hur binding to cytoplasmic mrna is perturbed by heat shock ogfod1, a novel modulator of eukaryotic translation initiation factor 2alpha phosphorylation and the cellular response to stress ribonomic analysis of human pum1 reveals cis-trans conservation across species despite evolution of diverse mrna target sets dendritic localization of the translational repressor pumilio 2 and its contribution to dendritic stress granules recruitment of the rna helicase rhau to stress granules via a unique rna-binding domain rpp20 interacts with smn and is redistributed into smn granules in response to stress survival motor neuron protein facilitates assembly of stress granules staufen recruitment into stress granules does not affect early mrna transport in oligodendrocytes zbp1 subcellular localization and association with stress granules is controlled by its z-dna binding domains a role for eif4e and eif4e-transporter in targeting mrnps to mammalian processing bodies cytoplasmic foci are sites of mrna decay in human cells the human lsm1-7 proteins colocalize with the mrna-degrading enzymes dcp1/2 and xrnl in distinct cytoplasmic foci ge-1 is a central component of the mammalian cytoplasmic mrna processing body multiple processing body factors and the are binding protein ttp activate mrna decapping a phosphorylated cytoplasmic autoantigen, gw182, associates with a unique population of human mrnas within novel cytoplasmic speckles the gw182 protein colocalizes with mrna degradation associated proteins hdcp1 and hlsm4 in cytoplasmic gw bodies decapping and decay of messenger rna occur in cytoplasmic processing bodies human retroviral host restriction factors apobec3g and apobec3f localize to mrna processing bodies antiviral protein apobec3g localizes to ribonucleoprotein complexes found in p bodies and stress granules quantitative analysis of argonaute protein reveals microrna-dependent localization to stress granules argonaute 2/risc resides in sites of mammalian mrna decay known as cytoplasmic bodies the translational regulator cpeb1 provides a link between dcp1 bodies and stress granules the dead-box rna helicase ddx3 associates with export messenger ribonucleoproteins as well as tip-associated protein and participates in translational control determination of the role of ddx3 a factor involved in mammalian rnai pathway using an shrna-expression library cellular microrna and p bodies modulate host-hiv-1 interactions rna-associated protein 55 (rap55) localizes to mrna processing bodies and stress granules the dsrna protein kinase pkr: virus and cell control pkr; a sentinel kinase for cellular stress identification and characterization of pancreatic eukaryotic initiation factor 2 alpha-subunit kinase, pek, involved in translational control protein translation and folding are coupled by an endoplasmic-reticulum-resident kinase regulation of protein synthesis by hypoxia via activation of the endoplasmic reticulum kinase perk and phosphorylation of the translation initiation factor eif2alpha uncharged trna activates gcn2 by displacing the protein kinase moiety from a bipartite trna-binding domain heme-regulated inhibitor kinase-mediated phosphorylation of eukaryotic translation initiation factor 2 inhibits translation, induces stress granule formation, and mediates survival upon arsenite exposure regulation of protein synthesis by heme-regulated eif-2 alpha kinase analysis of subcellular g3bp redistribution during rubella virus infection cell proteins tia-1 and tiar interact with the 3 0 stem-loop of the west nile virus complementary minus-strand rna and facilitate virus replication modulation of hepatitis c virus rna abundance and virus release by dispersion of processing bodies and enrichment of stress granules hepatitis c virus hijacks p-body and stress granule components around lipid droplets the polypyrimidine tract-binding protein affects coronavirus rna accumulation levels and relocalizes viral rnas to novel cytoplasmic domains different from replication-transcription sites mouse hepatitis coronavirus replication induces host translational shutoff and mrna decay, with concomitant formation of stress granules and processing bodies induction of stress granule-like structures in vesicular stomatitis virus-infected cells respiratory syncytial virus induces host rna stress granules to facilitate viral replication activation of protein kinase r is required for induction of stress granules by respiratory syncytial virus but dispensable for viral replication roles of the respiratory syncytial virus trailer region: effects of mutations on genome production and stress granule formation arenavirus infection induces discrete cytosolic structures for rna replication mammalian orthoreovirus escape from host translational shutoff correlates with stress granule disruption and is independent of eif2alpha phosphorylation and pkr reovirus induces and benefits from an integrated cellular stress response formation of antiviral cytoplasmic granules during orthopoxvirus infection colocalization of transcription and translation within cytoplasmic poxvirus factories coordinates viral expression and subjugates host functions modulation of stress granules and p bodies during dicistrovirus infection we were supported by the following grants: the ministry of education, culture, sports key: cord-312075-asbt0mcj authors: schulz, katharina s.; mossman, karen l. title: viral evasion strategies in type i ifn signaling – a summary of recent developments date: 2016-11-11 journal: front immunol doi: 10.3389/fimmu.2016.00498 sha: doc_id: 312075 cord_uid: asbt0mcj the immune system protects the organism against infections and the damage associated with them. the first line of defense against pathogens is the innate immune response. in the case of a viral infection, it induces the interferon (ifn) signaling cascade and eventually the expression of type i ifn, which then causes an antiviral state in the cells. however, many viruses have developed strategies to counteract this mechanism and prevent the production of ifn. in order to modulate or inhibit the ifn signaling cascade in their favor, viruses have found ways to interfere at every single step of the cascade, for example, by inducing protein degradation or cleavage, or by mediate protein polyubiquitination. in this article, we will review examples of viruses that modulate the ifn response and describe the mechanisms they use. the mammalian immune system evolved to detect and fight viral infections effectively. the induction of type i interferon (ifn), predominantly ifn-α and ifn-β, forms the first line of defense. the type i ifn response consists of two parts. first, the cell produces type i ifn, when triggered by a viral stimulus. the ifn is then secreted and, in the second part of the response, it is sensed by the producing, as well as neighboring cells, resulting in the production of ifn-stimulated genes (isgs) [reviewed in ref. (1) ]. viruses, which have coevolved with their host, develop strategies to counteract the signaling cascades of the innate immune system and ensure their replication. recently, several reviews were published, describing the innate immune evasion strategies of individual viruses or virus families, such as influenza virus (2, 3) , phleboviruses (4), herpes viruses (5-7), coronaviruses severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) (8) , human immunodeficiency virus (hiv) (9, 10) , as well as multiple rna viruses (11, 12) . moreover, there are recent articles that review how viruses prevent detection by pathogen recognition receptors (prrs) (13, 14) and how viruses modulate innate immune signaling by use of viral deubiquitinases (15) . in this review, we will compare the different strategies viruses have developed to suppress innate immune signaling of individual components of the innate immune signaling cascade. due to the tremendous amount of data in this field, we will focus on recent discoveries. older studies were summarized in ref. (16, 17) . (14) ]. the most important viral markers for the innate immune system are viral nucleic acids. the detection of viral dna through the cgas-sting pathway and the counter measurements taken by viruses have been reviewed recently (18) and are not part of this review. viral rnas, which are mostly double-stranded (ds-)rna, are recognized by three prrs: the endosomal toll-like receptor 3 (tlr3), the cytoplasmic retinoic acid-inducible gene i (rig-i)like receptors (rlrs), and the nucleotide-oligomerization domain (nod)-like receptors (nlrs) (19) . tlr3 and the rlrs are important for inducing the type i ifn response, whereas nlrs have been shown to regulate interleukin-1β (il-1β) maturation through activation of caspase-1 (20) . the group of rlrs consists of rig-i, melanoma differentiation-associated gene 5 (mda5), and laboratory of genetics and physiology 2 (lgp2). the three receptors have a similar structure, all containing a caboxyterminal domain, which functions as a repressor domain (rd) in rig-i and lgp2 (21) and a central helicase domain, but lgp2 lacks the caspase activation and recruitment domains (cards) that function in signaling [reviewed in ref. (19, 22) ]. both the helicase and the carboxy-terminal domain are required for rna binding. rig-1 and mda-5 detect specific viral rna pamps, while lgp2 negatively regulates rig-i signaling and promotes rna binding to mda5 [reviewed in detail in ref. (14) ]. in unstimulated cells, rig-i and mda-5 are kept in a repressed state due to phosphorylations on serine and threonine residues in the cards and carboxy-terminal domains (23, 24) . upon binding of rna, both rig-i and mda-5 undergo conformational changes, resulting in release of their cards (25, 26) . recruited phosphatases remove the phosphate residues, and e3 ubiquitin ligases attach lys63-linked ubiquitin polymers onto the cards and c-terminal domain of rig-i, which are important for rig-i tetramerization (27) (28) (29) (30) (31) . rna-bound rig-1 then interacts with 14-3-3ε, a mitochondrial trafficking protein, and the trim25 ubiquitin ligase, which together transport rig-i to the mitochondria (32) . there the cards of rig-i or mda-5 interact with the card of the mitochondrial activator of virus signaling (mavs, also known as ips-1, visa, and cardif), which is an essential signaling adaptor protein. the activation of mavs has recently been reviewed in detail in ref. (33) . tlr3 interacts with trif, which serves as a molecular platform and forms physical interactions with several adaptor molecules (34) . by interacting with upstream adaptors, trif undergoes conformational changes and recruits the downstream tnf receptor-associated factor (traf)3 and traf6 [reviewed in ref. (35) ]. the kinase receptor-interacting protein-1 (rip-1) is part of both the signaling pathways downstream of tlr3 and rig-i. it can interact with trif to induce nfκb activation (36) . moreover, the dsrna-activated tlr3 can recruit trif, rip-1, and caspase-8 and induce apoptosis (37) . also, rip-1 and its adaptor protein fas-associated protein with death domain (fadd) are part of the signaling cascade downstream of rig-i and mda-5 and involved in the activation of the transcription factors interferon regulatory factor (irf)3 and irf7 (38) . traf3 serves as a linker between the upstream adaptor proteins (trif or myd88 for tlrs and mavs for rlrs) and the downstream signaling kinases tbk1/ikkε or irak1/ikkα. the recruitment of traf3 to the tlr or rlr signaling complexes activates the e3 ligase activity of traf3, which then catalyzes its own k63-linked ubiquitinylation. subsequent traf3 activates tbk1/ikkε or irak/ikkα [reviewed in ref. (39) ] (figure 1) . viruses target rig-i directly or indirectly to block the type i ifn response. the phlebovirus toscana virus expresses a non-structural protein, which directly interacts with rig-i and induces its proteasomal degradation (40, 41) . foot-and-mouth disease virus (fmdv) proteins l pro , 3c pro , and 2b increase the rig-i mrna expression but decrease the protein expression of rig-i. l pro and 3c pro both induce rig-i degradation, whereas the mechanism of how 2b reduces rig-i protein levels has not been solved yet (42) . other viruses target rig-i indirectly. hepatitis b virus (hbv) induces mir146a, which then posttranscriptionally inhibits the expression of rig-i and suppresses the production of type i ifn (43) . the dengue virus ns3 protein binds to 14-3-3ε and prevents the translocation of rig-i to mavs. the binding site on ns3 is a highly conserved phosphomimetic motif, which was verified by generation of a virus containing a mutation in this motif (44) . it has been proposed that in certain cell types rig-i requires sentinels, such as the protein ddx60, which associates with rig-i and promotes the rig-i rna-binding activity (45, 46) . other studies question ddx60 acting as a broadly active enhancer of antiviral responses (47, 48) and instead suggest that ddx60 only functions in the antiviral response to specific viruses, such as hepatitis c virus (47) . however, there are data indicating that influenza a virus and hepatitis c virus attenuate ifnβ-promoter activation by targeting the sentinel ddx60. both viruses activate the epidermal growth factor (egf) receptor, which in turn phosphorylates ddx60 on tyr-793 and tyr-796. this results in the attenuation of ddx60-dependent rig-i activation. in addition, independent of its role as sentinel for rig-i viral rna recognition, ddx60 plays a role in viral rna degradation (46) (figure 1 ). mitochondrial activator of virus signaling is blocked by different viruses in various ways. the dengue virus protein ns4a targets mavs, and the interaction prevents the binding of mavs to rig-i (49) . the porcine reproductive and respiratory syndrome virus (prrsv) 3c-like protease (3clsp), by contrast, cleaves mavs in a proteasome-and caspase-independent manner at glu268 (e268/g269). both cleavage products fail to activate the type i ifn response (50) . likewise, the hepatitis c virus protein ns3-4a (51, 52) , as well as the highly pathogenic porcine reproductive and respiratory syndrome virus (hp-prrsv) protein nsp4 (53) have been shown to cleave mavs and block rlr signaling. the porcine epidemic diarrhea virus (pedv) also targets mavs in small intestinal epithelial cells (iecs). however, the exact mechanism has not been solved yet (54) (figure 1) . the sars coronavirus protein orf9b not only influences antiviral signaling but also alters host cell mitochondria morphology by inducing degradation of the dynamin-like protein (drp1). mavs becomes concentrated into small puncta in the presence (56) . in addition to mavs, also the levels of traf3 and traf6 are reduced by orf9b. however, it is unlikely that traf3 and traf6 are targeted directly. more likely, they are degraded due to their interaction with mavs (55) (figure 1) . human t-cell lymphotropic virus type i (htlv-1) protein tax disrupts innate immune signaling in multiple ways: it binds to the rip homotypic interaction motif (rhim) domains of rip-1 and disrupts the interaction between rip-1 and rig-i or mda-5 and the activation of the type i ifn promoter. tax also binds to trif and thereby interrupts the tlr3 signaling cascade. finally, tax blocks the association between rip-1 and irf7, which resulted in repression of the irf7 activity (57) (figure 1) . middle east respiratory syndrome coronavirus m protein interacts with traf3 and disrupts the interaction between traf3 and tbk1, which ultimately leads to a reduced irf3 activation. for the interaction with traf3, the n-terminal transmembrane domain of the mers-cov m protein is sufficient (58) , similar to what has been shown for sars-cov before (59) (figure 1 ). triggering of the tlr3-and rlr-signaling cascade results in the activation of the transcription factors nfκb and irf3/irf7. in its inactive state, the transcription factor nfκb is complexed with its inhibitor iκb (60) . upon stimulation, iκb is phosphorylated by the iκb kinase (ikk) complex, which is composed of two catalytic subunits, such as ikkα and ikkβ, and a regulatory subunit, such as nfκb essential modulator (nemo) (61) . the phosphorylation of iκbα induces its polyubiquitination through the e3 ubiquitin ligase β-transducin repeat-containing protein (β-trcp) and subsequent proteasomal degradation (62) , allowing nfκb to translocate into the nucleus and induce the expression of target genes (63) (figure 2) . encephalomyocarditis virus (emcv) protein 3c cleaves traf family member-associated nfκb activator (tank), which inhibits traf6-mediated nfκb activation, on gln291. as a result, nfκb is activated and the unstable c-terminal fragment of tank is subjected to proteasomal degradation (64) . also, other viruses express proteases that cleave tank, although on other residues, such as porcine reproductive and respiratory syndrome virus (prrsv) (tank is cleaved by nsp4), fmdv (protease 3c cleaves tank), and equine arteritis virus (eav) (tank is cleaved by nsp4). thus, tank seems to be a common target of several positive rna viral proteases (64) (figure 2) . several viruses have been shown to disrupt ifn signaling by cleaving nemo. pedv 3c-like protease, nsp5, cleaves nemo at gln231 (65), whereas the hepatitis a virus 3c protease (3c pro ) cleaves nemo at gln304 (66) and the picornavirus fmdv protease 3c pro at gln383, removing the c-terminal zinc finger domain from the protein (67) . the human rotavirus has developed another way. its non-structural protein 1 (nsp1) has been shown to inhibit the nfκb pathway by degrading β-trcp and consequently stabilizing iκb (68) (figure 2) . tank-binding kinase 1 (tbk1) and inhibitor of κb kinase ε (ikkε) are classified as non-canonical serine/threonine kinases and are both able to induce irf3 and irf7 phosphorylation and subsequent dimerization (69) (70) (71) (72) . however, while tbk1 is constitutively expressed in most cell types, the expression of ikkε is more restricted (73) . upon stimulation, tbk1 and ikkε are recruited by adaptor proteins to signaling complexes to be activated by phosphorylation on ser172 and both have been shown to be subjected to k63-linked polyubiquitination [reviewed in ref. (73, 74) ]. for tbk1, k63-linked polyubiquitination seems to be important for tlr-and rlr-induced ifn production, as ubiquitin chains might serve as a platform for the assembly of tbk1 signaling complexes. moreover, deubiquitinases are able to terminate the tbk1-mediated pathway by cleaving the k63linked ubiquitin chains [reviewed in ref. (74, 75) ]. activated tbk1/ikkε phosphorylates irf3 and/or irf7 in the cytosol at specific serine residues. this phosphorylation results in homo-or heterodimerization of irf3 and irf7 and nuclear translocation (76, 77) . interestingly, while irf3 is constitutively expressed, irf7 is expressed at low levels in most cell types and expression is induced upon ifn signaling. therefore, in most cells, irf7 strongly enhances the production of ifn [reviewed in ref. (78) ]. once phosphorylated irf3 and/or irf7 dimers have translocated into the nucleus, they bind to the transcription coactivator creb-binding protein (cpb)/p300 (79, 80) . together with other factors, such as nfκb, they form the enhanceosome on the ifnβ promoter and induce the expression of type i ifn [reviewed in ref. (76) ]. the viral proteins that target tbk1 act by either blocking activation of tbk1 by mavs or by inhibiting activation of irf3 by tbk1. the mers-cov protein orf4b blocks ifnβ production by binding to tbk1 and ikkε and suppressing the formation of a mavs/ikkε complex (81) . in addition to inhibiting tbk1/ikkε activation, orf4b can also inhibit the production of ifnβ in the nucleus; however, the mechanism has not been solved yet (81) . recently, two herpes simplex virus proteins have been shown to target tbk1/ikkε and inhibit the phosphorylation of irf3: icp27 (82) and vp24 (83) . also, dengue virus serotype 4 non-structural proteins ns2a and ns4b, as well as the ns2a and ns4b proteins of other dengue viruses, inhibit the phosphorylation of tbk1 (84) and pedv n protein has been shown to interact with tbk1, hampering the association of tbk1 with irf3 and preventing the activation of irf3 activation (85) . the human t-cell leukemia virus type 1 oncoprotein tax has been shown to also interact with tbk1. however, studies came to contradicting results on how that influences the production of ifnβ. while one group showed that tax activates tbk1 and the production of ifnβ (86), another group showed that tax suppresses the ifn production by interaction with tbk1 (87) . interestingly, when a recent study tested how the rabies virus p protein of street strains behaves compared to laboratory-adapted strains with regard to the induction of type i ifn, they found that both street strains and laboratory strains inhibit tbk1-mediated signaling, but only the p protein of street strains also interacts with and inhibits ikkε-inducible irf3dependent ifnβ expression (88) (figure 1) . interferon regulatory factor 3 is targeted by many viruses to impair innate immune signaling. most viruses inhibit the phosphorylation and thereby also the dimerization and translocation of irf3, such as the porcine deltacoronavirus (89) or poliovirus (90) . hepatitis e virus protein orf3 also suppresses irf3 phosphorylation, but in an indirect way. it activates the signal regulator protein α (sirp-α), which negatively regulates type i ifn induction (91) . in contrast, porcine bocavirus (pbov) np1 protein does not affect irf3 expression, phosphorylation, or nuclear translocation. instead, it interacts with the dna-binding domain of irf3 and inhibits the dna-binding activity (92) . a very interesting way of how to circumvent the host innate immune response was found when studying gammaherpesviruses kaposi's sarcoma-associated herpesvirus (kshv) and rhesus macaque rhadinovirus (rrv). they express several viral homologs to the irfs, called viral irfs (virfs). these virfs have found multiple ways to suppress type i ifn production. for kshv, different strategies have been reviewed in ref. (6) . recently, the rrv virf r6 has been shown to interact with the transcriptional coactivator cbp in the nucleus, similar to the kshv virf1. as a result, cbp cannot form a complex with the phosphorylated irf3, and the ifn expression is not induced (93) (94) (95) . interestingly, rrv r6 is the first virf for which an association with the viron could be shown. therefore, virf v6 can shut down the type i ifn response shortly after the cell was infected, rendering the cell more susceptible to infection (95) . the pedv protein nsp1 also targets cbp. nsp1 induces cbp degradation in a proteasome-dependent manner and thereby interrupts enhanceosome assembly and the production of type i ifn (96) (figure 1) . for most of these interactions, the molecular mechanisms have not been unraveled yet. a protein that has been shown to interact with and induce proteasomal degradation of irf3 some time ago is classical swine fever virus (csfv) npro (97, 98) . recently, the molecular mechanism has been published. irf3 and npro interact direct and form a soluble 1:1 complex. moreover, it was shown that npro interacts with the full-length irf3, not with individual domains, and that npro binds the constitutively active form of irf3 in the presence of cpb. thus, npro interacts with both the monomer and the active irf3 dimer and likely targets both species for ubiquitinylation and proteasomal degradation (99) . interferon regulatory factor 7 is targeted by two human enteroviruses, such as enterovirus 71 and enterovirus 68. they downregulate irf7 by cleaving it with their protease 3c, leaving the cleavage products unable to induce ifn expression. while enterovirus 71 cleaves irf7 once at gln189-ser190 (100), enterovirus 68 cleaves it twice, the cleavage sites being gln167 and gln189 (101) . moreover, megalocytivirus, a dna virus that infects marine and freshwater fish, induces the expression of the host microrna pol-mir-731, which then specifically suppresses the expression of irf7 (102) (figure 1) . the type i ifns act in an autocrine, paracrine, or systemic manner to stimulate antiviral responses. they are recognized by the ifnα/β receptor (ifnar), which consists of the subunits ifnar1 and ifnar2 expressed on virtually all cell types (103) . the interaction of type i ifn with the receptor results in the phosphorylation and activation of the ifnar1-and ifnar2-associated tyrosine kinases tyrosine kinase 2 (tyk2) and janus kinase 1 (jak1), which then phosphorylate ifnar tyrosine residues, resulting in the recruitment and activation of signaling molecules, such as the signal transducer and activator of transcription (stat) family of transcription factors (104, 105) . upon activation, stat1 and stat2, together with irf9, form the ifn-stimulated gene factor 3 (isgf3), which then translocates into the nucleus to induce transcription of isgs [reviewed in detail in ref. (106) (107) (108) ]. several viruses target ifnar to prohibit ifn binding and signaling. influenza virus induces the degradation of ifnar1. hemagglutinin (ha) triggers the phosphorylation and ubiquitinylation of ifnar1, thus promoting protein degradation (109) . encephalitic flaviviruses, such as tick-borne encephalitis virus or west nile virus, inhibit ifnar1 surface expression. their protein ns5 binds the cellular dipeptidase prolidase (pepd), which is involved in ifnar1 maturation and accumulation, activation of ifnβ-stimulated gene induction, and ifn-dependent viral control. this interaction inhibits ifnar1 intracellular trafficking and glycosylation but does not promote ifnar1 degradation (110) (figure 3) . both stat1 and stat2 are targeted by many viruses to suppress isg induction. pedv induces stat1 ubiquitinylation and targets it for degradation in the proteasomes (111) . (112) . similarly, human metapneumovirus (hmpv) protein sh impairs stat1 expression, phosphorylation, and activation (113) . simian varicella virus not only inhibits stat2 phosphorylation but also promotes degradation of irf9 in a proteasome-dependent manner through its protein orf63 (114) . also, infectious bronchitis virus (ibv) inhibits phosphorylation and nuclear translocation of stat1. however, despite detailed analyses, it is unclear which viral protein is responsible. it was, however, shown that the accessory protein 3a contributes to ibv resistance to type i ifn, although the target is unknown as well (115) . in case of the human parvovirus b19, it becomes evidently clear that both the virus and the immune system constantly evolve to prevail. while its protein ns1 suppresses stat phosphorylation, the immune system senses the protein and triggers the production of type i ifn (116) . sftsv, an emerging tick-borne pathogen, developed multiple ways to prevent isg induction. the viral non-structural protein ns impairs stat1 expression, phosphorylation, and activation (117) and interacts with stat2 and sequesters stat1 and stat2 into viral inclusion bodies, where they are trapped (118) (figure 3) . the jak-stat signal transduction pathway is negatively regulated by the suppressor of cytokine signaling (socs) family of proteins in form of a classical feedback loop (119, 120) . some viruses induce the expression of socs to take advantage of this mechanism to minimize the induction of isgs. japanese encephalitic virus (jev) downregulates the expression of micro-rna mir-432, which then results in upregulated socs5 levels (121) . varicella-zoster virus (vzv) infection induces the expression of socs3 (122) and respiratory syncytial virus (rsv) nonstructural proteins ns1 and ns2 induce upregulation of socs1 and socs3, which also inhibited the induction of chemokines (123) (figure 3 ). viruses fully depend on the translation machinery of the host cell for replication. accordingly, they have evolved multiple ways to hamper host protein synthesis [reviewed in ref. (124) ]. one way is to shut off host protein synthesis. for some time, it was thought that gamma-and deltacoronaviruses do not induce host shutoff, such as alpha-and betacoronaviruses do. however, a recent study showed that the infectious bronchitis gammacoronavirus induces host shutoff using its protein 5b. it seems like 5b is a functional equivalent of nsp1, the host shutoff protein of alpha-and betacoronaviruses (125) . viruses evolved to have various strategies to circumvent the innate immune response by blocking the production of type i ifn or the expression of isgs. while these diverse strategies may appear contradictory between viruses, several factors require consideration. for example, the use of clinical isolates versus viral evasion of interferon pathways frontiers in immunology | www.frontiersin.org november 2016 | volume 7 | article 498 laboratory-passaged strains could yield different results, particularly with rna viruses that rapidly accumulate mutations due to error-prone rna-dependent rna polymerases. moreover, the choice of cell line can greatly influence experimental outcomes, as many immortalized or transformed continual cell lines harbor mutations in critical innate immune signaling (126) . likewise, the use of genetic knockout versus knockdown cell lines or organisms can influence experimental outcomes, as can the experimental procedures themselves, particularly when endogenous interactions are disrupted with the use of overexpression approaches. studying the mechanisms used by viruses to prevent an immune response is of great importance for the development of new strategies to limit the sequelae of viral infections. identification of key immune evasion proteins allows development of antivirals to target these proteins. alternatively, identification of key cellular antiviral pathways allows development of strategies to enhance these pathways to overwhelm incoming viruses. information on key immune evasion factors further facilitates the engineering of safe and effective vaccine strains and designing strategies to target new emerging viruses from the same or closely related family. ks and km conceptualized the scope of the review article. ks wrote the review with input from km. this work was supported by a postdoctoral fellowship from the deutsche forschungsgemeinschaft (schu3011/1-1). work in the mossman laboratory on innate antiviral signaling is supported by the canadian institutes for health research. induction and function of ifnbeta during viral and bacterial infection functions of the influenza a virus ns1 protein in antiviral defense to conquer the host, influenza virus is packing it in: interferon-antagonistic strategies beyond ns1 phleboviruses and the type i interferon response the tiers and dimensions of evasion of the type i interferon response by human cytomegalovirus herpesviruses: interfering innate immunity by targeting viral sensing and interferon pathways evasion of host antiviral innate immunity by hsv-1, an update middle east respiratory syndrome and severe acute respiratory syndrome innate antiviral immune signaling, viral evasion and modulation by hiv-1 hiv replication: a game of hide and sense strategies of highly pathogenic rna viruses to block dsrna detection by rig-i-like receptors: hide, mask, hit molecular mechanisms of innate immune inhibition by non-segmented negative-sense rna viruses cytosolic sensing of viruses viral evasion of intracellular dna and rna sensing regulation of cellular innate antiviral signaling by ubiquitin modification recent advances in understanding viral evasion of type i interferon devasthanam as. mechanisms underlying the inhibition of interferon signaling by viruses the cgas-sting defense pathway and its counteraction by viruses rig-i in rna virus recognition intracellular nod-like receptors in host defense and disease regulation of innate antiviral defenses through a shared repressor domain in rig-i and lgp2 an evolving arsenal: viral rna detection by rig-i-like receptors phosphorylationmediated negative regulation of rig-i antiviral activity phosphorylation of rig-i by casein kinase ii inhibits its antiviral response structural basis for the activation of innate immune pattern-recognition receptor rig-i by viral rna a structure-based model of rig-i activation trim25 ringfinger e3 ubiquitin ligase is essential for rig-i-mediated antiviral activity reconstitution of the rig-i pathway reveals a signaling role of unanchored polyubiquitin chains in innate immunity conventional protein kinase c-alpha (pkc-alpha) and pkc-beta negatively regulate rig-i antiviral signal transduction a distinct role of riplet-mediated k63-linked polyubiquitination of the rig-i repressor domain in human antiviral innate immune responses dephosphorylation of the rna sensors rig-i and mda5 by the phosphatase pp1 is essential for innate immune signaling the mitochondrial targeting chaperone 14-3-3epsilon regulates a rig-i translocon that mediates membrane association and innate antiviral immunity how rig-i like receptors activate mavs antiviral responses induced by the tlr3 pathway a unique host defense pathway: trif mediates both antiviral and antibacterial immune responses rip1 is an essential mediator of toll-like receptor 3-induced nf-kappa b activation dsrna induces apoptosis through an atypical death complex associating tlr3 to caspase-8 a fadd-dependent innate immune mechanism in mammalian cells traf3: a novel tumor suppressor gene in macrophages. macrophage (houst) (2015) 2:e1009 toscana virus nss protein inhibits the induction of type i interferon by interacting with rig-i truncation of the c-terminal region of toscana virus nss protein is critical for interferon-beta antagonism and protein stability foot-and-mouth disease virus viroporin 2b antagonizes rig-i mediated antiviral effects by inhibition of its protein expression hepatitis b virus inhibits intrinsic rig-i and rig-g immune signaling via inducing mir146a a phosphomimetic-based mechanism of dengue virus to antagonize innate immunity ddx60, a dexd/h box helicase, is a novel antiviral factor promoting rig-i-like receptor-mediated signaling ddx60 is involved in rig-i-dependent and independent antiviral responses, and its function is attenuated by virus-induced egfr activation a diverse range of gene products are effectors of the type i interferon antiviral response mouse superkiller-2-like helicase ddx60 is dispensable for type i ifn induction and immunity to multiple viruses dengue virus subverts host innate immunity by targeting adaptor protein mavs porcine reproductive and respiratory syndrome virus 3c protease cleaves the mitochondrial antiviral signalling complex to antagonize ifn-beta expression hepatitis c virus protease ns3/4a cleaves mitochondrial antiviral signaling protein off the mitochondria to evade innate immunity hepatitis c virus ns3-4a inhibits the peroxisomal mavs-dependent antiviral signalling response highly pathogenic porcine reproductive and respiratory syndrome virus nsp4 cleaves visa to impair antiviral responses mediated by rig-i-like receptors porcine epidemic diarrhea virus inhibits dsrna-induced interferon-beta production in porcine intestinal epithelial cells by blockade of the rig-i-mediated pathway sars-coronavirus open reading frame-9b suppresses innate immunity by targeting mitochondria and the mavs/traf3/traf6 signalosome pcbp2 mediates degradation of the adaptor mavs via the hect ubiquitin ligase aip4 oncogenic human t-cell lymphotropic virus type 1 tax suppression of primary innate immune signaling pathways middle east respiratory syndrome coronavirus m protein suppresses type i interferon expression through the inhibition of tbk1-dependent phosphorylation of irf3 suppression of innate antiviral response by severe acute respiratory syndrome coronavirus m protein is mediated through the first transmembrane domain a firm hand on nfkappab: structures of the ikappabalpha-nfkappab complex ikappab kinases: key regulators of the nf-kappab pathway inducible degradation of ikappabalpha by the proteasome requires interaction with the f-box protein h-betatrcp shaping the nuclear action of nf-kappab encephalomyocarditis virus 3c protease relieves traf family member-associated nf-kappab activator (tank) inhibitory effect on traf6-mediated nf-kappab signaling through cleavage of tank porcine epidemic diarrhea virus 3c-like protease regulates its interferon antagonism by cleaving nemo hepatitis a virus 3c protease cleaves nemo to impair induction of beta interferon foot-and-mouth disease virus 3c protease cleaves nemo to impair innate immune signaling nsp1 of human rotaviruses commonly inhibits nf-kappab signalling by inducing beta-trcp degradation ikkepsilon and tbk1 are essential components of the irf3 signaling pathway triggering the interferon antiviral response through an ikk-related pathway regulation and function of ikk and ikk-related kinases involvement of the ubiquitin-like domain of tbk1/ikk-i kinases in regulation of ifn-inducible genes ikappab kinase epsilon (ikkepsilon): a therapeutic target in inflammation and cancer regulation of tbk1 activity by optineurin contributes to cell cycle-dependent expression of the interferon pathway negative regulation of tbk1-mediated antiviral immunity irfs: master regulators of signalling by toll-like receptors and cytosolic pattern-recognition receptors the irf family transcription factors in immunity and oncogenesis the irf family transcription factors at the interface of innate and adaptive immune responses direct triggering of the type i interferon system by virus infection: activation of a transcription factor complex containing irf-3 and cbp/p300 mechanism for transcriptional synergy between interferon regulatory factor (irf)-3 and irf-7 in activation of the interferon-β gene promoter middle east respiratory syndrome coronavirus orf4b protein inhibits type i interferon production through both cytoplasmic and nuclear targets hsv-1 icp27 targets the tbk1-activated sting signalsome to inhibit virus-induced type i ifn expression herpes simplex virus 1 serine protease vp24 blocks the dna-sensing signal pathway by abrogating activation of interferon regulatory factor 3 dengue virus ns proteins inhibit rig-i/mavs signaling by blocking tbk1/irf3 phosphorylation: dengue virus serotype 1 ns4a is a unique interferon-regulating virulence determinant porcine epidemic diarrhea virus nucleocapsid protein antagonizes beta interferon production by sequestering the interaction between irf3 and tbk1 htlv-1 tax protein recruitment into ikkepsilon and tbk1 kinase complexes enhances ifn-i expression suppression of type i interferon production by human t-cell leukemia virus type 1 oncoprotein tax through inhibition of irf3 phosphorylation contribution of the interaction between the rabies virus p protein and i-kappa b kinase to the inhibition of type i ifn induction signalling porcine deltacoronavirus (pdcov) infection suppresses rig-i-mediated interferon-beta production proteolysis of mda5 and ips-1 is not required for inhibition of the type i ifn response by poliovirus hepatitis e virus infection activates signal regulator protein alpha to down-regulate type i interferon porcine bocavirus np1 protein suppresses type i ifn production by interfering with irf3 dnabinding activity functional analysis of human herpesvirus 8-encoded viral interferon regulatory factor 1 and its association with cellular interferon regulatory factors and p300 viral interferon regulatory factor 1 of kaposi's sarcoma-associated herpesvirus (human herpesvirus 8) binds to, and inhibits transactivation of, creb-binding protein a rhesus rhadinovirus viral interferon (ifn) regulatory factor is virion associated and inhibits the early ifn antiviral response suppression of type i interferon production by porcine epidemic diarrhea virus and degradation of creb-binding protein by nsp1 classical swine fever virus npro interacts with interferon regulatory factor 3 and induces its proteasomal degradation the npro product of classical swine fever virus and bovine viral diarrhea virus uses a conserved mechanism to target interferon regulatory factor-3 pestivirus npro directly interacts with interferon regulatory factor 3 (irf3) monomer and dimer cleavage of interferon regulatory factor 7 by enterovirus 71 3c suppresses cellular responses 3c protease of enterovirus d68 inhibits cellular defense mediated by interferon regulatory factor 7 pol-mir-731, a teleost mirna upregulated by megalocytivirus, negatively regulates virus-induced type i interferon response, apoptosis, and cell cycle arrest the interferons and their receptors -distribution and regulation jak-stat signaling: from interferons to cytokines stats get their move on transcriptional regulation by stat1 and stat2 in the interferon jak-stat pathway stat2 and irf9: beyond isgf3. jakstat (2013) dynamic control of type i ifn signalling by an integrated network of negative regulators hemagglutinin of influenza a virus antagonizes type i interferon (ifn) responses by inducing degradation of type i ifn receptor 1 flavivirus antagonism of type i interferon signaling reveals prolidase as a regulator of ifnar1 surface expression porcine epidemic diarrhea virus infection inhibits interferon signaling by targeted degradation of stat1 la piedad michoacan mexico virus v protein antagonizes type i interferon response by binding stat2 protein and preventing stats nuclear translocation human metapneumovirus small hydrophobic (sh) protein downregulates type i ifn pathway signaling by affecting stat1 expression and phosphorylation varicella viruses inhibit interferon-stimulated jak-stat signaling through multiple mechanisms infectious bronchitis coronavirus inhibits stat1 signaling and requires accessory proteins for resistance to type i interferon activity nonstructural protein (ns1) of human parvovirus b19 stimulates host innate immunity and blunts the exogenous type i interferon signaling in vitro suppression of type i and type iii ifn signalling by nss protein of severe fever with thrombocytopenia syndrome virus through inhibition of stat1 phosphorylation and activation disruption of type i interferon signaling by the nonstructural protein of severe fever with thrombocytopenia syndrome virus via the hijacking of stat2 and stat1 into inclusion bodies the role of suppressors of cytokine signaling (socs) proteins in regulation of the immune response regulation of the immune system by socs family adaptor proteins japanese encephalitis virus exploits the microrna-432 to regulate the expression of suppressor of cytokine signaling (socs) 5 suppressor of cytokine signaling 3 expression induced by varicella-zoster virus infection results in the modulation of virus replication respiratory syncytial virus nonstructural proteins upregulate socs1 and socs3 in the different manner from endogenous ifn signaling viral subversion of the host protein synthesis machinery infectious bronchitis coronavirus limits interferon production by inducing a host shutoff that requires accessory protein 5b deregulation of interferon signaling in malignant cells the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-343824-00mqmpzw authors: qian, wei; wei, xiaoqin; guo, kelei; li, yongtao; lin, xian; zou, zhong; zhou, hongbo; jin, meilin title: the c-terminal effector domain of non-structural protein 1 of influenza a virus blocks ifn-β production by targeting tnf receptor-associated factor 3 date: 2017-07-03 journal: front immunol doi: 10.3389/fimmu.2017.00779 sha: doc_id: 343824 cord_uid: 00mqmpzw influenza a virus non-structural protein 1 (ns1) antagonizes interferon response through diverse strategies, particularly by inhibiting the activation of interferon regulatory factor 3 (irf3) and ifn-β transcription. however, the underlying mechanisms used by the ns1 c-terminal effector domain (ed) to inhibit the activation of ifn-β pathway are not well understood. in this study, we used influenza virus subtype of h5n1 to demonstrate that the ns1 c-terminal ed but not the n-terminal rna-binding domain, binds tnf receptor-associated factor 3 (traf3). this results in an attenuation of the type i ifn signaling pathway. we found that the ns1 c-terminal ed (named ns1/126-225) inhibits the active caspase activation and recruitment domain-containing form of rig-i [rig-i(n)]-induced ifn-β reporter activity, the phosphorylation of irf3, and the induction of ifn-β. further analysis showed that ns1/126-225 binds to traf3 through the traf domain, subsequently decreasing traf3 k63-linked ubiquitination. ns1/126-225 binding also disrupted the formation of the mitochondrial antiviral signaling (mavs)–traf3 complex, increasing the recruitment of ikkε to mavs; ultimately shutting down the rig-i(n)-mediated signal transduction and cellular antiviral responses. this attenuation of cellular antiviral responses leads to evasion of the innate immune response. taken together, our findings offer an important insight into the interplay between the influenza virus and host innate immunity. of caspase activation and recruitment domains (card). as the cascade continues, different residues in rig-i are ubiquitinated by the e3 ligases trim25 (5) and riplet (6) , resulting in rig-i oligomerization. subsequently, rig-i interacts with the adaptor protein mitochondrial antiviral signaling (mavs) via their card-card association, which in turn results in mavs oligomerization. through tnf receptor-associated factor 3 (traf3) or traf6, this leads to activation of the kinase complexes containing tbk1 or ikkε and ikkα/β/γ. through several final phosphorylation steps, these kinases ultimately elicit antiviral and pro-inflammatory responses through interferon regulatory factor 3 (irf3) and nuclear factor κb (nf-κb), respectively (7, 8) . influenza a virus belongs to the orthomyxovirus family, containing eight negative-sense rna segments in an enveloped viral particle encoding 14 or 17 proteins (9) . this array of proteins contributes to virulence; including the proteins associated with viral rna-dependent rna polymerase (10) and the nonstructural protein 1 (ns1). ns1 consists of 215-237 amino acids and comprises two functional domains: an n-terminal rnabinding domain (rbd) (aa1 to 73) and a c-terminal effector domain (ed) (aa74-end) (11) . the ns1 protein plays a crucial role in regulating the host antiviral response through various mechanisms. one important function of the ns1 protein involves inhibition of ifn production. the mechanism of this inhibition includes activation of the transcription factors irf3 (12) , nf-κb (13) , and ap-1 (14) , thus blocking ifn production. this efficient inhibitory effect is associated with an rig-i signaling pathway through the ns1-rig-i complex (15) (16) (17) . previous studies have indicated that ns1 is also related to two positive factors of rig-i, the e3 ligases trim25 (18) and riplet (19) . the residues e96/ e97 of ns1 mediate their interaction with the coiled-coil domain of trim25, thus blocking both trim25 multimerization and rig-i card domain ubiquitination. this subsequently induces lower levels of ifn-β (18) . ns1 can also interact with riplet preventing the activation of rig-i, although e96/e97 are not involved in that inhibition (19) . the dsrna binding ability of ns1 could also be playing a role in the pre-transcriptional inhibition of the interferon pathway by sequestering the pathogenassociated molecular patterns (pamps) that rig-i recognizes. two residues, r38 and k41, are required for the dsrna binding activity of ns1 (20) , thus highly impairing its ability to block interferon production. in another similar pathway, ns1 has been shown to inhibit host mrna synthesis by binding a cellular 3′ end-processing factor, the 30 kda subunit of the cleavage and polyadenylation specificity factor (cpsf30), thus attenuating type i interferon (ifn-α/β) and other interferon stimulated gene (isg) mrnas that are involved in the antiviral response (21) . the ns1 proteins encoded by the seasonal h1n1, h2n2, h3n2, and avian h5n1 viral subtypes strongly bind to cpsf30 (22) , whereas pr8, 2009 pandemic h1n1, and novel h7n9 virus do not efficiently bind cpsf30 (23) . it is noteworthy that cells infected with viruses expressing ns1 proteins in seasonal h3n2 and h2n2 viruses do not inhibit irf3 activation. however, activation is blocked in cells infected with viruses expressing ns1 proteins in some, but not all, seasonal h1n1 viruses, 2009 pandemic h1n1, and avian h5n1 viruses. trim25 was previously reported to interact with each of these ns1 proteins, whether or not they block irf3 activation, indicating that binding of trim25 by the ns1 protein does not necessarily lead to blocking of irf3 activation (22) . hence, binding of the ns1 protein to dsrna, rig-i, and trim25 has not established that these ns1 interactions are responsible for inhibiting the activation of irf3 and ifn transcription. in this case, one or more host factors may participate in the ns1 blocking of irf3 activation. in view of several yet undetermined roles of ns1 in the inhibition of interferon, we conducted a study aimed to determine the importance of the function of the n-and/or c-terminal domains of the ns1 protein in immune evasion. by using the luciferase reporter assay, we were able to demonstrate that the c-terminal ed (aa126 to 225, named ns1/126-225) of the ns1 protein was sufficient to inhibit the production of ifn-β driven by rig-i(n). mechanistically, ns1/126-225 was found specifically to interact with traf3, to dissociate mavs-traf3 complex, and to decrease k63-linked polyubiquitination of traf3. this was shown to result in reduced irf3-dependent production of ifn-β, with subsequent enhancement of virus replication. these data reveal a novel mechanism for how the influenza a virus ns1 protein induces inhibition of the host ifn production and may provide a potential target for antiviral drug development. the hpai h5n1 virus strain, a/duck/hubei/hangmei01/2006 (h5n1; designated h5n1/hm) was isolated from a duck. influenza a virus (strain a/puerto rico/8/1934 h1n1), a/ pr/8/34, was grown in our laboratories and stored until use. rns1-sd30 was constructed as previously described (24) . influenza virus stocks of h5n1/hm, pr8, and rns1-sd30 strains were amplified using 10-day-old embryonic chicken eggs and then titrated by determining log10 tcid50/ml values in mdck cells. all cell experiments with h5n1 virus were performed in an animal biosafety level 3 laboratory (bsl-3). this study was carried out in accordance with the recommendations of bsl-3, huazhong agricultural university (hzau). the protocol was approved by the bsl-3 of hzau. the recombinant vesicular stomatitis virus (vsv) encoding green fluorescence protein (vsv-gfp) was a gift from the harbin veterinary research institute (harbin, china). sendai virus (sev) was grown in 10-day-old embryonic chicken eggs and titrated using a hemagglutination assay as previously described (25) . human embryonic kidney 293t cells and hela cells were purchased from atcc (manassas, va, usa) and cultured at 37°c with 5% co2 in roswell park memorial institute-1640 medium (hyclone, china) supplemented with 10% fetal bovine serum (fbs) (pan-biotech, germany), containing 100 u/ml penicillin, and 100 mg/ml streptomycin (gnm15140 cell lysates and the immunoprecipitates were resolved by 10-12% sds-page and transferred to pure nitrocellulose membranes (ge). the membranes were blocked in 1% bovine serum albumin (bsa) in tbst buffer for 1 h at room temperature and probed with indicated primary antibodies for 1-2 h at room temperature. after hybridizing with either goat anti-rabbit or goat anti-mouse secondary antibodies at a dilution of 1:10,000 in tbst buffer, the membranes were washed with tbst buffer for four times (10 min each) before visualized with ecl reagents (advansta). hela cells were plated onto coverslips in 24-well plates and transfected with the indicated plasmids. at 24 h post transfection, cells were washed once with phosphate-buffered saline (pbs) and fixed in 4% paraformaldehyde for 15 min. cells were permeabilized with 0.1% triton x-100 for 15 min and blocked for 1 h at room temperature with 1% bsa in pbs, followed by incubation with primary antibody for 1 h. after three washes with pbs containing 0.1% tween 20, cells were incubated with fitc or cy3-conjugated secondary antibodies for 1 h at room temperature and then incubated with 4′,6-dapi for 10 min. finally, the coverslips were washed extensively and fixed onto slides. images were taken under a zeiss lsm510 meta confocal microscope (carl zeiss, zena, germany). quantitative real-time pcr (qrt-pcr) total rna was isolated from cells using trizol reagent (invitrogen) following manufacturer's instructions and cdna was prepared by using avian myeloblastosis virus reverse transcriptase (takara). cdna was used for quantification of the indicated mrna copy number on an abi viia 7 pcr system (applied biosystems, usa) by using sybr green master mix (rox). to detect and validate the specific amplification of pcr products, dissociation curve analysis of the products was conducted at the end of each pcr. transcript levels of each gene were normalized with the expression of β-actin, and the 2 −∆∆c t method was used to analyze gene expression in the samples (26) . the primers used in qrt-pcr are listed in table 1 . three sirna oligonucleotides against traf3 and the corresponding negative control sirna were obtained from genepharma. sequences are as follows: si-1, 5′-ccacuggagag augaauau-3′; si-2, 5′-guugugcagagcaguuaau-3′; and si-3, 5′-cugguuacuuuggcuauaa-3′. transfection of sirna into 293t cells was performed by lipofectamine 2000 according to manufacturer's instructions. 293t cells were seeded into 60-mm dishes and transiently transfected with the indicated plasmids. thirty-six hours after transfection, cells were harvested and the lysates were prepared in a 1% np-40 lysis buffer supplemented with a commercially available 0.1% protease inhibitor cocktail and a 10 mm deubiquitinase inhibitor n-ethylmaleimide (sigma-aldrich). samples were immunoprecipitated with 1 µg anti-flag antibodies along with 30 µl protein a/g plus-agarose. polyubiquitination was detected using anti-ha antibodies. influenza a virus infection of a549 cells a549 cells were transfected with the indicated plasmids for 24 h at 37°c. cells were washed twice with f12 medium and then infected with h5n1/hm or pr8 at an indicated moi (2 or 0.001, the results are expressed as means ± sd. statistical analyses were performed on data from triplicate experiments by using twotailed student's t-test. a p-value of less than 0.05 was considered significant and a p-value of less than 0.01 was considered highly significant. the h5n1 ns1 protein inhibits the rigi(n)-mediated activation of ifn-β via its c-terminal ed in an rna bindingindependent manner with the aim of elucidating the mechanism by which iav ns1 protein counteracts the host innate immune responses, we generated four truncated h5n1 ns1 proteins, ns1/1-73, ns1/74-225, ns1/1-125, and ns1/126-225 ( figure 1a) . in order to investigate the function of wtns1, and its truncated peptides, we assessed its effect on ifn-β promoter activity using a luciferase reporter assay in 293t cells. our results showed that wtns1, ns1/74-225, and ns1/126-225 significantly decreased the ifn-β reporter activities driven by rig-i or rig-i(n). conversely, ns1/1-73 did not change the activity of ifn-β reporter, and ns1/1-125 only slightly increased the activity compared to an empty vector control ( figure 1b) . in addition, we found that wtns1 and all truncated peptides had inhibitory effects on ifn-β reporter activities induced by sev or rns1-sd30 virus infection or transfection of poly(i:c) ( figure 1b) . this suggests that ns1 n-terminal rbd alone is sufficient to inhibit the activation of ifn-β only in the presence of dsrna; the c-terminal ed of ns1 could inhibit the activity of ifn-β reporter in all tested conditions. driven by rig-i(n), ns1/126-225 caused a dose-dependent inhibition of ifn-β promoter activity and ifn-β transcription ( figure 1c) . previous studies indicated that iav ns1 sequesters dsrna and binds rig-i at its rbd, subsequently inhibiting the activation of irf3 and preventing the induction of ifn-β (11, 16) . our findings reveal that c-terminal ed of ns1 (ns1/126-225) blocked rig-i(n)-mediated ifn-β induction in an rna binding-independent manner. transcription factor irf3 is a key innate immune system component that mediates ifn-β induction. once irf3 is phosphorylated, it forms a dimer, translocates into the nucleus from the cytoplasm, and induces the expression of ifn-β and isgs through specifically binding to their promoter regions (27) . in order to further investigate how ns1/126-225 inhibits the signaling that mediates type i ifn production, we used an irf3-luciferase reporter plasmid, allowing for the measurement of irf3 activation. as shown in figure 2a , irf3-luciferase reporter activation by rig-i(n) was blocked in 293t cells overexpressing ns1/126-225 or wtns1. we next addressed whether ns1/126-225 affected the dimerization and nuclear localization of endogenous irf3 mediated by rig-i(n). non-reduced sds-page and immunoblot analysis of cell lysates after rig-i(n) transfection showed that ns1/126-225 or wtns1 expression produced a considerable reduction in the activated dimer form of irf3 (figure 2b) . similarly, rig-i(n)induced phosphorylation of irf3 was strongly repressed by ns1/126-225 or wtns1 and that phosphorylated irf3 was largely distributed in nuclear fractions ( figure 2c) . elisa assays of ifn-β in the medium of cells transfected with plasmids encoding ns1/126-225 or wtns1 along with rig-i(n) showed that both ns1/126-225 and wtns1 inhibited the production of ifn-β ( figure 2d) . together, these data indicate that ns1/126-225 inhibits the expression of type i ifn induced by rig-i(n) through blocking the phosphorylation of irf3. previous studies have shown that a recombinant vsv-gfp system can be used as a strategy to screen proteins possessing ifn-antagonizing activity (28) . in the present study, we employed recombinant vsv-gfp to investigate if ns1/126-225 serves as an antagonist of ifn production. when 293t cells expressed ns1/126-225, a high level of vsv-gfp replication was present, consistent with wtns1 protein (figure 2e) , suggesting that the inhibitory effect of ns1/126-225 on ifn production is also present during actual viral infection. in order to test the effect of ns1/126-225 on various components of the rlr pathway, ns1/126-225 and expression (figure 3a) . by contrast, wtns1 significantly decreased the rlr adaptor-mediated ifn-β promoter activity. as expected, ns1/126-225 exhibited a decrease in the ifn-β mrna level induced by rig-i(n) or mavs ( figure 3a ). in addition, the secretion of ifn-β and the transcription level of isgs triggered by tbk1 or irf3(5d) in the presence of ns1/126-225 were tested. ns1/126-225 induced nearly a complete loss of the inhibition of ifn-β and isgs, including oasl, pkr, and mx1, whereas wtns1 strongly blocked the production of ifn-β and isgs mrna expression (figures 3b,c) . together, these data indicate that ns1/126-225 significantly inhibits the cellular antiviral response at the level between mavs and tbk1. in the rlr-mediated signaling pathway, traf3 serves as a critical link between the adaptor mavs and downstream regulatory kinases that are essential for irf3 activation (29, 30) . thus, we hypothesized that traf3 is the target of ns1/126-225. co-ip experiments revealed that ns1/126-225 interacted selectively with traf3 but not other components (figures 4a,b) . in another experiment, wtns1 and ns1/74-225 also interacted most potently with traf3 ( figure 4c ). this association was confirmed under physiological conditions in an experiment that detected this interaction by overexpression of flag-traf3 in infected a549 cells, where rig-i served as a positive control ( figure 4d) . furthermore, pr8 ns1 also interacted with traf3 in infected a549 cells (figure 4e ). to address whether the wtns1 protein physically interacts with endogenous traf3, we performed endogenous ip assays on h5n1/hm or pr8-infected cell lysates. our results showed that traf3 could be co-precipitated by the ns1 antibody ( figure 4f ). in addition, the ns1 proteins of the strain a/shanghai/02/2013(h7n9) and other avian the promoter activities were detected by the dual-luciferase assay system. all luciferase assays were repeated at least three times, and the data shown are mean ± sd from one representative experiment. significance was analyzed with a two-tailed student's t-test (*p < 0.05 or **p < 0.01, ***p < 0.001). h9n2 strain did not bind traf3 ( figure s1 in supplementary material). these results demonstrate that ns1/126-225 or wtns1 interacts with traf3 in a strain-specific manner. based on the findings that ns1/126-225 or wtns1 interacted with traf3, we next asked whether the two molecules co-localize in cells. confocal microscopy revealed the co-staining of ns1/ 126-225 or wtns1 and traf3 in cells, suggesting the co-localization of the two proteins ( figure 4g) . normally, expression of wtns1 in hela cells resulted in a nuclear localization with minor cytoplasmic staining. traf3 overexpression led to a marked increase in the cytoplasmic localization of wtns1 or ns1/126-225. these results indicate that ns1/126-225 or wtns1 colocalize with traf3 in cells and traf3 overexpression led to a marked increase of ns1/126-225 or wtns1 cytoplasmic localization. traf3 is essential for ns1/126-225 to downregulate ifn-β the above data showed that ns1/126-225 blocks ifn-β induction and interacts with traf3. hence, we used traf3 sirna to determine whether traf3 is essential for the regulatory function of ns1/126-225 in 293t cells. knockdown of traf3 by sirna diminished ifn-β promoter activity triggered by rig-i(n) (figures 5a,b) . in cells with silenced traf3, after transfection of rig-i(n), the induction of ifn-β was greatly reduced and the inhibitory effect of ns1/126-225 was markedly attenuated. nevertheless, this inhibitory effect was rescued successfully with a traf3 expression plasmid ( figure 5c) . these data suggest that traf3 is necessary for ns1/126-225 to decrease ifn-β activity. it is well-known that traf3 is modified with a polyubiquitin chain to provide a scaffold for complex formation. thus, in order to study the effect of ns1/126-225 on traf3 ubiquitination, co-ip experiments were conducted. flag-traf3, pub-ha, and ns1/126-225 along with rig-i(n) were cotransfected into 293t cells and the ubiquitination level of traf3 was monitored. compared to the control, ns1/126-225 reduced the rig-i(n)-induced ubiquitination of traf3 (figure 6, left) . to explore the type of traf3 ubiquitin chains, flag-traf3 was transfected into 293t cells with ubiquitin mutants, including the pub-k48-ha or pub-k63-ha expression plasmid. the results showed that ns1/126-225 could decrease the k63-linked ubiquitination of traf3 but not the k48-linked ubiquitination of traf3 (figure 6, right) . in summary, these results demonstrate that ns1/126-225 suppresses the k63-linked ubiquitination of traf3 that is important for the recruitment of the tbk1-ikkε kinase complex. expressing aa1 to 346 and containing the ring domain, the zinc-fingers domain, the isoleucine zipper domain, and flag-traf3-td, expressing aa347 to 568 and containing the traf domain ( figure 7a ). co-ip assays showed that the traf domain of traf3 was found to be the crucial region responsible for the association of traf3 with ns1/126-225 ( figure 7b ) as well as with wtns1 (data not shown). previously, it has been reported that the tim domain of mavs interacts with amino acid residues y440 and q442 within the traf domain of traf3 (31) . to examine whether ns1/126-225 affected ifn signaling at the level of mavs-traf3 interaction, mavs-traf3 association was determined in the presence of ns1/126-225. expression of mavs led to an interaction with traf3 and was increased by rig-i(n) transfection. however, ns1/126-225 and wtns1 markedly disrupted this interaction by 4.6-and 8.5-fold, respectively ( figure 7c) . in h5n1/hm infection of a549 cells, the mavs-traf3 complex was decreased by 3.3-fold compared to the control ( figure 7d) . as reported previously, ikkε is recruited to the c-terminal region of mavs following sev or vsv infection, mediated by lys63-linked polyubiquitination of mavs at lys500, resulting in the inhibition of downstream ifn signaling (31, 32) . therefore, it was necessary to test whether wtns1 or ns1/126-225 affect this process to accomplish its negative regulatory role in ifn-β production after sev infection. the interaction of mavs and ikkε was readily detected by co-ip, while sev infection resulted in a significant decrease in the mavs-ikkε interaction. interestingly, the interaction of mavs and ikkε was remarkably increased when cotransfected with ns1/126-225 or wtns1 (figure 7e) , indicating that ns1/126-225 or ns1 promotes the recruitment of ikkε to mavs. furthermore, we measured the secretion of ifn-β in cell supernatants. results showed that ns1/126-225 or ns1 inhibited the production of ifn-β mediated by mavs-traf3 or mavs-ikkε (figure 7f) , implying that ns1/126-225 functions in downregulating ifn expression. taken together, these results indicate that the association of the mavs-traf3 complex is disrupted by ns1/126-225, which, in turn, blocks ifn-β production. to determine whether the replication of iav is enhanced by the ns1/126-225 protein, a549 cells were transfected with ns1/126-225, or an empty vector, then infected with different titers of h5n1/hm or pr8 virus. upon infection with h5n1/hm or pr8 virus, ns1/126-225 could facilitate transcription of np of both viruses (figure 8a) , resulting in an increase in np and ha proteins observed in the ns1/126-225 group (figure 8b ). this was confirmed by the titers of h5n1/hm or pr8 virus, which significantly increased by 40-and 31-fold, respectively, in ns1/126-225 overexpressing cells compared with control cells (figure 8c) . these results demonstrate that ns1/126-225 enhances the capacity of iav to replicate in cells. prrs of host cells recognize a pamp and subsequently initiate a series of signaling cascades. the final step is activation of irf3 and nf-κb, thus inducing the transcription of ifn-β (33) . given the cascade of responses triggered by the host in response to infection, influenza viruses adapted different strategies to escape the ifn response. this survival tactic has proven successful in order for virus proliferation and infection. both pb2 and pb1-f2 limit ifn production by associating with mavs (10, 34, 35) . other structural proteins, such as pb1, pa, np, and even the genomic rna itself, also contribute to impairing rig-i-mediated antiviral responses (36) . moreover, ha (ha1) was recently shown to drive the degradation of the ifn receptor chain ifnar1, thereby suppressing ifn-triggered jak/stat signaling (37) . the most effective weapon influenza a viruses have at their disposal is ns1 protein. the ns1 protein acts as an antiviral antagonist protein capable of limiting ifn production. rig-i recognizes and binds dsrna structures with 5′-triphosphates upon infection to initiate the host antiviral response. during the course of viral infection, the ns1 protein of iav inhibits host ifn responses either by sequestering viral dsrna or by binding to rig-i and trim25 or riplet proteins required for rig-i activation and ifn signaling pathways (11, 16, 18, 19) . in this study, we found that the ns1 c-terminal ed (aa126 to 225) of h5n1 virus inhibits the activation of ifn-β pathway. to achieve a negative regulatory function in the cellular antiviral response, ns1/126-225 associates with traf3 to remove the lys63-polyubiquitin chains on traf3 and to disrupt the mavs-traf3 complex. ns1/126-225 also increases the recruitment of ikkε to mavs, releasing traf3 from the mitochondria. this further decreases the level of k63linked ubiquitination of traf3, impairing irf3 phosphorylation and reducing the production of ifn-β (figure 9) . interestingly, our study has shown that the ed of ns1 protein possesses the ability to suppress ifn response in the absence of rna. typically, the rbd of ns1 mediates the inhibition of ifn synthesis, and the ed of ns1 induces the inhibition of gene expression, together with its known interactors (11) . in this study, we have found that the ns1 c-terminal ed (ns1/74-225 and ns1/126-225), but not the rbd (ns1/1-73 and ns1/1-125) block ifn-β reporter activity induced by rig-i(n). expression of ns1/126-225 resulted in the inhibition of irf3 activation, indicating that the ns1 protein blocks ifn-β activation through an rna-independent manner. it was demonstrated in previous studies that influenza a viruses tx/98 and a/viet nam/1203/04 expressing c-terminally truncated ns1 proteins of 73, 99, or 126 amino acids were attenuated. the resultant reduced growth correlated with a high level of ifn-α/β induced by these mutant viruses (28, 38) . in addition, in both influenza b and c viruses, the c-terminal domains of the ns1 proteins were found to possess ifn antagonist activity (39, 40) . more importantly, the n terminus-truncated ns1 proteins encoded by pr8, which was to elucidate the mechanism of how the ns1 ed inhibits ifn-β activation, we speculated that the ns1 ed contacts its counterparts in rig-i signaling leading to inhibition. for this purpose, we examined a step within the signaling pathway that ns1/126-225 targets and found that ns1/126-225 acted downstream of mavs and upstream of tbk1. the co-ip assays showed unexpectedly that ns1/126-225 binds to traf3, which interacts with mavs forming a platform for rna virus signaling. we also tested the binding of traf3 to the full-length ns1 protein and found that this interaction exists, and co-localized in the cytoplasm. the ns1/126-225 protein mainly localized in the cytoplasm. traf3 expression led to a marked increase in the ns1/126-225 cytoplasmic localization, suggesting that ns1/126-225 inhibits the activation of the ifn-β pathway. although all types of influenza virus ns1 proteins interact with trim25, only part of ns1 prevents irf3 activation, indicating that trim25 is not required for the inhibition of irf3 activation. in this study, we did not observe the interaction between ns1/126-225 and rig-i, consistent with the results described in a previous publication (41) . consequently, rig-i seems to be non-essential for the optimal inhibition of ifn production in iav-infected cells. however, our study demonstrated that the influenza a virus ns1 ed targets traf3, subsequently inhibits ifn production, implying that traf3 is a key factor involved for iav to escape host innate immune responses. the mavs-traf3 complex is a focal point of rlr-directed signaling response (42, 43) . traf3 localizes to the endoplasmic reticulum (er) and needs to be recruited to mitochondrial mavs in order to activate tbk1 complexes (44) . many viral proteins, accessory and non-structural proteins in particular, hijack traf3 or the traf3 complex to mediate immune evasion. sars coronavirus m protein or open reading frame-9b prevents the formation of traf3-tank-tbk1/ikkε complex or mavs-traf3/traf6 signalosome to evade host innate immunity (45, 46) . sars-cov papain-like protease interacts with and disrupts sting-traf3-tbk1 complex, it also inhibits the tlr7-mediated innate immunity through removing lys63linked ubiquitin chains of traf3 and traf6 (47, 48) . herpes simplex virus 1 ubiquitin-specific protease ul36 deubiquitinates traf3 then counteracts the ifn-β pathway (49) . over the past 10 years, there have been major advances in understanding how influenza a viruses successfully escape the surveillance of the immune system. the current report furthers this research revealing the surprising finding that ns1/126-225 acts by targeting traf3; specifically, ns1/126-225 targets the traf domain of traf3. traf3 links the upstream ifn signaling responses of mavs to tbk1 relying on the traf domain. this report also shows that a specific interaction between traf3 and mavs was observed when traf3 and mavs were co-expressed in 293t cells. however, the interaction between traf3 and mavs was disrupted in the presence of ns1/126-225. interestingly, the interaction between mavs and ikkε was markedly increased in ns1/126-225-expressing cells. it has been previously demonstrated that, after sev infection, k63-linked polyubiquitination at lys500 of mavs recruits ikkε to the mitochondria, functionally causes release of traf3 from mavs initiating the signal to shutdown the ifn response (31) . the mavs-ikkε complex was enhanced when ns1/126-225 was present, indicating that ns1/126-225 can utilize this process to shut down further activation of ifn pathway. taken together, these data indicate that ns1/126-225 impedes the interactions between components of mavs-traf3 complex, preventing the phosphorylation of irf3, where it would activate the ifn-β response. ubiquitination has emerged as a key posttranslational modification that controls induction and shutdown of the interferon response. traf3, serving as a crucial functional link, is modified with a polyubiquitin chain providing a scaffold for complex formation, and, not surprisingly, many viruses encode proteins that inhibit ubiquitination processes to overcome host innate responses. previous studies showed that nairoviruses and arteriviruses encode for ovarian tumor domain-containing proteases that hydrolyze ubiquitin chains from host proteins (50, 51) . in this report, we have shown that ns1/126-225 suppresses the k63linked ubiquitination of traf3. it is likely that ns1/126-225 works through recruiting a deubiquitinase to cleave the traf3 ubiquitin chain since it has been shown that ns1/126-225 does not belong to any known deubiquitinase family. for example, duba, a member of the otubain (otub) family, has been shown to deubiquitinate traf3 and negatively regulate tlr3-and rig-i/ mda5-mediated ifn induction (52) . it was also shown that two otub deubiquitinating enzyme family members, otub1 and otub2, can deubiquitinate traf3 and traf6, leading to the inhibition of virus-induced ifn-β expression and cellular antiviral responses (53) . therefore, whether these proteins, or other dub proteins, are involved in this regulation, and the detailed regulatory mechanism of traf3 activity triggered by ns1/126-225 remains to be discovered. interestingly, strain-specific targeting of traf3 was demonstrated by specific interaction of ns1 proteins encoded by pr8 or avian h5n1 but not novel h7n9 or avian h9n2 viruses. this difference may be associated with strain-specific sequence variations. the ns1 protein most often occurs as a 230 residue peptide, including ns1 of seasonal h1n1 virus and avian h5n1 virus (80-84 residues have been deleted since 2000), which were used in this study. however, premature stop codons or, alternatively, suppression of the genuine stop codon (codon 231) resulted in length variations at ns1's c-terminus. abdelwhab et al. analyzed ns1 protein sequences of all aiv subtypes in birds from 1902 to 2015 to study the prevalence and distribution of carboxyl terminal end truncation (δcte). they found that ns217 proteins lacking amino acids 218-230 were the most prevalent form (88%). this truncation is prevalent in lpaiv of non-h5/h7 subtypes; particularly h9n2, h10, and h6 viruses that are known to be widespread and mostly (semi)endemic in land-based poultry (54) . similar truncations have also been observed in swine influenza viruses, which harbor a c-terminally truncated ns1 and have also been found in human h1n1 viruses that have been in public circulation since the 2009 pandemic (55) . hence, whether the interaction of ns1 and traf3 are associated with the δcte requires further investigation. in summary, the present study demonstrated that traf3 is a target of the c-terminal ed (aa126 to 225) of h5n1 ns1 protein, revealing a novel function of the ns1 protein in modulating host innate immunity and possibly facilitating iav infection. the physiological significance of the ns1 ed in iav replication and its pathological role in flu diseases warrant further investigation to probe the potential value of this molecule as a therapeutic and/or disease prevention target. viral rna detection by rig-i-like receptors sensing viral invasion by rig-i like receptors rig-i-mediated antiviral responses to single-stranded rna bearing 5'-phosphates rig-i detects viral genomic rna during negative-strand rna virus infection trim25 ringfinger e3 ubiquitin ligase is essential for rig-i-mediated antiviral activity riplet/rnf135, a ring finger protein, ubiquitinates rig-i to promote interferon-beta induction during the early phase of viral infection the roles of tlrs, rlrs and nlrs in pathogen recognition regulation of rig-i-like receptor signaling by host and viral proteins molecular mechanisms enhancing the proteome of influenza a viruses: an overview of recently discovered proteins the pb2 subunit of the influenza virus rna polymerase affects virulence by interacting with the mitochondrial antiviral signaling protein and inhibiting expression of beta interferon the multifunctional ns1 protein of influenza a viruses activation of interferon regulatory factor 3 is inhibited by the influenza a virus ns1 protein influenza a virus ns1 protein prevents activation of nf-kappab and induction of alpha/beta interferon the influenza a virus ns1 protein inhibits activation of jun n-terminal kinase and ap-1 transcription factors ns1 protein of influenza a virus inhibits the function of intracytoplasmic pathogen sensor, rig-i inhibition of retinoic acid-inducible gene i-mediated induction of beta interferon by the ns1 protein of influenza a virus ifnbeta induction by influenza a virus is mediated by rig-i which is regulated by the viral ns1 protein influenza a virus ns1 targets the ubiquitin ligase trim25 to evade recognition by the host viral rna sensor rig-i species-specific inhibition of rig-i ubiquitination and ifn induction by the influenza a virus ns1 protein rna binding by the novel helical domain of the influenza virus ns1 protein requires its dimer structure and a small number of specific basic amino acids influenza virus ns1 protein interacts with the cellular 30 kda subunit of cpsf and inhibits 3' end formation of cellular pre-mrnas influenza a virus strains that circulate in humans differ in the ability of their ns1 proteins to block the activation of irf3 and interferon-beta transcription a single amino acid substitution in the novel h7n9 influenza a virus ns1 protein increases cpsf30 binding and virulence effect on virulence and pathogenicity of h5n1 influenza a virus through truncations of ns1 eif4gi binding domain multiple anti-interferon actions of the influenza a virus ns1 protein analysis of relative gene expression data using realtime quantitative pcr and the 2(-delta delta c(t)) method immune signaling by rig-i-like receptors mutations in the ns1 protein of swine influenza virus impair anti-interferon activity and confer attenuation in pigs critical role of traf3 in the toll-like receptor-dependent and -independent antiviral response traf3: uncovering the real but restricted role in human a functional c-terminal traf3-binding site in mavs participates in positive and negative regulation of the ifn antiviral response ubiquitinregulated recruitment of ikappab kinase epsilon to the mavs interferon signaling adapter pathogen recognition and innate immunity the influenza virus protein pb1-f2 inhibits the induction of type i interferon at the level of the mavs adaptor protein influenza virus protein pb1-f2 inhibits the induction of type i interferon by binding to mavs and decreasing mitochondrial membrane potential to conquer the host, influenza virus is packing it in: interferon-antagonistic strategies beyond ns1 hemagglutinin of influenza a virus antagonizes type i interferon (ifn) responses by inducing degradation of type i ifn receptor 1 live attenuated influenza viruses containing ns1 truncations as vaccine candidates against h5n1 highly pathogenic avian influenza the n-and c-terminal domains of the ns1 protein of influenza b virus can independently inhibit irf-3 and beta interferon promoter activation influenza c virus ns1 protein counteracts rig-imediated ifn signalling role of n terminus-truncated ns1 proteins of influenza a virus in inhibiting irf3 activation triggering the innate antiviral response through irf-3 activation ikkepsilon and tbk1 are essential components of the irf3 signaling pathway proteomic profiling of the traf3 interactome network reveals a new role for the er-to-golgi transport compartments in innate immunity severe acute respiratory syndrome coronavirus m protein inhibits type i interferon production by impeding the formation of traf3.tank.tbk1/ikkepsilon complex sars-coronavirus open reading frame-9b suppresses innate immunity by targeting mitochondria and the mavs/traf3/traf6 signalosome sars coronavirus papain-like protease inhibits the type i interferon signaling pathway through interaction with the sting-traf3-tbk1 complex sars coronavirus papain-like protease inhibits the tlr7 signaling pathway through removing lys63-linked polyubiquitination of traf3 and traf6 herpes simplex virus 1 ubiquitin-specific protease ul36 inhibits beta interferon production by deubiquitinating traf3 viral evasion mechanisms of early antiviral responses involving regulation of ubiquitin pathways viral otu deubiquitinases: a structural and functional comparison duba: a deubiquitinase that regulates type i interferon production regulation of virustriggered signaling by otub1-and otub2-mediated deubiquitination of traf3 and traf6 prevalence of the c-terminal truncations of ns1 in avian influenza a viruses and effect on virulence and replication of a highly pathogenic h7n1 virus in chickens stop-codon variations in non-structural protein ns1 of avian influenza viruses the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.the reviewer, bn, and handling editor declared their shared affiliation, and the handling editor states that the process nevertheless met the standards of a fair and objective review. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-000125-uvf5qzfd authors: kenworthy, rachael; lambert, diana; yang, feng; wang, nan; chen, zihong; zhu, haizhen; zhu, fanxiu; liu, chen; li, kui; tang, hengli title: short-hairpin rnas delivered by lentiviral vector transduction trigger rig-i-mediated ifn activation date: 2009-09-03 journal: nucleic acids res doi: 10.1093/nar/gkp714 sha: doc_id: 125 cord_uid: uvf5qzfd activation of the type i interferon (ifn) pathway by small interfering rna (sirna) is a major contributor to the off-target effects of rna interference in mammalian cells. while ifn induction complicates gene function studies, immunostimulation by sirnas may be beneficial in certain therapeutic settings. various forms of sirna, meeting different compositional and structural requirements, have been reported to trigger ifn activation. the consensus is that intracellularly expressed short-hairpin rnas (shrnas) are less prone to ifn activation because they are not detected by the cell-surface receptors. in particular, lentiviral vector-mediated transduction of shrnas has been reported to avoid ifn response. here we identify a shrna that potently activates the ifn pathway in human cells in a sequenceand 5′-triphosphate-dependent manner. in addition to suppressing its intended mrna target, expression of the shrna results in dimerization of interferon regulatory factor-3, activation of ifn promoters and secretion of biologically active ifns into the extracellular medium. delivery by lentiviral vector transduction did not avoid ifn activation by this and another, unrelated shrna. we also demonstrated that retinoic-acid-inducible gene i, and not melanoma differentiation associated gene 5 or toll-like receptor 3, is the cytoplasmic sensor for intracellularly expressed shrnas that trigger ifn activation. a specific double-stranded rna (dsrna) structure, $21-22 bp dsrna with 3 0 overhangs, plays a critical role in initiating both microrna (mirna)-and small interfering rna (sirna)-mediated gene silencing, as it is the structure recognized by the rna interference (rnai) machinery, the rna-induced silencing complex (risc) (1) (2) (3) . except for preformed sirna duplexes of $21 bp, the risc-loaded small rnas are generated by a ribonuclease (rnase) iii-like enzyme that is found in virtually all eukaryotic organisms. this enzyme, aptly named dicer for its ability to cleave a variety of larger (>30 bp) dsrna molecules into the $21 bp dsrna with a characteristic 3 0 overhang of 2 nt, is a multidomain rna-binding protein and itself a component of risc. the primary sequence of the rnas is not important in risc formation, and rnai can suppress virtually any target as long as rules of sequence complementarities between the small rna and the target rna are satisfied. dsrnas are also a type of pathogen-associated molecular pattern (pamp) that are detected by cellular innate immunity sensors named pattern recognition receptors (prrs) (4) . the interaction between a pamp and a prr triggers activation of the interferon (ifn) pathway in mammalian cells, which significantly changes the gene-expression profile in the cells and contributes to the well-documented off-target effect of rnai. ifn induction is especially problematic in antiviral studies employing rnai, where the antiviral effect of ifn must be distinguished from that of rnai. typical ifn-inducing structure patterns include dsrna of certain length, single-stranded rna (ssrna) containing 5 0 -triphosphates (5 0 -ppp), the dsrna analogue polyinosinic-polycytidylic acid (poly i:c), and certain dsdna molecules. these rna patterns are generally believed to possess 'non-self' properties to allow the cell to recognize foreign (often viral) rnas specifically. various forms of sirna duplexes have been reported to trigger ifn induction both in vitro and in vivo (5) (6) (7) (8) (9) , probably through the cell surface-and/or endosomeexpressed toll-like receptors (tlrs), including tlr3 and tlr7 (6, 8, 9) . short-hairpin rnas (shrnas) expressed from a dna plasmid have also been shown to activate ifn (10) . the double-stranded form of these rnas is below the size limit of the stem-loop rnas that can be detected by the rna-activated protein kinase (pkr) (11) and is probably detected by other cytoplasmic prrs. two cytoplasmic rna helicases, retinoic-acid-inducible gene i (rig-i) and melanoma differentiation associated gene 5 (mda5), signal to the ifn-b promoter when activated by specific rna structures (12) (13) (14) . although both prrs signal through the mitochondrial antiviral signaling protein mavs/cardif/visa/ips-1 (15) (16) (17) (18) , studies of ligand specificity suggest that rig-i and mda5 are parallel sensors with overlapping substrates. for example, although both prrs are activated by poly i:c in cell culture systems (12, (19) (20) (21) (22) (23) , mda5 appears to be more important in mediating the poly i:c response in vivo (13, 14) . in addition, rig-i can bind and respond to ssrnas bearing 5 0 -ppp, whereas mda5 is not activated by 5 0 -ppp-containing rna (24, 25) . finally, several cytosolic sensors for dsdna has been recently reported (26) (27) (28) (29) (30) (31) . nevertheless, current data on what constitutes effective substrates for either prr are incomplete and sometimes controversial. here we report for the first time that shrnas delivered by lentiviral transduction triggered ifn activation and that rig-i and mavs, but not mda5 or tlr3, mediated the ifn activation triggered by intracellularly expressed shrna, which could activate both ifn-a and ifn-b promoters. ifn activation depended on sequence, a 5 0 -ppp and correct processing of the rna hairpin by dicer; it was independent of promoter choice, presence of blunt ends, route of delivery and rnai potency. gs5 and lh86 cells have been described earlier (32, 33) . huh-7 and 293ft cells were maintained in dmem supplemented with 10% fbs. we used the following antibodies: anti-cypa (biomol, plymouth meeting, pa, usa); anti-cypb (afenity bioreagents, rockford, il, usa); anti-ku80, anti-flag and anti-actin (sigma-aldrich, st louis, mo, usa); anti-ifn stimulate gene (isg)15 (rockland immunochemicals, gilbertsville, pa, usa); anti-ns5a (virogen, watertown, ma, usa) and anti-ns3 (in-house). gsb1 and h801 cells have been described earlier (34) . poly i : c was purchased from sigma-aldrich, and synthetic hairpin rna was purchased from integrated dna technologies (coralville, ia, usa). synthetic sirna was purchased from ambion (austin, tx, usa). protein contents of cell lysate were quantified with the bio-rad dc protein assay (bio-rad, hercules, ca, usa), and an equal amount of total protein was loaded in each lane. samples for irf-3 dimerization assay were run on a polyacrylamide gel under non-denaturing conditions (35) . other samples were denatured and separated by sodium dodecyl sulfate polyacrylamide gelelectrophoresis (sds-page). proteins were then transferred onto a nitrocellulose membrane and stained with the appropriate antibodies with the snap i.d. tm system (millipore, worcester, ma, usa) according to the manufacturer's instructions. for luciferase assays, cells were seeded to a confluency of 50%, and for all other assays, cells were seeded to a confluency of 30%. the next day, transfections of dna plasmids and synthetic rnas were performed with lipofectamine tm 2000 (invitrogen, carlsbad, ca, usa) according to the manufacturer's instructions. plasmids pgl3-ifna1, pgl3-ifnb, prl-tk, pcmv-flag-irf-3 and pcr3.1-irf-7a have been described earlier (36) . shrnas were expressed from a human immunodeeciency virus (hiv)-based lentiviral vector (32, 37) , and sh-pcaf was constructed on the basis of a previously reported sequence (38) . plasmid sh-b971/h1 was constructed by cloning of the dna fragment encoding the sh-b971 rna into psilencer 3.0-h1 (ambion, austin, tx, usa) according to the manufacturer's instructions. the rig-i and tlr3 constructs have been described (39, 40) . the rig-i c construct encodes flag-tagged, c-terminal 707 aa of human rig-i cloned into a bicistronic expression vector modified from pbicep-cmv-1 (sigma-aldrich, st louis, mo, usa), in which the cmv promoter was replaced with the elongation-factor-1 promoter. the mda5, mda5-c constructs were kindly provided by fujita (12) . hcv genotype 2a ns3-4a protease was expressed from the pcmv-3tag-1a plasmid (stratagene, la jolla, ca, usa). 293ft cells were seeded in 24-well plates and were transfected 16 h later with 400 ng of a shrna expression vector, 40 ng of pgl3-ifna1 or pgl3-ifnb, 20 ng of prl-tk and 50 ng of pcr3.1-irf-7a. cells were collected 48 h after transfection. luciferase assays were performed with the dual-glo õ luciferase assay system reagents (promega, madison, wi) and luminescence quantified with a modulus microplate reader (turner biosystems, sunnyvale, ca, usa). ratios of firefly luciferase (from the pgl3 vectors) to renilla luciferase (from the prl-tk vector) were calculated, and that of the sh-b971 sample was normalized to 100%. sequences of shrna are shown in table 1 . lentiviral vector production and transduction were performed as described earlier (37) . viral vectors were pelleted by ultracentrifugation at 50 000g at 4 c for 3 h and resuspended in a volume of pbs that was 1% of the original medium volume. the titers of the concentrated vectors were then measured with a p24 elisa kit (zeptometrix, buffalo, ny, usa). real-time reverse transcription pcr (rt-pcr) was performed as described earlier (32) . the primers used were oas1 forward, 5 0 -agg tgg taa agg gtg gct cc-3 0 and oas1 reverse 5 0 -aca acc agg tca gcg tca gat-3 0 ; rig-i forward 5 0 -gag gca gag gaa gag caa gag g-3 0 and rig-i reverse 5 0 -cgc ctt cag aca tgg gac gaa g-3 0 ; gapdh forward 5 0 -tca ctg cca ccc aga aga ctg-3 0 and gapdh reverse 5 0 -gga tga cct tgc cca cag c-3 0 . the primers for hcv detection were 5 0 -cgc tca atg cct gga gat ttg-3 0 and 5 0 -gca ctc gca agc acc cta tc-3 0 . for flow cytometry, gs5 cells were fixed 48 h after treatment in a solution of 2% paraformaldehyde and analyzed with a facscanto flow cytometer (bd biosciences, san jose, ca, usa). mean gfp intensity was plotted, and that of the sh-ntc sample was normalized to 100%. total rna from transiently transfected 293ft cells was extracted with rna stat-60 (tel-test, friendswood, tx, usa) and separated on a 7.5% urea polyacrylamide gel. the transfer of rna onto nitrocellulose membrane and hybridization were performed according to standard molecular biology protocols. the probe for detecting the expression of sh-b971 and its variants was a synthetic dna oligomer corresponding to the bottom strand of sh-b971. radioactive labeling of the probe was performed with an end-labeling protocol with t7 polynucleotide kinase (ambion, austin, tx, usa). the exposure and detection of the radioactive signal was performed with a typhoon imager (ge healthcare, piscataway, nj, usa) with quantity one software (bio-rad, hercules, ca, usa). a short-hairpin rna directed at cypb induces ifn production in human embryonic kidney cells to investigate the potential role of the cyclophilins (cyps) in hcv replication (41), we delivered several shrnas directed at mrnas of three cyps into hcv replicon cells by means of a lentiviral vector, using a murine u6 promoter to drive the expression of the shrna ( figure 1a ) (37) . we observed a discrepancy between two anti-cypb shrnas (b971 and b710) in their relative efficiency in knocking down cypb expression and in suppressing hcv. lentiviral vector sh-b971 was less efficient in knocking down cypb expression but potently inhibited hcv ns5a expression in a human hepatoma cell line containing replicating hcv rna ( figure 1b , left). viral inhibition was independent of cypb knockdown, as control medium from transfected 293ft cells that did not contain any lentiviral vector particles, generated by omission of the packaging plasmids during transfection, also inhibited hcv replication ( figure 1b , right) without affecting cypb expression. the fast kinetics of viral inhibition (complete inhibition with 48 h, data not shown) was also more consistent with ifn than with rnai-based inhibition. the presence of ifn in the lentiviral vector preparation of sh-b971 was confirmed by strong induction of 2 0 -5 0 -oligoadenylate synthetase 1 (oas1), a classic ifn-induced gene, in both naı¨ve huh-7 and the hcv replicon cell line (gs5) treated with the medium ( figure 1c ). in addition, hcv replication in an ifn-resistant hcv replicon cell line (h801), in contrast to that in a wildtype replicon cell line (gsb1) (34), was not inhibited by the sh-b971 medium ( figure 1d ), suggesting the lack of additional viral inhibiting agents in the sh-b971 medium. expression of sh-b971 in 293ft cells also induced dimerization of irf-3, confirming the activation of the ifn production pathway in these transfected cells ( figure 1e ). finally, sh-b971 was able to activate both ifn-a and ifn-b promoters, although the activation of the ifn-a promoter required coexpression of irf-7, which is normally expressed at very low levels in 293-based cells ( figure 1f ). these results demonstrate that sh-b971 is a potent activator of irf-3 and irf-7, master regulators of ifn expression in human cells. we next investigated the role of the different viral/ exogenous rna sensors, rig-i, mda5 and tlr3, in sh-b971-triggered ifn production. mammalian expression plasmids encoding each of these proteins, as well as the dominant negative (dn) mutants of rig-i and mda5, were transfected into 293ft cells with shrnas and an ifn-b promoter reporter construct. the signaling to ifn-b promoter and the expression of the prr proteins were then examined 48 h after transfection. in the absence of sensor proteins, the sh-b971 increased activation of the ifn-b promoter by 2.6-fold ( figure 2a ). coexpression of mda5 or tlr3 did not increase or decrease sh-b971's ability to activate ifn-b promoter relatively to the negative control shrna (sh-ntc), but in the presence of rig-i coexpression, the induction of ifn-b promoter by sh-b971 was increased to $30-fold. moreover, ectopic expression of a dn mutant of rig-i (rig-i c), but not that of mda5 (mda5-c), completely abrogated ifn promoter activation by sh-b971. with the exception of tlr3, which required prolonged exposure of the western blot to be detected, the cytoplasmic sensors and their mutants were expressed at comparable levels ( figure 2b ). moreover, activation of irf-3 ( figure 1e ) and ifn promoters ( figure 1f ) in 293ft cells, which do not contain a functional tlr3 signaling pathway (42) , indicates that tlr3 plays a negligible role, if any, in ifn induction by sh-b971. the combination of sh-b971 and rig-i produced the highest level of ifn-b promoter activity, which were confirmed by western blotting showing that endogenous isg15 induction was only detectable in cells cotransfected with sh-b971 and wild-type rig-i ( figure 2b ). to confirm further that biologically active ifn was released from these cells, we applied the culture medium of the transfected 293ft cells to an hcv replicon cell line (gs5) in which ns5a-gfp expression is used for monitoring viral rna replication (43) . hcv replication in this cell line is extremely sensitive to ifn, and the effect of the cytokine can be readily measured as the change in the mean gfp intensity of the treated cells. as shown in figure 2c , culture medium from sh-b971 efficiently suppressed hcv replication, resulting in a decrease in figure 1 . a small-hairpin rna directed at cypb induces ifn production in human embryonic kidney cells. (a) sequence of sh-b971, which was expressed from a self-inactivating human immunodeficiency virus (hiv) vector with a murine u6 promoter (59) . (b) inhibition of hcv expression by culture media of sh-b971-transfected 293ft cells. gs5 cells were treated with culture supernatant taken from 293ft cells transfected with various shrna plasmids with (left) or without (right) the packaging plasmids overnight. cells were then cultured in fresh media for an additional 6 days before being lysed for western blotting. (c) oas1 induction by culture supernatant from 293ft cells transfected with sh-b971. huh 7 and gs5 cells were treated with culture supernatant from 293ft cells transfected with either sh-luc or sh-b971 for 24 h before rna extraction and real-time rt-pcr analysis. oas1 rna level was normalized to that of gapdh rna. (d) transfected culture media failed to suppress hcv replication in an ifn-resistant cell line. hcv replicon cells were cultured as described earlier (34) and then treated with the indicated culture medium from transfected 293ft cells. hcv rna was analyzed with real-time rt-pcr. (e) irf-3 dimerization in response to sh-b971 expression. flag-irf-3 was cotransfected with a shrna into 293ft cells. cells were lysed 24 h after transfection, and total cell lysate was separated on a polyacrylamide gel under non-denaturing conditions, transferred and stained with an anti-flag antibody. (f) ifn-a and ifn-b promoter activation by sh-b971 expression. sh-ntc, sh-c454 (an shrna directed at cypc), or sh-b971 was cotransfected along with luciferase reporter plasmids with or without irf-7. the ratios of firefly luciferase readings to renilla luciferase readings were plotted. the ns5a-gfp intensity within 48 h of treatment. cotransfecting wild-type rig-i produced a medium with stronger inhibition, whereas the rig-c drastically suppressed the antiviral effect of the medium. finally, real-time rt-pcr analysis revealed that sh-b971, but not the negative control shrna, strongly activated expression of endogenous rig-i, a well-characterized isg whose induction requires paracrine/autocrine action of ifn (44, 45) . as expected, poly i : c activated rig-i expression in the same assay ( figure 2d ). these results, taken together, show that rig-i is the cellular sensor that mediates the ifn induction by sh-b971. the majority of the shrnas that we use in the lab do not activate rig-i expression and ifn signaling despite having essentially the same structure as sh-b971, so we wanted to determine whether the sequence of sh-b971 is distinctive enough to trigger the production of ifn. we first tested a synthetic sirna duplex with the same target sequence as sh-b971. this sirna (si-b971-syn) should resemble the final dicer product of sh-b971 except for the 5 0 -ends. the synthetic sirna contains 5 0 -oh groups, whereas the dicer products probably figure 3a ) while failing to activate ifn production, as measured by the gfp-hcv assay ( figure 3b ). to determine whether the sequence of the intact hairpin rna before dicer cleavage is sufficient to trigger ifn, we tested a synthetic shrna (sh-b971-syn) that had exactly the same sequence as the predicted intracellular sh-b971 transcript generated by the u6 promoter. again, the 5 0 -end of the synthetic sh-b971 had a 5 0 -oh group instead of any phosphate. sh-b971-syn behaved similarly to si-b971-syn in that it knocked down cypb expression without activating ifn response (figure 3) . these results suggest that the 5 0 -end status of sh-b971 is important for ifn activation, consistent with the previously finding that a 5 0 -triphosphate is required for rig-i activation (24, 25) . to determine the contribution of the individual residues of the sh-b971 sequence, we introduced a series of point mutations into the shrna and tested them for ifn induction. we changed the first nucleotide from a to g, c, or t while maintaining base-pairing between nucleotides +1 and +47. these mutant shrnas lacked the ability to activate ifn production (table 1) . changing the +1 nucleotide to g while leaving the +47 nucleotide intact also abolished ifn activation by the shrna (a1/g), as did the reciprocal mutation u47/c. the importance of the first nucleotide was further confirmed by the inability of sh-b971+1 to activate ifn. the target of sh-b971+1 was shifted 1 nt downstream on the cypb mrna, producing an shrna starting with a g at the +1 position. the presence of an a at the +1 position was not, however, sufficient to render a shrna competent for ifn activation, as replacing the first nucleotide of the sh-ntc with an a did not generate an ifn-inducing shrna (ntc-a and ntc+1). these results indicate that a protruding/unpaired a at the end of the hairpin or the rna duplex, a potential result of 'breathing' at the end of the dsrna, is not sufficient to trigger ifn induction as previously suggested (38) . two point mutations located farther into the stem structure of the shrna (9g9 and b18a1) also reduced its ability to induce ifn even though the base-pairing was perfectly maintained in these mutants. finally, replacing the 9-nt hairpin loop with a 7-nt loop that had been previously shown to abolish shrna-mediated rnai (loop a mutant) (46) eliminated sh-b971's ability to induce ifn, suggesting the importance of rna processing in the induction. to determine whether the inability of the mutant shrnas to induce ifn was due to lower expression levels, we performed northern blotting analysis of the shrna expression on the wild-type and two mutants. the mutants a1/g and loop a were chosen because their final sirna products have exactly the same sequence as that of the wild-type sh-b971 and can thus be detected with the same efficiency by the same probe. although sh-a/g and sh-loop a were clearly unable to activate ifn-b promoter ( figure 4a ), they were both expressed at levels comparable to those of the wild-type sh-b971 product ( figure 4b) . interestingly, the final sirna product of sh-loop a was slightly smaller than those of sh-b971 and sh-a1/g, suggesting that cleavage did occur and perhaps occurred one or 2 nt into the stem to compensate for the shorter loop. blunt-ended sirna has been previously reported to be stronger inducers of ifn than the sirnas with overhangs (47) . indeed, a previously reported ifn-inducing shrna, sh-pcaf (p300/creb-binding protein-associated factor), contains a blunt end (38) and was more potent in activating ifn than sh-b971 ( figure 5a ), which is predicted to form an overhang of 2-3 ts at each end of the final sirna. we therefore constructed a version of the sh-b971 that would be blunt at the end that is not processed by dicer by adding two extra as to the 5 0 -end of the shrna. this modification (blunt sh-b971) did not increase the ability of sh-b971 to activate ifn-b promoter ( figure 5a ). we confirmed, in two independent experiments, that ifn induction by sh-pcaf was also mediated by rig-i. first, cotransfection of dn rig-i resulted a 50-to 100-fold inhibition of ifn induction by sh-pcaf ( figure 5b ), whereas wild-type rig-i increased ifn induction by several fold in the same assay. second, when hcv ns3-4a protease, which cleaves mavs, thereby blocking the rig-i pathway, was coexpressed with either sh-b971 or sh-pcaf, ifn induction by these shrnas were severely compromised ( figure 5c ), further substantiating a role of the rig-i and mavs pathway in mediating ifn induction by both the blunt-ended sh-pcaf and the sh-b971 with overhang. the proper expression of ns3-4a protease was confirmed by western blotting ( figure 5d ). to assess the contribution of the promoter choice in ifn activation by intracellular expressed shrna, we expressed sh-b971 from another commonly used pol iii promoter, the human h1 promoter. both the original, mu6-driven sh-b971 and the h1-driven sh-b971 activated ifn-b promoter ( figure 6a ) and resulted in secretion of ifn into the transfected cell-culture media, which in turn suppressed hcv replication ( figure 6b ). proper expression of the sirna ( figure 6c ) and the subsequent knockdown of cypb expression ( figure 6d ) all appeared normal for sh-b971 expressed from the h1 promoter plasmid, which has a backbone different from that of our lentiviral vector carrying the mu6 promoter. these data suggest that ifn induction by sh-b971 is not restricted to a particular promoter or expression construct. further supporting this conclusion was the observation that the expression cassette by itself, removed and isolated from the lentiviral plasmid by restriction digestion, could also activate ifn production in transfected 293ft cells (data not shown). to this point, all the ifn induction experiments were done with transient transfection of dna vectors and it was possible that certain features of the double-stranded plasmid dna are responsible for ifn induction. we first tried to address this point by transfecting just the shrnaexpressing cassette, generated either by pcr or restriction enzyme digestion, into 293ft cells and confirming that these fragments of $200 bp were sufficient to trigger ifn induction (supplementary figure s1) . to definitively rule out any contribution by dsdna, we used a lentiviral transduction system which has been suggested to express shrnas that can escape detection by prrs and ifn activation (48) . we produced lentiviral particles containing shrnas from 293ft cells using standard . sh-b971 expressed from an h1 promoter triggers ifn activation. sh-b971 expressed from an h1 promoter was capable of (a) activating ifn-b promoter and (b) triggering ifn production to inhibit hcv replication in gs5 cells. (c) intracellular levels of u6-and h1-driven sh-b971 products. rna extraction and northern blotting were performed as described in figure 4b . (d) knockdown of cypb expression by sh-b971 expressed from an h1 promoter. methods, centrifuged them to separate the vectors from the ifn-containing media, and then used them to infect naı¨ve 293ft cells ( figure 7a ). both sh-b971 and sh-pcaf vectors induced ifn production when delivered as concentrated lentiviral particles, measured both by hcv suppression ( figure 7b ) and by oas induction ( figure 7c ) in huh-7 cells. to rule out the possibility that residual ifn in the concentrated viral particles was responsible for these results, we added 100 u/ml ifn to the negative control vector sample before the concentration step. this preparation, designated sh-ntc*, was not able to trigger ifn production in naı¨ve 293ft cells, suggesting that the concentration step effectively removed the soluble ifn from the viral particle pellet. proper knockdown of the sirna target of sh-b971 was confirmed by this route of shrna delivery ( figure 7d ). to prove definitively that ifn induction by the shrnas was mediated by the lentiviral infection route, we tested the effect of an inhibitor of hiv reverse transcriptase, nevirapine, on ifn induction by sh-b971 and sh-pcaf. as shown in figure 7e , inclusion of nevirapine at the time of transduction effectively blocked the ability of both shrnas to induce ifn in the transduced cells, suggesting the importance of the reverse transcription step in the expression of the shrnas delivered by the lentiviruses. to determine whether lentiviral vector-delivered shrna can trigger ifn induction in cells other than 293ft cells, we transduced a human hepatoma cell line, lh86, which has been reported to produce ifn upon viral infection (33) , and examined ifn induction in these cells. culture medium from lh86 cells transduced with sh-pcaf contained biologically active ifn, which suppressed hcv replication in gs5 cells ( figure 7f ), indicating that the ability of shrnas delivered by lentivirus to induce ifn response was not limited to 293ft cells. it has been reported that certain chemically synthesized and phage polymerase in vitro transcribed sirnas can non-specifically induce ifn responses and produce offtarget effect via various prrs, including tlrs. however, the induction of ifn response by shrnas and its underlying mechanisms have not been as well studied. the actual number of shrnas that are capable of triggering ifn response will certainly be larger than the few that have been reported in the literature, yet very little is known about the unique characteristics of the select shrnas and the pathway that they use to activate ifn production. the present study identifies rig-i, but not mda5 or tlr3, as the mediator for activation of ifn responses by two shrnas that are distinct in sequence and structure but both capable of ifn induction in human cells. this was demonstrated by induction of irf-3 dimerization, activation of ifn promoters, induction of endogenous isgs (isg15, oas and rig-i), and secretion of ifn, all of which depended on rig-i and its downstream adaptor, mavs. in addition, we show that delivery of these shrnas via lentiviral transduction does not reduce their ifn-inducing capacity, indicating that the ability of lentiviral vector transduction to avoid ifn induction by shrnas, as reported previously (48), may not be universally applicable to all the shrnas. specific recognition of dsrnas or ssrnas bearing 5 0 -triphosphates by rig-i is presumably determined mostly by structural features other than the nucleotide sequence of the rna. yet ifn activation by sh-b971 exhibited a stringent dependence on specific nucleotides at multiple positions of the shrna. an aa dinucleotide at the beginning of the u6 transcript has previously been suggested to result in aberrant transcription, and preserving a c/g sequence at positions à1/+1 suggested to avert ifn induction (38) . we indeed observed a strict requirement for an adenylate at the +1 position of sh-b971 for rig-i recognition and ifn activation, but we observed no difference in expression levels or the apparent sizes of the sh-b971 rnas bearing either an a or a g at the +1 position. furthermore, mutations introduced elsewhere in the shrna also abolished or diminished sh-b971's ability to activate ifn, suggesting additional sequence requirement for efficient rig-i recognition and ifn triggering. despite these results, because we were not successfully in cloning and sequencing the vectorexpressed sirna, we cannot exclude the possibility that the adenylate at the +1 position interferes with transcription and that the resultant abnormal transcript contributes to ifn induction. interestingly, the loop a mutant, which contains a predicted loop of 7 nt, generated a sirna duplex inside the cells that is slightly smaller than that of the shrnas with a wild-type hairpin loop, suggesting the processing by dicer into the stem, perhaps fulfilling the requirement of a length of 9 nt for the hairpin loop (46) . this mutant form of sh-b971 was not, however, able to trigger ifn activation. despite the abilities of both sh-b971 and sh-pcaf to activate the rig-i pathway, the two shrnas are unrelated in sequence. two short stretches of sirna sequences, guccuuccaa and ugugu, that have been previously defined as ifn-or cytokine-activating motifs (8, 9) are not found in either sh-b971 or sh-pcaf. any common sequence motifs of ifn-activating shrnas, if any, remain to be defined. the two shrnas also differ in that one is predicted to contain one blunt end and the other two ends with overhangs. these results suggest that, although blunt ends may increase sirna's ability to be recognized by rig-i (47), they are not required for ifn activation by an endogenously expressed shrna. the best-characterized rna structure motif recognized by rig-i is the 5 0 -ppp, which is absent from virtually all the cellular rnas as a result of either 5 0 -capping or internal cleavage before their appearance in the cytoplasm. a synthetic shrna that has the same sequence as sh-b971 but lacks the 5 0 -ppp failed to induce ifn, suggesting the 5 0 -end status of the intracellularly expressed sh-b971 contributes to ifn activation. whether or not the 5 0 -end of an shrna is capped has not been investigated. murine u6 rna does not contain the trimethylguanosine cap that is present on mrnas and other u small nuclear rnas; instead it contains a g-monomethyl phosphate cap at its 5 0 -end (49) . capping of heterologous transcripts produced from the mu6 promoter, however, requires a stem loop at the 5 0 -end of the transcript and an auauac sequence immediately after (50) . most shrnas, including sh-b971 and sh-pcaf, would not meet these requirements and thus should contain unmodified 5 0 -ppp. similarly, no evidence of a cap structure for h1 transcripts could be found in the literature. we attempted to express sh-b971 using a mirna expression cassette and the pol ii promoter (51) . the primary transcript generated with this construct would be capped at 5 0 -end by a trimethylguanosine cap and the final sirna duplex would bear a monophosphate at the 5 0 -ends of both strands because of drosha and dicer cleavage. this version of the sh-b971 vector was much weaker in its ability to trigger ifn activation. unfortunately the intracellular expression of the rna duplex was also much weaker and barely detectable by northern blotting. in addition, no knockdown of the target cypb mrna was seen with this mirna-based sh-b971 (data not shown). as a result, whether sh-b971, if expressed at higher level from this construct, could effectively activate ifn remains unclear. so far as we know, ours is the first report of ifn activation in the target cells by shrnas delivered by lentiviral transduction. a previous report of ifn induction by lentiviral vector-expressed shrna only examined the ifn generated in the vector-producing cells, which then up-regulated ifn-stimulated genes in the transduced cells (10) . the distinction is important as lentiviral vectors used in a gene-therapy setting will likely be purified and free of any ifn that has been generated during the vector preparation step, but ifn activation in the target cells would pose a more serious concern. our data suggest the importance of screening shrnas for ifn induction in the transduced cells in vitro before largescale studies. an hiv reverse transcriptase inhibitor efficiently blocked ifn production by both sh-b971 and sh-pcaf when delivered by transduction, indicating the virion-encapsulated rna was not able to trigger ifn activation. in this respect, it is interesting to note that positive-stranded rna viruses, which produce dsrna intermediates in the cytoplasm during replication (52) (53) (54) (55) , often replicate in membrane enclosed vesicles (56) , this sequestration of viral dsrna in membranous structures may shield the rna from the cytoplasmic prrs and contribute to a successful infection. ifn-induction and rnai by shrnas appear to be independent functions of the same rna (57). our results also showed that ifn-induction by sh-b971 is independent of its ability to suppress target mrna expression through rnai. on the other hand, it might be possible to screen for duel functional sirnas that confer therapeutic benefits by both rnai and immunostimulation (58) . for example, sirnas that target either viral genomes or cellular cofactors of the viruses can be screened for their ability to trigger ifn activation in hopes of find 'super sirnas' with increased efficacy against ifn-sensitive viruses. an rna-directed nuclease mediates post-transcriptional gene silencing in drosophila cells single-stranded antisense sirnas guide target rna cleavage in rnai human risc couples microrna biogenesis and posttranscriptional gene silencing pathogen recognition and innate immunity activation of the interferon system by short-interfering rnas small interfering rnas mediate sequence-independent gene suppression and induce immune activation by signaling through toll-like receptor 3 interferon induction by sirnas and ssrnas synthesized by phage polymerase sequence-specific potent induction of ifn-alpha by short interfering rna in plasmacytoid dendritic cells through tlr7 sequence-dependent stimulation of the mammalian innate immune response by synthetic sirna induction of an interferon response by rnai vectors in mammalian cells 0 -triphosphate-dependent activation of pkr by rnas with short stem-loops the rna helicase rig-i has an essential function in doublestranded rna-induced innate antiviral responses differential roles of mda5 and rig-i helicases in the recognition of rna viruses essential role of mda-5 in type i ifn responses to polyriboinosinic : polyribocytidylic acid and encephalomyocarditis picornavirus ips-1, an adaptor triggering rig-i-and mda5-mediated type i interferon induction cardif is an adaptor protein in the rig-i antiviral pathway and is targeted by hepatitis c virus identification and characterization of mavs, a mitochondrial antiviral signaling protein that activates nf-kappab and irf 3 visa is an adapter protein required for virus-triggered ifn-beta signaling the v proteins of paramyxoviruses bind the ifn-inducible rna helicase, mda-5, and inhibit its activation of the ifn-beta promoter shared and unique functions of the dexd/h-box helicases rig-i, mda5, and lgp2 in antiviral innate immunity polyinosinicpolycytidylic acid induces the expression of gro-alpha in beas-2b cells ebola virus vp35 protein binds double-stranded rna and inhibits alpha/beta interferon production induced by rig-i signaling expression of ip-10/cxcl10 is upregulated by double-stranded rna in beas-2b bronchial epithelial cells rig-i-mediated antiviral responses to single-stranded rna bearing 5 0 -phosphates aim2 recognizes cytosolic dsdna and forms a caspase-1-activating inflammasome with asc aim2 activates the inflammasome and cell death in response to cytoplasmic dna an orthogonal proteomic-genomic screen identifies aim2 as a cytoplasmic dna sensor for the inflammasome hin-200 proteins regulate caspase activation in response to foreign cytoplasmic dna dai (dlm-1/zbp1) is a cytosolic dna sensor and an activator of innate immune response rna polymerase iii detects cytosolic dna and induces type i interferons through the rig-i pathway characterization of hepatitis c virus subgenomic replicon resistance to cyclosporine in vitro hepatitis c virus triggers apoptosis of a newly developed hepatoma cell line through antiviral defense system defective jak-stat activation in hepatoma cells is associated with hepatitis c viral ifn-alpha resistance induction of irf-3/-7 kinase and nf-kappab in response to double-stranded rna and virus infection: common and unique pathways a kaposi's sarcoma-associated herpesviral protein inhibits virus-mediated induction of type i interferon by blocking irf-7 phosphorylation and nuclear accumulation identification of cellular cofactors for human immunodeficiency virus replication via a ribozyme-based genomics approach determinants of interferonstimulated gene induction by rnai vectors gb virus b disrupts rig-i signaling by ns3/4a-mediated cleavage of the adaptor protein mavs distinct poly(i-c) and virus-activated signaling pathways leading to interferon-beta production in hepatocytes cyclophilin a is an essential cofactor for hepatitis c virus infection and the principal mediator of cyclosporine resistance in vitro recognition of double-stranded rna and activation of nf-kappab by toll-like receptor 3 effect of cell growth on hepatitis c virus (hcv) replication and a mechanism of cell confluencebased inhibition of hcv rna and protein expression antitumor nk activation induced by the toll-like receptor 3-ticam-1 (trif) pathway in myeloid dendritic cells central role of interferon regulatory factor-1 (irf-1) in controlling retinoic acid inducible gene-i (rig-i) expression a system for stable expression of short interfering rnas in mammalian cells a structural basis for discriminating between self and nonself double-stranded rnas in mammalian cells stable expression of shrnas in human cd34+ progenitor cells can avoid induction of interferon responses to sirnas in vitro gamma-monomethyl phosphate: a cap structure in spliceosomal u6 small nuclear rna capping of mammalian u6 small nuclear rna in vitro is directed by a conserved stem-loop and auauac sequence: conversion of a noncapped rna into a capped rna a lentiviral microrna-based system for single-copy polymerase ii-regulated rna interference in mammalian cells visualization of double-stranded rna in cells supporting hepatitis c virus rna replication double-stranded rna is produced by positive-strand rna viruses and dna viruses but not in detectable amounts by negative-strand rna viruses subcellular localization and membrane topology of the dengue virus type 2 non-structural protein 4b sars-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum seeking membranes: positive-strand rna virus replication complexes sirna and isrna: two edges of one sword 0 -triphosphate-sirna: turning gene silencing and rig-i activation against melanoma design of hiv vectors for efficient gene delivery into human hematopoietic cells the authors thank dr andre irsigler and dr jason robotham for technical assistance and dr anne b. thistle for proofreading the manuscript. supplementary data are available at nar online.conflict of interest statement. none declared. key: cord-001129-gi2kswai authors: lemos de matos, ana; mcfadden, grant; esteves, pedro j. title: positive evolutionary selection on the rig-i-like receptor genes in mammals date: 2013-11-27 journal: plos one doi: 10.1371/journal.pone.0081864 sha: doc_id: 1129 cord_uid: gi2kswai the mammalian rig-i-like receptors, rig-i, mda5 and lgp2, are a family of dexd/h box rna helicases responsible for the cytoplasmic detection of viral rna. these receptors detect a variety of rna viruses, or dna viruses that express unusual rna species, many of which are responsible for a great number of severe and lethal diseases. host innate sentinel proteins involved in pathogen recognition must rapidly evolve in a dynamic arms race with pathogens, and thus are subjected to long-term positive selection pressures to avoid potential infections. using six codon-based maximum likelihood methods, we were able to identify specific codons under positive selection in each of these three genes. the highest number of positively selected codons was detected in mda5, but a great percentage of these codons were located outside of the currently defined protein domains for mda5, which likely reflects the imposition of both functional and structural constraints. additionally, our results support lgp2 as being the least prone to evolutionary change, since the lowest number of codons under selection was observed for this gene. on the other hand, the preponderance of positively selected codons for rig-i were detected in known protein functional domains, suggesting that pressure has been imposed by the vast number of viruses that are recognized by this rna helicase. furthermore, the rig-i repressor domain, the region responsible for recognizing and binding to its rna substrates, exhibited the strongest evidence of selective pressures. branch-site analyses were performed and several species branches on the three receptor gene trees showed evidence of episodic positive selection. in conclusion, by looking for evidence of positive evolutionary selection on mammalian rig-i-like receptor genes, we propose that a multitude of viruses have crafted the receptors biological function in host defense, specifically for the rig-i gene, contributing to the innate species-specific resistance/susceptibility to diverse viral pathogens. the mammalian innate immune system operates as the first line of defense against microbial pathogen invasion [1] [2] [3] . this system recognizes infectious agents through a limited number of germline-encoded pattern-recognition receptors (prrs) predominantly expressed on sentinel cells [2, [4] [5] [6] . the host prrs recognize and react with specific microbial components, known as pathogen-associated molecular patterns (pamps), which includes bacterial lipopolysaccharides, peptidoglycans, lipoteichoic acids and cell-wall lipoproteins, fungal β-glucan and viral nucleic acids [2, 3, 5, 6] . the host prrs exhibit distinct expression patterns and following sensing of their cognate ligands, activate specific signaling pathways that lead to the expression of a variety of inducible self-defense genes involved in the collective inflammatory and immune responses [2] . to date, four different classes of prrs have been identified, including the cell membrane-associated c-type lectin receptors (clrs), the toll-like receptors (tlrs) at the cell surface and at the membrane of intracellular vesicles (endosomes and lysosomes), and the cytoplasmic detection systems for intracellular pamps, namely the rig-i-like receptors (rlrs) and the nod-like receptors (nlrs) [3, [6] [7] [8] . the rlrs are a family of dexd/h box rna helicases critically and exclusively involved in the recognition of "nonself" rna from actively replicating viruses in the cytoplasm of infected cells [9] . this receptor family consists of three members, the retinoic acid-inducible gene-i (rig-i/ddx58), the melanoma differentiation associated factor 5 (mda5/ifih1) and the laboratory of genetics and physiology 2 (lgp2/dhx58) [10] [11] [12] [13] [14] . rig-i and mda5 share high sequence similarity and several structural features, including an n-terminal region consisting of tandem caspase activation and recruitment domains (cards), a central dexd/h box rna helicase domain and a c-terminal domain (ctd). the two n-terminally located cards function as a signaling and interaction domain with other card-containing proteins [13, 15, 16] . the helicase domain retains catalytic activity to bind and unwind double stranded rna (dsrna) in an atp hydrolysis-dependent manner [10, 17] . the ctd plays a predominant role in highaffinity binding with dsrna, encoding a repressor domain (rd) in rig-i, but not in mda5, which harbors an rd-like domain that does not participate in autoregulation [18] . these two rlrs detect a variety of both dna and rna viruses, particularly at early phase of viral replication, and signal the production of type i interferons (ifns) and induction of an antiviral response [10, 17] . the third element of the rlr family, the lgp2 protein, lacks any cards but contains the helicase domain and the ctd also harbors a rd. the role of lgp2 in anti-viral immunity is less clear, but it has been suggested in different studies to serve both as a negative and a positive regulator of rig-i and mda5 signaling [10, [19] [20] [21] . despite the similarities between rig-i and mda5, they were shown to play different roles in anti-viral immunity by recognizing and protecting from specific classes of viruses [22] . rig-i detects preferentially and most effectively short rna sequences marked with 5'-triphosphate group (5'-ppp) and blunt-end of short double-stranded rnas (dsrnas) or singlestranded rna (ssrna) hairpins [23] [24] [25] [26] [27] . as a key sensor of ssrna viruses, rig-i is implicated in the response to arenaviridae [28] , bunyaviridae [28] , filoviridae [28] , flaviviridae [18, 29] , orthomyxoviridae [22, 30] , paramyxoviridae [22, 28, 30, 31] and rhabdoviridae [22, 23] . on the other hand, mda5 is activated by high-molecular-weight poly(i:c) fragments [22, 32] , and also by long-duplex rnas from the genomes of dsrna viruses [30] or dsrna replication intermediates of positive-strand viruses, such as caliciviridae [33] , coronaviridae [34] and picornaviridae [22, 32] . regardless the virus recognition specificity by rig-i and mda5, some viruses are redundantly sensed by both rlrs, such as the west nile virus and the dengue virus [30, 35] . in addition to the extensively described recognition of rna viruses by rig-i and mda5, a role in anti-viral signaling in response to several dsdna viruses has also been observed. as an rna sensor, rig-i does not detect dna directly; however, not only do many dna viruses create dsrna products by virtue of convergent transcriptional units derived from opposite strands, but also the host rna polymerase iii can mediate the transcription of cytoplasmic dna templates (such as transfected poly da:dt) into dsrna containing 5'-triphosphate, which will activate rig-i and trigger the production of type i ifn [36, 37] . both epstein-barr virus and myxoma virus are detected by rig-i, while vaccinia virus is sensed by mda5 [38] [39] [40] . it is also likely that the precise rlrs utilized for the sensing of specific viruses also operate within cell-specific contexts as well. interaction between host and pathogen results in a dynamic arms race. whenever pathogens develop strategies to overtake the host immune system, the host proteins involved in pathogen recognition have to respond by evolving to avoid or reduce potential infections. these dynamics result in hostpathogen adaptation and counter-adaptation, which in turns lead to the rapid co-evolution of both parties. particularly for the host, this accelerated molecular evolution is often reflected in host defense genes that exhibit strong signatures of ongoing diversifying selection [41, 42] . because viruses are responsible for a great number of severe and lethal diseases, together with the important role that rlrs play in mammalian innate immune system, we expect that rig-i, mda5 and lgp2 genes may have been under intense selective pressures in all mammals. we have previously demonstrated that one other class of mammalian prrs, the tlrs, exhibit striking evidence of positive genetic selection as a result of selective pressures exerted by pathogens [43] . using six different codon-based maximum likelihood (ml) methods, we searched for evidence of long-term selective pressures in the three rlr genes present in the available sequenced mammalian genomes and, where possible, pinpoint positively selected residues that might be involved in the host-virus interactions that have shaped their rapid diversification. specific lineages subject to episodic positive selection have also been identified in the three rlr genes by using two different branch-site models. publicly available mammalian rig-i, mda5 and lgp2 gene sequences were collected from ensembl and ncbi databases (table s1 ) for phylogenetic and selection analyses. the nucleotide coding sequences for each of the three rlr gene orthologous were aligned and are represented in figure s1 (rig-i alignment), figure s2 (mda5 alignment) and figure s3 (lgp2 alignment). the translation into deduced protein sequences is also represented in figure s4 (rig-i alignment), figure s5 (mda5 alignment) and figure s6 ( lgp2 alignment). the inherent limitations of using solely publicly available mammalian rlrs sequences should be highlighted, although several studies have used the same source of data for general conclusions about other genes in mammals [43] [44] [45] [46] [47] [48] [49] . the analyses performed ahead use only an individual representative of each included species and therefore, any drawn conclusions should be carefully considered. in mammalian rig-i phylogenetic reconstruction, the monophyly of six of a total of eight taxonomic orders was observed ( figure 1 ). however, an interesting fact was registered for the two remaining orders, rodentia and lagomorpha, when the european rabbit (order lagomorpha) grouped with the rodent cluster. when looking carefully at the european rabbit rig-i deduced protein sequence ( figure s4 ), a great number of conserved regions between this species and the other mammalian species was observed, with the exception of a region between codons 840 and 879. this 40 amino acid domain region was unique to the european rabbit rig-i. we originally speculated that this difference might have been the result of a gene conversion event with adjacent genes. however, when we examined the genes that are chromosomally adjacent to european rabbit rig-i (ndufb6, topors and frp), no clear evidence of gene conversion was detected by the software gard [50, 51] . for the mammalian mda5 gene sequences alignment, a significant recombination breakpoint was detected (nucleotide 903; p<0.01). therefore, two ml trees were reconstructed for the resulting segments, one for the first 903 nucleotides ( figure 2a ) and another ml tree for the remaining 2211 nucleotides ( figure 2b ). the gene phylogeny was also reconstructed for the whole alignment without testing recombination ( figure 2c ) to compare its topology with the other two resulting trees. the monophyly of the eight taxonomic orders included in the mda5 alignment was roughly recovered, with the clear exception of chiroptera in figure 2a and primates in figure 2b . regarding the lgp2 gene, no clear evidence of recombination was detected. the ml tree obtained (figure 3 ) supported the monophyly of the nine mammalian orders collected for this gene. all the molecular evolutionary analyses in this study were performed for both the complete nucleotide alignments ( figure s1 , figure s2 and figure s3 ) and for a trimmed version of the same genes to remove alignment gaps. figure s7 (rig-i alignment), figure s8 (mda5 alignment) and figure s9 (lgp2 alignment) correspond to the alignments where gaps present in all sequences, with the exception of one or two, have been removed, while gaps present in only one or two sequences were kept. we observed no significant differences in the results when using one or the other alignment for each gene (data not shown), but ultimately only the results from the trimmed version are presented here. evidence for positive selection on mammalian orthologous for rig-i ( figure s7 ), mda5 ( figure s8 ) and lgp2 ( figure s9 ) genes was detected using paml package [54, 55] site-specific models m1a versus m2a and m7 versus m8. these models test at the codon level whether a hypothesis that allows for positive selection (models m2a and m8) is a better fit to the data when compared to a null neutral hypothesis (models m1a and m7). results on the likelihood ratio test (lrt) performed between the likelihood scores of the null neutral and alternative selection models for each gene is indicated in table 1 . models which allow for positive selection (m2a and m8) gave a significantly better fit to the data for both rig-i and lgp2 alignments, suggesting that at least some of the codons within each set of orthologous gene sequences are subject to positive selection [56] . since a recombination breakpoint was detected on the mda5 alignment, each resulting segment (identified as 1 st and 2 nd segments) was tested individually for paml package [54, 55] site-specific models. although the comparison between the null neutral site model m1a and the selection site model m2a did not allow for rejection of the null hypothesis of neutral selection, the comparison between the more powerful pair of site-specific models m7 (neutral) and m8 (selection) yielded significant lrts ( table 1) . the parris method [57] implemented in the datamonkey web server [52, 53] was also applied to each rlr trimmed gene alignment ( figure s7 , figure s8 and figure s9 ) to look for evidence of positive selection, but no selective pressures were detected in any of the three genes (table s2 ). for each orthologous gene sequences alignment, the tree length parameter is indicated in table 1 . higher values of tree length, i.e. the expected number of nucleotide substitutions per codon, correspond to higher sequence divergence [58, 59] . the tree length values registered for the three genes fell into an intermediate and realistic level of sequence divergence which confers power to the codon models indicated by the lrt scores and to the bayes empirical bayes (beb) approach for site-specific inference of positive selection [58, 60] . model m8 implemented in the paml package [54, 55] and datamonkey web server [52, 53] slac, fel, rel, meme and fubar methods [61] [62] [63] were used to detect specific codons under selection in the three rlr genes. based on the methodology adopted by other authors and in previous studies [43, 47, 48, 64] , only codons identified by at least three of the six used methods are considered to be under positive selection (table s3 ). since the breadth of species included in each alignment is wide, by applying several methods to detect codons under positive selection and by overlapping the results, we should be decreasing the incidence of false positives. a total of sixteen codons for rig-i (figure 4 ), twenty for mda5 ( figure 5 ) and ten for lgp2 ( figure 6 ) were identified as candidate codons under selective pressure. regarding their location in each corresponding protein, the greatest number of these codons are located in protein functional domains, more specifically, eleven out of the sixteen rig-i codons (~ 69%), ten out of the twenty mda5 codons (50%) and seven out of the ten lgp2 codons (70%). to estimate the percentage of positively selected codons in the three proteins, we used human deduced protein sequences as a reference. human lgp2 exhibited 1.47% (10/678) of codons under positive selection. higher values were obtained for human mda5 and rig-i, 1.95% (20/1025) and 1.73% (16/925) of codons under selective pressure, respectively. to detect signatures of episodic positive selection in specific lineages of each rlr orthologous gene sequences alignment we performed two branch-site model analyses. these models allow the selective pressure indicated by the nonsynonymous to synonymous substitution rate ratio ω (d n /d s ) to vary both across sites in the gene and across lineages on the tree [65] . since no biological hypothesis existed to specify a priori branches to be examined for positive selection, the branch-site model a implemented in the paml package [54, 55, 66] was applied to all species branches on each rlr gene phylogenetic tree. the lrt performed for each branch was significant for 2δlnl > 3.84 [55, 66] . our analyses suggest that nine species branches in rig-i are under selective pressure (table 2 and figure 4b ). branch-site model a was applied to the two mda5 trees resultant from recombination testing and, for each tree, positive selection has operated only in two species branches (table 3 and figure 5b ). for lgp2, a total of six species branches had significant lrts corresponding to candidate lineages under selection (table 4 and figure 6b ). some of the species branches recognized by the branch-site model a were also identified by the branch-site rel method [67] (table 5 ) available in the datamonkey web server [52, 53] . for both rig-i and mda5, two species branches were simultaneously identified by the two methods, consisting in dog (calu) and european rabbit (orcu) branches for rig-i ( figure 4b ) and giant panda (aime) and guinea pig (capo) branches for mda5 ( figure 5b ). only one lgp2 species branch, corresponding to the giant panda (aime), was simultaneously identified by the branch-site model a and the branch-site rel method ( figure 6b ). in a human population genetics context, the first study on rlrs evolutionary history and selective footprints has been recently published [68] . nevertheless, to the best of our knowledge, our study is the first that searches for selective pressure acting on mammalian orthologous of the three rlrs and, in fact, we provide strong evidence of positive selection as well as identify a significant number of codons under probable selective pressures for rig-i, mda5 and lgp2. furthermore, our results on the rig-i rd in specific hosts suggest that certain viruses might be exerting long-term selective pressures on this gene. tlrs adaptive evolution has been the most extensively characterized of all the prrs in several animal groups, such as echinoderms [69] , birds [70] and different mammals [43, 64, [71] [72] [73] [74] . studies on known viral-recognition tlrs (tlr3, 7, 8 and 9) of closely related animal groups, like birds [70] , or within species, like humans and chimpanzees [64] , demonstrated that this class of prrs exhibits a background of strong purifying selection to keep their functional integrity, albeit in the birds study [70] significant instances of positive selection acting on a few amino acid sites were identified. nevertheless, when different ml codon-based methods were applied to detect evidence of acting positive selection in broader groups where a great number of species are included, like primates [64] and mammals [43] , most of the viral tlrs exhibited strong evidence of positive selection and specific codons with a high probability of being under selection were identified. similarly, in our study the codon-based analyses strongly support that the three rlr genes, rig-i, mda5 and lgp2, have all been subject to long-term selective pressures during mammalian evolution. also, we applied several methods that identified specific rlr codons with a high probability of being under selection, which may directly perturb downstream immune responses in a particular host infected by a viral pathogen. one of the major concerns when using large scale divergent species to infer positive selection acting on a set of orthologous genes and across lineages on the phylogenetic tree is the effect of saturation in synonymous substitutions, since they may saturate quickly as sequences diverge [75, 76] . as codon models consider both synonymous and nonsynonymous substitutions, the saturation of the first could cloud the information provided by nonsynonymous substitutions. nevertheless, the sequence divergence in our study, inferred through rlrs tree length values, fit into intermediate and realistic levels that should confer power to the lrt used to compare nested codon-models and robustness to the branchsite models, and to the beb approach for codon-specific detection of positive selection [58] [59] [60] . also, in this study the mammalian species collected for each of the three rlr genes were nearly the same, thus this host species spectrum should not influence the codon-based analyses and our observations when comparing the level of selective pressure between genes. in our study, mammalian mda5 showed the highest number and percentage of positively selected codons. nonetheless, the percentage of mda5 codons under selection located in the known protein functional domains was the lowest. this should reflect the imposition of functional and structural constraints in mda5 defined domains. on the other hand, we observed that lgp2 is apparently less prone to evolutionary change with the lowest number and percentage of codons under selective pressures. for rig-i, the greatest number of codons identified as candidates under selective pressures were located in known protein functional domains, which might reveal the pressure imposed by the great number of viruses recognized by this rlr [13, 14] . vasseur and colleagues [68] came to different conclusions in their study, once they were focused on intraspecies (human populations) polymorphisms and on the comparison of nonsynonymous to synonymous rates ratio ω (d n /d s ) between human and chimpanzee lineages for the three rlr genes. rig-i exhibited a stronger evolutionary constraint [68] , as attested by its low levels of nucleotide diversity, population differentiation and low tolerance of amino acid-altering variation. it also exhibited a dramatic decay in the ω ratio when compared to the other two rlrs [68] . this is the expected outcome in evolutionary studies when using closely related species, or genetic information for population of the same species, which result in a background of strong purifying selection to keep the protein functional integrity. in the same study [68] , the strongest signatures of positive selection were found in mda5 and lgp2 by exhibiting higher ω ratios than (table 2 ) are colored in green; branches simultaneously identified by paml branch-site model a and hyphy branch-site rel method (table 5) rig-i. besides, mda5 and lgp2 also appear to have evolved adaptively in specific human populations, presenting a great number of nonsynonymous mutations in both helicase and cterminal domains [68] . rig-i and mda5 contain two n-terminal cards [10, 17] . the interaction of these domains with an adaptor protein named ips-1 (also known as mavs, visa or cardif) is a crucial process to activate a wide range of downstream response factors, including type i ifns and other essential anti-viral proteins to induce intracellular immune responses [77] . interestingly, in our study, the cards of both rig-i and mda5 concentrated a large number of the deduced codons under selection. some of these are radical in terms of their physicochemical properties changes across mammalian species (figure 4 and figure 5 ), strengthening the case for positive selection. since the two cards are fundamental for (table 3) are colored in green; branches simultaneously identified by paml branch-site model a and hyphy branch-site rel method (table 5) (table 4 ) are colored in green; branch colored in blue has been simultaneously identified by paml branch-site model a and hyphy branch-site rel method (table 5 ). (c) positively-selected codons are exhibited in the table and numbered according to the mammalian lgp2 deduced protein sequences alignment ( figure s6 downstream rig-i and mda5 signaling, which implies functional constraints, the observed variability across species can be perceived as a great structural plasticity for mammalian cards. the helicase domain in the rlr family is generally described as exhibiting affinity for dsrna [78] . the existence of six highly conserved sequence motifs within this domain is a characteristic of the helicase superfamily 2 which integrates dexd/h box rna helicases. also, different aspects of helicase functions have been assigned to specific motifs [79] . bamming and horvath [11] compared the amino acid sequences of the three human rlr helicase domains with the established consensus sequences of helicase families elements and, despite slight differences, the sequences in individual motifs are highly conserved within rig-i, mda5 and lgp2. indeed, in our study the six helicase motifs of the three proteins were evolutionary conserved (data not shown) in the mammalian species collected. minor alterations occur in some species, but the extent of those differences concerning the involvement in substrate interaction, signal transduction and/or the whole antiviral response profile, is difficult to predict. rig-i rd is responsible for recognizing and binding to its rna substrates in a 5'-triphosphate (5'-ppp)-dependent manner. besides, binding studies clearly established that the ppprna binding site resides within the rd [14, 26, 80] . the function described for rig-i rd makes our current results worthy of note, since the rd is the rig-i domain that exhibits the strongest evidence of trans-acting selective pressures (figure 4 ). whether these differences play a role in rig-i activation after binding to the rnas from different viral pathogens that infect distinct mammalian hosts is a complex question. nevertheless, we can assume that the rd variability in mammals is related to the fact that rig-i senses a large variety of viruses [13, 14] . the performance of branch-site models in our study imposes a careful interpretation of data, since only one representative element of each species was included. still, some branches of the three rlr phylogenetic trees exhibited evidence of positive selection. the two species under episodic positive selection on [23, 39] . such results suggest that these lethal pathogens, and possibly other re-occurring viral infections in these specific hosts, might be exerting longterm selective pressures on the susceptible host rig-i gene. therefore, the changes on rig-i sequences across species, with special focus on the rd as suggested above, should be the result of a co-evolutionary process between speciesspecific infecting viruses and this host rna sensor protein. by detecting the extension of acting positive selection on mammalian rlrs, this study provides further insights into their biological functions in host defense against viral pathogens in general. differences in these genes across mammalian species may consequently impact downstream immune responses and, as a result, contribute to the species-specific resistance/ susceptibility profiles against many diverse viral pathogens. the coding region of the three rlr genes, rig-i, mda5 and lgp2, were collected for different mammalian species from ncbi (http://www.ncbi.nlm.nih.gov) and ensembl (http:// www.ensembl.org/index.html) databases (table s1 ). each set of mammalian orthologous gene sequences was aligned with clustalw [81] implemented in bioedit v7.0.9 [82] . the nucleotide sequences alignment corresponding to each gene coding region is represented in figure s1 (rig-i alignment), figure s2 (mda5 alignment) and figure s3 ( lgp2 alignment). translation into protein sequences was performed using also bioedit [82] . figure s4 , figure s5 and figure s6 represent the alignments of the deduced protein sequences for rig-i, mda5 and lgp2, respectively. for the evolutionary analyses, representative alignment gaps in figure s1 , figure s2 and figure s3 had to be removed: gaps present in all sequences, with the exception of one or two, have been removed, while gaps present in only one or two sequences were kept. figure s7 (rig-i alignment), figure s8 (mda5 alignment) and figure s9 (lgp2 alignment) correspond to trimmed versions of the nucleotide sequences alignment of each rlr gene. the nucleotide sequences alignment of each gene was firstly tested for recombination, as this biological process can mislead phylogenetic and positive selection analyses [83] . we used the software gard (genetic algorithm for recombination detection) [50, 51] , implemented in the datamonkey web server [52, 53] , to detect possible recombination breakpoints on each alignment. the nucleotide substitution model for each phylogenetic reconstruction was indicated by the akaike information criterion (aic) implemented in jmodeltest v0.1.1 [84] . regarding the rig-i gene one breakpoint was identified, but it was not supported by the kishino-hasegawa test. therefore, the complete alignment was used for the gene phylogeny reconstruction and gtr+g nucleotide substitution model was indicated as the best-fitting model. on the other hand, the software gard found evidence of two breakpoints in the mda5 gene alignment. however, only one of the breakpoints (nucleotide 903) reflected a significant topological incongruence (kishino-hasegawa test, p<0.01), suggesting that the multiple tree model can be preferred over the single tree model. we reconstructed mda5 phylogeny for the first 903 nucleotides of the mammalian alignment as also for the remaining 2211 nucleotides. to compare the different mda5 trees topology, we also used the complete alignment (no recombination testing) to reconstruct the gene phylogeny and gtr+g nucleotide substitution model was indicated by the aic as the best-fit. for the mda5 segments which resulted from recombination detection, the best-fitting nucleotide substitution models were tim3+g (first segment) and tim3+i+g (second segment). finally, we found no evidence of recombination for the lgp2 gene alignment. the best-fitting nucleotide substitution model determined for this alignment was the tpm2uf+i+g model. ml phylogenetic reconstruction was performed for the three genes using garli v2.0 (genetic algorithm for rapid likelihood inference) [85] . the analyses were performed with 1,000,000 generations and 1,000 bootstrap searches. ml trees were displayed using figtree v1.3.1 (http://tree.bio.ed.ac.uk/). codon substitution models implemented in the codeml program in paml v4.4 (phylogenetic analysis by maximum likelihood) package [54, 55] were applied to the trimmed alignments of rig-i ( figure s7 ), mda5 ( figure s8 ) and lgp2 ( figure s9 ). the codon frequency model f3x4 was fitted to all the alignments. two pairs of site-specific models were used, m1a (nearly neutral) versus m2a (selection) and m7 (neutral, beta) versus m8 (selection, beta & ω). in these comparisons, m1a and m7 neutral models (null hypothesis) do not admit positive selection, while m2a and m8 alternative models allow positive selection. a lrt with 2 degrees of freedom was performed, where a significant lrt demonstrates that the selection model fits better than the neutral model [56, 86, 87] . from the hyphy software available on the datamonkey web server [52, 53] , the parris method [57] was also applied to detect if a proportion of sites in each rlr alignment evolved with ω (d n /d s ) > 1. six different codon-based ml methods were applied to detect codons under positive selection on mammalian rig-i, mda5 and lgp2 trimmed alignments, and based on the methodology adopted by other authors and in previous studies [43, 47, 64] , only codons identified by at least three of the six used methods were considered to be under positive selection. model m8 from paml package [54, 55] was one of the codonbased methods used to detect codons under positive selection, and a bayes empirical bayes (beb) approach was employed to detect codons with a posterior probability >90% of being under selection [88] . five other methods, using hyphy software implemented in the datamonkey web server [52, 53] , were also applied to detect sites under selection for the three genes: the single likelihood ancestor counting (slac) method, the fixed effect likelihood (fel) method, the random effect likelihood (rel) method [61] and the recently described mixed effects model of evolution (meme) [62] and fast unbiased bayesien approximation (fubar) [63] methods. to avoid a high falsepositive rate [61] , sites with p-values <0.1 for slac, fel and meme models, bayes factor >50 for rel model and a posterior probability >0.90 for fubar were accepted as candidates for selection. two branch-site models allowing ω ratios to vary both among lineages and amino acid sites were performed: the paml branch-site model a [66] and the hyphy branch-site rel method [67] . when performing paml branch-site model a [66] , every species branch was analyzed as a foreground branch independently. for each analysis of a foreground branch, the remaining lineages were denominated as background branches. in branch-site model a, three ω ratios are assumed for foreground (0 < ω 0 < 1, ω 1 = 1, ω 2 > 1) and two ω ratios for background (0 < ω 0 < 1, ω 1 = 1). the null model is the same as model a, but ω 2 = 1 is fixed [66] . the beb approach was also used to calculate the posterior probability of a specific codon site and to identify those most likely to be under positive selection (posterior probability >90%) [88] . on the other hand, the branch-site rel method [67] was applied to identify branches where a proportion of sites evolved under episodic diversifying selection. figure s1 . mammalian rig-i nucleotide coding region sequences alignment. rig-i nucleotide coding region sequences for twenty-six mammalian species were collected from ensembl and ncbi databases, and aligned with clustalw implemented in bioedit. the symbol "." represents the same nucleotide as the reference sequence of human rig-i gene, "?" symbolizes an undetermined nucleotide and "-" represents a gap or deletion in the alignment. the figure s2 . mammalian mda5 nucleotide coding region sequences alignment. mda5 nucleotide coding region sequences for twenty-six mammalian species were collected from ensembl and ncbi databases, and aligned with clustalw implemented in bioedit. the symbol "." represents the same nucleotide as the reference sequence of human mda5 gene, "?" symbolizes an undetermined nucleotide and "-" represents a gap or deletion in the alignment. the lgp2 nucleotide coding region sequences for thirty mammalian species were collected from ensembl and ncbi databases, and aligned with clustalw implemented in bioedit. the symbol "." represents the same nucleotide as the reference sequence of human lgp2 gene and "-" symbolizes a gap or deletion in the alignment. the figure s4 . mammalian rig-i deduced protein sequences alignment. rig-i deduced protein sequences for twenty-six mammalian species were collected from ensembl and ncbi databases, and aligned with clustalw implemented in bioedit. the symbol "." represents the same codon as the reference sequence of human rig-i protein, "?" symbolizes an undetermined codon and "-" represents a gap or deletion in the alignment. the figure s5 . mammalian mda5 deduced protein sequences alignment. mda5 deduced protein sequences for twenty-six mammalian species were collected from ensembl and ncbi databases, and aligned with clustalw implemented in bioedit. the symbol "." represents the same codon as the reference sequence of human mda5 protein, "?" symbolizes an undetermined codon and "-" represents a gap or deletion in the alignment. the lgp2 deduced protein sequences for thirty mammalian species were collected from ensembl and ncbi databases, and aligned with clustalw implemented in bioedit. the symbol "." represents the same codon as the reference sequence of human lgp2 protein and "-" symbolizes a gap or deletion in the alignment. the used abbreviations correspond, by order of appearance, to the following species: hosa -human; patr -chimpanzee; papa -bonobo; gogo -gorilla; poab -orangutan; mamu -rhesus macaque; sabo -blackcapped squirrel monkey; caja -marmoset; mimu -mouse lemur; otga -bushbaby; bota -cow; ovar -sheep; susc -pig; tutr -dolphin; mylu -little brown myotis; ptva -large flying fox; ptal -black flying fox; loaf -elephant; mupu -ferret; aime -giant panda; calu -dog; feca -cat; eqca -horse; ocpr -american pika; orcu -european rabbit; ictr -squirrel; crgr -chinese hamster; mumu -mouse; rano -rat; capo -guinea pig. (tif) figure s7 . mammalian rig-i nucleotide trimmed sequences alignment. rig-i nucleotide trimmed sequences for twenty-six mammalian species were collected from ensembl and ncbi databases, and aligned with clustalw implemented in bioedit. the symbol "." represents the same nucleotide as the reference sequence of human rig-i gene, "?" symbolizes an undetermined nucleotide and "-" represents a gap or deletion in the alignment. the mammalian mda5 nucleotide trimmed sequences alignment. mda5 nucleotide trimmed sequences for twenty-six mammalian species were collected from ensembl and ncbi databases, and aligned with clustalw implemented in bioedit. the symbol "." represents the same nucleotide as the reference sequence of human mda5 gene, "?" symbolizes an undetermined nucleotide and "-" represents a gap or deletion in the alignment. the lgp2 nucleotide trimmed sequences for thirty mammalian species were collected from ensembl and ncbi databases, and aligned with clustalw implemented in bioedit. the symbol "." represents the same nucleotide as the reference sequence of human lgp2 gene and "-" symbolizes a gap or deletion in the alignment. the the immune response of drosophila pathogen recognition and innate immunity the roles of tlrs, rlrs and nlrs in pathogen recognition approaching the asymptote? evolution and revolution in immunology innate immunity recognition of microorganisms and activation of the immune response dendritic cells and c-type lectin receptors: coupling innate to adaptive immune responses regulation of rlr-mediated innate immune signaling--it is all about keeping the balance innate immune recognition of viral infection shared and unique functions of the dexd/h-box helicases rig-i, mda5, and lgp2 in antiviral innate immunity regulation of signal transduction by enzymatically inactive antiviral rna helicase proteins mda5, rig-i, and lgp2 rig-i-like receptors: cytoplasmic sensors for non-self immune signaling by rig-i-like receptors intracellular pathogen detection by rig-i-like receptors activation of rig-i-like receptor signal transduction sensing of viral nucleic acids by rig-i: from translocation to translation the rna helicase rig-i has an essential function in doublestranded rna-induced innate antiviral responses regulation of innate antiviral defenses through a shared repressor domain in rig-i and lgp2 the rna helicase lgp2 inhibits tlr-independent sensing of viral replication by retinoic acid-inducible gene-i loss of dexd/h box rna helicase lgp2 manifests disparate antiviral responses lgp2 is a positive regulator of rig-i-and mda5-mediated antiviral responses differential roles of mda5 and rig-i helicases in the recognition of rna viruses ) 5'-triphosphate rna is the ligand for rig-i rig-i-mediated antiviral responses to single-stranded rna bearing 5'-phosphates recognition of 5' triphosphate by rig-i helicase requires short blunt double-stranded rna as contained in panhandle of negativestrand virus preference of rig-i for short viral rna molecules in infected cells revealed by nextgeneration sequencing structural basis of innate immune recognition of viral processing of genome 5' termini as a strategy of negativestrand rna viruses to avoid rig-i-dependent interferon induction regulating intracellular antiviral defense and permissiveness to hepatitis c virus rna replication through a cellular rna helicase, rig-i distinct rig-i and mda5 signaling by rna viruses in innate immunity cytosolic 5'-triphosphate ended viral leader transcript of measles virus as activator of the rig i-mediated interferon response essential role of mda-5 in type i ifn responses to polyriboinosinic:polyribocytidylic acid and encephalomyocarditis picornavirus mda-5 recognition of a murine norovirus murine coronavirus mouse hepatitis virus is recognized by mda5 and induces type i interferon in brain macrophages/microglia establishment and maintenance of the innate antiviral response to west nile virus involves both rig-i and mda5 signaling through ips-1 rig-i-dependent sensing of poly(da:dt) through the induction of an rna polymerase iii-transcribed rna intermediate rna polymerase iii detects cytosolic dna and induces type i interferons through the rig-i pathway eb virusencoded rnas are recognized by rig-i and activate signaling to induce type i ifn rig-i mediates the co-induction of tumor necrosis factor and type i interferon elicited by myxoma virus in primary human macrophages activation of mda5 requires higher-order rna structures generated during virus infection the evolution of parasite recognition genes in the innate immune system: purifying selection on drosophila melanogaster peptidoglycan recognition proteins two-stepping through time: mammals and viruses signatures of positive selection in toll-like receptor (tlr) genes in mammals adaptive evolution of young gene duplicates in mammals adaptive evolution of the matrix extracellular phosphoglycoprotein in mammals positive selection and the evolution of izumo genes in mammals evolution and divergence of the mammalian samd9/samd9l gene family maximumlikelihood approaches reveal signatures of positive selection in il genes in mammals computational analyses of an evolutionary arms race between mammalian immunity mediated by immunoglobulin a and its subversion by bacterial pathogens automated phylogenetic detection of recombination using a genetic algorithm gard: a genetic algorithm for recombination detection datamonkey: rapid detection of selective pressure on individual sites of codon alignments datamonkey 2010: a suite of phylogenetic analysis tools for evolutionary biology paml: a program package for phylogenetic analysis by maximum likelihood paml 4: phylogenetic analysis by maximum likelihood likelihood ratio tests for detecting positive selection and application to primate lysozyme evolution robust inference of positive selection from recombining coding sequences accuracy and power of the likelihood ratio test in detecting adaptive molecular evolution the branch-site test of positive selection is surprisingly robust but lacks power under synonymous substitution saturation and variation in gc a bayesian model comparison approach to inferring positive selection not so different after all: a comparison of methods for detecting amino acid sites under selection detecting individual sites subject to episodic diversifying selection fubar: a fast, unconstrained bayesian approximation for inferring selection adaptation and constraint at toll-like receptors in primates multiple hypothesis testing to detect lineages under positive selection that affects only a few sites evaluation of an improved branchsite likelihood method for detecting positive selection at the molecular level a random effects branch-site model for detecting episodic diversifying selection the selective footprints of viral pressures at the human rig-i-like receptor family dynamic evolution of toll-like receptor multigene families in echinoderms molecular evolution of the toll-like receptor multigene family in birds evolutionary dynamics of human toll-like receptors and their different contributions to host defense molecular evolution of bovine toll-like receptor 2 suggests substitutions of functional relevance natural selection in the tlr-related genes in the course of primate evolution a history of recurrent positive selection at the toll-like receptor 5 in primates synonymous nucleotide divergence: what is "saturation synonymous substitutions substantially improve evolutionary inference from highly diverged proteins ips-1, an adaptor triggering rig-i-and mda5-mediated type i interferon induction nonself rna-sensing mechanism of rig-i helicase and activation of antiviral immune responses sf1 and sf2 helicases: family matters the c-terminal regulatory domain is the rna 5'-triphosphate sensor of rig-i clustal w: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice bioedit: a user-friendly biological sequence alignment editor and analysis program for windows 95/98/nt the effect of recombination on the accuracy of phylogeny estimation jmodeltest: phylogenetic model averaging genetic algorithm approaches for the phylogenetic analysis of large biological sequence datasets under the maximum likelihood criterion likelihood models for detecting positively selected amino acid sites and applications to the hiv-1 envelope gene codonsubstitution models for heterogeneous selection pressure at amino acid sites bayes empirical bayes inference of amino acid sites under positive selection key: cord-301362-f3lp10lm authors: delgui, laura r.; colombo, maría i. title: a novel mechanism underlying the innate immune response induction upon viral-dependent replication of host cell mrna: a mistake of +srna viruses' replicases date: 2017-01-20 journal: front cell infect microbiol doi: 10.3389/fcimb.2017.00005 sha: doc_id: 301362 cord_uid: f3lp10lm viruses are lifeless particles designed for setting virus-host interactome assuring a new generation of virions for dissemination. this interactome generates a pressure on host organisms evolving mechanisms to neutralize viral infection, which places the pressure back onto virus, a process known as virus-host cell co-evolution. positive-single stranded rna (+srna) viruses are an important group of viral agents illustrating this interesting phenomenon. during replication, their genomic +srna is employed as template for translation of viral proteins; among them the rna-dependent rna polymerase (rdrp) is responsible of viral genome replication originating double-strand rna molecules (dsrna) as intermediates, which accumulate representing a potent threat for cellular dsrna receptors to initiate an antiviral response. a common feature shared by these viruses is their ability to rearrange cellular membranes to serve as platforms for genome replication and assembly of new virions, supporting replication efficiency increase by concentrating critical factors and protecting the viral genome from host anti-viral systems. this review summarizes current knowledge regarding cellular dsrna receptors and describes prototype viruses developing replication niches inside rearranged membranes. however, for several viral agents it's been observed both, a complex rearrangement of cellular membranes and a strong innate immune antiviral response induction. so, we have included recent data explaining the mechanism by, even though viruses have evolved elegant hideouts, host cells are still able to develop dsrna receptors-dependent antiviral response. a novel mechanism underlying the innate immune response induction upon viral-dependent replication of host cell mrna: a mistake of +srna viruses' replicases introduction eukaryotic cells are able to detect viruses at multiple steps and benefit from redundant mechanisms with the aim of limiting viral infections. recognition of viral double-strand rna (dsrna) molecules by intracellular toll-like receptors (tlrs) or retinoic acid inducible gene i-like receptors (rlrs) is a central event which entails the early steps of the immune response elicited during viral infections. a functional anti-viral role of tlr3 has been demonstrated for several animal and human viruses, recently reviewed in matsumoto et al. (2011) . the rlrs family, including the retinoic acid inducible gene i (rig-i), the melanoma differentiation-associated gene 5 (mda5) and laboratory of genetics and physiology 2 (lgp2), represent another powerful anti-viral tool parallel to that comprised by tlr3. rig-i, the leading member of the rlrs, is activated upon dsrna recognition generating the production of an anti-viral state in the infected-cell and in the surrounding tissue. its specific activity has been extended reviewed by kell and gale (2015) . since we consider tlr3, rig-i, and mda-5 as "major sentries" of viral dsrna molecules, a brief revision of their specific anti-viral action will be presented in this report. as forced intracellular parasites, viral agents relay on the host cell biosynthetic pathway in order to follow their replication program to generate new viral progeny. since +srna viruses generate dsrna molecules during their replication process, they have the necessity of hiding their genome from the host cellular dsrna sentries. so, the induction of specialized membranous niches, often forming organelle-like structures, is a common feature among these viruses. with the aide of pioneering classical electron microscopy (em) and, during the past few years the most sophisticated electron tomography, several 3-d architecture of viral replication factories have been deciphered (for a technical review on electron tomography, see frey et al., 2006) . despite several differences among host range, viral structure, genome organization or membrane-donor organelles from the cell, these analyses revealed that +srna viruses are able to induce two types of membranous modifications as replicative niches: invaginated vesicles or spherules or a double membrane vesicle type. in this review we will describe, employing a prototype, well-studied, viral agent for each type of membrane alteration, how the virus builds its hideout to shelter from dsrna receptors. however, a concern arises when observing that while +srna viruses build their replication niches associated to membranes, the host cells are still able to establish an antiviral response, mediated by the cellular receptors that the viruses are intended to hide from. regarding this important question, a recent proposed mechanism will be included to clarify this intriguing crossroad, a paradigmatic scenario of virus-host cell co-evolution process. tlrs are type i transmembrane domain family of proteins with a tripartite structure. they consist of an amino (n)terminal ectodomain containing leucine rich repeats responsible for ligand recognition, a single transmembrane spanning region and a carboxyl (c)-terminal globular cytoplasmic tolllike/interleukin-1 (il-1) receptor (tir) involved in downstream signaling activation (gay and gangloff, 2007) . tlrs recognize pathogen-associated molecular patterns (pamps) derived from microorganisms and induce an inflammatory response. due to its ability to recognize dsrna molecules, the tlr3 is the "cellular major sentinel" against these agents (thompson et al., 2011) . tlr3 recognizes genomic dsrna or dsrna replication intermediates present in virus-infected cells independently of the sequence (alexopoulou et al., 2001) . tlr3 is broadly expressed in immune and non-immune cells and has a high level of conservation among vertebrates (mikami et al., 2012) . after synthesis, tlr3 is retained in the endoplasmic reticulum (er) in unstimulated cells and translocate to the endolysosomal compartment when a dsrna-stimulation occurs, in a process where the er membrane protein uncoordinated 93b1 and the leucine-rich repeat containing-protein 59/p34 play an important role (kim et al., 2008; tatematsu et al., 2015) . although it has been shown that dsrna-sensing tlrs are translocated from the er to lysosomes, upon ligand stimulation, the molecular mechanism of ligand-dependent trafficking of the tlrs is largely unknown. once within the endolysosomal compartment, tlr3 is able to recognize dsrna longer than ∼40 bp for robust stimulation, largely through the minor groove and the nearby phosphate backbone explaining why recognition is independent of rna sequence. genomic nucleic acid material from internalized dsrna viruses such as reoviruses consisting of long stretches of dsrna represent molecular structures that are absent in non-infected eukaryotic cells. endogenous rnas forming secondary double-stranded structures that are released after necrosis and tissue damage after viral infection represent another source of dsrna molecules reaching the endosomes, inducing host-derived dsrna-mediated inflammatory responses through tlr-3 recognition (kawai and akira, 2010) . however, dsrna-tlr3 high affinity binding is strikingly dependent on the acidic environment since protonation of histidine on the tlr3 surface is required to allow ionic interaction . the dsrna-tlr3 structure has been recently elucidated revealing that the dsrna molecules induce the dimerization of tlr3 ectodomain (ecd) inside the vesicle (liu et al., 2008) . moreover, the proximity of two ecd generates the dimerization of the cytosolic tir domains . additionally to recognition and dimerization, phosphorylation of the tyr 759 and tyr 858 residues in the cytoplasmic domain of tlr3 are required for triggering the recruitment of tir domaincontaining adaptor protein interferon-β (trif) to the tir domain of tlr3 (sarkar et al., 2007) . finally, trif recruitment results in stimulation of the transcription factors irf3 (interferon regulatory transcription factor 3), nf-κβ (nuclear factor-κβ) and ap-1 (activator protein 1) thought two different branches (alexopoulou et al., 2001; sato et al., 2003) , which finally generates three major antivirals responses: (i) type i interferon production (inf-α/β); (ii) cytopathic effect or infected-cell death; and (iii) generation of pro-inflammatory environment by the activation of nf-κβ and ap-1. during co-evolution with the cell host, viruses have evolved mechanisms to avoid cell responses against viral infections for their own success. indeed, hiding dsrna molecules represents a powerful tool to avoid a harsh cellular war against viruses' replication that initiates after tlr3 recognition and activation. rig-i-like receptors (rlrs) mainly include the cytosolic retinoic acid-induced gene i (rig-i) and the melanoma differentiationassociated gene 5 (mda5), both sharing the same molecular architecture consisting in a conserved "helicase" core connected to two caspase activation and recruitment domains (cards) at the n-terminus, and an rna binding domain known as c-terminal domains (ctd) (yoneyama et al., 2005) . in the absence of an rna trigger, rig-i is in the cytoplasm in a resting state, in which the cards fold back to the c-terminal portion. upon binding of non-self duplex rna, rig-i hydrolyses atp and undergoes extensive structural rearrangements to reach the fully activated state displaying the n-terminal cards and initiating the antiviral signaling cascade (yoneyama et al., 2004; fujita et al., 2007; saito et al., 2007) . rna recognition by rig-i involves three different rig-i domains (hel1, hel2i, and the ctd) that together clasp the duplex rna, enwrapping it within a network of interactions that are dominated by polar contacts (luo et al., 2011) . accurately defining the rig-i stimulatory rna structure and sequence remains controversial. however, it seems clear that short (<300 bp) dsrna panhandle structures are stimulatory if they contain exposed 5 ′ -triphosphate (5 ′ -ppp) and blunted 5 ′ end (hornung et al., 2006; kato et al., 2006; pichlmair et al., 2006; marq et al., 2011) . an overview of rig-i interaction with viruses from different genera has been recently reviewed by kell and gale (2015) . on the other hand, mda5 binds to long dsrna (>1000 bp) with no end specificity (hornung et al., 2006; kato et al., 2006; pichlmair et al., 2006) and by a different mechanism, which leads mda5 to form a filament along dsrna, initiated from internal sites in the dsrna rather than from the ends (peisley et al., 2011 (peisley et al., , 2013 berke and modis, 2012) . both rlrs form large oligomeric structures around dsrna molecules that serve as platforms for recruitment and nucleation of mitochondrial antiviral signaling protein (mavs) (peisley et al., 2013; xu et al., 2014) . mavs form a polymeric structure as well, self-propagating drawing soluble monomers from the cytoplasm into the growing polymer (hou et al., 2011) . the polymeric form of mavs is tethered to the mitochondrial membrane where it triggers the activation of the cytosolic kinases ikb-ε (ikkε) and tank-binding kinase-i (tbki), which in turn activate nf-κβ and irf3, respectively (fitzgerald et al., 2003; yoneyama et al., 2004) . activated nfκβ and irf3 are translocated into the nucleus where they induce expression of type i interferon and other inflammatory antimicrobial molecules. viruses replicate in the host cell to generate new infectious virions. to overcome the innate antiviral response, viral particles include ways to circumvent inf-α/β production achieved by blocking the rlr pathway in its upstream portion to avoid dsrna-mediated activation of rlrs, further evidencing the potency of such pamp in triggering a robust innate immune response (bowie and unterholzner, 2008) . indeed, hiding dsrna molecules from rlrs in compartmentalized microenvironments inside the cytoplasm comprise a powerful strategy to tackle rlrs-induced antiviral response. semiliki forest virus (sfv) belongs to the togaviridae family, which comprises alphaviruses and the etiologic agent of rubella, the rubella virus. this family is, to date, the sole group of +srna viruses that modify endosomal and lysosomal membranes to replicate their genomes (froshauer et al., 1988; kujala et al., 1999) . alphaviruses are a genus of viruses generally transmitted by mosquito vectors, which replicate inside the cytoplasm of both, invertebrate and vertebrate cells. they can infect a variety of hosts including small and large mammals, birds, and humans (reviewed by kuhn, 2013) . among alphaviruses there are several important pathogens affecting human and other animals, including the encephalitogenic alphaviruses that affect horses (e.g., western, eastern, and venezuelan equine encephalitis viruses) and the recently re-emerging chikungunya virus (chikv). chikv re-emerged in 2004 to cause outbreaks of millions of cases in countries around the indian ocean area, in asia, and recently the caribbean (http://www.cdc.gov/ chikungunya/geo). chik causes painful arthritis with symptoms that can persist for years, and can also cause neurological complications and neonatal encephalitis (schwartz and albert, 2010) . sfv and chikv are very similar in terms of molecular and cell biology, e.g., regarding replication and molecular interactions, but are strikingly different regarding pathology: chikv is a relevant human pathogen, while sfv is a lowpathogenic model virus, albeit neuropathogenic in mice (atkins et al., 1999) .there are currently no effective vaccines or treatments for human alphavirus infections. alphaviruses are small-enveloped particles that enter the cell by clathrin-mediated endocytosis (reviewed by kielian et al., 2010) , followed by fusion of the virus envelope with early endosomal membranes leading to nucleocapsid core delivery into the cytoplasm (gibbons et al., 2004) . the viral nucleocapsid is disassembled with the aide of ribosomes, which have affinity for the capsid protein (singh and helenius, 1992) . the sfv genome, ∼11.5 kb long with a 5 ′ cap structure and 3 ′ poly (a) sequence, is translated into a replicase polyprotein, which consists in four non-structural proteins (nsp1-nsp4), involved in viral rna synthesis, and five structural proteins. the replicase complex [rna-dependent rna polymerases (rdrp)] is remodeled by the viral protease nsp2 through sequential cleavages to give rise to the four different units nsp1, nsp2, nsp3, and nsp4 . these four units form a macromolecular arrangement responsible of viral genome replication, which also contains rna originated from newly synthesis (kujala et al., 2001) . however, the rdrp core is formed by nsp4, which harbors a conserved catalytic gly-asp-asp triad (kamer and argos, 1984) . together, they give rise to replication complexes (rcs) colocalizing to bulb-shaped membrane invaginations designated spherules (kujala et al., 2001; salonen et al., 2003; spuul et al., 2010) . these spherules, thanks to their homogenous size, defined morphology and electron density in infected-cells, were firstly described between late 1960s and early 1970s (friedman and berezesky, 1967; grimley et al., 1968; friedman et al., 1972) . at that time, the spherules were described to have a diameter of ∼50 nm and were found located in the membranes of large cytoplasmic compartments, which were termed virus-induced cytopathic vacuole of type i (cpv-i) (grimley et al., 1968 ). subsequently, froshauer et al. demonstrated that the spherules contained endosomal and lysosomal markers and, employing electron microscopy (em), they observed that the luminal side of the spherule was linked to the cytoplasm by a pore from which electron-dense structures seems to diffuse to the cytoplasm (froshauer et al., 1988) . during the subsequent decades, a great amount of effort has been done to address the biogenesis and dynamics of the cpv-i and, nowadays, a whole picture of the mechanism involved in endosomal and lysosomal membrane modification by sfv has been nicely depicted. the nsps are synthesized from the viral positive-sense rna genome as one polyprotein, which gives rise to four nonstructural proteins generated by cleavages catalyzed by nsp2. of the four nsps, only nsp1, thanks to an amphipathic helix spotted in the central part of the polypeptide, is the only non-structural protein that interacts with membranes (peranen et al., 1995; ahola et al., 1999; lampio et al., 2000) . nsp1 has specific affinity for negatively charged phospholipids explaining its predominant localization to plasma membrane (pm), where those lipids are enriched. the membrane association of nsp1 is mediated through direct interaction of an amphipathic helix with anionic phospholipids and is increased by post-translational palmitoylation of one to three cysteine residues at positions 418-420 (laakkonen et al., 1996; ahola et al., 1999) . it has recently been demonstrated that nsp1 can only become palmitoylated after associating with membranes via the amphipathic peptide and that this interaction is essential for virus replication (spuul et al., 2007) . with the aim of following the distribution of sfv rcs spuul et al. performed double labeling and em studies in a time course infection (spuul et al., 2010) . the authors discovered that the rcs were predominantly at the pm where numerous typical spherules on the cell surface were observed starting from 1 h post-infection (p.i.), demonstrating that these structures were forming from the pm. from 2 to 4 h p.i., the rcs components were localized to small intracellular vesicles, and then later in the infection, the dsrna localized to large vacuoles in the perinuclear area, the so called cpv-i. the authors also observed that pmassociated spherules trafficking was strongly dependent upon the activity of class i phosphatidylinositol 3-kinase and a functional actin-myosin network, suggesting that the spherules were an unusual type of endocytic cargo (spuul et al., 2010) . related to this critical step, these authors, together with others, have previously published, employing em, the presence of spherules associated to pm-derived vesicles morphologically similar to endocytic vesicles at the stage of internalization (froshauer et al., 1988; kujala et al., 2001) . employing a fluorescent recombinant sfv (sfv-zsg), and lysotracker stained cells, spuul et al. observed that the spherules were internalized from the pm in neutral vesicles which underwent several fusion events to be delivered, via a microtubule-based transport, to larger acidic organelles located in the perinuclear area to generate the final stable and static compartment cpv-i, containing hundreds of rcs on their surfaces. the average size of cpv-i reaches 2 µm at 12 h p.i. significantly exceeding the sizes of late endosomes and lysosomes in non-infected cells (luzio et al., 2007) indicating that alphaviruses have evolved a mechanism to generate and stabilize membranes of the endolysosomal compartment for their replication. a nice graphic schematizing a model for the alphavirus rcs trafficking and biogenesis of cpv-i has been depicted by spuul et al. (2010) . members of the togaviridae family, as mentioned above, induce viral replication factories with spherule morphology usurping the endosomal and lysosomal pathway from the cell. even though no 3-d reconstruction of an alphavirus replication niche has been published to date, spherule structures associated to virus replication has been nicely described for sindbis virus (frolova et al., 2010) and rubella virus (fontana et al., 2010) , in addition to sfv. the first 3-d reconstruction coming from a +srna viral replication niche was published by kopek et al. (2007) . electron tomography of flock house virus (fhv)-infected cells uncovered invaginations or spherules on the external mitochondrial membrane (omm). similar to alphaviruses, the spherules detected in fhv-infected cells were about 50 nm in diameter (miller et al., 2001) and contains a membranous neck with an internal diameter of around 10 nm connecting the spherule lumen with the cytoplasm (kopek et al., 2007) . contrary to the replication niches of these +srna viruses, replication factories of two members of the flaviviridae family, west nile virus (wnv) and dengue virus (denv), are derived from the endoplasmic reticulum (er). recently, welsch et al. reported a detailed study deciphering the 3-d architecture of virus-induced membrane rearrangements involved in denv replication (welsch et al., 2009 ). the authors employed several em techniques including electron tomography (et) to obtain the 3-d analysis of the virus-induced vesicles revealing that they are invaginations of the er membrane, connected to the cytosol through a pore that may regulate import of factors required for rna replication as well as export of newly synthesized genomes to be used for translation or virus assembly. additionally, and thanks to the powerful et technique, the authors demonstrated the presence of virus budding sites in close proximity to the pores of replication vesicles, providing for the first time a direct visualization, in 3d, of this process (welsch et al., 2009 ). vesicle formation is probably induced by the non-structural protein 4a (ns4a), which appears to contain a central peripheral membrane domain that intercalates into the luminal leaflet of the er membrane . regarding plant viruses, even though 3-d structural information on plant +srna virus-infected cells is limited, virus-host interactions have been extensively studied for brome mosaic virus (bmv), a member of the bromoviridae family, or the beet black scorch virus (bbsv), a member of the tombusviridae family, both generating convolution and invagination of the er membrane and neck-like channels connecting the interiors of spherules to the cytoplasm to replicate inside (bamunusinghe et al., 2011; cao et al., 2015) . polioviruses belong to the genus enterovirus of picornaviridae family. viruses in this family have nonenveloped particles with a tightly packaged, non-segmented, single-stranded, ssrna. among its many members are numerous important human and animal pathogens, such as poliovirus, hepatitis a virus, foot and mouth disease virus (fmdv), enterovirus 71, and rhinovirus (racaniello, 2013) . poliovirus particles, as other members of the family, consist of an icosahedral protein shell surrounding the naked rna genome of around 7500 nucleotides. the basic building block of the picornavirus capsid is a protomer, which contains one copy each of four structural proteins: vp1, vp2, vp3, and vp4. the shell is formed by vp1-vp3, and vp4 lies on its inner surface (fry and stuart, 2010) . the viral arn encodes a single poliprotein, which is cleaved by virus-encoded proteinases to yield 11-15 final polypeptides. the polyprotein contains three regions: p1, p2, and p3. the p1 region encodes the viral capsid proteins, whereas the p2 and p3 regions encode proteins involved in protein processing and genome replication (stanway, 1990) . the initial attachment of the virion to the host cell plasma membrane involves the receptor cd155, a type i transmembrane protein member of the immunoglobulin superfamily of proteins (mendelsohn et al., 1989) , causing a conformational change in the capsid which leads to viral internalization via a clathrinindependent endocytic process (fricks and hogle, 1990; tuthill et al., 2006) . upon infection, the virus genome replication occurs in the cytoplasm associated to complex membranous replication factories. the first step in genome replication is copying of the positive stranded rna to form a negative stranded intermediate; this step is followed by the production of additional positive strands (for a revision see paul and wimmer, 2015) . it is believed that the dsrna functions as replicative intermediate during the synthesis of viral rna. a hallmark of this type of virus is the remarkably rearrangement of cellular membranes into organellelike replicative factories. interestingly, it has been determined that newly synthetized membranous structures, but not preexisting cell membranes, are required for viral replication. thus, the formation of the complex replication factories requires coupled viral translation, lipid synthesis, new membranes generation and viral rna synthesis (reviewed by rossignol et al., 2015) . early in the 70's, based on the incorporation of modified lipids into the membranes of poliovirus replication sites (mosser et al., 1972) indicated that these structures are different from pre-existing membranous compartments, clearly demonstrating that the virus replication factories are "self-tailored" (mosser et al., 1972) . it was shown that several poliovirus and host proteins are involved in the membrane rearrangements that are essential for virus rna replication (reviewed by jackson, 2014) . to explore the role of individual viral proteins, cells were transfected or microinjected and visualized by electron microscopy to study the complex cellular changes that take place during viral infection. the viral protein 2bc (a p2 proteolytic precursor of 2b and 2c proteins) is responsible for the generation of 50-350 nm clusters of empty vesicles limited by a single membrane, usually in peripheral regions of the cell containing a high concentration of the 2c epitope (suhy et al., 2000) . in contrast, when 2bc and 3a proteins are expressed, membrane vesicles that displayed double membranes, cytoplasmic luminal contents, and substantial immunolabeling by anti-2c antibody were observed resembled those observed during poliovirus infection and consistent with the idea of an autophagic origin for these membranes (schlegel et al., 1996; suhy et al., 2000, please, see below) . regarding the participation of host proteins it was found that the golgi-resident small g protein arf1 (adp-ribosylation factor), as well as its activator gbf1 [a guanine nucleotide exchange factor (gef)], were recruited to sites of poliovirus replication (belov et al., 2005) . it is well known that arf1 is critical for the proper functioning of the secretory pathway, thus, its translocation toward the viral replication complexes may account for the inhibition of protein secretion observed in infected-cells (for a revision see belov et al., 2007) . in addition, it was shown that the release of copii-coated vesicles that bud from eres (er exit sites) increases in poliovirus-infected cells (trahey et al., 2012) , and that expression of a dominant negative mutant of the er-resident gtpase sar1confirmed that pv requires functional er exit sites for normal levels of rna production and expression (hsu et al., 2010) . both observations suggest that the virus replication vesicles may be associated or derived from copii-vesicles. however, more recent studies indicate that classical copii vesicles do not seem to be the site for rna replication, supporting again the idea that specialized "self-tailored" membrane vesicles are involved in the poliovirus factories. the initial ultrastructural studies of dales et al. in 1965 showed a marked increase in single membrane vesicles at 3 h p.i. and double-membrane structures associated to viral particles at 7 h p.i., suggesting that autophagic vesicles are involved in biogenesis of viral replication factories (dales et al., 1965) . we now know that double-membrane compartments constitute the hallmark vesicles of the constitutive degradative process known as autophagy, or "self-eating" (schneider and cuervo, 2014) . autophagy is an essential and constitutive cellular process that regulates turnover of organelles, lipid, and proteins, and plays a role in viral infections (shi and luo, 2012) . in later studies, kirkergaard and collaborators were able to identify, using a high pressure freezing and freeze substitution technique, doublemembrane structures in infected cos-1 cells at early infection time points (i.e., 4 h p.i.) (schlegel et al., 1996; suhy et al., 2000) . the virions were in between clustered vesicles and also within double-membrane vesicles labeled with the autophagic protein lc3 where rna replication was taking place (belov et al., 2012; richards et al., 2014) . belov and collaborators performed a threedimensional analysis showing that indeed the vesicles seemed to be interconnected forming a network of tubular structures (belov et al., 2012) . these recent studies have revealed that the poliovirus factories morphology are indeed complex structures that at early times p.i. consist in clusters of single membrane vesicles, as previously described by dales et al. (1965) but at later times p.i. most of them are compose by double membrane vesicles and that some of these double membrane structures are not completely closed. these structures may serve to protect double-stranded rna intermediates during rna replication (for a comprehensive review see rossignol et al., 2015) . the kikergaard's group was the first demonstrating that autophagy benefit poliovirus replication since treatment with autophagy inducers such as rapamycin increased viral particles production (jackson et al., 2005) . in addition, it was also shown that viruses traffic into the mature acidic autophagic vesicles and that maturation of infectious poliovirus particles requires intracellular vesicle acidification (richards and jackson, 2012) . cumulative evidence indicate that polioviruses hijack autophagic components to allow their assembly, maturation and exit from the host cell, via a process known as awol (autophagosome mediated exit without cell lysis) (arita et al., 2012) . it has been shown that lc3 silencing with a sirna leads to a decrease of viral cell-to-cell spread whereas autophagy induction favors this non-lytic release in both cultured cells and mice (bird et al., 2014) . the release of viral particles via a non-lytic process was also previously suggested by a study in the spinal cords of bonnet monkeys (ponnuraj et al., 1998) . in a recent publication it was also shown the release of enwrapped virus via autophagosomallike vesicles, enriched in phosphatidylserine, which were highly efficient in infection (chen et al., 2015) . other +srna viruses such as the enterovirus coxsackievirus (kemball et al., 2010) , hepatitis c virus (flaviviridae family) (sir et al., 2012) , or coronavirus such as mvh (reggiori et al., 2010) also usurp the autophagy pathway and induce remarkably alterations in intracellular membranous components to harbor the sites for viral rna replication. however, it is important to take into account that significant differences emerge in the mode that different virus hijack cellular components to establish their replication niches (for more comprehensive revisions see paul and bartenschlager, 2013; harak and lohmann, 2015) . using semliki forest virus (sfv) as a prototype to analyze the innate immune response of host-cell to +srna viruses, nikonov et al. (2013) have conspicuously described and proposed a novel mechanism of action for type i interferons induction upon rdpp activity detection inside the cell. based on previous observations that expression of the viral replicase from others +srna viruses, without the replication-competent viral genome, can initiate inf-β promoter; the authors approached their study uncoupling the sfv replicase expression from the viral rna expression. for achieving this, they generated a plasmid construction bearing the coding sequence of the rdrp from a pathogenic sfv strain, sfv4 prep. as a control, they generated a plasmid coding an inactive version harboring two changes in the rdrp specific catalytic domain of the nsp4, prep-ga (kamer and argos, 1984) . they observed that active sfv replicase is able of inducing inf-β without the replicationcompetent viral rna. employing interfering experiments they demonstrated that rig-i is the major sensor mediating infβ induction with mda-5 acting as an additional sensor, also contributing to inf-β enhancement. since rig-i was involved, they suspected that expression of sfv replicase was responsible for generating pamps so they characterized these molecules and observed the generation of non-polyadenilated rna species, larger than 200 nucleotides, containing dsrna regions and a terminal 5 ′ -phosphate. moreover, they showed that those dsrna structures generated by sfv replicase associated to endosomes and lysosomes were the strongest inf-β inducers from those present in the cell, reinforcing the notion that these organelles serve as the sites of sfv replicase docking and viral dsrna intermediates generation. based on these and other results obtained through elegant approaches, the authors proposed a novel mechanism of pamps generation and inf-β induction by the viral replicase transcription of non-viral host-cell rna templates. at the same time, the viral replicase molecules would anchor to endosomes membranes to build up membranous spherules where viral dsrna or 5-ppp rna intermediates keep inaccessible to host sensors any longer, as suggested by nikonov and nikonov et al. (2013) , shown in figure 1 . accordingly, employing adenoviral vectors as delivery method into murine and human hepatocytes, yu and colleagues demonstrated that the hepatitis c virus (hcv) rdrp ns5b was capable of inducing the innate immune response in the absence of other hcv rna replication components and/or other nonstructural proteins. they further showed that this induction was dependent upon the ns5b enzymatic activity and on the adaptor protein mavs that functions downstream of rig-i and mda5 (yu et al., 2012) . supporting this novel mechanism of innate immune antiviral response activation, painter and colleagues observed that the ectopic expression of rdrp from theiler's murine encephalomyelitis virus (tmev, member of the picornaviridae family), in the complete absence of other viral structures, is able to induce isg activation in an mda5-dependent fashion. the authors showed the presence of endogenous dsrna molecules in un-infected tissues of rdrp transgenic mice, which sustained the mda5-activation. at this point, the authors did not observe a role of rig-i in this process, but neither rule out this possibility (painter et al., 2015) . thus, despite the few +srna viruses for which the induction of the innate immune response has been observed to be thanks to rdrp activity, it seems likely that it may be a universal mechanism employed by the host cell to withstand the viral conquest. in this context, since +srna viruses conceal the entire replication machinery in membrane-bound cytoplasmic compartments achieved by extensive re-organization of host organelle membranes, it seems to be a matter of time lapsed between virus uncoating with rdrp emergence into the host's cytoplasm and the hideout of the replication machinery. finally, and as a consequence, illustrating the virus-host cell co-evolution process once more, virus have evolved with weapons to be protected from innate immune response recognition irrespective of the origin of pamps generated during the course of infection. for many +srna viruses, mentioned throughout this review, rna replication occurs in association with 50-70 nm diameter membranous vesicles or spherules that form in the lumen of specific cellular organelles, or in double membrane vesicles, reminiscent to that of the autophagic pathway. although important discoveries on the 3-d architecture of +srna virus replication factories have been made, current knowledge is largely descriptive and important information about mechanisms is figure 1 | proposed model of host-cell mrna replication by +srna viruses' replicases. inspired by the "model of mutant semiliki forest virus replication restriction in fibroblasts," proposed by nikonov et al. (2013) . after viral internalization and uncoating, the genomic rna serves as mrna recognized by the host cell machinery to translate the viral replication complex (rdrp), which binds to the plasma membrane to build up the membranous niche called spherule. once there, the replicase activity generates new viral genome copies producing double-stranded rna (dsrna) molecules as intermediates, which remain hided from the cytoplasmic sentries inside the spherules (1). nevertheless, in the meantime, the rdrp is able to take cellular mrna as template originating dsrna molecules in the cytoplasm, exposed to the pamp recognition receptors [prrs (mda-5 and rig-i)] to initiate the innate immune response, which produces the viral restriction (2). missing. for instance, the exact topology of rna replication sites for dmv-type replication factories is yet uncharacterized. indeed, novel experimental techniques such as metabolic in situ labeling of nascent viral rna and its visualization by employing high resolution and specific microscopy methods will help tackling this important feature of +srna viruses replication. membrane-remodeling events responsible for the biogenesis of replication factories are also mostly unknown. it is likely that a viral protein interplaying with cellular factors might be responsible for that, but precise contributions of individual factors and their temporal and spatial coordination remain to be discovered. nevertheless, cellular host is able to develop an innate immune response mediated by prrs without needing viral replication, which have placed the pressure back on viral agents to evolve specific strategies to counteract its action, while allowing the viral genome to replicate inside their hideouts. ld and mc together built up the idea and structure of the review. ld wrote the majority of the text. mc wrote a section of the review and helped performing the revision of the manuscript to its final version. semliki forest virus mrna capping enzyme requires association with anionic membrane phospholipids for activity recognition of double-stranded rna and activation of nf-kappab by toll-like receptor 3 valosin-containing protein (vcp/p97) is required for poliovirus replication and is involved in cellular protein secretion pathway in poliovirus infection the molecular pathogenesis of semliki forest virus: a model virus made useful? subcellular localization and rearrangement of endoplasmic reticulum by brome mosaic virus capsid protein hijacking components of the cellular secretory pathway for replication of poliovirus rna poliovirus proteins induce membrane association of gtpase adp-ribosylation factor complex dynamic development of poliovirus membranous replication complexes mda5 cooperatively forms dimers and atp-sensitive filaments upon binding double-stranded rna nonlytic viral spread enhanced by autophagy components viral evasion and subversion of pattern-recognition receptor signalling morphogenesis of endoplasmic reticulum membrane-invaginated vesicles during beet black scorch virus infection: role of auxiliary replication protein and new implications of three-dimensional architecture phosphatidylserine vesicles enable efficient en bloc transmission of enteroviruses electron microscopic study of the formation of poliovirus ikkepsilon and tbk1 are essential components of the irf3 signaling pathway three-dimensional structure of rubella virus factories electron tomography of membrane-bound cellular organelles cell-induced conformational change in poliovirus: externalization of the amino terminus of vp1 is responsible for liposome binding cytoplasmic fractions associated with semliki forest virus ribonucleic acid replication membrane-associated replication complex in arbovirus infection functional sindbis virus replicative complexes are formed at the plasma membrane alphavirus rna replicase is located on the cytoplasmic surface of endosomes and lysosomes virion structure triggering antiviral response by rig-i-related rna helicases structure and function of toll receptors and their ligands conformational change and protein-protein interactions of the fusion protein of semliki forest virus cytoplasmic structures associated with an arbovirus infection: loci of viral ribonucleic acid synthesis ultrastructure of the replication sites of positive-strand rna viruses 5'-triphosphate rna is the ligand for rig-i mavs forms functional prion-like aggregates to activate and propagate antiviral innate immune response viral reorganization of the secretory pathway generates distinct organelles for rna replication poliovirus-induced changes in cellular membranes throughout infection subversion of cellular autophagosomal machinery by rna viruses primary structural comparison of rnadependent polymerases from plant, animal and bacterial viruses differential roles of mda5 and rig-i helicases in the recognition of rna viruses the role of pattern-recognition receptors in innate immunity: update on toll-like receptors rig-i in rna virus recognition coxsackievirus infection induces autophagy-like vesicles and megaphagosomes in pancreatic acinar cells in vivo alphavirus entry and membrane fusion unc93b1 delivers nucleotide-sensing toll-like receptors to endolysosomes three-dimensional analysis of a viral rna replication complex reveals a virus-induced mini-organelle togaviridae intracellular distribution of rubella virus nonstructural protein p150 biogenesis of the semliki forest virus rna replication complex the effects of palmitoylation on membrane association of semliki forest virus rna capping enzyme membrane binding mechanism of an rna virus-capping enzyme the tlr3 signaling complex forms by cooperative receptor dimerization structural basis of toll-like receptor 3 signaling with double-stranded rna structural insights into rna recognition by rig-i lysosomes: fusion and function short double-stranded rnas with an overhanging 5' ppp-nucleotide, as found in arenavirus genomes, act as rig-i decoys antiviral responses induced by the tlr3 pathway cellular receptor for poliovirus: molecular cloning, nucleotide sequence, and expression of a new member of the immunoglobulin superfamily molecular evolution of vertebrate toll-like receptors: evolutionary rate difference between their leucine-rich repeats and their tir domains flock house virus rna replicates on outer mitochondrial membranes in drosophila cells the non-structural protein 4a of dengue virus is an integral membrane protein inducing membrane alterations in a 2k-regulated manner incorporation of lipid precursors into cytoplasmic membranes of poliovirus-infected hela cells rig-i and mda-5 detection of viral rna-dependent rna polymerase activity restricts positive-strand rna virus replication antiviral protection via rdrpmediated stable activation of innate immunity initiation of protein-primed picornavirus rna synthesis architecture and biogenesis of plus-strand rna virus replication factories cooperative assembly and dynamic disassembly of mda5 filaments for viral dsrna recognition rig-i forms signalingcompetent filaments in an atp-dependent, ubiquitin-independent manner the alphavirus replicase protein nsp1 is membrane-associated and has affinity to endocytic organelles rig-i-mediated antiviral responses to single-stranded rna bearing 5'-phosphates cell-to-cell spread of poliovirus in the spinal cord of bonnet monkeys (macaca radiata) picornaviridae: the viruses and their replication coronaviruses hijack the lc3-i-positive edemosomes, erderived vesicles exporting short-lived erad regulators, for replication intracellular vesicle acidification promotes maturation of infectious poliovirus particles generation of unique poliovirus rna replication organelles the role of electron microscopy in studying the continuum of changes in membranous structures during poliovirus infection regulation of innate antiviral defenses through a shared repressor domain in rig-i and lgp2 properly folded nonstructural polyprotein directs the semliki forest virus replication complex to the endosomal compartment two tyrosine residues of toll-like receptor 3 trigger different steps of nfkappa b activation toll/il-1 receptor domain-containing adaptor inducing ifn-beta (trif) associates with tnf receptor-associated factor 6 and tank-binding kinase 1, and activates two distinct transcription factors, nf-kappa b and ifn-regulatory factor-3, in the toll-like receptor signaling cellular origin and ultrastructure of membranes induced during poliovirus infection autophagy and human disease: emerging themes biology and pathogenesis of chikungunya virus interplay between the cellular autophagy machinery and positive-stranded rna viruses role of ribosomes in semliki forest virus nucleocapsid uncoating replication of hepatitis c virus rna on autophagosomal membranes phosphatidylinositol 3-kinase-, actin-, and microtubule-dependent transport of semliki forest virus replication complexes from the plasma membrane to modified lysosomes role of the amphipathic peptide of semliki forest virus replicase protein nsp1 in membrane association and virus replication structure, function and evolution of picornaviruses remodeling the endoplasmic reticulum by poliovirus infection and by individual viral proteins: an autophagy-like origin for virus-induced vesicles lrrc59 regulates trafficking of nucleic acid-sensing tlrs from the endoplasmic reticulum via association with unc93b1 pattern recognition receptors and the innate immune response to viral infection poliovirus infection transiently increases copii vesicle budding characterization of early steps in the poliovirus infection process: receptordecorated liposomes induce conversion of the virus to membrane-anchored entry-intermediate particles regulation of the sequential processing of semliki forest virus replicase polyprotein composition and three-dimensional architecture of the dengue virus replication and assembly sites structural basis for the prion-like mavs filaments in antiviral innate immunity shared and unique functions of the dexd/h-box helicases rig-i, mda5, and lgp2 in antiviral innate immunity the rna helicase rig-i has an essential function in doublestranded rna-induced innate antiviral responses hepatic expression of hcv rna-dependent rna polymerase triggers innate immune signaling and cytokine production the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2017 delgui and colombo. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-252485-cxi3cr15 authors: yoshida, asuka; kawabata, ryoko; honda, tomoyuki; tomonaga, keizo; sakaguchi, takemasa; irie, takashi title: ifn-β-inducing, unusual viral rna species produced by paramyxovirus infection accumulated into distinct cytoplasmic structures in an rna-type-dependent manner date: 2015-08-04 journal: front microbiol doi: 10.3389/fmicb.2015.00804 sha: doc_id: 252485 cord_uid: cxi3cr15 the interferon (ifn) system is one of the most important defensive responses of mammals against viruses, and is rapidly evoked when the pathogen-associated molecular patterns (pamps) of viruses are sensed. non-self, virus-derived rna species have been identified as the pamps of rna viruses. in the present study, we compared different types of ifn-β-inducing and -non-inducing viruses in the context of sendai virus infection. we found that some types of unusual viral rna species were produced by infections with ifn-β-inducing viruses and accumulated into distinct cytoplasmic structures in an rna-type-dependent manner. one of these structures was similar to the so-called antiviral stress granules (avsgs) formed by an infection with ifn-inducing viruses whose c proteins were knocked-out or mutated. non-encapsidated, unusual viral rna harboring the 5′-terminal region of the viral genome as well as rig-i and typical sg markers accumulated in these granules. another was a non-sg-like inclusion formed by an infection with the cantell strain; a copyback-type di genome, but not an authentic viral genome, specifically accumulated in the inclusion, whereas rig-i and sg markers did not. the induction of ifn-β was closely associated with the production of these unusual rnas as well as the formation of the cytoplasmic structures. eukaryotic cells are equipped with various defense mechanisms to detect and respond to viral infections rapidly. the interferon (ifn) system is one of the most important natural defenses of mammalian cells in the early phase of viral infection. host cells sense the invasion of viruses by recognizing their pathogen-associated molecular patterns (pamps), including the structural characteristics of viral rnas that differentiate them from cellular rnas . viral rnas are detected by non-self rna sensors such as toll-like receptors (tlr), and a family of cytosolic rna helicases termed rig-i-like receptors (rlrs), including retinoic-acid inducible gene-i (rig-i), melanoma differentiation-associated gene 5 (mda5), and laboratory of physiology and genetics gene 2 (lgp2). this is followed by the subsequent induction of ifn-β (gitlin et al., 2006; kaisho and akira, 2006; saito et al., 2007) . autocrine or paracrine ifns bind to ifn receptors on the cell surface, leading to the expression of 100s of ifn-stimulated genes (isgs) through the jak/stat signaling pathway, which ultimately exerts various antiviral effects . translational arrest is one of the ifn responses of host cells triggered by viral infections. various eukaryotic translation initiation factor 2 (eif2) kinases such as protein kinase r (pkr) are activated in response to ifns, and the accumulation of phosphorylated eif2α inhibits the translation of both cellular and viral mrnas kedersha and anderson, 2002; holcik and sonenberg, 2005) . cytoplasmic stress granules (sgs), which are the foci of concentrated 48s translation preinitiation complexes and defined by certain marker rna binding proteins such as t-cell intracellular antigen-1 (tia-1), tia-1-related protein (tiar), and ras-gap-sh3 domain-binding protein (g3bp1), are formed under these conditions kedersha and anderson, 2002) . because they contain stable inert mrna, sgs are believed to serve as temporary sites at which mrnaprotein complexes are stored to pause active translation or be decayed in adjacent processing bodies (anderson and kedersha, 2006; balagopal and parker, 2009; buchan and parker, 2009) . a number of viruses have been shown to induce the formation of sgs in infected cells, and this may be related to the virus-induced shut-off of cellular protein translation. for example, hepatitis c virus, poliovirus, semliki forest virus, and mammalian orthoreovirus promote the shut-off of cellular proteins and the assembly of sgs at the early phase of infection, and this is inhibited as the infection progresses (mcinerney et al., 2005; white et al., 2007; qin et al., 2009 qin et al., , 2011 ariumi et al., 2011; white and lloyd, 2011; garaigorta et al., 2012; panas et al., 2012; ruggieri et al., 2012; fitzgerald and semler, 2013; pager et al., 2013; carroll et al., 2014) . two rna viruses, respiratory syncytial virus and coronavirus, are also known to utilize sgs as part of the machinery to inhibit host cellular protein translation (raaben et al., 2007; lindquist et al., 2010 lindquist et al., , 2011 . sgs were not detected during infection by influenza a virus (iav); however, a recombinant iav lacking non-structural protein 1 (ns1), an inhibitor of pkr, efficiently induced the formation of sgs and the production of ifn-β in a pkr-dependent manner (khaperskyy et al., 2012; mok et al., 2012; onomoto et al., 2012) . in this case, sgs were suggested to play an important role as the sites of viral rna sensing and subsequent anti-viral responses, because rlrs localized together with viral nucleoproteins and rna as well as anti-viral proteins in sgs (onomoto et al., 2012; ng et al., 2013) . the rna species produced during the course of rna viral replication, such as mrna, dsrna, and 5 -triphosphate (5 -ppp) rna including leader, trailer, genome, and antigenome rnas as well as defective interfering (di) genomes, have been shown to trigger the production of type i ifns (yoneyama et al., 2004; hornung et al., 2006; pichlmair et al., 2006; strahle et al., 2007; hausmann et al., 2008; baum et al., 2010; baum and garcia-sastre, 2011; kato et al., 2011; marq et al., 2011; davis et al., 2012; bowzard et al., 2013; weber et al., 2013; runge et al., 2014; schmolke et al., 2014) . we and other groups have recently reported that recombinant viruses of sendai virus (sev), a prototype of the family paramyxoviridae, in which the c proteins are knocked-out or mutated, generate dsrna in infected cells at levels similar to the production of ifn-β (takeuchi et al., 2008; irie et al., 2010) . previous studies also reported that, in the cases of sev and iav, copyback (cb)-and internal deletion (id)-type di genomes, respectively, rather than full-length viral genomes, preferentially associated with rig-i and strongly induced the production of ifn-β (baum et al., 2010; baum and garcia-sastre, 2011) . in spite of the large number of studies conducted in this field, it remains unknown what kinds of viral rna species are recognized by rlrs and where the sites of recognition are in real infections by rna viruses. in the present study, we compared some types of ifn-β-non-inducing and ifn-β-highly inducing viruses in the context of sev infection. one of the biggest advantages of this study is that the comparison can be performed within the context of the same viral species. we found that some types of unusual rna species that were distinguishable according to specific detectability by the anti-dsrna antibody or fish analysis were produced during the viral replication of ifn-inducing sevs, but not ifn-non-inducing sev strains, and accumulated into distinct cytoplasmic structures in an rna-type-dependent manner. these unusual rnas exhibited distinct properties in infected cells in terms of encapsidation with the viral n protein and subcellular distribution with sg marker proteins and rlrs. our results suggest that rna-typedependent mechanisms recognize and accumulate virus-derived, ifn-β-inducible, unusual rnas into specific compartment to trigger the production of ifn-β, and that sev may evade detection by the host innate immune system by preventing the production of these rna species. llc-mk2 cells (macaque monkey kidney-derived cells, described in kiyotani et al., 1990) and hela cells (ccl-2; purchased from atcc) were maintained as described previously (irie et al., 2012) . all of the sevs, even the wt of strain z and hamamatsu (hmt), used in this study were recovered from cdna using a reverse genetics technique as described previously (kato et al., 1996; fujii et al., 2002) , except for the cantell (cnt; vr-907; atcc), fushimi (fsm), and nagoya (ngy) strains. the sev recombinants, c /c(-), 4c(-), and v(-), were kindly provided by kato (national institute of infectious diseases, japan; kato et al., 1997; kurotani et al., 1998) . all of the sevs as well as virulent and avirulent newcastle disease virus (ndv) miyadera and d26 strains (toyoda et al., 1987) , respectively, were propagated in embryonated chicken eggs. sev and ndv titers were determined by an immunofluorescent infectious focus assay in llc-mk2 cells and expressed as cell infectious units (cius)/ml, as described previously (kiyotani et al., 1990) . one-step growth kinetics of the viruses was determined as described previously (irie et al., 2014) . plasmids encoding the p, c, and v proteins of sev strain z in the pcaggs. mcs vector have been described previously (sakaguchi et al., 2005 (sakaguchi et al., , 2011 irie et al., 2008b irie et al., , 2012 . the polyclonal antibodies (pabs) against whole virions of sev and ndv were described previously (kiyotani et al., 1990) . the pabs against the sev p and c proteins were kindly provided by kato. the monoclonal antibody (mab) against the sev n protein was kindly provided by suzuki (national institute of infectious disease, japan). the mab and pab against g3bp1 (sc-365338, santa cruz biotechnology; and ab39533, abcam, respectively), pab against rig-i (28137; immuno-biological laboratories, japan), mab against tiar, and dsrna (#8509, cell signaling technology; and j2, scicons, hungary, respectively) were used according to the protocols of the suppliers. hela cells cultured on glass coverslips were infected with the indicated viruses or transfected with the indicated plasmids. the culture medium was replaced by serum-free dmem 24 h postinfection (p.i.) or post-transfection (p.t.), and cells were treated with sodium arsenite (naaso 2 ; sigma) at a final concentration of 0.5 mm for 30 min or with ifn-α (1,000 iu/ml; r&d systems) for 6 h. hela cells cultured on glass coverslips were transfected with the indicated plasmids using fugene hd transfection reagent (promega) or infected with the indicated viruses at an moi of 5. at 24 h p.t. or p.i., cells were fixed and immunostained as described previously using appropriate combinations of primary and secondary antibodies (irie et al., 2013) . to detect rig-i, and dsrna, the tyramide signal amplification (tsa) kit with hrp-goat anti-mouse igg and alexa fluor 488 tyramide (molecular probes) were used to increase the detection sensitivity. coverslips were mounted on glass slides with the slowfade gold antifade reagent with or without dapi (molecular probes) and observed using an lsm 5 confocal microscope (carl zeiss). hela cells were infected with the indicated viruses at an moi of 5. at 24 h p.i., total rna was prepared using the high pure rna isolation kit (roche diagnostics). viral rna in the working viral stocks was prepared using the high pure viral rna kit (roche diagnostics). quantitative (q) rt-pcr was performed as described previously (irie et al., 2010 (irie et al., , 2014 . qrt-pcr samples were analyzed using the eco real-time pcr system (illumina). for direct comparison, all of the indicated samples were analyzed in the same experiments. to detect the genome-length viral rnas and cbdi genomes of sev stocks, qrt-pcr was performed using the primer sets of 5sevz1683 + 3sevz1843, as described previously (irie et al., 2008a (irie et al., , 2014 , and 5cbdidetect15,312-15,293 + 3cbdidetect15,033-15,014, which were complementary to the regions of the indicated positions of sev genome rna, as described recently by baum et al. (2010) . hela cells were lysed in ripa buffer (0.5% np-40, 20 mm tris-hcl [ph 7.4], 150 mm nacl) after 24 h of infection by the indicated viruses or 30 min of the arsenite treatment, and the insoluble fraction was removed by high-speed centrifugation. the viral proteins in the lysates were removed by three consecutive immunoprecipitation steps using anti-sev or anti-ndv pabs and protein g sepharose beads (ge healthcare life sciences). supernatants were harvested from the final immunoprecipitation samples, and were then subjected to rna preparation, as described above. hela cells cultured on glass coverslips were transfected with 1 μg of the indicated rna samples prepared above, together with 0.5 μg of an empty puc19 vector, using the fugene hd transfection reagent. cantell samples (1.4 × 10 9 ciu/ml) that were 10 8∼9 -fold serially diluted were inoculated into the allantoic cavity of 10-days-old embryonated chicken eggs, and incubated for 72 h at 34 • c. allantoic fluid was harvested from the eggs, and the titers of each fluid stock were determined as described above. rna samples were prepared from 100 μl of each fluid stock, and the ratios of the cbdi genomes to viral genomes were determined by qrt-pcr as described above. hela cells cultured on glass coverslips were infected with the indicated viruses. at 24 h p.i., cells were fixed with 3% paraformaldehyde solution in pbs, and then subjected to fish analysis using the fish tag rna green kit with alexa fluor 488 dye (molecular probes) according to the protocol of the supplier. the rna probe was designed to be complementary to the region of 14,761-15,384 in sev genome rna. after fish, some samples were further subjected to fluorescent immunodetection as described above using the indicated antibodies. final samples were observed using an lsm 5 confocal microscope. we first examined whether g3bp1-positive granular structures were formed during infections by a series of c knockedout and mutated sev recombinants and the parental z strain by immunofluorescence microscopy (figure 1 ; supplementary figure s1 ). treating hela cells with sodium arsenite (naaso 2 ) caused the formation of granular structures in the cytosol, which were figure 1 | subcellular distribution of g3bp1 and tiar in hela cells treated with or without sodium arsenite (a), and infected with a series of c-deficient rsevs, dy1, dy2, d2y, c /c(-), and 4c(-), and a v-deficient rsev, v(-) (b). hela cells treated with ethanol (etoh) or sodium arsenite (naaso 2 ) or infected with the indicated virus were immunostained with anti-g3bp1 mab, anti-tiar mab, and anti-sev pab. (c) the g3bp1 foci in the samples of (b) were observed under a fluorescent microscope, and the number of g3bp1 granule-positive cells was counted and is presented as percentages against the number of sev antigen-positive cells (closed bars). the relative amounts of ifn-β mrna in cells infected with the indicated viruses were determined by qrt-pcr, and normalized to those of β-actin mrna (open bars). (d) similar experiments were performed for a series of c-mutated sev recombinants, and the results are presented as bar graphs, similarly to (c). defined as sgs based on the expression of related proteins such as g3bp1 and tiar. g3bp1 and tiar are well-established sg-associated proteins that are typically and diffusely present throughout the cytoplasm and dominantly present in the nucleus, respectively. however, treating cells with arsenite markedly changed the localization to form sgs containing these proteins in nearly all cells ( figure 1a) . when cells were infected with 4c(-), g3bp1-positive granular structures were observed in almost 90% of sev antigen-positive cells, and were considered to be sg-like structures since tiar was also detected in the majority of the granules (figures 1b,c) . in this situation, tiar was mostly in the cytoplasmic structures, whereas a larger part of tiar was still observed in the nucleus in the arsenite-treated cells. this difference is probably due to the different exposure time to the stimuli: 30 min. for the treatment with arsenite and 24 h for the infection. in contrast, the percentages were only 8% or less in cells infected with the parental z-wt as well as the dy1, dy2, and d2y recombinants, which lacked the smaller c proteins, y1, y2, and both y1 and y2, respectively ( figure 1c; supplementary figure s1a ). an infection by c /c(-) and v(-), lacking the larger c proteins, c and c, and the v protein, respectively, resulted in a slight increase in the number of granules ( figure 1c ; supplementary figure s1a ). of note, unlike the viruses reported previously, such as ns1-deficient iav and vesicular stomatitis virus (mok et al., 2012; onomoto et al., 2012; dinh et al., 2013) , the fluorescence of the sev antigen was not colocalized with that of the representative sg marker g3bp1 in the granules (figure 1b ; supplementary figure s1 ). ifn-β mrna levels in the infected cells were also compared between the viruses ( figure 1c) . strong correlations were observed between ifn-β mrna levels and the percentages of granular structure-forming cells against infected cells. similar strong correlations were observed for a series of c mutant viruses that possessed single-to triple-amino-acid substitutions of highly conserved, charged amino acids within the c proteins, which diminished their ability to antagonize the host ifn system to various degrees (figure 1d ; supplementary figure s1b ; irie et al., 2010) . the marked difference observed in granular formation and ifn-β mrna levels among the viruses was not due to their different growing abilities. the one-step growth kinetics of all the recombinants used above was previously reported to be similar to that of the parental z-wt, except for 4c(-) and c /c(-), the titers of which were 1-2 logs lower than that of z-wt throughout the time course, and viral protein synthesis in the infected cells did not significantly differ among the viruses examined (kurotani et al., 1998; irie et al., 2008a irie et al., , 2010 . treatment of hela cells with ifn-α did not induce g3bp1-positive granules, indicating that the formation of sglike structures observed by sev infection was triggered by sev infection, but not by sev-induced type i ifns (supplementary figure s1c) . taken together, these results strongly suggested that a relationship may exist between the formation of g3bp1-positive granules and the induction of ifn-β in the c recombinants, and also that c proteins may suppress the formation of granules. we examined whether expression of the c protein alone and infection of the non-granule-forming sev could inhibit the formation of granules in cells treated with arsenite or infected with ndv (figure 2; supplementary figure s2 ). ndv, another prototypic paramyxovirus, which was considered to be an ifn-β-inducing virus, could induce g3bp1-positive granules in nearly all cells infected with virulent as well as avirulent strains (miyadera and d26 strains, respectively; figure 2b ). the c protein alone failed to inhibit the formation of granules in cells treated with arsenite (figure 2a) as well as infected with ndv ( figure 2b) , although the number of granules was slightly reduced in the c-expressing cells treated with arsenite compared to that observed in the neighboring non-c-expressing cells (figure 2a) . in addition to c, the other p gene products, p and v proteins, also failed to inhibit the formation of both types of granule (supplementary figure s2) . infection by sev-z-wt and c-deficient recombinant 4c(-) also failed to inhibit the arsenite-induced formation of granules (figures 2c,d) . small granules dispersed in the cytoplasm were induced by arsenite in both cases of infection by wt and 4c(-). of note, the g3bp1-positive granules induced by arsenite and viruses, such as 4c(-) and ndv, differed in size; the granules induced by the infection were apparently larger than those induced by arsenite (figures 1 and 2) , implying a possible difference in cellular pathways leading to these two types of granular structure. these results demonstrated that sev did not have the ability to inhibit the formation of both types of granules. a marked difference was noted in the abilities of the sev strains to induce ifn-β. although most of the sev strains including z have been characterized by their strong ability to counteract the innate immune system, the cnt strain has been widely used as a virus that induces high levels of ifn-β (baum et al., 2010; tapia et al., 2013) . therefore, we compared the abilities of some sev strains to induce ifn-β and sg-like structures (figure 3 ; supplementary figure s3 ). in all cases, g3bp1-positive granular structures were only detected in less than 9% of sev antigen-positive cells (figure 3 ; supplementary figure s3a ). ifn-β mrna was not highly induced in cells infected with the sev strains, except for cnt, whereas cnt induced ifn-β mrna at a level that was 46-fold higher than that by z (figure 3) . regarding the sev strains, unlike that observed in the c recombinants, a correlation was not observed between ifn-β mrna levels and the percentage of granular structure-forming cells against infected cells (figure 3) . the different levels of ifn-β mrna induced by the strains could not be attributed to differences in viral growth (supplementary figure s3b) . these results suggested that the formation of g3bp1-positive granules was not necessarily required to sense the sev cnt infection, followed by the production of ifn-β, unlike the c recombinants. we attempted to identify the reason for the difference in granular formation between these two types of ifn-β-inducing virus, 4c(-) and cnt. to address this issue, we first examined the ability of total rna prepared from virus-infected as well as arsenite-treated cells to induce g3bp1-positive granules ( figure 4a; supplementary figure s4a ). total rna samples prepared from cells infected with any of the ifn-β-inducing viruses, sev-cnt, 4c(-), and ndv, were able to induce the granules as well as ifn-β in hela cells despite no formation of the granules by the cnt infection, whereas those from cells infected with the ifn-β-non-inducing sev-z-wt or treated with arsenite were not (figure 4a) . we then examined the content rates of cbdi genomes, a potent ligand for rig-i, against viral genome-length rnas in the rna samples used above ( figure 4b ). the cnt sample contained cbdi genomes at a level that was ∼15,700-fold higher than that in the z-wt sample, although the sample of 4c(-) contained similar levels to the z-wt sample ( figure 4b) . we further examined the effect of removal of encapsidated viral rna species, such as viral full-length and di genomes, by three consecutive rounds of immunoprecipitation using antisera against the whole virions of sev and ndv prior to the preparation of rna samples (supplementary figure s4b) . total rna prepared from the postimmunoprecipitation samples of 4c(-) and ndv was still able to induce the granules as well as ifn-β, but that of cnt lost these abilities (figure 4c ). since the naked cbdi genomes have been reported readily to form an ideal structure as the rig-i ligands of 5 -triphosphated, blunt-ended dsrna (kolakofsky, 1976) , these results indicated that the major ifn-β-inducing viral rna species produced in the cells infected with cnt was encapsidated cbdi genomes, whereas those for sev-4c(-) and ndv were not. the results above suggested that there may be at least two types of ifn-β-inducing viral rna species with a marked difference in the formation of sg-like granules. to elucidate this difference, we examined the subcellular distribution of unusual viral rna species produced by 4c(-) and cnt (figures 5 and 6) . we and other groups previously reported a strong correlation between the production of dsrna detected by the anti-dsrna antibody j2 (j2-dsrna) and the induction of ifn-β in infections of the c-mutated sev recombinants and ndv (takeuchi et al., 2008; irie et al., 2010) . therefore, we first examined the production and subcellular distribution of j2-dsrna in the cells infected with the ifn-β-inducing viruses by immunofluorescence microscopy (figure 5) . dsrna fluorescent signals were absent in cells infected with ifn-non-inducing z-wt as well as in those with the ifn-β-inducing strain cnt ( figure 5a ). in contrast, dsrna fluorescent signals were clearly observed in the cytoplasm of cells infected with ifn-β-inducing sev-4c(-) and ndv with dispersed and granular distributions, respectively ( figure 5b) . a previous study reported that ns1-deficient iav infections caused rig-i to form granular aggregates that contained sg markers as well as viral rna (onomoto et al., 2012) . therefore, the cells infected with 4c(-) and ndv were co-stained with anti-rig-i and anti-g3bp1 antibodies ( figure 5c ). similar to iav, rig-i almost perfectly colocalized with the virus-induced g3bp1-positive granules in both cases of infection ( figure 5c) ; however, unlike iav, these granules did not colocalize with the viral antigen or viral j2-dsrna ( figure 5b, arrowheads) , as shown in figures 1 and 2. together with the results of figure 4 , j2-dsrna appeared to be not or less encapsidated by viral nucleoproteins because the access of the antibody to and the formation of dsrna by tightly encapsidated viral rna, such as viral genomes, was unlikely. we further attempted to visualize the subcellular distribution of the unusual viral rna species that were not fully encapsidated, unlike the genome-length viral rnas, by fish analysis (figure 6) . infected cell samples were prepared without a protease treatment to exclude fully encapsidated viral rna species, and were then stained with an rna probe complementary to the 14,761-15,384 region of the (-)-sense viral genome rna, designed by referring to two wellcharacterized cbdi genomes of sev (calain et al., 1992; -gil et al., 2013) . fluorescence-positive cytoplasmic inclusions were observed in the 4c(-)-as well as in the cntinfected samples, while an apparent signal was not detected in z-wt-infected or uninfected samples ( figure 6a ). when the z-wt-infected sample was treated with proteinase k, apparent signals were observed throughout the cytoplasm ( figure 6a) . these results strongly suggested that fully encapsidated viral genomes were not detected, whereas nonor partially encapsidated viral rna species were detectable in this system. the fish-positive inclusions observed in cntinfected cells were no longer detected in the cells infected with the cnt-lowdi (figure 6a) , which contained ∼10-fold fewer cbdi genomes than the original cnt sample ( figure 4b) . together with the results of figure 4 , the fish signals observed in the cnt-infected cells were considered as the cbdi genomes. the fish samples of 4c(-) and cnt-infected cells were further immunostained to examine the subcellular colocalization of fish signals, the sev n protein, rig-i, and g3bp1 (figures 6b-d, respectively) . the sev n protein was detected in the fish-positive inclusions of cnt-infected cells, suggesting that the rna species detected in the cnt samples were only partially, not fully, encapsidated, unlike the fully encapsidated, full-length, intact viral genomes ( figure 6b ). in contrast, the n protein was not detected in the inclusions of 4c(-)-infected cells, which suggested that the rna species detected in the 4c(-) samples were not encapsidated ( figure 6b) . the fish-positive inclusions of cnt-infected cells were not apparently colocalized with rig-i or g3bp1, whereas most of those in the 4c(-)-infected cells colocalized obviously with rig-i and g3bp1, unlike the j2-dsrna (figures 6c,d) . these results indicated that unusual viral rna species harboring the 5 -region of (-)-sense sev genome rna, which was not produced during infection by ifn-β-non-inducing z-wt, was produced in infections by , and were selectively formed into distinct cytoplasmic inclusions in an rna-type-dependent manner. the inclusions in the 4c(-)-infected cells could be identified as avsgs, and may be the site to detect viral rna by rig-i, whereas those in cnt-infected cells were not avsgs, but as-yet-unidentified structures. although a number of studies have examined host innate immune responses against pathogenic microbes including rna viruses, the virus-derived rna species that serve as pamps in real viral infections and the sites at which pamps are recognized by rlrs have yet to be clarified in detail. two important insights were reported recently. regarding the real pamps in infections by rna viruses, the cb and id types of di genomes were identified as the ligands of rig-i in sev-and iav-infected cells, respectively, both of which could form ideal structures as the rig-i ligands (baum et al., 2010; baum and garcia-sastre, 2011; martinez-gil et al., 2013) . the sg-like structures have been suggested to serve as the sites at which the rlrs encounter viral rna and subsequently activate the ifn signaling pathways in infections by rna viruses (onomoto et al., 2012; yoo et al., 2014) . in order to establish what and where viral rna species were detected by rlrs, in the present study, we compared two types of ifnβ-inducing sev, a recombinant 4c(-) and a strain cnt, with a non-ifn-β-inducing z strain, in terms of the formation of sglike granules and the production of unusual viral rna species. a major advantage of our study is that the comparison can be performed within the context of the same viral species. several types of unusual viral rna species were found to be generated in cells infected with the ifn-inducing sevs but not those with the ifn-non-inducing sevs (summarized in table 1 ). one was a dsrna (j2-dsrna) that was detected by the anti-dsrna antibody, j2 (type i in table 1) . as for sev, the generation of j2-dsrna may have been restricted by the c proteins because it was only detected in cells infected with c-deficient or mutated recombinants, but not in those with intact sevs (figure 5 ; takeuchi et al., 2008; irie et al., 2010) . we and other groups demonstrated that j2-dsrna activated pkr, and this was followed by the phosphorylation of eif2 and the production of ifn-β, both of which resulted in antiviral effects in the host cells. the activation of pkr has been reported to induce the formation of sg-like structures during infections by some rna viruses, such as iav and measles virus (mev; mok et al., 2012; onomoto et al., 2012; okonski and samuel, 2013) , and this also appears to be the case for the sev c recombinants. unlike these viruses, the sg-like structures formed during 4c(-) infections did not include the j2-dsrna, which was dispersed in the cytoplasm, although they contained rig-i (figure 5) . this may lead to the assertion that the sg-like structures induced during infections by c recombinants are not the sites at which to detect sev infections. however, the sg-like structures formed by 4c(-) were revealed to contain another type of unusual viral rna species by fish analysis, in which an rna probe targeting the 600 nt region of the 5 -end of the (-)-sense sev genome was used (type ii in table 1 ; figure 6 ). this now strongly suggests that the sg-like structures found in the 4c(-) infection are defined as avsgs. unlike the iav infection, the type i and ii rna species seemed to be not or less encapsidated, given that they were not colocalized with viral antigens and were not removed from the infected cell lysates by immunoprecipitation using anti-sev antibody (figures 1 and 4-6) . sev trailer rna, which is transcribed from the 3 -ends of (+)-sense antigenome rna, was previously reported to interact with tiar to inhibit apoptosis and the formation of sgs induced by infection (iseni et al., 2002) . the 4c(-) virus was shown to induce apoptosis more quickly and severely in infected cells than the wt virus (irie et al., 2010) . although appearing to contain the nonencapsidated 5 -end of a (-)-sense genome rna, at least in the part that is concordant with the trailer rna, it is unlikely that the type ii rna observed in the 4c(-)-infected cells has the ability of the trailer rna to inhibit apoptosis and sg formation. although details of the type i and ii rnas remain to be solved, given the cytoplasmic replication of sev rna without forming inclusion bodies and the differences of the rna species in subcellular distribution and reactivity with the j2 and fish probe, the type i dsrna somewhat unwound actively or incidentally into the type ii rna might be accumulated into the avsgs. although the sg-like structures and j2-dsrna were not detected during cnt infections, fish-positive non-granularshaped inclusions were observed (type iii in table 1 ; figure 6 ). these cnt inclusions were markedly different from those of 4c(-). the inclusions did not include rig-i or g3bp1, but contained the n protein (figure 6) , suggesting that the inclusions were not avsgs, and that the type iii rna was at least partially encapsidated. the cnt stock used in the present study contained a larger amount of cbdi genomes than the other viral stocks ( figure 4b) . the sev cbdi genomes have been shown to be partially and/or more loosely encapsidated than intact genomes (kolakofsky, 1976; strahle et al., 2006) . the sev cbdi genomes were recently identified as strong ligands for rig-i (baum et al., 2010; tapia et al., 2013) . indeed, the cnt-lowdi had lost the ability to induce ifn-β and the inclusions had not been found in the infected cells (figures 4b and 6) . taken together, the type iii rna was identified as the cbdi genome. these results indicated that the process of detecting infections and the subsequent induction of ifn-β differed largely between 4c(-) and cnt: avsg-dependent and -independent mechanisms for 4c(-) and cnt, respectively. most viruses have been shown to possess the ability to antagonize host ifn pathways in order to avoid activating host antiviral actions, and this strategy is mostly based on a counteraction against the molecules involved in these pathways (versteeg and garcia-sastre, 2010) . however, a recent study reported that encephalomyocarditis virus (emcv) has the ability to disrupt sgs by cleaving g3bp1 in order to avoid the innate immune detection of its infection and subsequent induction of ifn-β (ng et al., 2013) , suggesting that another effective strategy for viral evasion from the ifn system by preventing avsg formation exists. generation of the unusual viral rna species triggering the production of ifn-β seems to be suppressed during intact rna viral replication. similarly to sev, another paramyxovirus mev c protein was recently shown to impair the production of j2-dsrna and the activation of pkr coupled with the formation of sg-like structures (okonski and samuel, 2013; pfaller et al., 2014) . unlike the case of sev, in which the knockout of c resulted in the production of j2-dsrna (figures 5 and 6 ), but not cbdi genomes, the j2-dsrna produced during the infection by a c-deficient mev recombinant was reported to be a cbdi genome (pfaller et al., 2014) . although the cbdi genomes were more dominantly produced by sev-cnt than by the other strains and c-recombinants tested ( figure 4b and data not shown), this unique property of cnt might be attributed to its c protein that possibly have a functional difference with those of the other sevs. the c proteins of both sev and mev have been shown to play critical roles in modulating viral rna synthesis and maintaining its integrity by potentially stabilizing the ribonucleoprotein (rnp)-polymerase complex (tapparel et al., 1997; reutter et al., 2001; bankamp et al., 2005; irie et al., 2008a irie et al., , 2014 ito et al., 2013) . dysfunctions in the c proteins may result in a disturbance in integrity, leading to the production of the unusual, ifn-β-inducing rna species. the results of the present study indicate that several types of ifn-β-inducible, unusual viral rna species may be produced during sev infections and included in avsg-like and non-avsg-like cytoplasmic inclusions, which suggests that rna-typedependent mechanisms recognize and accumulate such unusual viral rnas in specific compartments. in addition, the production of these unusual rna species may be restricted during intact viral replication in order to avoid detection by host innate immunity. pathogen recognition and innate immunity visibly stressed: the role of eif2, tia-1, and stress granules in protein translation rna granules hepatitis c virus hijacks p-body and stress granule components around lipid droplets polysomes, p bodies and stress granules: states and fates of eukaryotic mrnas identification of naturally occurring amino acid variations that affect the ability of the measles virus c protein to regulate genome replication and transcription differential recognition of viral rna by rig-i preference of rig-i for short viral rna molecules in infected cells revealed by nextgeneration sequencing rig-i goes beyond naked recognition eukaryotic stress granules: the ins and outs of translation molecular cloning of natural paramyxovirus copy-back defective interfering rnas and their expression from dna amino acids 78 and 79 of mammalian orthoreovirus protein microns are necessary for stress granule localization, core protein lambda2 interaction, and de novo virus replication the 3' untranslated regions of influenza genomic sequences are 5'ppp-independent ligands for rig-i induction of stress granule-like structures in vesicular stomatitis virus-infected cells poliovirus infection induces the colocalization of cellular protein srp20 with tia-1, a cytoplasmic stress granule protein involvement of the leader sequence in sendai virus pathogenesis revealed by recovery of a pathogenic field isolate from cdna hepatitis c virus (hcv) induces formation of stress granules whose proteins regulate hcv rna replication and virus assembly and egress essential role of mda-5 in type i ifn responses to polyriboinosinic:polyribocytidylic acid and encephalomyocarditis picornavirus rig-i and dsrna-induced ifnbeta activation translational control in stress and apoptosis 5'-triphosphate rna is the ligand for rig-i inhibition of interferon regulatory factor 3 activation by paramyxovirus v protein conserved charged amino acids within sendai virus c protein play multiple roles in the evasion of innate immune responses paramyxovirus sendai virus c proteins are essential for maintenance of negative-sense rna genome in virus particles recruitment of alix/aip1 to the plasma membrane by sendai virus c protein facilitates budding of virus-like particles sendai virus c proteins regulate viral genome and antigenome synthesis to dictate the negative genome polarity clustered basic amino acids of the small sendai virus c protein y1 are critical to its ran gtpase-mediated nuclear localization sendai virus trailer rna binds tiar, a cellular protein involved in virus-induced apoptosis measles virus nonstructural c protein modulates viral rna polymerase activity by interacting with host protein shcbp1 toll-like receptor function and signaling the paramyxovirus, sendai virus, v protein encodes a luxury function required for viral pathogenesis initiation of sendai virus multiplication from transfected cdna or rna with negative or positive sense rig-i-like receptors: cytoplasmic sensors for non-self rna stress granules: sites of mrna triage that regulate mrna stability and translatability influenza a virus inhibits cytoplasmic stress granule formation immediate protection of mice from lethal wild-type sendai virus (hvj) infections by a temperaturesensitive mutant, hvjpi, possessing homologous interfering capacity isolation and characterization of sendai virus di-rnas sendai virus c proteins are categorically nonessential gene products but silencing their expression severely impairs viral replication and pathogenesis respiratory syncytial virus induces host rna stress granules to facilitate viral replication activation of protein kinase r is required for induction of stress granules by respiratory syncytial virus but dispensable for viral replication short double-stranded rnas with an overhanging 5' ppp-nucleotide, as found in arenavirus genomes, act as rig-i decoys a sendai virus-derived rna agonist of rig-i as a virus vaccine adjuvant importance of eif2alpha phosphorylation and stress granule assembly in alphavirus translation regulation the ns1 protein of influenza a virus interacts with cellular processing bodies and stress granules through rna-associated protein 55 (rap55) during virus infection encephalomyocarditis virus disrupts stress granules, the critical platform for triggering antiviral innate immune responses stress granule formation induced by measles virus is protein kinase pkr dependent and impaired by rna adenosine deaminase adar1 critical role of an antiviral stress granule containing rig-i and pkr in viral detection and innate immunity modulation of hepatitis c virus rna abundance and virus release by dispersion of processing bodies and enrichment of stress granules sequestration of g3bp coupled with efficient translation inhibits stress granules in semliki forest virus infection measles virus c protein impairs production of defective copyback double-stranded viral rna and activation of protein kinase r rig-i-mediated antiviral responses to single-stranded rna bearing 5'-phosphates mammalian orthoreovirus escape from host translational shutoff correlates with stress granule disruption and is independent of eif2alpha phosphorylation and pkr mammalian orthoreovirus particles induce and are recruited into stress granules at early times postinfection mouse hepatitis coronavirus replication induces host translational shutoff and mrna decay, with concomitant formation of stress granules and processing bodies mutations in the measles virus c protein that up regulate viral rna synthesis dynamic oscillation of translation and stress granule formation mark the cellular response to virus infection in vivo ligands of mda5 and rig-i in measles virus-infected cells regulation of innate antiviral defenses through a shared repressor domain in rig-i and lgp2 analysis of interaction of sendai virus v protein and melanoma differentiation-associated gene 5 aip1/alix is a binding partner of sendai virus c protein and facilitates virus budding rig-i detects mrna of intracellular salmonella enterica serovar typhimurium during bacterial infection sendai virus defective-interfering genomes and the activation of interferon-beta activation of the beta interferon promoter by unnatural sendai virus infection requires rig-i and is inhibited by viral c proteins sendai virus c protein plays a role in restricting pkr activation by limiting the generation of intracellular double-stranded rna defective viral genomes arising in vivo provide critical danger signals for the triggering of lung antiviral immunity inhibition of sendai virus genome replication due to promoterincreased selectivity: a possible role for the accessory c proteins structural comparison of the cleavage-activation site of the fusion glycoprotein between virulent and avirulent strains of newcastle disease virus viral tricks to grid-lock the type i interferon system incoming rna virus nucleocapsids containing a 5'-triphosphorylated genome activate rig-i and antiviral signaling inhibition of cytoplasmic mrna stress granule formation by a viral proteinase poliovirus unlinks tia1 aggregation and mrna stress granule formation the rna helicase rig-i has an essential function in double-stranded rna-induced innate antiviral responses dhx36 enhances rig-i signaling by facilitating pkrmediated antiviral stress granule formation we thank the staff of the analysis center of life science, hiroshima university, for the use of their facilities. we also thank dr. k. takeuchi (university of tsukuba) for fruitful discussions. this work was supported by jsps kakenhi (grant numbers 23790505 and 25460569). the supplementary material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fmicb. 2015.00804 key: cord-007689-0vpp3xdl authors: schlee, m.; barchet, w.; hornung, v.; hartmann, g. title: beyond double-stranded rna-type i ifn induction by 3prna and other viral nucleic acids date: 2007 journal: interferon: the 50th anniversary doi: 10.1007/978-3-540-71329-6_11 sha: doc_id: 7689 cord_uid: 0vpp3xdl production of type i ifn is the key response to viral infection. since the discovery of type i ifns in 1957, long double-stranded rna formed during replication of many viruses was thought to be responsible for type i ifn induction, and for decades double-stranded rna-activated protein kinase (pkr) was thought to be the receptor. recently, this picture has dramatically changed. it now became evident that not pkr but two members of the toll-like receptor (tlr) family, tlr7 and tlr9, and two cytosolic helicases, rig-i and mda-5, are responsible for the majority of type i ifns induced upon recognition of viral nucleic acids. in this review, we focus on the molecular mechanisms by which those innate immune receptors detect viral infection. based on the recent progress in the field, we now know that tlr7, tlr9, and rig-i do not require long double-stranded rna for type i ifn induction. type i ifns (ifn-α isoforms and ifn-β) are regarded as the dominant mediators of antiviral defense in vertebrates. since their initial discovery half a century ago as acid-stable, soluble factors "interfering" with viral proliferation in cultured cells (isaacs and lindenmann 1957; nagano and kojima 1958) , intense research has focused on type i ifn receptor signaling and the plethora of type i ifn-mediated effects (theofilopoulos et al. 2005) . for the host, an intact type i ifn response is critical for the survival of many viral infections (gresser et al. 1976; muller et al. 1994) . sensing of viral replication has been proposed to be responsible for triggering the production of type i ifns by infected host cells. however, the specific host immune receptors and their respective molecular ligands remained elusive until very recently. moreover, to mount an appropriate antiviral response, the innate immune system must distinguish viruses from bacteria, fungi, and multicellular parasites. charles janeway was the first to propose that the detection of highly conserved pathogen-associated molecular patterns (pamps) may be mastered by a limited number of germline-encoded pattern recognition receptors (prrs) (janeway 1989) . a few years later, the first experimental evidence of such a receptor came from the fruit fly (lemaitre et al. 1996) . shortly afterwards, a member of the family of toll-like receptors (tlr), the mammalian homolog of drosophila toll, was demonstrated to be responsible for detecting lipopolysaccharides (lps), a characteristic component of the cell walls of gram-negative bacteria (medzhitov et al. 1997; poltorak et al. 1998 ). this observation was confirmed by the subsequent generation of tlr4-deficient mice (hoshino et al. 1999) . parasites, bacteria, and fungi rely on a multitude of molecules that are distant in evolutionary terms from the mammalian organism, and are thus readily discernible as non-self by members of the toll-like receptor (tlr) and nodlike receptor (nlr) families (reviewed in meylan et al. 2005) . in sharp contrast, all components of viruses are produced within the infected host cell, and therefore lack distinguishable non-self molecular patterns. nevertheless, viruses are promptly recognized by the innate immune system and elicit pronounced antiviral type i interferon and cytokine responses. shortly after the discovery of type i interferons, it was proposed that viral nucleic acids could be stimulating the type i ifn response (isaacs et al. 1963) . many viruses synthesize double-stranded rna (dsrna) during their replication cycle (baltimore et al. 1964; montagnier and sanders 1963) , whereas dsrna was thought to be absent in uninfected cells. therefore dsrna formed during viral infection was postulated to be the molecular signature of viral infection. in support of this hypothesis, the enzymatically generated double-stranded rna polynucleotide polyinosinic:polycytidylic acid (poly i:c) was found to be a potent inducer of type i ifn (field et al. 1967) . although the authors carefully emphasized that all other double-stranded polynucleotides were inactive, the notion that long viral double-stranded rna elicits type i ifn became commonplace, and poly i:c has been used as an interferon-inducing mimic of viral dsrna ever since. in early attempts to uncover the inducers of interferon and of other mediators of antiviral activity, ifn-α and poly i:c-treated or reticulocyte extracts were analyzed. chromatographic separation of lysates revealed proteins that were increased by preincubation with ifn-α, and whose enzymatic activity depended on the presence of dsrna (usually poly i:c) (farrell et al. 1978; hovanessian et al. 1977; zilberstein et al. 1978) . two proteins, interferon inducible double-stranded rna-activated protein kinase (pkr) and the 2′,5′ oligoadenylate synthetase (oas) could be affinity purified using poly i:c-cellulose (farrell et al. 1978; hovanessian et al. 1977) . both activated pkr and oas were found to block translation of viral rna by distinct mechanisms. in the presence of poly i:c, oas catalyzes the synthesis of 2′,5′ oligomers of adenosine (2-5as) (hovanessian et al. 1977; zilberstein et al. 1978) , which activate rnase l (farrell et al. 1978) . rnase l in turn degrades single-stranded viral and cellular rnas (farrell et al. 1978 ) in a sequence-independent manner (minks et al. 1979) . consequently rnase l-deficient mice displayed a reduced antiviral activity of ifn-α, as well as impaired apoptosis (zhou et al. 1997) . in contrast, the serine threonine kinase pkr was found to more specifically block the translation of viral rna (farrell et al. 1978 ) by phosphorylation of the eukaryotic translation initiation factor eif2a. besides its function in limiting translation of viral protein, pkr was also reported to activate nf-κb (kumar et al. 1994) . pkr was therefore proposed as a key receptor mediating virus-and dsrna-induced production of type i interferons (kumar et al. 1994 ). however, these findings remained controversial, as other studies that examined pkr-deficient mice and cells (chu et al. 1999; iordanov et al. 2001; maggi et al. 2000; smith et al. 2001; yang et al. 1995) found no defects in the induction of interferon in response to poly i:c or viral infection that could not be overcome with type i ifn pretreatment. further analysis revealed that pkr is not only activated by poly i:c but is able to interact with dsrna as short as 11 bp. however, at least 30 bp are required to activate pkr kinase activity (manche et al. 1992) . in another study (zheng and bevilacqua 2004) , recombinant pkr could also be activated by rna oligonucleotides containing a 16-bp dsrna stem loop in combination with a more than 11bp-long single-stranded rna part at the 5′ or 3′ end. all these studies question the often quoted requirement of a dsrna molecule longer than 30 bp; furthermore, it became evident that the translational shut-down by pkr is not linked to the induction of type i ifn synthesis and secretion. the finding that pkr -/cells still produce type i ifns spurred further research on receptors capable of recognizing long double-stranded rna. such investigations led to a member of the toll-like receptor (tlr) family, tlr3, which was proposed to bind to long dsrna and to induce ifn-β (alexopoulou et al. 2001) . tlrs are transmembrane receptors that were shown to recognize a variety of conserved pathogenassociated molecular patterns (pamps) of bacterial, fungal, and parasitic origin. the study of alexopoulou et al. was the first to demonstrate a role for tlrs in the recognition of viruses. tlr9 was found to be the receptor for unmethylated cpg motifs in dna (hemmi et al 2000) ; however, cpg-dna at first was thought to be characteristic for bacterial dna, and the role of tlr9 in detecting dna viruses was only proposed later (krug et al. 2004a (krug et al. , 2004b tabeta et al. 2004) . upon engagement with their specific ligands, tlrs trigger signaling pathways that lead to the activation of nf-κb and irfs (signaling of tlrs reviewed in moynagh 2005) . tlr3 was found to induce type i ifns upon poly i:c stimulation by activation of the kinase tbk1, which phosphorylates the transcription factor irf3, resulting in the induction of ifn-β (doyle et al. 2002; fitzgerald et al. 2003; sharma et al. 2003) . another group reported that tlr3 is activated by ssrna (kariko et al. 2004b ); however, tlr3-deficient mice and mice deficient in the signaling adapter trif (gitlin et al. 2006; kato et al. 2006 ) still responded to poly i:c. moreover, dendritic cells derived from tlr3-deficient mice were still stimulated by dsrna transfected into the cytosol (diebold et al. 2003 ). unlike tumor cell lines, which were examined in early studies on type i ifn and dsrna, primary immune cells such as peripheral blood mononuclear cells (pbmcs) express a wide spectrum of functional tlrs. different immune cell subsets express distinct patterns of tlrs (hornung et al. 2002) . plasmacytoid dendritic cells (pdcs) (reviewed in colonna et al. 2004 ) are the major producers of early type i ifn production upon viral infection. pdcs express tlr7 and tlr9 but not tlr3. both tlr7 and tlr9 are located in the endosomal compartment and signal via the adaptor molecules myd88, irak1, and traf6, leading to activation of irf7 and the induction of type i interferons as reviewed by moynagh (2005) . in addition, recent studies show that traf3 plays a crucial role in the myd88-dependent signaling cascade (hacker et al. 2005; oganesyan et al. 2005) . single-stranded dna and the small antiviral compound r848 had been shown to induce ifn in pdcs dependent on tlr9 and tlr7, respectively (hemmi et al. 2000 (hemmi et al. , 2002 jurk et al. 2002; krug et al. 2001b; rothenfusser et al. 2002) . tlr9 detects unmethylated so-called cpg motifs in single-stranded dna (hemmi et al. 2000) . different classes of synthetic cpg oligodeoxynucleotides (odn) were developed based on the distinct effects on the two tlr9-expressing immune cell types: pdcs and b cells (hartmann et al. 2003; hartmann and krieg 2000; krug et al. 2001a) . in contrast to tlr3, both tlr7 and 9 depend on the signaling adapter myd88. accordingly, pdcs derived from tlr9-or myd88-deficient mice are unable to produce type i ifn in response to dna viruses such as herpes simplex viruses (hsv) and murine cytomegalovirus (mcmv) (krug et al. 2004a (krug et al. , 2004b lund et al. 2003; tabeta et al. 2004) . while tlr9 was responsible for detecting viral dna, tlr7 was shown to recognize rna: tlr7 detects synthetic short (20-27 bases) single-stranded rna (diebold et al. 2004; heil et al. 2004 ) and short interfering double-stranded rna (sirna) (hornung et al. 2005; judge et al. 2005; sioud 2005 ; reviewed in schlee et al. 2006 ). the amount of type i interferon induction was dependent on the rna sequence. ironically, hornung and colleagues came across a very potent type i interferon inducing small rna sequence core motif (5′-guccuucaa-3′) in the attempt to knock down the interferon inducer tlr9 in pdcs using the sirna technology (hornung et al. 2005) . it was demonstrated that these sirnas induce systemic immune activation in mice, and that the immunological activity required tlr7. of note, the same sirna did not induce type i interferon in immortalized human embryonic kidney cells (hek293), which produced type i interferon in response to poly i:c. in subsequent studies, similar findings were reported by judge et al. (2005) (identifying a core motif 5′-ugugu-3′) and sioud (2005) . in all three studies, transfection with cationic lipids (e.g., dotap, lipofectamine) or cationic polymers (e.g., pei, polyethylenimine) was essential for the immunological activity of sirna. the same applies for the immunological activity of single-stranded rna (diebold et al. 2004; heil et al. 2004; scheel et al. 2005 ). while for short rna oligonucleotides the immunological activity is clearly sequence dependent (hornung et al. 2005; judge et al. 2005) , for long rna molecules such as mrna, sequence specificity of immunological activity is less prominent (scheel et al. 2005) . this raises the question of how the immune system is able to distinguish between self and non-self (for example viral) rna. this question was addressed recently by kariko and colleagues (2005) who showed that human mitochondrial rna, when transfected into monocyte-derived dendritic cells, provoked secretion of tnf-α at similar quantities compared to total rna isolated from escherichia coli . in contrast, rna of other cellular compartments showed no immunological activity. the authors proposed that mammalian rna is masked by naturally occurring nucleoside modifications that are expected to be similar in closely related species. according to this concept, mitochondrial rna is stimulatory since it resembles bacterial rather than mammalian rna. in healthy cells, mitochondrial rna will not be released. in contrast to other self-rna, mitochondrial rna never enters the cytosolic compartment. as a consequence, mitochondrial rna under healthy conditions is not detected by cytosolic mechanisms of detection. only if the cell is lysed can mitochondrial rna enter the endosomal compartment of immune cells via phagocytosis. indeed, the stimulatory effect of in vitro rna transcripts composed of unmodified nucleotides in their study could be abrogated by incorporation of modified nucleosides such as pseudouridine, 5-methylcytidine, n6-methyladenosine, inosine, and n7methylguanosine. in order to examine modification sensitivity of different tlrs, hek293 cells expressing tlr3, tlr7, tlr8, or tlr9 were transfected with rna containing modified nucleosides. transfection of unmodified rnas stimulated il-8 production (sensitive readout for immunoactivation of hek293 cells) in hek293 cells overexpressing tlr3, 7, and 8. interestingly, rna recognition by tlr3, tlr7, and tlr8 is suppressed by the presence of different types of modified nucleotides within the rna ligand. tlr3 was the least sensitive receptor with regard to suppression by nucleoside modifications. furthermore, the authors showed that in monocyte-derived dendritic cells, 5%-10% of modified nucleosides were sufficient to inhibit tnf-α secretion by 75%-90%. together, these results show that rna modification contributes to the distinction of self versus non-self rna by the immune system. further insight into the properties that render rna molecules stimulatory to the immune system is driven by sirna technology. based on studies by tuschl and colleagues (elbashir et al. 2001) , sirna is now used worldwide as a robust tool for target-specific gene silencing in cell lines and human primary cells. however, depending on the mode of synthesis and the sequences used to generate sirna, also nonspecific, so-called nonspecific off-target effects of sirnas were observed. to overcome limitations with the transfection of synthetic sirna, vectorbased (e.g., lentiviral) expression systems for the introduction of short hairpin sirnas (shrna) mimicking sirnas were developed (brummelkamp et al. 2002; harborth et al. 2003; paddison et al. 2002) . the most commonly used shrna expression system consists of a rna-polymerase iii dependent promoter driving the expression of two complementary 19-to 29-bp rna sequences linked by a short loop of 4-10 nt. the resulting transcript is exported to the cytoplasm and processed by dicer. lentiviral vectors haboring the pol iii-shrna expression cassette (li et al. 2003; rubinson et al. 2003; tiscornia et al. 2003) allow rnai-mediated gene silencing via sirna in cells that are otherwise difficult to transfect. sequence specificity of gene silencing by such shrna was questioned by bridge et al. (bridge et al. 2003) , who demonstrated that infection of human lung fibroblasts with pol iii-shrna containing lentivirus directed against the gene morf4l1 not only silenced morf4l1 but also stimulated interferoninducible genes such as 2′,5′-oas, an indicator of type i interferon. the ifninducing effect was dependent on the sequence and the dose of the vector; seven of 23 shrnas targeting different genes exhibited ifn induction. in contrast, transfection of synthetic sirna with the same putative ifn-inducing sequences led to sequence-specific silencing without triggering an ifn response. northern blot analysis of shrna showed that the majority of shrna transcripts were correctly processed to 20 nt transcripts. the authors speculated that remaining unprocessed transcripts could be detected by cytosolic rna sensing receptors. in the follow-up paper, the group of iggo (pebernard and iggo 2004) , further correlated the u6 promoter sequence with oas induction. this study revealed that the region between -2 (the end of the promoter) and +2 (the start of rna transcript) is crucial for the immune stimulatory effect, which was lost when they used the endogenous human sequence (ccga). further mutations leading to a partial mismatch in the shrna (predicted to create a 14-bp duplex) suggested that stimulation required more than a 14-bp duplex. william´s group (sledz et al. 2003 ) described the induction of ifn target genes by transfection of synthetic sirnas into a human glioblastoma cell line (t98g) or a renal carcinoma cell line (rcc). when comparing the two studies from bridge and colleagues and from sledz and colleagues, it is important to note that different cell lines (bridge, human lung fibroblasts; sledz, rcc and t98g) and different ways of sirna generation (bridge, synthetic sirnas and shrnas; sledz, synthetic sirna and t7-phage-polymerase sirna) were used. using mouse embryonic fibroblasts (mefs) with different gene deficiencies related to the ifn response system, sledz et al. proposed that pkr was the interferon-inducing receptor for sirnas. later, the same group postulated a different sirna receptor (rig-i, see below) in t98g cells (marques et al. 2006) . of note, in the two studies published by bridge et al. (2003) and sledz et al. (2003) , type i interferon was not analyzed at the protein level. kariko et al. (2004a) suggested that tlr3 was responsible for the induction of type i ifn by sirna. these data are based on keratinocyte (hacat) and hek 293 cells, which responded to synthetic sirna but not to the singlestranded components (ssrna) by secretion of low amounts of ifn-β that was comparable to stimulation with poly i:c. overexpression of tlr3 in hek 293 cells resulted in fourfold higher induction of type i ifn secretion in response to transfected sirna. however, overexpression of nf-κb-inducing receptors such as tlr3 may also contribute indirectly to the enhanced type i ifn response induced by sirna, for example by upregulating ifn-inducible cytosolic rna receptors. for example, tlr3 overexpressing hek 293 cells secrete more il-8 than empty vector or tlr9 overexpressing hek 293 cells (kariko et al. 2005) ; consequently, such studies do not necessarily provide evidence for a direct interaction between sirna and tlr3 . kim et al. (2004) showed that the induction of type i ifn by sirna depended on the use of t7-rna polymerase (t7 rnap) for sirna generation. in contrast to bridge et al. (2003) and sledz et al. (2003) , in the study by kim and colleagues, type i ifn was measured at the protein level, which is less sensitive than measuring ifn-dependent responses on the transcriptional level and thus underscores the magnitude of the ifn response they reported. in their study, kim and colleagues examined sirnas targeting the early icp4 gene of hsv-1. only t7 rnap-derived transcripts but not synthetic sirna elicited a potent antiviral activity when transfected into hek 293 cells. the same antiviral activity was observed by transfection of t7 transcripts with unrelated sequences. analysis of supernatants revealed the presence of substantial amounts of ifn-α and ifn-β protein. these results were reproduced in hela cells, as well as k562, cem, and jurkat cells. it is well known that unlike capped mammalian mrna, the 5′ ends of t7 transcripts harbor a triphosphate gtp-nucleotide. treatment of t7 transcripts with rnase t1 (with the 5′ end p-ggg removed, which was single-stranded in their case) and alkaline phosphatase was sufficient to completely abrogate interferon -inducing activity. additional experiments using t3 and sp6 phage rna polymerases demonstrated similar induction of type i ifn. the examination of multiple cell lines by kim et al. (2004) pointed to a powerful ubiquitously expressed sensor for short triphosphate rna. yoneyama and colleagues identified the interferon-inducing cytoplasmic dexd/ h box rna helicase rig-i, containing a caspase recruitment domain (card) (yoneyama et al. 2004) . expression of the card domain sensitized cells to activate the transcription factor irf3, leading to the induction of the ifn-β promoter. later on it was shown that this pathway involves the irf3 kinase tbk1, which is activated by the newly characterized adaptor protein ips-1, also known as cardif, mavs, or visa (kawai et al. 2005; meylan et al. 2005; seth et al. 2005; xu et al. 2005 ; reviewed in sen and sarkar 2005) . in overexpression experiments, rig-i was shown to bind poly i:c. however, overexpression of a dominant negative mutant of rig-i impaired irf3 activation by newcastle disease virus (ndv), a negative-strand rna virus, while irf3 activation by poly i:c was not inhibited. subsequent studies with rig-i -/mice and mefs showed no defect in the response to poly i:c. hornung and colleagues (2006) demonstrated that rig-i detects in vitro transcribed rna. rna with a triphosphate at the 5′ end (now termed 3prna), which is generated during in vitro transcription, was identified to be the ligand for rig-i. the minimal length of 3prna was 19 nucleotides. the activity of 3prna was independent of double-strand formation. both exogenous 3prna transfected into the cell and endogenously formed 3prna (expression of t7 rna polymerase) activated rig-i. genomic rna prepared from a negative-strand rna virus and rna prepared from virus-infected cells, but not rna from noninfected cells, triggered a potent ifn-α response in a 5′-triphosphate-dependent manner. binding studies of rig-i and 3prna revealed a direct molecular interaction. the 5′ capping or incorporation of modified nucleotides such as pseudouridine, 2-thiouridine, and 2′-o-methylated uridine in place of uridine in short 3prna strongly diminished ifn-α induction. in a parallel study, pichlmair et al. (2006) attributed the inhibitory effect of the influenza virus protein ns1 to its binding and inhibition of the rig-i triphosphate rna complex. these results provide evidence that uncapped unmodified 3prna is detected by rig-i in the cytosol of eukaryotic cells. of note, all primer-independent rna transcripts in a normal uninfected cell initially contain a 5′-triphosphate end. however, most if not all self-rna species entering the cytosol lack a free 5′triphosphate end. before self-rna leaves the nucleus, rna is further processed, which applies to rna transcripts of all three dna-dependent rna polymerases (pol) in eukaryotes. pol i transcribes a large polycistronic precursor of ribosomal rna (rrna) that contains the sequences for the mature rrnas (18, 5.8s, 25-28s rrna), two external transcribed spacers, and two internal transcribed spacers. this primary transcript is subjected to endo-and exonucleolytic processing steps to produce the mature rrnas. the net result of this maturation process is a monophosphate group at the 5′ end of all pol i transcribed rrnas (fromont-racine et al. 2003) . messenger rnas (mrnas) and small nuclear rnas (snrnas), which are transcribed by pol ii, receive a 7-methyl guanosine group that is attached to the 5′-triphosphate of the nascent rna by a process called capping (shatkin and manley 2000) . thus, upon export into the cytoplasm, no free triphosphate groups are found in pol ii transcripts. all mature trnas (pol iii) have a 5′-monophosphate (xiao et al. 2002) , as it is likely to apply to 5s rrna. u6 rna receives a γ-monomethylphosphate cap structure following transcription. however, 7sl rna (pol iii) has a triphosphate at the 5′ end, and is present at high copy numbers in the cytosol. therefore, the presence or absence of a 5′ triphosphate might not be the only structural feature of rna responsible for the distinction of self and viral rna. it is well known that eukaryotic rna undergoes significant modifications to its nucleosides and its ribose backbone. among all nucleoside modifications, pseudouridinylation is one of the most common post-transcriptional modifications of rna that appears to be universal among rrnas and small stable rnas such as splicing small nuclear rnas (snrnas), trnas, and small nucleolar rnas (snornas). however, the frequency and location of pseudouridinylated nucleotides vary phylogenetically. intriguingly, eukaryotes contain far more nucleoside modifications within their rna species. human ribosomal rna, for example, the major constituent of cellular rna, contains ten times more pseudouridine and 25 times more 2-o-methylated nucleosides than e. coli rrna (rozenski et al. 1999) . the same applies to eukaryotic trnas, the most heavily modified subgroup of rna with up to 25% of modified nucleosides. the host machinery that guides nucleoside modifications and 2′-o-methylation of the ribose backbone is located in the nucleolus, and consists of rna-protein complexes containing snornas and several associated proteins (snornps) (decatur and fournier 2003) . information on nucleolus-specific nucleoside modifications or ribose 2′-o-methylation of viral rna genomes is limited. since most rna viruses do not replicate in the nucleus and modification is tightly confined to the sequence and structure of their target, extensive modification of viral rna seems unlikely. altogether, post-transcriptional modifications of eukaryotic rna such as 5′ processing or capping, as well as nucleoside modifications or ribose backbone methylation, provide the molecular basis for the distinction of self-rna generated in the nucleus from viral rna of cytosolic origin containing 5′-triphosphate (3prna). the mrnas of viruses infecting eukaryotic cells also commonly contain 7-methyl guanosine cap-structures at their 5′ ends and poly(a) tails at their 3′ends (furuichi and shatkin 2000) . some viruses make use of the host transcription machinery to acquire caps and poly(a) tails. rna viruses that do not rely on the host transcriptional machinery produce their own capping enzymes or utilize other mechanisms such as snatching the 5′terminal regions of host mrnas. despite these adaptations of viruses to the host transcriptional system, viral rna synthesis leads to transient cytosolic rna intermediates with an uncapped 5′-triphosphate end. with notable exceptions such as the picornavirus family (see below), viral rna-dependent rna polymerases (rdrp) initiate polymerase activity de novo, without a specific primer (kao et al. 2001) . as a consequence, these rdrp-dependent transcripts start with an uncapped 5′-triphosphate. this has been studied in great detail for the replication of positive-strand rna viruses of the family of flaviviridae (including the genera flavivirus , pestivirus , and hepacivirus ); members of all of these virus genera were reported as being recognized via rig-i (honda et al. 1998; kato et al. 2006; sumpter et al. 2005) . segmented nsv rely on a cap-snatched primer for mrna transcription, yet initiate genomic and the complementary antigenomic rna replication by a primer-independent de novo mechanism resulting in a 5′-triphosphateinitiated transcript (honda et al. 1998; neumann et al. 2004) . nsv with a nonsegmented genome (order mononegavirales), including the paramyxoviruses and rhabdoviruses, initiate both replication and transcription de novo leading to 5′-triphosphate rna in the cytosol. both the full-length replication products, vrna and crna, and a short leader rna, which is abundantly synthesized during initiation of transcription, maintain their 5′triphosphate (colonno and banerjee 1978; whelan et al. 2004 ), while the virus-encoded mrna transcripts are further modified at their 5′ ends by capping and cap methylation. consequently, genomic rna from nsvs per se is expected to trigger an ifn-response without the need for replication and presumed dsrna formation. consistent with this notion, not only live virus but also rna purified from nsv virions (vsv) has been shown to trigger strong type i interferon responses depending on rig-i ). hornung and colleagues confirmed and extended these observations by demonstrating that dephosphorylation of the viral rna isolates completely abolished the ifn-response, thereby indicating that the 5′-triphosphate moiety is strictly required for recognition . a notable exception are the viruses in the picornavirus-like supergroup (picornavirus, potyvirus, comovirus, calicivirus, and other viruses), which exclusively employs a protein known as viral genome-linked protein (vpg) as a primer for both positive-and negative-strand rna production. this protein primer is part of the precursor rdrp and is cleaved off as elongation of the initial complex occurs, usually to become a 5′-genome-linked protein (lee et al. 1977) . thus during the life-cycle of picornaviruses uncapped, triphosphorylated 5′ ends are absent. consequently, based on our studies, rig-i is expected to be involved in the detection of flaviviridae and nsv but not picornaviruses. this is confirmed in a recent study . a number of studies suggested that the helicases mda-5 and rig-i recognize dsrna (andrejeva et al. 2004; rothenfusser et al. 2005; yoneyama et al. 2004) . the results in the work of hornung and colleagues (2006) demonstrated that double-strand formation of rna is not required for rig-i-rna interaction, and that dsrna is not sufficient for rig-i activation. these results further demonstrate that mda-5 is not involved in 5′-triphosphate rna recognition. although there is convincing evidence that mda-5 is activated by the long dsrna mimic poly i:c, activation of mda-5 by natural long dsrna is still controversial . taken together, tlr3 is so far the only receptor that induces type i ifn upon binding of the natural molecule long dsrna, but the contribution of tlr3 to type i ifn induction and viral clearance in vivo seems to be weak (rudd et al. 2006) . there is good evidence that short dsrna such as sirna generated by dicermediated cleavage of long dsrna does not elicit a type i ifn response in nonimmune cells (elbashir et al. 2001; hornung et al. 2005; kim et al. 2004) . a recent study suggests that the two-nucleotide overhang at the 3′ end of dicer cleavage products are essential for the lack of immunorecognition of short dsrna (marques et al. 2006) . the same study proposed that synthetic blunt-end short dsrna is recognized via rig-i. the conclusion that rig-i is the receptor for blunt end short dsrna is based on experiments using rig-i overexpression and using anti-rig-i sirna (short dsrna with two-nucleotide 3′overhangs) on top of stimulation with blunt end short dsrna stimulation. rig-i-deficient cells have not been examined in this study. this experimental design does not provide clear-cut evidence for the primary involvement of rig-i in type i ifn induction by blunt-end short dsrna. furthermore, in the study by hornung and colleagues, 5′ triphosphate blunt-end rna and 5′ triphosphate 2-nt overhang rna showed identical rig-i ligand activity, suggesting that the molecular feature 2-nt overhang does not inhibit rig-i-mediated recognition ). mda-5 is structurally related to rig-i, as it also contains two card domains and a helicase domain. mda-5 was originally identified as a type i ifn-inducible molecule mediating cell cycle arrest and apoptosis in melanoma cells (hence the name melanoma differentiation antigen 5) (kang et al. 2002 (kang et al. , 2004 kovacsovics et al. 2002) . a first indication of a role for mda-5 in virus recognition came from the observation that a paramyxoviral protein that mediated immune evasion bound to mda-5 (andrejeva et al. 2004 ). in overexpression experiments, mda-5 was shown to bind poly i:c, and enhanced the interferon response to poly i:c as well as several viruses. conversely, sirna mediated knock-down blocked type i ifn induction in response to these stimuli . mda-5 was then shown to play an essential role in the detection of picornaviruses such as encephalomyocarditis virus (emcv) or theiler's virus (gitlin et al. 2006; kato et al. 2006 ). in addition, mice deficient in mda-5 were found to be highly susceptible to emcv. although the nature of the natural rna ligand that engages mda-5 has so far remained obscure, a surprising observation was that cells derived from mda-5-deficient mice, as well as mda-5 -/mice stimulated in vivo were found unable to mount a type i ifn response to poly i:c, establishing mda-5, rather than the several other receptors that bind, or have been shown to be activated by poly i:c, as the dominant receptor mediating the interferon response to poly i:c (gitlin et al. 2006; kato et al. 2006 ). however, the natural viral ligand for mda-5 has not yet been identified. in addition to rig-i and mda-5, another cytosolic receptor may exist for detecting dna. until recently tlr9 was the only innate sensor for detecting microbial dna. recent studies indicate that dna is detected in the cytosol independently of tlr9 (okabe et al. 2005; stetson and medzhitov 2006) , but the receptor has not been identified yet. the cytosolic receptor mediating recognition of b-form dna, unlike rig-i and mda-5, signals independently of ips-1. as discussed in the previous sections, immune and nonimmune cell types express characteristic patterns of nucleic acid receptors (table 1 ) . for example, melchjorsen and colleagues reported that activation of innate defense against a paramyxovirus is mediated by rig-i, tlr7, and tlr8 in a cell-type-specific manner (melchjorsen et al. 2005) . they found that nonimmune cells relied entirely on rna recognition through rig-i for activation of an antiviral response. in contrast, immune cells such as myeloid cells utilized tlr7 and tlr8. unlike modifications that have been shown to prevent detection by the receptors indicated and that are frequently found in mammalian nucleic acids in nonimmune cells, rna sensing in paramyxovirus-infected myeloid cells was independent of rig-i, tlr3, and pkr. kato and colleagues also found celltype specific involvement of rig-i in antiviral immune response. in their study type i ifn induction in both fibroblasts and myeloid dendritic cells was rig-i-dependent, while type i ifn induction in pdc was rig-i-independent . it is important to note that the mechanisms used for rna sensing may not only be cell-type-dependent but may also depend on the type of virus and its strategy to enter the target cell and to evade immune recognition. in contrast, recognition of synthetic rna or of rna transcribed from vector systems is more predictable because there is no immune evasion and because the mode of delivery is known. of note, the use of cationic lipids and polycationes leads to both endosomal and cytosolic delivery (almofti et al. 2003; boussif et al. 1995) and thus both tlr-and rig-i-mediated rna sensing is triggered, provided these receptors are expressed in the cell type examined, and the appropriate rna ligand is delivered. of note, subcellular localization of tlr3 is cell-type-specific (matsumoto et al. 2003) : in fibroblasts, tlr3 is located on the cell surface, and the tlr3-mediated activity can be blocked by anti-tlr3 antibodies. in myeloid dendritic cells, tlr3 is found in the cytosolic compartment. a more detailed analysis in tlr3-transfected b cells revealed that tlr3 is detectable in multivesicular bodies, a subcellular compartment situated in the endocytic trafficking pathway (matsumoto et al. 2003) . bacteria, fungi, or cellular parasites are recognized via conserved molecules typical for the respective type of pathogen. in contrast, all virus components are formed within the infected host cell; consequently, a virus-specific detection system is more difficult to achieve. it is now evident that host cells are equipped to detect viral nucleic acids. for viral infection in vivo, the following picture is evolving: large parts of the early type i ifn response upon viral infection are due to tlr7 and tlr9 expressed in pdcs; in fact, pdcs are the only considerable source of tlr7-and tlr9-induced type i ifn production upon viral infection. the major advantages of this pdc response are that the presence of viral particles is sufficient for recognition, that viral infection of cells is not required for detection, and that viruses are recognized before viral proteins have a chance to mediate immune evasion. this first wave of type i ifn production plays an important role in limiting viral spread by pdc-derived direct antiviral mechanisms early on, and by sensitizing yet uninfected cells for cytosolic recognition of viral nucleic acid via strong upregulation of the two cytosolic helicases, rig-i and mda-5. these two cytosolic receptors are then responsible for the second and prolonged wave of type i ifn production and for the induction of apoptosis of virally infected cells. for all four receptors, distinction of self from viral nucleic acid is based on a combination of localization and molecular structure. in this sophisticated system of virus detection, the following situations signal viral danger: 1. appearance of unmodified rna in the endosomal compartment of pdcs 2. appearance of dna containing unmethylated cpg motifs in the endosomal compartment of pdcs 3. unmodified rna with a triphosphate group at the 5′ end (3prna) in the cytosol of any cell type 4. dna in the cytosol of any cell type it is still unclear whether long dsrna in the cytosol is sufficient to elicit an antiviral response via one of the receptors known to date. although poly i:c is a ligand for mda-5, long double-stranded rna seems insufficient as a ligand, and the natural ligand still needs to be identified. in addition to rnadetecting receptors, the cytosolic receptor for dna may add new perspectives in therapeutic viral mimicry. with regard to viruses that perform inside the nucleus such as hbv and hiv, uncovering molecular mechanisms of sensing viral nucleic acids in the nucleus appears on the radar of scientific challenges. recognition of double-stranded rna and activation of nf-kappab by toll-like receptor 3 cationic liposome-mediated gene delivery: biophysical study and mechanism of internalization the v proteins of paramyxoviruses bind the ifn-inducible rna helicase, mda-5, and inhibit its activation of the ifn-beta promoter virus-specific double-stranded rna in poliovirus-infected cells dendritic cells respond to influenza virus through tlr7-and pkrindependent pathways a versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine induction of an interferon response by rnai vectors in mammalian cells a system for stable expression of short interfering rnas in mammalian cells control of coronavirus infection through plasmacytoid dendritic-cell-derived type i interferon jnk2 and ikkbeta are required for activating the innate response to viral infection plasmacytoid dendritic cells in immunity complete nucleotide sequence of the leader rna synthesized in vitro by vesicular stomatitis virus rna-guided nucleotide modification of ribosomal and other rnas innate antiviral responses by means of tlr7-mediated recognition of single-stranded rna viral infection switches non-plasmacytoid dendritic cells into high interferon producers irf3 mediates a tlr3/tlr4-specific antiviral gene program duplexes of 21-nucleotide rnas mediate rna interference in cultured mammalian cells interferon-beta is required for interferon-alpha production in mouse fibroblasts interferon action: two distinct pathways for inhibition of protein synthesis by double-stranded rna inducers of interferon and host resistance. ii. multistranded synthetic polynucleotide complexes lps-tlr4 signaling to irf-3/7 and nf-kappab involves the toll adapters tram and trif ribosome assembly in eukaryotes viral and cellular mrna capping: past and prospects essential role of mda-5 in type i ifn responses to polyriboinosinic:polyribocytidylic acid and encephalomyocarditis picornavirus role of interferon in the pathogenesis of virus diseases in mice as demonstrated by the use of antiinterferon serum. i. rapid evolution of encephalomyocarditis virus infection specificity in toll-like receptor signalling through distinct effector functions of traf3 and traf6 sequence, chemical, and structural variation of small interfering rnas and short hairpin rnas and the effect on mammalian gene silencing rational design of new cpg oligonucleotides that combine b cell activation with high ifn-alpha induction in plasmacytoid dendritic cells mechanism and function of a newly identified cpg dna motif in human primary b cells species-specific recognition of single-stranded rna via tolllike receptor 7 and 8 small anti-viral compounds activate immune cells via the tlr7 myd88-dependent signaling pathway a toll-like receptor recognizes bacterial dna identification of the 5′ terminal structure of influenza virus genome rna by a newly developed enzymatic method role of a transductional-transcriptional processor complex involving myd88 and irf-7 in toll-like receptor signaling 5′-triphosphate rna is the ligand for rig-i sequence-specific potent induction of ifn-alpha by short interfering rna in plasmacytoid dendritic cells through tlr7 quantitative expression of toll-like receptor 1-10 mrna in cellular subsets of human peripheral blood mononuclear cells and sensitivity to cpg oligodeoxynucleotides cutting edge: toll-like receptor 4 (tlr4)-deficient mice are hyporesponsive to lipopolysaccharide: evidence for tlr4 as the lps gene product synthesis of low molecular weight inhibitor of protein synthesis with enzyme from interferon-treated cells activation of nf-kappab by doublestranded rna (dsrna) in the absence of protein kinase r and rnase l demonstrates the existence of two separate dsrna-triggered antiviral programs foreign nucleic acids as the stimulus to make interferon virus interference. i. the interferon approaching the asymptote? evolution and revolution in immunology sequencedependent stimulation of the mammalian innate immune response by synthetic sirna human tlr7 or tlr8 independently confer responsiveness to the antiviral compound r-848 expression analysis and genomic characterization of human melanoma differentiation associated gene-5, mda-5: a novel type i interferon-responsive apoptosisinducing gene mda-5: an interferon-inducible putative rna helicase with double-stranded rna-dependent atpase activity and melanoma growth-suppressive properties de novo initiation of viral rna-dependent rna synthesis small interfering rnas mediate sequence-independent gene suppression and induce immune activation by signaling through toll-like receptor 3 suppression of rna recognition by toll-like receptors: the impact of nucleoside modification and the evolutionary origin of rna cell type-specific involvement of rig-i in antiviral response differential roles of mda5 and rig-i helicases in the recognition of rna viruses interferon-alpha induction through tolllike receptors involves a direct interaction of irf7 with myd88 and traf6 ips-1, an adaptor triggering rig-i-and mda5-mediated type i interferon induction interferon induction by sirnas and ssrnas synthesized by phage polymerase overexpression of helicard, a card-containing helicase cleaved during apoptosis, accelerates dna degradation tlr9-dependent recognition of mcmv by ipc and dc generates coordinated cytokine responses that activate antiviral nk cell function herpes simplex virus type 1 activates murine natural interferon-producing cells through toll-like receptor 9 identification of cpg oligonucleotide sequences with high induction of ifn-alpha/beta in plasmacytoid dendritic cells toll-like receptor expression reveals cpg dna as a unique microbial stimulus for plasmacytoid dendritic cells which synergizes with cd40 ligand to induce high amounts of il-12 double-stranded rnadependent protein kinase activates transcription factor nf-kappa b by phosphorylating i kappa b a protein covalently linked to poliovirus genome rna the dorsoventral regulatory gene cassette spatzle/toll/cactus controls the potent antifungal response in drosophila adults inhibition of hiv-1 infection by lentiviral vectors expressing pol iiipromoted anti-hiv rnas toll-like receptor 9-mediated recognition of herpes simplex virus-2 by plasmacytoid dendritic cells recognition of single-stranded rna viruses by toll-like receptor 7 potential role of pkr in double-stranded rna-induced macrophage activation interactions between doublestranded rna regulators and the protein kinase dai differential viral induction of distinct interferonalpha genes by positive feedback through interferon regulatory factor-7 a structural basis for discriminating between self and nonself double-stranded rnas in mammalian cells subcellular localization of toll-like receptor 3 in human dendritic cells a human homologue of the drosophila toll protein signals activation of adaptive immunity activation of innate defense against a paramyxovirus is mediated by rig-i and tlr7 and tlr8 in a cell-type-specific manner cardif is an adaptor protein in the rig-i antiviral pathway and is targeted by hepatitis c virus structural requirements of doublestranded rna for the activation of 2' ,5'-oligo(a) polymerase and protein kinase of interferon-treated hela cells replicative form of encephalomyocarditis virus ribonucleic acid tlr signalling and activation of irfs: revisiting old friends from the nf-kappab pathway functional role of type i and type ii interferons in antiviral defense inhibition of vaccinia infection by a liquid factor in tissues infected by homologous virus orthomyxovirus replication, transcription, and polyadenylation critical role of traf3 in the toll-like receptor-dependent and -independent antiviral response toll-like receptor-independent gene induction program activated by mammalian dna escaped from apoptotic dna degradation short hairpin rnas (shrnas) induce sequence-specific silencing in mammalian cells determinants of interferon-stimulated gene induction by rnai vectors rig-i-mediated antiviral responses to single-stranded rna bearing 5'-phosphates defective lps signaling in c3h/hej and c57bl/10sccr mice: mutations in tlr4 gene the rna helicase lgp2 inhibits tlrindependent sensing of viral replication by retinoic acid-inducible gene-i plasmacytoid dendritic cells: the key to cpg the rna modification database: 1999 update a lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by rna interference deletion of tlr3 alters the pulmonary immune environment and mucus production during respiratory syncytial virus infection positive feedback regulation of type i ifn genes by the ifn-inducible transcription factor irf-7 toll-like receptor-dependent activation of several human blood cell types by protamine-condensed mrna sirna and isrna: two edges of one sword hitching rig to action identification and characterization of mavs, a mitochondrial antiviral signaling protein that activates nf-kappab and irf 3 triggering the interferon antiviral response through an ikk-related pathway the ends of the affair: capping and polyadenylation induction of inflammatory cytokines and interferon responses by double-stranded and single-stranded sirnas is sequence-dependent and requires endosomal localization activation of the interferon system by short-interfering rnas irf3 and irf7 phosphorylation in virus-infected cells does not require double-stranded rna-dependent protein kinase r or ikappa b kinase but is blocked by vaccinia virus e3l protein recognition of cytosolic dna activates an irf3-dependent innate immune response regulating intracellular antiviral defense and permissiveness to hepatitis c virus rna replication through a cellular rna helicase, rig-i toll-like receptors 9 and 3 as essential components of innate immune defense against mouse cytomegalovirus infection type i interferons (alpha/ beta) in immunity and autoimmunity a general method for gene knockdown in mice by using lentiviral vectors expressing small interfering rna transcription and replication of nonsegmented negative-strand rna viruses eukaryotic ribonuclease p: a plurality of ribonucleoprotein enzymes visa is an adapter protein required for virus-triggered ifn-beta signaling deficient signaling in mice devoid of double-stranded rnadependent protein kinase shared and unique functions of the dexd/h-box helicases rig-i, mda5, and lgp2 in antiviral innate immunity the rna helicase rig-i has an essential function in double-stranded rna-induced innate antiviral responses activation of the protein kinase pkr by short double-stranded rnas with single-stranded tails interferon action and apoptosis are defective in mice devoid of 2' ,5'-oligoadenylate-dependent rnase l isolation of two interferon-induced translational inhibitors: a protein kinase and an oligo-isoadenylate synthetase key: cord-261532-q923xxn2 authors: chen, huihui; jiang, zhengfan title: the essential adaptors of innate immune signaling date: 2012-09-21 journal: protein & cell doi: 10.1007/s13238-012-2063-0 sha: doc_id: 261532 cord_uid: q923xxn2 microbial components and the endogenous molecules released from damaged cells can stimulate germ-line-encoded pattern recognition receptors (prrs) to transduce signals to the hub of the innate immune signaling network-the adaptor proteins myd88/trif/mavs/sting/caspase-1, where integrated signals relay to the relevant transcription factors irf3/irf7/nf-κb/ ap-1 and the signal transducer and activator of transcription 6 (stat6) to trigger the expression of type i interferons and inflammatory cytokines or the assembly of inflammasomes. most pleiotropic cytokines are secreted and bind to specific receptors, activating the signaling pathways including jak-stat for the proliferation, differentiation and functional capacity of immune cells. this review focuses on several critical adaptors in innate immune signaling cascades and recent progress in their molecular mechanisms. the innate immune response is initiated by germline-encoded pattern recognition receptors (prrs) that serve as sensors to pathogen-associated molecular patterns (pamps) and damage-associated molecular patterns (damps), providing the first line of defense against microbial infection or endogenous danger signals . pamps refer to molecules released by pathogenic bacteria (e.g. lipopolysaccharides, lps) or viruses (e.g. dsrna/ ssrna/dsdna/ssdna), whereas damaged or dying cells release endogenous molecules termed damps (such as adenosine triphosphate, atp; uric acid crystals and s100 proteins) that activate the immune system in a way analo-gous to pamps. currently identified prrs include the membrane-bound toll-like receptors (tlrs) and c-type lectin receptors (clrs), as well as cytosolic retinoic acid-inducible gene i (rig-i)-like receptors (rlrs), nucleotide-binding oligomerization domain (nod)-like receptors (nlrs) and dna sensors (e.g. dnadependent activator of interferon regulatory factor, dai; absent in melanoma-2, aim2) . prrs have evolved in different cell types to detect pamps or damps, including hematopoietic cells like dendritic cells (dcs) and macrophages as well as epithelial cells and fibroblasts. these receptors can recruit specific adaptor proteins, like myeloid differentiation primary response gene 88 (myd88) or toll/interleukin-1 receptor (tir) domain-containing adaptor inducing ifn-β (trif) in the tlr pathway, mitochondrial antiviral signaling protein (mavs) downstream of rlrs, stimulator of interferon genes (sting) in the cytosolic dna response pathway and, cysteine aspartic protease 1 (caspase-1) as part of the inflammasome, all of which orchestrate the host innate responses, through activation of transcriptional factors such as nuclear factor κb (nf-κb), activator protein 1 (ap-1) and interferon regulatory factors (irfs), to trigger the production of type і interferons (ifns), inflammatory cytokines and chemokines. upon secretion, the cytokines function in an auto-or paracrine manner to induce the expression of hundreds of effecter genes involved in the generation of the innate anti-infection state (e.g. the janus kinase (jak)-signal transducer and activator of transcription (stat) pathway activated by secreted type i interferons) (brierley and fish, 2005) . many of these innate immune-stimulated proteins can initiate adaptive immune responses involving antibody production and cytotoxic t-cell activity (hebenstreit et al., 2006) . it is amazing that such a complicated signal network is exquisitely regulated through only a few adaptors, which properly reflects the essential roles of these molecules. in this review, we summarize the signaling pathways mediated by such essential adaptors, and focus on the most recent findings regarding the interplay between stat6 and the adaptor sting. myd88 consists of a death domain (dd) and a tir domain (vogel et al., 2003) . it is crucial for the proper responses of il-1, il-18 and nearly all tlrs except tlr3 in the activation of transcription factors nf-κb and ap-1 followed by the induction of proinflammatory genes. in addition, myd88-dependent signaling downstream of tlr7 and tlr9 elicits the production of ifn-α and ifn-β1 through the activation of irf7 and/or irf1 ( fig. 1) (watters et al., 2007; kumar et al., 2011) . in most cases, the receptors associate with myd88 through homotypic interactions between their respective tir domains, with the aid of tir domain-containing adaptor protein (tirap)/myd88 adaptor-like (mal) in case of tlr2 and tlr4. this interaction allows myd88 to recruit il-1r-associated kinase (irak) family (irak4, irak1 and irak2) via dd interaction (cao et al., 1996; marta et al., 1997) . irak1 is phosphorylated by irak4 and, after separated from myd88, interacts with tumor necrosis factor (tnf)r-associated factor 6 (traf6), an e3 ubiquitin ligase that functions together with the ubc13/uev1a complex to generate dissociative lysine-63 (k 63 )-linked poly-ubiquitin chains (horng et al., 2001) . unanchored k 63 poly-ubiquitin chains bind and activate a complex comprising transforming growth factor β (tgf-β)-activated kinase 1 (tak1) and tak1-binding proteins (tab1, -2 and -3) to phosphorylate inhibitor of kappab kinase beta (ikbkb or ikkβ) and mitogen-activated protein kinase kinase 6 (map2k6 or mkk6) (wang et al., 2001) . then the ikk complex, made up of ikk-α, ikk-β and nf-κb essential modulator (nemo), phosphorylates iκbα, leading to its degradation by the proteasome and the release of nf-κb, while phosphorylated mkk6 activates mapks including c-jun n-terminal kinase 1/2 (jnk1/2), p38 and extracellular signal-regulated kinase 1/2 (erk1/2) to stimulate ap-1 activity. active nf-κb and ap-1 translocate to the nucleus to turn on the expression of proinflammatory cytokines. individually, tlr7 and tlr9 recruit myd88 followed by the formation of a complex containing irak1, traf6, traf3, ikk-α and irf7/irf1 , which results in the production of type i interferons (brown et al., 2011) . myd88 also recruits irf5 and triggers a traf6 signaling pathway that leads to the activation of nf-κb, where irf4 competes with irf5 for myd88 binding (chen, 2005; lee and kim et al., 2007) . the fact that lps and poly (i:c) (a viral dsrna agonist) is still capable of activating nf-κb and mkk6 in myd88-deficient cells indicates the existence of a myd88-independent pathway ( fig. 1) (kawai et al., 1999) . in such contexts, trif/tir domain-containing adaptor molecule 1 (ticam-1) was found to be involved in the myd88-independent responses. trif comprises 712 amino acids with three domains: a traf binding domain, a tir domain and a c-terminal rhim -receptor-interacting protein (rip) homotypic interaction motif-domain (yamamoto et al., 2003) . tlr3 directly interacts with trif, while tlr4 recruits trif through trif-related adaptor molecule (tram). the n-terminus of trif interacts with traf3 and traf6 while its c-terminal rhim domain recruits rip1 and rip3. as an e3 ubiquitin ligase, traf3 undergoes k 63 -linked self-ubiquitination to activate traf family member-associated nf-κb activator (tank)-binding kinase-1 (tbk1) and/or inducible ikk (ikk-i)/ikk-ε. then, phosphorylated tbk1 or ikk-i activates irf3 and irf7. the dimerization and nuclear translocation of irf3 and irf7 lead to the induction of type i interferons and ifn-stimulated genes (isgs). traf6 activates nf-κb through multiple ways in complex with rip1, tnfr-associated death domain protein (tradd) and fas-associated death domain protein (fadd). traf3 undergoes k 48 -and k 63 -linked ubiquitination in either myd88-dependent or trif-dependent signaling pathway, indicating its critical roles in both pathways, fine-tuning the production of proinflammatory cytokines and type i interferons ( häcker et al., 2006; tseng et al., 2010) . signaling specificity of individual tlr can be imparted to downstream signaling molecules via the interaction of its tir domain with myd88, trif, tram or tirap. tlr stimulation can induce the production of proinflammatory cytokines, co-stimulatory molecules, type і interferons (ifn-α/β), type ii interferon (ifn-γ) and chemokines. all tlrs except tlr3 recruit myd88 to elicit inflammatory expression. tlr2 and tlr4 recruit tirap and myd88 to activate nf-κb and mapks for the production of inflammatory cytokines, and tlr5 directly interacts with myd88 to trigger nf-κb and mapk signaling pathways. in addition, tlr4 (utilizes tram) and tlr3 activate irf3 via trif for the induction of type і interferons. in plasmacytoid dendritic cells (pdcs), the tlr7/tlr9-myd88 axis elicits the expression of ifn-α and ifn-related genes. myd88-dependent signaling pathway: upon specific ligand recognition, the tir of tlr2 and tlr4 interacts with tirap and myd88. sequentially, myd88 re-cruits and activates irak4, which in turn activates another irak family member-irak1. the downstream activation of traf6 by irak4/1 leads to the activation of tak1 and tab complex, which can stimulate both nf-κb and mapk signaling pathways. activated ikk complex (consisting of ikk-α, ikk-β and nemo) causes the phosphorylation and degradation of iκbα, releasing nf-κb for its nuclear translocation and transcription initiation. in pdcs, myd88 recruits irak4/1 to activate traf6 and ultimately the transcriptional factor irf7. trif-dependent signaling pathway: upon ligand binding, tlr3 directly recruits trif via its tir domain while tlr4 utilizes tram to interact with trif. trif-recruited traf3 mediates the activation of tbk1 and/or ikk-i, which phosphorylate irf3. irf3 then dimerizes and translocates to the nucleus for the induction of cytokines such as type і interferons and il-10. meanwhile, trif also recruits traf6 and rip1, which in turn activate the tak1/tab complex responsible for the activation of nf-κb. mavs, also named interferon promoter stimulator-1 (ips-1)/caspase recruitment domain (card) adaptor inducing ifn-β (cardif) /virus-induced signaling adaptor (visa) (meylan et al., 2005; kawai et al., 2005; seth et al., 2005; xu et al., 2005) , is a mitochondrial outer membrane protein participating in the intracellular rlr pathways (thompson and locarnini, 2007) . it is also implicated to locate to peroxisomes, and both localizations seem necessary for a maximal antiviral response. peroxisomal mavs activates irf1 to induce the expression of isgs as the primary antiviral responses, while mitochondrial mavs works in a delayed stage for the vast production of isgs and type i interferons via the activation of irf3 (dixit et al., 2010) . rlrs belong to the dexd/h-box protein family, and serve as cytosolic rna sensors. to date, there exist three members: rig-i, melanoma differentiation associated factor 5 (mda-5) and laboratory of genetics and physiology 2 (lgp2) (yoneyama and fujita, 2008) . rig-i and mda-5 comprise of two n-terminal card domains, a dead-box helicase/ atpase domain in the middle and a c-terminal regulatory domain. rig-i mainly recognizes paramyxovirus such as newcastle disease virus (ndv) and vesicular stomatitis virus (vsv). in addition, rig-i recognizes flaviviridae, such as japanese encephalitis virus (jev) and hepatitis c virus (hcv). meanwhile, rig-i only senses relatively short dsrna (about 1 kb), and the shortest one (19-mer or 21-mer) ended with 5' triphophorylation can stimulate host cells to induce massive production of type i interferons. however, chemosynthetic 5' triphophorylated ssrna has no effect on the upregulation of inflammatory cytokines or type i interferons, while dsrna ended with 5' tri-or single phosphorylated, enable to activate the rig-i signaling hornung et al., 2006) . picornavirus like encephalomyocarditis virus (emcv) and polio virus are largely recognized by mda-5. mda-5 prefers to sense long strand dsrna (more than 2 kb), and the intermediates of rna virus replication in emcv-infected cells including the higher-structured rna consisted of dsrna and ssrna is able to activate mda-5 signaling pathway. moreover, both west nile virus and dengue virus can activate rig-i and mda-5 signaling pathway simultaneously (kato et al., 2008) . lgp2, with no card, was originally recognized as a negative regulator for rig-i and mda-5 (saito et al., 2007; kato et al., 2008; rothenfusser et al., 2012) . controversially, studies on lgp2 -/mice and at-pase-inactivated mice unraveled a positive role for lgp2 in response to rna virus, with the capability of promoting the interaction of rig-i/mda-5 with rna (satoh et al., 2010) . in rna virus-invaded cells, rig-i and mda-5 experience conformational changes during rna-binding and expose their cards to recruit mavs, which activates traf3/tbk1/ ikk-i and then irf3/irf7, resulting in the expression of type i interferons. meanwhile, mavs induces nf-κb-dependent expression of proinflammatory factors by recruiting the tradd/fadd/caspase-8/caspase-10 complex (fig. 2) . during activation, rig-i undergoes k 63 -linked ubiquitination on its card by tripartite motif containing 25 (trim25) and rnf135 (zeng et al., 2010) , whereas rnf125 downregulates rig-i signaling through k 48 -linked ubiquitination and degradation (arimoto et al., 2007) . the card domains of the rna sensors rig-i /mda-5 bind to k63-polyubiquitin chains, and sequentially form the rig-i or mda-5 oligomers, which is essential for irf3 activation and signaling regulation (jiang et al., 2012) . k63-polyubiquitinanted rig-i oligomers are highly potent in aggregating and activating mavs on the mitochondrial memberane through a prion-like manner, propagating the antiviral signaling cascade (hou et al., 2011) . according to a recent work, a ser/thr phosphatase eyes absent 4 (eya4), is found to interact with mavs and is esfigure 2 . rlr-mavs signaling pathways induced by rna viruses. rig-i and mda-5 detect short dsrna with 5' triphosphate ends and long dsrna, respectively. by card domain interactions, they recruit mavs and in turn activate irf3/irf7 through eya4, traf3, nf-κb activating kinase-associated protein 1 (nap1)/similar to nap1 tbk1 adaptor (stintbad) and tbk1/ikk-i signaling cascades, leading to the expression of type і interferon genes. meanwhile, mavs activates nf-κb via the tradd, fadd, caspase-8/-10 signaling cascades, resulting in the production of proinflammatory cytokines. lgp2 functions as a positive regulator of rig-i and mda-5 signaling pathways. activation of rig-i is positively and negatively regulated by ubiquitin ligases trim25 and rnf125, respectively. rlr signaling requires polyubiquitination of traf3, which can be removed by a deubiquitinase, deubiquitinating enzyme a (duba). cell sential for the production of ifn-β (okabe et al., 2009) . a member of the nlr family, nlrx1 (or nod9) colocalizes with mavs at mitochondria and negatively regulates mavs-dependent antiviral immune responses (moore et al., 2008) . besides, the rlr pathway can be downregulated by autophagosome-related proteins such as autophagy-related gene 5 (atg5) and atg16l1. when mouse embryonic fibroblasts (mefs) and conventional dcs (cdcs) lacking autophagosome are infected with rna viruses, mavs-resident mitochondria will dysfunction and produce massive reactive oxygen species (ros), the release of which in turn triggers the induction of type i interferons. stimulator of interferon genes sting, also named mediator of irf3 activation (mita)/endoplasmic reticulum (er) interferon stimulator (eris)/membrane tetraspanner associated with mhc class ii (mpys) that locates to er or/and mitochondria, is an important adaptor with a potent ability to induce type i interferons, interleukins and other proinflammatory factors (ishikawa and barber, 2008; zhong et al., 2008; sun et al., 2009) . homo sapiens sting consists of 379 amino acids, with a signal peptide and four transmembrane domains at its n-terminus. in the vicinity of these domains there exist two er retention sequences, r 76 ir 78 and r 178 yr 180 , which are essential for the proper localization and stability of sting (sun et al., 2009) . sting-deficient mice are highly sensitive to both rna viruses, e.g. vesicular stomatitis virus (vsv) and sendai virus (sev) and dna viruses, e.g. herpes simplex virus type i (hsv-1), underscoring the physiological function of sting in antiviral immune responses (ishikawa et al., 2009 ). however, sting cannot bind directly to rna or dna (barber, 2011) . in rna-triggered pathway, sting is mainly involved in rig-i rather than mda-5 signaling. our published data show that sting interacts with rig-i but not mda-5, consistent with the fact that sting-deficiency seldom affects poly (i:c) (recognized by mda-5)-mediated signaling (ishikawa et al., 2009 ). on the other hand, besides ifi16 and ddx41, cytosolic dna sensor(s) that utilize sting for the induction of type i interferons remain to be identified or validated. intracellular localization is a primary factor for the function of sting, since it is transported into er lumen from er-bond ribosomes after translation, depending on the interaction between sting and a four-subnuit (α-δ) complex, the translocon associated protein (trap) complex ssr2/trapβ. individually, the trap complex can bind with translocons, a three-subunit component comprising sec61α/sec61β/ sec61γ required for protein folding and secretion. as part of the exocyst component, sec5 is found to colocalize with sting, essential for its transportation from post-golgi to plasma membrane. in virus-infected cells, sting translocates from er to golgi, and then to cytoplasmic punctate structures, where it recruits tbk1 in the aid of ralb gtpase and sec5. irf3/irf7 is thus activated to induce the expression of type i interferons. importantly, stat6 is also activated to trigger the production of chemokines, which is discussed in details later (fig. 3) . the regulation of sting turns out very complicated. sting can be ubiquitinated and phosphorylated, and under the stimulation of sev or poly (i:c)/poly dadt, sting undergoes apparent oligomerization (sun et al., 2009) . and in vitro, sting interacts with tbk1 and irf3, and stimulates the phosphorylation of irf3 by tbk1 in the cytosolic dna signaling pathway (tanaka and chen, 2012) . a research claims that there is colocalization between autophagy-related gene 9a (atg9a) and sting that negatively regulates sting signaling. in addition, 3' repairing exonuclease 1 (trex1) plausibly downregulates sting-mediated signal pathways, for reverse transcribed human immunodeficiency virus type i (hiv-1) dna activates sting to suppress its own replication or to eliminate other dna viruses (yan et al., 2011) . interestingly, sting is found to share homology with proteins of flavivirus, such as dengue virus, yellow fever virus and hepatitis c virus, all of which localize to er (ishikawa et al., 2009) . current evidence shows that human coronavirus (hcov) nl63 and severe acute respiratory syndrome (sars) cov papain-like protease (plp) interact with sting to negatively regulate signaling by disrupting the sting dimmers and preventing the ubiquitination of rig-i/tbk1/irf3 (sun et al., 2012) . it is yet-to-be validated if there are other viruses that inhibit this pathway to escape the host immune responses. undoubtedly, such work would provide insights into therapeutic approaches of autoimmune diseases. the inflammasome is a multimeric danger-sensing platform that mainly exists in macrophages and dendritic cells. activation of the inflammasome initiates the secretion of il-1β/il-18 and induces a caspase-1-dependent cell death named "pyroptosis" to protect the host against pathogens (schroder and tschopp, 2010) . most inflammasomes are composed of nlrs or pyrin and hematopoletic ifn-inducible nuclear proteins with 200 amino acids (hin-200) domain (pyhin) family members and the protease caspase-1 (schroder and tschopp, 2010) . the nlrs is a large receptor family characterized by n-terminal card or pyrin domains (pyd), a central nucleotide-binding and oligomerization domain(nod), and c-terminal leucine-rich repeats (lrrs) responsible for ligand sensing. to date, three groups of the nlr family: nlr family pyd-containing 1 (nlrp1), nlrp3 and nlr family cardcontaining 4 (nlrc4) are found to be involved in the formation of inflammasomes (kanneganti, 2010) . nlrp3 inflammasome has been studied most extensively. numerous pamps, damps and several environment stimulating factors can activate the nlrp3 inflammasome. the figure 3 . sting-mediated dna sensing pathways. dna viruses, bacteria genomic dna and unprocessed endogenous dna can be captured by high-mobility group protein b1 (hmgb1) and then recognized by dai or unidentified dna sensor(s). alternatively, dsdna is transcribed into dsrna by polymerase iii and recognized by rig-i to induce type і interferons. upon dna stimulation, sting translocates from er membrane to the cytoplasmic punctate structure, where it interacts with tbk1 and activates irf3/irf7. oligomerization of activated nlrp3 enables the pyd domains coupling together and recruit asc sequentially. the card domain of asc then recruits pro-caspase-1 which in turn self-cleave to form the activated caspase-1, namely p10/p20 tetramer, which consequently cut pro-il-1β into its mature form. recent work suggests that nlrc5 also participates in the formation of nlrp3 inflammasome, because bacteria-induced nlrp3 inflammasome is inhibited when silencing nlrp5 (fig. 4) (pétrilli et al., 2007) . nlrc4 (or ipaf) inflammasome is mainly comprised of nlrp4 and pro-caspase-1, and is activated via direct interaction with the cards of caspase-1 and nlrp4 to induce caspase-1-dependent pyroptosis. the bacterial type iii secretion systems (t3ss) (such as salmonella typhimuurium, listeria, francisella or shigella flexneri) is essential for the activation of nlrc4 inflammasome (zhao et al., 2011; kofoed and vance, 2011) . nlrp1 is the first receptor in nlr family that is found to be involved in inflammasome assembly and caspase-1 activation. homo sapiens nlrp1 inflammasome is composed of nlrp1, asc, caspase-1 and caspase-5, which mainly recognizes muramyl dipeptide (mdp) deriving from bacterial peptidoglycan. as the only nlr receptor containing an extra c-terminal card domain, nlrp1 can recruit pro-caspase-1 directly, but asc can upregulate the activation of human nlrp1 inflammasome. murine nlrp1 has three homology proteins: nlrp1a, nlrp1b and nlrp1c, among which, nlrp1b can be activated by lethal factor (lf) from anthrax toxin. hence, it is still contorversial whether mdp can activate nlrp1 directly, and it is reported that nod2 (nlrc2) serves as the intracellular receptor for mdp and participates in nlrp1 inflammasome assembly. the pyhin family comprises of four members: ifninducible protein x (ifix), ifi16, myeloid nuclear differentiation antigen (mnda) and aim2, and their c-terminal hin domain is in charge of sensing dna and n-terminal pyd domain recruits downstream adaptor asc (bürckstümmer et al., 2009; fernandes-alnemri et al., 2009; hornung et al., 2009; roberts et al., 2009 .) aim2 acts as a intracellular receptor for alien or endogenous dsdna to induce caspase-1-dependent maturation of il-1β/il-18 and cell death like pyroptosis. the immune functions of other hin-200 family members (mnda, ifi16 and ifix) remain unclear. . nlrp1、 nlrp3、 nlrc4 and aim2 inflammasomes. nlrp1, nlrp3 and aim2 recruit and activate caspase-1 through the bridging molecule asc, and the card domain of caspase-1 is removed by autocleavage resulting in the formation of the activated caspase-1 p10/20 tetramer. nlrc4 directly binds to caspase-1 and induces the inflammasome assembly, yet there are several reports that stress the requirement for asc in the nlrc4 inflammasome. currently, there mainly exists three models for nlrp3 inflammasome activation: first, the nlrp3 agonist, atp, triggers p2x7-dependent pore formation or pore forming toxins initiate the efflux of potassium, which directly elicit the assembly of nlrp3 inflammasome by a yet-unknown mechanism. second, engulfed crystalline and the lysosomal protease cathepsin b plays an important role in the formation of nlrp3 inflammasome. third, all of damps and pamps, including atp and crystalline activators can induce the formation of reactive oxygen species (ros), which indirectly activate nlrp3 inflammasome by a yet-unidentified manner. caspase-1 clustering induces autoactivation and caspase-1-dependent maturation and secretion of proinflammatory cytokines, such as il-1β. stat6 belongs to the signal transducer and activator of transcription family of proteins. in mammals, there are seven members: stat1, -2, -3, -4, -5a, -5b and -6 (brierley and fish, 2005) . these proteins transmit signals from a receptor complex to the nucleus and activate gene expression, known as the classical jak-stat pathways．stat6 mainly participates in il-4/il-13-mediated allergic reaction, playing a vital role in the differentiation of t-helper type 2 (th2) cells (hebenstreit et al., 2006; chapoval et al., 2010) . the homo sapiens stat6 gene maps to chromosome 12q13.3-q14.1, while mus musculus stat6 gene lies clustered in the distal region of murine chromosome 10. this protein is ubiquitously expressed, especially plentiful in b and t cells, and constantly active in most cell types. human stat6 consists of 847 amino acids (murine 837 amino acids), which specifies a size of 94 kda in an unmodified state. it contains an n-terminal α-helix domain, a dna binding domain (dbd), a linker domain (ld), a src homology 2 (sh2) domain and a c-terminal transactivation domain (tad) (fig. 5) (hebenstreit et al., 2006) . the n-terminus of stat6 enriches α-helix motif, namely coiled-coil domain. recent publications have demonstrated that the n-terminus of stats is involved in its interaction with receptors, and the phosphorylation, nuclear translocation of stats, however, it remains to be seen whether these findings have functional relevance to stat6, as an isoform of stat6 truncated at n-terminus appears to be fully functional. the dbd of stat6 extends from posi-tions 268 to 430, allowing dna binding when stat6 is present as a dimer. we previously reported that ser 407 in dbd is phosphorylated by tbk1 after virus infection, but the biological significance of this modification remains unclear . the sh2 domain of stat6 has been shown essential for its binding to phosphorylated tyrosine residues on the cytoplasmic tail of the receptor and its own dimerization, followed by the reciprocal interaction of the sh2 domain with a phosphorylated tyrosine at position 641. our work suggested that when the l 551 in sh2 is mutated, stat6 can still be activated by il-4 but not virus infection. stat6 has the longest tad among stat family members, which is 147 amino acids in length and is rich of proline residues. stat6 displays a dominant-negative feature without tad. s 756 in tad can be phosphorylated after il-4 stimulation, but further studies are needed to unravel its functional significance. the canonical il-4/il-13 signaling pathways stat6 is classically involved in the il-4/il-13 signaling (fig. 6 right) . the cytokines il-4/il-13 are produced by th2 cells, mast cells and basophils. these two cytokines share similar physiological functions and are implicated in pathological conditions such as asthma, allergy and autoimmune diseases. nevertheless, il-4 is more active regulating th2 cells development, whereas il-13 is more involved in the regulation on respiratory hypersensitivity, mucus hypersecretion and th2-type inflammation of the bowel. the receptors for il-4 and il-13 share several features including four conserved cysteine residues, a wsxws motif, fibronectin type iii modules in the extracellular domain and proline-rich box regions in the intracellular domain, which are critical for the binding to tyrosine kinases of jak family. il-4 receptors consist of two transmembrane proteins. in hematopoietic cells, the il-4rа chain binds to a common gamma chain (γc) to form the type i il-4r receptor. most notably, il-4rα chain has high affinity with il-4, and the γc chain is also a component of the receptors for il-2, il-7, il-9, il-15 and il-21. in nonhematopoietic cells, the type ii il-4 receptor is formed by interaction of il-4rα with il-13rα1 instead of γc and recognizes both il-4 and il-13. il-13 binds to il-13rα1 with higher affinity (hebenstreit et al., 2006) . in contrast, il-13α2 has higher affinity for il-13 but fails to induce a signal, indicating that il-13α2 acts as a decoy receptor. the cytoplasmic tails of il-4/il-13 receptor subunits associate with tyrosine kinase of the janus family: il-4rα binds to jak1, γc with jak3 whereas il-13rα1 with jak2 or tyk2. upon il-4/il-13 binding, heterodimerized receptors activate jaks. subsequently, jaks phosphorylate three core tyrosine residues (y 575 , y 603 and y 631 ) in il-4rα, providing the docking sites for stat6. intriguingly, any one of the three tyrosine residues is competent to activate stat6. then jaks further phosphorylate stat6 at the receptor: activated stat6 dimerizes through sh2 domain and translocates into nucleus to function as a transcriptional factor, regulating il-4/il-13-dependent signaling such as th2 differentiation, immunoglobulin e (ige) and chemokine production and mucus generation. stat6 prefers sites with a 4-bp spacer (ttcnnnngaa, the n4 site), which are mainly found in il-4 responsive promoters. it is notable that the activation of stat6 by cytokines is very transient, peaking at 10-30 min, and nearly undetectable after 4 h, indicating a quick downregulating mechanism. it is reported that tyrosine phosphatase shp-1 is involved in this dephosphorylating process. other studies also indicate that stat6 induces the expression of some negative regulators such as the suppressor of cytokine signaling (socs). besides il-4/il-13, many other cytokines have been reported to activate stat6 in a similar way. il-3 affects the proliferation of hematopoietic cells via tyrosine phosphorylation of stat6. il-15 activates stat6 in different cells including t cell, nk cell, and mast cell to initiate the expression of anti-apoptosis genes like bcl-xl. in some b cell lines, ifn-α was found to activate stat6, which can even lead to the formation of stat2/stat6 heterodimer. in addition, platelet-derived growth factor (pdgf), a major mitogen and chemotactic factor for mesenchymal cells such as fibroblasts and smooth muscle cells, also exerts an effect on jak-mediated stat6 activation in nih 3t3 fibroblasts and 3t3-l1 preadipocytes. on the contrary, experiments using fibroblasts derived from stat6 knockout mice do not support any dependence of stat6 on pdgf. researches on murine mastocytes demonstrate that il-15 induces tyk2-mediated stat6 activation consequently s u p p r e s s i n g t h e a p o p t o s i s i n m a s t o c y t e s . upon binding to il-4/il-13, jaks located in the c terminal of il-4rα is phosphorylated and activated. sequentially, jaks phophorylate three tyrosine residues of the receptors, which provide docking sites for stat6. afterwards, stat6 is phosphorylated by jaks, leading to the dimerization and nuclear translocation to induce the expression of genes related to th2 differentiation. left: the stat6-dependent antiviral signaling pathways. upon dna virus infection, activated sting recruits stat6. tbk1 is also recruited by sting to phosphorylate stat6 on s 407 , which in turn activates another unidentified tyrosine kinase to phosphorylate stat6 on y 641 , leading to the homodimerization and nuclear translocation of stat6. stat6 dimer then binds to its target sites to initiate transcription. on the other hand, rna virus infection triggers sting activation through sting-mavs interaction on mavs-resident mam or peroxisomal (pex) membrane; activated sting then dissociates with mavs and recruits stat6 and tbk1, leading to stat6 activation. moreover, in mast cells, kit ligand (kl), which is recognized by the receptor tyrosine kinase c-kit, is implicated in hematopoiesis by activating stat6 (hundley et al., 2004) . in cardiomyocytes, angiotensin ii (ang ii) activates jak2 and stat6 to regulate cardiovascular and renal homeostasis. moreover, one leptin receptor obese receptor (ob-r) splice variant preferentially expressed in the hypothalamus codes for the activation of stat6. although these cytokines/factors showed to activate stat6 in specific types of cells, the physiological relevance of these activation need to be further verified. previous studies suggest that stat6 functions only in adap-tive immune responses, but our recent publication raised the possibility that stat6 is also involved in antiviral innate immunity . we found that rna and dna viruses trigger the mavs/sting/tbk1 axis, resulting in the expression of both type i interferons and stat6-regulating genes, including chemokines like chemokine (c-c motif) ligand 2 (ccl2), ccl20 and ccl26 etc. specifically, viruses or intracellular nucleic acids triggers sting to recruit stat6 to endoplasmic reticulum, leading to the phosphorylation, dimerization and nuclear translocation of stat6 for the induction of specific target genes responsible for immune cell homing. it was also found that activated stat6 is phosphorylated at multiple sites, among which ser 407 is phosphorylated by tbk1 whilst tyr 641 is phosphorylated inde-pendent of jaks, possibly by a thus unidentified tyrosine kinase. importantly, the multi-site phosphorylation of stat6 mentioned above is different from the classical il-4/13 pathways. for instance, leu 551 is essential for stat6 in response to virus infection, but it is not phosphorylated in activated stat6 in the classical pathways. stat6 -/mice are remarkably susceptive to virus infection, and all these phenomena revealed the vital role of stat6 in antiviral immune responses. since stat6 is activated by a wide variety of cytokines, and also by viral infection, its physiological function is highly diversified. previous work demonstrated that stat6 performs its main role in mediating the biologic functions of il-4/13, and therefore connects to the th2 polarization of the immune system. it has been shown that more than 150 genes were direct targets of activated stat6 and therefore up-regulated by il-4/13 treatment, most of these genes are involved in human th2 cell programming. stat6 is also shown to be crucial in the development of protective immunity against gastrointestinal nematode parasites. infection of mice with intestinal nematode parasites induces a strong th2 cytokine response, thus triggers worm expulsion through an il-4rα-activated, stat6-dependent mechanism (hoeck and woisetschläger, 2001) . besides its role in th2 cell development and immune response against nematode infection, studies from animal models and knockout mice demonstrated that stat6 also actively functions in the pathogenesis of allergic diseases, like airway hyperresponsiveness (ahr), eosinophilic inflammation, or mucus production. asthma is a chronic inflammatory disorder on remodeled airways, characterized by mast cell and eosinophil infiltration due to polarized th2 response, leading to the abnormal secretion of cytokines like il-3, -4, -5, -9, -13, and gm-csf (sehra et al., 2010) . it is one of the top health problems, about three billion people are suffering from it worldwide. recent studies on the pathogenic mechanism demonstrate that stat6-dependent activation of th2 response and the produced cytokines/chemokines is essential for the initiation of this chronic inflammation. stat6 deficiency also leads to impaired macrophage development and functional responses, including morphological changes and the decreased mhc class ii expression and nitric oxide production. in addition, stat6 has been found to be constitutively activated in transformed cell lines, suggesting that inappropriate activation of stat6 is involved in uncontrolled cell growth in an oncogenic state. human t-lymphotropic virus type i (htlv-1) transformed cells constitutively activate the jak-stat6 pathway. however, solid evidence for the involvement of stat6 in cellular transformation is still lacking. recent work found that stat6 plays an important role in antiviral innate immunity. in response to virus infection, stat6 was activated by sting-tbk1/ikkε. activated stat6 translocated to nuclei to regulate the expression of chemokines for homing of various immune cells including lymphocytes, monocytes, basophils, neutrophils, and eosinophils. the physiological function of stat6 in this pathway was highlighted as stat6 −/− mice are sensitive to both dna and rna viral infections. unlike other stat6 activating pathways that are restricted to specific cell types, virus-induced stat6 activation is found in all tested cell types, suggesting the fundamental importance of this signaling cascade . viral infection-activated stat6 regulates the expression of chemotactic factors including ccl2, ccl20 and ccl26. these chemokines have been well documented for capable of attracting many types of cells including monocytes, macrophages, eosinophils, basophils, neutrophils and t cells, nk cells, and therefore implicated in the pathology of many diseases characterized by immune cell infiltration. ccl2 contributes to the sustained inflammation in chronic inflammatory disorders like atherosclerosis. it is required for initiating some autoimmune diseases (mahad and ransohoff, 2003) , and functions in host defense against various pathogens (winter et al., 2007) . ccl20 could function as both an inflammatory and a homeostatic chemokine; its function in allergic airway diseases has been well documented (weckmann et al., 2007) . recent work also suggested that ccl20 was implicated in the pathogenesis of psoriasis. ccl26 has multiple roles in allergic inflammation such as atopic dermatitis and asthma and also in the pathogenesis of eosinophilic esophagitis and churg-strauss syndrome (bhattacharya et al., 2007) . since cx3cr1 is involved in th1-mediated diseases such as rheumatoid arthritis, diabetes, lichen planus, and psoriasis, ccl26 is therefore likely involved in these diseases as well (nakayama et al., 2010) . given the fact that viral-infection-activated stat6 regulates the expression of genes for various immune cells homing, the physiological function of stat6 on this side, especially in development of the microbial-triggered allergic diseases, is just start to emerge. the past two decades have witnessed great progress in the understanding of molecular mechanisms underlying innate immune activation, providing important clues to how host cells recognize and resist the microbial harassment. various pathogenic microorganisms invading the host can be identified by different prrs, and through the innate immune signaling network, these diverse signals go to the corresponding hub molecules including myd88, trif, mavs, sting and caspase-1, which drive the relevant downstream transcriptional factors irf3/7, nf-κb, ap-1 and/or stat6 to induce the expression of innate immune-related genes, leading to the elimination of the microbe and the activation of the adaptive immunity. since pprs, their relevant signaling adapters and downstream molecules locate in different cellular position, so it is of fundamental importance to elaborate how different signaling pathways were utilized in different cellular contexts. it is found that almost of the signaling molecules studied distributed differently, exemplified that the mrna expression of 10 tlrs and 21 related genes performed quite variously in different tissues and cell lines (masuhiro and shinsaku, 2005) . besides, the influence of myd88 on the gene expression during sepsis is strikingly organ-specific (heike et al., 2006) . the ability of prrs to engage different adaptor proteins, crosstalk with other regulatory pathways and suppress or activate a plethora of kinases involved in a multitude of signaling pathways is an essential factor in shaping the type, strength, magnitude, timing and duration of inflammation and antiviral responses. this multidimensional signaling network precisely regulates both the innate and adaptive immune systems. nevertheless, our knowledge of inflammation and antiviral biology is still in its infancy and many problems remain obscure. specifically, the substantial antimicrobial mechanisms actually are beyond our understanding. furthermore, we do not know much about the dynamic regulation of the entire immune system caused by crosstalk of multiple signaling cascades as well as the mechanisms fine-tuning the innate and adaptive immunity. future investigations urgently need the introduction of more advanced and effective approaches, such as imaging techniques that can be used to define the dynamics of immune cell behavior and the system biology strategy to analyze and integrate accumulating knowledge (chevrier et al., 2011) . in future, these sophisticated methods will definitely enable scientists to indentify unknown prrs exemplified the unidentified dna sensors and uncover the initiation innate immunity and the integration of the signaling cascades. how to apply to use theoretical knowledge to clinic drug design and therapy for autoimmune diseases will be a huge challenge. abbreviations γc, gamma chain; ahr, airway hyperresponsiveness; ang ii, angiotensin ii; ap-1, activator protein 1; atg, autophagy-related gene; card, caspase recruitment domain; cardif, card adaptor inducing ifn-β; cdc, conventional dc; dbd, dna binding domain; dc, dendritic cell; duba, deubiquitinating enzyme a; emsv, encephalomyocarditis virus; er, endoplasmic reticulum; eris, er interferon stimulator; eya4, eyes absent 4; fadd, fas-associated death domain protein ifn, interferon; hcov, human coronavirus; hcv, hepatitis c virus; htlv-1. t-lymphotropic virus type i; hiv, human immunodeficiency virus; hmgb1, high-mobility group protein b1; hsv, herpes simplex virus type; ifix, ifn-inducible protein x; ig, immunoglobulin; irak, il-1r-associated kinase; irf, interferon regulatory factors; ips-1, interferon promoter stimulator-1; isg, ifn-stimulated gene; jev, japanese encephalitis virus; kl, kit ligand; ld, linker domain; lf, lethal factor; lgp2, laboratory of genetics and physiology 2; lrr, leucine-rich repeats; map2k6 (mkk6), mitogen-activated protein kinase kinase 6; mavs, mitochondrial antiviral signaling protein; mdp, muramyl dipeptide; mef, mouse embryonic fibroblast; mnda, myeloid nuclear differentiation antigen; myd88, myeloid differentiation primary response gene 88; ndv, newcastle disease virus; nemo, nf-κb essential modulator; nf-κb, nuclear factor κb; nlrp1, nlr family pyd-containing 1; nlrc4, nlr family card-containing 4; nod, nucleotide-binding and oligomerization; ob-r, obese receptor; pdc, plasmacytoid dc; pdgf, platelet-derived growth factor; plp, papain-like protease; prr, pattern recognition receptor; pyd, pyrin domain; rip, rhim-receptor-interacting protein; ros, reactive oxygen species; sars, severe acute respiratory syndrome; sev, sendai virus; stat, signal transducer and activator of transcription; sh2, src homology 2; socs, suppressor of cytokine signaling; sting, stimulator of interferon genes; t3ss, type iii secretion system; tad, transactivation domain; tak1, (tfg-β)-activated kinase 1; tbk1, traf family member-associated nf-κb activator (tank)-binding kinase; tgf-β, transforming growth factor β; ticam-1, trif/tir domain-containing adaptor molecule 1; tir, toll/interleukin-1 receptor; th2, t-helper type 2; tnf, tumor necrosis factor; tram, trif-related adaptor molecule; tradd, tnfr-associated death domain protein; traf6, (tnf)r-associated factor 6; trap, translocon associated protein; trif, tir domain-containing adaptor inducing ifn-β; visa, virus-induced signaling adaptor; vsv, vesicular stomatitis virus; negative regulation of the rig-i signaling by the ubiquitin ligase rnf125 innate immune dna sensing pathways: sting, aimii and the regulation of interferon production and inflammatory responses increased expression of eotaxin-3 distinguishes between eosinophilic esophagitis and gastroesophageal reflux disease stats: multifaceted regulators of transcription tlr-signaling networks: an integration of adaptor molecules, kinases, and cross-talk an orthogonal proteomic-genomic screen identifies aim2 as a cytoplasmic dna sensor for the inflammasome irak: a kinase associated with the interleukin-1 receptor regulation of the t helper cell type 2 (th2)/t regulatory cell (treg) balance by il-4 and stat6 activation of stat6 by sting is critical for antiviral innate immunity ubiquitin signalling in the nf-κb pathway systematic discovery of tlr signaling components delineates viral-sensing circuits peroxisomes are signaling platforms for antiviral innate immunity aim2 activates the inflammasome and cell death in response to cytoplasmic dna specificity in toll-like receptor signalling through distinct effector functions of traf3 and traf6 signaling mechanisms, interaction partners, and target genes of stat6 stat6 mediates eotaxin-1 expression in il-4 or tnf-alpha-induced fibroblasts tirap: an adaptor molecule in the toll signaling pathway aim2 recognizes cytosolic dsdna and forms a caspase-1-activating inflammasome with asc 5'-triphosphate rna is the ligand for rig-i mavs forms functional prion-like aggregates to activate and propagate antiviral innate immune response kit and fcepsilonri mediate unique and convergent signals for release of inflammatory mediators from human mast cells sting regulates intracellular dna-mediated, type i interferon-dependent innate immunity ubiquitin-induced oligomerization of the rna sensors rig-i and mda5 activates antiviral innate immune response differential roles of mda5 and rig-i helicases in the recognition of rna viruses unresponsiveness of myd88-deficient mice to endotoxin ips-1, an adaptor triggering rig-i-and mda5-mediated type i interferon induction innate immune recognition of bacterial ligands by naips determines inflammasome specificity pathogen recognition by the innate immune system signaling pathways downstream of pattern-recognition receptors and their cross talk the role of mcp-1 (ccl2) and ccr2 in multiple sclerosis and experimental autoimmune encephalomyelitis (eae) irak (pelle) family member irak-2 and myd88 as proximal mediators of il-1 signaling cardif is an adaptor protein in the rig-i antiviral pathway and is targeted by hepatitis c virus nlrx1 is a regulator of mitochondrial antiviral immunity eotaxin-3/cc chemokine ligand 26 is a functional ligand for cx3cr1 tissue-specific mrna expression profiles of human toll-like receptors and related genes regulation of the innate immune response by threonine-phosphatase of eyes absent activation of the nalp3 inflammasome is triggered by low intracellular potassium concentration the rna helicase lgp2 inhibits tlr-independent sensing of viral replication by retinoic acid-inducible gene-i regulation of innate antiviral defenses through a shared repressor domain in rig-i and lgp2 lgp2 is a positive regulator of rig-i-and mda5-mediated antiviral responses the inflammasomes il-4 is a critical determinant in the generation of allergic inflammation initiated by a constitutively active stat6 il-4 regulates skin homeostasis and the predisposition toward allergic skin inflammation identification and characterization of mavs, a mitochondrial antiviral signaling protein that activates nf-κb and irf 3 coronavirus papain-like proteases negatively regulate antiviral innate immune response through disruption of sting-mediated signaling eris, an endoplasmic reticulum ifn stimulator, activates innate immune signaling through dimerization sting specifies irf3 phosphorylation by tbk1 in the cytosolic dna signaling pathway pattern recognition receptors and inflammation toll-like receptors, rig-i-like rna helicases and the antiviral innate immune response different modes of ubiquitination of the adaptor traf3 selectively activate the expression of type i interferons and proinflammatory cytokines tlrs: differential adapter utilization by toll-like receptors mediates tlr-specific patterns of gene expression tak1 is a ubiquitin-dependent kinase of mkk and ikk structure, function and regulation of the toll/il-1 receptor adaptor proteins critical link between trail and ccl20 for the activation of th2 cells and the expression of allergic airway disease organ-secific role of myd88 for gene regulation during polymicrobial peritonitis lung-specific overexpression of cc chemokine ligand (ccl) 2 enhances the host defense to streptococcus pneumoniae infection in mice: role of the ccl2-ccr2 axis visa is an adapter protein required for virus-triggered ifn-β signaling role of adaptor trif in the myd88-independent toll-like receptor signaling pathway the cytosolic exonuclease trex1 inhibits the innate immune response to human immunodeficiency virus type 1 structural mechanism of rna recognition by the rig-i-like receptors reconstitution of the rig-i pathway reveals a signaling role of unanchored polyubiquitin chains in innate immunity the nlrc4 inflammasome receptors for bacterial flagellin and type iii secretion apparatus the adaptor protein mita links virus-sensing receptors to irf3 transcription factor activation key: cord-306533-lvm11o4r authors: woo, bean; baek, kwang-hyun title: regulatory interplay between deubiquitinating enzymes and cytokines date: 2019-06-08 journal: cytokine growth factor rev doi: 10.1016/j.cytogfr.2019.06.001 sha: doc_id: 306533 cord_uid: lvm11o4r deubiquitinating enzymes (dubs) are cysteine protease proteins that reverse the ubiquitination by removing ubiquitins from the target protein. with over 100 dubs identified and categorized into at least 7 families, many dubs interact with one or more cytokines, influencing cellular processes, such as antiviral responses, inflammatory responses, apoptosis, etc. while some dubs influence cytokine pathway or production, some dubs are cytokine-inducible. in this article, we summarize a list of dubs, their interaction with cytokines, target proteins and mechanisms of action. ubiquitination and deubiquitination are post-translational modifications for numerous proteins, which in turn affect many physiological processes. ubiquitination is defined as an attachment of one or more ubiquitin (ub) molecules onto the target protein through the function of a series of proteins: e1, e2 and e3 ( fig. 1 ) [1] . seven lysine residues have been identified (k6, k11, k27, k29, k33, k48 and k63) on the ubiquitin molecule [2] . also, additional ub molecules can be attached onto one of the seven lysine residues or the n-terminal methionine to form polyubiquitin chains (polyub) [2, 3] . perhaps, ub is most well-known as the crucial marker of the ubiquitin-proteasome system (ups), in which ubiquitinated proteins enter proteasomal degradation via 26s proteasome ( fig. 1 ) [3] . however, ub also affects many other aspects of tagged proteins, such as localization, protein interaction, function, etc. [3] . on the other hand, deubiquitination refers to the process that reverses ubiquitination via deubiquitinating enzymes (dubs) (fig. 1 ). little less than 100 human dubs have been identified so far [4] . dubs were categorized into five different families in the past [5] , but at least seven different families are identified as of now, which include the ubiquitin-specific protease (usp), ubiquitin carboxyl-terminal hydrolase (uch), machado-josephin disease protein (mjd), ovarian tumor (otu), jab1/mpn/mov34 (jamm), permutated papain fold peptidases of dsrna viruses and eukaryotes (pppde) and miu-containing novel dub family (mindy) [6, 7] . cytokines are groups of small proteins that play a role in cell signaling and immune system by binding to their respective receptors. since dubs regulate diverse physiological processes, it was to be expected that dubs and cytokines affect one another. as anticipated, the more studies were performed regarding the functions of dubs, the more interaction between dubs and cytokines were revealed. recently, several reviews dealing with how dubs affect pathway of a specific cytokine were published [8] [9] [10] , but none has yet introduced as a whole the interaction between dubs and cytokines. in this review, we wish to provide a brief overview of the dubs discovered to regulate cytokine signaling pathways and cytokine-inducible dubs. we will discuss the dubs that influence the pathways of interferons (ifn), tumor necrosis factors (tnf), tnf-related apoptosis-inducing ligand (trail), interleukins (il) and chemokines. ifn-α and ifn-β cytokines belong to type i ifn family that are essential for antiviral responses, cancer, inflammation, etc. [11] . when a cell recognizes a viral infection through detecting ifn-stimulating signaling molecules or foreign double stranded dna in the cytosol, retinoic acid-inducible gene-i (rig-i) is activated, triggering the cascade of the second messenger system to activate and translate ifn-α and ifnβ signaling pathways (fig. 2) [12] . dubs interact with some of the key molecules in the ifn signaling pathway, which include, but are not limited to, rig-i, stimulator of interferon genes (sting), tumor necrosis factor receptor-associated factors (trafs), interferon regulatory factor are summarized in table 1 . we will discuss the dubs in the order of ifn signaling pathway shown in fig. 1 . the first group of dubs are those that deubiquitinate rig-i to inhibit the production of ifn. rig-i is a cytosolic protein that plays a significant role in ifn signaling by detecting viral dna and rna [13, 14] , which is then ubiquitinated by tripartite motif (trim) to activate the signaling cascade to synthesize ifn [15] . orf64, usp25, usp21, usp15, usp3, cylindromatosis (cyld), porcine epidemic diarrhea virus papain-like protease 2 (pedv plp2) and transmissible gastroenteritis virus papain-like protease1 (tgev pl1) are the dubs found to deubiquitinate rig-i. we will discuss them one by one. in a study using hek293 t cells and sendai virus (sev), knockdown of the gene transcribing usp15 resulted in upregulation of type i ifn, while overexpression of usp15 decreased type i interferon as usp15 showed dose-dependent inhibition of ifn-β [16] . further experiment supported the idea that usp15 deubiquitinates k63-polyub from rig-i [16] . however, when the effects of usp15 with a mutated catalytic site and wild type usp15 were compared, the results were surprisingly similar, indicating that usp15's catalytic activity is not necessary for it to inhibit ifn synthesis [16] . orf64 is a dub activity containing tegument protein, found within kaposi's sarcoma-associated herpesvirus (kshv) and murine gamma herpesvirus 68 (mhv68) [17, 18] . the expression of kshv orf64 in hek293 t cells led to suppression of both rig-i-induced and sev infection-induced ifn-β promoter activation [18] . on the contrary, kshv orf64-c29 g mutant, with defective deubiquitinating activity, resulted in lesser to no suppression, confirming the influence of orf64 on the ifn synthesis [18] . also, the overexpression of trim25, but not the mutant trim25, reversed the orf64's effect on ifn production and reverted the ubiquitination of rig-i, further confirming the result [18] . it is also noteworthy that orf64 was not capable of suppressing mavs-induced activation of ifn-β production [18] . when mhv68 infected bone marrow derived dendritic cells (bmdc) were induced by mcmv and hsv-1 for type i ifn induction, no ifn was detected, but tnf-α, il-6 and il-1β were expressed upon high dose stimulation [19] . this provided evidence that mhv68 induced innate immunity of the host to a lesser extent [19] . on the other hand, orf64 mutant mhv68 stimulated innate immune response [19] . by utilizing dub activity of orf64, mhv68 blocked viral dna induced, sting-mediated ifn production [19] . in a study performed by zhong et el, usp25, too, was found to deubiquitinate rig-i and reduce sev-induced ifn-β production in hek293 t cell line [20] . knockdown of usp25 gene also led to augmentation of isre promoter upon sev induction [20] . mutating the catalytic residue of usp25 was sufficient to block usp25's effect on ifnβ induction, supporting that ifn-β suppression via usp25 is dub activity dependent [20] . usp25 targeted not only rig-i for deubiquitination, but also extended to traf2 [20] , traf3 [21, 22] and traf6 [20] and to affect ifn signaling. usp25 also deubiquitinated traf5 and traf6 to regulate in il-17 signaling [23] . however, some studies have given different results regarding usp25's ability to deubiquitinate traf6. lin et al. stimulated usp25 knockout bmdc with sev or hsv-1 infection and added wild type (wt) usp25 or mutant usp25, but k48-ub of traf6 did not differ from one another [21] . however, zhong et al.'s study using hek293 t cells supported deubiquitination of traf6 by usp25 [20] . this variance in the result may be caused by the difference of the cell line used for the studies. furthermore, usp25 showed its ability to suppress phosphorylation of interferon regulatory factor 3 (irf3) and p65, also contributing to inhibition of ifn promoter activation [20] . fan et al., knowing that usp21 inhibits rig-i-induced ifn-β production, searched for its mechanism [24] . they unveiled that usp21 fig. 1 . mechanism of action of ubiquitin proteasome system and deubiquitinating enzymes. ub attaches to the target protein by going through a series of reaction with e1 (ubiquitin activating), e2 (ubiquitin conjugating) and e3 (ubiquitin ligating) enzymes. a target protein could be ubiquitinated once or multiple times on lysine residues. 26s proteasome identifies target proteins with polyub chain and degrades them into amino acid segments and reusable ub. ubiquitinated proteins could also be deubiquitinated by dubs, resulting in a different fate. inhibited isre reporter activity induced by sev and rig-i-card, but not by tank-binding kinase 1 (tbk1) in mouse embryonic fibroblasts (mef) cells [24] . usp21 deubiquitinated rig-i in hek293 t cells [24] . also, they found that usp21's function regarding antiviral response is compatible in mef and hek293 t cell lines by introducing each cell line's usp21 to the other cell line and observing the effect [24] . usp21's specificity to rig-i was also confirmed in hela cells through coimmunoprecipitation (co-ip) of usp21 with rig-i, using rabbit polyclonal antibodies against usp21 [24] . usp21 also deubiquitinated mda5 to inhibit antiviral response [24] . usp3 is also a dub that deubiquitinates k63-polyub chain of both rig-i and mda5 and suppresses ifn-β activation [25] . usp3's effect was found viable in 293 t, thp-1, human peripheral blood mononuclear cells (pbmcs) and raw264.7 cells, supporting that usp3's activity is viable in both human and murine cells [25] . usp3 did not inhibit mavs, sting, tbk1, irf3 and tirf, as demonstrated by isre-luc activity induction test [25] . also co-ip demonstrated the interaction between usp3 and stimulated rig-i or mda5, but not the unstimulated ones, supporting that ligand stimulation is required for usp3 to interact with rig-i or mda5 [25] . more specifically, poly(i:c) (lmw) stimulation leads usp3 to have a strong interaction with rig-i, but a weak one with mda5, while poly(i:c) (hmw) stimulation leads usp3 to have a strong interaction with mda5, but a weak one with rig-i [25] . cyld is another dub that removes k63-ub chain from rig-i to decrease the ifn production [26, 27] , but tbk1 and ikkε were also identified as the target of the deubiquitination of cyld in 293 ebna cells [27] , resulting in the same effect. cyld also interacted with ips-1 to negatively regulate it, but did not deubiquitinate it [27] . schmid et al. found that in brain and peripheral blood of c57bl/6, the mrna level of ifn-γ gene decreased with the knockdown of cyld, while the serum concentration of ifn-γ increased [28] . a study conducted using human kidney mesangial cells (mc) showed slightly different results: silencing cyld in mc cells and stimulating them with poly ic increased the toll-like receptor 3 (tlr3)-induced activation of rig-i and mda5 [26] ; however, the level of mrna of rig-i and mda5 actually decreased [26] . the authors speculated this difference to be caused by the change in cell line used [26] , but further study is necessary to determine the cause. cyld also decreased ifn promotor activation by deubiquitinating traf2 and traf6 in hek293 t cells, respectively [29, 30] . cyld in u2os/nod2 cells were found to deubiquitinate k63-ub of ripk proteins, especially ripk2, to suppress nod2-induced nf-κb activation [31] . when cyld was suppressed, ubiquitinated receptor interacting protein kinase 1 (ripk1), also called rip1, and ripk2 proteins accumulated within cells [31] . plps, first discovered in coronavirus in 2005 [32] , are multifunctional proteins with dub activity that are synthesized by many families of viruses that regulate ifn signaling pathway by interacting with rig-i [33] [34] [35] [36] . recently, the mechanism by which pedv plp2 suppresses ifn production in the host cell was identified. in hek293 t cells, pedv plp2 was found to deubiquitinate rig-i and sting, thereby affecting its downstream pathway, resulting in suppression of ifn production [35] . tgev pl1 also was revealed to bind and deubiquitinate both rig-i and sting in hek293 t cells [36] . studies on middle east respiratory syndrome coronavirus encoded papain-like protease (mers-cov pl pro ) showed that it also has a dub function [37] . this was supported by a study by bailey-elkin et al., in which they obtained the crystal structure of pl pro -ub complex and showed that wt mers-cov pl pro , but not the dub mutant pl pro , suppresses ifn-β promotor activity [33] . the targets of mers-cov pl pro were identified as rig-i, mda5 and mavs [33, 34] . mers-cov pl pro and severe acute respiratory syndrome coronavirus (sars)-cov pl pro inhibited the proinflammatory signaling in hek293 t cells upon mda5 stimulation, which included decreased expression of ccl5 and ifn-β and decreased level of cxcl10 mrna [34] . another study on sars-cov pl pro found that irf3 is ubiquitinated and that deubiquitinating activity of sars-cov pl pro was required for it to affect irf3 [38] . sars-cov pl pro did not affect irf3 in other means, such as dimerization or nuclear translocation [38] . dub domain mutated plp2 of equine arteritis virus (eav) also increased in expression of ifn-β and il-8 in equine long fibroblasts (elf) [39] . plp domain was also found in nsp3 protein, which will be discussed in a later section. sting is a transmembrane protein found in mitochondria and endoplasmic reticulum that regulates ifn-promotor activation at the downstream of rig-i [40] . zhang et al. studied the effect of usp18 (also known as ubp43) on sting and revealed that usp18 interacts with sting to affect ifn-promotor activity [41] . however, when usp18 -/-mef cells with either wt usp18 or dub activity-mutated usp18 were induced with hsv-1, hcmv or cytosolic dna, ifnb, ifna4, tnf, il-6 or cxcl1 genes increased in expression, indicating that the deubiquitinating activity of usp18 is not responsible for this phenomenon [41] . subsequently, they searched for dubs that interact with usp18 and found that knockdown of usp20 inhibited usp18-induced deubiquitination of sting and knockdown of usp18 inhibited usp20-induced deubiquitination of sting [41] . immunoprecipitation revealed that sting, usp18 and usp20 are arranged as usp20-usp18-sting, but both usp20 and usp18 were associated with the n-terminus of sting [41] . usp20 deubiquitinated k33-or k48-linked ubiquitin of sting [41] . these results together supported that although usp18 does not deubiquitinate sting itself, usp18 recruits usp20 to deubiquitinate sting to suppress ifn synthesis [41] . another way that usp18 inhibited nf-κb activation is by deubiquitinating k63-ub of tak1 and nemo [42] . usp18 strongly interacted with tak1-tab1 and dub activity dependently deubiquitinated k63-ub of tak1 in 293 t cells [42] and in th17 cells [43] . usp18 also fig. 3 . tlrs, ifnari and ifnarii induced ifn production pathway. pamps, ifn-α and ifn-β stimulate tlr4 and ifnar i & ii receptors respectively to induce ifn production as well as nf-κb activation. dubs that play a role in these pathways are indicated in the figure to show the mechanism of their action. decreased k63-ub of nemo [42] . in a study by malakhova et al., usp18 inhibited ifn-induced gene activation by affecting jak-stat signaling pathway in 293 t cells [44] . their study showed that usp18 does not interact with ifnar1, but with box1-box2 region of ifnar2 to disrupt its interaction with jak to inhibit jak's tyrosine kinase activity in a dub activity independent manner [44] . consistent with this, usp18 knockout murine cells displayed hyperactivity towards type i ifn signaling, resulting in the increase of the level of phosphorylation of stat1 and stat2 [45] . usp18's interaction with ifnar2 also interfered with ifnar2's ability to recruit ifnar1, hindering ifn i signaling [46] . herpes simplex virus 1 (hsv-1) invades a host and escapes its ifn-mediated innate immunity by encoding a large tegument protein, ul36, which has a motif with dub activity, named ul36 ubiquitin-specific protease (ul36usp) [47] . when hek293 t cells were transfected with markers of co-ip and ul36usp or the c40a (a dub motif mutant) and then infected with sev, the result showed reduction of ubiquitination of traf3 in cells with wt ul36usp, while c40a has no reduction of ul36usp's ubiquitination [48] . the result supported that ul36usp deubiquitinates traf3 molecules to inhibit ifn-promotor activation [48] . in a different study regarding the function of ul36usp, it was found that ul36usp inhibits cgas and sting dependent ifn-β production [49] . nf-κb activation from overexpressing sting, tbk1, ikkα and ikkβ was also inhibited, but not from overexpressing p65 [49] . in this study, human foreskin fibroblast (hff) cells were infected with either hsv-1 or hsv-1 c40a mutant and stimulated with ifn stimulatory dna [49] . as a result, the level of endogenous iκbα in hff cells with mutant hsv-1 significantly decreased compared to hff cells with wt hsv-1, supporting that ul36usp decreases the degradation of iκbα in a dub activity-dependent manner [49] . additionally, co-ip study in hek293 t cells showed decrease in ubiquitination of iκbα in those transfected with wt ul36usp, but not in those transfected with c40a mutant [49] . taken together, ul36usp deubiquitinates iκbα to inhibit its degradation, suppressing nf-κb activity [49] . a study by lin et al. revealed that usp25 is required for both dna and rna virus-induced signaling [21] . supporting this claim, silencing usp25 in mefs or mouse lymphatic fibroblasts (mlf) led to inhibition of expression of ifna4, tnf, and il-6 upon triggering them with sev, vesicular stomatitis virus (vsv) or poly(i:c) [21] . also, the level of ifnα and il-6 was reduced in mlfs, bmdcs or flt3lpdc cells with usp25 knockdown [21] . usp25's dub activity was also found to be necessary for virus-induced signaling, as usp25 knockdown mefs with wt usp25 reconstitution allowed expression of ifnb, ifna4 and il-6 upon sev or hsv-1 induction, while those with dub activity mutant usp25 did not [21] . overexpressing usp25 in hek293 t cells resulted in reduction of irf3 phosphorylation when stimulated with sev, leading to inhibition of nf-κb activity [20] . isre reporter activity was also inhibited by usp25 in a dose-dependent manner [20] . taking it one step further, lin et al. uncovered that usp25 stabilizes traf3 in a dub activity dependent manner by deubiquitinating k48-ub of traf3 in bone marrow-derived macrophages (bmdm) cells, inhibiting tlr4 signalinginduced innate immune responses [21] . the nonstructural protein 3 (nsp3) is a viral protein with deubiquitinating activity in its plp2 domain, which was found in scov and in mouse hepatitis virus a59 (mhv-a59) [50, 51] . infecting mef cells with mhv-a59 did not result in detectable ifn-β induction, while infecting them with sev (the control) did result in ifn-β responses [51] . when cells were given variants of nsp3, the ifn-β induction only took place in cells that lacked wt plp2 domain [51] . when the plp2 domain was present, ifn-β induction was suppressed upon viral infection [51] . moreover, polyub of irf3, which is necessary for ifn-β induction, was deubiquitinated in the presence of plp2, which was further confirmed by co-ip indicated formation of a complex of plp2 and irf3 [51] . this deubiquitination inhibited nuclear translocation of irf3 [51] . in hek293 t cells and mef cells, k63-polyub was also deubiquitinated by the plp2 domain of nsp3, inactivating tbk1-irf3 complex in the cytoplasm [52] . monocyte chemotactic protein-inducing protein 1 (mcpip1) is a protein, common to human and mouse, with dub activity toward traf2, traf3 and traf6, thereby inhibiting jnk and nf-κb signaling [53] . a more recent study has uncovered through co-ip in sev infected hek293 t and hela cells that mcpip1 interacts with irf3 and through confocal microscopy that transfection with mcpip1 inhibited nuclear translocation of irf3 [54] . additionally, the presence of mcpip1 inhibited traf3 and tbk1 activated ifn-β expression [54] . co-ip also revealed possibility of mcpip1 to interact with ips-1 and ikkε as well [54] . a20 (also known as tnfaip3) inhibited lps-induced nf-κb activity in mef cells in a study by boone et al. [30] . upon further testing in hek293 t, they found that wt a20, but not dub activity domain mutant a20 removed k63-ub from traf6 [30] . in raji cells, the n-terminal dub domain of a20 interacted with and deubiquitinated irf7 [55] . however, in vitro study showed no interaction between a20 and irf7, which is likely due to requirement of other intracellular proteins [55] . irf7 has been known to be activated by epstein-barr virus (ebv)'s oncoprotein called latent membrane protein 1 (lmp1) [56] . a20 has been known for a long time as a negative regulator of nf-κb pathway mediated by rig-i. a20 does interact with rig-i, and suppresses rig-i-mediated nf-κb pathway [57] , but whether a20 deubiquitinates rig-i or not still requires confirmation. similar to orf64 in mhv68 [17] , bplf1 is an ebv encoded large tegument protein with dub activity that opposes tlr signaling in the host [58] . gent et al.'s research revealed that bplf1 deubiquitinates traf6 and nemo in 293 t cells [58] . immunoprecipitation in 293 t cells revealed that k63-ub of traf6 and nemo was reduced when wt bplf1 was expressed, while mutant bplf1 did not [58] . k48-ub of iκbα was also identified as a target of bplf1's dub activity [58] . the brcc36 isopeptidase complex (brisc) is a nuclear dub complex, composed of abraxas, brcc36, brcc45 and merit40, capable of deubiquitinating k63-ub [59] . in a study by zheng et al., serine hydroxymethyltransferase (shmt) formed a complex with brisc to form brisc-shmt complex [59] . shmt allowed interaction of brisc with ifnar1 to deubiquitinate k63-ub of ifnar1, reducing ifnar1's internalization and degradation by lysosome [59] . taken together, brisc is the first dub complex we discussed that works to actually increase responses to ifn. tnf is a cytokine that plays a significant role in inflammation and regulation of immune cells. since tnf shares some of its pathway with ifn, studies that focused on tnf rather than ifn are included in this section for the purpose of this review (fig. 4) (table 1) . usp4 was identified by studies to negatively regulate both tnf-αand il-1β-induced nf-κb activation [60] [61] [62] . jiang et al. observed that introducing small interfering usp4 (siusp4) to decrease usp4 level in microglia from the spinal cord of sprague-dawley rats led to an increase in p-p65 and traf6 expression as well as secretion of tnf-α and il-1β, all of which decreased upon introduction of ha-usp4 plasmid [60] . xiao et al.'s study added on to this by demonstrating usp4's interaction and deubiquitination of traf2 and traf6, but not traf3, both in vivo, in hek293 t cells, and in vitro [61] . as a result, usp4 negatively influenced tnf-α-induced-nf-κb activation-mediated cytokine induction, including il-6 and il-8 in a549 and h1229 cells [61] . usp4 also deubiquitinates tak1 in hek293 t cells [62] . usp4 also protected iκbα from degradation, [61] , a necessary step for tnf-α-induced nf-κb activation [63] . this was further supported by knockout of usp4 aiding iκbα degradation [61] . l. infantum otubain (otuli) has been shown to induce inflammatory responses in peritoneal macrophages from c57bl/6, shown by production of tnf-α and il-6 as well as lipid droplet synthesis [64] . also otuli demonstrated strong dub activity on k48-ub and weak activity on k63-ub in vitro at ph 7.5 [64] . we mentioned in a previous section that usp25 deubiquitinates traf3 and interacts with traf6, increasing expression of tnf in hek293 t cells [21, 22] , while in mef cells, usp25 negatively affects tnf-α-induced nf-κb activation [20] . a20 also affects tumor necrosis factor receptor 1 (tnfr1) signaling pathway. in bmdms and bmdc, a20 worked together with tax1bp1 to interact with ubc13, an e2 enzyme, resulting in the inhibition of e3 ligase activities of traf6, traf2 and ciap1 [65] . futhermore, a20 and tax1bp1 participated in degrading ubc13 upon il-1 and tnf-α stimulation in mef cells [65] . a20's zf4 motif was found to recruit a20 dimers to bind with ripk1 in the tnfr signaling complexes and inhibit ubiquitination of k48-ub and k63-ub chains of ripk1, hindering tnf signaling [66] . in intestinal epithelial cells (iec), a20 dimer interacted with the ripoptosome (also known as complex iia), which allowed ubiquitins on ripk1 to sustain, increasing caspase-8 activation to promote tnf-induced apoptosis [67] . however, this effect was not dub activity dependent [67] . cezanne is a dub that belongs to the a20 subgroup of otu family. similar to a20, cezanne also was shown to suppress nf-κb signaling by deubiquitinating k63-polyub from ripk1 [68, 69] and traf6 [70] . also, cezanne was recruited to the activated tnfr prior to deubiquitinating ripk1, which was dependent on the ubiquitin-associated (uba) domain of cezanne [68, 69] . consistnent with the findings, inhibiting cezanne production via sirna resulted in an increased production of il-8 upon tnf-α stimulation [68] . cezanne's dub activity was required for inhibiting phosphorylation and degradation of iκbα [68] . usp48 (also known as usp31) interacted with and deubiquitinated traf2 in beas2b cells [71] . a noteworthy fact is that traf2 in jnk pathway was targeted by usp48, but not traf2 in nf-κb signaling [71] . trail is a cytokine that binds to death receptors (dr) and induces apoptosis, especially in tumor cells. its specificity for tumor cells have made trail and its receptor as the targets for anti-cancer therapeutics. trail inducing dubs are also summarized in table 1 . in a study of malignant mesothelioma, a loss of function mutation of brca associated protein 1 (bap1) resulted in increased sensitivity fo trail induction [72] . when testing for domains that play a role in trail sensitivity in h226 mm cells, only asxl1/2 binding site-mutated bap1 and dub domain-mutated bap1 resulted reduction in rtrail sensitivity, indicating that asxl1/2 binding sites play a role in trail sensitivity [72] . this was in congruence with the fact that bap1 binds to asxl to form the polycomb repressive deubiquitinase complex (pr-dub) that deubiquitinates histone h2a [73] . also, flow cytometry analysis confirmed that the mutation of c91a, or the deubiquitinating domain, of dub resulted in decreased expression of dr4 and dr5 in h226 cells [72] . only dr4 expression increased in h2818 cells upon bap1 knockout [72] . on the same line with bap1, usp35 knockout also increased trail sensitivity [74] . a study by leznicki et al. introduced three isoforms of usp3 in hek293 cells: usp35 iso1 , usp35 iso2 and usp35 iso3 , although the focus was on the first two [74] . usp35 iso2 was revealed as an integral membrane protein on endoplasmic reticulum, while usp35 iso1 was identified as a cytosolic protein [74] . moreover, the proteins that they interacted with also varied [74] . usp35 iso2 led to er stress, bap31 cleavage and activation of caspase-8 and caspase-3, resulting in apoptosis [74] . usp35 iso2 upregulated c/ebp homologous protein (chop) and dr5 in u2osfipin and hela fipin cells [74] . on the other hand, overexpressing usp35 iso1 exerted an opposite effect of delaying caspase-8 processing in trail-induced apoptosis [74] . this effect was dependent on dub activity [74] . b-ap15 is a small therapeutic molecule that inhibits usp14 and uchl5 [75] . b-ap15 has been identified as an agent that increases trail receptors on many types of cancer cells, increasing their likelihood to enter apoptosis via nk cells [76] . introduction of b-ap15 in a549, hct116 and calu-1 cells increased the level of dr5, but not the other death-inducing signaling complex (disc) components [77] . the increase in the level of dr5 protein was due to reduction in the degradation of dr5, leading to an increase in trail-induced apoptosis [77] . in a different study, caspase-denependent apoptosis was increased in mantle cell lymphoma (mcl) cells when exposed to b-ap15, which was confirmed with addition of pan-caspase inhibitor zvad-fmk, which resulted in an inhibition of apoptosis [78] . this study indirectly demonstrated that usp14 and/or uchl5 partakes in decreasing dr5 expression. mcpip1 is another dub that decreases dr5 [79] . exposing mda-mb-231 cells to doxycycline (dox) led to induction of mcpip1, which then led to a decrease in dr5 [79] . similarly, when a549 human lung cancer cells were exposed to mcp1, mcpip1 level increased in a dosedependent manner, also resulting in a decrease in dr5 [79] . likewise, dr5 level increased when mcpip was knocked down via short hairpin rna (sirna) [79] . mcpip1 successfully achieved this by deubiquitinating dr5, thereby stimulating lysosomal degradation of dr5 [79] . also, the increase in dr5 level following mcpip1 knockdown catalized the formation of disc during the dr5-induced apoptosis [79] . we will now discuss dubs that induce interleukins, which are listed in table 2 . usp25 has been shown to deubiquitinate traf3 and tlr4 and to interact with traf6, increasing expression of il-6 in mef cells [21, 22] . on top of decreasing production of ifn [17, 18] , orf64 in mhv68 also induced the production of il-1β [19] . il-1β production was dependent on nlrp3 and asc, rather than aim2 [19] . usp4 negatively regulates il-1β-induced nf-κb activation [60] [61] [62] . increases (c57bl/6 lung homogenate) [19] increase = inc. in production, + = induce positive effect. eeyarestatin i (esi), a small molecule that inhibits deubiquitination, has been found responsible for blocking il-1β release [80] . lopex-castejon et al., who reported this finding speculated uch37 or usp14 was responsible for this phenomenon, but futher study showed that they do not regulate il-1β secretion individually or cooperatively [80] . this result left possibility of an uncharacterized dubs or an additional dub(s) partaking in the process. usp18 knockout murine splenocytes and naïve t cells produced more il-2 compared to the wt splenocytes [43] . under th17 polarizing condition, il-2 production was significantly higher in the usp18 knockout naïve cd4 + t cells compared to the wt naïve cd4 + t cells [43] . also usp18 knockout naïve cd4 + t cells underwent hyperproliferation under th17 polarizing condition, which was reversed by adding il-2 neutralizing antibody [43] . taken together, usp18 downregulates il-2 synthesis and tcr-induced t cell proliferation [43] . additional mechanism of action has been discussed in the previous seciton. dufner et al. found that usp8 was essential in t cell maturation and homeostasis, although it was not required for negative selection [81] . inhibiting usp8 leads to decrease in il-7ra mrna as well as ccr7 [81] . also, il-6, il-12p70, ifn-γ and tnf levels were increased in the blood of usp8 f/f cd4-cre mice than usp8 f/f [81] . deleting otulin gene in mouse immune cells, t, b, natural killer cells (nk), dendritic cells (dc) and macrophage cells, resulted in production of cytokines specifically responsible for acute systemic inflammation, such as tnf, il-1β, il-6, mcp-1, mip-1α and g-csf [82] . cytokines responsible for adaptive immunity were not affected by the deletion [82] . it is noteworthy that this study suggests that although many cytokines partake in the inflammatory response in murine cells without otulin, the primarily responsible cytokine is tnf [82] . unlike in immune cells, deleting otulin in myeloid cells resulted in activation of cytokines responsible for acute and chronic inflammation as well as autoimmunity, showing an increase in the level of 16 out of 25 cytokines tested [82] . deficiency of otulin in macrophages resulted in nf-κb activation without an induction, which was due to the inability to manage polyub chains synthesized by linear ubiquitin chain assembly complex (lubac) [82] . another study found that otulin overexpression inhibited tnf-α-induced nuclear translocation of p65 in hek293 t cells [83] . interestingly, both wt and dub domain mutant otulin disabled lubac-induced nf-κb activation, indicating that otulin-mediated met1-polyub is not the only factor influencing nf-κb activation [83] . otulin has also been identified to deubiquitinate met1-polyub of ripk1 and inhibited the binding of nemo and ripk1, blocking the tnf-α-induced nf-κb response [83] . ebv bplf1 suppresses ifn production, but il-8 production by cells upon malp-2 ligand stimulation was also abrogated upon bplf1 expression [58] . ebv bplf1 suppressed production of proinflammatory cytokine il-8 in 293-tlr2/cd14 cells [58] . trabid is a dub from otu family, translated from the gene zranb1 [84] . upon zranb1 knockout in mice, il-6, tnf, il-12a, il-12b and il-23a displayed a decrease in expression [84] . trabid's deubiquitinating activity was necessary for recruiting c-rel and p50 to il-12 promoter by influencing histone modifications [84] . knockout of trabid rendered bmdc incapable of producing il-12 and il-23, leading to a decrease in the number of differentiation of cd4 + t cells to t h 1 and t h 17 cells, which was reversible with adding il-12 and il-23 [84] . not many studies have focused their study objectives on discovering relationship between chemokines and dubs. some discovered interactions are listed in table 2 . we have discussed how dubs induce cytokine production, signaling and effects. compared to dub's effects on cytokines, cytokine-inducible dubs are far less studied due to the difficult nature of planning such studies. however, this information can be as important as dub-induced cytokines. we will now discuss some known examples of cytokine-inducible dubs, as listed in table 3 . dub-1 is one of the early identified cytokine-inducible dubs. studying the sequence of dub-1 gene, unveiled that it contained a il-3 inducible enhancer in ba/f3 murine lymphocyte cell line [85] . the timing of il-3 induced dub-1 mrna increase was identified as early g1 phase [85] . moreover, when dub-1 was constitutively expressed, majority of ba/f3 cells were arrested in g 1 phase of the cell cycle, which was dub activity dependent [85] . induction of dub-1 was dependent on viable jak2 and raf-1, but not stat5, suggesting that dub-1 expression is dependent on two pathways: jak2 and ras/raf-1/mapk pathway [86] . il-5 and granulocyte-macrophage colony-stimulating factor (gm-csf) also induced dub-1 transcription, which supported that β common (βc) subunit plays a part [87] . dub-1a decreased in expression when jak2 was suppressed, also suggesting dub-1a to be affected by il-3 and jak2 pathway [88] . dub-2 is similar to dub-1 in its sequence of amino acids and is also induced by jak2/stat5 pathway [89] . however, unlike dub-1, dub-2 was induced only by il-2 in t cells, but not by il-3 [90] . dub-2 also increase = inc. in production. + = induce positive effect. showed increase in jak/stat signaling pathway products by decreasing il-2 induced dephosphorylation of stat5 [89] . also, dub-2 decreased apoptosis in ba/f3 cells upon withdrawal of cytokines [89] . the mechanism by which dub-2 achieves these effects needs to be further studied. in myeloid 32d cells, dub-2 stabilized csf3r and increased its signaling activity by decreasing lysosomal degradation of csf3r by deubiquitinating it, leading to prolongation of stat5 phosphorylation in csf3 signaling pathway [91] . on the other hand, dub-2a is a dub expressed in hematopoietic cells, such as b and t cells [92] . unlike dub-2, which was more expressed by il-2, dub-2a was further expressed upon exposure to il-3 [92] . although similar to dub-1 induction by il-3 [85] , dub-2a induction by colony-stimulating factor 3 (csf3) in myeloid 32d cells did not require erk [91] . dub-3 (also known as usp17) is a cytokine-inducible human dub that was found to deubiquitinate sds3 and block proliferation in hela cells [93] . in mrna level, dub-3 expression increased in raji cells when treated with il-4 and in u937 cells when treated with il-6 [94] . dub-3 also influenced the ras/mek/erk signaling pathway and deubiquitinated ras converting enzyme 1 (rce1), decreasing proliferation of cells [95] . otud-6b, a dub from otu family, was upregulated in a mouse pro-b cells, ba/f3 cells, upon cytokine stimulation [96] . stimulation with il-3, il-4, il-13 or gm-csf resulted in a dose-dependent increase of otud-6b mrna in the first 0 to 2 h of stimulation, but quick decrease was observed from 4 to 6 h [96] . overexpressing otud-6b in ba/f3 cells resulted in downregulation of proliferation and increased the frequency of apoptosis [96] . usp18 has been known to be induced by viral infection, genotoxic stress or interferon [97] . consistent with this, usp18's mrna level increased in thp-1 cells and thp-1-derived macrophages upon exposure to ifn-β [42] . furthermore, tlr ligands, lps, pam3csk4 and cl097 all gave the same result of increased mrna level of usp18, supporting that tlr-induced signaling pathway induces usp18 expression [42] . exposure to tnfα caused gsk3β-mediated phosphorylation of usp48, leading to deubiquitination of traf2 in beas2b cells, increasing jnk signaling upon tnf-α-induction [71] . a20 was identified since 1990 as a tnf-α-induced dub in human umbilical vein endothelial cells (huvec) [98] . mrna of cezanne quickly increased in hek293 and huvec cells upon exposure to tnf-α, but not upon shear stress [68] . we have discussed some known cases of dub-regulated cytokines and cytokine-inducible dubs. cytokines are intertwined in numerous cellular processes and dubs are closely related to cytokines. this review dealt with many dubs, but less than half of all known dubs are discussed. moreover, we cannot say for certain that all the functions of the dubs discussed here are discovered. this grants us to further investigate the molecular mechanism and their effects. studying dubs and their effects could enlighten us with a novel therapeutic approach to various diseases, including but not limited to immunological diseases and cancer. structural and functional insights to ubiquitin-like protein conjugation ubiquitin modifications the ubiquitin code in the ubiquitin-proteasome system and autophagy a genomic and functional inventory of deubiquitinating enzymes regulation and cellular roles of ubiquitin-specific deubiquitinating enzymes an atlas of altered expression of deubiquitinating enzymes in human cancer mindy-1 is a member of an evolutionarily conserved and structurally distinct new family of deubiquitinating enzymes broad and diverse mechanisms used by deubiquitinase family members in regulating the type i interferon signaling pathway during antiviral responses the roles of ubiquitin modifying enzymes in neoplastic disease viral deubiquitinases: role in evasion of anti-viral innate immunity type i interferon in chronic virus infection and cancer triggering antiviral response by rig-i-related rna helicases double-stranded dna and doublestranded rna induce a common antiviral signaling pathway in human cells rna recognition and signal transduction by rig-i-like receptors trim25 ring-finger e3 ubiquitin ligase is essential for rig-i-mediated antiviral activity ubiquitin-specific protease 15 negatively regulates virus-induced type i interferon signaling via catalytically-dependent and -independent mechanisms a functional ubiquitin-specific protease embedded in the large tegument protein (orf64) of murine gammaherpesvirus 68 is active during the course of infection inhibition of rig-i-mediated signaling by kaposi's sarcoma-associated herpesvirusencoded deubiquitinase orf64 evasion of innate cytosolic dna sensing by a gammaherpesvirus facilitates establishment of latent infection ubiquitin-specific proteases 25 negatively regulates virus-induced type i interferon signaling induction of usp25 by viral infection promotes innate antiviral responses by mediating the stabilization of traf3 and traf6 ubiquitin-specific protease 25 regulates tlr4-dependent innate immune responses through deubiquitination of the adaptor protein traf3 negative regulation of il-17-mediated signaling and inflammation by the ubiquitinspecific protease usp25 usp21 negatively regulates antiviral response by acting as a rig-i deubiquitinase usp3 inhibits type i interferon signaling by deubiquitinating rig-i-like receptors cylindromatosis (cyld), a deubiquitinase, attenuates inflammatory signaling pathways by activating toll-like receptor 3 in human mesangial cells the tumour suppressor cyld is a negative regulator of rig-i-mediated antiviral response the deubiquitinating enzyme cylindromatosis dampens cd8(+) t cell responses and is a critical factor for experimental cerebral malaria and blood-brain barrier damage loss of the cylindromatosis tumour suppressor inhibits apoptosis by activating nf-kappab the ubiquitinmodifying enzyme a20 is required for termination of toll-like receptor responses cyld limits lys63-and met1-linked ubiquitin at receptor complexes to regulate innate immune signaling deubiquitination, a new function of the severe acute respiratory syndrome coronavirus papain-like protease? crystal structure of the middle east respiratory syndrome coronavirus (mers-cov) papain-like protease bound to ubiquitin facilitates targeted disruption of deubiquitinating activity to demonstrate its role in innate immune suppression mers-cov papain-like protease has deisgylating and deubiquitinating activities the papain-like protease of porcine epidemic diarrhea virus negatively regulates type i interferon pathway by acting as a viral deubiquitinase transmissible gastroenteritis virus papain-like protease 1 antagonizes production of interferon-beta through its deubiquitinase activity proteolytic processing, deubiquitinase and interferon antagonist activities of middle east respiratory syndrome coronavirus papain-like protease the sars coronavirus papain like protease can inhibit irf3 at a post activation step that requires deubiquitination activity deubiquitinase function of arterivirus papain-like protease 2 suppresses the innate immune response in infected host cells modulation of stimulator of interferon genes (sting) expression by interferongamma in human keratinocytes usp18 recruits usp20 to promote innate antiviral response through deubiquitinating sting/mita usp18 negatively regulates nf-kappab signaling by targeting tak1 and nemo for deubiquitination through distinct mechanisms usp18 inhibits nf-kappab and nfat activation during th17 differentiation by deubiquitinating the tak1-tab1 complex ubp43 is a novel regulator of interferon signaling independent of its isg15 isopeptidase activity microarray analysis reveals that type i interferon strongly increases the expression of immuneresponse related genes in ubp43 (usp18) deficient macrophages receptor dimerization dynamics as a regulatory valve for plasticity of type i interferon signaling a deubiquitinating activity is conserved in the large tegument protein of the herpesviridae herpes simplex virus 1 ubiquitin-specific protease ul36 inhibits beta interferon production by deubiquitinating traf3 herpes simplex virus 1 ubiquitin-specific protease ul36 abrogates nf-kappab activation in dna sensing signal pathway the papainlike protease of severe acute respiratory syndrome coronavirus has deubiquitinating activity plp2, a potent deubiquitinase from murine hepatitis virus, strongly inhibits cellular type i interferon production plp2 of mouse hepatitis virus a59 (mhv-a59) targets tbk1 to negatively regulate cellular type i interferon signaling pathway mcpinduced protein 1 deubiquitinates traf proteins and negatively regulates jnk and nf-kappab signaling mcpip1 negatively regulate cellular antiviral innate immune responses through dub and disruption of traf3-tbk1-ikkepsilon complex the a20 deubiquitinase activity negatively regulates lmp1 activation of irf7 intracellular signaling molecules activated by epstein-barr virus for induction of interferon regulatory factor 7 negative regulation of the retinoic acid-inducible gene i-induced antiviral state by the ubiquitin-editing protein a20 epstein-barr virus large tegument protein bplf1 contributes to innate immune evasion through interference with toll-like receptor signaling a brisc-shmt complex deubiquitinates ifnar1 and regulates interferon responses downregulation of usp4 promotes activation of microglia and subsequent neuronal inflammation in rat spinal cord after injury ubiquitin-specific protease 4 (usp4) targets traf2 and traf6 for deubiquitination and inhibits tnfalpha-induced cancer cell migration usp4 targets tak1 to downregulate tnfalpha-induced nf-kappab activation ubiquitin signalling in the nf-kappab pathway revealing a novel otubain-like enzyme from leishmania infantum with deubiquitinating activity toward k48-linked substrate inhibition of nf-kappab signaling by a20 through disruption of ubiquitin enzyme complexes dimerization and ubiquitin mediated recruitment of a20, a complex deubiquitinating enzyme elevated a20 promotes tnf-induced and ripk1-dependent intestinal epithelial cell death nf-kappab suppression by the deubiquitinating enzyme cezanne: a novel negative feedback loop in pro-inflammatory signaling the n-terminal ubiquitin-associated domain of cezanne is crucial for its function to suppress nf-kappab pathway cezanne regulates inflammatory responses to hypoxia in endothelial cells by targeting traf6 for deubiquitination the deubiquitinating enzyme usp48 stabilizes traf2 and reduces e-cadherin-mediated adherens junctions loss of functional bap1 augments sensitivity to trail in cancer cells histone h2a deubiquitinase activity of the polycomb repressive complex pr-dub expansion of dub functionality generated by alternative isoforms -usp35, a case study proteasome deubiquitinases as novel targets for cancer therapy a novel inhibitor of proteasome deubiquitinating activity renders tumor cells sensitive to trail-mediated apoptosis by natural killer cells and t cells the proteasome deubiquitinase inhibitor b-ap15 enhances dr5 activation-induced apoptosis through stabilizing dr5 the novel deubiquitinase inhibitor b-ap15 induces direct and nk cell-mediated antitumor effects in human mantle cell lymphoma monocyte chemotactic protein-induced protein-1 enhances dr5 degradation and negatively regulates dr5 activation-induced apoptosis through its deubiquitinase function deubiquitinases regulate the activity of caspase-1 and interleukin-1beta secretion via assembly of the inflammasome the ubiquitin-specific protease usp8 is critical for the development and homeostasis of t cells the deubiquitinase otulin is an essential negative regulator of inflammation and autoimmunity otulin antagonizes lubac signaling by specifically hydrolyzing met1-linked polyubiquitin epigenetic regulation of the expression of il12 and il23 and autoimmune inflammation by the deubiquitinase trabid dub-1, a deubiquitinating enzyme with growth-suppressing activity jak2 is required for induction of the murine dub-1 gene the murine dub-1 gene is specifically induced by the betac subunit of interleukin-3 receptor dub-1a, a novel deubiquitinating enzyme subfamily member, is polyubiquitinated and cytokine-inducible in blymphocytes the deubiquitinating enzyme dub-2 prolongs cytokine-induced signal transducers and activators of transcription activation and suppresses apoptosis following cytokine withdrawal dub-2 is a member of a novel family of cytokine-inducible deubiquitinating enzymes the deubiquitinating enzyme dub2a enhances csf3 signalling by attenuating lysosomal routing of the csf3 receptor dub-2a, a new member of the dub subfamily of hematopoietic deubiquitinating enzymes lys-63-specific deubiquitination of sds3 by usp17 regulates hdac activity dub-3, a cytokine-inducible deubiquitinating enzyme that blocks proliferation usp17 regulates ras activation and cell proliferation by blocking rce1 activity evidence for otud-6b participation in b lymphocytes cell cycle after cytokine stimulation ubp43 (usp18) specifically removes isg15 from conjugated proteins the a20 cdna induced by tumor necrosis factor alpha encodes a novel type of zinc finger protein we would like to thank members of baek's laboratory for their critical comments this study was funded by the korea ministry of environment (moe) as 'the environmental health action program (2016001360008)'. he has been serving as an editorial board member of more than a dozen of international journals. current research and clinical interests are in the molecular genetics of ubiquitination and deubiquitination systems relevant to various cancers. he first coined terms ubiquitomics and deubiquitomics in the field of the ubiquitin-proteasome system. in addition, he is also interested in genomics and proteomics during stem cell proliferation, differentiation, and reprogramming. key: cord-254492-42d77vxf authors: heaton, steven m.; borg, natalie a.; dixit, vishva m. title: ubiquitin in the activation and attenuation of innate antiviral immunity date: 2016-01-11 journal: j exp med doi: 10.1084/jem.20151531 sha: doc_id: 254492 cord_uid: 42d77vxf viral infection activates danger signals that are transmitted via the retinoic acid–inducible gene 1–like receptor (rlr), nucleotide-binding oligomerization domain-like receptor (nlr), and toll-like receptor (tlr) protein signaling cascades. this places host cells in an antiviral posture by up-regulating antiviral cytokines including type-i interferon (ifn-i). ubiquitin modifications and cross-talk between proteins within these signaling cascades potentiate ifn-i expression, and inversely, a growing number of viruses are found to weaponize the ubiquitin modification system to suppress ifn-i. here we review how hostand virus-directed ubiquitin modification of proteins in the rlr, nlr, and tlr antiviral signaling cascades modulate ifn-i expression. the frontline in the cellular response to viral infection is comprised of the specific and general effectors of the innate immune system. effector molecule production is initiated by immune sentinels known as pattern recognition receptors (prrs), which screen the intra-and extracellular environment for molecular motifs uniquely associated with pathogens. prr engagement transduces pro-immune signals into the nucleus via protein signaling cascades that self-limit to mitigate autoimmunity as the infection clears (crampton et al., 2012) . protein posttranslational modifications (ptms) form part of this exquisite system of regulation, with ubiquitin and ubiquitin-like modifications key among them. the retinoic acid-inducible gene 1 (rig-i)-like receptors (rlrs) and nucleotide-binding oligomerization domain (nod)-like receptors (nlrs) are intracellular prrs. the rlrs sense invasive rna produced during infection by both rna and dna viruses (schlee, 2013) . rlr engagement up-regulates type-i ifn (ifn-i) expression, which in turn stimulates transcription of hundreds of ifn-stimulated genes (isgs) that commit host and nearby cells to an antiviral posture. recognized for their role in antibacterial immunity, the nlrs are emerging as antiviral mediators that regulate both ifn-i and nf-κb activation. these are also activated by tlrs, a cell-specific class of extracellular and endosomal transmembrane prrs that sense a broad spectrum of pathogenic motifs. rlr, nlr, and tlr signaling proteins must be spatially and temporally coordinated for efficient immune signal transduction. ubiquitination is a ptm involving the covalent attachment of the 8.6-kd protein ubiquitin to target proteins. ubiquitination is catalyzed by the ubiquitin-activating enzyme (e1), ubiquitin-conjugating enzyme (e2), and ubiquitin protein ligase (e3). the e3 largely dictates substrate specificity, with at least 617 genes encoding putative ubiquitin and ubiquitin-like e3s annotated in the human genome (li et al., 2008) . ubiquitin can undergo ubiquitination itself at its seven lysine residues (k6/k11/k27/k29/k33/k48/k63), building lysine-linked polyubiquitin chains, or its n-terminal methionine (m1), forming linear polyubiquitin chains. alternatively, ubiquitin chains may be noncovalently associated with target proteins. furthermore, ubiquitin chains may be remodeled by deubiquitinating enzymes (dubs; fig. 1 ). the function, abundance, or subcellular distribution of proteins involved in almost every cellular process is regulated in this way, with an increasingly clear role in regulating innate immunity. viruses are obligate intracellular parasites that facilitate their own replication by manipulating the host cell environment. thus, the ubiquitin modification system presents a key manipulation target for viruses to circumvent antiviral signaling pathways. methods for this include substrate molecular mimicry, binding and blocking e3-substrate pairs, expressing virally encoded e3s/dubs, and hijacking host e3s/dubs. additionally, a novel mechanism involving ubiquitin chain packaging into nascent virions for subsequent redeployment viral infection activates danger signals that are transmitted via the retinoic acid-inducible gene 1-like receptor (rlr), nucleotide-binding oligomerization domain-like receptor (nlr), and toll-like receptor (tlr) protein signaling cascades. this places host cells in an antiviral posture by up-regulating antiviral cytokines including type-i interferon (ifn-i). ubiquitin modifications and cross-talk between proteins within these signaling cascades potentiate ifn-i expression, and inversely, a growing number of viruses are found to weaponize the ubiquitin modification system to suppress ifn-i. here we review how host-and virus-directed ubiquitin modification of proteins in the rlr, nlr, and tlr antiviral signaling cascades modulate ifn-i expression. localization changes from the cytosol to distinct subcellular compartments depending on upstream signaling events (goncalves et al., 2011) . in promoting ifn-i signaling, tbk1 associates with rig-i as well as key adaptor proteins, including tank and nf-κb essential modulator (nemo; guo and cheng, 2007; zhao et al., 2007; wang et al., 2012a) . this facilitates interactions between tbk1, inhibitor of nf-κb kinase subunit ε (ikkε) and trafs, particularly traf3. upon rlr activation, these proteins are colocalized at the cytosolic surface of the mitochondrial outer membrane (parvatiyar et al., 2010; van zuylen et al., 2012) , coordinated by the mitochondrial antiviral-signaling protein (mavs; also termed visa/cardif/ips-1). at rest, the rlr cascade is maintained in an inactive but tensioned state through an intricate negative feedback system involving protein expression levels, conformational changes, compartmentalization, and ptms. part of this system operates at the receptor through conformational auto-inhibition of the rig-i cards. the rig-i c-terminal repressor domain (rd) audits the cytosol for viral rna, binding of which induces a major structural rearrangement in the rd and card (saito et al., 2007) . conversely, mda5 oligomerizes along the length of rna ligands, forming immunogenic filaments that are potentiated by atp hydrolysis and interaction with lgp2 (peisley et al., 2012; bruns et al., 2014) . the unfurled rig-i cards undergo tetramerization upon k63-linked polyubiquitination or unanchored polyubiquitin chain association (peisley et al., 2014) . these modifications drive mitochondrial accumulation of rig-i, promoting card-card interac(1) ubiquitin (ub) expresses as an inactive polyprotein, encoded by the ubb and ubc genes. dubs cleave this polyprotein into monomers that are activated by the e1-activating enzyme, involving the energy-dependent adenylation of the ubiquitin c-terminal glycine. the ubiquitin-adenylate intermediate (dashed line) converts into a covalent thioester bond (solid line). (2) ubiquitin transfers to the active site cysteine residue of an e2-conjugating enzyme. (3) the e3 directly or indirectly transfers the e2-bound ubiquitin to a substrate acceptor residue, forming an isopeptide bond. (4) dubs remodel ubiquitin modifications and antagonize ubiquitin-driven functional outcomes. tions with mavs and inducing its oligomerization and filamentation. the rig-i cards contain a high proportion of hydrophobic residues and are prone to aggregation, thus oligomerization and polyubiquitination may stabilize the activated cards or elicit a separate mitochondrial targeting signal. conversely, ubiquitination has no known role in mda5 or lgp2 activation. the first virus-triggered rig-i ubiquitination site described, k172, depends on the e3 activity of tripartite motif protein 25 (trim25; gack et al., 2007) . plausibly as a means of restricting escape mutant selection, this activation mechanism now appears to have evolved with partial redundancy using alternate e3s. trim4 was recently described to modify this same site in addition to two other card residues: k154 and k164 (yan et al., 2014) . furthermore, these same three residues are reportedly ubiquitinated by really interesting new gene (ring) finger protein-135 (rnf135; also termed riplet/reul; gao et al., 2009) , although this is controversial ( fig. 3 ; oshiumi et al., 2010) . underscoring the importance of these modifications, ubiquitin-specific protease 3 (usp3) and ubiquitin c-terminal hydrolase are dubs that inhibit ifn-i production by removing such chains from rig-i (friedman et al., 2008; cui et al., 2014) . both trim25 and rnf135 are targets of the influenza a virus (iav) nonstructural protein 1 (ns1), which blocks their e3 activity and ubiquitin-dependent rig-i activation ( fig. 3 ; gack et al., 2009; rajsbaum et al., 2012) . iav-ns1 binds the central coiled-coil domain (ccd) of trim25 and is postulated to prevent ccd-mediated homooligomerization. although the ns1-binding site on rnf135 is unknown, rnf135 and trim25 share a similar ring-ccd-b30.2/spry (sp1a and ryanodine receptors) domain figure 2 . schematic of the tlr, rlr, and nlr antiviral protein signaling cascades and modes of cross-talk. prrs (blue) screen the intracellular and extracellular environment for pathogenic motifs. ligand-activated prrs bind adaptor proteins (purple) and recruit protein kinases (yellow) and ubiquitin-protein ligases (green). these regulate immune signal transduction to transcription factors (orange) through ptm of signaling cascade proteins. other regulatory proteins (gray) support or sequester these signaling proteins. immune signaling scaffolds such as mitochondria typically coordinate these actions. activated transcription factors translocate into the nucleus and bind to promoter response elements, stimulating appropriate antiviral gene transcription. blue and green circles represent ubiquitination and phosphorylation, respectively. black arrows, activation; red lines, deactivation. organization. however, the ccds differ in size and sequence markedly, suggesting that iav-ns1 may bind multiple sites on trim25 and rnf135. alternatively, given that ccds often mediate protein-protein interactions, iav-ns1 may sense and subvert ccd-interacting domains more broadly. notably, the iav -ns1 :rnf135 interactions observed by rajsbaum et al. (2012) were strain dependent. rnf135 enables rig-i card activation by trim25 upon ubiquitinating rd residues k849 and k851. rnf135 knockdown inhibits interaction between rig -i :trim25 and eliminates tbk1 recruitment (oshiumi et al., 2009) , revealing an ordered functional interplay between ubiquitination and phosphorylation in coordinating rig-i activation. hepatitis c virus (hcv) ns3-4a protease exploits this concept by targeting rnf135 for proteolytic cleavage (oshiumi et al., 2013) . furthermore, numerous herpesviruses encode their own dubs that inhibit ifn-i expression by stripping ubiquitin modifications from activated rig-i (inn et al., 2011b) . accordingly, hcv and herpesvirus infections are treatable with ifn (oberman and panet, 1988; nguyen et al., 2014) , although this can carry significant side effects. endogenous ifn-i expression and self-regulation may be restored by defeating such mechanisms of viral antagonism. ubiquitin in the return to homeostasis. rlr signaling is also counterbalanced and diminished through ubiquitin modification as the antiviral posture becomes unnecessary. rnf125 forms part of this process, ligating k48-linked polyubiquitin chains to the activated card of rig-i and mda5, leading to proteasome-mediated degradation of both receptors and diminished ifn-i signaling. usp4 is a dub that sustains rlr signaling by specifically removing such chains (wang et al., 2013a) . in the same way, rnf125 ubiquitinates and degrades activated mavs (arimoto et al., 2007) , suggesting that rnf125 is an e3 that destabilizes proteins containing activated cards. given how commonly card-containing proteins and their homotypic interactions feature in immune signaling pathways (bouchier-hayes and martin, 2002) , rnf125 may represent a general immune signaling antagonist. conversely, the 52-kd repressor of the inhibitor of the protein kinase (p52ripk) binds and enhances the stability of rig-i by blocking its ubiquitin-mediated degradation (now and yoo, 2011) . accordingly, the properties of p52ripk or rnf125 may be exploitable in the treatment of viral infections or autoimmune disorders. the linear ubiquitin assembly complex (lub ac), containing sha nk-associated rh domain-interacting protein (sha rpin), heme-oxidized irp2 ubiquitin ligase 1l (hoil-1l), and hoil-1-interacting protein (hoip), was proposed to negatively regulate rlr-mediated ifn-i expression via two independent mechanisms (inn et al., 2011a) . first, hoil-1l competes with trim25 for rig-i card binding, abrogating the rig -i :mavs interaction. second, hoip promotes m1-and k48-linked polyubiquitination of trim25 and induces its proteasomal degradation, thereby decreasing trim25-mediated activation of rig-i. if lub ac were capable of ligating k48-linked polyubiquitin chains to substrates, trim25 would be the first example to our knowledge. another route of rlr inhibition involves tetraspanin-6 ubiquitination by an unknown e3. during rlr activation, polyubiquitinated tetraspanin-6 is recruited to mavs and blocks the rlr :mavs interaction, thereby impeding recruitment of the downstream signaling apparatus (wang et al., 2012b) . mitochondria, peroxisomes, and endoplasmic reticulum function as immune signaling platforms linking viral pattern recognition with effector molecule production ( fig. 2 , middle). although this process remains poorly characterized, the nature and context of viral ligands detected by prrs drives accumulation of the downstream signaling apparatus to these platforms. adaptor proteins mediate this accumulation: mitochondria and peroxisomes by mavs (dixit et al., 2010) and endoplasmic reticulum by stimulator of ifn genes (sti ng; ishikawa and barber, 2008) . cyclic gmp-amp synthase (cgas) and aim2-like receptors (alrs), which include aim2 and human ifn-inducible protein 16 (ifi16), have also emerged as important dna virus prrs. cgas signals via sti ng and aim2 generates inflammasome oligomers, whereas ifi16 can stimulate both signaling mechanisms (diner et al., 2015) . rna-activated rig-i and mda5 colocalize with mavs, inducing its filamentation. it remains unclear why these filaments are potent inducers of downstream signaling; however, rlr cascade proteins including nemo, ikkε, and various trafs possess mavs-targeting signals (paz et al., 2011) . furthermore, tbk1 and other key rlr cascade proteins interact with these proteins but are activated only upon oligomerization. thus, steady state isolation of mavs may represent a spatiotemporal barrier that restrains innate immune signaling, overcome through coordinating these proteins into signaling complexes upon mavs multimerization. in this way, it is conceivable how immunomodulating e3s/dubs may be compartmentalized together with their substrates. ubiquitin stringently regulates the mavs signalosome. the central position that mavs occupies within the rlr cascade is commensurate with the many ptms that modulate its role. to our knowledge, mavs ubiquitination has not been observed in resting cells using a variety of proteomic and biochemical approaches, indicating that mavs ubiquitination occurs specifically during viral infection. at least seven e3s ubiquitinate mavs, leading to mavs degradation in almost every case, as described later in this section (fig. 3) . at least five of these modify other substrates within the same cascade, highlighting mavs as a crucial locus of rlr regulation. accordingly, mavs is targeted by numerous viruses in a variety of ways; however, with the exception of hbv and severe acute respiratory syndrome-coronavirus (sars-cov; fig. 3 ; wei et al., 2010; shi et al., 2014) , this is usually achieved by means other than manipulating mavs ubiquitination, likely given the extensive ubiquitin-mediated negative regulatory systems already in place. mavs aggregation is a key feature of rlr cascade activation, but how these aggregates are resolved during deactivation is only beginning to be clarified. in addition to ubiquitinating rig-i and enhancing its association with mavs, trim25 ubiquitinates mavs at lys7 and lys10 and induces its partial proteolysis (castanier et al., 2012) . this was proposed as a means of discharging the activated rlr signalosome from the mitochondrial recruitment platform and would begin to address how irf3 and other rlr signalosome components traffic correctly after activation. more recently, lys7 and lys500 were shown to be polyubiquitinated by membrane-associated ring finger protein 5 (mar ch5), a mitochondrial membrane-bound e3 that effectively dissolves mavs aggregates by specifically targeting them for degradation. mar ch5 is an important regulator of mitochondrial fission and fusion whose expression is up-regulated during infection (yoo et al., 2015) . these mechanisms of mavs aggregate resolution may be nonredundant, with the trim25 mechanism occurring throughout the immune response and the mar ch5 mechanism amplifying gradually in an ifn-i negative feedback loop. complete mavs degradation is independently promoted by the e3s rnf5, rnf125, atrophin-1-interacting protein 4 (aip4; also termed itch), smad ubiquitination regulatory factor 1 (smurf1), and smurf2 (fig. 3) . rnf5 polyubiquitinates mavs at lys362 and lys461, whereas the adjacent residues lys371 and lys420 are polyubiquitinated by aip4 upon recruitment by poly(rc)-binding protein 1 (pcbp1) or pcbp2 (you et al., 2009; zhong et al., 2010; zhou et al., 2012) . aip4 additionally inhibits ifn-i as well as nf-κb activation by ubiquitinating the inhibitor of apoptosis protein 1 (ciap1), targeting it for lysosomal degradation (tigno-aranjuez et al., 2013) . ciap1 is an e3 that activates traf3/6 during viral infection (mao et al., 2010) , revealing that aip4 broadly and multiply inhibits nlr-, rlr-, and tlr-mediated immune signaling. the acceptor site or sites for rnf125-induced mavs ubiquitination are unknown; however, given that rnf125 also ubiquitinates the activated cards of rig-i and mda5 (arimoto et al., 2007) , the mavs card appears a likely candidate. nedd4 family-interacting protein 1 (ndfip1) binds mavs and recruits smurf1 and possibly smurf2, facilitating ubiquitination of unknown sites within mavs (wang et al., 2012c; pan et al., 2014) . moreover, numerous trafs, including traf3 and traf6, interact with mavs, and smurf1 also targets these for degradation (li et al., 2010a) . finally, lys500 was reported as a single site of ifn-i-activating polyubiquitination by an unknown e3, inhibiting nf-κb activation by sequestering ikkε (paz et al., 2009 ). the trafs are six multifunctional adaptor proteins that regulate both nf-κb activation and ifn-i expression via the rlr, nlr, and tlr protein signaling cascades (fig. 2) . traf-mediated signaling outcomes are augmented by ubiquitin, and, excepting traf1, all trafs possess a ring finger domain and multiple zinc coordination sites, features typical of ubiquitin e3s. k63-linked autoubiquitination at lys124 is a key activation mechanism of traf6 (lamothe et al., 2007; jiao et al., 2015) and possibly traf2 (habelhah et al., 2004) , traf4 (marinis et al., 2012) , and traf5 (zhong et al., 2012) . in vitro traf3 ubiquitination assays and analysis of recombinant traf3δri ng isolated from mammalian cell lysates are also consistent with an autoubiquitination activation mechanism for traf3 (kayagaki et al., 2007; zeng et al., 2009) . traf3 and traf6 are among the first molecules activated by mavs in the rlr pathway (fig. 2, middle) . furthermore, there is increasing evidence of ubiquitin-mediated cross-talk between trafs. traf3 promotes ifn-i expression by activating tbk1/irf3 (parvatiyar et al., 2010) , whereas traf6 activates mitogen-activated protein kinase kinase kinase 1 (mekk1) to activate nf-κb, which also enhances ifn-i expression (yoshida et al., 2008) . simultaneously, traf3 suppresses nf-κb by inhibiting ikk activation upon binding traf2 (zarnegar et al., 2008) , likely as a mecha-nism to skew innate immune effector molecule expression as required. inversely, the e3 ciap2, after itself being ubiquitinated by traf6, promotes traf3 degradation by ligating k48-linked polyubiquitin chains to traf3 at residues k107 and k156, thereby restoring nf-κb activation (tseng et al., 2010) . however, as well as degrading traf3, ciap1/2 can also activate traf3 by catalyzing its k63-linked polyubiquitination ( fig. 3 ; mao et al., 2010) . this suggests that the context-dependent ubiquitination state of ciap1/2 determines its effect on traf3. the e3 rnf166 was recently reported to ubiquitinate and activate both traf3 and traf6 . finally, the rig-i-activating e3 trim25 was reported to enhance mda5-mediated nf-κb activation at the level of traf6 , although mechanistic details remain unclear. traf-mediated signaling is also terminated by ubiquitin in numerous ways. hsv encodes the dub ul36usp, which strips k63-linked polyubiquitin chains from traf3 to prevent downstream protein recruitment ( fig. 3 ; wang et al., 2013b) , possibly antagonizing ciap1/2-mediated ubiquitination. traf3 and traf6 are both deactivated by the dubs otubain 1 (otub1) and otub2, which remove k63-linked polyubiquitin chains (li et al., 2010b) . traf3 is further deactivated by the deubiquitinase duba, which removes k63-linked polyubiquitin chains from traf3 (kayagaki et al., 2007) . furthermore, the e3 triad3a redirects traf3 to the proteasome by ligating k48-linked polyubiquitin chains (nakhaei et al., 2009 ). altogether, this constitutes a ubiquitin-dependent feedback mechanism that enables trafs to dictate the direction of immune signal transmission in a context-dependent manner. in contrast to the three rlr receptors, the 22 nlrs have diverse expression patterns and largely under-characterized functions. the nlrs are well recognized for their roles in regulating nf-κb activation and antibacterial immunity; however, at least five members have emerging roles in antiviral immune signaling: nod1, nod2, nlrc5 (nlr family card domain-containing protein 5), nlrp4 (nac ht, lrr, and pyd domain-containing protein 4), and nlrx1 (nlr family member x1; fig. 2, right) . although nlrs recruit e3s and modulate the ubiquitination of other proteins, including several in the rlr cascade, the role of ptms in nlr regulation remains under-defined. nlr regulation and innate immune signaling cross-talk. the prrs nod1 and nod2 are the best characterized nlrs. nod1 is expressed ubiquitously, whereas nod2 is expressed mainly in cells of myeloid and lymphoid origin and is up-regulated during bacterial and viral infection. the classic nod2-activating ligand is bacterial muramyl dipeptide (mdp), which promotes nf-κb activation. however, nod2 also promotes ifn-i expression during infection by numerous rna viruses, in part through recognizing single-stranded rna (ssrna) and interacting with mavs. nod2 may also promote ifn-i expression during infection by particular dna viruses by an undefined mechanism (sabbah et al., 2009; kapoor et al., 2014) . accordingly, nod2 dysfunction leads to inefficient innate and adaptive immune responses to viral infection (lupfer et al., 2014) . nod2 features regularly in the immune signaling landscape, yet mechanisms of nod2 regulation and cross-talk are only beginning to be revealed. upon activation by mdp, nod2 is ubiquitinated by trim27, leading to nod2 degradation and nf-κb inhibition (zurek et al., 2012) . nod2 signaling is further suppressed by aip4, which ubiquitinates lys209 of receptor-interacting serine/threonine protein kinase 2 (ripk2), the immediate downstream interacting partner of nod2 (fig. 2, right; tao et al., 2009) . conversely, nod2-driven nf-κb activation is enhanced by lub ac, a negative regulator of rlr signaling, as well as x-linked iap (xiap), which respectively ligate m1-and k63-linked polyubiquitin chains to nod2 and ripk2 (damgaard et al., 2012) . these activating ubiquitin chains may be antagonized by the ubiquitin-editing enzyme a20 , which also disrupts ubiquitin-mediated tbk1 activation in the rlr signaling cascade as well as ubiquitin-mediated traf6 activation in the tlr cascade parvatiyar et al., 2010) . another mitochondrial link between the nlr and rlr signaling pathways is the ubiquitously expressed nlrx1 (fig. 2, right) , whose role in ifn-i regulation is controversial. nlrx1 is localized to the mitochondrial outer membrane and was reported to inhibit mavs-dependent ifn-i signaling by blocking the interaction between activated rig-i/mda5 and mavs, although viral replication experiments using gene knockout cells have produced conflicting results (soares et al., 2013) . nlrx1 also potentiates nf-κb signaling by promoting reactive oxygen species production during bacterial infection, linking the mitochondrial immune signaling platform with proinflammatory cytokine generation (tattoli et al., 2008) . nlrc5 was initially described to enhance ifn-γ and ifn-α expression and inhibit nf-κb and ifn-β, in the latter case through sequestering the activated effector domains of rig-i and mda5 (cui et al., 2010; kuenzel et al., 2010) . nlrc5 has also been shown to bind and inhibit tbk1mediated ifn-β induction in hek-293t cells, although nlrc5 −/− mice show relatively normal cytokine responses upon exposure to rlr-, tlr-, and nlr-activating stimuli (kumar et al., 2011) . still other findings indicate that the rig -i :nlrc5 interaction also positively regulates ifn-β expression, and this interaction is targeted by the iav-ns1 protein (fig. 2, right; neerincx et al., 2010; ranjan et al., 2015) . these disparate conclusions may reflect cell-specific differences given that nlrc5 is predominantly expressed in hematopoietic cells or differences between mouse and human signaling pathways, suggesting that the nlrc5 regulatory framework is complex. adding to this, ubiquitination plays an uncharacterized role in regulating nlrc5 upon lps stimulation and may be induced by nlrc5 overexpression (cui et al., 2010; kuenzel et al., 2010) . given the diversity of interactions that nlrc5 takes part in, it is likely that further ptms will be shown to regulate nlrc5 during viral infection. nlrp4 has gained prominence as another negative regulator of multiple immune signaling pathways that is more widely expressed than nlrc5. nlrp4 was initially described to inhibit ikkα-mediated nf-κb activation (fiorentino et al., 2002) . upon rlr cascade activation, nlrp4 also inhibits irf3 activation by recruiting the e3 deltex-4 (dtx4) to ubiquitinate and degrade tbk1 (cui et al., 2012) , revealing yet another route for rlr/nlr cross-talk (fig. 2, right) . tlrs are differentially expressed in a wide range of cell populations. tlr3, tlr7, tlr8, and tlr9 are expressed in endosomal vesicles, whereas tlr2 and tlr4 are expressed on the cell surface. tlr3 recognizes double-stranded rna, activating nf-κb-mediated proinflammatory cytokine production and strongly up-regulating tbk1/irf3-dependent ifn-i expression. tlr7 and tlr8 recognize ssrna, up-regulating ifn-α and proinflammatory cytokine production. tlr9 recognizes unmethylated cytosine-phosphate-guanine (cpg) dna, a common feature of nonmammalian genomes, and stimulates ifn-α production. tlr2 and tlr4 are activated by a variety of microbial ligands, including specific viral proteins, resulting in proinflammatory cytokine expression (fig. 2, left) . nf-κb activation and ifn-i up-regulation. tlr2, tlr4, tlr7, tlr8, and tlr9 signaling is mediated through the adaptor protein myeloid differentiation primary response gene 88 (myd88; fig. 2 , left). myd88 recruits nf-κb and ifn-i signaling components, including interleukin-1 receptor-associated kinase 1 (irak1), irak4, traf6, and irf7. activated traf6 ubiquitinates irf7, leading to ifn-α expression (kawai et al., 2004) . traf6 also promotes k63linked polyubiquitination of nemo, enabling recruitment of the tgf-β-activated kinase (tab)-tak1 kinase complex (tseng et al., 2010) . subsequent association between nemo and m1-polyubiquitin chains induces tak1-mediated phosphorylation of ikkα and ikkβ, priming them for full transactivation through autophosphorylation . activated ikkα phosphorylates the iκbα subunit, leading to its ubiquitination and proteasomal degradation and releasing nf-κb for nuclear translocation. furthermore, myd88, irak1/4, and tab2/3 are also modified and activated by k63-linked polyubiquitin chains. such chains were recently described as substrates for m1-polyubiquitination by hoip, resulting in hybrid chains that may connect the myd88/ irak and tak1/ikk signaling apparatus (emmerich et al., 2013) . tlr3 signaling is mediated by tir domain-containing adaptor-inducing ifn-β (trif). trif activates traf3, which promotes irf3/irf7 activation (tseng et al., 2010) , and also traf6, which promotes ikk activation (fig. 2, left; jiang et al., 2004) . ubiquitin regulates the myd88-and trif-dependent pathways. although tlrs undergo extensive ptm, ubiquitination performs no known role in regulating tlrs directly. instead, ubiquitination modulates their downstream signaling targets, particularly myd88, trif, and traf6, and it is often here that viruses terminate tlr-mediated immunity. nrdp1 is an e3 that promotes ifn-i expression at the expense of proinflammatory cytokines. tbk1 polyubiquitination by nrdp1 activates tbk1 in trif-mediated ifn-i expression, which simultaneously k48-polyubiquitinates and down-regulates myd88-mediated nf-κb activation . conversely, the ubiquitin-editing enzyme a20 inhibits ubiquitin-mediated activation of traf6, inhibiting nf-κb activation via the tlr and nlr cascades, as well as tbk1, inhibiting ifn-i expression turer et al., 2008; parvatiyar et al., 2010) . additional avenues of signaling cross-talk include smurf1 and smurf2, which degrade mavs and inhibit ifn-i activation. smurf1 and smurf2 also degrade myd88, inhibiting tlr-mediated nf-κb activation (lee et al., 2011) . ciap2 is an e3 that, after itself being ubiquitinated by traf6, targets traf3 for degradation (tseng et al., 2010) . this is important in promoting tlr4-mediated signaling and cytokine production at the expense of type i ifn production . furthermore, traf6 itself promotes proinflammatory cytokine production at the expense of ifn-i. traf6 is activated by trans-autoubiquitination at k124, abolition of which eliminates nemo ubiquitination and tak1 activation (lamothe et al., 2007) . this mechanism is exploited by hsv, which uses the virally encoded e3 infected cell polypeptide 0 (icp0) to recruit usp7 to deubiquitinate nemo and traf6 (daubeuf et al., 2009 ). in addition, icp0 directly catalyzes ubiquitination and degradation of myd88 and tir ap (toll/interleukin-1 receptor domain-containing adaptor protein; van lint et al., 2010) . kaposi's sarcoma-associated herpesvirus (kshv) encodes replication and transcription factor (rta), an e3 that activates latent virus. this activation process involves suppression of antiviral cytokines, partly involving rta-catalyzed ubiquitination and degradation of myd88 and trif (ahmad et al., 2011; zhao et al., 2015) , thereby impairing all tlr-mediated immune signaling pathways. the final relay: irfs transmit danger signals into the nucleus tlr, nlr, and rlr ifn-i signaling converges at irf activation, the penultimate step toward ifn-i transcription. irf3 is constitutively expressed in most cell types, residing inactive in the cytosol until phosphorylation by tbk1/ikkε within two activation clusters (ser385/ser386 and ser396/ ser398/ser402/thr404/ser405), resulting in homodimerization, nuclear accumulation, dna binding, and participation in ifnb gene transcription (lin et al., 1998) . ifn-β acts in an autocrine and paracrine manner upon its cognate receptor, ifn-α/β receptor (ifn ar), thereby activating jak/stat signaling and isg expression. irf7 expression is up-regulated in this way, which in turn activates ifna transcription and additional isg expression by a similar mechanism. ubiquitin is an irf master toggle. the irfs are among the most tightly controlled ifn-i signaling proteins through an interplay of ptms, including phosphorylation, ubiquitination, and ubiquitin-like modifications. phosphorylation of irf7 at ser477 and ser479 by tbk1/ikkε is required for its activation (tenoever et al., 2004) . however, ubiquitination by traf6 at nearby residues lys444, lys446, and lys452 appears to be a prerequisite to this and serves as a link between the nf-κb and ifn-i activation pathways (ning et al., 2008) . furthermore, trim28 binds active irf7 and ligates small ubiquitin-like modifier (sumo) at two of these ubiquitination sites, lys444 and lys446, negatively regulating virus-triggered ifn-α production (liang et al., 2011) , indicating that as yet unidentified dubs or desumoylating enzymes participate in regulating irf7. reminiscent of irf7, irf3 residues lys70 and lys87 accept both polyubiquitin chains and sumo, and competition between these modifications can determine the fate of ifn-i signal transduction. at steady state, the sumo-conjugating enzyme ubiquitin carrier protein 9 (ubc9) protects irf3 from ubiquitin-mediated degradation by occupying these sites with sumo. alternatively, the desumoylating enzyme sentrin-specific protease 2 (senp2) removes sumo from irf3, enabling its k48-linked polyubiquitination (ran et al., 2011) . subsequent work identified trim26 as an e3 that conjugates k48-linked polyubiquitin chains to these same sites ( fig. 3 ; wang et al., 2015) , triggering degradation of the active, nuclear-localized form of irf3. furthermore, activated irf3 undergoes phosphorylation at ser339. this promotes interaction with peptidyl-prolyl cis/ trans isomerase nima-interacting 1 (pin1), a nuclear-localized protein that promotes irf3 degradation (saitoh et al., 2006) . the e3 recruited by pin1 for this purpose is unknown; however, trim26 is also localized to the nucleus and seems a strong candidate. irf3 degradation is undesirable at early stages of the innate immune response and is limited in several ways. the irf3-pin1 interaction is inhibited by the hect (homologous to the e6-ap c terminus) domain and rcc1-like domain-containing protein 5 (herc5), which ligates another ubiquitin-like protein, isg15, onto irf3 at lys193, lys360, and lys366, thereby sustaining irf3 activation (shi et al., 2010) . trim21 is a ubiquitin e3 described to both inhibit the irf3-pin1 interaction and target irf3 for proteasomal degradation (higgs et al., 2008; yang et al., 2009) . trim21 reportedly also targets irf7 for degradation upon tlr7 or tlr9 activation (higgs et al., 2010) , although the trim21-dependent irf3/ifr7 ubiquitin acceptor sites remain undefined (fig. 3) . jem vol. 213, no. 1 several additional e3s regulate irf abundance. the skp-cullin-f-box (scf)-containing complex, of which cullin1 (cul1) is a core component, catalyzes irf3 degradation as well as iκb degradation, promoting nf-κb activation ( fig. 3 ; bibeau-poirier et al., 2006) . ranbp-type and c3hc4-type zinc finger-containing protein 1 (rbck1) catalyzes k48-linked polyubiquitination and degradation of irf3 during viral infection ( fig. 3 ; zhang et al., 2008) . finally, the forkhead box protein o1 (foxo1), a regulator of insulin signaling, binds irf3 and promotes its degradation by recruiting an unknown e3. foxo1 also negatively regulates irf7 transcription (lei et al., 2013) , altogether implying a link between metabolism and innate immune induction. expression of cul1 and the e3s rbck1, trim21, trim26, and herc5 is ifn-i inducible (henig et al., 2013) , constituting a multiply redundant negative feedback web in which ifn-i expression is self-restraining. not so fast: seizing the penultimate step toward antiviral gene transcription. irfs are a significant target of viral disruption, usually resulting in their proteasome-mediated degradation. rotavirus (rov) nonstructural protein 1 blocks nf-κb signaling and usurps the ubiquitin modification system to redirect irf3/5/7/9 to the proteasome in a strain-specific manner ( fig. 3 ; morelli et al., 2015) . cells produce trace quantities of ifn-i at rest through basal activation of endogenous irf3/irf7, the intracellular concentration of which are regulated by the e3 rta-associated ubiquitin ligase (raul). kshv exploits this mechanism to diminish immune signaling, recruiting usp7 to deubiquitinate raul and thereby maintain raul-mediated irf3/irf7 degradation (yu and hayward, 2010) . the raul-dependent ubiquitin acceptor sites on irf3/irf7 remain unknown (fig. 3) , but better characterization of the raul-irf interaction may have implications for antiviral and autoimmunity treatments. furthermore, the kshv rta protein catalyzes polyubiquitination and degradation of irf7 and myd88 (yu et al., 2005) . thus, kshv effectively terminates several signaling pathways at multiple stages. similar to kshv, hiv infection fails to stimulate activation of irf3, endogenous levels of which are quickly reduced upon infection. underscoring the importance for hiv to disrupt early ifn-i-mediated immunity, irf3 degradation is independently promoted by two viral accessory proteins, viral infectivity factor (vif) and viral protein r (vpr). the e3s hijacked for this purpose are unknown (fig. 3) , although vif and vpr recruit scf-related components to degrade other antiviral proteins (okumura et al., 2008) . the innate immune signaling architecture is complex and has coevolved with the pathogens it guards against, meanwhile restraining autoimmunity through an elaborate negative feedback scheme. a cornerstone of this dynamic regulatory framework is the ubiquitin modification system, which is manipulated by viruses relevant to human disease. going forward in understanding mechanisms of infection and autoimmunity, we must address significant gaps in knowledge regarding the specificity and context-dependent regulation of e3s and dubs and the consequences of ubiquitin modification. this will expose further cross-talk between the immune signaling cascades, revealing a functional and self-regulating whole. in the search for a new generation of antiviral and autoimmune treatments, we continue to learn from the pathogens that have long adapted to exploit this ready-made system of functional regulation; humans possess hundreds of specific-and general-effect e3s and dubs, many of which could be harnessed for therapeutic use. kaposi sarcoma-associated herpesvirus degrades cellular toll-interleukin-1 receptor domain-containing adaptor-inducing β-interferon (trif) negative regulation of the rig-i signaling by the ubiquitin ligase rnf125 influenza a virus uses the aggresome processing machinery for host cell entry involvement of the iκb kinase (ikk)-related kinases tank-binding kinase 1/ikki and cullin-based ubiquitin ligases in ifn regulatory factor-3 degradation card games in apoptosis and immunity the innate immune sensor lgp2 activates antiviral signaling by regulating mda5-rna interaction and filament assembly mavs ubiquitination by the e3 ligase trim25 and degradation by the proteasome is involved in type i interferon production after activation of the antiviral rig-i-like receptors ring finger protein 166 potentiates rna virus-induced interferon-β production via enhancing the ubiquitination of traf3 and traf6 activation of the canonical ikk complex by k63/m1-linked hybrid ubiquitin chains a novel paad-containing protein that modulates nf-κb induction by cytokines tumor necrosis factor-α and interleukin-1β the tumour suppressor cyld is a negative regulator of rig-imediated antiviral response trim25 ring-finger e3 ubiquitin ligase is essential for rig-i-mediated antiviral activity influenza a virus ns1 targets the ubiquitin ligase trim25 to evade recognition by the host viral rna sensor rig-i reul is a novel e3 ubiquitin ligase and stimulator of retinoic-acid-inducible gene-i functional dissection of the tbk1 molecular network modulation of the interferon antiviral response by the tbk1/ikki adaptor protein tank ubiquitination and translocation of traf2 is required for activation of jnk but not of p38 or nf-κb interferon-beta induces distinct gene expression response patterns in human monocytes versus t cells the e3 ubiquitin ligase ro52 negatively regulates ifn-β production post-pathogen recognition by polyubiquitin-mediated degradation of irf3 self protection from anti-viral responses-ro52 promotes degradation of the transcription factor irf7 downstream of the viral toll-like receptors the ubiquitinediting enzyme a20 restricts nucleotide-binding oligomerization domain containing 2-triggered signals linear ubiquitin assembly complex negatively regulates rig-iand trim25-mediated type i interferon induction inhibition of rig-i-mediated signaling by kaposi's sarcoma-associated herpesvirus-encoded deubiquitinase orf64 sti ng is an endoplasmic reticulum adaptor that facilitates innate immune signalling toll-like receptor 3-mediated activation of nf-κb and irf3 diverges at toll-il-1 receptor domaincontaining adapter inducing ifn-β the kinase mst4 limits inflammatory responses through direct phosphorylation of the adaptor traf6 activation of nucleotide oligomerization domain 2 (nod2) by human cytomegalovirus initiates innate immune responses and restricts virus replication interferon-α induction through toll-like receptors involves a direct interaction of irf7 with myd88 and traf6 duba: a deubiquitinase that regulates type i interferon production the nucleotidebinding oligomerization domain-like receptor nlrc5 is involved in ifn-dependent antiviral immune responses nlrc5 deficiency does not influence cytokine induction by virus and bacteria infections site-specific lys-63-linked tumor necrosis factor receptorassociated factor 6 auto-ubiquitination is a critical determinant of iκb kinase activation regulation of mda5-mavs antiviral signaling axis by trim25 through traf6-mediated nf-κb activation smad6-specific recruitment of smurf e3 ligases mediates tgf-β1-induced degradation of myd88 in tlr4 signalling foxo1 negatively regulates cellular antiviral response by promoting degradation of irf3 ubiquitin ligase smurf1 targets traf family proteins for ubiquitination and degradation regulation of virus-triggered signaling by otub1-and otub2-mediated deubiquitination of traf3 and traf6 genome-wide and functional annotation of human e3 ubiquitin ligases identifies mul an, a mitochondrial e3 that regulates the organelle's dynamics and signaling tripartite motif-containing protein 28 is a small ubiquitin-related modifier e3 ligase and negative regulator of ifn regulatory factor 7 virus-dependent phosphorylation of the irf-3 transcription factor regulates nuclear translocation, transactivation potential, and proteasome-mediated degradation nucleotide oligomerization and binding domain 2-dependent dendritic cell activation is necessary for innate immunity and optimal cd8 + t cell responses to influenza a virus infection virus-triggered ubiquitination of traf3/6 by ciap1/2 is essential for induction of interferon-β (ifn-β) and cellular antiviral response iκb kinase α phosphorylation of traf4 downregulates innate immune signaling putative e3 ubiquitin ligase of human rotavirus inhibits nf-κb activation by using molecular mimicry to target β-trcp the e3 ubiquitin ligase triad3a negatively regulates the rig-i/mavs signaling pathway by targeting traf3 for degradation a role for the human nucleotide-binding domain, leucine-rich repeat-containing family member nlrc5 in antiviral responses meta-analysis of patients with hepatitis c virus genotype 6: 48 weeks with pegylated interferon and ribavirin is superior to 24 weeks traf6 and the three c-terminal lysine sites on irf7 are required for its ubiquitination-mediated activation by the tumor necrosis factor receptor family member latent membrane protein 1 a protein-kinase, ifn-inducible doublestranded rna dependent inhibitor and repressor of p58 (prk rir) enhances type i ifn-mediated antiviral response through the stability control of rig-i protein inhibition of transcription of herpes simplex virus immediate early genes in interferon-treated human cells hiv-1 accessory proteins vpr and vif modulate antiviral response by targeting irf-3 for degradation riplet/ rnf135, a ring finger protein, ubiquitinates rig-i to promote interferon-β induction during the early phase of viral infection the ubiquitin ligase riplet is essential for rig-i-dependent innate immune responses to rna virus infection a distinct role of riplet-mediated k63-linked polyubiquitination of the rig-i repressor domain in human antiviral innate immune responses smurf2 negatively modulates rig-i-dependent antiviral response by targeting visa/mavs for ubiquitination and degradation tax1bp1 and a20 inhibit antiviral signaling by targeting tbk1-ikki kinases ubiquitin-regulated recruitment of iκb kinase ε to the mavs interferon signaling adapter a functional c-terminal traf3-binding site in mavs participates in positive and negative regulation of the ifn antiviral response kinetic mechanism for viral dsrna length discrimination ubiquitin in antiviral immunity by mda5 filaments. proc. natl. acad. sci. usa structural basis for ubiquitin-mediated antiviral signal activation by rig-i species-specific inhibition of rig-i ubiquitination and ifn induction by the influenza a virus ns1 protein senp2 negatively regulates cellular antiviral response by desumoylating irf3 and conditioning it for ubiquitination and degradation nlrc5 interacts with rig-i to induce a robust antiviral response against influenza virus infection activation of innate immune antiviral responses by nod2 regulation of innate antiviral defenses through a shared repressor domain in rig-i and lgp2 negative regulation of interferon-regulatory factor 3-dependent innate antiviral response by the prolyl isomerase pin1 master sensors of pathogenic rna -rig-i like receptors recognition of 5′ triphosphate by rig-i helicase requires short blunt double-stranded rna as contained in panhandle of negative-strand virus sars-coronavirus open reading frame-9b suppresses innate immunity by targeting mitochondria and the mavs/ traf3/traf6 signalosome positive regulation of interferon regulatory factor 3 activation by herc5 via isg15 modification nlrx1 does not inhibit mavs-dependent antiviral signalling itch k63-ubiquitinates the nod2 binding protein, rip2, to influence inflammatory signaling pathways nlrx1 is a mitochondrial nod-like receptor that amplifies nf-κb and jnk pathways by inducing reactive oxygen species production activation of tbk1 and ikkε kinases by vesicular stomatitis virus infection and the role of viral ribonucleoprotein in the development of interferon antiviral immunity a discrete ubiquitinmediated network regulates the strength of nod2 signaling different modes of ubiquitination of the adaptor traf3 selectively activate the expression of type i interferons and proinflammatory cytokines homeostatic myd88-dependent signals cause lethal inflammation in the absence of a20 herpes simplex virus immediate-early icp0 protein inhibits toll-like receptor 2-dependent inflammatory responses and nf-κb signaling proteomic profiling of the traf3 interactome network reveals a new role for the er-to-golgi transport compartments in innate immunity the e3 ubiquitin ligase nrdp1 'preferentially' promotes tlr-mediated production of type i interferon nemo binds ubiquitinated tankbinding kinase 1 (tbk1) to regulate innate immune responses to rna viruses usp4 positively regulates rig-i-mediated antiviral response through deubiquitination and stabilization of rig-i trim26 negatively regulates interferon-β production and antiviral response through polyubiquitination and degradation of nuclear irf3 herpes simplex virus 1 ubiquitin-specific protease ul36 inhibits beta interferon production by deubiquitinating traf3 tetraspanin 6 (tsp an6) negatively regulates retinoic acid-inducible gene i-like receptor-mediated immune signaling in a ubiquitinationdependent manner ndfip1 negatively regulates rig-idependent immune signaling by enhancing e3 ligase smurf1-mediated mavs degradation the hepatitis b virus x protein disrupts innate immunity by downregulating mitochondrial antiviral signaling protein trim4 modulates type i interferon induction and cellular antiviral response by targeting rig-i for k63-linked ubiquitination trim21 is essential to sustain ifn regulatory factor 3 activation during antiviral response the mitochondrial ubiquitin ligase mar ch5 resolves mavs aggregates during antiviral signalling traf6 and mekk1 play a pivotal role in the rig-i-like helicase antiviral pathway pcbp2 mediates degradation of the adaptor mavs via the hect ubiquitin ligase aip4 the ubiquitin e3 ligase raul negatively regulates type i interferon through ubiquitination of the transcription factors irf7 and irf3 the kshv immediate-early transcription factor rta encodes ubiquitin e3 ligase activity that targets irf7 for proteosome-mediated degradation control of canonical nf-κb activation through the nik-ikk complex pathway key role of ubc5 and lysine-63 polyubiquitination in viral activation of irf3 an unexpected twist to the activation of ikkβ: tak1 primes ikkβ for activation by autophosphorylation negative feedback regulation of cellular antiviral signaling by rbck1-mediated degradation of irf3 kaposi's sarcoma-associated herpesvirus-encoded replication and transcription activator impairs innate immunity via ubiquitin-mediated degradation of myeloid differentiation factor 88 the nemo adaptor bridges the nuclear factor-κb and interferon regulatory factor signaling pathways the e3 ubiquitin ligase rnf5 targets virus-induced signaling adaptor for ubiquitination and degradation negative regulation of il-17-mediated signaling and inflammation by the ubiquitin-specific protease usp25 ubiquitin-specific protease 25 regulates tlr4-dependent innate immune responses through deubiquitination of the adaptor protein traf3 poly(c)-binding protein 1 (pcbp1) mediates housekeeping degradation of mitochondrial antiviral signaling (mavs) trim27 negatively regulates nod2 by ubiquitination and proteasomal degradation ribose 2′-o-methylation provides a molecular signature for the distinction of self and non-self mrna dependent on the rna sensor mda5 we are grateful to dr. sarah atkinson for critical review of the manuscript. n.a. borg is an australian research council (arc) future fellow (ft110100223). the authors declare no competing financial interests. accepted: 2 december 2015 key: cord-284156-btb4oodz authors: liu, yiliu; olagnier, david; lin, rongtuan title: host and viral modulation of rig-i-mediated antiviral immunity date: 2017-01-03 journal: front immunol doi: 10.3389/fimmu.2016.00662 sha: doc_id: 284156 cord_uid: btb4oodz innate immunity is the first line of defense against invading pathogens. rapid and efficient detection of pathogen-associated molecular patterns via pattern-recognition receptors is essential for the host to mount defensive and protective responses. retinoic acid-inducible gene-i (rig-i) is critical in triggering antiviral and inflammatory responses for the control of viral replication in response to cytoplasmic virus-specific rna structures. upon viral rna recognition, rig-i recruits the mitochondrial adaptor protein mitochondrial antiviral signaling protein, which leads to a signaling cascade that coordinates the induction of type i interferons (ifns), as well as a large variety of antiviral interferon-stimulated genes. the rig-i activation is tightly regulated via various posttranslational modifications for the prevention of aberrant innate immune signaling. by contrast, viruses have evolved mechanisms of evasion, such as sequestrating viral structures from rig-i detections and targeting receptor or signaling molecules for degradation. these virus–host interactions have broadened our understanding of viral pathogenesis and provided insights into the function of the rig-i pathway. in this review, we summarize the recent advances regarding rig-i pathogen recognition and signaling transduction, cell-intrinsic control of rig-i activation, and the viral antagonism of rig-i signaling. to ensure successful antiviral defenses and to avoid aberrant or dysregulation of host immune signaling, antiviral pathways need to be tightly regulated at each level. in this review, we will summarize the cell-intrinsic regulation of rig-i receptor activity, as well as the viral strategies to subvert the rig-i signaling machinery. the three members of the rlr family: rig-i, mda5 (melanoma differentiation factor 5), and lgp2 (laboratory of genetics and physiology 2) are expressed in most cell and tissue types. they function as cytoplasmic sensors for the recognition of a variety of rna viruses and subsequent activation of downstream signaling to drive type i ifn production and antiviral gene expressions. these three rlr proteins are rna-dependent atpases belonging to the dexd/h-box family of helicases (11) . structurally, rlrs have a similar central helicase core that is comprised of two helicase domains, hel1 and hel2 with an insertion termed hel2i. in addition, they all have a c-terminal domain (ctd). however, only rig-i and mda5 contain two n-terminal caspase activation and recruitment domains (cards) (3) (figure 1a ). among these three, rig-i is the founding member and hence the most intensively studied member of this family. each domain of rig-i plays unique roles during rig-i autorepression and activation. in brief, the ctd and helicase domain are involved in rna ligand binding and atp hydrolysis-involved conformational changes (12) (13) (14) , whereas the rig-i cards facilitate interaction with other downstream card containing molecules (15) . retinoic acid-inducible gene-i has been shown to be involved in the recognition of a variety of rna viruses in the cytoplasm, such as the sendai virus, influenza a and b viruses (iav, ibv), vesicular stomatitis virus, measles virus (mv), newcastle disease virus, ebola virus (ebov), dengue virus (denv), and hepatitis c virus (hcv) (16) (17) (18) (19) . the short double-stranded (ds) rna with a triphosphate (ppp) motif at the 5′-end, as found in these viral genomes, were shown to be a key signature recognized by rig-i (20, 21) . the 5′ppp dsrna of viral nucleocapsids has also been characterized as stimulating rig-i (22) . 5′-diphosphate-bearing rna (5′pprna), either naturally contained in viruses, produced by in vitro transcription, or via chemical synthesis, were all shown to bind to rig-i and were sufficient to activate rig-i (20, 23) . physiologically, the control of in vitro and in vivo infections of reoviruses, which bear the 5′pprna genome, relies on rig-i functionality (24) . it is worth noting that the in vitro-synthesized 5′ppprna sequences also trigger rig-i activation (25) . these agonists have demonstrated their therapeutic potential as broadspectrum antiviral agents and could be optimized as vaccine adjuvant candidates (26) (27) (28) (29) (30) . furthermore, the recognition of several dna viruses, including herpes simplex virus type 1 (hsv-1), epstein-barr virus (ebv), vaccinia virus (vacv), and adenovirus, via the rna polymerase iii were found to be rig-idependent (31, 32) . interestingly, the rig-i-mediated upregulation of sting is required for protection against the hsv-1 by the rig-i agonist, offering new evidence of the overlapping between rig-i signaling and the host response to dna viral infection (33) . notably, viral rna triggered rig-i signaling also mediates the inflammatory response via distinct pathways. the first involves the formation of the rig-i inflammasome through interactions between rig-i, asc, and caspase-1 and the stimulation of il-1β release. the second involves the adaptor proteins card9, bcl-10, mitochondrial antiviral signaling protein (mavs), and the activation of nuclear factor-κb (nf-κb) (34, 35) . upon rna ligand binding, rig-i undergoes a series of conformational changes and posttranslational modifications (ptms) to achieve full activation (further detail below). activated rig-i recruits its downstream adaptor molecule mavs (also known as ips-1, cardif, and visa) through card-card-mediated interactions (36, 37) . the oligomeric rig-i card assembly and the polymeric formation of mavs, together serve as a signaling platform for protein complexes that mediate the bifurcation of signaling into two branches. one branch recruits tumor necrosis factor receptor-associated factors (traf)-2/6 and the receptor-interacting protein 1 to subsequently activate the ikk complex, resulting in nf-κb activation (38) . the other branch signals through traf3 and activates the tank/ikkγ/ikkϵ/tbk1 complex, leading to the phosphorylation and dimerization of interferon regulator factors (irf)-3 and -7 (39, 40) . activated irf3/7 and nf-κb then translocate to the nucleus, together with atf2, c-jun, and the transcription coactivator creb-binding protein/p300, to coordinate the ifn and pro-inflammatory gene expressions (41) . once secreted, ifns bind to specific cell surface receptors and activate the jak-stat pathway. the activated transcription factors stat1, stat2, and irf9 form the interferon-stimulated gene factors (isgf3) complex. isgf3 then translocates to the nucleus and coordinates the transcription of hundreds of isgs including rig-i, thus generating an amplifying loop leading to the accumulation of rig-i during several types of infections (8) (figure 1b ). structural and biochemical studies have demonstrated that the activation of rig-i is a multi-step process and is primarily regulated by conformational changes and ptms. when initially identified as a dsrna sensor, it was hypothesized that rig-i was under negative regulation in physiological conditions. the over expression of the card domain of rig-i alone demonstrated superior signaling activity than full length rig-i in absence of viral pamps (2) . studies by saito et al. showed that the deletion of card was dominant-negative for rig-i signaling. by contrast, the deletion of repressor domain (rd) resulted in constitutive signaling, whereas rd expression alone ablated rig-i signaling actions. together, these findings provided the model of rig-i autoregulation in which the rd is predicted to mask cards for signaling transduction in uninfected cells (42) . the crystal structural analysis further delineated the models of autorepressed and ligand activated states of rig-i, respectively. in a ligand-free state, cards and hel2i interactions hinder dsrna binding and inactivate rig-i (14) . the binding of 5′ppp dsrna to rd leads to a conformational switch of rig-i, which releases the autorepressed cards and exposes the helicase domain for atp binding (14, 43) . atp hydrolysis is essential for rig-i signaling. it enables rig-i to translocate along the dsrna, and further promotes the oligomerization of rig-i cards. these processes assemble rig-i into a filamentous architecture which facilitates the card-card interactions with the mitochondrial mavs, leading to the subsequent signaling transduction for ifn production (44, 45) . importantly, rig-i atpase activity also plays a role in distinguishing self-rna from non-self-rna (46) . it was reported that rig-i atp hydrolysis increases the binding affinity of rig-i and dsrna ligands; whereas the rig-i mutants deficient in atp hydrolysis promotes the interaction of rig-i and self-dsrna and results in unintentional immune signaling (47) . one of the first ptms of rig-i following the initial ligand recognition is performed by the robust ubiquitination machinery (figure 2) . mass spectrometry analysis revealed that trim25, a member of the tripartite motif (trim) protein family possessing e3 ligase activity, induces the covalent lys63-linked ubiquitination of rig-i. mechanistically, the c-terminal spry domain of trim25 interacts with card1 and facilitates the ubiquitination of card2 at k172 (48) . the rig-i-trim25 ubiquitination complex, associates with the adaptor protein 14-3-3ϵ and translocates to mitochondria for mavs binding (49) . mutation figure 2 | regulation of retinoic acid-inducible gene-i (rig-i) activation. (a) in resting cells, rig-i is kept inactivated through the phosphorylation of caspase activation and recruitment domains (cards) and c-terminal domain (ctd) mediated by casein kinase ii and protein kinase c-α/β, respectively. (b) following the binding of 5′ triphosphate (5′ppp) rna and atp hydrolysis, rig-i is dephosphorylated by phosphoprotein phosphatase 1-α/γ and results in a conformational change that opens cards. hdac6-mediated deacetylation of rig-i ctd is critical for rig-i and 5′ppprna binding. the lys63-linked ubiquitination of rig-i mediated by trim25, riplet, oligoadenylate synthetases-like protein, and mex3c at both cards and ctd further activate rig-i and facilitate its tetramerization. (c) interactions between rig-i-trim25 complex and 14-3-3ϵ promote rig-i translocation to mitochondrial mitochondrial antiviral signaling protein (mavs) for downstream signaling, leading to interferon production. interactions between trim25, rig-i, and mavs are further negatively regulated by the lys48-linked ubiquitination, which is meditated by lubac, rnf125, and rnf122. sec14l1 and atg5-atg12 both inhibit the signaling by interrupting rig-i-mavs interactions, whereas sumoylation promotes rig-i-mavs binding. of k172 disrupts the interaction between rig-i and mavs thus abrogating downstream signaling and ifns production (50) . furthermore, a rig-i splice variant which lacks the trim25 interaction domain acts as a feedback inhibitor of rig-i signaling transduction upon viral infections (48) . in addition, riplet (ring-finger protein leading to rig-i activation, also named rnf135 or reul), another e3 ubiquitin ligase, also promotes rig-i ubiquitination. multiple sites within the cards, as well as within the ctd of rig-i, were identified as the crucial ubiquitin anchoring residues (51-53). among which, k63-linked polyubiquitination (pub) at lys788, is demonstrated as being critical for rig-i activation. however, unlike trim25-induced ubiquitination, riplet induced rig-i pub is dispensable for rig-i-rna binding but is essential for releasing card from its autorepressed state. this enhances trim25 functionality as well as promoting the oligomerization of rig-i and the activation of mavs (54) . mex3c (mex-3 rna binding family member c), another recently identified e3 ligase, also mediates lys63-ub at k99 and k169 of card, playing a critical role in rig-i activation (55) . in addition, the oligoadenylate synthetases-like (oasl) protein, although not an e3 ubiquitin ligase itself, contains a dsrna-binding groove and enhances rig-i activation by mimicking the k63-linked pub through its ubiquitin-like (ubl) domain (56, 57) . non-covalent binding of k63-ubiquitin chains to cards also potently activates rig-i (58) . recent structural analysis suggests that covalent and non-covalent binding of ubiquitin synergistically stabilize rig-i tetramerization and enhance polymerization of mavs cards (59) . on the other hand, several deubiquitinating enzymes (dubs) were identified to remove k63-linked pub chains from rig-i, thus dampening rig-i signaling. the tumor suppressor protein cylindromatosis (cyld) removes k63-linked pub chains from rig-i as well as tbk1 and ikkϵ to inhibit the irf3 response, serving as a pathway negative regulator (60) . syndecan-4, a newly identified negative regulator of rig-i, functions through attracting cyld to rig-i complex, thus potentiating the k63-mediated deubiquitination of rig-i (61) . in addition, the ubiquitin-specific protease (usp) family members, such as usp3 and usp21, were also identified as inhibitors of rig-i activation by deubiqutinating rig-i (62, 63) . in contrast to k63-linked ubiquitination, which promotes protein activation, k48-linked ubiquitination triggers proteasomal degradation of its target. for instance, the ring-finger protein 125 (rnf125), together with the ubiquitin e2 ligase ubch5, conjugate k48-linked ubiquitin to rig-i and mavs, targeting them for proteasomal degradation and thereby inhibiting downstream signaling (64) . similarly, rnf122 was recently demonstrated to mediate the proteasomal degradation of rig-i by delivering the k48-linked ubiquitin to rig-i cards (65) . the linear ubiquitin assembly complex (lubac) has been shown to promote k48 pub of trim25, leading to its degradation (66). conversely, the deubiquitinase usp15 antagonizes lubac by removing k48linked ubiquitin from trim25, leading to its stabilization and thereby promoting rig-i-mediated antiviral signaling (67) . in parallel with ubiquitination, phosphorylation has emerged in the past several years as a critical regulator of the rig-i signaling transduction (figure 2) . protein purification and mass spectrometry analysis identified that phosphorylation of thr170 in the cards antagonizes rig-i signaling by inhibiting trim25-mediated lys172 ubiquitination and mavs binding (68) . ser8 phosphorylation of cards also serves as a negative regulator of rig-i (69). in addition, the ctd of rig-i is constitutively phosphorylated at thr770 and ser854/855 by casein kinase ii to promote intermolecular interactions between ctd and cards, thereby maintaining rig-i at an autorepressive state to prevent premature downstream signaling (70) . a recent mass spectrometry analysis revealed that ikk phosphorylates rig-i at ser855, thereby providing a negative feedback regulation of rig-i (71). furthermore, conventional protein kinase c-α (pkc-α) and pkc-β have also been shown to phosphorylate cards, thus suppressing rig-i-trim interaction and subsequent antiviral responses (72) . in fact, rig-i signaling activity is controlled by a dynamic balance between phosphorylation and dephosphorylation. dephosphorylation of rig-i occurs rapidly with the presence of viral rna. a functional sirna screen identified phosphoprotein phosphatase 1-α (pp1α) and pp1γ as essential phosphatases responsible for cards dephosphorylation at ser8 and thr170, leading to rig-i signal activation and viral inhibition (73) . in addition to the ubiquitination and phosphorylation described above, acetylation modulation has recently started to gain more acknowledgment for controlling rig-i activity (figure 2) . mass spectrometry has identified the acetylation of two lysine residues (k858 and k909) in the ctd of rig-i at its inactivate state and are deacetylated during viral infection (74) . the mutation of these two sites restricts rig-i from undergoing the virusinduced interaction with mavs. k858 and k909 acetylation of rig-i has also been shown to control the pamp rna-induced rig-i oligomerization (75) . the cytoplasmic deacetylase hdac6-mediated removal of k909 acetylation has been shown as critical for rig-i binding to dsrna during viral infections (76) . furthermore, hdac6-dependent rig-i deacetylation also regulates rig-i oligomerization upon ligand binding, thus facilitating rig-i activation (75) . rig-i signal transduction is further regulated by additional ptms, regulatory proteins, and other cellular processes (figure 2) . it is worth noting that a number of ubl proteins including sumo, isg15, fat10, and atg8-atg12 are involved in these positive or negative regulatory mechanisms (77) . sumoylation serves as a positive regulator of rig-i by enhancing the rig-i and mavs binding (78) . on the contrary, the hla-f adjacent transcription 10 (fat10), an ubl modifier protein, was shown to negatively regulate rig-i by modulating rig-i solubility through a non-covalent association with cards (79) . in addition, ifn-induced isg15 negatively regulates the rig-i mediated signaling in a feedback-loop control manner (80) . sec14l1 has been observed competing with mavs for rig-i card binding (81) . furthermore, autophagy has been reported to be involved in rig-i modulation through its key regulator, the atg5-atg12 conjugate. atg5-atg12 has been found to suppress rig-i-mavs interaction, thereby inhibiting downstream signaling (82) . recently, deamidation of ctd has been described as a distinct means to induce rig-i activation. for examples, vgat (glutamine amidotransferase), from kshv (kaposi's sarcomaassociated herpesvirus) and γhv68 (murine gamma herpesvirus 68), recruits cellular phosphoribosylformyglycinamide synthase to deamindate and activate rig-i (83, 84). in order to establish infections, viruses have developed sophisticated mechanisms to counteract host immune responses. with regard to rig-i signaling, these include mechanisms such as altering viral genomes and their intermediate transcripts to avoid detection, manipulating the activation and degradation of rig-i and mavs, as well as modulating downstream signaling cascades. studying these antagonistic viral strategies has greatly broadened our understanding of rig-i activation and regulation. since 5′ triphosphate (5′ppp) is an important feature recognized by rig-i, modification of this motif has long been described as one of the major mechanisms for viruses to antagonize rig-i signaling. crimean-congo hemorrhagic fever virus, borna disease virus (bdv), and hantavirus (htnv) remove the 5′ppp group on their genome posttranscriptionally, make rig-i unable to bind to viral rna, and therefore incapable of triggering rig-i activation (85) . mechanistically, htnv uses the "prime and realign" strategy to generate a 5′-terminal monophosphorylate (86, 87) . bdv on the other hand, employs genome trimming to form a 3′-terminal overhang as well as convert 5′ppp to 5′p to avoid detection by rig-i (88) . the arenavirus presents an unpaired 5′ppp-nucleotide overhang to evade recognition by rig-i (89) . the 5′-end of viral rna can also be modified through rna-capping pathways. for example, the genomic rna of polioviruses linked to vpg (viral protein genome-linked) to cap the 5′-end from exposure to rig-i (90). the 5′-end capping with 7-methyl guanosine and methylation of 5′ppp dsrna at the 2′-o position makes viral rna non-distinguishable from the host mrnas, and therefore does not stimulate rig-i (91, 92) . by contrast, some viruses encode viral proteins to prevent rna recognition. the ebov utilizes its vp35 protein to sequester viral rna (18) . the crystal structural analysis indicates that the vp35 interferon inhibitory domain competes with rig-i for dsrna binding by forming an "end-cap" complex with dsrna, resulting in substantially diminished activation of rig-i (93) . similarly, the marburg virus vp35 spirals around the dsrna backbone and end-caps the dsrna to escape from rig-i detection (94, 95) . the iav non-structural protein 1 (ns1) possesses dsrna-binding properties to shield viral rna from rig-i (96). iav has also been shown to antagonize rig-i activation via its viral polymerase subunit pb2. pb2 position 627k in the mammalian strain increases pb2-nucleocapids binding affinity, thus inhibiting rig-i interaction with the nucleoprotein-encapsidated 5′ppp rna (22, 97) . in addition to altering and concealing their genome to prevent rna binding, viruses also re-localize viral rna to specific cellular compartments, such as mitochondria, endoplasmic reticulum (er), and golgi, to avoid cytosolic surveillance by rig-i. for instance, the denv conceals dsrna in the intracellular membrane as an escape strategy (98) . er is an important organelle for viral entry, replication, and assembly. the severe acute respiratory syndrome (sars) coronavirus (sars-cov) has been shown to induce a modified er to hide its replicating rna from detection (99) . these viral antagonism strategies highlight the importance of cellular organelle localization in viral-host interactions during innate antiviral responses. as reviewed above, ubiquitination represents one critical ptm mechanism of rig-i activation and, not surprisingly, is an attractive target for viral manipulation (figure 3a) . viruses have evolved ways to inhibit k63-linked ubiquitination of rig-i by interacting with the e3 ligases trim25 and riplet. for instance, iav ns1 from various strains has been shown to suppress trim25-mediated rig-i cards ubiquitination. among all the trim25 binding amino acids identified in ns1, r38/k41 and e96/e97 were described as critical in interfering with the coil-coiled domain of trim25. these interactions resulted in an inhibition of trim25 multimerization and therefore blocked the rig-i cards ubiquitination (100) . intriguingly, ns1-trim25 binding is found to be preserved in human and avian, but lost in mouse, indicating a species-specific manner of inhibition. this study further demonstrates that the ns1 suppression of rig-i ubiquitination in mouse is riplet-dependent (101) . conversely, phosphorylation of ns1 at thr49 was recently identified as impairing the ns1-trim25 interaction, thereby suppressing its antagonistic activity of rig-i signaling (102) . phosphorylation of another site on ns1, thr80, has also been reported to disrupt ns1 binding affinity with rig-i (103) . similar to iav, the ibv non-structural ns protein (ns1-b) has recently been described as inhibiting rig-i ubiquitination, which involves trim25-ns1 c-terminal effector domain interaction and the rig-i/trim25/ ns1-b complex formation (104) . by contrast, the protease ns3-4a of hcv functions differently, rather than inhibiting trim25, it is thought to target the e3 ligase riplet. ns3-4a directly disrupts riplet, abolishes riplet-mediated rig-i ubiquitination, and further reduces the interaction between trim25 and rig-i (54). on the other hand, some viruses encode enzymes that directly deubiquitinate rig-i. for instance, kshv encoded deubiquitinase orf64 cleaves lys63-ubiquination chains on cards, blocks cards interaction between rig-i and mavs, thereby downregulating rig-i signaling (105) . other viruses including arterivirus, nairovirus, sars-cov, and foot-and-mouth disease virus (fmdv) have also been reported to downregulate rig-i ubiquitination through their viral encoded dubs (106, 107) . few viruses have been shown to manipulate rig-i regulation with regards to targeting the phosphorylation or dephosphorylation process of rig-i. nevertheless, it was reported that mv efficiently escapes antiviral response via suppressing rig-i dephosphorylation in dendritic cells (dcs). in this study, the growth arrest and dna damage protein (gadd34) was shown to form complexes with pp1 to facilitate rig-i activation. the mv infection induced dc-sign signaling results in an inhibition of gadd34-pp1 phosphatases activity and thereby impairs rig-i activation (108). another distinct strategy used by viruses to antagonize rig-i signaling is the direct cleavage or degradation of the receptor and multiple members of the signaling cascade ( figure 3a) . rig-i has been reported in some studies to be cleaved by the proteinase 3c pro during infections with picornavirus, coxsackievirus b3 (cvb3), and enterovirus 71 (ev71) (109, 110) . the encephalomyocarditis virus directs both caspase-and proteasome-dependent degradation of rig-i (111) . intriguingly, the ns1-ns2 degradasome of the respiratory syncytial virus (rsv) has been shown to mediate the proteasomal degradation of rig-i (112) . mitochondrial antiviral signaling protein is also a well-studied molecule which is often targeted by many types of viral-induced cleavage. for example, the hepatitis a virus (hav) cleaves mavs for proteolysis by its protease 3c pro (113) . both cvb3 proteinase 2a pro and 3c pro trigger mavs cleavage at different sites during infection, and the cleavage of mavs by ev71 is accomplished via its 2a pro activity (110, 114) . in addition, serine protease ns3-4a of hcv cleaves mavs, removing it from the mitochondria, thereby inhibiting downstream signaling (36, 115) . in a parallel fashion, many viruses mediate cellular proteolytic degradation of mavs to attenuate rig-i antiviral responses. hepatitis b virus viral protein hbx triggers the proteasome-mediated degradation of mavs through lys136 ubiquitination (116) . another study reported that the hav cysteine protease abc targets mavs for proteolysis at mitochondrial membrane (113) . additionally, viral modulation of cellular organelles such as mitochondria also affects rig-i-mavs signaling. the pb1-f2 of iav, for instance, has been described as decreasing the mitochondrial membrane potential, resulting in the acceleration of mitochondrial fragmentation, thereby inhibiting rig-i-mavs signaling (117) (118) (119) . it is important to note that the proper localization of rig-i and mavs is a prerequisite for effective signaling transduction. mavs resides on the mitochondrial membrane, peroxisomes, and mitochondria-associated membranes for antiviral signaling. in fact, a rig-i translocon has been identified to direct rig-i redistribution from cytosol to membranes during viral infection (49) . studies have shown that several viruses encode proteins to disrupt the proper localization of rig-i or mavs as a novel mechanism of regulation, such as ns3 of denv (113) , nucleoprotein of rsv (120), and non-structural proteins of thrombocytopenia syndrome virus (sftsv) (121) . to ensure successful rig-i signaling transduction, the kinase activities of tbk1 and ikkϵ are tightly controlled via various regulatory mechanisms and are common targets of viruses ( figure 3b) . for example, both the leader proteinase (l pro ) of fmdv (122) and the non-structural protein 3 (ns3) of the mouse hepatitis virus a59 (123) inhibit ubiquitination of tbk1. dengue virus serotype4 non-structural proteins, ns2a and ns4b, as well as the flips proteins encoded by the molluscum contagiosum virus (mcv), all reduce tbk1 phosphorylation, thereby preventing its activation (124, 125) . several viruses have been shown to prevent the formation of functional tbk1-containing complexes. the k7 protein of the vacv prevents tbk1/ikkϵ complex-induced irf activation by targeting host dead box protein 3 (ddx3) (126) . two other viruses, the ny-1 htnv and sars-cov, disrupt the tbk1-traf3 and tank-tbk1/ikkϵ complex, respectively (127, 128) . moreover, sftsv has been shown to irreversibly re-localize tbk1 and ikk from mitochondria and sequester the tbk1/ikkϵ/irf3 complex via the formation of inclusion bodies, causing signaling cascade termination (129) . viral regulation of the transcription factors, irfs and nf-κb, further serve as points of control in rig-i signaling ( figure 3b) . one of the best studied examples is the inhibition of irf3 activity by the iav ns1 protein (130) . besides this, the hsv-1, rabies virus, sars-cov, as well as several paramyxoviruses have been demonstrated to interfere with the phosphorylation state of irf3, thereby blocking ifn induction (131) (132) (133) (134) . the ebv conjugates sumo to irf7 at lysine 452 to decrease irf7 transcriptional activity (135) . the rotavirus ns1, targets both irf3 and irf7 for degradation to prevent irfs from undergoing dimerization (136) . viruses have also developed various means to suppress the irf3 dna binding ability. herpes simplex virus, thogoto virus, and kshv, all developed strategies to downregulate irf3 transcriptional activity by either disrupting irf3 binding complex formations or competing binding regions on the ifnb promoter (137) (138) (139) . viral strategies in inhibiting cytoplasmic or transcriptional activities of nf-κb have been extensively studied during the vacv infection. studies reported that multiple proteins encoded by vacv and hsv-1 suppress nf-κb activation (140) (141) (142) (143) . viruses have also developed multiple inhibitory mechanisms to counteract the ifn stimulation of isgs by targeting stat1 and/or stat2 ( figure 3b ). for example, the langat virus was shown to inhibit the phosphorylation of both stat1 and stat2 (144) . varicella viruses and the japanese encephalitis virus, both block the jak/stat1 pathway through multiple mechanisms including inhibiting stat proteins phosphorylation and nucleotranslocation (145, 146) . the non-structural protein ns5 of several flaviviruses, have been shown to target stat proteins via distinct mechanisms. for example, mnv ns5 inhibits stat1 phosphorylation, whereas denv ns5 interacts with ubr4 to promote stat2 degradation (147, 148) . by contrast, the zika virus ns5 induced proteasomal degradation of stat2 was recently identified as ubr4 independent (149) . furthermore, other viruses, such as hcv (150) , rsv (151) , and paramyxovirus (152) , also demonstrate negative regulation of the jak-stat pathway. studies from the past decade have well established rig-i as one of the principal prrs for the recognition of cytoplasmic viral rna, as well as defining its critical role in the induction of ifns during viral infections. our understanding of the rig-i-mediated antiviral response has been greatly expanded with the key discoveries made regarding the molecular mechanism of rig-i regulation, such as ubiquitination, phosphorylation, and acetylation. meanwhile, investigating viral strategies to manipulate rig-i responses not only allow us to understand the viral pathogenesis, but also significantly contributed to our knowledge of how rig-i is activated and regulated. these new insights into the viral-mediated rig-i regulations are important for vaccine and drug development aiming to suppress infectious diseases and enhance immune responses. yl wrote the manuscript. rl and do revised and approved the manuscript. the authors would like to thank alexandre sze for his critical reading and editing of the manuscript. this research was supported by grant from canadian institutes of health research (mop130401) to rl; do was supported by a peter quinlan mcgill postdoctoral fellowship. the authors would like to acknowledge all the colleagues in the field and apologies to those whose important contributions could not be included in the review due to space constraints. the figures of the review were illustrated using the servier medical art library, http://www. servier.com/powerpoint-image-bank. the role of pattern-recognition receptors in innate immunity: update on toll-like receptors the rna helicase rig-i has an essential function in double-stranded rna-induced innate antiviral responses immune signaling by rig-i-like receptors tlr signaling pathways regulation of the antimicrobial response by nlr proteins cyclic gmp-amp synthase is a cytosolic dna sensor that activates the type i interferon pathway ifi16 is an innate immune sensor for intracellular dna mechanisms of type-i-and type-ii-interferon-mediated signalling viral evasion and subversion of patternrecognition receptor signalling viral evasion of intracellular dna and rna sensing pattern recognition receptors and inflammation structural insights into rna recognition by rig-i structural basis of rna recognition and activation by innate immune receptor rig-i structural basis for the activation of innate immune pattern-recognition receptor rig-i by viral rna orchestrating the interferon antiviral response through the mitochondrial antiviral signaling (mavs) adapter recognition of viral nucleic acids in innate immunity rna recognition and signal transduction by rig-i-like receptors ebola virus vp35 protein binds double-stranded rna and inhibits alpha/beta interferon production induced by rig-i signaling rig-i, mda5 and tlr3 synergistically play an important role in restriction of dengue virus infection 5′-triphosphate rna is the ligand for rig-i recognition of 5′ triphosphate by rig-i helicase requires short blunt double-stranded rna as contained in panhandle of negative-strand virus incoming rna virus nucleocapsids containing a 5′-triphosphorylated genome activate rig-i and antiviral signaling rig-imediated antiviral responses to single-stranded rna bearing 5′-phosphates antiviral immunity via rig-i-mediated recognition of rna bearing 5′-diphosphates systems analysis of a rig-i agonist inducing broad spectrum inhibition of virus infectivity inhibition of dengue and chikungunya virus infections by rig-i-mediated type i interferon-independent stimulation of the innate antiviral response enhanced influenza virus-like particle vaccination with a structurally optimized rig-i agonist as adjuvant defining new therapeutics using a more immunocompetent mouse model of antibody-enhanced dengue virus infection sequence-specific modifications enhance the broad-spectrum antiviral response activated by rig-i agonists cutting edge: the rig-i ligand 3prna potently improves ctl cross-priming and facilitates antiviral vaccination early innate recognition of herpes simplex virus in human primary macrophages is mediated via the mda5/mavs-dependent and mda5/mavs/ rna polymerase iii-independent pathways rna polymerase iii detects cytosolic dna and induces type i interferons through the rig-i pathway rig-i mediated sting up-regulation restricts hsv-1 infection recognition of rna virus by rig-i results in activation of card9 and inflammasome signaling for interleukin 1 beta production type i ifn triggers rig-i/tlr3/nlrp3-dependent inflammasome activation in influenza a virus infected cells cardif is an adaptor protein in the rig-i antiviral pathway and is targeted by hepatitis c virus identification and characterization of mavs, a mitochondrial antiviral signaling protein that activates nf-kappab and irf 3 regulation and function of nf-kappab transcription factors in the immune system a functional c-terminal traf3-binding site in mavs participates in positive and negative regulation of the ifn antiviral response the nemo adaptor bridges the nuclear factor-kappab and interferon regulatory factor signaling pathways direct triggering of the type i interferon system by virus infection: activation of a transcription factor complex containing irf-3 and cbp/p300 regulation of innate antiviral defenses through a shared repressor domain in rig-i and lgp2 the c-terminal regulatory domain is the rna 5′-triphosphate sensor of rig-i atpase-driven oligomerization of rig-i on rna allows optimal activation of type-i interferon rig-i forms signaling-competent filaments in an atp-dependent, ubiquitin-independent manner rig-i atpase activity and discrimination of self-rna versus non-self-rna correction: atp hydrolysis by the viral rna sensor rig-i prevents unintentional recognition of self-rna roles of rig-i n-terminal tandem card and splice variant in trim25-mediated antiviral signal transduction the mitochondrial targeting chaperone 14-3-3epsilon regulates a rig-i translocon that mediates membrane association and innate antiviral immunity trim25 ringfinger e3 ubiquitin ligase is essential for rig-i-mediated antiviral activity riplet/rnf135, a ring finger protein, ubiquitinates rig-i to promote interferon-beta induction during the early phase of viral infection the ubiquitin ligase riplet is essential for rig-i-dependent innate immune responses to rna virus infection reul is a novel e3 ubiquitin ligase and stimulator of retinoic-acid-inducible gene-i a distinct role of ripletmediated k63-linked polyubiquitination of the rig-i repressor domain in human antiviral innate immune responses pivotal role of rna-binding e3 ubiquitin ligase mex3c in rig-i-mediated antiviral innate immunity antiviral activity of human oasl protein is mediated by enhancing signaling of the rig-i rna sensor structural and functional analysis reveals that human oasl binds dsrna to enhance rig-i signaling reconstitution of the rig-i pathway reveals a signaling role of unanchored polyubiquitin chains in innate immunity structural basis for ubiquitin-mediated antiviral signal activation by rig-i the tumour suppressor cyld is a negative regulator of rig-i-mediated antiviral response syndecan-4 negatively regulates antiviral signalling by mediating rig-i deubiquitination via cyld usp3 inhibits type i interferon signaling by deubiquitinating rig-i-like receptors usp21 negatively regulates antiviral response by acting as a rig-i deubiquitinase negative regulation of the rig-i signaling by the ubiquitin ligase rnf125 rnf122 suppresses antiviral type i interferon production by targeting rig-i cards to mediate rig-i degradation linear ubiquitin assembly complex negatively regulates rig-i-and trim25-mediated type i interferon induction the ubiquitin-specific protease usp15 promotes rig-i-mediated antiviral signaling by deubiquitylating trim25 phosphorylationmediated negative regulation of rig-i antiviral activity negative role of rig-i serine 8 phosphorylation in the regulation of interferon-beta production phosphorylation of rig-i by casein kinase ii inhibits its antiviral response ikk negatively regulates rig-i via direct phosphorylation conventional protein kinase c-alpha (pkc-alpha) and pkc-beta negatively regulate rig-i antiviral signal transduction dephosphorylation of the rna sensors rig-i and mda5 by the phosphatase pp1 is essential for innate immune signaling lysine acetylation targets protein complexes and co-regulates major cellular functions regulation of retinoic acid inducible gene-i (rig-i) activation by the histone deacetylase hdac6 regulates cellular viral rna sensing by deacetylation of rig-i ubiquitin-like proteins sumoylation of rig-i positively regulates the type i interferon signaling ubiquitin-like modifier fat10 attenuates rig-i mediated antiviral signaling by segregating activated rig-i from its signaling platform negative feedback regulation of rig-i-mediated antiviral signaling by interferon-induced isg15 conjugation negative regulation of rig-i-mediated innate antiviral signaling by sec14l1 the atg5 atg12 conjugate associates with innate antiviral immune responses viral pseudo-enzymes activate rig-i via deamidation to evade cytokine production emerging roles of protein deamidation in innate immune signaling processing of genome 5′ termini as a strategy of negative-strand rna viruses to avoid rig-i-dependent interferon induction the 5′ ends of hantaan virus (bunyaviridae) rnas suggest a prime-and-realign mechanism for the initiation of rna synthesis old world hantaviruses do not produce detectable amounts of dsrna in infected cells and the 5′ termini of their genomic rnas are monophosphorylated genome trimming: a unique strategy for replication control employed by borna disease virus unpaired 5′ ppp-nucleotides, as found in arenavirus double-stranded rna panhandles, are not recognized by rig-i a protein covalently linked to poliovirus genome rna conventional and unconventional mechanisms for capping viral mrna structural basis for m7g recognition and 2′-o-methyl discrimination in capped rnas by the innate immune receptor rig-i structural basis for dsrna recognition and interferon antagonism by ebola vp35 marburg virus vp35 can both fully coat the backbone and cap the ends of dsrna for interferon antagonism structural basis for marburg virus vp35-mediated immune evasion mechanisms a recombinant influenza a virus expressing an rna-binding-defective ns1 protein induces high levels of beta interferon and is attenuated in mice influenza virus adaptation pb2-627k modulates nucleocapsid inhibition by the pathogen sensor rig-i the dengue virus conceals double-stranded rna in the intracellular membrane to escape from an interferon response sars-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum influenza a virus ns1 targets the ubiquitin ligase trim25 to evade recognition by the host viral rna sensor rig-i species-specific inhibition of rig-i ubiquitination and ifn induction by the influenza a virus ns1 protein phosphorylation of influenza a virus ns1 protein at threonine 49 suppresses its interferon antagonistic activity threonine 80 phosphorylation of non-structural protein 1 regulates the replication of influenza a virus by reducing the binding affinity with rig-i robust lys63-linked ubiquitination of rig-i promotes cytokine eruption in early influenza b virus infection inhibition of rig-i-mediated signaling by kaposi's sarcoma-associated herpesvirusencoded deubiquitinase orf64 regulation of rig-i-like receptor signaling by host and viral proteins arterivirus and nairovirus ovarian tumor domain-containing deubiquitinases target activated rig-i to control innate immune signaling measles virus suppresses rig-i-like receptor activation in dendritic cells via dc-sign-mediated inhibition of pp1 phosphatases rig-i is cleaved during picornavirus infection enterovirus 2apro targets mda5 and mavs in infected cells the viral rna recognition sensor rig-i is degraded during encephalomyocarditis virus (emcv) infection viral degradasome hijacks mitochondria to suppress innate immunity disruption of innate immunity due to mitochondrial targeting of a picornaviral protease precursor the coxsackievirus b 3c protease cleaves mavs and trif to attenuate host type i interferon and apoptotic signaling hepatitis c virus protease ns3/4a cleaves mitochondrial antiviral signaling protein off the mitochondria to evade innate immunity the hepatitis b virus x protein disrupts innate immunity by downregulating mitochondrial antiviral signaling protein influenza virus protein pb1-f2 inhibits the induction of type i interferon by binding to mavs and decreasing mitochondrial membrane potential the influenza virus protein pb1-f2 inhibits the induction of type i interferon at the level of the mavs adaptor protein influenza a virus protein pb1-f2 translocates into mitochondria via tom40 channels and impairs innate immunity human respiratory syncytial virus nucleoprotein and inclusion bodies antagonize the innate immune response mediated by mda5 and mavs hijacking of rig-i signaling proteins into virus-induced cytoplasmic structures correlates with the inhibition of type i interferon responses the leader proteinase of foot-and-mouth disease virus negatively regulates the type i interferon pathway by acting as a viral deubiquitinase plp2, a potent deubiquitinase from murine hepatitis virus, strongly inhibits cellular type i interferon production dengue virus ns proteins inhibit rig-i/mavs signaling by blocking tbk1/irf3 phosphorylation: dengue virus serotype 1 ns4a is a unique interferon-regulating virulence determinant inhibition of interferon gene activation by death-effector domain-containing proteins from the molluscum contagiosum virus viral targeting of dead box protein 3 reveals its role in tbk1/ikkepsilon-mediated irf activation the ny-1 hantavirus gn cytoplasmic tail coprecipitates traf3 and inhibits cellular interferon responses by disrupting tbk1-traf3 complex formation sars coronavirus papain-like protease inhibits the type i interferon signaling pathway through interaction with the sting-traf3-tbk1 complex evasion of antiviral immunity through sequestering of tbk1/ikkepsilon/irf3 into viral inclusion bodies activation of interferon regulatory factor 3 is inhibited by the influenza a virus ns1 protein inhibition of interferon regulatory factor 3 activation by paramyxovirus v protein the sars coronavirus papain like protease can inhibit irf3 at a post activation step that requires deubiquitination activity genetic dissection of interferon-antagonistic functions of rabies virus phosphoprotein: inhibition of interferon regulatory factor 3 activation is important for pathogenicity herpes simplex virus 1 serine/threonine kinase us3 hyperphosphorylates irf3 and inhibits beta interferon production epstein-barr virus latent membrane protein 1 regulates the function of interferon regulatory factor 7 by inducing its sumoylation rotavirus nsp1 mediates degradation of interferon regulatory factors through targeting of the dimerization domain thogoto virus ml protein suppresses irf3 function binding of kaposi's sarcoma-associated herpesvirus k-bzip to interferon-responsive factor 3 elements modulates antiviral gene expression recruitment of activated irf-3 and cbp/p300 to herpes simplex virus icp0 nuclear foci: potential role in blocking ifn-beta induction the vaccinia virus k1 ankyrin repeat protein inhibits nf-kb activation by preventing rela acetylation vaccinia virus protein c4 inhibits nf-kappab activation and promotes virus virulence herpes simplex virus 1-encoded tegument protein vp16 abrogates the production of beta interferon (ifn) by inhibiting nf-kappab activation and blocking ifn regulatory factor 3 to recruit its coactivator cbp herpes simplex virus 1 protein kinase us3 hyperphosphorylates p65/rela and dampens nf-kappab activation inhibition of interferon-stimulated jak-stat signaling by a tick-borne flavivirus and identification of ns5 as an interferon antagonist blocking of interferon-induced jak-stat signaling by japanese encephalitis virus ns5 through a protein tyrosine phosphatase-mediated mechanism varicella viruses inhibit interferon-stimulated jak-stat signaling through multiple mechanisms the ns5 protein of the virulent west nile virus ny99 strain is a potent antagonist of type i interferon-mediated jak-stat signaling dengue virus co-opts ubr4 to degrade stat2 and antagonize type i interferon signaling zika virus targets human stat2 to inhibit type i interferon signaling hepatitis c virus targets the interferon-alpha jak/stat pathway by promoting proteasomal degradation in immune cells and hepatocytes respiratory syncytial virus ns1 protein degrades stat2 by using the elongin-cullin e3 ligase paramyxovirus disruption of interferon signal transduction: status report key: cord-342653-bpyc2gbl authors: wang, hai-tao; hur, sun title: substrate recognition by trim and trim-like proteins in innate immunity date: 2020-10-20 journal: semin cell dev biol doi: 10.1016/j.semcdb.2020.09.013 sha: doc_id: 342653 cord_uid: bpyc2gbl trim (tripartite motif) and trim-like proteins have emerged as an important class of e3 ligases in innate immunity. their functions range from activation or regulation of innate immune signaling pathway to direct detection and restriction of pathogens. despite the importance, molecular mechanisms for many trim/trim-like proteins remain poorly characterized, in part due to challenges of identifying their substrates. in this review, we discuss several trim/trim-like proteins in rna sensing pathways and viral restriction functions. we focus on those containing pry-spry, the domain most frequently used for substrate recognition, and discuss emerging mechanisms that are commonly utilized by several trim/trim-like proteins to tightly control their interaction with the substrates. the innate immune system is the first line of defense against a broad range of microbial pathogens. innate immune receptors, so called pattern recognition receptors (prrs), recognize conserved pathogenassociated molecular patterns (pamps) and activate a variety of innate immune responses that mediate immune cell recruitment and pathogen restriction. over the last three decades or so, several families of germline encoded prrs have been identified and extensively characterized. these include membrane-bound toll like receptors (tlrs) that survey extracellular or endosomal space for the presence of infection, and soluble receptors, such as rig-i like receptors (rlrs), cgas and nod-like receptors (nlrs), that monitor the cytosolic compartment [1] . upon recognition of cognate pamps, these receptors engage with their downstream adaptor proteins, such as mavs (also known as visa, ips-1, and cardif [2] [3] [4] [5] ), sting (also known as mita, mpys, eris and tmem173 [6] [7] [8] [9] ) and asc, to initiate signaling cascades that culminate in transcriptional or post-translational activation of antiviral and proinflammatory cytokines [10] . while their functions are critical for proper defense against pathogens, accumulating evidence suggests that inappropriate activation of these receptors or inefficient suppression of their signaling pathways can lead to a spectrum of autoimmune and inflammatory disorders. as such, multiple regulatory mechanisms are in place to tightly regulate the activities of these innate immune pathways, while ensuring rapid amplification of the immune signaling cascades upon pathogen detection. one primary mechanism of immune regulation is through posttranslational modifications, which allows rapid and often reversible modulation of the activities of immune signaling molecules. among those, ubiquitin (ub) and ub-like (ubl) protein modifications play central roles in regulating nearly all innate immune signaling pathways [11, 12] . ub/ubl modification is mediated by sequential actions of an e1 activating enzyme, e2 conjugating enzyme and e3 ligase. the e3 ligase is the one that carries out the final step of ub transfer from e2 to the substrate, and thus dictates the substrate specificity. there are over 600 e3 ligases in primates, as opposed to 2 e1s and ~40 e2s [13] . accordingly, much effort has been made to identify e3 ligases that modulate any given immune pathway. tripartite motif proteins (trims) are a family of e3 ligases that are emerging as key players in innate immunity. one of the first clues for their innate immune functions came from the fact that many trims are induced by interferons or pro-inflammatory cytokines [14] . while e3 ligases are often thought to negatively regulate the stability of the target molecule by ub-mediated proteasomal targeting, many trims have been shown to enhance innate immune signaling pathways [15] , through both proteasome-dependent and -independent mechanisms. furthermore, some trims were shown to play more active immune functions as pathogen sensors or restriction factors [16, 17] , instead of simply up-or down-regulating the activities of immune signaling molecules. despite their importance in a broad range of innate immune functions, precise modes of action of many trim proteins remain elusive. this limitation largely reflects the challenge of identifying substrate proteins, which is the key to understanding their functions and mechanisms. we here review several trim and related e3 ligases (broadly defined as trim-like proteins) in innate immunity, with a particular focus on their substrate recognition mechanisms. we first begin by introducing the general properties of trim and trim-like proteins. we here apologize in advance to those whose studies were not included in the review due to the space limitation. trims are characterized by three distinct domains at the n-terminus: a ring domain that binds e2 conjugating enzymes, followed by one or two b-box domains and a coiled-coil (cc) domain (fig. 1a) . they are also known as rbcc (ring-b-box-coiled-coil) proteins because the order at which these domains appear is conserved [18] . the trim family is one of the largest subclasses of ring-e3 ligases, consisting of more than 70 members in human [19] . it is also an ancient family of e3 ligases as members of the family can be found in almost all metazoans [19] . not all trim proteins, however, have all three domains; some trims have linkers replacing one of the three domains, while preserving the order of the rest of the domains [18, 20] . there are also many ligases (to be called trim-like proteins) not formally categorized into the trim family, but harboring two out of three domains with the same conserved order. the strict conservation of the domain orders implicates that functions of these domains are intimately coordinated with one another and their spatial arrangement in three dimensions is also likely conserved. as ring-e3 ligases, trim/trim-like proteins function as an adaptor that bridges e2 and substrate. the ring domain carries out the e2 binding activity, while the c-terminal variable domain is often responsible for substrate binding (see section 3). while most trim/trim-like proteins have been shown to function as ub e3 ligases, some can conjugate ubl proteins, such as sumo and isg15 [21, 22] . many ring domains in trim/trim-like proteins require homo-dimerization to induce the "closed" e2~ub conformation that stimulates ub transfer from e2 to the substrate [23, 24] (fig. 1b) . while some rings form a constitutive dimer, others may form the dimer transiently when bound with the e2~ub complex. b-box is a zinc-finger domain, and is further categorized into b-box1 and b-box2 depending on the residues coordinating the zn ions [20] ( fig. 1b) . while many trims have both b-boxes, with b-box1 preceding b-box2, some harbor only a single b-box, in which case it is always b-box2 [18] (fig. 1a) . while this suggests an essential function of b-box2 that cannot be replaced by b-box1, the precise function of b-box2 remains unclear. in trim5α, b-box2 was found to form a trimer (fig. 1b) , which leads to higher-order multimerization of trim5α [25] . it remains to be examined whether this is a generalizable property of b-box2 and what role b-box1 plays for those trim/trim-like proteins with both bboxes. previous studies have shown that ccs in many trim proteins form a homodimer in vitro [26] (fig. 1b) , but its function appears to be more complex than simple dimerization. structures of several trim cc's showed an antiparallel dimeric architecture, which would place the two ring domains near the opposite ends of the cc [26, 27] . since ring dimerization is required for many trims, this geometric restraint raises the question of whether a ring:ring contact occurs within a trim dimer or through an inter-dimeric interaction. for certain trims, the two rings may be too far apart to allow an intra-dimeric ring:ring contact, in which case higher-order oligomerization is likely required for their e3 ligase activities. additionally, trim cc also appears to play roles in positioning the c-terminal variable domain. many trims harbor a characteristic linker following cc, which folds back onto the cc [23, [26] [27] [28] [29] [30] (fig. 1b) and tethers the c-terminal domain near the center of cc [23, [26] [27] [28] [29] [30] . such tethering appears important at least in some cases (e.g. trim5α [30] and trim25 [29] ), as further evidenced by viral antagonists that target this function of cc [29] . finally, cc was also proposed to promote higher-order oligomerization [23, 31, 32] or to form hetero-oligomeric complex with other related trims [18, 33] . thus, trim cc plays diverse functions in regulating the overall architecture and functions of trim proteins. the c-terminal variable domain of trim/trim-like proteins often play important roles in substrate recognition. the pry-spry domain is the most common c-terminal domain, accounting for that of about half of all known trims [34, 35] (fig. 2a) . trims with pry-spry are vertebrate-specific, emerged recently, evolves more rapidly, and are more intimately involved in host-pathogen interactions than other trims [19, 27, 36] . the pry-spry (a.k.a b30.2) domain is a ~200 amino acids-long, single globular domain (fig. 2b ). it is formed by appendage of the nterminal pry (~60 amino acids) to the c-terminal spry (~140 amino acids). the spry domain is widely conserved from yeast to human. by contrast, the pry-spry domain is only found in vertebrates [37] . previous structural analyses showed that pry-spry domains display a highly conserved beta sandwich structure where two beta sheets are packed against each other [38] (fig. 2b ). while the sequences in the core β-strands are well-conserved, those of loops between β-strands are highly variable [39] (fig. 2c) . it is the cluster of these variable loops that bind substrates [39] [40] [41] [42] (fig. 2b) . the high sequence variability in these loops allows recognition of a diverse set of substrates, ranging from a linear peptide to three dimensional protein structure [39, [41] [42] [43] and to even an rna molecule [44] . the utilization of variable loops for substrate recognition is akin to that of immunoglobulins, which utilize the complementary determine regions (cdrs) for substrate specificity. below, we focus on pry-spry-containing trim/trim-like proteins and their substrate recognition mechanisms. we begin with trim/ trim-like proteins involved in innate immune signaling pathways and expand our discussion to include those involved in antiviral effector immunity. [25] ) and coiled-coil domains (pdb: 6fln, [29] ). yellow regions in cc (orange) indicate the linker c-terminal to trim cc that often folds back onto cc. rig-i is the founding member of the rlr family that recognizes viral rnas and activates the signaling pathways to upregulate type i/iii interferons and pro-inflammatory cytokines [45] . rig-i consists of two tandem caspase activation recruitment domains (2card) at the n-terminus, a dexd/h helicase domain in the middle, and a characteristic cterminal domain (ctd) (fig. 3a) . 2card is responsible for downstream signal activation, while the helicase domain and ctd cooperate to recognize viral rnas. rig-i relies on the presence of 5'-triphosphate (5'ppp) or 5'-diphosphate and dsrna structure for distinguishing viral rnas from cellular rnas [46, 47] . for cellular rnas, 5'ppp is typically removed from nascent transcripts during 5'-processing before they are exported to the cytoplasm, but this does not occur for many viral rnas that are synthesized in the cytoplasm. in addition to 5'ppp, dsrna length also plays an important role in self vs. non-self-discrimination by rig-i. dsrna duplex of > 20 bp is required and ~40-150 bp are ideal for activating rig-i [31] (to be discussed more later). although a short hairpin rna with a ~10 bp stem was found to activate rig-i [48] , <~20 bp dsrna without a hairpin loop do not [48] . given our current understanding that rig-i does not bind the loop, the precise reason for the distinct rig-i-stimulatory activities of hairpin vs. standard dsrna remains unclear. it is possible that concatemerization of a hairpin rna extends the duplex region, allowing it to mimic longer dsrnas, as was shown before [49] . in addition to 5'ppp and 5'pp dsrna, there are several other features of rna that have been reported to stimulate rig-i, such as features of 3' end or sequence composition [50] [51] [52] . in many cases, however, the mechanisms remain unclear and require further investigations. previous biochemical and structural studies provided detailed pictures of how rig-i selectively recognizes viral rnas and how it activates the downstream antiviral immune response. upon viral rna binding, rig-i undergoes at least two distinct conformational changes (fig. 3b ). before rna engagement, rig-i is in the auto-repressed state, in which 2card is bound by the helicase domain [53] . the auto-repression is released by viral dsrna binding as rna competes with 2card for the helicase domain [53] . release of 2card, however, is insufficient to activate rig-i signaling. the second necessary conformational change is homo-tetramerization of 2card [54, 55] (fig. 3b ). only tetramerized 2card can stably interact with the downstream adaptor mavs [56] . the 2card tetramer then acts as a nucleus to induce filament formation of mavs, which serves as the signaling scaffold for activating the further downstream signaling pathway [56, 57] . studies showed that the tetramerization of rig-i 2card is greatly stimulated by k63-linked ub chains (k63-ub n ), which wraps around the 2cards tetramer to stabilize the core 2card tetramer [54] (fig. 3c ). recent genetic and biochemical studies showed that the e3 ligase rip-let (a.k.a. rnf135) is responsible for rig-i ubiquitination and activation [31, 58, 59] . while it has long been thought that another e3 ligase, trim25, was responsible for rig-i activation, this notion was established largely based on the effect of trim25 on isolated 2cards, an artificial construct that also activates mavs upon overexpression [60] . in fact, few studies have demonstrated the positive impact of trim25 on the signaling activity of full-length rig-i. furthermore, trim25 does not directly bind or ubiquitinate full-length rig-i in a manner dependent on rna ligand, the condition that must be satisfied in order to be functionally relevant [31] . this is because any e3 ligase would have a nonspecific ubiquitination activity at high protein concentrations. thus, specificity control, such as rig-i in the absence of stimulatory rna, is essential to validate the assay condition. consistent with the notion that riplet is the bona fide e3 ligase for rig-i, riplet binds and ubiquitinates rig-i only in the presence of rig-i-stimulatory rna, recapitulating the cellular activation condition [31] . interestingly, multiple studies have shown that trim25 can inhibit a broad range of viruses in a manner independent of rig-i; trim25 binds influenza a nucleocapsid and blocks its replication [61] , and cooperates with the antiviral protein zap to inhibit viral gene expression [62, 63] . trim25 was also shown to directly interact with the linear ubiquitin chain assembly complex (lubac), which often plays a negative regulatory role in innate immune signaling [64] . altogether, the antiviral functions of trim25 appears complex, but is not directly involved in the rig-i signaling pathway. instead, riplet mediates ubiquitination and activation of rig-i. another area of controversy has been on the importance of covalent ub conjugation vs. non-covalent binding in rig-i signaling. previous biochemical and structural studies [54, 55] have showed that unanchored k63-ub n alone can induce 2card tetramerization and activate rig-i signaling in vitro. however, whether non-covalent interaction alone is indeed sufficient in cells has been controversial. in order to address this issue, multiple studies have examined the effect of mutations of potential conjugation sites of rig-i, but such efforts frequently involved mutations of multiple lysines, which alone can significantly affect protein folding and other activities besides ubiquitination. multiple lines of evidence support the importance of covalent ub conjugation as well as non-covalent interaction. first, non-covalent interaction between k63-ub n and rig-i 2card is weak, while covalent conjugation greatly stimulates the stability of their complex and the resultant 2card tetramer [54] . second, a recent biochemical analysis suggests that riplet can constitutively generate unanchored k63-ub n without rig-i, but the presence of rig-i filaments converts its activity to the rig-i conjugation mode, suggesting that the conjugated k63-ub n is more likely to be relevant to rig-i functions [31] . third, in a reconstituted signaling system, riplet-dependent rig-i signaling was maintained even after treatment with isot, which selectively degrades unanchored ub chains [31] . while another previous study reported an abrogation of rig-i signaling upon isot treatment [65] , one important difference is that the latter study utilized trim25, which unlike riplet, only generates unanchored k63-ub n with or without rig-i. trim25 in this in vitro system likely generates a sufficient level of unanchored k63-ub n to activate rig-i. since trim25 does not bind rig-i [31] , its activity to constitutively synthesize unanchored k63-ub n is unlikely to be relevant for rig-i signaling. in summary, these observations collectively suggest the importance of both anchored and unanchored k63-ub n for rig-i signaling, and may serve as a model to study the importance of covalent ub conjugation in other systems. riplet is a trim-like e3 ligase, harboring ring, cc and pry-spry, but not b-boxes (fig. 3b ). as mentioned above, riplet binds and ubiquitinates rig-i only in the presence of dsrna [31] , which parallels dsrna-dependent rig-i signaling in cells. a more detailed analysis showed that riplet:rig-i binding requires dsrna to be at least ~20 bp in length, again in agreement with the cellular requirement for rig-i activation [31] . this is because individual pry-spry has low affinity for monomeric rig-i, and a high affinity interaction requires both pry-spry domains of dimeric riplet to simultaneously bind multimeric rig-i assembled on > 20 bp dsrna [31] . this avidity-driven binding explains why rig-i signaling is more efficient with longer dsrna (~40-150 bp), on which rig-i forms filaments [31, 66] . rig-i forms filaments through two steps (fig. 3d) : initial recruitment of rig-i to a activated mavs in turn induces type i interferon production by triggering traf2/3/5/6, tbk1 and irf3. b. three steps involved in rig-i activation. rig-i binding to dsrna releases 2card auto-repression, but this is not sufficient for signal activation. 2card must be tetramerized in order to activate mavs. riplet promotes 2card tetramerization by conjugating k63-ub n to rig-i, which binds 2card and stabilizes its tetramer structure (see c). c. the crystal structure of rig-i 2card (purple-shade colors) in complex with k63-ub n (yellow) (pdb:4nqk, [54] ). k63-ub n wraps around the 2card tetramer, stabilizing its assembly architecture. d. in the absence of viral rna, rig-i is in the auto-repressed state. in the presence of viral dsrna, rig-i binds dsrna end recognizing 5'ppp. dsrna binding triggers atp hydrolysis, which in turn stimulates translocation along dsrna, and subsequent recruitment of additional rig-i molecules. iterations of the end recruitment and translocation of rig-i molecules then results in rig-i filament formation near dsrna ends. riplet binds rig-i only in the filamentous state, leading to k63-ub n conjugation, 2card tetramerization and mavs activation. e. riplet binding also leads to rig-i filament bridging and clustering, which results in the amplification of rig-i signaling. negative stain electron micrographs are taken from [31] . dsrna end via 5'ppp moiety, and subsequent translocation of rig-i from the dsrna end to the interior through an atp hydrolysis-driven motor activity. iterations of this process with multiple rig-i molecules result in accumulation of filamentous oligomers of rig-i near the dsrna end [31, 66] . the reason rig-i and riplet's activity declines on >~0.5 kb dsrna is that rig-i filament formation is dependent on 5'ppp, which is diluted with the increasing length of dsrna [31] . intriguingly, riplet binding to rig-i filaments can also lead to filament cross-bridging, creating aggregate-like assemblies with a heightened signaling potential [31] (fig. 3e) . the cc domain of riplet plays an important role in filament bridging, presumably by forming higher-order oligomers [31] . in this clustered assembly structure, rig-i signaling is further amplified, likely because there are more riplet ring domains to form an active ring:ring dimer, and/or there is higher concentration of rig-i molecules to form the active 2card tetramer. the study of rig-i and riplet interaction provides a detailed example of how trim-like proteins utilize bivalency and cc for regulating substrate selectivity, higher-order oligomerization and innate immune function. mda5 is another member of the rlr helicase family that functions as a cytosolic receptor for viral dsrnas. mda5 shares with rig-i the same domain architecture and the downstream signaling pathway, including the adaptor molecule mavs. however, mda5 and rig-i play nonredundant roles as they recognize largely distinct groups of viruses and viral rnas [67] . while rig-i detects a broad range of negative strand rna viruses by recognizing 5'ppp-containing dsrna with a preference for mid-long lengths (~40-150 bp), mda5 detects several positive strand rna viruses, such as picornaviruses and coronaviruses [68, 69] through recognition of much longer (>~1 kb) dsrnas independent of 5'ppp [70, 71] . while mda5 also forms filaments along the length of dsrna, it utilizes different mechanisms for assembling filaments and regulating its stability; while rig-i filament propagates from a dsrna end to an interior in a 5'ppp-and atp-dependent manner, mda5 filament is nucleated directly from a dsrna interior, and disassembles from its termini during atp hydrolysis [66, 72] . these differences ensure mda5 to form filaments more efficiently on much longer dsrnas than those preferred by rig-i, and its signaling activity to progressively increase with dsrna length. like rig-i, mda5 signaling also requires homo-tetramerization of its 2card, and the efficiency of 2card tetramerization is enhanced by k63-ub n [54, 73] . mda5 signaling and ubiquitination is independent of riplet [31] , but instead relies on trim65 [74, 75] . knocking out trim65 impairs mda5 signaling activity without affecting other antiviral signaling pathways, such as that of rig-i. analogous to how rip-let binds rig-i, trim65 utilizes the pry-spry domain to bind the helicase domain of mda5 [75] . furthermore, trim65 also binds mda5 only in its filamentous form on long dsrna, and this filament-specific recognition stems from the requirement for bivalent pry-spry engagement, as was the case for riplet [76] . thus, for both riplet and trim65, avidity-dependent substrate recognition allows more precise control of immune activation and ensures immune signaling to occur only in the filamentous form, i.e. in the presence of the viral rna ligand. given that an increasing number of receptors and signaling molecules in the innate immune system are shown to multimerize upon activation [77] , it is tempting to speculate that trim/trim-like proteins may utilize multimer-specific substrate recognition as a common mechanism for regulating their immune functions. besides riplet and trim65, several other trim/trim-like proteins have been proposed to act on rlrs and modulate their stabilities and/or antiviral signaling activities. one such e3 ligase is trim38, which was reported to promote antiviral signaling of both rig-i and mda5 through sumo conjugation [22] . trim38 uses its pry-spry domain to interact with both 2cards and the helicase domains of rig-i and mda5, and these interactions were found to be dependent on viral infection. this study further reports that sumo modification is dynamically regulated over the course of infection, and delays k48-linked ubiquitination of rig-i and mda5, thereby temporarily stabilizing these receptors and potentiating their signaling activities [22] . it is yet unclear how sumoylation competes with k48-linked ubiquitination and how the sumoylation of rlrs changes during the course of infection. intriguingly, trim38-mediated sumoylation was later found to also regulate cgas, a foreign dna sensor that functions parallel to rlrs [78] . considering that cgas also forms filament-like structure during viral dna sensing [79] , it is possible that a multimer-specific substrate recognition mechanism, akin to those of riplet and trim65, is involved in virus-dependent sumoylation of cgas and rlrs by trim38. besides rlrs, several other dexd/h-box helicases have been reported to play roles in sensing viral nucleic acids. one such helicase is ddx41, which is an rna helicase previously known to be involved in splicing, translation and many other cellular rna processes [80, 81] . recent studies found that ddx41 is also involved in innate immune response to foreign dsdna [82] [83] [84] . a systematic sirna screen of dexd/h helicases showed that knock-down of ddx41 greatly reduces the level of ifn-β induction in response to poly(da:dt) or hsv-1 [85] . the dead domain of ddx41 can directly bind both dsdna and sting, the signaling adaptor molecule for cgas [85] . the interaction between ddx41 and sting was proposed to result in the activation of tbk1 and the ikk complex for the induction of type i ifns and pro-inflammatory cytokines. interestingly, the stability of ddx41 was reported to be regulated by trim21 [85] . trim21 utilizes pry-spry to bind the dead domain and conjugates ddx41 with k48-linked ub chains (k48-ub n ) for proteasomal degradation [85] . knocking down trim21 enhances the ifn expression upon dsdna stimulation or dna virus infection [85] . much remains to be investigated as to whether trim21 also regulates the stability of ddx41 during its constitutive function in rna processes, and whether their interaction is regulated differently during rna processing vs. dna sensing. more detailed analyses of how trim21 acts on ddx41 and whether this interaction depends on the conformational or oligomeric state of ddx41 upon dna or rna binding may bring new mechanistic insights. mavs is the signaling adaptor molecule for rig-i and mda5. upon interaction with activated rig-i and mda5, mavs forms filaments on the surface of the mitochondria, serving as a signaling platform to recruit and activate further downstream molecules, such as trafs and tbk1 [56, 86] . at least six trim proteins have been reported to regulate mavs. among them, trim21, trim25, and trim14 utilize pry-spry for interaction with mavs [87] [88] [89] [90] . trim21 was reported to conjugate mavs with k27-linked ub chains to facilitate recruitment of tbk1 [88] , while trim25 was found to modify mavs with k48-linked ub chain to promote its degradation [87, 91] . unlike trim21 and trim25, trim14 lacks the ring domain and does not ubiquitinate mavs. instead, trim14 was proposed to function as an adaptor molecule to recruit nf-kb essential modulator (nemo), whip and ppp6c to mavs to potentiate its signaling activity [89, 90] . viral infection was shown to promote trim14's interaction with mavs [90] , raising the possibility that their interaction may depend upon mavs filament formation. additionally, trim40, trim44 and trim31, which do not contain pry-spry, were also reported to interact with mavs and alter mavs's stability and/or signaling activity in both ub-dependent and -independent manners [92] [93] [94] . however, given that mavs forms filaments, which could attract a large number of molecules through specific or nonspecific binding, more detailed investigation is necessary to understand precisely how each trim protein acts on mavs, and how these trim proteins collectively work together for proper functioning of mavs. traf6 is an e3 ligase involved in multiple immune signaling pathways, including those of rlrs, tlrs, il-1 receptor, tgfβ receptors and b-cell receptors. upon activation of these upstream receptors, traf6 is recruited to the receptors or downstream signaling complexes [95, 96] , which triggers its e3 ligase activity to synthesize k63-ub n . while the precise activation mechanism and ubiquitination target of traf6 has been unclear, the current model is that k63-ub n conjugation within the signaling complex (either on traf6 or other targets) enables recruitment of various signaling molecules, leading to the activation of nf-kb, mapk and/or irf pathways [96, 97] . recent studies suggest that trim38 is an important negative regulator of traf6 [98] . trim38 utilizes its pry-spry domain to recognize traf6 and conjugates traf6 with k48-ub n for proteasomal degradation [98] . how the activity of trim38 on traf6 is regulated remains to be further investigated. one potential mechanism is a temporal control of the level of trim38. trim38 is known to be up-regulated by a variety of immune signals, including rlr and tlr pathway activation, which may ensure trim38 to act on traf6 only upon immune signal activation [98] [99] [100] . alternatively, traf6 was proposed to form a higherorder assembly upon its activation [101, 102] , which may function as a signal for trim38 to engage and ubiquitinate traf6 only upon its activation. it also remains to be addressed how, during rlr signaling, trim38 functions as an ub e3 ligase for traf6, while acting as a sumo e3 ligase for rig-i/mda5, and how its positive effect on rig-i/mda5 interplays with its negative effect on traf6. mechanistic details of trim38 functions in the context of the rlr pathway require future studies. irf3 is an essential transcription factor for expressing type i ifns and is broadly involved in many prr signaling pathways in innate immunity, including rlrs, tlrs and cgas pathways. upon activation of these receptors, their respective downstream adaptor molecules (mavs, trif, and sting, respectively) recruit tbk1, which then phosphorylates the c-terminal tail of irf3 [103] [104] [105] . phosphorylated irf3 in turn forms a homodimer and translocates to the nucleus, which results in the transcriptional activation of type i ifns [106] . irf3 has long been known to undergo rapid turnover upon its activation [106] [107] [108] . one mechanism by which the stability of irf3 is regulated is through trim26. a study showed that, upon tlr activation, trim26 interacts with irf3, and conjugates irf3 with k48-ub n for proteasomal degradation [105] . intriguingly, trim26 is translocated to the nucleus upon infection or ifn-β treatment, which allows trim26 to specifically target only the activated irf3, leaving inactive cytosolic irf3 intact. similarly, constitutively active irf3 variants are targeted by trim26, while phosphorylation-defective irf3 or that lacking the nuclear localization signal is not [105] . by targeting only the activated irf3, cells may be able to rapidly shut down the immune signal, while still maintaining the ability to elicit a subsequent immune response in the case of uncontrolled infection or re-infection. the spatial control of trim26 thus shows yet another mechanism by which trim/trim-like proteins are regulated. some trim proteins can function as a direct sensor of viruses and a restriction factor. one well-known example is trim5α, an isoform of trim5 that contains the pry-spry domain [32] . previous studies showed that trim5α from rhesus monkey restricts hiv-1 replication by inducing premature capsid disassembly and inhibiting reverse transcription [109, 110] . it was later found that the restriction activity of trim5α is highly dependent on the species and viral strains/types; human trim5α cannot restrict hiv-1, but it can restrict another retrovirus, n-tropic murine leukemia virus (n-mlv) [111, 112] . the viral specificity of trim5α is determined by the viral capsid and the pry-spry domain of trim5α [109, 113] . a single amino acid change in the hiv-2 capsid rendered it more susceptible to human trim5α [114] . similarly, three amino acid substitution in pry-spry to mimic those of rhesus trim5α allowed human trim5α to restrict hiv-1 [113, 114] . more detailed study of trim5α revealed avidity-dependent binding of hiv-1 capsid, analogous to that of riplet and trim65. hiv-1 capsid protein forms a fullerene-shaped cone, through hexagonal assembly of the capsid hexamers and pentamers [32, 115] . trim5α does not bind free capsid molecules, and instead, recognizes only the pre-assembled capsid lattice [110, 116] (fig. 4a ). viral capsid binding further induces higher-order oligomerization of trim5α into a hexagonal lattice with the symmetry that matches that of the viral capsid (fig. 4a & fig. 4b ), further strengthening their avidity-driven interaction [117, 118] . the lattice formation of trim5α appears to be driven by b-box2, which forms a trimer at the three-fold symmetric vertex of the hexagonal lattice [25, 117] (fig. 4a) . mutations in the b-box2 or cc of trim5α impair its lattice formation and its viral restriction function [119] [120] [121] . precise mechanism by which trim5α's capsid binding leads to viral restriction is yet unclear. upon capsid binding and lattice formation, the e3 ligase activity of trim5α was proposed to be stimulated as the lattice formation would cluster the ring domain and promote its dimerization, the requirement for efficient ub transfer from e2 to the substrate [122] . although whether trim5α can ubiquitinate the capsid is unclear, trim5α binding and lattice formation on the viral capsid is important for proteasomal or autophagic degradation of the capsid proteins and capsid disassembly [123] [124] [125] [126] . trim5α has been shown to conjugate k63-ub n to itself or generate unanchored ub chains [123, 127] , which was proposed to assemble signaling complexes to induce antiviral signaling cascades. while much of these proposed mechanisms remain to be further investigated, trim5α serves as an intriguing example of trims that can function as both a prr and a restriction factor. trim21 (a.k.a. ro52 or ss-a) was first identified as an autoantigen in autoimmune diseases, such as sjogren's syndrome and systemic lupus erythematosus [128] , but more recent studies showed various other immune functions of trim21 in the cytoplasm. when non-enveloped viruses or bacteria are opsonized by antibody, they can be neutralized and cleared by phagocytes. however, opsonized microbial pathogens, especially those partially coated with antibody, can evade extracellular neutralization and can still invade cells. trim21 appears to act on such microbes that enter the cytosolic compartment with antibody attached to their surface [129] (fig. 4c ). trim21 tightly binds fc of igg antibodies, although it also binds fc's of igm and iga with lower affinities [130] . this engagement leads to the auto-ubiquitination of trim21, first with k63-ub n , but gradually involving k48-ub n in the form of mixed or branched chains [131] . auto-ubiquitinated trim21 then recruits the proteasome for degradation of pathogen-associated molecules, such as viral capsid, and neutralization of the pathogen infectivity [129, 131] (fig. 4c ). in parallel, the proteasome-associated deubiquitinating enzyme poh1 liberates k63-ub n from trim21, allowing unanchored k63-ub n to serve as a signal to activate antiviral signaling pathways [131] (fig. 4c) . thus, as with trim5α, trim21 also displays a dual function as a prr and an effector. they, however, differs in that trim5α directly recognizes pamps, while trim21 indirectly recognizes pamps through antibodies. as with other pry-spry containing trim/trim-like proteins, trim21 utilizes pry-spry to bind fc. while individual pry-spry has a low affinity for fc, its bivalency within the dimeric trim21 dramatically increases the affinity for dimeric fc (k d decreases from ~200 to 0.6 nm) [129] . thus, as a dimer, trim21 is the tightest fc receptor known to date. the crystal structure of pry-spry in complex with fc showed that pry-spry binds the hinge interface between the c h 2 and c h 3 domains, recognizing the three-dimensional structure of fc [39, 130] (fig. 4d ). this is unlike other pry-spry domains that recognize a linear peptide sequence [41] . more detailed study suggests that trim21 pry-spry recognizes both the sequence and structure of fc that are conserved across a broad range of vertebrate species [40] . it would be interesting to examine the evolutionary origin of the immune defense mechanism of trim21 that utilizes both innate and adaptive immune components. another intriguing question is whether the fc binding activity of trim21 is also involved in the pathogenesis of the autoimmune diseases, that are associated with anti-trim21 autoantibodies. note that anti-trim21 autoantibodies target the ring and b-box domains of trim21, not the pry-spry domain [132] . the multiple possible modes of interaction between trim21 and anti-trim21 antibodies would undoubtedly endow their immune complex with the unique property to form large insoluble aggregates, which may contribute to the pathogenesis. we here summarized several trim/trim-like proteins that are involved in innate immunity, in particular in rna sensing and viral restriction pathways. from the survey of trim/trim-like proteins with pry-spry, the domain most widely used by trim/trim-like proteins for substrate recognition, a common mechanism for substrate recognition emerges. that is, trim/trim-like proteins often utilize bivalency or multivalency to tightly control their substrate specificity and activity during immune response. many targets of trim/trim-like proteins are the host immune signaling molecules that form oligomers or large assemblies in their activated state [77, 133] . trim/trim-like proteins also target viral molecules that form large assemblies (e.g. capsid) in their pathogenic state. the avidity-driven substrate recognition mechanism of trim/trim-like proteins would thus ensure more precise control of innate immune signaling and restriction functions. furthermore, we also reviewed trim/trim-like proteins that utilize spatial and temporal control of their protein levels as an additional measure to control their activities. these observations thus highlight the complex layers of mechanisms that regulate "the regulators" of innate immunity. despite the increasing appreciation of the importance of trim/ trim-like proteins in innate immunity, molecular mechanisms for many trim/trim-like proteins remain enigmatic. in particular, substrate identity, which is the key to understanding their functions, has been one of the most controversial topic for many trims. while coimmunoprecipitation and in vitro or cellular ubiquitination assay have often been used for validating e3 ligase-substrate relationships, it is important to recognize caveats and limitations in each of these methods. in our own experience, pulling-down full-length e3 ligase to identify or validate a substrate is less ideal than pulling-down ubiquitination-deficient variant (e.g. ring-deletion trim). this is because once ubiquitinated, substrates often recruit many additional partners that would confound efforts to identify direct substrates of the trim/trim-like protein. for cellular ubiquitination assay, one often pulls down a target protein of interest under non-denaturing or mildly denaturing condition (e.g. ripa) followed by anti-ub blot to examine its ubiquitination state. however, proteins purified under such conditions are often fig. 4 . trim5α and trim21 have dual functions as innate immune receptors and effectors. a. schematic of trim5α function. trim5α binds incoming retroviral capsid in a sequence-specific and avidity-dependent manner. capsid then serves as a template to further assemble hexagonal lattice of trim5α. this results in both the effector function of capsid disassembly and degradation as well as the receptor function of antiviral signal activation. ubiquitination activity of trim5α is thought to mediate both of these functions, although some studies proposed a direct role of trim5α as an autophagy receptor [125, 126] . b. cryo-electron microscope reconstruction map of trim5α hexagonal lattice bound to hiv-1 capsid lattice (emd-20565, [32] ). c. schematic of trim21 function. microbial pathogen partially coated with antibody may evade neutralization, and enter the cytosolic compartment. trim21 recognizes the fc portion of the antibody, which triggers the autoubiquitination activity and subsequently its antiviral functions similar to those of trim5α.(d.) crystal structure of trim21 pry-spry in complex with a dimeric fc portion of igg (pdb:2iwg, [39] ). not pure enough to allow confident assignment of anti-ub signal to the target protein of interest. thus, more stringent, fully-denaturing condition is necessary to disrupt large and stable assemblies that are expected for trim's substrates. for in vitro ubiquitination assays, proper specificity control is crucial as the assays often require high protein concentrations to achieve robust ubiquitination signal. while one may choose any arbitrary protein for the control, the best control, in our opinion, is the same target protein in a different conformational or activity state that allows direct comparison of substrate specificity in cells vs. in vitro. such close controls allow more confident assessment of whether the in vitro ubiquitination indeed recapitulates cellular events. besides substrate identity, many exciting questions remain to be addressed. for example, some trim/trim-like proteins have been reported to bind one another [111, 134, 135] or with other e3 ligases (e.g. trim25:lubac, and trim38:traf6), raising a question of how their interactions modify functions of individual e3 ligases. additionally, the role of trim/trim-like proteins has so far been attributed predominantly to ub/ubl modification. however, recent studies suggest that ub/ubl-independent activities also exist [31, 123] . we anticipate many new discoveries on functions, mechanisms and regulations of trim/ trim-like proteins as the field continues to move forward. authors declare no conflict of interests. pathogen recognition by the innate immune system ips-1, an adaptor triggering rig-i-and mda5-mediated type i interferon induction cardif is an adaptor protein in the rig-i antiviral pathway and is targeted by hepatitis c virus identification and characterization of mavs, a mitochondrial antiviral signaling protein that activates nf-kappab and irf 3 visa is an adapter protein required for virus-triggered ifn-beta signaling sting is an endoplasmic reticulum adaptor that facilitates innate immune signalling the adaptor protein mita links virus-sensing receptors to irf3 transcription factor activation mpys is required for ifn response factor 3 activation and type i ifn production in the response of cultured phagocytes to bacterial second messengers cyclic-di-amp and cyclic-di-gmp eris, an endoplasmic reticulum ifn stimulator, activates innate immune signaling through dimerization prion-like polymerization in immunity and inflammation dynamic regulation of innate immunity by ubiquitin and ubiquitin-like proteins ubiquitin signaling in immune responses the demographics of the ubiquitin system human trim gene expression in response to interferons the e3-ligase trim family of proteins regulates signaling pathways triggered by innate immune pattern-recognition receptors rhesus trim5alpha disrupts the hiv-1 capsid at the inter-hexamer interfaces surveillance for intracellular antibody by cytosolic fc receptor trim21 the tripartite motif family identifies cell compartments genomic analysis of the trim family reveals two groups of genes with distinct evolutionary properties the tripartite motif: structure and function the interferon-inducible ubiquitin-protein isopeptide ligase (e3) efp also functions as an isg15 e3 ligase innate immunity to rna virus is regulated by temporal and reversible sumoylation of rig-i and mda5 functional role of trim e3 ligase oligomerization and regulation of catalytic activity mechanism of trim25 catalytic activation in the antiviral rig-i pathway mechanism of b-box 2 domain-mediated higher-order assembly of the retroviral restriction factor trim5alpha structural determinants of trim protein function structural insights into the trim family of ubiquitin e3 ligases the tripartite motif coiled-coil is an elongated antiparallel hairpin dimer molecular mechanism of influenza a ns1-mediated trim25 recognition and inhibition trim5alpha spry/coiled-coil interactions optimize avid retroviral capsid recognition ubiquitin-dependent and -independent roles of e3 ligase riplet in innate immunity hierarchical assembly governs trim5alpha recognition of hiv-1 and retroviral capsids trim family: pleiotropy and diversification through homomultimer and heteromultimer formation trim family proteins and their emerging roles in innate immunity trim family proteins: roles in autophagy, immunity, and carcinogenesis genomics and evolution of the trim gene family relationship between spry and b30.2 protein domains. evolution of a component of immune defence structure and function of the spry/b30.2 domain proteins involved in innate immunity structural basis for pryspry-mediated tripartite motif (trim) protein function trim21 is an igg receptor that is structurally, thermodynamically, and kinetically conserved the trim14 pryspry domain mediates protein interaction via its basic interface structure of the rhesus monkey trim5alpha pryspry domain, the hiv capsid recognition module structural basis for protein recognition by b30.2/spry domains rna-binding activity of trim25 is mediated by its pry/spry domain and is required for ubiquitination the rna helicase rig-i has an essential function in double-stranded rna-induced innate antiviral responses the chase for the rig-i ligand-recent advances antiviral immunity via rig-i-mediated recognition of rna bearing 5'-diphosphates a minimal rna ligand for potent rig-i activation in living mice rna dimerization promotes pkr dimerization and activation innate immunity induced by composition-dependent rig-i recognition of hepatitis c virus rna nucleotide sequences and modifications that determine rig-i/ rna binding and signaling activities rnase l amplifies interferon signaling by inducing protein kinase r-mediated antiviral stress granules structural basis for the activation of innate immune patternrecognition receptor rig-i by viral rna structural basis for ubiquitin-mediated antiviral signal activation by rig-i ubiquitin-induced oligomerization of the rna sensors rig-i and mda5 activates antiviral innate immune response molecular imprinting as a signal-activation mechanism of the viral rna sensor rig-i mavs recruits multiple ubiquitin e3 ligases to activate antiviral signaling cascades riplet, and not trim25, is required for endogenous rig-i-dependent antiviral responses the ubiquitin ligase riplet is essential for rig-i-dependent innate immune responses to rna virus infection trim25 ring-finger e3 ubiquitin ligase is essential for rig-i-mediated antiviral activity nuclear trim25 specifically targets influenza virus ribonucleoproteins to block the onset of rna chain elongation trim25 is required for the antiviral activity of zinc finger antiviral protein trim25 enhances the antiviral action of zinc-finger antiviral protein (zap) linear ubiquitin assembly complex negatively regulates rig-i-and trim25-mediated type i interferon induction reconstitution of the rig-i pathway reveals a signaling role of unanchored polyubiquitin chains in innate immunity rig-i forms signaling-competent filaments in an atp-dependent, ubiquitin-independent manner distinct rig-i and mda5 signaling by rna viruses in innate immunity mda5 is critical to host defense during infection with murine coronavirus coronavirus nonstructural protein 15 mediates evasion of dsrna sensors and limits apoptosis in macrophages length-dependent recognition of double-stranded ribonucleic acids by retinoic acid-inducible gene-i and melanoma differentiation-associated gene 5 differential roles of mda5 and rig-i helicases in the recognition of rna viruses kinetic mechanism for viral dsrna length discrimination by mda5 filaments structural basis for dsrna recognition, filament formation, and antiviral signal activation by mda5 the salmonella effector protein sopa modulates innate immune responses by targeting trim e3 ligase family members trim65-catalized ubiquitination is essential for mda5-mediated antiviral innate immunity structural analysis of rig-i-like receptors reveals ancient rules of engagement between diverse rna helicases and trim ubiquitin ligases smocs: supramolecular organizing centres that control innate immunity sumoylation promotes the stability of the dna sensor cgas and the adaptor sting to regulate the kinetics of response to dna virus hopfner, cgas senses long and hmgb/tfam-bound u-turn dna by forming protein-dna ladders inherited and somatic defects in ddx41 in myeloid neoplasms the dead-box rna helicase ddx41 is a novel repressor of p21(waf1/cip1) mrna translation the helicase ddx41 senses intracellular dna mediated by the adaptor sting in dendritic cells the helicase ddx41 recognizes the bacterial secondary messengers cyclic di-gmp and cyclic di-amp to activate a type i interferon immune response the cytosolic sensor, ddx41, activates antiviral and inflammatory immunity in response to stimulation with double-stranded dna adherent cells of the olive flounder, paralichthys olivaceus the e3 ubiquitin ligase trim21 negatively regulates the innate immune response to intracellular doublestranded dna how rig-i like receptors activate mavs cyclophilin a-regulated ubiquitination is critical for rig-i-mediated antiviral immune responses trim21 promotes innate immune response to rna viral infection through lys27-linked polyubiquitination of mavs assembly of the whip-trim14-ppp6c mitochondrial complex promotes rig-i-mediated antiviral signaling trim14 is a mitochondrial adaptor that facilitates retinoic acid-inducible gene-ilike receptor-mediated innate immune response mavs ubiquitination by the e3 ligase trim25 and degradation by the proteasome is involved in type i interferon production after activation of the antiviral rig-i-like receptors novel function of trim44 promotes an antiviral response by stabilizing visa the ubiquitin e3 ligase trim31 promotes aggregation and activation of the signaling adaptor mavs through lys63-linked polyubiquitination the e3 ubiquitin ligase trim40 attenuates antiviral immune responses by targeting mda5 and rig-i traf molecules in cell signaling and in human diseases immune control by traf6-mediated pathways of epithelial cells in the eime (epithelial immune microenvironment) activation of the ikappab kinase complex by traf6 requires a dimeric ubiquitin-conjugating enzyme complex and a unique polyubiquitin chain e3 ubiquitin ligase tripartite motif 38 negatively regulates tlr-mediated immune responses by proteasomal degradation of tnf receptor-associated factor 6 in macrophages tripartite motif-containing protein 38 negatively regulates tlr3/4-and rig-i-mediated ifn-beta production and antiviral response by targeting nap1 trim38 negatively regulates tlr3/4-mediated innate immune and inflammatory responses by two sequential and distinct mechanisms e2 interaction and dimerization in the crystal structure of traf6 oligomerization-primed coiled-coil domain interaction with ubc13 confers processivity to traf6 ubiquitin ligase activity triggering the interferon response: the role of irf-3 transcription factor triggering the interferon antiviral response through an ikk-related pathway trim26 negatively regulates interferon-beta production and antiviral response through polyubiquitination and degradation of nuclear irf3 virus-dependent phosphorylation of the irf-3 transcription factor regulates nuclear translocation, transactivation potential, and proteasome-mediated degradation involvement of the ikappab kinase (ikk)-related kinases tank-binding kinase 1/ikki and cullin-based ubiquitin ligases in ifn regulatory factor-3 degradation negative regulation of interferon-regulatory factor 3-dependent innate antiviral response by the prolyl isomerase pin1 the cytoplasmic body component trim5alpha restricts hiv-1 infection in old world monkeys specific recognition and accelerated uncoating of retroviral capsids by the trim5alpha restriction factor unique features of trim5alpha among closely related human trim family members role of human trim5alpha in intrinsic immunity species-specific variation in the b30.2(spry) domain of trim5alpha determines the potency of human immunodeficiency virus restriction a single amino acid change in the spry domain of human trim5alpha leads to hiv-1 restriction the structural biology of hiv assembly trim5alpha selectively binds a restriction-sensitive retroviral capsid the trim5alpha b-box 2 domain promotes cooperative binding to the retroviral capsid by mediating higher-order self-association hexagonal assembly of a restricting trim5alpha protein modulation of retroviral restriction and proteasome inhibitor-resistant turnover by changes in the trim5alpha b-box 2 domain a b-box 2 surface patch important for trim5alpha selfassociation, capsid binding avidity, and retrovirus restriction determinants for the rhesus monkey trim5alpha-mediated block of the late phase of hiv-1 replication ring dimerization links higher-order assembly of trim5alpha to synthesis of k63-linked polyubiquitin trivalent ring assembly on retroviral capsids activates trim5 ubiquitination and innate immune signaling trim5alpha-mediated ubiquitin chain conjugation is required for inhibition of hiv-1 reverse transcription and capsid destabilization trim proteins regulate autophagy and can target autophagic substrates by direct recognition trim proteins regulate autophagy: trim5 is a selective autophagy receptor mediating hiv-1 restriction trim5 is an innate immune sensor for the retrovirus capsid lattice the immunobiology of ro52 (trim21) in autoimmunity: a critical review antibodies mediate intracellular immunity through tripartite motif-containing 21 (trim21) trim21: a cytosolic fc receptor with broad antibody isotype specificity sequential ubiquitination and deubiquitination enzymes synchronize the dual sensor and effector functions of trim21 structural, functional and immunologic characterization of folded subdomains in the ro52 protein targeted in sjogren's syndrome filament-like assemblies of intracellular nucleic acid sensors: commonalities and differences ubiquitination of e3 ubiquitin ligase trim5 alpha and its potential role transcription cofactors trim24, trim28, and trim33 associate to form regulatory complexes that suppress murine hepatocellular carcinoma the authors acknowledge nih r01s (ai154653 and ai111784 to sh) and roche post-doctoral fellowship (to hw) for supporting this work. key: cord-311823-85wj08gr authors: katze, michael g.; fornek, jamie l.; palermo, robert e.; walters, kathie-anne; korth, marcus j. title: innate immune modulation by rna viruses: emerging insights from functional genomics date: 2008 journal: nat rev immunol doi: 10.1038/nri2377 sha: doc_id: 311823 cord_uid: 85wj08gr although often encoding fewer than a dozen genes, rna viruses can overcome host antiviral responses and wreak havoc on the cells they infect. some manage to evade host antiviral defences, whereas others elicit an aberrant or disproportional immune response. both scenarios can result in the disruption of intracellular signalling pathways and significant pathology in the host. systems-biology approaches are increasingly being used to study the processes of viral triggering and regulation of host immune responses. by providing a global and integrated view of cellular events, these approaches are beginning to unravel some of the complexities of virus–host interactions and provide new insights into how rna viruses cause disease. viruses can have a devastating effect despite their small genomes. all rna viruses encode proteins that are essential for structural components and replication, and most encode proteins that function to circumvent host antiviral responses [1] [2] [3] . this limited number of proteins is sufficient to ensure the entry, replication and subsequent spread of the virus. however, viruses do not self-propagate and depend on various host-cell functions to complete their life cycle. the processes of viral entry, the triggering and regulation of the host antiviral response and subsequent viral replication together result in an intricate series of interactions between virus and host. much can be learnt about the nature and complexities of these interactions by global profiling of the transcriptional changes in host cells that occur during viral infection (box 1) . in this review, we discuss how functional genomic and systems-biology approaches are contributing to our understanding of interactions between rna viruses and the host, of viral pathogenesis and of host immunity to infection. rather than providing a comprehensive literature review, we present examples of how these approaches are providing insight into the interaction of viruses with innate immune defence mechanisms, the evaluation of therapeutics that target these pathways and the crucial balance between protective immune responses and immunopathology. in addition, we describe how genomic approaches are being applied to vaccine evaluation and design, and how these approaches can be combined with other high-throughput technologies to provide an improved and integrated systems-biology view of virus infection. although genomic approaches are being used to study a wide variety of viruses, we highlight the current literature through discussion of a select few. among these is influenza virus, for which the looming threat of a new pandemic and concerns regarding therapeutic and vaccine preparedness have stimulated exciting new research efforts. we also review findings relating to hepatitis c virus (hcv) infection, for which genomic analyses are being used to shed light on the response of patients to treatment with type i interferons (ifns) and the relationship between hcv replication and liver disease. in addition, we highlight studies of west nile virus, severe acute respiratory syndrome-associated coronavirus (sars-cov) and ebola virus, all of which have revealed previously undescribed strategies used by these viruses to regulate innate immunity. finally, we discuss how genomic approaches are being applied to vaccine evaluation and how genomics is being combined with other high-throughput approaches to provide a systems-biology view of virus-host interactions. viruses and innate immunity a variety of cellular signalling networks have evolved in host cells to detect and respond to viral infection. one area in which genomics-based analyses are being put to abstract | although often encoding fewer than a dozen genes, rna viruses can overcome host antiviral responses and wreak havoc on the cells they infect. some manage to evade host antiviral defences, whereas others elicit an aberrant or disproportional immune response. both scenarios can result in the disruption of intracellular signalling pathways and significant pathology in the host. systems-biology approaches are increasingly being used to study the processes of viral triggering and regulation of host immune responses. by providing a global and integrated view of cellular events, these approaches are beginning to unravel some of the complexities of virus-host interactions and provide new insights into how rna viruses cause disease. these genes contain interferon (ifn)-responsive promoters and are responsible for the antiviral, antiproliferative and immunomodulatory properties of ifn. over 400 such genes have been identified by microarray analysis. some, such as protein kinase r, ribonuclease l, mx1 (myxovirus resistance 1) and isg15 (ifn-stimulated protein of 15 kda), have well documented antiviral activities, but the precise biological function of the majority of these genes is unknown. particularly good use is in shedding new light on the components of innate antiviral defence mechanisms and the viral strategies used to overcome them. in this section, we review recent studies in which genomic approaches have been used to provide new information on how viruses trigger and regulate innate immune pathways, and to evaluate the use of type i ifn-based therapy as a means to enhance the innate immune response to hcv. mammalian cells have specialized proteins that are responsible for the recognition of virus infection, and other proteins that elicit responses to combat the invading virus. the antiviral response is triggered when host pathogen-recognition receptors (prrs) are engaged by pathogen-associated molecular patterns (pamps) in viral proteins and nucleic acids (reviewed in refs 4, 5) . prrs that function in virus recognition include the cytosolic double-stranded rna helicases retinoic-acid-inducible gene i (rig-i) and mda5 (melanoma differentiation-associated gene 5) and certain toll-like receptors (tlrs) that are present on the cell surface or in endosomal membranes. after binding to viral pamps, prrs initiate intracellular signalling cascades that result in the activation of transcription factors, including ifn-regulatory factors (irfs) and nuclear factor-κb (nf-κb). these transcription factors in turn regulate the expression of hundreds of genes, such as ifns and ifn-stimulated genes (isgs) 6, 7 , and pro-inflammatory cytokines and chemokines that are involved in the orchestration of the adaptive immune response (fig. 1) . one way in which gene-expression profiling has been used to examine this aspect of the antiviral response is through the use of mouse embryonic fibroblasts deficient in rig-i or mda5. a recent study demonstrated that west nile virus infection of wild-type cells led to the induction of irf3 target genes and isgs, including several subtypes of ifnα (ref. 8) . this was followed by a second phase of ifn-dependent antiviral gene expression that occurred at a later stage of infection. by contrast, cells lacking rig-i had delayed or inhibited initial and secondary gene-expression responses to the virus, indicating that rig-i has an essential but not exclusive role in initiating innate immune responses to west nile virus (fig. 2) . the additional deletion of mda5 in these cells was found to further block their ability to respond to infection, indicating that the host immune response to west nile virus also involves mda5. this is a noteworthy finding, as previous studies suggested that rig-i and mda5 recognized a specific subset of viruses, rather than acting cooperatively as found in the response to west nile virus 9 . the role of rig-i in the response to influenza virus infection has also been assessed 10 . similar to west nile virus, genomic analysis of influenza virus-infected wild-type and rig-i-deficient mouse embryonic fibroblasts revealed that rig-i is necessary for the type i ifn response to this virus (fig. 2) . in rig-i-deficient cells, influenza virus fails to elicit the expression of ifnβ and of many isgs, including key antiviral mediators such as irf3, stat1 (signal transducer and activator of transcription 1), ifit1 (ifn-induced protein with tetratricopeptide repeats 1; also known as isg56) and isg54 (also known as ifit2). this study also showed that, unlike during infection with west nile virus, mda5 does not function as a secondary mediator of the response to infection with influenza virus 10 . important next steps in these studies will be to compare the profiles of genes induced by each of these viruses -and to determine whether some genes are specific for rig-i or mda5 signalling -and to begin to define the involvement of these genes in innate immunity. although this biological validation process will be necessary to follow-up genomic analyses, few studies so far have included such experiments. functional genomic analyses have also been helpful in elucidating the complex transcriptional events triggered following tlr signalling. tlrs are expressed by various immune cells, including macrophages, dendritic cells and lymphocytes, and a subset of these receptors are involved in viral recognition. so far, genomic studies have largely focused on the analysis of macrophages treated with tlr ligands, such as lipopolysaccharide (lps; a component of the cell wall of gram-negative bacteria) or polyinosinic-polycytidylic acid (a synthetic mimic of viral double-stranded rna, dsrna) [11] [12] [13] . to obtain a comprehensive view of the transcriptional programmes that are induced by tlr activation, elkon et al. used a computational approach to analyse geneexpression data sets derived from four studies in which human or mouse macrophages were stimulated with pathogen-mimetic agents that engage various tlrs 14 . this analysis identified one transcriptional profile that is universally activated by all tlrs and a second profile that is specific to both tlr3 (which specializes in the recognition of viral dsrna) and tlr4 (which recognizes genomics is broadly defined as the study of genomes. the term was first adopted nearly 20 years ago to describe the emerging discipline of using nucleotide sequencing, gene mapping and computational biology to define the structure and organization of a genome 110 . as ever increasing amounts of nucleotide sequence information have become available, the focus of genomics has expanded to include gene function 93 . the human genome project was a driving force in advancing both structural and functional genomics, and the nucleotide sequence information generated by this project has fuelled tremendous advances in our understanding of human health and disease. one way in which this has occurred is through the convergence of comprehensive genome sequence information with advances in high-throughput technology. today, the standard technology in functional genomics is the oligonucleotide microarray [111] [112] [113] . several alternative platforms are available, with the most common being microarrays for which thousands of oligonucleotide 'probes', each corresponding to an mrna transcript, are synthesized in situ directly on a glass slide. such microarrays enable researchers to simultaneously measure the expression of virtually all genes in a genome. for 'target' preparation, mrna is extracted from experimental samples and labelled with fluorescent dyes by reverse transcription. the labelled target is then hybridized with the microarray, and the fluorescence of the features is determined using an array scanner. following image analysis, the data are subjected to a variety of bioinformatic processes to identify statistically significant changes in gene expression between samples. because each comparison yields tens of thousands of data points, mining the data for biological meaning is a formidable challenge. a variety of sophisticated commercial and open-source analysis tools are therefore used to find relationships between differentially expressed genes, to identify networks or signalling pathways that are activated or repressed and to compare gene-expression profiles between experimental samples. envelope components of viruses and cell-surface components of bacteria (such as lps)). a computational analysis of promoter sequences identified nf-κb as the key regulator of the universal response, which occurs early after tlr stimulation, and the ifn-stimulated response element (isre) as the key component of the tlr3 and tlr4 response, which is induced after the nf-κb response. this computational approach provided additional knowledge regarding the kinetics of the tlr3 and tlr4 response, the regulatory circuitry involved and the identity of the genes figure 1 | stimulation of interferon-stimulated gene expression and initiation of antiviral activity. pathogenassociated molecular patterns (pamps) in viral proteins and nucleic acids are recognized by cellular pathogen-recognition receptors (prrs) that include rig-i (retinoic-acid-inducible gene i), mda5 (melanoma differentiation-associated gene 5) and certain toll-like receptors (tlrs). prr-pamp interactions trigger signalling cascades that result in the activation of transcription factors, including interferon (ifn)-regulatory factor 3 (irf3) and nuclear factor-κb (nf-κb), which induce the production of type i ifns, ifn-stimulated genes (isgs) and pro-inflammatory cytokines and chemokines. the specific process differs between antigen-presenting cells, in which both the tlr pathway and the rig-i or mda5 pathway are operative, and other cell types, in which only the rig-i or mda5 pathway is present. activation of prr signalling induces an antiviral state in all cell types, and in antigen-presenting cells it can also induce the production of pro-inflammatory cytokines and chemokines. this normally results in an innate antiviral response that controls infection until it is resolved by the adaptive immune response. however, some viruses, such as the 1918 pandemic influenza virus, elicit an aberrant or disproportional response that results in immunopathology. alternatively, viruses that suppress the type i ifn response can subvert the mechanisms of innate surveillance and diminish the potential adaptive immune response, resulting in a chronic infection. for vaccine strategies, the best induction of a broad adaptive immune response might require some degree of type i ifn response in the initial stages of infection. dcs, dendritic cells; dsrna, double-stranded rna; ifnar, ifnα receptor; il, interleukin; ips1, ifnb-promoter stimulator 1; oas, 2′,5′-oligoadenylate synthetase; pkr, protein kinase r; ssrna, single-stranded rna; stat, signal transducer and activator of transcription; tap1, transporter associated with antigen processing 1; tnf, tumour-necrosis factor. chimeric scid-alb/upa mouse model a chimeric mouse model of severe combined immunodeficient (scid) mice that contain a urokinase plasminogen activator transgene driven by an albumin promoter (alb/upa). these mice can be transplanted with human hepatocytes to generate chimeric mousehuman livers, providing the only small-animal infection model for hepatitis c virus infection. activated in both the universal and tlr3-and tlr4mediated responses. although these studies have provided considerable information regarding the genes activated downstream of tlr activation, it will be advantageous to extend genomic analyses in the context of viral infection using cells lacking the expression of specific tlrs. the ability of a virus to establish an infection depends, at least to some extent, on its ability to block the host innate immune response or to modulate the activity of antiviral effector proteins. hcv is one example of a virus that has devised a means to block the initial triggering of the host innate immune response. several studies have shown that the hcv ns3-ns4a serine protease blocks the tlr3-dependent activation of irf3 (refs 17, 19) . this is achieved by ns3-ns4a-mediated cleavage of trif (toll/interleukin-1 (il-1) receptor-domain-containing adaptor protein inducing ifnβ), an adaptor protein that links tlr3 to kinases that are responsible for activating irf3 and nf-κb 17, 19 . hcv also inhibits the ability of rig-i to activate irf3 (refs 15,16,18,20), which is achieved through ns3-ns4a-mediated cleavage of ips1 (ifnb-promoter stimulator 1; also known as visa, cardif, mavs), a recently identified rig-i adaptor protein [21] [22] [23] [24] [25] . in light of these findings, it is both perplexing and paradoxical that virtually all gene-expression profiling carried out using hcv-infected tissue shows the induction of isg expression, including irf3 target genes [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] . the induction of isg expression is observed in liver tissue from hcv-infected patients 30, 32, 37 and during the initial host response in acutely infected chimpanzees 26, 28 , and is a major part of the transcriptional response to hcv infection in the chimeric scid-alb/upa mouse model 33 . this poses an interesting question about the source of both type i ifns and isg expression. it is possible that isgs are mainly expressed in uninfected hepatocytes and are induced in response to exogenous type i ifn released from adjacent hcv-infected cells. alternatively, it has been suggested that t cells and plasmacytoid dendritic cells that infiltrate the liver are a possible source of hepatic type i ifns 37 . although this is possible, it is relevant to note that hcv infection in the scid-alb/upa mouse model is also associated with the induction of hepatic isg expression in the absence of these immune cell types 33 . other genomic studies have revealed examples of highly virulent viruses that are relatively successful at inhibiting isg expression. perhaps the best example is a characterization of the host transcriptional response of human liver cells infected with filoviruses 38 . this study demonstrated the marked suppression of genes in key innate antiviral pathways, including those mediated by irf3. interestingly, this study also suggested a correlation between the antagonism of the type i ifn response and filovirus virulence. highly virulent viruses, such as zaire ebola virus and marburgvirus, inhibit the expression of most isgs that are induced in uninfected ifn-treated cells. by contrast, the relatively non-pathogenic reston ebola virus is less inhibitory and induces the expression of more than 20% of these genes. the suppression of the type i ifn response by the pathogenic viruses is associated with more rapid viral spread and higher rate of viral replication than that observed during reston ebola virus infection. a comparable trend was seen in a study evaluating the host transcriptional response and inflammation in the brains of mice infected with rabies virus 39 . this study revealed that infection with an attenuated virus results in both inflammation and the induction of expression of key isgs. however, these events are either absent or diminished during infection with a highly pathogenic rabies virus. on the basis of results with filoviruses, it would follow that attenuation of the type i ifn response would be associated with higher viral replication and spread in the case of pathogenic infection with rabies virus; however, this was not measured in the study. similarly, infection with highly virulent pseudorabies virus suppressed the induction of a subset of isgs, even in type i ifn-treated cells 40 . together, these data suggest that the virulence of acute, highly pathogenic viruses is at least partially related to their ability to suppress the host antiviral response, which seems to allow higher levels of viral replication. genomic analyses using cells that lack rig-i (retinoic-acid-inducible gene i) show the requirement for this pathogen-recognition receptor in the induction of interferonregulatory factor 3 (irf3) target genes and interferon-stimulated genes (isgs) by west nile virus and influenza virus. a | the infection of rig-i-deficient cells by west nile virus results in the delay and partial inhibition of isg expression. deletion of mda5 (melanoma differentiation-associated gene 5) further blocks the response to infection (not shown), indicating that the response to west nile virus also involves mda5. b | by contrast, the infection of rig-i-deficient cells by influenza virus results in a near complete inhibition of isg expression that is not further blocked by the absence of mda5, suggesting that mda5 does not mediate influenza virus-induced gene-expression changes. pamps, pathogen-associated molecular patterns. images generated from data in refs 8, 10. suppression of innate immunity and persistent infection. evidence discussed in this review suggests that suppression of elements of the innate immune response enables extensive viral replication and increased pathogenesis. does the converse hold true for a virus such as hcv, which typically establishes a persistent infection characterized by mild (or slowly progressing) disease? some evidence suggests that this might be the case; for example, studies using the chimeric scid-alb/upa mouse model indicate that an attenuated type i ifn response is associated with higher levels of intrahepatic hcv replication together with a greater induction of lipid metabolism and oxidative-stress genes, which have the potential to cause cytopathic effects 33 . similarly, gene-expression profiling of serial liver biopsies obtained from patients that had received an hcv-infected liver transplant shows that rapid progression of fibrosis following transplantation is associated with the suppression of genes involved in the type i ifn response, antigen presentation and the cytotoxic t-cell response 30 . although in these studies the apparent defect in the host antiviral response is probably related to host genetics rather than viral factors, the concept that a defective innate immune response correlates with enhanced pathogenesis is still evident. it is possible that the selective pressures on persistent viruses never resulted in a need for a complete subversion of host innate antiviral responses, so such viruses use these responses to limit their replication to a level that does not significantly affect the normal functions of the host cell. conversely, acute viruses, such as filoviruses, highly pathogenic influenza virus and rabies virus, seem to have evolved to antagonize these responses following cell entry to allow immediate, high levels of replication, which subsequently facilitate virus spread and transmission. given the importance of the innate immune response in regulating virus infection, there is considerable interest in enhancing or modulating this response for therapeutic benefit. one role for genomics in this area is assisting in the evaluation of type i ifn treatment of hcv infection. combination therapy with ifnα and the antiviral drug ribavirin results in virus clearance in only ~50% of individuals infected with hcv genotype 1 and ~80% of individuals infected with hcv genotypes 2 or 3 (refs 41-44). as ifnα is the only approved treatment for chronic hcv, there is strong interest in improving this therapy, in understanding the molecular mechanisms that underlie treatment failure and in identifying markers to accurately predict a patient's response to treatment (that is, responders or non-responders). several groups that have used transcriptional profiling of patient hepatic tissue to address these issues have found that higher levels of expression of isgs before treatment are associated with treatment failure. for example, chen et al. carried out microarray experiments on pretreatment liver tissue obtained from a cohort of 31 patients with chronic hcv infection who subsequently underwent ifnα and ribavirin therapy 45 . this analysis identified a set of 18 genes, many of which are known isgs; in general these genes were more highly induced in the livers of patients that did not respond to therapy. although the authors suggest that this set of genes could therefore be used to predict the response to therapy, it remains to be determined whether they can be used to accurately predict the response in other patient cohorts. similarly, feld et al. showed that non-responders have significantly higher intrahepatic pretreatment expression levels of isgs than patients who respond to type i ifn therapy 46 . although these studies are intriguing, it is still unclear whether there is a causal relationship between higher pretreatment levels of isgs and therapy failure. other factors, such as viral quasispecies diversity, may also be important. owing to the technical and ethical issues of obtaining sufficient liver material for gene-expression studies, investigators have also used peripheral-blood mononuclear cells (pbmcs) to evaluate the response to treatment 47,48 . an example is virahepc, a multicentre study designed to define the differences in response rates among caucasian and african americans and to identify host and viral parameters associated with a lack of response to treatment 48 . overall, this study showed that, during the first 28 days of treatment, a lower level of induction of known isgs is associated with non-responsiveness to type i ifn treatment. however, in many cases, these differences are not strikingly dissimilar between responders and non-responders. the implication of such minor differences with respect to antiviral function is uncertain and the feasibility of using them for predicting a patient's response is questionable. in addition, analyses using pbmcs should be interpreted with caution, as a recent study showed that the transcriptional response to type i ifn treatment is significantly different in the blood and the liver of hcv-infected chimpanzees, presumably owing to the absence of hcv replication in pbmcs 49 . although it has not yet been evaluated, this will almost certainly hold true for humans as well. an alternative mechanism of a failed response to type i ifn treatment could involve the induction of genes associated with ifn inhibitory pathways 46 . walsh et al. found significantly increased intrahepatic expression of the gene encoding suppressor of cytokine signalling 3 (socs3) in patients who did not respond to type i ifn treatment 50 . enhanced intrahepatic socs3 expression is also thought to contribute to the non-responsiveness of hcv-infected chimpanzees to type i ifn therapy 51 . however, a separate evaluation of 21 patients for intrahepatic socs3 mrna expression before antiviral therapy actually found higher levels of expression in those patients who went on to respond successfully to type i ifn treatment 51 . therefore, the relationship between treatment failure and induction of type i ifn inhibitory pathways is currently less clear than that between higher pretreatment levels of expression of isgs and treatment failure. there are still surprisingly few answers to the fundamental question of how virus infection results in disease pathology. although the mechanisms are certain to be different for each virus, a common theme is that there is abarrently high and sustained nature reviews | immunology infection resolves immunopathology a crucial balance between protective immune responses and immunopathology 52,53 . although the innate immune response is designed to target and eliminate invading pathogens, genomic analyses have indicated that some viruses, such as the highly virulent influenza virus that was responsible for the 1918 pandemic, elicit aberrant or disproportional innate immune responses that may also harm the host. the 1918 influenza virus pandemic (known as the spanish flu) killed as many as 50 million people worldwide 54 , and several studies have begun to provide clues to what made this virus so deadly (reviewed in . although genomic analyses have previously been carried out using engineered viruses containing one or more genes from the 1918 pandemic virus 58,59 , a major advance in the ability to study this virus came from its reconstruction based on nucleotide sequence information 60 . genomic analyses of lung or bronchial tissue derived from mice or macaques that were infected with the reconstructed 1918 virus indicate how the beneficial role of the innate immune response can be tipped towards immunopathology. mice infected with the reconstituted 1918 influenza virus show severe pulmonary pathology and an increased and accelerated transcriptional activation of immuneresponse genes 61 . this includes a marked activation of genes associated with pro-inflammatory and cell-death pathways by 24 hours after infection (fig. 3) , which remain unabated until the death of the animals. this response is in contrast to the less dramatic and delayed host immune responses (and less severe disease pathology) in mice that were infected with influenza viruses containing only subsets of genes from the 1918 virus, including the haemagglutinin (ha) and non-structural protein (ns) genes, or the ha, neuraminidase (na), matrix (m) and nucleoprotein (np) genes. these findings suggest that enhanced pro-inflammatory and cell-death responses can contribute to severe immunopathology. an additional study that evaluated the host response to the 1918 influenza virus using a cynomologus macaque (macaca fascicularis) infection model produced similar results 62 . in macaques, the 1918 virus replicates to high levels and spreads rapidly throughout the respiratory tract of infected animals, causing severe lung damage and the massive infiltration of immune cells throughout the course of infection. functional genomic analyses of bronchial tissue revealed that the 1918 virus triggers the aberrantly high and sustained expression of numerous genes involved in the innate immune response, including pro-inflammatory cytokines and chemokines. although the timing of the response is somewhat different, the increased and sustained host response in macaques that were infected with the 1918 virus is similar to that observed in mice. these studies reveal similarities and differences in the host response to contemporary and 1918 pandemic influenza virus infection. first, contemporary and 1918 viruses each trigger an innate immune response that includes the expression of nf-κb and irf3 target genes, which is expected to occur if the virus triggers the rig-i pathway in infected respiratory cells. second, both viruses trigger a robust cytokine response that probably attracts immune-cell infiltration to infected tissues. unlike contemporary virus strains, in which the early response to infection is resolved, the innate immune response triggered by the 1918 virus is characterized by a strong and sustained induction that is associated with massive tissue damage and death of the infected animal. however, in preliminary genomic analyses carried out with lung tissue from macaques that were infected with avian h5n1 viruses, we have found that there are significant differences in the regulation of antiviral responses by the 1918 pandemic and h5n1 viruses (j. c. kash and m.g.k., unpublished observations). therefore, there may be differences in the ways in which highly pathogenic influenza viruses regulate the innate immune response and cause disease. the enhanced pathogenicity of the 1918 and h5n1 influenza viruses might be attributed to distinct components of their genomes. although much emphasis has been placed on the ns1 protein of the 1918 virus acting as an inhibitor of the type i ifn response, recent evidence suggests that the viral proteins pb1 (a polymerase), ha and na contribute to its pathogenicity 63 . likewise, the polymerases of h5n1 viruses have been linked to increased viral pathogenesis 64 , suggesting that the increased pathogenesis of these viruses may be related to their replicative fitness. another respiratory virus, sars-cov, has emerged recently and has caused great concern among the public health and research communities. it has been suggested that disease pathology associated with sars-cov is caused by a disproportional immune response, illustrated by increased levels of pro-inflammatory cytokines and chemokines [65] [66] [67] . studies carried out in our laboratory have combined the use of functional genomics with a cynomologus macaque infection model to study the host response to this virus 68 . we observed that sars-cov-infected macaques show a strong increase in the expression of innate immune response genes early after infection and that this response wanes after 4 days. conversely, genes that are induced later in infection tend to be involved in the cell cycle and in cell repair. none of the animals used in this study succumbed to infection, and sars-cov-induced pathology in these macaques resembled the pathological changes seen in the majority of human patients with sars who recover from the disease 68 . unlike the findings of the 1918 pandemic influenza virus study, these data suggest that early immune responses to sars-cov infection are productive and enable the host to properly fight the virus, allowing a return to cellular homeostasis. however, in the 10% of human infections in which sars-cov infection is fatal (mostly in the elderly), it is possible that the timing or magnitude of the response results in immunopathology. studies using aged macaques might help to address this possibility. viruses such as sars-cov, h5n1 influenza virus and 1918 influenza virus are all zoonotic infections, in which a virus that was adapted to another host was transferred to humans. because the type i ifn response is somewhat different in different hosts, it is possible that these viruses, which have adapted to their normal animal hosts, elicit an aberrant response when infecting a human host in which adaptation has not occurred, resulting in immunopathology. this possibility also raises the question of how appropriate the various animal infection models (such as mice and macaques) are for the understanding of human pathogenesis. as reviewed elsewhere 56 , there are both advantages and disadvantages associated with different animal models, and it is important to keep in mind that responses observed using an animal model may not always accurately reflect the response in humans. genomics in vaccine evaluation and design genomic information and high-throughput technologies are beginning to have an impact on the field of vaccine development, but the main focus has been directed towards identifying important conserved features of pathogens that could serve as immunogens and characterizing host genotypes associated with strong protective responses [69] [70] [71] . in recent years, it has become evident that the type i ifn response has a significant role in the development of the adaptive immune response. this commences with the influence of type i ifns on the activation, maturation and migration of dendritic cells 72, 73 . the development of the antibody response is also enhanced by type i ifns through the direct effect of ifn on b cells and on the priming or function of cd4 + t helper cells 74 . there is now also evidence that type i ifns act directly on cd8 + t cells to promote clonal expansion and indirectly by stimulating cross-priming by antigen-presenting cells that have engulfed infected cells to acquire antigen [75] [76] [77] . so, viruses that suppress the type i ifn response not only subvert the mechanisms of innate surveillance, but also diminish the potential adaptive immune response that could mediate viral clearance or establish a quiescent, non-pathogenic state. for vaccine strategies, the implication is then that the best induction of a broad adaptive immune response will require some degree of type i ifn response in the initial stages. just as dna microarray technology spurred the development of functional genomics, the development of immunomic microarray technology is driving the emerging field of functional immunomics (reviewed in ref. 114) . the goal of immunomics is to provide a detailed understanding of host immunological responses to foreign antigens through the use of high-throughput technologies and computational methods. the technologies that are central to this effort include antibody microarrays (consisting of antibodies as probes and antigens as targets), peptide microarrays (consisting of antigen peptides as probes and serum antibodies as targets) and more recently peptide-mhc microarrays (consisting of recombinant peptide-mhc complexes and co-stimulatory molecules as probes and populations of t cells as targets). antibody microarrays are used to measure the concentration of specific antigens (such as cancer antigens), whereas peptide-mhc microarrays can map mhc-restricted t-cell epitopes which are involved in helper and regulatory functions of the immune system. peptide microarrays are used in various applications, including b-cell epitope mapping and detection and diagnostic assays. peptide microarrays are also being used in vaccine studies for mapping epitopes associated with effective immune responses and for testing the ability of experimental vaccines to generate specific antibody responses against those epitopes after immunization and challenge 115 . studies of immune responses that are associated with different clinical outcomes, such as those of patients who are hiv positive and who rapidly progress to aids and those of infected long-term survivors, can also provide direction for the development of vaccines 116 . it is probable that immunomics will become an increasingly integral part of a systems-biology approach to vaccine development and of obtaining a better understanding of host immunity to virus infection. animal models. we have used functional genomics to evaluate a live influenza virus vaccine in a macaque model, in which attenuation of the virus was accomplished by truncation of the gene encoding ns178. this modification eliminates or reduces the ability of the ns1 protein to antagonize type i ifn production 79 and, in mouse and swine models, such attenuated live viruses are immunogenic and protective 80, 81 . gene-expression profiling of tracheal and bronchial epithelial cells from macaques immunized with the ns1-truncated virus show clear evidence of a robust type i ifn response. compared with immunization with a traditional killedvirus vaccine, the attenuated live-virus-vaccine group had higher antibody titres before and after challenge and a broader range of influenza virus-specific t-cell responses. following challenge with infective virus, the protection afforded by the attenuated live-virus vaccine was evident by the limited viral replication and minor pathology observed in treated animals. in addition, gene-expression profiles of lung tissue from animals that received the attenuated live-virus vaccine show less upregulation of innate and pro-inflammatory response genes compared with animals immunized with the killed-virus vaccine or untreated animals. at the same time, the transcriptional profiles for the attenuated live-virus-vaccine animals showed a stronger induction of genes that are associated with b-cell and t-cell responses. the general picture overall is that the truncated-ns1containing influenza virus vaccine undergoes minimal replication but induces sufficient type i ifns to galvanize the adaptive immune response, leaving the host in a state of adaptive preparedness after just one immunization. the early induction of type i ifns in response to the truncated-ns1-containing vaccine might be especially important in the local b-cell response that is crucial for viral clearance. a relevant observation in this regard is that early stimulation of the respiratory-tract b cells (within 48 hours of influenza virus infection) was shown to be strongly driven by virus-induced type i ifns 82, 83 . human studies. at present, there are only limited examples in which gene-expression profiling applied to vaccine design supports a picture consistent with that described above for the influenza virus model. the standards for prevention of measles and yellow fever are immunizations with attenuated live-virus vaccines. to assess the impact of infection on primary target cells, gene-expression profiling was carried out in tissue-culture systems comparing wild-type and vaccine strains. for both measles and yellow fever, it was clear that the attenuated vaccine strains led to a greater induction of the type i ifn response than the pathogenic wild-type virus 84, 85 . although in the case of measles virus this disparity in the ifn response has previously been shown by serological techniques 86 , expression analysis indicated that the antagonism of the response by the wild-type virus originated at the level of transcription. this early induction of the type i ifn response was also evident in microarray studies examining chimeras of the yellow fever vaccine strain that were devised as attenuated live-virus vaccines against other flaviviruses such as dengue virus 87 . this contrasted with the low-level induction of type i ifns by dengue virus infection as seen by expression profiling using infection of primary cells or macaque disease models 87, 88 . it is interesting to note that the measles and yellow fever vaccine strains are attenuated by passage in cells from other species. therefore, with suitable molecular understanding, the ability of some viruses to induce type i ifns might be optimized by directed molecular techniques, as was done for the truncated-ns1 influenza virus strain. as an alternative, one might consider using recombinant type i ifns as vaccine adjuvants instead of inducing them with the vaccine constituents 89 , but at our present level of understanding, these approaches have yet to prove clinically tenable 90 . functional genomics for the evaluation of immunological memory. functional genomic studies have been more equivocal in assessing the significance of type i ifn production during the immunological memory response. in the aforementioned macaque influenza virus study, animals receiving the attenuated live-virus vaccine showed upregulation of type i ifn pathways in tracheobronchial cells 2 days after challenge, and this coincided with the development of a strong memory response 78 . this type i ifn induction seems to be weaker than that observed at the corresponding time after the primary exposure to the vaccine, but is far lower than the type i ifn induction observed after challenge of animals receiving the killed-virus vaccine or of naive animals. this would suggest some role of this innate pathway in stimulating immunological recall. in contrast to this, examination of transcriptional profiles observed shortly after rechallenge of human pbmcs from individuals previously immunized against influenza virus are more in accord with early production of ifnγ, possibly arising from antigenic stimulation of memory cells 91 . dhiman et al. also did not see evidence of a type i ifn response in a microarray study of whole blood taken from individuals immunized with measles virus after rechallenge with an attenuated live-virus vaccine strain, although genes associated with lymphocyte activation and survival were upregulated 92 . it could be considered that technical issues might hamper the relevance of these studies in assessing the role of type i ifns in the memory response. in the case of the first study 91 , pbmcs are not a primary target of influenza virus, so virus internalization might have been inefficient and a type i ifn response might have been poor. in the measles study 92 , the earliest time point examined was 7 days after rechallenge rather than early, when the type i ifn response would be expected to be strongest. therefore, further functional genomic experiments, with appropriately designed models, are required to address whether an early innate immune response is a key stage in triggering immunological memory. functional genomics has proven to be a highly efficient method for providing broad views of the host response in studies of virus-host interactions. as we have discussed, these techniques have revealed the activation or nature reviews | immunology repression of innate immune signalling pathways, crosstalk between pathways, the timing and magnitude of the immune response and, depending on the experimental system, the degree to which the immune response varies among individuals. conversely, functional genomics has been less effective in pinpointing the role of specific host genes in the antiviral response or, somewhat surprisingly, in identifying previously undiscovered genes and pathways that are important in the infection process, despite this being one of its early goals 93 . indeed, the early assumption that functional genomics would provide quick answers to the complexities of virus-host interactions has proved naive. how then can greater benefits be gained from using functional genomics to study virus-host interactions? rather than being used as a singular approach, the future of functional genomics in virology will be in the integration of genomic data with data derived from other high-throughput technologies (fig. 4) . the obvious complementary approach to functional genomics is proteomics, which will provide much needed information regarding the correlation of gene expression with protein abundance [94] [95] [96] . our group has begun to integrate genomic and proteomic data to better understand the host response to influenza virus infection 97 . other possibilities for data integration are also beginning to unfold. for example, micrornas, which regulate both transcription and translation, might have an important role in mediating virus-host interactions 98 . the discovery of micrornas in certain large dna viruses, such as herpesvirus, suggests that some viruses may encode micrornas to regulate cellular functions 99 . in addition, immunomic strategies (box 2) will provide additional opportunities to interrogate the host immune response; screens using small interfering rnas are currently being combined with genomic data to identify specific cellular proteins that are used by viruses during infection [100] [101] [102] . together with virology, clinical and pathology data, this integrated set of information might provide the systems-biology view that will be needed to clearly understand the role of specific host genes and pathways involved in the development of immunity or disease after virus infection. another use for genomics that will no doubt expand is expression quantitative trait loci (eqtl) mapping 103 . the combination of global gene-expression data with eqtl mapping provides greater power in elucidating complex genetic traits in addition to providing insights into specific genes or mutations that might be responsible for the trait in question. this approach is currently being used to better understand the genetic basis for various disease conditions in mice [104] [105] [106] , and it is likely that it will also be useful in increasing our understanding of virus-host interactions. for example, using recombinant inbred strains of mice derived from parental strains that react differently to infection with a given virus, it should be possible to use eqtl mapping to determine chromosomal locations for potential traitcontributing factors and highlight genes of interest for the trait. with this increased level of complexity, however, it will be important to work closely with the bioinformatics and computational-modelling communities, and to make best use of the sophisticated bioinformatics tools, data-mining schemes and mathematical-modelling strategies that are continually being developed 107, 108 . it might also be necessary to take a step back to simpler experimental systems (such as cell-culture models) to dissect cellular events before moving on to more complex in vivo models. the use of combined computational approaches that can account for gene-regulatory networks and cell-to-cell interactions will also facilitate the move to whole animal physiological modelling. functional genomics is clearly providing advances in our understanding of virus-host interactions, and the evolution to an integrated systems-biology approach holds even greater promise for the field. in addition to providing new insights into viral pathogenesis and host immunity, this approach provides a host-oriented antiviral discovery paradigm with the potential for discovering the benefits of functional genomics will be further enhanced by integrating genomic data with data derived from other high-throughput technologies. the potential information and biological insights provided by these technologies are shown. together, these approaches will help to provide a systems-biology view of virus-host interactions that spans the flow of biological information from dna (genetics) to mrna (genomics) to protein (proteomics) to protein function (immunomics). new targets for broad-spectrum antiviral therapies 109 and for improving vaccine evaluation and design. we are optimistic about continuing advancements in the technologies and computational methods used to study virus-host interactions and in improved capabilities to identify, characterize and circumvent the strategies used by viruses to outsmart their long-suffering hosts. recent studies have shown that rig-i preferentially recognizes single-stranded rna (ssrna) with polyu motifs, whereas mda5 recognizes long dsrna molecules. these differences might help to explain the differential recognition and innate immune signalling induced by different rna viruses [117] [118] [119] . pathogenic viruses: smart manipulators of the interferon system pathogen subversion of cell-intrinsic innate immunity viruses and interferon: a fight for supremacy principles of intracellular viral recognition toll-like receptors, rig-i-like rna helicases and the antiviral innate immune response functional classification of interferon-stimulated genes identified using microarrays identification of genes differentially regulated by interferon α, β, or γ using oligonucleotide arrays establishment and maintenance of the innate antiviral response to west nile virus involves both rig-i and mda5 signaling through ips-1 in this study, genomic analyses show that rig-i and mda5 operate cooperatively to establish an antiviral state and mediate an ifn amplification loop that supports immune effector gene expression during west nile virus infection differential roles of mda5 and rig-i helicases in the recognition of rna viruses distinct rig-i and mda5 signaling by rna viruses in innate immunity systems biology approaches identify atf3 as a negative regulator of toll-like receptor 4 transcriptional profiling of the lps induced nf-κb response in macrophages human macrophage activation programs induced by bacterial pathogens functional genomic delineation of tlrinduced transcriptional networks regulation of interferon regulatory factor-3 by the hepatitis c virus serine protease control of antiviral defenses through hepatitis c virus disruption of retinoic acid-inducible gene-i signaling immune evasion by hepatitis c virus ns3/ 4a protease-mediated cleavage of the toll-like receptor 3 adaptor protein trif regulating intracellular antiviral defense and permissiveness to hepatitis c virus rna replication through a cellular rna helicase, rig-i molecular determinants of trif proteolysis mediated by the hepatitis c virus ns3/4a protease inhibition of rig-i-dependent signaling to the interferon pathway during hepatitis c virus expression and restoration of signaling by ikkε viral and therapeutic control of ifn-β promoter stimulator 1 during hepatitis c virus infection ips-1, an adaptor triggering rig-i-and mda5-mediated type i interferon induction identification and characterization of mavs, a mitochondrial antiviral signaling protein that activates nf-κb and irf3 visa is an adapter protein required for virus-triggered ifn-β signaling cardif is an adaptor protein in the rig-i antiviral pathway and is targeted by hepatitis c virus dna microarray analysis of chimpanzee liver during acute resolving hepatitis c virus infection intrahepatic gene expression during chronic hepatitis c virus infection in chimpanzees genomic analysis of the host response to hepatitis c virus infection hepatitis c virus and liver disease: global transcriptional profiling and identification of potential markers gene expression patterns that correlate with hepatitis c and early progression to fibrosis in liver transplant recipients protective immunity and susceptibility to infectious diseases: lessons from the 1918 influenza pandemic use of functional genomics to understand influenza-host interactions a question of self-preservation: immunopathology in influenza virus infection cellular transcriptional profiling in influenza a virus-infected lung epithelial cells: the role of the nonstructural ns1 protein in the evasion of the host innate defense and its potential contribution to pandemic influenza global host immune response: pathogenesis and transcriptional profiling of type a influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus characterization of the reconstructed 1918 spanish influenza pandemic virus genomic analysis of increased host immune and cell death responses induced by 1918 influenza virus aberrant innate immune response in lethal infection of macaques with the 1918 influenza virus genomic analyses indicate that cynomologus macaques infected with the 1918 influenza virus mount an immune response that is characterized by dysregulation of the antiviral response and that is insufficient for protection. this indicates that atypical host innate immune responses might contribute to lethality single gene reassortants identify a critical role for pb1, ha, and na in the high virulence of the 1918 pandemic influenza virus molecular basis for high virulence of hong kong h5n1 influenza a viruses interferon-mediated immunopathological events are associated with atypical innate and adaptive immune responses in patients with severe acute respiratory syndrome expression profile of immune response genes in patients with severe acute respiratory syndrome an interferon-γ-related cytokine storm in sars patients functional genomics highlights differential induction of antiviral pathways in the lungs of sars-cov-infected macaques immunomics: discovering new targets for vaccines and therapeutics heterogeneity in vaccine immune response: the role of immunogenetics and the emerging field of vaccinomics bridging the knowledge gaps in vaccine design these authors show the importance of type i ifns in plasmacytoid dendritic-cell activation and migration using an elegant series of in vivo mouse models that exploited ifnα-receptor-deficient mice and synthetic tlr ligands type i ifns enhance the terminal differentiation of dendritic cells cutting edge: enhancement of antibody responses through direct stimulation of b and t cells by type i ifn type i interferons act directly on cd8 t cells to allow clonal expansion and memory formation in response to viral infection innate inflammatory signals induced by various pathogens differentially dictate the ifn-i dependence of cd8 t cells for clonal expansion and memory formation a role for the transcription factor relb in ifn-α production and in ifn-α-stimulated crosspriming functional genomic and serological analysis of the protective immune response resulting from vaccination of macaques with an ns1-truncated influenza virus influenza virus evades innate and adaptive immunity via the ns1 protein immunogenicity and protection efficacy of replication-deficient influenza a viruses with altered ns1 genes efficacy of intranasal administration of a truncated ns1 modified live influenza virus vaccine in swine type i ifn receptor signals directly stimulate local b cells early following influenza virus infection influenza virus infection causes global respiratory tract b cell response modulation via innate immune signals measles virus-induced modulation of host-cell gene expression despite a limited technical capacity to measure only 1,176 genes host-cell interaction of attenuated and wild-type strains of yellow fever virus can be differentiated at early stages of hepatocyte infection evasion of host defenses by measles virus: wild-type measles virus infection interferes with induction of α/β interferon production innate immune responses in human dendritic cells upon infection by chimeric yellow-fever dengue vaccine serotypes 1-4. am transcriptional activation of interferon-stimulated genes but not of cytokine genes after primary infection of rhesus macaques with dengue virus type 1 type i ifn as a vaccine adjuvant for both systemic and mucosal vaccination against influenza virus the relevance of cytokines for development of protective immunity and rational design of vaccines transcriptional analysis of human peripheral blood mononuclear cells after influenza immunization immune activation at effector and gene expression levels after measles vaccination in healthy individuals: a pilot study virology in the 21st century: finding function with functional genomics hepatoproteomics: applying proteomic technologies to the study of liver function and disease viral proteomics: global evaluation of viruses and their interaction with the host integrated molecular signature of disease: analysis of influenza virus-infected macaques through functional genomics and proteomics microarray analysis shows that some micrornas downregulate large numbers of target mrnas identification of micrornas of the herpesvirus family identification of host proteins required for hiv infection through a functional genomic screen copi activity coupled with fatty acid biosynthesis is required for viral replication identification of host genes involved in hepatitis c virus replication by small interfering rna technology genetical genomics: combining genetics with gene expression analysis an integrative genomics strategy for systematic characterization of genetic loci modulating phenotypes integrating genetic and gene expression data: application to cardiovascular and metabolic traits in mice uncovering regulatory pathways that affect hematopoietic stem cell function using 'genetical genomics' computational methodologies for modelling, analysis and simulation of signalling networks the model organism as a system: integrating 'omics' data sets systems biology and the host response to viral infection a new discipline, a new name microarray analysis: basic strategies for successful experiments dna microarray technology for the microbiologist: an overview chips with everything: dna microarrays in infectious diseases from functional genomics to functional immunomics: new challenges, old problems, big rewards microarray profiling of antibody responses against simian-human immunodeficiency virus: postchallenge convergence of reactivities independent of host histocompatibility type and vaccine regimen microarray profiling of antiviral antibodies for the development of diagnostics, vaccines, and therapeutics nonself rna-sensing mechanism of rig-i helicase and activation of antiviral immune responses innate immunity induced by compositiondependent rig-i recognition of hepatitis c virus rna the length-dependent recognition of double-stranded ribonucleic acids by retinoic acidinducible gene-i and melanoma differentiationassociated gene 5 we thank b. paeper and s. proll for discussions and assistance with preparation of the original figures. research in the authors' laboratory is supported by public health service grants (r01ai022646, r01hl080621, r21ai017892, r 2 4 r r 01 6 3 5 4 , p 01 a i 0 5 210 6 , p 01 a i 0 5 811 3 , p30da015625 and p51rr000166) from the national institutes of health, usa. this study uses gene-expression profiling of serial liver-biopsy samples from patients that had received a liver transplant to demonstrate that rapidly progressive fibrosis is associated with an impaired immune response, as indicated by a lack of induction of genes associated with the ifnmediated antiviral response, antigen presentation and cytotoxic t-cell response. 31 key: cord-254895-ym0jsir5 authors: eisenächer, katharina; steinberg, christian; reindl, wolfgang; krug, anne title: the role of viral nucleic acid recognition in dendritic cells for innate and adaptive antiviral immunity date: 2008-01-18 journal: immunobiology doi: 10.1016/j.imbio.2007.09.007 sha: doc_id: 254895 cord_uid: ym0jsir5 abstract dendritic cells which are located at the interface of innate and adaptive immunity are targets for infection by many different dna and rna viruses. dendritic cell subpopulations express specific nucleic acid recognition receptors belonging to the toll-like receptor family (tlr3, 7, 8, 9) and the cytosolic rna helicase family (rig-i, mda5, lgp2). activation of dendritic cells by viral dna and rna via these receptors is essential for triggering the innate antiviral immune response and shaping the ensuing adaptive antiviral immunity. this review will summarize our current knowledge of viral nucleic acid recognition and signaling by toll-like receptors and rna helicases focusing on recent evidence for their specific functions in antiviral defense in vivo. dendritic cells (dcs) , which are present in the tissues of all organs as well as in the circulation and in lymphatic organs, are targets for infection by many different viruses. viruses entering the body through epithelial surfaces or directly via the blood stream encounter dcs at early time points during infection. dcs are unique in their capacity to migrate from the periphery to lymphoid organs or from the blood stream into lymphatic and peripheral tissues. virus-infected dcs can therefore transport viruses to other parts of the organism and thus contribute to the spreading of the pathogens to many organs during systemic infection. however, dcs are also equipped with a range of receptors (for example toll-like receptors, tlrs) that recognize conserved molecular patterns of viruseseither viral proteins or viral nucleic acids with specific distinguishing features (lund et al., 2003; krug et al., 2004a, b; schulz et al., 2005) . triggering of these pattern recognition receptors in virus-infected dcs initiates the innate antiviral immune response, which is essential for limiting further viral dissemination (dalod et al., 2003; krug et al., 2004a) . the dcs themselves produce inflammatory cytokines and antiviral interferons in response to viral infection. in addition dcs express ligands for activating receptors on the surface of natural killer (nk) cells leading to activation of this innate antiviral effector cell type (andoniou et al., 2005) . dcs are experts in the processing and presentation of viral antigens on mhc class i as well as crosspresentation on mhc class ii. the antigen presenting function of dcs is critically involved in the generation of efficient adaptive immunity against viruses (belz et al., 2004) . dc activation by viruses via pattern recognition receptors provides the 2nd and 3rd signals required for the priming of naı¨ve-specific t lymphocytes and their differentiation into effector t cells. dc subpopulations fulfill specific tasks in the generation of antiviral immune responses. plasmacytoid dcs (pdc), which have also been described as natural interferon producing cells (perussia et al., 1985; ronnblom et al., 1983 ) represent a specialized subset of dcs characterized by their ability to express large amounts of type i interferons and interferoninduced genes in response to viruses as well as synthetic tlr7 and tlr9 ligands (cella et al., 1999; krug et al., 2004a krug et al., , 2001 siegal et al., 1999) . in response to many viruses pdcs produce 10-to 100fold more type i interferon on a single cell basis than other cell types such as conventional dc or macrophages (colonna et al., 2002) . in many viral infections pdcs have been shown to release the first wave of type i interferons and thus support subsequent steps of antiviral immunity, including nk cell activation, th1 cell and ctl differentiation (dalod et al., 2002 (dalod et al., , 2003 krug et al., 2004a; cervantes-barragan et al., 2006) . it has been shown in vivo and in vitro however, that cells other than pdcs (for example conventional dcs, macrophages and non-immune cells), also significantly contribute to type i ifn responses during viral infection despite the lower amount of type i interferon produced per cell barchet et al. 2005; krug et al., 2004a) . in contrast to other dc populations, pdcs are less efficient in presenting antigens. several studies report that pdcs are capable of presenting antigens to t cells and triggering t cell proliferation and differentiation to effector cells (boonstra et al., 2003; dalod et al., 2003; salio et al., 2004; schlecht et al., 2004) . however, initial priming of t cell responses in many viral infections relies on conventional dcs, especially the cd8a + dcs in the murine system (belz et al., 2004) . in addition it has been reported recently that pdcs activated via tlrs may also induce regulatory t cells (moseman et al., 2004) , which may inhibit the expansion and differentiation of antiviral t lymphocytes to prevent overstimulation of the immune system. what is the molecular basis for the rapid and robust interferon production in pdcs? pdcs recognize viruses via tlrs, particularly via the endosomally located tlr7, tlr8 and tlr9. downstream signaling of tlr7/8 and tlr9 leads to the activation of irf7, which is the central transcription factor for expression of type i interferon and interferon inducible genes (honda et al., 2005b) . in contrast to most other cell types pdcs show constitutive expression of irf7 which is required for the rapid and potent production of the full range of type i ifns (izaguirre et al., 2003; kerkmann et al., 2003) . pdc are unique in that ifn-a expression is induced by ligands of tlr7/8 and tlr9 via a supramolecular complex formed between myd88, traf6, irak1/4 and irf7, which leads to direct activation of the constitutively expressed irf7 only in this specialized cell type (honda et al., 2004; kawai et al., 2004) . the cytoplasmatic rna helicases rig-i or mda5 do not contribute to viral recognition in pdcs, because the response of pdcs from mice deficient in rig-i or the essential signaling adaptor of rig-i and mda5 to rna viruses is comparable to that of wild-type mice . it has been demonstrated in several studies that pdcs recognize herpes simplex virus type 1 and 2 (hsv-1 and -2) (krug et al., 2004b; lund et al., 2003) , murine cytomegalovirus (mcmv) (krug et al., 2004a) and also recombinant replication-deficient adenovirus (basner-tschakarjan et al., 2006) , via tlr9, whereas influenza virus (barchet et al., 2005; diebold et al., 2004) , vesicular stomatitis virus (vsv), newcastle disease virus (ndv) and sendai virus (sev) (lund et al., 2004) are recognized by tlr7 in pdcs. many viruses that enter pdcs and trigger tlrs are prevented from expressing viral genes and replicating their genome. thus, the activation of the type i ifn response in pdcs most often is not counteracted by viral immune evasion strategies. accordingly, active viral replication is not required for the induction of type i ifn production in pdcs for many enveloped viruses, for example influenza virus or herpes viruses (krug et al., 2004b; lund et al., 2003) . following endocytic uptake it is assumed that these viruses are retained in acidified endosomal compartments, where tlr7 and tlr9 are localized, so that the viral particles are degraded and viral nucleic acids come into contact with tlr7 and tlr9. a recent report, however, clearly demonstrates that cytosolic viral replication and a transport mechanism involving autophagy is necessary for triggering the tlr7-dependent response to other rna viruses, such as vsv or sev in pdcs (lee et al., 2007) . after infection of pdcs with these rna viruses, viral replication intermediates in the cytosol are internalized in autophagic vesicles which are then directed towards the tlr7-containing lysosomes, where recognition of the viral rna occurs (lee et al., 2007) . the toll-like receptors are a family of type i transmembrane glycoproteins characterized by the extracellular leucine-rich-repeat domain and the cytoplasmatic tir domain for downstream signaling, which is homologous to the tir domain of the il-1 and il-18 receptors. tlr expression is predominantly found in various immune cells like dendritic cells, macrophages, b cells and some types of t cells. moreover tlrs can be expressed by non-immune cells such as fibroblasts and epithelial cells. among the tlr family a subgroup of mainly intracellularly localized tlrs (tlr3, 7, 8, 9) can be differentiated from tlrs which are expressed on the cell surface (tlr1, 2, 4, 5, 6, 5) . whereas surfaceexpressed tlrs are mainly involved in the recognition of bacterial and fungal cell wall components as well as some viral proteins, the intracellular/endosomal tlrs have the capacity to detect microbial nucleic acids, particularly viral dna and rna. apart from activating the nfkb and mapk signaling pathways leading to inflammatory cytokine and chemokine production as well as costimulatory molecule expression, the intracellularly localized nucleic acid recognition receptors tlr3, 7, 8 and 9 specifically trigger type i interferon production via myd88-and trif-dependent signaling pathways. tlr3 binds double-stranded (ds) rna which is found in dsrna viruses, such as reovirus, or is generated during replication of single-stranded (ss) rna viruses such as west nile virus (wang et al., 2004) or respiratory syncytial virus (rudd et al., 2006) or as a by-product of symmetrical transcription of viral dna, for example from herpes viruses. another ligand for tlr3 is poly(i:c) which is a synthetic dsrna mimicking viral infection (alexopoulou et al., 2001) . studies in tlr3 à/à mice identified poly(i:c) and dsrna as ligands for tlr3, which induce type i ifn and proinflammatory cytokine production. up-regulation of tlr3 expression by type i ifn amplifies the response to tlr3 ligands. tlr3 is expressed in conventional dc (cdcs) subpopulations and macrophages as well as nonimmune cells, such as fibroblasts and epithelial cells, and the cellular localization of tlr3 varies between different cell types. in conventional dcs tlr3 is thought to be localized in intracellular vesicular structures (matsumoto et al., 2003) . high expression of tlr3 is seen in the subset of cd8a + cd4 à dc which phagocytose apoptotic cells, including dying cells that are infected by rna viruses (edwards et al., 2003) . in other cell types such as fibroblasts or epithelial cells tlr3 is expressed on the cell surface (matsumoto et al., 2003) . epithelial expression of tlr3 can be found in many different organs including the airways as well as the gastrointestinal and urogenital system. furthermore, strong expression of tlr3 is detectable in the brain suggesting a specific role in response to viral infections of the central nervous system. despite this wide expression pattern tlr3 does not appear to be essential for the initial antiviral immune response in several mouse models of viral infection (edelmann et al., 2004; lopez et al., 2004) . downstream signaling of tlr3 is unique among all tlrs, because it is entirely myd88-independent and is mediated by the cytosolic tir-domain containing adaptor protein trif. trif is recruited to the cytoplasmatic tir domain of tlr3 and interacts with a set of different signaling molecules and kinases which in turn initiate activation of nfkb or irf3 and irf7 leading to type i interferon induction. interaction of trif with traf3 allows complex formation with the non-canonical ikks-tbk1 and ikke leading to the activation of irf3 and irf7, which form homo-or heterodimers upon phosphorylation and are then translocated to the nucleus to induce type i ifn and ifn inducible gene expression sharma et al., 2003; yamamoto et al., 2003) . nfkb activation by tlr3 ligands is mediated in two waysvia association of trif with rip1 or via interaction of trif with traf6, which in turn activates tak1. both rip1 and tak1 mediate activation of canonical ikks (ikka, ikkb) resulting in ikb degradation and nfkb translocation to the nucleus (meylan et al., 2004; sato et al., 2007) . the role of tlr3 in antiviral immunity remains unclear so far. most studies did not find an essential role of tlr3 for the generation of effective antiviral immune responses. tlr3-deficient mice are as susceptible to reovirus, vsv and lcmv infection as wild-type mice and there was no significant difference in generating specific cd4 and cd8 t cell responses to these viruses (edelmann et al., 2004) . interestingly, however, virally induced cns injury was improved by tlr3-deficiency suggesting that inflammatory responses mediated by tlr3 are at least partially responsible for a breakdown of the blood-brain barrier which facilitates and enhances virus entry into the brain. tlr3-deficient mice survived viral cns infection with west nile virus (wang et al., 2004) , which is characterized by meningitis and encephalitis induced by inflammatory mediators, for longer time periods than wild-type mice. similarly, in murine influenza a virus infection tlr3-mediated inflammatory responses in the lung contribute significantly to host morbidity and lethality. despite higher pulmonary viral loads tlr3 à/à mice showed less inflammation and better survival (le goffic et al., 2006) . interestingly, human lung epithelial cells express proinflammatory cytokines including il-6 and il-8 upon infection with influenza a virus in a tlr3dependent manner (le goffic et al., 2007) , suggesting that tlr3-mediated inflammatory responses may also contribute to influenza virus-induced lung pathology in humans. the role of tlr3 in mcmv infection is more controversial. whereas edelmann et al. (2004) claimed no impairment in antiviral response to mcmv in tlr3 à/à mice, another publication suggested a higher susceptibility to mcmv in the absence tlr3 (tabeta et al., 2004) . in this study tlr3 deficiency results in higher splenic viral titers compared to wild-type mice. similar observations were made when infecting trif lps2/lps2 mice with mcmv (hoebe et al., 2003) . the increased viral load was accompanied by a decreased cytokine response which mostly affected type i ifn and to a lesser extent il12p40 and ifn-g produced by nk and nkt cells (tabeta et al., 2004) . crosspriming occurs most efficiently in the specialized subpopulation of cd8a + dcs, which have a high capacity for internalization of apoptotic cells. tlr3 which is expressed at high levels in cd8a + dcs (edwards et al., 2003) plays a critical role for crosspriming of ctls directed against viruses that do not infect dcs directly. cd8a + dcs are activated by viral rna contained in internalized virally infected apoptotic cells and crosspresent viral antigens to specific cd8 + t cells in a tlr3-dependent manner (schulz et al., 2005) . toll-like receptor 9 tlr9 has been described to recognize bacterial dna or synthetic oligodesoxyribonucleotides (odn) containing specific unmethylated cpg sequence motifs. these can also be found in the genome of dna viruses. viruses recognized by tlr9 are hsv type 1 and 2 krug et al., 2004b) , mcmv (krug et al., 2004a; lund et al., 2003; tabeta et al., 2004) , adenovirus (basner-tschakarjan et al., 2006) and baculovirus (abe et al., 2005) . recognition of hsv or mcmv by tlr9 of pdcs results in robust induction of type i interferon and inflammatory cytokines (krug et al., 2004a, b; lund et al., 2003) . cpg odn class a, b and c have been designed to trigger primarily type i ifn response (a) or costimulatory molecule expression and inflammatory cytokine production (b) or both (c) in pdcs. cpg-b and cpg-c additionally trigger b cell stimulation via tlr9 (krug et al., 2001; verthelyi et al., 2001; hartmann et al., 2003) . induction of type i ifns by tlr9 ligands in pdcs depends on retention of the ligand-receptor complex within early endosomes at a ph value between 6.2 and 5.5 (guiducci et al., 2006) . delivery of tlr9 ligands to late endosome with lower ph values (o4.5) impairs the induction of type i ifns and promotes inflammatory cytokine and costimulatory molecule expression in pdcs (guiducci et al., 2006) . early endosomal retention is achieved by using cpg-a, which forms aggregates or by transfecting cpg-b with cationic liposomes or delivering cpg dna in the form of immune complexes (kerkmann et al., 2005; means et al., 2005; guiducci et al., 2006) . tlr9 ligands delivered within herpes virus particles also seem to be retained long enough in the early endosomal compartment to trigger type i ifn responses efficiently. upon ligand binding the tir domain of tlr9 recruits myd88 which forms a supramolecular complex with traf6, irak1, irak4 and irf7 (honda et al., 2004; kawai et al., 2004) . irf7 becomes activated upon phosphorylation which results in homodimerization of irf7 or heterodimerization of irf3 and irf7. these dimers then translocate to the nucleus and induce expression of type i interferon and interferon inducible genes. several proteins were implicated in the phosphorylation of irf7 including irak-1, ikka, and a precursor of osteopontin (hoshino et al., 2006; shinohara et al., 2006; uematsu et al., 2005) . lack of irak1 abolishes ifn-a production and irf7 activation in response to tlr7, tlr8 and tlr9 ligands . irf7 plays the role of a ''master regulator'' in the induction of type i interferon and ifn inducible genes in response to viruses (honda et al., 2005a) . the currently accepted two step model of positive feedback regulation of type i interferon gene expression consists of an initial phase with activation of irf7 expressed constitutively at low levels, formation of irf7-homodimers or irf7/irf3-heterodimers and induction of small amounts of ifn-b/ifn-a and chemokines. during the second phase secreted type i ifns signal via type i ifn receptor in an autocrine and paracrine manner. downstream signaling of the type i ifn receptor induces a strong up-regulation of irf7 production leading to full expression of type i ifn genes (positive feedback loop). although pdcs constitutively express irf7 at higher levels than other cells, they also depend on further up-regulation of irf7 by type i ifn signaling to be able to sustain high level production of type i ifns honda et al., 2005b) . in contrast to pdcs, type i ifn production in myeloid dcs in response to dna viruses is mediated by both tlr9-dependent and tlr9/myd88-independent pathways. downstream signaling of tlr9 in murine myeloid dcs shows differences compared to pdcs. in myeloid dcs irf1 plays a crucial role in the downstream signaling of tlr9. irf1 à/à mice show an impaired induction of ifn-b, inos and il-12p35 upon stimulation with tlr9 ligand cpg-b, whereas type i interferon response of pdcs is not affected by lack of irf1 (negishi et al., 2006; schmitz et al., 2007) . what is the role of tlr9 for antiviral immunity in vivo? several studies have described increased susceptibility of tlr9 à/à mice to systemic murine cytomegalovirus (mcmv) infection (tabeta et al. 2004; krug et al., 2004a; delale et al., 2005) . we could show that tlr9-dependent recognition of mcmv by pdcs and conventional dc is clearly involved in the innate immune response to mcmv. in the very early phase of systemic mcmv infection pdcs and conventional dcs release the first wave of type i interferons and inflammatory cytokines including il-12. the expression of these cytokines peaks between 36 and 38 h postinfection (dalod et al., 2002; orange and biron, 1996b) and triggers non-specific nk cell activation at this time point. control of viral replication and clearance of the virus from infected organs during the first week of mcmv infection relies mostly on nk cells, which produce ifn-g (mediated by il-12 and il-18) and kill infected cells (orange and biron, 1996a) . we found a significant reduction in ifn-a and il-12 serum levels in tlr9-and myd88-deficient mice at 36 h after infection. however, ifn-a serum levels comparable to those of wild-type mice could be observed in tlr9-and myd88-deficient mice at later time points suggesting the existence of an additional tlr9/myd88-independent type i ifn induction mechanism with delayed kinetics. loss of the early ifn-a peak is due to an impaired function of pdcs in tlr9 à/à mice as mcmv recognition and type i ifn production are tlr9dependent in this cell type. pdc depletion in vivo led to markedly reduced ifn-a levels at 36 h after infection reflecting their almost exclusive role for ifn-a production at this early time point. in contrast to ifn-a, il-12 production was severely impaired in tlr9-and myd88-deficient mice at all time points reflecting the requirement of tlr9 for il-12 responses to mcmv in all dc subpopulations. ifn-g production by nk cells which is induced by il-12 and il-18 was also significantly reduced in the absence of tlr9 or myd88. these defects in the innate immune response to mcmv correlated with higher viral titers in spleen and liver of tlr9 à/à and myd88 à/à mice on the c57bl/6 background reflecting increased susceptibility to mcmv in the early phase of the infection krug et al., 2004a) . a recent report showed that pdcs which are recruited to the vaginal mucosa after local infection with hsv-2 are activated to produce type i ifn in a tlr9dependent manner, thus reducing local viral replication and pathology (lund et al., 2006) . in two mouse models of local infection with hsv-1 (footpad or eye infection), however, we did not find a significant difference in viral titers between tlr9 à/à and myd88 à/à mice and wildtype mice (krug et al., 2004b) . the requirement for tlr9 in the innate immune response to herpes viruses in vivo is influenced by the site of virus entry and the recruitment of dc subpopulations to the infected tissue. all of the described in vivo studies show that in addition to tlr9 other pattern recognition receptors and signaling pathways responding to herpes virus infection must exist, which can partially compensate for the lack of tlr9 and other myd88-dependent receptors (krug et al., 2004b; hochrein et al., 2004; lund et al., 2006) . the cytosolic dna receptor which has been described recently (stetson and medzhitov, 2006; ishii et al., 2006; takaoka et al., 2007) may also be involved in the tlr9independent component of the immune response to herpes virus infections. toll-like receptor 7 and 8 tlr7 and tlr8 are both located on the x chromosome and are homologous to each other. gu-rich ssrna sequences of viral or host origin, poly-u rna and specific sirna sequences are ligands for both receptors, whereas synthetic imidazoquinoline derivatives have been designed to specifically activate tlr7 or tlr8 or both receptors (heil et al., 2004; hemmi et al., 2002; diebold et al., 2004; jurk et al., 2002; hornung et al., 2005; gorden et al., 2005) . tlr7 is expressed in human and murine pdcs, conventional dcs and b cells, whereas tlr8 seems to be functional mainly in human monocytes and myeloid dcs. the role of tlr8 in the murine immune system remains to be elucidated. similar to what has been described for tlr9, activation of tlr7 and tlr8 by specific ligands occurs in the acidified endosomal compartment. the myd88-dependent signal transduction pathway downstream of tlr7 and 8 is very similar to tlr9-mediated signaling. several viruses are recognized by pdcs in a tlr7dependent manner including influenza virus (diebold et al., 2004; barchet et al., 2005) , newcastle disease virus (ndv) , vesicular stomatitis virus (vsv) (lund et al., 2004) , coronaviruses (cervantesbarragan et al., 2006) and rna viruses (heil et al., 2004; beignon et al., 2005) . tlr7 does not seem to play an essential role in the innate immune response to rna virus infection in vivo. we could not find a significant difference between myd88 à/à and wild-type mice in the susceptibility to intranasal influenza virus infection and type i ifn response to intravenously injected influenza virus was only partially reduced in the myd88-deficient animals (barchet et al., 2005) . at high viral doses type i ifn response to systemic vsv infection is tlr7dependent and mediated by pdcs, whereas at lower doses tlr7-independent type i ifn induction pathways play the major role. a recent study demonstrates an essential function of tlr7 for the innate immune response to systemic coronavirus infection in mice (cervantes-barragan et al., 2006) . pdcs, but not conventional dcs are capable of producing significant amounts of type i ifn in response to the rapidly replicating coronaviruses and this response is entirely tlr7-dependent. pdc depletion led to abrogation of type i ifn response and increased disease severity (cervantesbarragan et al., 2006) . it can be concluded that tlr7 (and possibly tlr8 in the human system) contribute to the initiation of the antiviral immune response against viruses which specifically target pdcs. in addition the ubiquitously expressed rna helicases provide protection against rna viruses in all cell types (see below). depending on the route and kinetics of infection, cellular tropism, viral entry and replication as well as immune evasion mechanisms of individual virus strains one or the other viral rna recognition pathway may dominate the innate antiviral immune response in vivo. as suggested by the murine in vivo studies there is considerable redundancy in tlr-dependent and tlr-independent viral pattern recognition mechanisms also in the human system. irak-4-deficient patients are prone to invasive infection with pneumococci, but are resistant to natural viral infection (picard et al., 2003) . irak4-deficient pbmc do not respond to any of the myd88-dependent tlr ligands, but are capable of type i ifn production in response to many dna and rna viruses. there also seems to be considerable redundancy between different tlrs, because in contrast to the irak4-deficient patients, which show no significant defects in antiviral responses (picard et al., 2003) , individuals with autosomal recessive deficiency in the intracellular protein unc-93b have an impaired antiviral type i ifn response and are susceptible to hsv-1 encephalitis (casrouge et al., 2006) . unc-93b-deficiency prevents signaling in response to ligands of the endosomally localized tlr3, tlr7, tlr8 and tlr9 . in addition, the unc-93b protein, which is localized in the endoplasmic reticulum, is required for efficient crosspresentation and mhc class ii presentation of exogenous antigens . therefore, unc-93b-deficiency may affect innate and adaptive antiviral immune responses at the same time. no human deficiencies or mutation in the rig-i/ mda5 pathway of rna virus recognition have been described so far. however, the critical role of this major pathway for innate antiviral immune responses in humans is greatly supported by the existence of viral immune evasion strategies, which, for example, are employed by hepatitis c virus and paramyxoviruses and are directed against several signaling molecules in this pathway (see below). in addition to the tlrs, a new family of viral pattern recognition receptors consisting of rna helicases retinoic acid inducible gene i (rig-i), melanoma differentiation antigen 5 (mda5) and laboratory of genetics and physiology 2 (lgp2) -was discovered and characterized in the last 3 years. these molecules, which are localized in the cytosol, bind specific rna molecules derived from the genome of different rna viruses and, with the exception of lgp2 which does not signal itself, trigger a signaling cascade leading to the production of type i ifns and of proinflammatory cytokines in response to viral infection. retinoic-acid-inducible protein i (rig-i) is a dexd/ h box-containing rna helicase that was originally identified as an enhancer of type i ifn expression in response to dsrna poly (i:c) (yoneyama et al., 2004) . dexd/h box rna helicases are defined by their ability to unwind dsrna using their intrinsic atpase activity. in addition to the c-terminal helicase domain rig-i contains two caspase recruitment domains (card) at its n terminus (zhang et al., 2000) . cards are found in a number of caspases, but also in other proteins such as nod1 and nod2, involved in sensing intracellular bacterial products (inohara and nunez, 2003) . the functions of rig-i have been analyzed in detail in the first publication by yoneyama et al. (2004) using deletion constructs of full length rig-i. overexpression of the n-terminal region of rig-i (delta rig-i) comprising the two tandem cards is sufficient to induce irf3 and nfkb activation even in the absence of a dsrna stimulus or viral challenge. the mutant of rig-i lacking the card domain is not capable of transmitting signals. this n-terminally truncated molecule even has a dominant negative effect since expression of this domain alone prevents activation of irf3 by dsrna transfection or ndv infection (yoneyama et al., 2004) . since cards are known to engage in homophilic protein-protein interactions in other signal transduction pathways, it was therefore likely that the cards of rig-i interact with other card containing molecules to activate downstream signaling molecules. the adaptor protein that links rig-i to the activation of tbk1/ikke and ikkb was identified and functionally characterized by four independent groups and designated as ifn-b promoter stimulator 1 (ips-1) , mitochondrial antiviral signaling protein (mavs) , virus-induced signaling adapter (visa) (xu et al., 2005) and card adapter inducing ifn-b (cardif) (meylan et al., 2005) , respectively. ips-1 contains an n-terminal card domain that interacts with the tandem card domains of rig-i and a c-terminal hydrophobic transmembrane (tm) domain that localizes it to the outer mitochondrial membrane . deletion analyses have shown that both the card and the transmembrane domain are essential for the function of ips-1. the mitochondrial localization of ips-1 is essential for its activity because deletion of the tm domain, which leads to cytosolic expression of the protein, abolishes the signaling function of ips-1. the binding of dsrna to the helicase domain of rig-i likely induces a conformational change that exposes the n-terminal card domains to recruit its signaling adaptor ips-1. this interaction subsequently induces ifn-a/inf-b and ifn-induced antiviral effector mechanisms that suppress virus replication (meylan et al., 2006) . in a recent publication saito et al. reported that an internal repressor (or regulatory) domain (rd) at the c-terminus controls rig-i multimerization and ips-1 interaction during virus infection and rna binding (saito et al., 2007) . expression of the c-terminal rd domain, encompassing the amino acids 735-925 of the rig-i protein, prevents signaling to the ifn-b promoter and increased cellular permissiveness to hepatitis c virus, whereas deletion of the rig-i rd results in constitutive signaling. saito et al. suggest a model of rig-i autoregulation and signaling predicting that in resting cells the c-terminal rd mediates a conformation of rig-i that masks the cards from signaling. once the cell is infected with virus, rig-i activation and signaling occurs in a stepwise manner involving dsrna binding and conformational changes that subsequently facilitate self-association and interaction with the ips-1 signaling adaptor. these conformational changes comprising displacement of the rd and unmasking of the cards for signaling via ips-1 might be triggered by atp hydrolysis. saito et al. also identified an analogous rd within the c terminus of lgp2 suggesting that lgp2 might inhibit rig-i through their rd interactions. lgp2 is a close relative of rig-i, which lacks the cards, but is capable of binding dsrna . thus, lgp2 may act as a postinduction repressor of rig-i signaling . from several studies it is known that rig-i is essential for antiviral responses to a specific set of rna viruses belonging to flaviviridae, paramyxoviridae, orthomyxoviridae and rhabdoviridae sumpter et al., 2005; yoneyama et al., 2004) . since rna is a fundamental entity of most living organisms and is found in abundance in host cells, a molecular pattern must exist that enables rig-i to discriminate between viral rna and host rna species in the cytoplasm of infected cells to prevent constant induction of type i ifns which would lead to autoimmune responses. two recent studies have identified features of viral rna that are structurally different to host rna. hornung et al. (2006) demonstrated that ssrna synthesized in vitro acquires a 5 0 -triphosphate moiety which is crucial for ifn production in host cells. furthermore, they demonstrated that rna isolated from rabies virus (rv) infected cells is a potent ifn inducer, whereas rna from non-infected cells and dephosphorylated rna isolates abrogated this ifn response. the 5 0 -phosphorylation status and the absence of a 7-methyl-guanosine cap provided by the viral polymerase is critical for recognition by rig-i. taken together, the results of hornung et al. (2006) show that rig-i directly recognizes 5 0 -triphosphate single stranded or double stranded rna independently of viral replication. in the second study, pichlmair et al. (2006) found that influenza a virus, which is known to be sensed by both rig-i and tlr3, did not generate dsrna upon infection of bone-marrow-derived dendritic cells. moreover, they observed that influenza virus ssrna, which is uncapped and bears 5 0 -triphosphates, associated with and activated rig-i. ips-1 functions as critical link between viral detection by rig-i and the downstream signaling events leading to interferon production. this receptor-adapter interaction results in the activation of the noncanonical kinases tbk1/ikke, which in turn induces dimerization of phosphorylated irf3 and irf7 and translocation to the nucleus where they activate the transcription of type i ifn genes . coimmunoprecipitation experiments suggest that ips-1 interacts with tbk1 and recruits endogenous irf3 in a virus-inducible manner (xu et al., 2005) . recently, traf3 was shown to be critically involved in ips-1-mediated ifn-a production and antiviral responses through a direct interaction between the traf domain of traf3 and a traf interaction motif within ips-1 (saha et al., 2006) . there is evidence that the tbk1/ikke adaptor protein tank plays a role in ips-1-traf3-mediated activation of tbk1/ikke (guo and cheng, 2007) . rip1 and fadd are additional molecules that have been reported to be required for type i ifn production in response to dsrna (balachandran et al., 2004) . another branch of ips-1 signaling leads to the activation of the ikk complex resulting in activation of nfkb that controls the expression of genes encoding inflammatory responses, but also expression of ifn-b. previous work showed that ips-1 activates a nfkbdependent reporter construct in cultured cells (matsuda et al., 2003) . one of the key signaling proteins in the nfkb pathway is traf6, an essential upstream regulator of the ikk complex. ips-1 binds to traf6 upon overexpression in mammalian cells and in a yeasttwo hybrid screen xu et al., 2005) . xu et al. (2005) reported that endogenous ips-1 also interacts with traf6. in addition, they showed that ips-1 fails to activate nfkb in the absence of traf6. seth et al. (2005) reported that virus-mediated induction of the ifn-b gene is not abolished in traf6 deficient cells and that a mutant ips-1 protein lacking the traf6 binding domain is still capable of inducing ifn-b. thus, traf6 seems to be required for nfkb activation but not ifn-b induction downstream of ips-1 which is mainly mediated by tbk1/ikke. in vitro studies performed with primary cells obtained from rig-i knockout mice confirmed that rig-i plays an essential role in eliciting immune responses against specific negative strand and positive strand rna viruses such as ndv, sev, vsv, japanese encephalitis virus (jev) and influenza virus in various cell types with the exception of pdcs . the experiments demonstrated that type i ifn production by rig-i deficient fibroblasts and conventional dcs is severely impaired in response to these rna viruses. in contrast, pdcs lacking rig-i show normal type i ifn responses to ndv or vsv for example, as the pdc response to these viruses is mediated by tlr7 . the studies performed so far indicate that the rna helicases rig-i and mda5 as well as their common signaling adaptor ips-1 are dispensible for viral triggering of type i ifn responses in pdcs . mda5 is another dexd/h-box-containing rna helicase that is involved in the sensing of intracellular dsrna and the induction of type i ifn in response to rna viruses (andrejeva et al., 2004) . it is the closest relative of rig-i, exhibiting 23 and 35% amino acid homology in the n-terminal card and c-terminal helicase domain, respectively. it was reported in a previous publication, that mda5, which was then called helicard, is cleaved by caspases upon induction of apoptosis, thereby separating the card domains from the c-terminal helicase domain, which localizes to the nucleus where it is involved in dna degradation and nuclear remodelling during apoptotic cell death (kovacsovics et al., 2002) . furthermore, mda5 has been implicated in the regulation of the growth and differentiation of melanoma cells (kang et al., 2002) . mda5 is ubiquitously expressed in low abundance and similarly to rig-i and lgp2 its expression is induced by type i ifn. like rig-i, mda5 interacts with the adapter protein ips-1 upon ligand binding leading to activation of protein kinases that subsequently activate transcription factors irf3, irf7 and nfkb, respectively. it was found recently by sato et al. (2007) that in contrast to rig-i, the c-terminal regulatory domain of mda5 did not exert a repressor function on mda5 signaling, which was also not inhibited by lgp2. mda5 overexpression in cell lines even at low levels induces ifn-b promoter activity in the absence of ligand binding. thus, mda5 expression which is upregulated in response to type i ifn signaling may function as an amplifier of type i ifn production even in the absence of specific mda5 ligands. although rig-i and mda5 are similar proteins inducing synthesis of type i ifn via the same signaling pathway, they are specialized in the recognition of different viruses. in vitro studies performed with embryonic fibroblasts and conventional dcs derived from mda5 knockout mice have shown that mda5 is specifically required for the recognition of intracellular poly (i:c) dsrna (but not in vitro transcribed ppp-rna) and picornaviruses such as encephalomyocarditis virus (emcv), theiler's virus and mengo virus, whereas rig-i was not necessary for the response to poly (i:c) or picornaviruses gitlin et al., 2006) . in addition, it was shown that mda5 might also play a role in the measles virus (mv) induced activation of ifn-b mrna synthesis since a549 cells transfected with mda5 showed a strong activation of the ifn-b promoter upon infection with mv. in contrast, the virus did not enhance reporter gene activity in cells that overexpressed rig-i (berghall et al., 2006) . mda5 was not required for recognition of vsv, ndv, jev, sev and influenza virus, indicating that rig-i was the predominant pattern recognition receptor for these viruses. it is currently unknown which specific viral rna motifs in picornaviruses are recognized by mda5 and how ligand specificity is determined on the molecular level. the in vivo relevance of the rig-i and mda5 pathways for innate antiviral immune responses was addressed by the generation of knockout mice. the specific susceptibility of mda5-deficient mice to encephalomyocarditis virus (emcv) infection was shown in two studies gitlin et al., 2006) . in a direct comparison with ifnar à/à and myd88 à/à mice kato et al. (2006) could show that survival upon emcv infection is as dramatically reduced in mda5 à/à mice as in ifnar à/à mice. myd88-deficient mice were only slightly more susceptible to emcv infection than wildtype mice. survival after emcv infection was not affected by rig-i-or tlr3-deficiency. studies in the ips-1 knockout mouse confirmed the dramatic reduction in the innate immune response to emcv infection, which correlated with increased viral titers and decreased survival, in the absence of the central downstream signaling adaptor of mda5 and rig-i (kumar et al., 2006) . only few in vivo experiments with rig-ideficient mice have been published so far, because for unknown reasons most of the rig-i-knockout mice (on a 129/c57bl/6 mixed background) died in utero or within a few weeks after birth . after crossing with icr mice healthy rig-i à/à mice were obtained and compared with littermate controls in the jev and vsv infection models. rig-i à/à mice were more susceptible to jev and vsv infection than wildtype mice and this correlated with reduced type i ifn responses. myd88-dependent type i ifn production induced by jev and vsv played only a minor role in these infection models . in accordance with these results ips-1-deficient mice succumbed rapidly to vsv infection (sun et al., 2006) . as pointed out by the study of sun et al. (2006) , type i ifn responses to systemic vsv infection were not entirely abrogated in ips-1-deficient mice, suggesting that tlr7-dependent pdc-mediated type i ifn release at least partially compensates for the dysfunctional rna helicase pathway, but this compensatory mechanism cannot prevent death after infection with high doses of vsv. the rna helicase pathway as target for viral immune evasion strategies viruses have adapted strategies to evade or inhibit key elements of antiviral immunity. a number of viral proteins inhibit host innate immune responses, prevent viral antigen presentation and abrogate induction of cell death. since the cytoplasmic rig-i/mda5 system is critical for host defense against rna viruses, the signaling cascades induced by these sensors are also targeted by viruses. various proteins encoded by rna viruses have been shown to antagonize the cytoplasmic rna helicase pathways. ifn antagonists of negative strand rna viruses can interfere with this pathway by hiding their rna (influenza a virus ns1 protein) or binding to the dsrna receptor (paramyxovirus v proteins) or preventing activation of downstream factors such as irf3 (ebola virus vp35, rhabdovirus p), respectively. the non-structural protein 1 (ns1) of the influenza viruses is a dsrna-binding protein that acts as an ifn antagonist (garcia-sastre et al., 1998) . by binding to dsrna ns1 disguises the viral dsrna pattern from the cytoplasmic receptors and inhibits ifn-a/inf-b induction via irf3 (garcia-sastre, 2004; talon et al., 2000) . rig-i was recently demonstrated to be essential for the induction of ifn-b by influenza virus in murine cells. furthermore, it was observed that ns1 colocalizes with rig-i (mibayashi et al., 2007) , suggesting that ns1 forms a complex with rig-i and ips-1 during viral infection, resulting in inhibition of further downstream signaling. respiratory syncytial virus (rsv) specifically interferes with irf3 activation and ifn-b response: two viral proteins of rsv, ns1 and ns2, are involved in blocking the pathway leading to irf3 phosphorylation, although the activation of nfkb and ap-1 is unaffected (bossert et al., 2003) . irf3 phosphorylation by tbk1 was identified as target of the rabies virus phosphoprotein p (brzozka et al., 2005) . recently it was shown that the ny-1 hantavirus g1 cytoplasmic tail inhibits rig-i-and tbk1-directed interferon responses (alff et al., 2006) . the v proteins of paramyxoviruses target mda5, but not rig-i. the v proteins of this diverse group of viruses bind mda5 via their highly conserved cysteinerich c-terminal domain. this suggests that paramyxoviruses use this interaction to reduce the amount of ifn released by infected cells (andrejeva et al., 2004; childs et al., 2007) . furthermore, it was shown that mda5 is also a target of picornaviruses since mda5 is degraded in poliovirus-infected cells. interestingly, mda5 is not directly cleaved by virus-encoded proteinases. degradation of mda5 in poliovirus-infected cells occurs in a proteasome-and caspase-dependent manner and correlates with the induction of apoptosis in poliovirus-infected cells. poliovirus-induced mda5 cleavage attenuates the production of type i ifn, thereby allowing higher levels of viral replication and dissemination in the host (barral et al., 2007) . hepatitis c virus (hcv) encodes the protease ns3/4a which targets ips-1 by cleaving it at position cys-508, thereby dislocating it from the mitochondrial membrane and thus abrogating further downstream signaling to type i ifn and nfkb-dependent target gene expression meylan et al., 2005) . interestingly, ips-1 was also found to be localized to the cytosol and not the mitochondria in liver tissue from patients chronically infected with hcv (loo et al., 2006) . inhibitors of the hcv ns3/4a, which have originally been designed to inhibit hcv replication, are able to prevent ips-1 cleavage and restore the rig-imediated innate immune response to hcv . therefore, ns3/4a inhibitors which are already being tested in clinical trials may have therapeutic potential for chronic hepatitis c infection. the fact that rna viruses have developed so many effective strategies to interfere with the rna helicase pathway of viral recognition during coevolution with their host provides further proof for the central role of this pathway in antiviral immune defense. involvement of the toll-like receptor 9 signaling pathway in the induction of innate immunity by baculovirus recognition of double-stranded rna and activation of nf-kappab by toll-like receptor 3 the pathogenic ny-1 hantavirus g1 cytoplasmic tail inhibits rig-i-and tbk-1-directed interferon responses interaction between conventional dendritic cells and natural killer cells is integral to the activation of effective antiviral immunity the v proteins of paramyxoviruses bind the ifn-inducible rna helicase, mda-5, and inhibit its activation of the ifn-beta promoter a fadddependent innate immune mechanism in mammalian cells dendritic cells respond to influenza virus through tlr7-and pkrindependent pathways mda-5 is cleaved in poliovirus-infected cells adenovirus efficiently transduces plasmacytoid dendritic cells resulting in tlr9-dependent maturation and ifnalpha production endocytosis of hiv-1 activates plasmacytoid dendritic cells via toll-like receptor-viral rna interactions cutting edge: conventional cd8 alpha+ dendritic cells are generally involved in priming ctl immunity to viruses the interferoninducible rna helicase, mda-5, is involved in measles virus-induced expression of antiviral cytokines flexibility of mouse classical and plasmacytoid-derived dendritic cells in directing t helper type 1 and 2 cell development: dependency on antigen dose and differential toll-like receptor ligation nonstructural proteins ns1 and ns2 of bovine respiratory syncytial virus block activation of interferon regulatory factor 3 identification of the rabies virus alpha/beta interferon antagonist: phosphoprotein p interferes with phosphorylation of interferon regulatory factor 3 herpes simplex virus encephalitis in human unc-93b deficiency plasmacytoid monocytes migrate to inflamed lymph nodes and produce large amounts of type i interferon control of coronavirus infection through plasmacytoid dendritic cell-derived type i interferon mda-5, but not rig-i, is a common target for paramyxovirus v proteins interferon-producing cells: on the front line in immune responses against pathogens interferon alpha/beta and interleukin 12 responses to viral infections: pathways regulating dendritic cell cytokine expression in vivo dendritic cell responses to early murine cytomegalovirus infection: subset functional specialization and differential regulation by interferon alpha/beta myd88-dependent and -independent murine cytomegalovirus sensing for ifn-alpha release and initiation of immune responses in vivo viral infection switches non-plasmacytoid dendritic cells into high interferon producers innate antiviral responses by means of tlr7-mediated recognition of single-stranded rna does toll-like receptor 3 play a biological role in virus infections? toll-like receptor expression in murine dc subsets: lack of tlr7 expression by cd8 alpha+ dc correlates with unresponsiveness to imidazoquinolines ikkepsilon and tbk1 are essential components of the irf3 signaling pathway identification and characterization of viral antagonists of type i interferon in negative-strand rna viruses influenza a virus lacking the ns1 gene replicates in interferondeficient systems essential role of mda-5 in type i ifn responses to polyriboinosinic:polyribocytidylic acid and encephalomyocarditis picornavirus synthetic tlr agonists reveal functional differences between human tlr7 and tlr8 properties regulating the nature of the plasmacytoid dendritic cell response to toll-like receptor 9 activation modulation of the interferon antiviral response by the tbk1/ikki adaptor protein tank rational design of new cpg oligonucleotides that combine b cell activation with high ifn-alpha induction in plasmacytoid dendritic cells species-specific recognition of single-stranded rna via toll-like receptor 7 and 8 small anti-viral compounds activate immune cells via the tlr7 myd88-dependent signaling pathway herpes simplex virus type-1 induces ifn-alpha production via toll-like receptor 9-dependent and -independent pathways identification of lps2 as a key transducer of myd88-independent tir signalling irfs: master regulators of signalling by toll-like receptors and cytosolic patternrecognition receptors role of a transductional-transcriptional processor complex involving myd88 and irf-7 in toll-like receptor signaling spatiotemporal regulation of myd88-irf-7 signalling for robust type-i interferon induction irf-7 is the master regulator of type-i interferon-dependent immune responses sequence-specific potent induction of ifn-alpha by short interfering rna in plasmacytoid dendritic cells through tlr7 0 -triphosphate rna is the ligand for rig-i ikappab kinase-alpha is critical for interferon-alpha production induced by toll-like receptors 7 and 9 nods: intracellular proteins involved in inflammation and apoptosis a toll-like receptorindependent antiviral response induced by double-stranded b-form dna comparative analysis of irf and ifn-alpha expression in human plasmacytoid and monocyte-derived dendritic cells functional and therapeutic analysis of hepatitis c virus ns3/4a protease control of antiviral immune defense human tlr7 or tlr8 independently confer responsiveness to the antiviral compound r-848 mda-5: an interferoninducible putative rna helicase with double-stranded rna-dependent atpase activity and melanoma growthsuppressive properties cell type-specific involvement of rig-i in antiviral response differential roles of mda5 and rig-i helicases in the recognition of rna viruses interferonalpha induction through toll-like receptors involves a direct interaction of irf7 with myd88 and traf6 ips-1, an adaptor triggering rig-i-and mda5-mediated type i interferon induction activation with cpg-a and cpg-b oligonucleotides reveals two distinct regulatory pathways of type i ifn synthesis in human plasmacytoid dendritic cells spontaneous formation of nucleic acid-based nanoparticles is responsible for high interferonalpha induction by cpg-a in plasmacytoid dendritic cells overexpression of helicard, a card-containing helicase cleaved during apoptosis, accelerates dna degradation identification of cpg oligonucleotide sequences with high induction of ifn-alpha/beta in plasmacytoid dendritic cells tlr9-dependent recognition of mcmv by ipc and dc generates coordinated cytokine responses that activate antiviral nk cell function herpes simplex virus type 1 activates murine natural interferon-producing cells through toll-like receptor 9 essential role of ips-1 in innate immune responses against rna viruses detrimental contribution of the toll-like receptor (tlr)3 to influenza a virus-induced acute pneumonia cutting edge: influenza a virus activates tlr3-dependent inflammatory and rig-i-dependent antiviral responses in human lung epithelial cells autophagy-dependent viral recognition by plasmacytoid dendritic cells hepatitis c virus protease ns3/4a cleaves mitochondrial antiviral signaling protein off the mitochondria to evade innate immunity viral and therapeutic control of ifn-beta promoter stimulator 1 during hepatitis c virus infection tlr-independent induction of dendritic cell maturation and adaptive immunity by negative-strand rna viruses toll-like receptor 9-mediated recognition of herpes simplex virus-2 by plasmacytoid dendritic cells recognition of single-stranded rna viruses by toll-like receptor 7 cutting edge: plasmacytoid dendritic cells provide innate immune protection against mucosal viral infection in situ large-scale identification and characterization of human genes that activate nf-kappab and mapk signaling pathways subcellular localization of toll-like receptor 3 in human dendritic cells human lupus autoantibody-dna complexes activate dcs through cooperation of cd32 and tlr9 rip1 is an essential mediator of toll-like receptor 3-induced nfkappa b activation cardif is an adaptor protein in the rig-i antiviral pathway and is targeted by hepatitis c virus intracellular pattern recognition receptors in the host response inhibition of retinoic acid-inducible gene i-mediated induction of beta interferon by the ns1 protein of influenza a virus human plasmacytoid dendritic cells activated by cpg oligodeoxynucleotides induce the generation of cd4+cd25+ regulatory t cells evidence for licensing of ifngamma-induced ifn regulatory factor 1 transcription factor by myd88 in toll-like receptor-dependent gene induction program an absolute and restricted requirement for il-12 in natural killer cell ifn-gamma production and antiviral defense. studies of natural killer and t cell responses in contrasting viral infections characterization of early il-12, ifn-alphabeta, and tnf effects on antiviral state and nk cell responses during murine cytomegalovirus infection a leukocyte subset bearing hla-dr antigens is responsible for in vitro alpha interferon production in response to viruses rig-i-mediated antiviral responses to single-stranded rna bearing 5 0 -phosphates properties of human natural interferon-producing cells stimulated by tumor cell lines the rna helicase lgp2 inhibits tlr-independent sensing of viral replication by retinoic acid-inducible gene-i deletion of tlr3 alters the pulmonary immune environment and mucus production during respiratory syncytial virus infection regulation of antiviral responses by a direct and specific interaction between traf3 and cardif regulation of innate antiviral defenses through a shared repressor domain in rig-i and lgp2 cpg-matured murine plasmacytoid dendritic cells are capable of in vivo priming of functional cd8 t cell responses to endogenous but not exogenous antigens toll/il-1 receptor domain-containing adaptor inducing ifn-beta (trif) associates with tnf receptor-associated factor 6 and tank-binding kinase 1, and activates two distinct transcription factors, nf-kappa b and ifn-regulatory factor-3, in the toll-like receptor signaling murine plasmacytoid dendritic cells induce effector/memory cd8+ t-cell responses in vivo after viral stimulation interferon-regulatory-factor 1 controls toll-like receptor 9-mediated ifn-beta production in myeloid dendritic cells toll-like receptor 3 promotes cross-priming to virus-infected cells identification and characterization of mavs, a mitochondrial antiviral signaling protein that activates nf-kappab and irf 3 triggering the interferon antiviral response through an ikk-related pathway osteopontin expression is essential for interferon-alpha production by plasmacytoid dendritic cells the nature of the principal type 1 interferon-producing cells in human blood recognition of cytosolic dna activates an irf3-dependent innate immune response regulating intracellular antiviral defense and permissiveness to hepatitis c virus rna replication through a cellular rna helicase, rig-i the specific and essential role of mavs in antiviral innate immune responses toll-like receptors 9 and 3 as essential components of innate immune defense against mouse cytomegalovirus infection the unc93b1 mutation 3d disrupts exogenous antigen presentation and signaling via toll-like receptors 3, 7 and 9 dai (dlm-1/zbp1) is a cytosolic dna sensor and an activator of innate immune response activation of interferon regulatory factor 3 is inhibited by the influenza a virus ns1 protein interleukin-1 receptorassociated kinase-1 plays an essential role for toll-like receptor (tlr)7-and tlr9-mediated interferon-{alpha} induction human peripheral blood cells differentially recognize and respond to two distinct cpg motifs toll-like receptor 3 mediates west nile virus entry into the brain causing lethal encephalitis visa is an adapter protein required for virus-triggered ifn-beta signaling role of adaptor trif in the myd88-independent toll-like receptor signaling pathway the rna helicase rig-i has an essential function in double-stranded rna-induced innate antiviral responses shared and unique functions of the dexd/h-box helicases rig-i, mda5, and lgp2 in antiviral innate immunity an rna helicase, rhiv-1, induced by porcine reproductive and respiratory syndrome virus (prrsv) is mapped on porcine chromosome 10q13 key: cord-283096-qm7h4qui authors: jeon, young joo; yoo, hee min; chung, chin ha title: isg15 and immune diseases date: 2010-02-12 journal: biochim biophys acta mol basis dis doi: 10.1016/j.bbadis.2010.02.006 sha: doc_id: 283096 cord_uid: qm7h4qui isg15, the product of interferon (ifn)-stimulated gene 15, is the first identified ubiquitin-like protein, consisting of two ubiquitin-like domains. isg15 is synthesized as a precursor in certain mammals and, therefore, needs to be processed to expose the c-terminal glycine residue before conjugation to target proteins. a set of three-step cascade enzymes, an e1 enzyme (ube1l), an e2 enzyme (ubch8), and one of several e3 ligases (e.g., efp and herc5), catalyzes isg15 conjugation (isgylation) of a specific protein. these enzymes are unique among the cascade enzymes for ubiquitin and other ubiquitin-like proteins in that all of them are induced by type i ifns or other stimuli, such as exposure to viruses and lipopolysaccharide. mass spectrometric analysis has led to the identification of several hundreds of candidate proteins that can be conjugated by isg15. some of them are type i ifn-induced proteins, such as pkr and rig-i, and some are the key regulators that are involved in ifn signaling, such as jak1 and stat1, implicating the role of isg15 and its conjugates in type i ifn-mediated innate immune responses. however, relatively little is known about the functional significance of isg15 induction due to the lack of information on the consequences of its conjugation to target proteins. here, we describe the recent progress made in exploring the biological function of isg15 and its reversible modification of target proteins and thus in their implication in immune diseases. since the discovery of type i interferons (ifnα and ifnβ) in 1957, they have widely been used as clinical drugs [1, 2] . for example, ifnα has been used for the treatment of chronic hepatitis b and hepatitis c viruses and of several cancers, such as leukemia, and ifnβ is effective for treating multiple sclerosis. type i ifns exert their effects through the activation of janus tyrosine kinase (jak)/signal transducer and activator of transcription (stat) signaling pathway [3] . upon binding of the ifns to their cell surface receptors (ifnar), the receptorassociated kinases jak1 and tyrosine kinase 2 (tyk2) become activated and phosphorylate tyrosine residues in the cytoplasmic tails of the receptor subunit, ifnar1. the phosphorylated subunit provides specific docking sites for the activation of stats by jak1/tyk2mediated phosphorylation [4, 5] . activated stats dissociate from the receptor and translocate into the nucleus, where they act as transcription factors that bind to the promoter regions of ifn-stimulated genes (isgs) [6] . stat1/stat2 heterodimers associate with ifn regulatory factor 9 (irf-9) to form the transcription complex ifn-stimulated gene factor 3 (isgf3), which binds to ifn stimulatory response elements (isres) within the promoters of isgs [7] . on the other hand, homodimers and heterodimers of stat1 and stat3 bind to gammaactivated sequence (gas) response elements [8] . the isg proteins generated by these pathways play key roles in the induction of innate and adaptive immune responses [9, 10] . protein modifications by ubiquitin and ubiquitin-like proteins (ubls), including sumo and nedd8, have emerged as critical regulatory processes, such as in the control of cell cycle, stress response, signaling transduction, and immune response [11] [12] [13] [14] [15] . moreover, deregulation of the modification systems gives rise to numerous human diseases, such as cancers, neurodegenerative diseases, and immune diseases. conjugation of ubiquitin and ubls to target proteins involves threestep cascade enzymes: ubiquitin-and ubl-activating e1 enzymes, ubiquitin-and ubl-conjugating e2 enzymes, and ubiquitin-and ubl e3 ligases. protein modifications by ubiquitin and ubls are reversible processes that are catalyzed by isopeptidases, called deubiquitinating enzymes (dubs) and ubl-specific proteases (ulps), respectively. isg15, a member of the ubl family, shows a significant sequence homology to ubiquitin. like ubiquitin, isg15 is conjugated to numerous cellular proteins via isopeptide bonds. this isg15 conjugation (isgylation) utilizes ube1l as e1 enzyme, ubch8 as e2 enzyme, and some ubiquitin e3 ligases, such as efp and herc5, as e3 enzymes (fig. 1) . on the other hand, ubp43 (also called usp18) acts as a major isg15-specific deconjugating enzyme. isg15 is not present in yeast, nematode (caenorhabditis), or insect (drosophila), indicating that the functions of isg15 and its modification are restricted to higher animals with evolved ifn signaling. isg15 is strongly induced by type i ifns [16, 17] . viral infection also strongly induces isg15 [18, 19] because one of its major host responses is the production of type i ifns. a number of proteins that are involved in antiviral signaling pathways, including rig-i, mda-5, mx1, pkr, stat1, and jak1, have been identified as target proteins for isgylation. moreover, recent studies have explored the biological functions of isg15 and its conjugation to target proteins. isg15 and its conjugation impair viral replication in vivo [20] [21] [22] [23] [24] [25] [26] . conversely, certain viruses induce viral specific proteins that can deconjugate isg15 from its target proteins or prevent the genera-tion of isgylated proteins, thus abrogating the antiviral response [18, 27, 28] . however, functional consequences of reversible isg15 modification of most target proteins, which are induced by viral infection, are still largely unknown. one of the key components of the innate immune response in regulating cancer development is the activation of type i ifn signaling pathways [29, 30] . type i ifns suppress cell proliferation and promote apoptosis and, therefore, have been used in the treatment of several cancers, such as leukemia [31] . notably, isg15 appears to function as an oncogenic protein as well as a tumor suppressor protein [32] . the findings that cancer chemotherapeutics cause an increase in the level of isg15 conjugates suggest the role of isg15 as a tumor suppressor [33] [34] [35] [36] [37] . conversely, the observations that deregulated overexpression of isg15 and enhanced isgylation are positively correlated with carcinogenesis implicate the oncogenic potential of isg15 protein [38] [39] [40] . however, since ifns are induced on the development of many cancers and isg15 is an interferon-inducible protein, the enhanced expression of isg15 and its conjugation could be a side product of ifn response to cancer and may not play a general role in carcinogenesis. thus, further studies are required for clarification of the functional relationship between isg15 and carcinogenesis. 2. isg15, the product of interferon-stimulated gene 15 isg15 was originally identified by farrell et al. [16] . because some antibodies directed against ubiquitin also react with isg15, it was initially named as an ubiquitin cross-reactive protein (ucrp) [17] . this cross-reactivity is explained by the fact that the 15-kda isg15 protein consists of two domains, each of which bears high sequence homology to ubiquitin. the primary sequences of the two ubiquitinlike domains that correspond to the n-and c-terminal regions of isg15 share 29% and 31% identities with ubiquitin, respectively (fig. 2) . furthermore, both the n-and c-terminal domains of isg15 show a striking similarity in their tertiary structures to ubiquitin (fig. 3 ) [41] . like isg15, fat10, which is also a member of the ubl family, fig. 1 . protein modification by isg15. isg15-specific proteases cleave off the c-terminal extensions from isg15 precursors to generate matured isg15 molecules. isg15 is activated by ube1l (e1) at the expense of atp and is subsequently linked to the activating enzyme via thiosester bond. isg15 linked to ube1l is transferred to ubch8 (e2) and then to a target protein with the aid of an isg15 e3 ligase, such as efp and herc5. ubp43 functions in the reversal of the isgylation process by cleaving off isg15 molecules that are conjugated to the substrate proteins via isopeptide bonds. comprises two ubiquitin-like domains [42] . fat10 is conjugated to a limited number of cellular proteins [43, 44] . like ubiquitin, isg15 proteins in some species are synthesized as precursors that need to be processed before conjugation to target proteins (fig. 2) [45] . while isg15 molecules in human, mouse, and rat are translated as precursors containing the extensions of5-8 amino acids in their c-termini, isg15 proteins in most other species, including fish and bovine, are synthesized as matured forms [46] . the c-terminal lrlrgg sequence of isg15, as that of ubiquitin, is essential not only for its conjugation to substrates but also for its recognition by the relevant processing proteases. however, this hexapeptide sequence is absent in the analogous position of the n-terminal domain. instead, a pro residue is present at the position 81 of isg15, thus preventing the incorrect processing that might generate the two cleaved ubiquitinlike domains of the 15-kda polypeptide [47] . the c-terminal ubiquitin-like domain is sufficient for its activation by ube1l and subsequent thioesterification of ube1l and ubch8 [48] . when overexpressed, the c-terminal domain of isg15 can be conjugated to cellular proteins. however, the level of cellular proteins conjugated by the c-terminal domain alone is much lower than that by full-length isg15, suggesting that both domains of isg15 are required for efficient conjugation to cellular proteins. interestingly, the replacement of arg153 in human isg15 (arg151 in mouse) by ala ablates the binding of isg15 to ube1l and subsequent transesterification of ubch8 [49] . accordingly, the ability of the mutant isg15 to form protein conjugates in 293t cells is markedly diminished. furthermore, while expression of wild-type isg15 protects ifnar −/− mice from lethality after sindbis virus (snv) infection, expression of the arg-to-ala mutant form of isg15 confers no survival benefit. this mutation also attenuates the ability of isg15 to decrease snv replication in ifnar −/− mice or prolong the survival of isg15 −/− mice. thus, arg153 appears to serve as a binding site for ube1l. type i ifns are capable of inducing isg15 [47] . notably, the induction of isg15 shows biphasic kinetics. after ifnβ treatment, the increase in the level of free isg15 molecules is first observable within 2 h, continues for the next 10 h, and becomes maximal by about 18 h. on the other hand, isg15 conjugates are observable at least 12 h after the exposure to ifnβ, and their level dramatically increases between 12 and 18 h and continuously increases thereafter although at a slower rate. this delayed induction of isg15 conjugates indicates the requirement of isg15-conjugating machinery that is expressed in the later periods after the treatment with type i ifns. isg15 is strongly induced by viral infection [18, 19] . like other ifn-stimulated genes, the isg15 gene has a 5′-cis-regulatory sequence called isre (interferon-stimulated response element) [50] . a number of irfs, including irf-3 and irf-9, bind to the isre for isg15 induction (fig. 4) [50] [51] [52] . irf-3 forms a complex with cbp/p300 coactivators for isg15 induction [53, 54] . on the other hand, irf-9 interacts with stat1 and stat2, leading to the formation of isgf3 complex that also induces isg15. upon dsrna treatment, however, irf-3 induces isg15 independently of ifns [55] . isg15 is also strongly induced by lipopolysaccharide (lps) [56] [57] [58] . when macrophages are stimulated by lps, isg15 can be detected as early as 1 h and its level becomes maximal at about 4 h [59] . infection of mice with bacille calmette-guérin (bcg) markedly induces isg15 in macrophages. moreover, this bacterial infection leads to isgylation of serpin2a (serine protease inhibitor 2a), which was the first identified target protein, although its biological significance remains unknown. isgylation of serpin2a may protect macrophages from lysosomal enzymes because a variety of lysosomal proteases are upregulated by ifnγ for the destruction of ingested bacteria and other pathogens. in addition, it has been shown that trif/ irf-3 signaling pathway is responsible for lps-mediated induction of isg15 and its conjugation to target proteins [60] . isg15 is a target gene of nf-κb. lps induces isg15 in 70z/3 cells that recapitulate aspects of the pre-b to immature b-cell transition in response to nf-κb activation, but not in 1.3e, which is a nf-κb signaling defective mutant cell line, indicating that nf-κb activation is required for isg15 induction [56] . ataxia telangiectasia (at) is a multifaceted autosomal recessive genetic disorder, characterized by the loss of coordination, progressive neuronal degeneration, immunodeficiency, and cancer proneness [61] . nf-κb is constitutively activated in human fibroblasts derived from at patients [62] . moreover, the level of isg15 is constitutively elevated in the at cells [63] . because activated nf-κb is capable of inducing the expression of type i ifns, it appears that the elevation of isg15 level in the at cells is due to abnormal production of type i ifns. isg15 also is a target gene of p53 [64] [65] [66] . activation of temperaturesensitive p53 leads to isg15 induction in the absence of exogenous stimuli. since cycloheximide treatment does not influence the increase in the level of isg15 mrna, isg15 induction appears to be a primary response to p53. while type i ifns induce the accumulation of isg15 mrna in both wild-type or p53-deficient cells, dsrna induces it only in wild-type cells, indicating that p53 is required for isg15 induction by dsrna but not for that by type i ifns. in addition, sequence analysis of the human isg15 gene has led to the identification of a putative p53-responsive element that is located adjacent to the isg15-specific isre [50] . thus, in virus-infected cells, p53 seems to exert its antiviral function by the induction of isg15 in addition to the induction of apoptotic signaling [67] . jnk induces the expression of isgs, such as isg15, isg12, and igtp [68] . swiss 3t3 cells expressing constitutively active mkk7-jnk1β fusion protein show increased resistance to apoptosis induced by vesicular stomatitis virus (vsv) infection, suggesting the involvement of jnk signaling pathway in antiviral response. recently, pi3k has been shown to control both ifnα-and ifnγ-induced expression of isgs, including isg15, at both transcriptional and translational levels [69] . ifn-mediated antiviral response is defective in cells lacking p85α/p85β, the regulatory subunits of pi3k, suggesting the involvement of pi3k signaling pathway in innate immune response. isg15 is induced by certain genotoxic stresses. for example, treatment with camptothecin, an inhibitor of topoisomerase i, leads to an increase in the level of isg15 mrna, and this increase requires protein synthesis and a functional p53 protein [35] . interestingly, ifns and jak/stat are dispensable for camptothecin-mediated induction of isg15. isg15 conjugates generated by camptothecin are distinct from those produced by type i ifns, suggesting that different protein substrates are targeted for isgylation. however, camptothecin markedly increases ifn-induced isgylation of cellular proteins. furthermore, treatment with both ifns and camptothecin causes synergistic killing of colorectal cancer xenografts in nude mice, suggesting that the combination of the drugs can be an effective therapeutic strategy [36] . retinoic acid upregulates the levels of isg15, its conjugates, and ube1l in acute promyelocytic cells [33, 34] . the retinoic acid-mediated accumulation of isg15 and its conjugates occurs in retinoic acid-sensitive leukemic cells but not in retinoic acid-resistant cells and the pattern of accumulated isg15 conjugates is similar to that observed by the exposure to type i ifns. in addition, several of the type i ifninduced proteins, such as irf-1 and (2′-5′) oligoadenylate synthetase, are induced by retinoic acid [70] [71] [72] . treatment with retinoic acid also leads to an increased secretion of type i ifns into culture media. blockade of ifnar with a neutralizing antibody prevents retinoic acidmediated accumulation of isg15 and its conjugates. thus, retinoic ns1b inhibits the thioesterification of ube1l and thereby the generation of isg15 conjugates that are required for antiviral response. plpro, otu protease, and viral e3 protein mediate the removal of isg15 from its conjugates. acid seems to elevate the levels of isg15 and its conjugates by stimulating cells to secrete ifns. ube1l is a 112-kda protein that shows a 45% identity in amino acid sequence to the human ubiquitin-activating e1 enzyme (ube1) [73] . ube1l expressed in baculovirus forms a thioester bond with isg15 but not with ubiquitin [18] , indicating that it is a specific isg15activating e1 enzyme. in addition, ube1l has a c-terminal ubiquitinfold domain that is required for the transfer of isg15 from ube1l to ubch8 as well as for the binding of ube1l to ubch8 [74] . as expected, ube1l-deficient mice are not capable of producing isg15 conjugates upon stimuli [75] . in ube1l −/− macrophages treated with lps and in ube1l −/− mouse embryonic fibroblasts (mefs) treated with ifns, ubiquitination of cellular proteins occurs normally, but isgylation of proteins is completely abolished, confirming the strict requirement of ube1l for isgylation. the ube1l gene is located in the d3f15s2 locus of chromosome 3q21 [76] . ube1l can be detected in the jejunum, colon, lung, liver, tonsils, testis, and skin but not in the spleen and pancreas. significantly, ube1l is not detectable in all tested human lung cancer cell lines [73, 77] , implicating a tumor-suppressive role of ube1l. this repressed expression in lung cancer cell lines is intriguing because a region of chromosome 3q, in which the ube1l gene resides, is often deregulated in preneoplastic or neoplastic epithelial tissues [78] . however, upon generation of ube1l −/− /k-ras la2 mice, it has recently been shown that the loss of isgylation does not affect tumor spectrum, tumor pathology, or survival of k-ras la2 mice [79] . nonetheless, it is possible that ube1l deficiency and k-ras mutation are two separate pathways in lung cancer development. additional works with other lung cancer mouse models are necessary to clarify the potential tumor suppressor function of ube1l in k-ras mutation-independent human lung cancers. ubch8, an isg15-conjugating e2 enzyme, is induced by type i ifns and viral infection [80] [81] [82] . ubch6 is also induced by type i ifns and forms a thioester intermediate with isg15, suggesting that ubch6 has the potential to function as an isg15-conjugating e2 enzyme [83] . however, the amount of the thioester intermediate formed by ubch6 is much lower than that by ubch8, indicating that ubch8 is a major e2 enzyme for isg15. ubch8 was originally identified as an ubiquitin-conjugating e2 enzyme by a yeast two-hybrid screening for its interaction with e6ap [84] . ubch8 was later shown to also interact with other ubiquitin e3 ligases, such as parkin, dorfin, staring, efp, and rlim and function as an ubiquitin-conjugating e2 enzyme [85] [86] [87] [88] [89] [90] . thus, ubch8 serves as an e2 enzyme for both ubiquitin and isg15. however, although ubch7 is the most closely related e2 to ubch8, it does not function in isg15 conjugation [74] . moreover, ubch8 shows a higher affinity to ube1l than ubch7, while ubch7 binds more strongly to ube1 than ubch8. in addition, it has been shown that two structural elements within the e2 n-terminal region are responsible for the differential interaction of ubch8 and ubch7 with ube1l. thus, ubch8 may preferentially, but not exclusively, function as an e2 enzyme for isg15. interestingly, ubch8 and isg15 can act as functional regulators of rnf125, an ubiquitin e3 ligase of rig-i [91] . in the absence of isg15, ubch8 interacts with rnf125 and interferes with rig-i ubiquitination. upon overexpression of isg15, however, the interaction of ubch8 with isg15 leads to the dissociation of ubch8 from rnf125, thus allowing the association of rnf125 with ubch5 for rig-i ubiquitination. the overlapping function of ubch8 as an e2 enzyme for both ubiquitin and isg15 raises a possibility that some ubch8-interacting ubiquitin e3 ligases can also function as isg15 e3 ligases. indeed, the ubch8-interacting protein efp (estrogen-responsive finger protein) that has a ring finger domain serves as an isg15 e3 ligase [92] . in the absence of isg15, ubch8 and efp may function as e2 and e3 enzymes for ubiquitination, respectively. upon isg15 induction, however, isg15 may compete with ubiquitin for binding to ubch8, allowing ubch8 and efp to serve as the enzymes for isgylation of 14-3-3σ. replacement of the active site cys residue in the ring domain disrupts efp-mediated isgylation of 14-3-3σ. like ubch8, efp is a type i ifninducible protein [93] . interestingly, efp can isgylate itself on the lys117 residue and this auto-isgylation negatively regulates the isg15 e3 ligase activity of efp toward 14-3-3σ, suggesting that a feedback loop is operating for the control of the protein isgylation [94, 95] . an additional ring-containing ubiquitin e3 ligase, called hhari (human homolog of drosophila ariadne), also serves as an isg15 e3 ligase for 4ehp [96] . herc5 (hect domain and rcc1-like domain containing protein 5) is an isg15 e3 ligase that contains the hect domain [97] [98] [99] . like efp, herc5 is a type i ifn-inducible protein. knockdown of herc5 by using sirna prevents ifn-mediated increase in the total level of isgylated cellular proteins, unlike that of efp, which blocks the isgylation of 14-3-3σ with little or no effect on the total level of isgylated proteins. thus, it appears that herc5 serves as a general ifn-induced isg15 e3 ligase. like efp, herc5 itself is a target for isgylation, but its functional significance is unknown. 3.4.1. ubp43 and other isg15-specific proteases ubp43 cleaves off isg15 from its protein conjugates that are linked via isopeptide bonds [100] . ubp43 can also cleave the peptide bonds immediately after the lrlrgg sequence in isg15 fusions [100] . thus, ubp43 appears to also function in the processing of isg15 precursors to generate matured isg15 molecules. however, apart from ubp43, additional isg15-specific proteases must exist because isg15 precursor is processed to its matured form in ubp43-deficient mice [101] . the overlapping function of some e2 and e3 enzymes in the conjugation of both isg15 and ubiquitin also implies the existence of promiscuous dubs that can serve as isg15-specific proteases. indeed, several dubs, including usp2, usp5, usp13, and usp14, have been identified as the candidates that can function as isg15-processing and/or deconjugating enzymes [102] . in addition, it has been reported that recombinant isg15 precursors can be properly processed by a 100-kda enzyme in the extracts of human lung fibroblasts cell line a549, whose activity is unaffected by type i ifn stimulation [103] . this 100-kda enzyme is a cysteine protease and shows a partial similarity in its amino acid sequence with that of the human ortholog of yeast ubp1 or ubp1-related protein. however, deletion of the ubp43 gene in mouse leads to a massive increase of isg15 conjugates in tissues without affecting the level of ubiquitin conjugates, indicating that ubp43 is the major isg15-specific protease that deconjugates isg15 from its target proteins. thus, it appears likely that the above-mentioned dubs as well as the 100-kda cysteine protease function primarily in the processing of isg15 precursors to generate matured isg15 molecules. ubp43 is induced by type i ifns, and this induction requires a functional jak/stat signaling pathway [104] . ifnβ induces ubp43 more strongly than ifnα and dsrna, but ifnγ barely induces it [105] . ubp43 is also induced by lps [58] . irf-3 is responsible for lps-mediated induction of ubp43, while irf-2 is involved in its induction to a basal level. however, both irf-2 and irf-3 are required for optimal responsiveness to lps. interestingly, ubp43 induction is upregulated by the acute myelogenous leukemia (aml1)-eto fusion protein that is created by t(8;21), suggesting a possible involvement of ubp43 in hematopoiesis [106] . however, it is unknown how aml1-eto affects the up-regulation of ubp43 induction. in addition, ubp43 has been identified as a substrate for skp2 [107] . skp2 promotes the ubiquitination of ubp43 and subsequent degradation by the proteasomes, suggesting that the scf skp2 may be involved in the regulation of type i ifn signaling by controlling the stability of ubp43. the human ubp43 gene maps to the chromosome 22q11.2 [108] . this region, known as digeorge syndrome critical region, is consistently deleted in digeorge syndrome and related disorders, suggesting that ubp43 may be involved in the development of thymus or differentiation of t cells. ubp43-deficient mice are viable and resistant to the fatal lymphocytic choriomeningitis and myeloencephalitis that develop in wildtype mice upon intracerebral inoculation of lymphocytic choriomeningitis virus (lcmv) and vsv, respectively [101, 109] . furthermore, survival of ubp43 −/− mice after lcmv infection correlates with severe inhibition of lcmv replication as well as with an increase in the level of isg15 conjugates in the brain. none of the ubp43 −/− mice infected with lcmv died or developed clinical symptoms by day 11 after infection, whereas all lcmv-infected wild-type mice died by day 7 infection. these findings strongly suggest that ubp43 deficiency causes an unfavorable environment for lcmv replication. in addition, ubp43 −/− mef cells exhibit enhanced type i ifn-mediated resistance to cytopathic effect caused by vsv and snv. these findings strongly suggest that ubp43 serves as a negative regulator of innate immune response against viral infection. however, the enhanced resistance to viral infection in ubp43 −/− mice cannot be rescued in ubp43 −/− / isg15 −/− or ubp43 −/− /ube1l −/− double knockouts, indicating that the phenotypic alterations are not associated with the protein modification by isg15 [75, 110] . thus, ubp43, independently of its isopeptidase activity, may have another biological function. ubp43 −/− cells are hypersensitive to type i ifns, resulting in a dramatic increase in the level of isgylated proteins, which is associated with the enhanced and prolonged jak/stat signaling [111] . however, ubp43, independently of its catalytic activity, also functions as a negative regulator of type i ifn signaling [112] . this ubp43 action is achieved through a direct interaction between ubp43 and ifnar2, a subunit of type i ifn receptor. binding of ifnar2 to ubp43 interferes with the interaction between jak and the receptor, leading to the inhibition of downstream phosphorylation cascade and other signaling events. in addition, complementation of ubp43 −/− cells with a catalytically inactive mutant of ubp43 leads to the inhibition of stat1 phosphorylation to a level seen by that with wild-type ubp43. moreover, down-regulation of the total level of isg15 conjugates by sirnamediated knockdown of ube1l in ubp43 −/− cells shows little or no effect on stat1 phosphorylation in comparison with that seen in ubp43 +/+ cells, indicating that the deisgylating activity of ubp43 is not required for its inhibitory effect on type i ifn signaling. this conclusion is further corroborated with the findings that phenotypic alterations in ubp43 −/− mice are not influenced by the lack of isg15 in ubp43 −/− /isg15 −/− double knockout mice [75, 110] . the isopeptidase-independent action of ubp43 is also involved in the replication of hbv (hepatitis b virus) [113] . ubp43 −/− cells show an increased induction of isgs in response to type i ifns, indicating that the lack of ubp43 results in a strengthened immune response. consistently, the steady-state level of hbv dna is substantially reduced in ubp43 −/− mice in comparison with that in ubp43 +/+ mice. thus, approaches that modulate ubp43 level could be used as therapeutic potentials in treating viral infection, especially for viruses sensitive to type i ifn signaling. in addition to the protection against hbv, ubp43 deficiency increases the resistance to oncogenic transformation by bcr-abl (breakpoint cluster region abelson leukemia protein), a fusion protein consisting of the n-terminal portion of bcr joined to most of the abl tyrosine kinase [114] . this resistance to leukemia development is heavily dependent on the activation of type i ifn signaling pathway. loss of type i ifn signaling through the loss of ifnar results in a reversal of the original resistance to the leukemia development observed in mice that received a transplant of bcr-ablexpressing ubp43-deficient donor cells. thus, it appears that inhibition of the negative effect of ubp43 on type i ifn signaling could enhance innate immune response against cancer development. by using a combination of affinity purification and mass spectrometry, hundreds of target proteins for isgylation have been identified. some of them are type i ifn-induced proteins, including pkr, mxa, hup56, and rig-i [115] . some are key regulators that are involved in type i ifn signaling, such as plcγ1, jak1, erk1, and stat1 [116] . most other targets are constitutively expressed and function in diverse cellular pathways, including translation, glycolysis, cell motility, protein modification, intracellular protein trafficking, rna splicing, chromatin remodeling, cytoskeletal organization, and stress responses [97, 115, 117] . these findings implicate the role of protein isgylation in type i ifn-induced immune responses as well as in the control of numerous fundamental cellular pathways. isg15 is synthesized in many cell types and secreted from human monocytes and lymphocytes [118] . both native and recombinant isg15 induce the synthesis and secretion of ifnγ from b-cell-depleted lymphocytes, implicating the role of isg15 as a cytokine that modulates immune response [119] . treatment with human isg15, but not its precursor form, leads to an increase in dna synthesis in cultured primary blood lymphocytes in a dose-dependent fashion, suggesting that isg15 can act as a mitogen and that the processing of isg15 precursor is required for the generation of biologically active isg15 [120] . furthermore, isg15 has been identified as a member of the cytokine cascades. upon viral infection, type i ifns produced in infected cells induce the synthesis of isg15. these isg15 molecules can be secreted or released by lysis of the infected cells. they may then induce the production of ifnγ from t cells, augment nk cell proliferation, activate non-major histocompatibility complex-restricted cytolytic lymphocytes, and activate monocytes and macrophages via the induced ifnγ. filamins are nonmuscle actin-binding proteins that comprise a family of three members: filamin a, b, and c [121, 122] . these filamin isoforms are large cytoplasmic proteins that play important parts in cross-linking cortical actin filaments into a dynamic three-dimensional structure. recently, it has been shown that filamin b functions as a scaffold that links between activated rac1 and a jnk cascade module for mediating type i ifn signaling [123, 124] . filamin b interacts with rac1, mekk1, mkk4, and jnk and enhances their sequential activation in response to type i ifns, thereby promoting jnk activation and apoptosis. this acceleration of jnk-mediated apoptosis provides a biological basis for antitumor and antiviral functions of type i ifns. furthermore, it has been revealed that type i ifns induce isgylation of filamin b, which leads to dissociation of rac1, mekk1, and mkk4 from the scaffold protein and thus to the prevention of prolonged activation of type i-induced jnk signaling pathway [124, 125] . in contrast, blockade of filamin b isgylation by substitution of the isg15 acceptor site lys2467 with arg or by sirna-mediated knockdown of ubel1 prevents the release of the signaling molecules from filamin b, resulting in persistent promotion of jnk activation and jnk-mediated apoptosis. these findings indicate that isgylation of filamin b serves as a negative feedback regulatory gate for desensitization of type i ifn-induced jnk signaling, thus providing an important mechanism for the survival of uninfected bystander cells. pp2cβ dephosphorylates tak1 and suppresses tak1/tab1mediated iκbα degradation, thus controlling nf-κb signaling pathway, which plays a critical role in innate and adaptive immunity and cancer [126] [127] [128] [129] . pp2cβ is a target for isgylation, and this modification blocks the suppressive function of the protein phosphatase against tak1/ tab1-mediated nf-κb activation [130] . this conclusion is based on the observation that overexpression of ube1l, ubch8, and isg15 blocks the suppressive effect of pp2cβ on nf-κb activation, but not that of its mutant, in which the isg15 acceptor sites lys12 and lys142 are replaced by arginine. thus, isgylation of pp2cβ seems to play a role in the control of nf-κb pathway. ubc13, an ubiquitin-conjugating e2 enzyme, is a target for isgylation [131, 132] . isg15 is conjugated to lys92 of ubc13, which is very closely located to its active site, thus preventing the formation of a thioester bond with ubiquitin. since mms2, which forms a heteromeric complex with ubc13, interacts with both unmodified and isgylated ubc13, the inhibitory effect of ubc13 isgylation on the thioesterification of ubiquitin seems to be achieved by the inability of isgylated ubc13 to accept ubiquitin to its active site or by blocking the recognition of ubiquitin e1 enzyme and subsequent transfer of ubiquitin from the e1 enzyme to ubc13. ubc13 is known to mediate the generation of lys63-linked poly-ubiquitin chains that are conjugated to a variety of signaling molecules [133, 134] . thus, it is possible that isgylation of ubc13 may play an important role in the control of signal transduction pathways, such as nf-κb pathway, which are associated with lys63-linked poly-ubiquitination [132] . ubch8 and ubch6 also are targets for isgylation [83] . isgylation of ubch6 occurs in response to type i ifns and blocks the thioesterification of ubiquitin, like that of ubc13, suggesting that isgylation of ubch6 may also lead to the suppression of its ubiquitin-conjugating activity. initial efforts that intend to determine the role of isg15 in antiviral responses appeared unsuccessful. ube1l −/− mice are fertile and exhibit normal antiviral responses against vsv and lcmv infection, indicating that ube1l and protein isgylation may not be essential for ifn signaling [75] . furthermore, isg15 −/− mice are also fertile and display no obvious abnormalities [135] . lack of isg15 does not affect the development and composition of the main cellular immune system. the ifn-induced antiviral and immune responses directed against vsv and lcmv are not significantly altered in the absence of isg15. in addition, ifn-or endotoxin-induced stat1 phosphorylation as well as the expression of typical stat1 target genes remain unaffected by the lack of isg15, indicating that isg15 is dispensable for stat1-mediated ifn signaling. however, the function of isg15 as an antiviral effector has come to the front. unlike the infection by vsv and lcmv, ube1l −/− mice display markedly increased susceptibility to influenza b virus infection, with only 25% survival of ube1l −/− mice for 21 days, compared to 86% survival of ube1l +/+ mice [20] . furthermore, both of the kinetics of lethality and the overall survival of ube1l −/− mice are identical to those of isg15 −/− mice, indicating that the antiviral activity of isg15 against influenza b virus is mediated by its conjugation to target proteins. the predominant site of isg15 action during influenza virus infection resides within a stromal cell population. a likely candidate is the respiratory epithelium, since it is the site of influenza virus replication. this is the first phenotype described for ube1l −/− mice, which were previously found to have no defect in response to vsv or lcmv infection [75] . in addition, it has recently been shown that both the synthesis of influenza a virus protein and the early rate of the virus replication are inhibited by isg15 conjugation of cellular proteins [21] . isg15 −/− mice also exhibit increased susceptibility to infection by a number of other viruses, such as influenza a/wsn/33 and b/lee/40 viruses, herpes simplex virus type 1 and murine gamma herpes virus 68, and snv [22] . in addition, isg15 is induced after snv infection, and this induction is markedly attenuated in ifnar −/− mice, indicating that induction of isg15 by snv infection is dependent on type i ifns [136] . isg15 induction protects against snv-induced lethality and decreases the virus replication in multiple organs. the increased susceptibility of ifnar −/− mice to snv infection can be rescued by the expression of wild-type isg15 having the c-terminal diglycine residues but not by that of an isg15 mutant, of which the c-terminal diglycine is replaced by dialanine, again indicating that the antiviral action of isg15 requires its conjugation to target proteins. type i ifn-mediated inhibition of human immunodeficiency virus (hiv) assembly and release has been shown to correlate with the induction of isg15 [23] . furthermore, overexpression of isg15 mimics the ifn effect and inhibits the release of hiv-1 virions without having any effect on the synthesis of the viral proteins in infected cells [24] . in cells infected with hiv-1 provirus, overexpression of isg15 and ube1l causes a complete inhibition of hiv-1 replication. on the other hand, coexpression of ubp43 can partially rescue the release of hiv-1 in isg15-expressing cells. intriguingly, isg15 expression specifically inhibits the ubiquitination of gag and tsg101 and disrupts their interaction, thereby preventing assembly and release of virions from infected cells. expression of isg15 alone (i.e., without ube1l) does not block the viral release, indicating that isgylation of target proteins, but not isg15 itself, is required for the inhibition of gag ubiquitination, although isgylation of either gag or tsg101 could not be detected. thus, isgylation of certain unknown viral and/or host proteins appears to play a critical role in the ifn-mediated inhibition of hiv-1 assembly and release. overexpression of isg15 or ube1l with ubch8 has been shown to inhibit budding of ebola virus vp40 virus-like particles (vlps) [25, 26] . ebola virus is a member of the filoviral family of negative-sense rna viruses. the vp40 matrix protein is a key structural protein that is critical for the virion release. the efficient budding of vlps requires the interaction of overlapping l-domains (late-budding domains) in the vp40 protein with nedd4, an ubiquitin e3 ligase, as well as with other members of the escrt pathway (e.g., tsg101) [137] [138] [139] [140] . isg15 overexpression decreases not only the level of vp40 detected in vlps but also the levels of endogenous nedd4 incorporated into budding vp40 vlps. nedd4 interacts with and is conjugated by isg15. moreover, isg15 overexpression blocks the ability of nedd4 to ubiquitinate vp40, leading to the prevention of l-domain-mediated budding of vp40 vlps. in addition, it has been shown that free isg15 specifically binds to nedd4 and inhibits the interaction of the e3 ligase with ubiquitin e2 enzymes (e.g., ubch6), thus preventing the transfer of ubiquitin from the e2 enzymes to nedd4 [26] . these findings reveal a mechanism for the antiviral function of isg15 that involves the isgylation and inactivation of the host nedd4 e3 ligase. irf-3 is a target for isgylation, which can be induced by both type i ifns and viral infection [141] . this isgylation prevents the ubiquitination and degradation of irf-3 in ndv (newcastle disease virus)-infected human fibrosarcoma 2ftgh cells, and promotes the translocation of irf-3 to the nucleus, where it binds to the ifnβ promoter. the relative level of irf-3 is significantly lower in ndvinfected isg15 −/− cells than in isg15 +/+ cells, indicating that the subversion of antiviral response is mediated by proteolysis of irf-3. moreover, the degradation of irf-3 can be counteracted by the induction of isg15. this finding provides a positive feedback mechanism for the induction of host antiviral response by isgylation-dependent stabilization of irf-3. however, isg15 is typically conjugated to only a small fraction of target proteins. therefore, an important issue is how the small fractions of isgylated proteins, including irf-3, can exert their biological functions. it has been proposed that if the small fractions of proteins that are modified by ubls (e.g., sumo and isg15) are localized to some functionally unique cellular site or the transient modification is sufficient to switch the protein into new state, their functions could efficiently operate [124, 142, 143] . intriguingly, overexpression of isg15 leads to the appearance of irf-3 as punctates in the nucleus [141] . this nuclear retention could allow irf-3 to exert its profound antiviral function, although only a small portion of irf-3 is isgylated upon the viral infection. an additional example is that upon overexpression of filamin b, ubch8 is recruited to membrane ruffles where filamin b is also concentrated with actin [123, 125] . this recruitment of ubch8, which otherwise resides throughout the cytoplasm and the nucleus, should allow the e2 enzyme to efficiently function in filamin b isgylation and thus in desensitization of type i ifn-induced jnk signaling (see above). the inducible nitric oxide synthase (inos) is not expressed under normal conditions, but like isg15, it is induced by various stimuli, such as exposure to cytokines, microbes, or microbial products, resulting in the sustained production of no [144] . upon stimuli, no as well as the products generated by its interaction with ros, such as peroxynitrite and peroxynitrous acid, are accumulated and utilized for the antibacterial or antiviral effects [144] [145] [146] . no is also covalently attached to the thiol group of cysteine residues of proteins, causing their s-nitrosylation. this posttranslational modification serves as an important mechanism that mediates antibacterial and antiviral effects through the alteration in enzymatic activity, protein-protein interaction, and signal transduction [147] . interestingly, the cysteine residue in isg15 can also be modified by no and this s-nitrosylation decreases the dimerization of isg15, thereby increasing the availability as well as the solubility of monomeric isg15 for its conjugation to cellular proteins [148] . moreover, treatment with s-ethylisothiourea, an inos inhibitor, reduces the level of isg15 conjugates, whereas overexpression of inos increases it. thus, inos may play a role in the enhancement of innate immune responses by mediating isg15 s-nitrosylation. 4ehp is an mrna 5′cap structure-binding protein and acts as a translation suppressor by competing with eif4e for binding to the cap structure [149] . 4ehp is a target protein for isgylation and this modification substantially increases its cap structure-binding activity [96] . this is the first report that shows "gain of function" by ifn-induced isgylation, thus providing a mechanism by which a small fraction of any isgylated protein can generate profound biological effects in response to ifns or pathogen infections. interestingly, 4ehp fused with isg15 at either of its n-or c-terminus dramatically enhances the cap structure-binding activity, indicating the isg15 fusion protein can mimic the isgylated 4ehp. since ifns inhibit the translation of viral mrnas while allowing normal translation of the majority of cellular mrnas [150] , it is possible that isgylated 4ehp acts as a viral mrnaspecific translation inhibitor in a cap binding-dependent manner. rig-i senses intracellular virus-specific nucleic acid structures and as an early antiviral response induces the production of type i ifns, which in turn activates the expression of rig-i, thus generating a positive feedback loop for further accumulation of rig-i [151] [152] [153] [154] [155] . intracellular dsrna activates nf-κb and irf-3/irf-7 through rig-i and the mitochondrial adaptor protein ifn promoter stimulator 1 (ips-1) [156] [157] [158] [159] . furthermore, rig-i is a target protein for isgylation [115, 160] . however, isgylation of rig-i leads to a reduction in the levels of both basal and virus-induced ifn production. consistently, the basal mrna and protein levels of rig-i are significantly higher in ube1l −/− cells than in ube1l +/+ cells. based on these observations, it has been proposed that a negative feedback loop is operating for fine-tuning the strength of rig-i-mediated signaling to maintain a balance between innate immune response and hypersensitivity during antiviral responses. in addition, ubiquitination of rig-i by trim25 is required to mediate antiviral signaling responses, whereas that by rnf125 results in the proteasomal degradation of rig-i [161] . thus, multiple positive and negative mechanisms appear to contribute to the maintenance of optimal rig-i level and thus to the control of rig-i-mediated signaling. viruses escape from the antiviral activity of isg15 by using different mechanisms. influenza b virus strongly induces isg15 during infection but specifically blocks isgylation of host proteins [18] . this inhibition is mediated by the viral ns1b protein, which interacts with isg15 and prevents the generation of isg15 conjugates under in vitro conditions as well as in infected cells (fig. 4) . purified ns1b inhibits the formation of isg15-ube1l-like thioester intermediate. isg15 conjugates accumulate in ifn-treated cells, but not in cells infected by influenza b virus, although similar amounts of isg15 are synthesized in both cells. moreover, isg15 conjugates are not detectable in cells infected with the virus expressing full-length ns1b but are markedly accumulated in cells infected with the virus expressing truncated ns1b, which cannot bind to isg15, indicating that the interaction of ns1b with isg15 is responsible for the inhibition of isg15 conjugation and thus of the antiviral function of isg15. sars-coronavirus (sars-cov) produces a papain-like protease, called plpro, which can generate an authentic isg15 molecule by cleaving off a protein that is fused to the c-terminus of isg15 [162] . it is possible that the activity of plpro may mimic the isg15-deconjugating activity of ubp43, thus favoring viral replication by counteracting protein isgylation. vaccinia virus (vacv) e3 protein is an early protein that interacts with isg15 through its c-terminal domain [28] . whereas sirnamediated knockdown of isg15 enhances the viral replication, complementation of isg15 to isg15 −/− cells attenuates it. notably, the level of isg15 conjugates is much higher in isg15-complemented isg15 −/− cells infected with the e3-deficient virus than that with the wild-type virus, suggesting that vacv e3 protein is directly or indirectly involved in deconjugation of isg15 from cellular proteins. moreover, the virus lacking e3 protein that is unable to grow in isg15 +/+ cells can replicate in isg15 −/− cells. these findings suggest a new strategy for poxviruses to evade the host antiviral response through the viral e3 protein-mediated control of protein isgylation. the ovarian tumor domain (otu)-containing proteases from nairoviruses and arterviruses, two unrelated groups of rna viruses, are capable of deconjugating ubiquitin and isg15, but not sumo, from cellular target proteins [27] . purified otu protease cleaves off ubiquitin from both lys48-and lys63-linked poly-ubiquitin chains in vitro. remarkably, the expression of otu protease antagonizes the antiviral effects of isg15 and enhances the susceptibility to snv infection. it has been shown that approximately 70% of ifnar −/− mice survive after infection of the virus expressing both isg15 and an otu variant, of which the catalytic cys residue is replaced by ala. in contrast, only 20% of the mice survive when infected with the virus expressing both isg15 and wild-type otu protease, indicating that the antiviral response is mediated by isg15, but not by other isgs, and that the immune-evading effect of otu protease requires its catalytic activity. these findings indicate that snv uses otu protease as a unique strategy to evade the host antiviral response. hepatitis c virus (hcv) is an enveloped virus that causes liver diseases, including cancer [163, 164] . rig-i signaling induces a host response that controls hcv rna replication [165] . however, hcv can evade this response in part through its ability to antagonize the relay of rig-i signaling to irf-3 [166] . ns3/4a, a major serine protease expressed by hcv [164] , cleaves ips-1, causing the release of cleaved ips-1 from the mitochondrial membrane [167, 168] . this cleavage results in the subcellular redistribution of ips-1 and loss of its interaction with rig-i, thereby preventing downstream activation of irf-3 and ifnβ induction. intriguingly, in liver tissues chronically infected by hcv, the ips-1 cleavage and its subcellular redistribution are associated with the lack of isg15 and its conjugates. among the hcvinfected tissues, isg15 and its conjugates can be detected only in the liver from patients, which has predominantly uncleaved, full-length ips-1 protein. these findings indicate that ns3/4a plays a critical role in evading the host antiviral response by attenuating the ifn amplification loop of rig-i and thus the expression of isgs, including isg15, which are normally induced by rig-i-and irf-3-dependent pathways. 4.6. isg15 in tumorigenesis 4.6.1. isg15 as a tumor suppressor ube1l has been shown to play an important role in the suppression of lung cancer growth. ube1l overexpression inhibits the growth of human bronchial epithelial cells and lung cancer cells. furthermore, ube1l promotes cyclin d1 isgylation and this modification leads to the destabilization of cyclin d1, indicating that ube1l confers growth suppression by targeting cyclin d1 [169] . these findings provide a mechanism for the tumor-suppressive role of ube1l [37, 73, 77, 170] , although it is unknown how the stability of cyclin d1 is affected by its isgylation. acute promyelocytic leukemia (apl) is characterized by the accumulation of oncogenic pml/rarα fusion protein [171, 172] . retinoic acid induces ube1l and subsequent isgylation of pml/rarα [33, 34, 37] . moreover, ube1l-mediated isgylation of pml/rarα leads to a decrease in the level of pml/rarα, thus overcoming the oncogenic effects of the fusion protein [173, 174] . on the other hand, treatment with n-acetyl-leu-leu-norleucinal, a proteasome inhibitor, prevents the degradation of isgylated pml/rarα, indicating that the proteasomes are involved in the control of pml/rarα stability. these findings implicate an important role of ube1l in the suppression of leukemia. in many tumors and tumor cell lines, isg15 is highly elevated and extensively conjugated to cellular proteins [39] . the level of isg15 mrna is significantly higher in cancerous mammary epithelial cells lines, such as bt20, mda-mb468, mda-mb231, t47d, and mcf7, than in nonmalignant mammary cell lines, including hmec and mcf10a. the level of isg15 protein is also higher in breast tumors than in normal tissues [175] . in addition, a correlation between increased isg15 level and unfavorable prognosis in the survival of patients has been reported, suggesting a potential role of isg15 in breast cancer development, although the role of isg15 in breast carcinogenesis is unknown. significantly, the increased level of isg15 in tumor cells is associated with the decreased levels of ubiquitinated proteins, suggesting that isg15 plays an antagonistic role against ubiquitin-mediated proteolysis and that deregulation of ubiquitin-proteasome pathway is related with tumorigenesis [39] . since some of the e2 (e.g., ubch8) and e3 enzymes (e.g., efp) can utilize both ubiquitin and isg15 for conjugation to target proteins, it seems possible that isg15 may potentially interfere with the ubiquitination pathway at the level of e2 and e3 enzymes, thus decreasing the level of ubiquitinated proteins in tumor cells. however, it has been shown that the level of ubiquitin conjugates in ubp43 −/− cells is nearly the same as that in ubp43 +/+ cells [116] , despite the fact that treatment with type i ifns leads to a dramatic accumulation in the level of isg15 conjugates in ubp43 −/− cells as compared with that in ubp43 +/+ cells. furthermore, treatment with lactacystin, an inhibitor of the proteasome, shows little or no effect on the level of isg15 conjugates in either of ubp43 +/+ or ubp43 −/− cells whether or not exposed to ifnβ, while as expected the level of ubiquitinated proteins markedly increases in both cells. thus, it appears that isg15 and its conjugates by themselves do not interfere with the ubiquitination of cellular proteins and their subsequent degradation. the reason for the tumor-specific overexpression of isg15 in different tumors is unclear. one possibility is that the elevated expression of isg15 may be due to constitutively activated nf-κb in many tumor cells. this possibility is supported by an observation that isg15overexpressing zr-75-1 cells show a greater nf-κb activity than isg15-underexpressing bt474 breast cancer cells [176] . alternatively, the deregulated expression of ube1l and ubp43 in certain tumors could contribute to the variation in the levels of isg15 conjugates in tumor biopsy samples. androgen receptor has been implicated in the initiation and progression of prostate cancer [177, 178] . significantly, ube1l and ubch8 are upregulated in prostate cancer lesions as compared to those in corresponding nonmalignant tissues [179] . moreover, overexpression of ube1l in lncap cells leads to a marked increase in the mrna and protein levels of androgen receptor in addition to an increase in the rate of cell proliferation. on the other hand, sirna-mediated knockdown of isg15 and ube1l in lncap cells results in a significant decrease in the transcript and protein levels of androgen receptor. these findings suggest that isgylation machinery participates in a positive control of androgen receptor expression during the onset of prostate cancers, thereby promoting the tumor growth. since the discovery of ifns, a vast knowledge on the role of the cytokines in innate immune responses has been accumulated. isg15 is one of the most strongly induced isgs upon exposure to type i ifns, virus, lps, and other stresses, including certain genotoxic stresses [16, 17, 35, 56, 64, 180] . considering that type i ifns play critical roles in innate immune responses regulating the antiviral responses as well as the cancer development, there is no doubt that isg15 and its conjugation to target proteins play critical roles in the type i ifninduced immune responses. however, the biological significance of protein modification by isg15 has been established only in several cases. therefore, much studies are required for clarification of the role of isg15 in innate immune responses against viral infection and cancer and thus for the control of immune diseases as well as for resolving the contradictory findings, such as the role of isg15 as a tumor suppressor versus an oncogenic protein. clinical uses of interferons virus interference: i. the interferon jak-stat pathways and transcriptional activation in response to ifns and other extracellular signaling proteins contribution of stat sh2 groups to specific interferon signaling by the jak-stat pathway a single phosphotyrosine residue of stat91 required for gene activation by interferon-gamma stat proteins and transcriptional responses to extracellular signals the jak-stat pathway: cytokine signalling from the receptor to the nucleus stats and gene regulation antiviral actions of interferons type i interferons in host defense evolution and function of ubiquitin-like protein-conjugation systems isg15, not just another ubiquitin-like protein the ubiquitin system protein regulation by monoubiquitin ubiquitin and its kin: how close are the family ties? accumulation of an mrna and protein in interferon-treated ehrlich ascites tumour cells interferon induces a 15-kilodalton protein exhibiting marked homology to ubiquitin influenza b virus ns1 protein inhibits conjugation of the interferon (ifn)-induced ubiquitin-like isg15 protein induced expression of the endogenous beta interferon gene in adenovirus type 5-transformed rat fibroblasts mice lacking the isg15 e1 enzyme ube1l demonstrate increased susceptibility to both mouse-adapted and non-mouse-adapted influenza b virus infection interferon-induced isg15 conjugation inhibits influenza a virus gene expression and replication in human cells virgin, ifn-stimulated gene 15 functions as a critical antiviral molecule against influenza, herpes, and sindbis viruses role of interferon-stimulated gene isg-15 in the interferon-omega-mediated inhibition of human immunodeficiency virus replication innate antiviral response targets hiv-1 release by the induction of ubiquitin-like protein isg15 isg15 inhibits ebola vp40 vlp budding in an l-domain-dependent manner by blocking nedd4 ligase activity isg15 inhibits nedd4 ubiquitin e3 activity and enhances the innate antiviral response ovarian tumor domain-containing viral proteases evade ubiquitin-and isg15-dependent innate immune responses vaccinia virus e3 protein prevents the antiviral action of isg15 interferons alpha and beta as immune regulators-a new look apoptosis and interferons: role of interferon-stimulated genes as mediators of apoptosis cancer immunotherapy: the interferon-alpha experience the interferon regulated ubiquitin-like protein, isg15, in tumorigenesis: friend or foe? involvement of ube1l in isg15 conjugation during retinoid-induced differentiation of acute promyelocytic leukemia ube1l is a retinoid target that triggers pml/ raralpha degradation and apoptosis in acute promyelocytic leukemia camptothecin induces the ubiquitinlike protein, isg15, and enhances isg15 conjugation in response to interferon interferon potentiates antiproliferative activity of cpt-11 against human colon cancer xenografts ube1l represses pml/rar{alpha} by targeting the pml domain for isg15ylation stage-associated overexpression of the ubiquitin-like protein, isg15, in bladder cancer elevated expression of isg15 in tumor cells interferes with the ubiquitin/26s proteasome pathway exploration of global gene expression patterns in pancreatic adenocarcinoma using cdna microarrays crystal structure of the interferon-induced ubiquitin-like protein isg15 a mhcencoded ubiquitin-like protein (fat10) binds noncovalently to the spindle assembly checkpoint protein mad2 the ubiquitin-like protein fat10 forms covalent conjugates and induces apoptosis fat10, a ubiquitinindependent signal for proteasomal degradation a 15-kda interferon-induced protein is derived by cooh-terminal processing of a 17-kda precursor molecular cloning of the fish interferon stimulated gene, 15 kda (isg15) orthologue: a ubiquitin-like gene induced by nephrotoxic damage the interferon-inducible 15-kda ubiquitin homolog conjugates to intracellular proteins different roles for two ubiquitin-like domains of isg15 in protein modification virgin, isg15 arg151 and the isg15-conjugating enzyme ube1l are important for innate immune control of sindbis virus interferoninduced transcription of a gene encoding a 15-kda protein depends on an upstream enhancer element gene induction pathways mediated by distinct irfs during viral infection identification of a member of the interferon regulatory factor family that binds to the interferonstimulated response element and activates expression of interferon-induced genes characterization of specific dna-binding factors activated by double-stranded rna as positive regulators of interferon alpha/beta-stimulated genes interferon regulatory factor 3 and crebbinding protein/p300 are subunits of double-stranded rna-activated transcription factor draf1 direct induction of interferon-gamma-and interferon-alpha/beta-inducible genes by double-stranded rna novel nemo/ikappab kinase and nf-kappa b target genes at the pre-b to immature b cell transition bacterial lipopolysaccharide and gamma interferon induce transcription of beta interferon mrna and interferon secretion in murine macrophages lipopolysaccharide activates the expression of isg15-specific protease ubp43 via interferon regulatory factor 3 serpin 2a is induced in activated macrophages and conjugates to a ubiquitin homolog enhanced antibacterial potential in ubp43-deficient mice against salmonella typhimurium infection by up-regulating type i ifn signaling ataxia-telangiectasia: an inherited disorder of ionizing-radiation sensitivity in man. progress in the elucidation of the underlying biochemical defect correction of radiation sensitivity in ataxia telangiectasia cells by a truncated i kappa b-alpha elevation of interferon beta-inducible proteins in ataxia telangiectasia cells 14-3-3 sigma is a p53-regulated inhibitor of g 2 /m progression a model for p53-induced apoptosis role for p53 in gene induction by doublestranded rna integration of interferon-alpha/beta signalling to p53 responses in tumour suppression and antiviral defence differential gene regulation by specific gain-offunction jnk1 proteins expressed in swiss 3t3 fibroblasts dual regulatory roles of phosphatidylinositol 3-kinase in ifn signaling gene expression profiling during all-trans retinoic acid-induced cell differentiation of acute promyelocytic leukemia cells gene expression networks underlying retinoic acid-induced differentiation of acute promyelocytic leukemia cells stat1 is induced and activated by all-trans retinoic acid in acute promyelocytic leukemia cells a gene in the chromosomal region 3p21 with greatly reduced expression in lung cancer is similar to the gene for ubiquitin-activating enzyme the basis for selective e1-e2 interactions in the isg15 conjugation system ube1l and protein isgylation are not essential for alpha/beta interferon signaling deletions of the short arm of chromosome 3 in solid tumors and the search for suppressor genes the ubiquitin-activating enzyme e1-like protein in lung cancer cell lines alteration of tumor spectrum by isgylation in p53-deficient mice deficiency of a potential 3p21.3 tumor suppressor gene ube1l (uba7) does not accelerate lung cancer development in k-rasla2 mice proteome analysis reveals ubiquitin-conjugating enzymes to be a new family of interferon-alpharegulated genes interferon-inducible ubiquitin e2, ubc8, is a conjugating enzyme for protein isgylation the ubch8 ubiquitin e2 enzyme is also the e2 enzyme for isg15, an ifn-alpha/beta-induced ubiquitin-like protein link between the ubiquitin conjugation system and the isg15 conjugation system: isg15 conjugation to the ubch6 ubiquitin e2 enzyme physical interaction between specific e2 and hect e3 enzymes determines functional cooperativity staring, a novel e3 ubiquitin-protein ligase that targets syntaxin 1 for degradation parkin suppresses unfolded protein stress-induced cell death through its e3 ubiquitin-protein ligase activity the histone deacetylase inhibitor valproic acid selectively induces proteasomal degradation of hdac2 a novel centrosomal ring-finger protein, dorfin, mediates ubiquitin ligase activity efp targets 14-3-3 sigma for proteolysis and promotes breast tumour growth parkin functions as an e2-dependent ubiquitin-protein ligase and promotes the degradation of the synaptic vesicle-associated protein, cdcrel-1 ubch8 regulates ubiquitin and isg15 conjugation to rig-i the interferon-inducible ubiquitin-protein isopeptide ligase (e3) efp also functions as an isg15 e3 ligase novel growth and death related interferon-stimulated genes (isgs) in melanoma: greater potency of ifn-beta compared with ifn-alpha2 a ubiquitin e3 ligase efp is up-regulated by interferons and conjugated with isg15 negative regulation of isg15 e3 ligase efp through its autoisgylation isg15 modification of the eif4e cognate 4ehp enhances cap structure-binding activity of 4ehp identification and herc5-mediated isgylation of novel target proteins herc5 is an ifn-induced hect-type e3 protein ligase that mediates type i ifn-induced isgylation of protein targets herc5, an interferon-induced hect e3 enzyme, is required for conjugation of isg15 in human cells ubp43 (usp18) specifically removes isg15 from conjugated proteins role of isg15 protease ubp43 (usp18) in innate immunity to viral infection screen for isg15-crossreactive deubiquitinases precursor processing of pro-isg15/ucrp, an interferon-beta-induced ubiquitin-like protein rnase-l-dependent destabilization of interferon-induced mrnas. a role for the 2-5a system in attenuation of the interferon response cloning and characterization of human ubiquitin-processing protease-43 from terminally differentiated human melanoma cells using a rapid subtraction hybridization protocol rash a novel ubiquitin-specific protease, ubp43, cloned from leukemia fusion protein aml1-eto-expressing mice, functions in hematopoietic cell differentiation the isg15 isopeptidase ubp43 is regulated by proteolysis via the scfskp2 ubiquitin ligase cloning and characterization of a novel human ubiquitin-specific protease, a homologue of murine ubp43 (usp18) dysregulation of protein modification by isg15 results in brain cell injury reexamination of the role of ubiquitin-like modifier isg15 in the phenotype of ubp43-deficient mice protein isgylation modulates the jak-stat signaling pathway ubp43 is a novel regulator of interferon signaling independent of its isg15 isopeptidase activity the level of hepatitis b virus replication is not affected by protein isg15 modification but is reduced by inhibition of ubp43 (usp18) expression ubp43 regulates bcr-abl leukemogenesis via the type 1 interferon receptor signaling human isg15 conjugation targets both ifn-induced and constitutively expressed proteins functioning in diverse cellular pathways high-throughput immunoblotting. ubiquitiin-like protein isg15 modifies key regulators of signal transduction proteomic identification of proteins conjugated to isg15 in mouse and human cells ifn-induced 15-kda protein is released from human lymphocytes and monocytes a human 15-kda ifn-induced protein induces the secretion of ifn-gamma immunoregulatory properties of isg15, an interferon-induced cytokine filamins as integrators of cell mechanics and signalling structural and functional aspects of filamins filamin b serves as a molecular scaffold for type i interferon-induced c-jun nh2-terminal kinase signaling pathway filamin b: a scaffold for interferon signalling isg15 modification of filamin b negatively regulates the type i interferon-induced jnk signalling pathway regulation of the tak1 signaling pathway by protein phosphatase 2c protein phosphatase 2cbeta association with the ikappab kinase complex is involved in regulating nf-kappab activity signaling to nf-kappab nf-kappab: linking inflammation and immunity to cancer development and progression negative regulation of protein phosphatase 2cbeta by isg15 conjugation isg15 modification of ubiquitin e2 ubc13 disrupts its ability to form thioester bond with ubiquitin isg15 modification of ubc13 suppresses its ubiquitinconjugating activity molecular insights into polyubiquitin chain assembly: crystal structure of the mms2/ubc13 heterodimer crystal structure of the human ubiquitin conjugating enzyme complex isg15, an interferonstimulated ubiquitin-like protein, is not essential for stat1 signaling and responses against vesicular stomatitis and lymphocytic choriomeningitis virus virgin, identification of interferon-stimulated gene 15 as an antiviral molecule during sindbis virus infection in vivo a ppxy motif within the vp40 protein of ebola virus interacts physically and functionally with a ubiquitin ligase: implications for filovirus budding ebola virus-like particles produced in insect cells exhibit dendritic cell stimulating activity and induce neutralizing antibodies role for amino acids 212klr214 of ebola virus vp40 in assembly and budding overlapping motifs (ptap and ppey) within the ebola virus vp40 protein function independently as late budding domains: involvement of host proteins tsg101 and vps-4 isg15 enhances the innate antiviral response by inhibition of irf-3 degradation modification of proteins by ubiquitin and ubiquitin-like proteins protein modification by sumo inos (nos2) at a glance perspectives series: host/pathogen interactions. mechanisms of nitric oxide-related antimicrobial activity reactive oxygen and nitrogen intermediates in the relationship between mammalian hosts and microbial pathogens protein snitrosylation: purview and parameters nitrosylation of isg15 prevents the disulfide bond-mediated dimerization of isg15 and contributes to effective isgylation a new paradigm for translational control: inhibition via 5′-3′ mrna tethering by bicoid and the eif4e cognate 4ehp mechanisms of type-i-and type-ii-interferon-mediated signalling pathogen recognition and innate immunity species-specific recognition of single-stranded rna via toll-like receptor 7 and 8 small anti-viral compounds activate immune cells via the tlr7 myd88-dependent signaling pathway 5′-triphosphate rna is the ligand for rig-i tlr3 in antiviral immunity: key player or bystander? ips-1, an adaptor triggering rig-i-and mda5-mediated type i interferon induction cardif is an adaptor protein in the rig-i antiviral pathway and is targeted by hepatitis c virus identification and characterization of mavs, a mitochondrial antiviral signaling protein that activates nf-kappab and irf 3 visa is an adapter protein required for virus-triggered ifn-beta signaling negative feedback regulation of rig-imediated antiviral signaling by interferon-induced isg15 conjugation negative regulation of the rig-i signaling by the ubiquitin ligase rnf125 the papain-like protease from the severe acute respiratory syndrome coronavirus is a deubiquitinating enzyme global epidemiology of hepatitis c virus infection unravelling hepatitis c virus replication from genome to function regulating intracellular antiviral defense and permissiveness to hepatitis c virus rna replication through a cellular rna helicase, rig-i regulation of interferon regulatory factor-3 by the hepatitis c virus serine protease control of antiviral defenses through hepatitis c virus disruption of retinoic acid-inducible gene-i signaling viral and therapeutic control of ifnbeta promoter stimulator 1 during hepatitis c virus infection ube1l causes lung cancer growth suppression by targeting cyclin d1 proteasomes modulate conjugation to the ubiquitinlike protein, isg15 pulsed-field gel electrophoresis analysis of retinoic acid receptor-alpha and promyelocytic leukemia rearrangements. detection of the t(15;17) translocation in the diagnosis of acute promyelocytic leukemia chromosomal translocation t(15;17) in human acute promyelocytic leukemia fuses rar alpha with a novel putative transcription factor accelerated degradation of pml-retinoic acid receptor alpha (pml-rara) oncoprotein by all-trans-retinoic acid in acute promyelocytic leukemia: possible role of the proteasome pathway caspases mediate retinoic acid-induced degradation of the acute promyelocytic leukemia pml/raralpha fusion protein the ubiquitinlike molecule interferon-stimulated gene 15 (isg15) is a potential prognostic marker in human breast cancer isg15 as a novel tumor biomarker for drug sensitivity prostatic intraepithelial neoplasia in mice expressing an androgen receptor transgene in prostate epithelium androgeninduced differentiation and tumorigenicity of human prostate epithelial cells expression, regulation and function of the isgylation system in prostate cancer identification of genes differentially regulated by interferon alpha, beta, or gamma using oligonucleotide arrays we thank mr. sangman michael kim for his critical reading of this article. this work was supported by grants from the krf (krf-2005-084-c00025) and kosef (m10533010001-05n3301). we apologize for the event that any relevant publications were inadvertently omitted. key: cord-312892-p72zwmtb authors: chen, nanhua; xia, pengpeng; li, shuangjie; zhang, tangjie; wang, tony t.; zhu, jianzhong title: rna sensors of the innate immune system and their detection of pathogens date: 2017-04-04 journal: iubmb life doi: 10.1002/iub.1625 sha: doc_id: 312892 cord_uid: p72zwmtb the innate immune system plays a critical role in pathogen recognition and initiation of protective immune response through the recognition of pathogen associated molecular patterns (pamps) by its pattern recognition receptors (prrs). nucleic acids including rna and dna have been recognized as very important pamps of pathogens especially for viruses. rna are the major pamps of rna viruses, to which most severe disease causing viruses belong thus posing a tougher challenge to human and animal health. therefore, the understanding of the immune biology of rna prrs is critical for control of pathogen infections especially for rna virus infections. rna prrs are comprised of tlr3, tlr7, tlr8, rig‐i, mda5, nlrp3, nod2, and some other minorities. this review introduces these rna prrs by describing the cellular localizations, ligand recognitions, activation mechanisms, cell signaling pathways, and recognition of pathogens; the cross‐talks between various rna prrs are also reviewed. the deep insights of these rna prrs can be utilized to improve anti‐viral immune response. © 2017 iubmb life, 69(5):297–304, 2017 the innate immune system represents the first line of defense against pathogens through its continuous monitoring of the pathogen associated molecular patterns (pamps), and subsequent activation of a series of defense mechanisms to eliminate the infections. the concept and model of innate immune sensing was first proposed by charles janeway jr. who predicted that there must exist a group of innate immune receptors responsible for recognition and sensing of non-self from self, and triggering subsequent adaptive immunity (1) . later studies confirmed his prediction and more and more innate immune receptors called pattern recognition receptors (prrs) have been found since then. based on protein domain homology, prrs have been divided into several families; they are toll-like receptors (tlrs), rig-i like receptor (rlrs), nod-like receptors (nlrs), c-type lectin receptors (clrs), aim2-like receptors (alrs), and the recently discovered cytosol dna sensing prr cyclic gmp-amp synthase (cgas). these prrs recognize and sense a variety of pamps from viruses, bacteria, fungi, and protozoa, which range from lipoproteins, carbohydrates, lipopolysaccharide to nucleic acids. prrs also recognize endogenous damage associated molecular patterns (damps) from host, which is related with both homeostasis and autoimmune diseases. upon sensing of pamps or damps, the prrs trigger intracellular cell signaling, leading to transcriptional activation and expression of cytokines, chemokines, mhc, and co-stimulatory molecules. additionally, prr triggered cell signaling induces several transcription-independent cell processes such as phagocytosis, autophagy, cell death, and inflammasome/cytokine processing, which work together with the transcriptional innate responses (2) . the nucleic acids rna and dna have drawn much attention as important pamps (3, 4) . different from non-pathogens, the pathogens including viruses and intracellular bacteria replicate in cells, and nucleic acids rna or dna represent the signature of pathogens in particular of viruses which accumulate large amount of nucleic acids during replication in cells. all the nucleic acid detecting prrs are localized intracellularly. for example, dna sensing prrs are endosomal tlr9, cytosolic aim2, ifi16, and cgas; rna sensing prrs are endosomal tlr3, tlr7, tlr8, and cytosolic rig-i, mda5, nlrp3, and nod2. rna prrs play more important roles than dna prrs in recognition of rna virus infections and initiation of protective immune responses. rna viruses exhibit rapid replication kinetics, high mutation rates, and complex evolutionary dynamics, thus rna viruses pose unique challenges to human and animal health (5) . most severe disease causing viruses are rna viruses, such as ebola virus, influenza virus, human immunodeficiency virus (hiv), foot-mouth disease virus (fmdv), etc. therefore, investigation and understanding of rna prrs are critical for control of virus infections and protection of host. following are the description of individual rna prr. the family of toll like receptors (tlrs) are the earliest discovered prrs. currently human and mouse have 10 and 12 tlrs, respectively: both human and mouse have tlr1-9; in addition, human has tlr10 whereas mouse has tlr11-13 (6) . all tlrs are type i transmembrane proteins and comprised of nterminal ectodomain or extracellular domain (ecd), middle transmembrane domain (tm) and c-terminal cytoplasmic toll/ il-1 receptor (tir) domain (supporting information fig. 1 ). the ecd contains 20-26 leucine rich repeats (lrr) motifs, which are juxtaposed into a horseshoe-shaped solenoid or a ring-like structure. the a-helix of each lrr is located on the convex surface of solenoid structure, and b-sheet of each lrr assembles and forms into the concave surface of the solenoid structure. different from other lrr containing proteins, tlrs bind their ligands including agonists on the lateral convex surface instead of concave surface (7) . the formation of m-shaped dimer or multimer is needed for all tlr activation, so that the c-terminal regions of the two tlr ecds are brought into proximity. it in turn causes the multimerization of cytoplasmic tir domains, which will recruit downstream adaptors trif or myd88 through homotypic interaction, further forming signaling complex called signalosome and activating downstream transcription factors: one is nf-jb that induces proinflammtory cytokines, another is interferon regulatory factor (irf) that induces anti-viral type i interferon (ifn) (6) . tlr3 is widely distributed in all innate immune cells except neutrophils and plasmacytoid dendritic cells (pdcs), and localized in the endosomes of these cells (8) . tlr3 recognizes the double-stranded rna (dsrna) of viruses and the synthetic dsrna analog poly i:c (9). the crystal structure of tlr3 ecd was the first resolved one among tlr proteins, existing as a monomer of solenoid structure. upon binding to dsrna, tlr3 forms a dimer with the backbone phosphates and sugars of dsrna binding to the lateral positive charged regions of n-and c-terminal ecd (10) . the downstream adaptor trif is recruited by activated tlr3 through homotypic interaction, which further forms trif signaling complex involving other signaling components, such as traf6, traf3, tbk1, ikke, and ikk. the transcription factors irf3/irf7 and nf-jb are subsequently activated by the signaling complex and induce the expression of ifn and proinflammatory cytokines, respectively (fig. 1) . tlr3 recognizes the genomic dsrna of reoviruses, and the intermediate rnas generated during replication of different viruses including mouse cytomegalovirus (mcmv), herpes simplex virus-1 (hsv-1), encephalomyocarditis virus (emcv), flaviviruses, and enteroviruses (table 1 ). in these cases, the activated tlr3 signaling restricts the virus replication; however, the tlr3 induced inflammation also contributes to the breaching of blood-brain barrier, leading to neuropathologies exemplified as west nile virus (wnv) infection in mice (11, 12) . tlr7 and tlr8-belong to the tlr7/8/9 subfamily whose members are all localized at endosomes of the cells. tlr7 and tlr8 are very similar in terms of ligand recognition and intracellular signaling. both are activated by small molecular agonists and gu or u rich single-stranded rna (ssrna) (fig. 1 ). small molecular compounds resiquimod (r848) and cl097 are agonists for both tlr7 and tlr8; however, imiquimod (r837) and loxoribine only for tlr7, and cl075 (3m002) only for tlr8. in addition, tlr7 and tlr8 may also recognize short dsrna such as sirna from rna interference (rnai), and some mirna such as mirna-21 and mirna-29a secreted by tumor cells (13) . the ecds of tlr7 and tlr8 both have 26 lrr motifs that are more than those of tlr1-6; additionally there are several insertions including the one between lrr14 and lrr15 called undefined region or z-loop, therefore, the ecds are larger and exhibit the ring-like crystal structures (14) . the tlr8 and tlr7 at endosome are proteolytically cleaved along the z-loop by cathepsins and arginine endopeptidase; nevertheless, the two cleaved fragments are still stuck together by multiple intermolecular interactions, and both are required for receptor's activation (15, 16) . the z-loop cleavage is necessary for tlr8/7 dimerization which is essential for activation (17) . newly recent crystal structures showed that there are dual agonist binding sites in both tlr7 and tlr8 dimers (18, 19) : one is located within dimer interfaces which binds small chemical agonists or degrade products of ssrna agonists; another is located on the concave surface of the tlr horseshoe structures and for binding of ssrna oligonucleotides. the first sites are enough for small chemical agonist induced tlr7 and tlr8 activation, whereas both sites are necessary for ssrna induced tlr7 and tlr8 activation. at steady state, tlr7 and tlr8 exist as dimers; upon binding to agonists, the conformation of dimers change such that the cytoplasmic tir domains multimerize and recruit downstream adaptor myd88 through homotypic interaction. the signaling complex called myddosome is formed involving irak4, irak1, traf6, traf3 and downstream transcription factors nf-jb and irf7 are activated to induce proinflammatory cytokines and ifns, respectively. despite the high similarity between tlr7 and tlr8, the cell distributions of these two are almost opposite: tlr7 is exclusively expressed by plasmacytoid dendritic cells (pdcs) and b cells, whereas tlr8 is expressed mainly in monocytes, macrophages and conventional dendritic cells (cdcs), very low level in pdcs and b cells (20) . both tlr7 and tlr8 are able to recognize multiple virus infections, including sendai virus (sev), influenza virus, coxsackie virus, vaccinia virus, measle virus (mv), respiratory syncytial virus (rsv), and retrovirus (11) . furthermore, tlr7 recognizes streptococcus group b (sgb) rna (21, 22) , and tlr8 recognizes the rnas from escherichia coli, mycobacteria bovis, helicobacter pylori, and borrelia burgdorferi (23) ( table 1) . rlrs are expressed in almost all mammalian cell types, and as the main family of cytosolic rna sensors play key roles in the rna sensors in pathogen detection. the rna viruses and some bacteria can be recognized by the main rna sensors inside cells including endosomal tlr3, tlr7, tlr8, cytosolic rig-i, mda5, nlrp3, and nod2. except nlrp3 which initiates inflammasome formation and processes cytokines for maturation, all others activate downstream nf-jb and irf signaling pathways, and thus cytokine gene transcription. all these rna sensor induced signaling together combats pathogen infections. chen et al. the summary of cellular localizations and distributions, ligand recognitions, activation mechanisms, cell signaling, recognition of pathogens, and cross-talks for rna prrs (2) tlr3 (2) rig-i (2) the "(1)" and "(2)" denote positive and negative regulation by the corresponding prrs of the first row of the fig. 1 ). the optimal rna recognized by rig-i is the 5 0 ppp-dsrna, whereas the one mda5 prefers is long dsrna (fig. 1) . the third member lgp2, lacks of n-terminal 2cards, has no signaling activity, but is able to regulate rig-i and mda5 signaling due to the capability of binding rna (24, 25) . rig-i, as the prototypic member of rlrs, has been subjected to extensive research. currently, there have been intensive investigations and clear understanding of rig-i activation mechanism: under steady state, rig-i cards binds to the hel-2i region of the helicase and is subjected to auto-inhibition (25) . upon binding of rig-i c-terminal rd to rna, the conformation of rig-i changes so that the rna further binds to hel-2i, and in turn the auto-inhibition of 2cards is released. next, through k63-polyubiquitination/polyubiquitin chain binding of the 2cards and/or the filament formation by rig-i rd-helicase along the dsrna chain, the 2cards are tetramerized into stable lock-washer structure. the effective 2card tetramer will nucleate downstream adaptor masv, which aggregates on the mitochondria into prion-like signaling complex (28) . the downstream traf3/tbk1/ikke and traf6/ikk further activate transcription factors irf3 and nf-jb, which drive ifn and proinflammtory cytokine expression, respectively (29) (fig. 1) . consistent with the recognition of short dsrna ligands, rig-i specifically recognizes most single-negative rna viruses which generate lots of short 5 0 ppp-dsrna during replication. these viruses include but are not limited to ebola virus (ebov) of filovirus family, measle virus (mv), sendai virus (sev), newcastle disease virus (ndv), respiratory syncytial virus (rsv) of paramyxovirus family, influenza virus of othomyxovirus family, hantavirus of bunyavirus family, vesicular stomatitis virus (vsv), and rabies virus (rv) of rhabdovirus family. rig-i also recognizes positive single rna viruses such as hepatitis c virus (hcv) and japanese encephalitis virus (jev) of flavivirus family. in addition, rig-i is able to sense some dna viruses such as adenovirus, vaccinia virus, herpes simplex virus (hsv) in that these viruses produce small dsrna through their type iii rna polymerase during replication. there was also report showing that rig-i can detect the rna from bacteria such as listeria monocytogenes, helicobacter pylori, and shigella flexneri (30) . mda5 has a very similar activation mechanism to that of rig-i: rd-helicase binds with rna, and conformational change exposes the n terminal 2cards, which form tetramer structure. because mda5 binds long dsrna, the 2card tetramer is stabilized mainly through the filament formation along the dsrna, and thus less dependent on k63-polyubiquitination/polyubiquitin chain binding (28) . the effective 2card tetramer will nucleate downstream adaptor mavs, activate traf3/ tbk1/ikke/irf3 and traf6/ikk/nf-jb, which drive ifn and proinflammtory cytokine expression, respectively (29) . mda5 recognizes long dsrna, and accordingly senses the single positive rna viruses such as the encephalomyocarditis virus (emcv), poliovirus and coxasackie virus of picornavirus family (30) . on the other hand, both rig-i and mda5 crossdetect the same viruses; these viruses are double rna rotavirus of reovirus family, dengue virus, and west nile virus (wnv) of flavivirus family, murine hepatitis virus of coronavirus family (30) ( table 1) . nlr is also the cytosolic receptor family, which is mainly utilized for detection of bacteria. human has 22 members, whereas mouse has 34 members. all nlr members have similar domain structures: n-terminal effector domain, middle nucleotide-binding and oligomerization domain (nod) and cterminal leucine rich repeats (lrrs) (supporting information fig. 1 ). based on the n-terminal effector domains, the nlr is divided into five subfamilies: nlra (acid activation domain), nlrb (baculovirus inhibitor of apoptosis repeats), nlrc (caspase activation and recruitment domain card), nlrp (pyrin domain pyd), and nlrx (unknown domain) (31) . except nod1 and nod2, all the nlrs upon activation induce the inflammasome formation other than gene transcription. nlrp3, as one of the most extensively studied nlrs, is able to recognize a very broad and distinct set of ligands including atp, uric acid, silica, adjuvant aluminum, cholesterol crystals, nigericin, pore-forming proteins, mitochondrial dna, and pathogen mrna. common among these stimulators, nlrp3 is activated directly by potassium efflux coupled with downstream nek7 (32, 33) . nevertheless, there existed potassium efflux independent non-canonical nlrp3 inflammasome formations under conditions of disrupted glycolytic flux or by stimulation of extracellular lps (33) . probably, nlrp3 would favor a general sensor of homeostatic disruption such as lysosomal rupture, mitochondria damage, reactive oxygen species (ros), and ionic imbalance (31) . however, the exact mechanisms of nlrp3 action need to be further investigated and determined. under steady state, nlrp3 is likely subjected to auto-inhibition; upon activation, nlrp3 binds downstream adaptor asc and substrate procaspase-1, forming the wheellike structure inflammasome. next the caspase-1 is activated, processing the pro-il-1 and pro-il-18 into active il-1 and il-18 (fig. 1) . nlrp3 recognizes the cytosolic dsrna/ssrna of influenza virus and sendai virus, and bacteria mrna, activating the inflammasome and inducing il-1 and il-18 (34-36) ( table 1) . nod2 and nod1 (also called nlrc2 and nlrc1), different from other nlrs, recognize bacterial glycopeptides mdp and ie-dap, respectively, recruit downstream adaptor rip2 through homotypic interaction forming signaling complex, and induce nf-jb activation and proinflammatory cytokine expression (31) . nod2 receptors are found mostly in macrophages, monocytes, paneth intestinal cells, and dendritic cells (37) . additional studies suggested that nod2 plays an important role in the restriction of respiratory syncytial virus (rsv), influenza a virus (iav), and human cytomegalovirus (hcmv), likely through the recognition of virus rna and subsequent ifn induction (38, 39) (table 1) . similar to rlrs, nod2 has nterminal 2cards, and may tetramerize into washer-locker structure to initiate the adaptor mavs aggregation and downstream ifn production (38, 40) (fig. 1) . to this end, the crystal structure of nod2 cards has to be solved in the future (31) . tlr13 is only expressed in mouse, and localized in the endosome compartment. once activated by rna, tlr13 recruits adaptor myd88 to trigger transcription factor nf-jb activation and induce proinflammtory cytokine production (22) . mouse tlr13 was reported to recognize a conserved cggaaagacc motif in staphylococcus aureus 23s rrna and e. coli 23s rrna (41, 42) . furthermore, tlr13 also recognizes streptococcus pyogenes (43) . in addition to rlrs rig-i and mda5, there exists a group of non-rlr rna helicases which mediate cytosolic rna recognition and signaling. these are ddx3, dhx9, dhx33, ddx60, and ddx1/ddx21/dhx36 (30, 44) . ddx3 binds poly i:c or vesicular stomatitis virus (vsv) rna, associates with rlr-mavs signaling complex, and enhances ifn response. dhx9 is expressed in mouse spleen dendritic cells and mouse bone marrow dendritic cells, recognizes poly i:c, influenza virus and reovirus rna, binds with mavs activating downstream signaling. dhx33 binds poly i:c and reovirus rna, activates mavs mediated signaling or nlrp3 mediated inflammasome. ddx60 can be induced by the virus infection, and in the meantime binds the virus rna, further binds rig-i, mda5, and lgp2, enhancing rlr signaling and downstream ifn response. ddx1/ddx21/dhx36 are expressed in myeloid dendritic cells, in which ddx1 binds poly i:c through helicase domain, whereas dhx36 and ddx21 bind trif tir domain through their ha2-duf and prk domain, respectively. the complex enhances ifn response and exhibits inhibitory effect to influenza virus and reovirus infections. the third minor rna prr, leucine-rich repeat flightless-interacting protein 1 (lrrfip1) was reported to bind both rna and dna and be involved in the recognition of vesicular stomatitis virus (vsv) and listeria monocytogene, activate b-catenin, which then translocates into nucleus and promotes irf3 transcription activity and ifn production (45) . interactions of microbes with the innate immune system involve the parallel recognition of different pamps of the whole pathogen by multiple prrs and simultaneous induction of multiple prr signaling pathways, for which evidence has been accumulated in the past years (46) (47) (48) . likewise, there are interactions between different rna prrs and mutual influence of different rna prr signaling. understanding of the enormous complexity of these processes helps provide insights into in vivo innate immune activation. within the rna sensing tlrs, synergistic effects were observed between tlr3 and tlr8 in monocyte-derived macrophages and dcs (49, 50) ; in contrast, tlr8 inhibits tlr7 signaling in both human and mice (51, 52) . between three subfamilies of rna prrs, tlr7/ 8 cooperates with nod2 in dc activation and results in a synergistic release of pro-inflammatory mediators which promote the activation of th 17 cells (53) . rlrs suppress the gene transcription of il-12p40 induced by the activation of toll-like receptors (tlrs) including tlr3 and subsequent tlr3 induced th 1 and th 17 responses (47) . furthermore, rlr rig-i crossinterferes with nod2 in regulating downstream signaling by direct interaction with each other (54) . the interactions between different prrs need to be considered in the contexts of distinct species, various cell types, and different agonists used for stimulation. the ability to devise strategies to control virus infections will be improved with the knowledge of rna prrs the understanding of rna prr immune biology including the ligand recognitions, cellular localizations, cell signaling pathways, mechanisms of activation, recognized pathogens and the interactions between different rna prrs will definitely be helpful to improve the anti-viral immune response. we give some examples here: first, based on the ligand recognitions and activation mechanisms of rna prrs, rna varieties and small-molecular agonists of higher potency can be developed for either direct anti-viral therapy or effective viral vaccine adjuvants. similarly, elucidation of the rna prr triggered cell signaling pathways will potentially lead to the discovery of novel innate immune modulators with higher anti-viral efficacy. second, for certain virus infections, only the rna prrs involved in the viral recognition will likely play an important role in the anti-viral immune response; in this case, knowledge of the virus recognition by rna prrs becomes critical. third, tlr3, 7, 8 are primarily expressed by macrophages and dcs and recognize viral rna within the endosomal compartment, whereas rlrs (rig-i, mda5) and nlrs (nlrp3, nod2) are ubiquitously expressed and sense viral rna within the cytoplasm of infected cells (table 1 ). therefore, it should be considered how to deliver the rna prr agonists to host to maximize the anti-viral immune response, such as delivery routes, with or without transfection etc. fourth, as seen in table 1 , tlrs have general positive cross-talks with nlrs, whereas rlrs have negative cross-talks with tlrs and nlrs. even though information is incomplete, this knowledge of crosstalks between different rna prrs will provide a rationale to develop appropriate combinations of various agonists/ modulators to control viruses which are simultaneously recognized in vivo by several rna prrs. with the information becoming complete, and additional knowledge on how the rna prr signaling influences downstream adaptive immune response, more and more effective therapeutics or vaccine adjuvants to control virus infections will be on the horizon. approaching the asymptote: 20 years later innate immune pattern recognition: a cell biological perspective nucleic acid recognition by the innate immune system molecular requirements for sensing of intracellular microbial nucleic acids by the innate immune system oasl-a new player in controlling antiviral innate immunity the role of pattern-recognition receptors in innate immunity: update on toll-like receptors variation matters: tlr structure and species-specific pathogen recognition toll-like receptors stimulate human neutrophil function does toll-like receptor 3 play a biological role in virus infections crystal structure of human toll-like receptor 3 (tlr3) ectodomain pattern recognition receptors and the innate immune response to viral infection toll like receptor 3 and viral infections of nervous system human toll-like receptor 8 can be cool too: implications for foreign rna sensing structural reorganization of the toll-like receptor 8 dimer induced by agonistic ligands toll-like receptor 9 processing: the key event in toll-like receptor 9 activation? structure and function of toll-like receptor 8 autoinhibition and relief mechanism by the proteolytic processing of toll-like receptor 8 tolllike receptor 8 senses degradation products of single-stranded rna structural analysis reveals that toll-like receptor 7 is a dual receptor for guanosine and single-stranded rna interferon-alpha and interleukin-12 are induced differentially by toll-like receptor 7 ligands in human blood dendritic cell subsets bacterial recognition by tlr7 in the lysosomes of conventional dendritic cells microbial sensing by toll-like receptors and intracellular nucleic acid sensors. cold spring harbor perspect tlr8: the forgotten relative revindicated master sensors of pathogenic rna -rig-i like receptors rig-i in rna virus recognition the rna helicase rig-i has an essential function in double-stranded rna-induced innate antiviral responses regulating intracellular antiviral defense and permissiveness to hepatitis c virus rna replication through a cellular rna helicase, rig-i filament assemblies in foreign nucleic acid sensors how rig-i like receptors activate mavs sensing microbial rna in the cytosol international union of basic and clinical pharmacology. xcvi. pattern recognition receptors in health and disease structural mechanisms in nlr inflammasome assembly and signaling recent insights into the molecular mechanisms of the nlrp3 inflammasome activation the nlrp3 inflammasome mediates in vivo innate immunity to influenza a virus through recognition of viral rna critical role for cryopyrin/nalp3 in activation of caspase-1 in response to viral infection and double-stranded rna detection of prokaryotic mrna signifies microbial viability and promotes immunity understanding the regulation of pattern recognition receptors in inflammatory diseases -a 'nod' in the right direction activation of innate immune antiviral responses by nod2 activation of nucleotide oligomerization domain 2 (nod2) by human cytomegalovirus initiates innate immune responses and restricts virus replication snapshot: nucleic acid immune sensors, part 1. immunity 41 sequence specific detection of bacterial 23s ribosomal rna by tlr13 tlr13 recognizes bacterial 23s rrna devoid of erythromycin resistanceforming modification cutting edge: tlr13 is a receptor for bacterial rna intracellular detection of viral nucleic acids the cytosolic nucleic acid sensor lrrfip1 mediates the production of type i interferon via a beta-catenin-dependent pathway rig-i like receptors and their signaling crosstalk in the regulation of antiviral immunity cross-interference of rlr and tlr signaling pathways modulates antibacterial t cell responses collaboration of toll-like and rig-i-like receptors in human dendritic cells: triggering antiviral innate immune responses tlr-tlr cross talk in human pbmc resulting in synergistic and antagonistic regulation of type-1 and 2 interferons, il-12 and tnf-alpha multiple signaling pathways contribute to synergistic tlr liganddependent cytokine gene expression in human monocyte-derived macrophages and dendritic cells the functional effects of physical interactions among toll-like receptors 7, 8, and 9 tlr8 deficiency leads to autoimmunity in mice tlr8 and nod signaling synergistically induce the production of il-1beta and il-23 in monocyte-derived dcs and enhance the expression of the feedback inhibitor socs2 retinoic acid-induced gene-i (rig-i) associates with nucleotidebinding oligomerization domain-2 (nod2) to negatively regulate inflammatory signaling the work was partly supported by the priority academic program development of jiangsu higher education institutions and jiangsu co-innovation center for prevention and control of important animal infectious diseases and zoonoses, china, by natural science foundation of china (31672523) to j.z. and by china postdoctoral science foundation (2016m590510) to n.c. key: cord-278523-djjtgbh6 authors: zhou, bei-xian; li, jing; liang, xiao-li; pan, xi-ping; hao, yan-bing; xie, pei-fang; jiang, hai-ming; yang, zi-feng; zhong, nan-shan title: β-sitosterol ameliorates influenza a virus-induced proinflammatory response and acute lung injury in mice by disrupting the cross-talk between rig-i and ifn/stat signaling date: 2020-06-05 journal: acta pharmacol sin doi: 10.1038/s41401-020-0403-9 sha: doc_id: 278523 cord_uid: djjtgbh6 β-sitosterol (24-ethyl-5-cholestene-3-ol) is a common phytosterol chinese medical plants that has been shown to possess antioxidant and anti-inflammatory activity. in this study we investigated the effects of β-sitosterol on influenza virus-induced inflammation and acute lung injury and the molecular mechanisms. we demonstrate that β-sitosterol (150–450 μg/ml) dose-dependently suppresses inflammatory response through nf-κb and p38 mitogen-activated protein kinase (mapk) signaling in influenza a virus (iav)-infected cells, which was accompanied by decreased induction of interferons (ifns) (including type i and iii ifn). furthermore, we revealed that the anti-inflammatory effect of β-sitosterol resulted from its inhibitory effect on retinoic acid-inducible gene i (rig-i) signaling, led to decreased stat1 signaling, thus affecting the transcriptional activity of isgf3 (interferon-stimulated gene factor 3) complexes and resulting in abrogation of the iav-induced proinflammatory amplification effect in ifn-sensitized cells. moreover, β-sitosterol treatment attenuated rig-i-mediated apoptotic injury of alveolar epithelial cells (aec) via downregulation of pro-apoptotic factors. in a mouse model of influenza, pre-administration of β-sitosterol (50, 200 mg·kg(−1)·d(−1), i.g., for 2 days) dose-dependently ameliorated iav-mediated recruitment of pathogenic cytotoxic t cells and immune dysregulation. in addition, pre-administration of β-sitosterol protected mice from lethal iav infection. our data suggest that β-sitosterol blocks the immune response mediated by rig-i signaling and deleterious ifn production, providing a potential benefit for the treatment of influenza. the annual spread of seasonal influenza a virus (iav), particularly the sporadic transmission of highly pathogenic avian influenza (hpai) viruses (e.g., the h5n1 and h7n9 subtypes) continues to constitute a major threat to public health. as of december 2018, avian influenza a h7n9 viruses have caused an estimated 1567 individual infections, with an overall mortality rate of 39.2% (http://www.fao.org/ag/againfo/programmes/en/empres/h7n9/ situation_update.html). recently, the na r292k and ns g540a substitutions in the h7n9 virus have been reported to be associated with increased resistance to oseltamivir and to confer enhanced viral replication in mammalian cells [1, 2] . this raises concerns about a potential human pandemic. iav initiates infection in humans via entry through the upper respiratory tract, eliciting symptoms that can be mild and self-limited, but in some patients, it can lead to acute respiratory distress syndrome (ards), which is characterized by the impairment of gas exchange resulting in a fatal outcome [3, 4] . a series of reports have indicated that a robust and dysregulated innate immune response is primarily responsible for the high mortality rate associated with influenza [5, 6] . however, there are few alternative medicine strategies available for the treatment of influenza virus-infected patients who have an intense inflammatory response. the innate immune system is armed with an array of pattern recognition receptors (prrs) that provide the principal barrier defense against infectious agents [7] . during the process of viral replication, influenza virus synthesizes viral rna containing a 5′triphosphate end and is then transported to the cytoplasm [8] . this pathogen-associated molecule (pam) is detected by the cytosolic sensor retinoic acid-induced gene i (rig-i) and subsequently activates multiple cellular signal cascades [9] . ultimately, these signaling pathways lead to the activation of transcription factors including nf-κb, ap-1 (atf2:c-jun), and irf3, which together bind to specific sites of the ifn-β promoter and initiate ifn-β synthesis for the antiviral response [10] . targeting p38 map kinase with a specific inhibitor affects the induction of ifn-β [11] , which inhibits the downstream phosphorylation of atf2 by p38 map kinase [12] . studies have demonstrated the crucial role of ifn production mediated by rig-i signaling in protecting against lethal iav challenge [13, 14] . in vivo studies have shown that the increased susceptibility of both rig-i-and ifn-deficient mice to several rna viruses [15] [16] [17] , including vesicular stomatitis virus (vsv), newcastle disease virus (ndv), and iav, is associated with the failure of ifns levels to increase or a lack of ifn signaling. there is evidence that infection with hpai h5n1 viruses triggers the hyperinduction of proinflammation via rig-i signaling cascades despite the crucial role of rig-i signaling in defense against iav [18] . the induction of downstream genes of rig-i signaling, such as il-6, tnf-α, il-8, and il-1β, is significantly upregulated in the bronchoalveolar lavage fluid (balf) of patients with sustained ards [3, 19, 20] . type i and type iii ifns secreted by infected cells bind to distinct receptors but trigger similar signal transduction through the jak/ stat cascades [21, 22] . upon ifn stimulation, the phosphorylation of the stat1:stat2 heterodimer leads to its interaction with irf9 and the formation of the interferon-stimulated gene factor 3 (isgf3) complex [23] . then, the complex moves from the cytoplasm to the nucleus where it binds to the interferonstimulated response element (isre) and drives the expression of interferon-stimulated genes (isgs). these genes possess direct antiviral and immunomodulatory activity [24] . for example, the mx protein, which was the first isg identified to restrict influenza virus infection to be identified, has been demonstrated to impair vrnp nuclear import [25] . however, despite the abundant production of ifns and hundreds of isgs in response to viral infection, the ifnmediated antiviral response is blocked by various viral products. the non-structural (ns1) protein of iav is a typical multifunctional protein involved in the blockade of the antiviral effect of isgs, such as 2′,5′-oligoadenylate synthetase (oas) and dsrnadependent protein kinase r (pkr) [26, 27] . in addition, iav infection has also been shown to suppress the antiviral response mediated by type i and iii ifns by upregulating the negative regulators socs3 and socs1 [28, 29] . given that viruses can overcome the ifn-mediated antiviral response through various mechanisms, it is possible that excessive ifn production during the antiviral response may contribute to the harmful effects. surprisingly, studies have found that increased host susceptibility to secondary bacterial co-infections correlates with ifn induction during iav infection [30] . moreover, a deficiency in ifn-α/β signaling increases survival through a reduction in excessive inflammation and apoptotic injury to lung epithelial cells [31] . similarly, high levels of trail in balf collected from patients with influenza-associated ards may be a result of increased trail expression induced by macrophage-derived ifn-β [32] , which contributes to lung alveolar epithelial cell injury. in addition, recent evidence has also revealed that ifn-mediated signaling drives immunopathologic injury in response to various viral infections, including respiratory syncytial virus infection (rsv) [33] and severe acute respiratory syndrome cov (sars-cov) [34] . given these findings, it is clear that the disease-promoting effects of ifn contribute to deleterious outcomes, and should be limited by pharmacological intervention. the identification of agents derived from medicinal plants that can prevent influenza is valuable. chinese medicinal plants, including lonicera japonica [35] , chrysanthemum morifolium [36] , taraxacum mongolicum [37] , forsythia suspense [38] , and isatis indigotica [39] have been prescribed for the common cold, heatclearing, and detoxication for thousands of years, but the bioactive ingredients of these plants that mediate these pharmacological effects is unknown. phytosterols contain structural features that resemble those of cholesterol and are abundant in vegetables, fruits, and medicinal plants [40, 41] . among phytosterols, β-sitosterol (24-ethyl-5-cholestene-3-ol) is the most common sterol and has been shown to possess antioxidant, antiinflammatory, antitumor, and antiasthmatic effects [42] [43] [44] [45] . in the present study, we hypothesized that β-sitosterol is the bioactive component of five types of medicinal plants. to test this hypothesis, we investigated the effects of β-sitosterol and the underlying mechanisms by which it may exert a therapeutic effect against influenza-mediated injury and dysregulated inflammation. preparation of extracts and quantitative analysis of β-sitosterol samples of four kinds of different heat-clearing and detoxifying traditional chinese medicines samples (l. japonica, c. morifolium, t. mongolicum, and f. suspense) were purchased from local markets in bozhou, china. i. indigotica was supplied by hutchison whampoa guangzhou baiyunshan chinese medicine co., ltd (guangzhou, china). a β-sitosterol standard was purchased from sigma (san francisco, usa), and hplc-grade methanol was purchased from fisher scientific (fisher, usa). a sample of each of the five medicinal materials was crushed into a coarse powder, and 2.0 g was placed in a 100-ml flask. extraction was performed using ultrasonic waves for 15 min and the addition of 50 ml of chloroform and was repeated three times. the samples were then centrifuged at 2500 × g for 10 min. the supernatants were combined and condensed to a proper volume under reduced pressure, and then the concentrates were dissolved with chloroform. the samples were transferred to 5-ml volumetric flasks, diluted with chloroform to 5 ml, and mixed. a total of 2.0 mg of the β-sitosterol standard was accurately weighed and dissolved in 5 ml of chloroform to produce individual stock solutions. hplc analysis of β-sitosterol was performed at 28°c on an hplc instrument (shimadzu 20a, japan) with a dad detector at 205 nm. chromatographic separation was performed on a shimadzu ods column (4.6 × 150 mm, 5 μm, tokyo, japan). the mobile phase was methanol, and the injection volume was 10 μl. the samples were subjected to quantitative analysis, which was performed using the external standard method. the results are expressed as mg/g, and all analyses were performed in triplicate. influenza a/puerto rico/8/34 (h1n1) and a/fm/1/47(h1n1) mouse-adapted viruses were stored in our laboratory and propagated in the allantoic cavities of 9-day-old specific pathogen-free embryonated chicken eggs at 37°c. freshly collected allantoic fluids were clarified by low-speed centrifugation at 72 h postinoculation and then stored in small aliquots at −80°c. the virus titers were determined using a plaque forming assay in monolayers of madin-darby canine kidney (mdck) cells as previously described. mouse experiments and viral challenge four-to six-week-old female balb/c mice (weighing 16-18 g) were purchased from guangdong medical laboratory animal center. all mice were housed and cared for under specific pathogen-free conditions at the state key laboratory of respiratory disease or guangdong laboratory animal monitoring institute. all animal experimental procedures in this study were approved by the ethics committee of the first affiliated hospital of guangzhou medical university and conducted in strict accordance with the approved guidelines. the 50% lethal dose (ld 50 ) of the mouse-adapted h1n1 virus was estimated in mice after the stock virus was serially diluted. the mice were treated intragastrically with β-sitosterol (50 mg·kg −1 ·d −1 , 200 mg·kg −1 ·d −1 ) or pbs (vehicle group) 2 days prior to viral challenge. the mice were anesthetized (5% isoflurane inhalation) and challenged intranasally with 5 ld 50 of mouse-adapted h1n1 virus. cell culture and viral infection human alveolar epithelial a549 cells and 293t human embryonic kidney cells were grown in dulbecco's modified eagleʼs medium (dmem/f12, 1:1 mixture) (gibco) supplemented with 10% fetal bovine serum (fbs) (gibco) in a humidified incubator at 37°c and 5% co 2 . for the viral infection, a549 cells (5 × 10 6 cells/ml) grown in 6well tissue culture plates (guangzhou jet bio-filtration co., ltd, tcp-010-006) were inoculated with a/pr/8/34 (h1n1) in serumfree dmem/f12 medium at an indicated multiplicity of infection (moi). after 2 h absorption, the inoculum was discarded and replaced with fresh serum-free dmem/f12 medium containing diluted compounds. antibodies and recombinant protein the following primary antibodies were used in the current study: anti-rig-i, anti-nf-κb p65, anti-phospho-nf-κb p65 (ser 536 ), anti-p38 mapk, anti-phospho-p38 mapk ( protein preparation and immunoblot analysis excised lungs and cells were lysed using ice-cold ripa buffer (25 mm tris·hcl ph 7.6, 150 mm nacl, 1% np-40, 1% sodium deoxycholate, and 0.1% sds) supplemented with protease inhibitors (sigma). the crude lysates were centrifuged at 14,000 rpm (13,000 × g) for 15 min at 4°c, and the supernatant was collected. equal amounts of cell or tissue extracts were loaded onto 10% sds-polyacrylamide gels for separation. then, proteins were transferred to 0.2 μm pvdf membranes (bio-rad), which allowed subsequent probing with primary antibodies. after overnight incubation at 4°c, the membranes were washed with 0.1% tbst (tbs/0.1% tween 20) and incubated with corresponding horseradish peroxidase (hrp)conjugated secondary antibodies (multisciences). the signals were visualized using enhanced chemiluminescence (ecl) reagents (perkin-elmer). quantification of the relative band intensities was performed using imagej software version 1.43. ppprna generation, rna isolation, and real-time rt-pcr viral rna (5′-triphosphate rna, 5′ppp-rna) and cellular rna were generated as previously described [46] . briefly, total rna was isolated from a549 lung epithelial cells infected with a/pr/8/34(h1n1) for 24 h (viral rna) and uninfected a549 lung epithelial cells (cellular rna) using trizol reagent (takara, usa). then, the 5'-phosphate group was dephosphorylated by treatment with calf-intestinal alkaline phosphatase (ciap) (takara). a549 lung epithelial cells were transfected with the indicated rna using lp2000 (thermo, usa). for qpcr, total rna (1 μg) was reverse transcribed into cdna and subjected to real-time quantitative pcr with gene-specific primers and probes on an abi7500 real-time pcr system. relative gene expression was calculated using the 2 −δδct method [47] . the specific primer and probe sets are shown in supplementary table s1 . small interfering rna (sirna), plasmid transfection, and reporter gene assays transient transfection of plasmids into cells, including an isre luciferase reporter plasmid (beyotime) and a flag-rig-i overexpression plasmid (geneppl), was performed by using lipofectamine 2000 (invitrogen). after isre luciferase reporter plasmid (0.5 μg) and flag-rig-i (0.5 μg) overexpression plasmid were transfected into a549 cells for 6 h, the transfected cells were stimulated with ifns or iav (moi = 0.1). in the other experiment, hek293 cells stably co-transfected with pnf-κb-tata-f-luci and pqcxip-egfp plasmid were stimulated with tnf-α (20 ng/ml) or iav for the indicated times. firefly luciferase activity was assayed using the luciferase reporter system (promega, usa) and normalized to renilla luciferase activity or the levels of gfp expression. rig-i-specific sirnas (rig-i #1 sirna and, rig-i #2 sirna) were purchased from guangzhou ribobio co., ltd. and transfected into cells with lipofectamine 2000 according to the manufacturer's instructions. histology and immunohistology lung tissue was excised, fixed in 10% formalin and embedded in paraffin using routine procedures. tissue sections (4 μm) were stained with hematoxylin-eosin for histological examination. for immunohistochemical staining, the sections were deparaffinized with xylene and rehydrated in a graded alcohol series. endogenous peroxidase was blocked with 3% hydrogen peroxide (h 2 o 2 ) in methanol for 20 min at room temperature and antigen retrieval was performed in 0.01 mm citrate buffer (ph 6.0). afterward, the sections were blocked with 5% normal serum and incubated with primary antibody at 4°c overnight. the sections were then incubated for 1 h in hrp-labeled secondary antibody solution and then visualized using a dab reagent kit (maixin, china). the sections were counterstained with mayer's hematoxylin for 2 min before mounting. apoptosis detection by annexin v and flow cytometry cells collected from both suspension and adherent were washed twice with cold pbs, and subsequently resuspended in 1× annexin binding buffer (bioscience, usa) at a concentration of 2 × 10 6 cells/ml. one hundred microliters of cell suspension was stained with 5 μl of annexin v-fitc and 5 μl of propidium iodide (pi) for 15 min at room temperature in the dark. the cells were analyzed using a novocyte flow cytometer within 4 h. bronchoalveolar lavage and flow cytometry mice were euthanized intraperitoneally (i.p.) with an overdose of sodium pentobarbital at the indicated time point. the trachea was cannulated via ventral middle incision. the lungs were lavaged three times with 600 μl of sterile saline. bronchoalveolar lavage fluid (balf) was centrifuged at 300 × g for 10 min at 4°c and the supernatants were collected and stored at −80°c for biochemical analysis. the balf pellets were resuspended in pbs with 0.5% bsa and stained with fluorochrome-conjugated monoclonal antibodies (mabs) (bioscience, usa) that bound to surface molecules for 20 min at 4°c. all samples were analyzed on a novocyte flow cytometer. statistical analysis all data are presented as the mean ± sem. statistical analysis was performed using spss software version 18.0. one-way anova followed by the student-newman-keuls (snk) test was carried out to assess differences between the study groups, and p values less than 0.05 were considered significant. quantitative analysis of β-sitosterol in five medicinal materials medicinal plants including l. japonica, c. morifolium, t. mongolicum, f. suspense, and i. indigotica are traditionally used for heatclearing and detoxification. we hypothesized that β-sitosterol is the common substance in these medicinal plants that mediates their pharmacological actions against respiratory diseases, such as β-sitosterol ameliorates iav-induced inflammation and ali bx zhou influenza. first, we quantified the content of β-sitosterol in these plants. as shown in table 1 , β-sitosterol was detected in all samples at concentrations ranging from 0.51 to 2.23 mg/g. β-sitosterol treatment inhibits the activation of the cellular signaling pathway in iav-infected cells to clarify whether β-sitosterol possesses antiviral activity for the treatment of iav infection, cytopathic effect (cpe) inhibition assays and plaque reduction assays were performed to investigate the antiviral effects of β-sitosterol. as shown in supplementary table s2 , β-sitosterol showed no activity against a/gz/gird07/ 09(h1n1), a/pr/8/34(h1n1), a/hk/8/68(h3n2), a/hk/y280/97 (h9n2), or b/lee/1940 (flub). these findings were further confirmed by plaque reduction assays ( supplementary fig. s1 ). the activation of multiple cellular signaling events has been implicated in the molecular pathogenesis of iav infection [48] . to identify the pharmacological effects exerted by β-sitosterol during iav infection, we assessed the impact of β-sitosterol on the activation of cellular signaling pathway in cells infected with iav. hek293 cells in which an nf-κb-luc reporter was stably expressed were stimulated with either tnf-α (20 ng/ml) (fig. 1a) or a/pr/8/ 34 (h1n1) (fig. 1b) , and then incubated with β-sitosterol for 24 h. we observed that hek293 cells stimulated with tnf-α or a/pr/8/ 34 (h1n1) exhibited robust nf-κb activation, on which β-sitosterol treatment had a dose-dependent suppressive effect ( fig. 1a, b) . furthermore, to test whether β-sitosterol possesses other pharmacological properties, we assessed the activation of signaling pathways in iav-infected a549 cells in the presence or absence of β-sitosterol by immunoblotting. nf-κb and mapk (p38, erk1/2, and jnk/spak) signaling was activated by iav infection (fig. 1c) . as expected, β-sitosterol inhibited the iav-induced p38 mapk activation and p65 phosphorylation, which is consistent with the data gathered using the nf-κb-luc reporter cell line. however, βsitosterol had no inhibitory effect on the erk1/2 or jnk mapk pathway. together, these data demonstrate that β-sitosterol has the potential to inhibit the activation of nf-κb and p38 mapk signaling in response to iav infection. β-sitosterol treatment decreases the expression of iav-induced proinflammatory mediators both the nf-κb and p38 mapk signaling cascades have been implicated as major contributors to hypercytokinemia during human hpaiv h5n1 infection [49, 50] . therefore, we next investigated the effect of β-sitosterol on the expression of proinflammatory mediators in iav-infected cells. a549 cells were infected with the pr8/h1n1 virus (moi = 0.1) in the presence of increasing concentrations of β-sitosterol (150-450 μg/ml) for 24 h. subsequently, the transcript levels of proinflammatory mediators were measured by qpcr. our data showed that the gene expression of an array of cytokines and chemokines, including il-6, tnf-α, ip-10, il-8, mcp-1, mip-1β, and rantes, was elevated dramatically following iav infection, and that this elevation was attenuated by β-sitosterol treatment (fig. 2a) . the kinetics of iavinduced proinflammatory mediator release showed that the protein levels of these proinflammatory mediators (including il-6, tnf-α, il-8, ip-10, rantes, and mcp-1) reached their expression peak at 24 h after viral infection (fig. 2b) , and that the increases in the levels of these mediators were reversed by β-sitosterol treatment (fig. 2c) . the production of cyclooxygenase-2 (cox-2) and its derivative prostaglandin e2 (pge2), has been shown to occur via nf-κb and p38 mapk signaling and play a pathogenic role during iav infection [51, 52] . indeed, increasing the expression of iav-induced cox-2 at both the mrna and protein levels at 24 h p.i., while treatment with β-sitosterol profoundly reduced cox-2 production (fig. 2d, e) . furthermore, we measured the effect of β-sitosterol on the iav-induced expression of iav-induced cox-2 and carried out elisa to quantify cox-2-derived pge2 in the culture supernatants. as expected, β-sitosterol treatment of iav-infected cells led to a significant dose-dependent reduction in pge2 levels (fig. 2f) . these results indicate that β-sitosterol treatment decreases the expression of iav-induced proinflammatory mediators through the inactivation of the nf-κb and p38 mapk signaling pathways. β-sitosterol treatment suppresses the iav-mediated induction of interferon expression and signal transduction by targeting rig-i in response to iav infection, transcription factors including nf-κb, atf2/c-jun, and irf3/7 bind cooperatively to the promoter regions of ifn genes, which are secreted to establish an antiviral state, to initiate their expression [10] . the activation of p38, which mediates the activation of its downstream target atf2, is a prerequisite for optimal ifn-β induction [12] . since β-sitosterol inhibits the iav-induced activation of p38, we hypothesized that β-sitosterol treatment might reduce the expression of ifns. to test this hypothesis, culture supernatants from iav-infected a549 cells treated with or without β-sitosterol were collected at 24 h p.i. and transferred to uninfected a549 cells. after 15 min of incubation, we assessed ifn levels in the culture supernatant by monitoring the phosphorylation and downstream signaling of ifns by immunoblot analysis. as shown in fig. 3a , supernatants from infected a549 cells stimulated the phosphorylation of stat1 tyr701 and stat2 tyr690 , while supernatants from donor cells treated with β-sitosterol attenuated the phosphorylation of stat1 tyr701 . our results indicated that the suppression of stat1 tyr701 phosphorylation by β-sitosterol was associated with reduced ifns production. to further confirm that β-sitosterol inhibits the iav-induced expression of ifns, ifn concentrations in supernatants collected at 24 h p.i. were measured using luminex. as demonstrated in fig. 3b , the iav-induced elevation of ifns, including type i ifn (ifnβ), type ii ifn (ifn-γ), and type iii ifn (ifn-λ1), was significantly decreased in the supernatants of β-sitosterol-treated cells. these observations led us to ask whether β-sitosterol directly affects the ifn signaling transduction pathway. to address this question, we tested whether signal transduction induced by exogenous ifn-β is altered by β-sitosterol treatment. then, we pretreated iav-infected a549 cells with β-sitosterol for 4 h before stimulating them with ifn-β for 15 min. the phosphorylation of stat1 tyr701 , but not stat2, was markedly inhibited by β-sitosterol treatment (fig. 3c) . however, at later time points (8 h p.i.), the ifnβ-mediated phosphorylation of stat1 tyr701 was reduced in iavinfected a549 cells regardless of whether they were pretreated with β-sitosterol or not (fig. 3d) . our data demonstrated that β-sitosterol reduced ifn production through the inhibition of nf-κb and p38 map kinase and that it blocks ifn-mediated signal transduction. we next sought to elucidate the mechanism by which β-sitosterol disrupts ifn production and signaling. the intracellular prr rig-i senses iav 5′ppp-rna, resulting in the activation of nf-κb and p38 map kinase which preferentially promote the initiation of ifn transcription [53] . it is worth mentioning that rig-i, an interferon-stimulated gene (isg), has been reported to augment stat1 activation and inhibit leukemia cell proliferation [54] . thus, it is reasonable to speculate that cross-talk between the rig-i and ifn signaling pathways is affected by β-sitosterol. to confirm this assumption, we investigated the effect of β-sitosterol on the iavinduced expression of rig-i. our results show that β-sitosterol decreased the induction of rig-i in infected a549 cells (fig. 3e, f) . furthermore, cells transfected with the flag-rig-i overexpression plasmid transfection were found to have elevated p-stat1 levels, which were diminished by β-sitosterol treatment (fig. 3g) . the tyrosine kinases jaks are well recognized to be involved in the activation of stat1. therefore, it is possible that β-sitosterol affected stat1 activation by inhibiting jaks. interestingly, we found that a549 cells pretreated with β-sitosterol for 12 h prior to ifn-β (20 ng/ml) stimulation did not exhibit decreased ifn-βmediated activation of jak1, stat1, and stat2 levels (fig. 3h) . together, these data indicate that β-sitosterol can exert an inhibitory effect on rig-i, which leads to decreased iav-induced ifn production and ifn-β signal transduction. β-sitosterol treatment attenuates the ifn-mediated amplification of the proinflammatory response during iav infection via the inhibition of rig-i it is widely recognized that ifn signaling plays a vital role in host antiviral immunity [55] . furthermore, ifns possess immunomodulatory activities on the induction of both chemokines and cytokines and on the recruitment of various immune cells [56] . to examine the effect of rig-i inhibition by β-sitosterol on the transcription of ifn signaling-related molecules, we analyzed iav-mediated ifn-stimulated response element (isre) activation with an assay that utilized a transient isre reporter plasmid. the results presented in fig. 4a show that iav-mediated isre-dependent transcription was significantly inhibited by βsitosterol treatment. rig-i knockdown in iav-infected cells by specific sirnas in iav-infected cells was confirmed to significantly inhibit the iav-mediated isre transcriptional activity (fig. 4b, c) . to further determine the effects of β-sitosterol on ifn-induced proinflammatory responses during iav infection, isre reporter plasmid-transfected cells were stimulated with ifn-β (500 ng/ml) 4 h prior to iav infection. prestimulation with ifn-β led to much higher isre activity than iav infection alone (fig. 4d) . similarly, ifn-β stimulation following iav infection also resulted in increased isre activity, but to a lesser extent than what was seen after ifn-β prestimulation (fig. 4d) . this indicates that ifn-β signaling contributes to the amplification of isre activity during iav infection. nevertheless, this increase in isre activity was diminished in a dose-dependent manner by β-sitosterol treatment representative results from at least three independent experiments are shown. d the band intensities of p-p65, p-p38, p-erk1/2, and p-jnk were semiquantified using imagej (normalized to the loading control gapdh). the data are presented as the mean ± sem (n = 3-5). **p < 0.01, ***p < 0.001 versus the group treated with tnf-α or iav. β-sitosterol ameliorates iav-induced inflammation and ali bx zhou immunoblot analysis was performed to evaluate the protein expression of cox-2. gapdh was used as the internal control (e). f quantification of pge2 in the culture supernatants using elisa. *p < 0.05, **p < 0.01, ***p < 0.001 versus the iav control group. ( fig. 4d) . we next assessed the effects of β-sitosterol on the expression of proinflammatory genes in iav-infected cells pretreated with or without ifn-β. consistent with the previously observed increase in isre activity, the mrna and protein levels of cytokines and chemokines, including il-6, ip-10, tnf-α, il-8, mcp-1 and gm-csf, were robustly increased in iav-infected cells after stimulation with ifn-β (fig. 4e, f) . a similar cytokine and chemokine expression pattern was observed in response to stimulation with ifn-λ1 (data not shown), which signals through the jak/stat pathway leading to isre activation. as expected, the elevation in cytokine and chemokine levels induced by ifn stimulation was decreased by β-sitosterol treatment (fig. 4e, f) . to understand whether the attenuation of the amplified proinflammatory response in ifn-β pretreated cells was solely due to a reduction in ifn-β levels, cells with ifn-β prestimulated for 4 h were treated with an ifn-β neutralizing antibody (2.5 μg/ml) prior to virus infection. however, we observed that the amplification effects of proinflammatory mediators (il-6 and ip-10) in cells prestimulated with ifn-β were not abrogated by ifn-β neutralizing antibody treatment (fig. 4g) . this indicates that cells prestimulated with ifn-β become sensitized and thereby amplify proinflammatory responses. to understand whether the observed inhibitory effect of βsitosterol was due to stat1 inhibition, we measured the phosphorylation of stat1 tyr701 in a549 cells stimulated with ifn-β (500 ng/ml). stat1 was significantly activated in cells pretreated with ifn-β (lane 4), but not in cells infected with iav prior to ifn-β stimulation (lane 8). treatment with β-sitosterol abrogated stat1 activation in a dose-dependent manner (lanes 5-7) (fig. 4h) , indicating that the inhibitory effect of β-sitosterol on stat1 reduced the ifn-mediated amplification of cytokine and chemokine expression. notably, pretreatment with ifn-β significantly increased the iav-triggered expression of rig-i in a549 cells, which was inhibited by β-sitosterol treatment (fig. 4i, j) . together, these data demonstrate that β-sitosterol blocks the iav-induced amplification of the proinflammatory response in ifn-β-activated a549 cells, which is due to inhibition of rig-i levels by β-sitosterol, leading to the inactivation of stat1, and thereby diminishes the transcriptional activity of interferon-stimulated gene factor 3 (isgf3). in addition to playing a role in ifn induction, rig-i signaling has been demonstrated to be involved in apoptosis [57] , which is implicated in iav-induced lung epithelial cell damage and injury. therefore, we investigated whether β-sitosterol affects rig-imediated apoptosis using intracellular viral rna (vrna, 5′ppp-rna) stimulation. stimulation with cellular rna (crna) or vrna and treatment with calfintestine alkaline phosphatase (ciap) to fig. 3 effect of β-sitosterol treatment on iav-induced ifn production and ifn signal transduction. a after 24 h, iav-infected cells with or without the indicated concentration of β-sitosterol treatment were harvested, and the supernatants were transferred to uninfected cells. after 15 min of incubation, the cells were lysed, and the expression of phosphorylated stat1 and stat2 was analyzed by immunoblotting. equal loading of protein was verified by immunoblotting for gapdh. b ifns (ifn-β, ifn-γ, and ifn-λ1) secretion into the culture media was measured at 24 h p.i. using a multiplex luminex assay. c, d after allowing 2 h for iav absorption, a549 cells were incubated with the indicated concentration of β-sitosterol for 4 h (c) or 8 h (d). then, the cells were stimulated for an additional 15 min with human ifn-β (20 ng/ml). the cells were lysed, and total extracts were processed for immunoblotting. e, f effect of β-sitosterol on the expression of rig-i. rig-i mrna expression levels were assayed by quantitative real-time pcr (e). rig-i protein levels were assessed by western blotting (f). g a549 cells were transfected with a flag-rig-i overexpression plasmid, and then treated with β-sitosterol. after 24 h, the cell lysates were collected for immunoblotting. h a549 cells were pretreated with β-sitosterol for 12 h, stimulated with ifn-β (20 ng/ml) for 15 min, and immunoblotted for the indicated proteins. *p < 0.05, **p < 0.01, ***p < 0.001 versus the iav control group. dephosphorylate 5′-triphosphate did not induce apoptosis, excluding the possibility that crna or the non-phosphate at the 5′ end of rna has a pro-apoptotic effect (fig. 5a) . strikingly, the apoptosis of vrna-transfected cells was reduced by β-sitosterol treatment (fig. 5a) . we confirmed the anti-apoptotic effect of βsitosterol by measuring the active caspase-3 and its substrate parp, observing that these products were found in cells transfected with vrna but not in those treated with β-sitosterol fig. 4 (continued) β-sitosterol ameliorates iav-induced inflammation and ali bx zhou (fig. 5b) . to determine whether the inhibitory effect of β-sitosterol on rig-i-mediated apoptosis is related to alterations in the expression of pro-apoptotic factors, we quantified trail and sfas ligand levels in the supernatants of viral rna-transfected cells. trail and sfas ligand levels were significantly reduced by βsitosterol treatment (fig. 5c ). next, we determined the effect of βsitosterol on the apoptosis of iav-infected cell. β-sitosterol treatment reduced iav-mediated apoptosis and active caspase-3 and parp (fig. 5d, e) . last, we observed that β-sitosterol treatment blocked the increase in the release of trail and sfas ligand in iavinfected cells (fig. 5f) . furthermore, rig-i knockdown by specific sirnas was demonstrated to significantly decrease iav-mediated apoptosis and the release of trail (fig. 5g, h) . however, the combination of rig-i sirnas and β-sitosterol did not have an additive effect on the inhibition of iav-mediated apoptosis and trail release, which indicated that β-sitosterol decreased iavmediated apoptosis via the inhibition of rig-i. given that vrna (5′ ppp-rna) recognition is associated with the rapid induction of ifns, we wondered whether β-sitosterol treatment affects ifn induction in the context of vrna transfection. as shown in fig. 5i , the upregulated expression of both type i ifn (ifn-β) and type iii ifn (ifn-λ1) in response to vrna was reversed by β-sitosterol treatment in a dose-dependent manner. to further explore whether the signaling cascade underlying rig-i-mediated apoptosis and ifn induction is affected by βsitosterol, we performed immunoblot analysis 24 h after the fig. 4 effect of β-sitosterol on the ifn-β-mediated amplification of iav-induced proinflammatory mediators. a the effect of β-sitosterol on isre luciferase reporter activity in iav-infected cells. a549 cells were co-transfected with 0.5 μg of pisre-ta-luc reporter plasmid and 0.05 μg of prl-tk plasmid as described in the materials and methods. at 6 h posttransfection, the cells were infected with iav and treated with βsitosterol. after 24 h, the cells were lysed, and luciferase activity was measured. ## p < 0.01 versus the control group. *p < 0.05, **p < 0.01 versus the iav control group. b rig-i knockdown by specific sirnas in iav-infected cells was confirmed by immunoblotting. c the effect of rig-i knockdown by specific sirnas on isre luciferase reporter activity in iav-infected cells. ### p < 0.001 versus the control group. ***p < 0.001 versus the iav control group. d the effect of β-sitosterol on isre luciferase reporter activity induced by stimulation with a combination of ifn-β and iav. after 6 h of transfection, a549 cells were pretreated with ifn-β (500 ng/ml) (columns 4-7) for 4 h or infected with iav prior to ifnβ stimulation (columns 8-11) for 4 h. ifn-β-pretreated cells were infected with iav in the presence or absence of β-sitosterol (150-450 μg/ml). iav-infected cells were stimulated with ifn-β (500 ng/ml) in the presence or absence of β-sitosterol (150-450 μg/ml). the cells were harvested and subjected to a luciferase assay at 24 h p.i. # p < 0.05 versus the iav control group (column 3). *p < 0.05 versus the ifn-β-pretreated group (column 4). § p < 0.05 versus the group infected with iav before being stimulated with ifn-β (500 ng/ml) stimulation group (column 8). e the effect of β-sitosterol on the ifn-β-mediated amplification of iav-induced proinflammatory cytokines and chemokines at the mrna levels was determined by real-time pcr. ### p < 0.001 versus the iav control group (column 2). *p < 0.05, **p < 0.01, ***p < 0.001 versus ifn-β-pretreated group (column 3). § p < 0.05, § § p < 0.01 versus the group infected with iav before being stimulated with ifn-β (500 ng/ml) (column 7). f the effect of β-sitosterol on the ifn-β-mediated amplification of iav-induced proinflammatory cytokines and chemokines at the protein level was determined by a multiplex luminex assay. # p < 0.05, ## p < 0.01 versus the iav control group (column 3). *p < 0.05, **p < 0.01, ***p < 0.001 versus ifn-β-pretreated group (column 4). § p < 0.05, § § p < 0.01, § § § p < 0.001 versus the group infected with iav before being stimulated with ifn-β (500 ng/ml) (column 8). g the effect of ifn-β neutralization on the ifn-β-mediated amplification of the iav-induced proinflammatory response. a549 cells prestimulated with ifn-β (500 ng/ml) for 4 h were treated with an ifn-β neutralizing antibody (2.5 μg/ml) prior to infection with iav. after 24 h, the culture supernatants were collected to measure proinflammatory mediator levels by a multiplex luminex assay. ***p < 0.001 versus iav control group. ns, not significant. h the effect of β-sitosterol on the ifn-β-mediated activation of stat1. lanes 1-3: a549 cells were treated with either ifn-β (500 ng/ml) or with iav for 24 h. lanes 4-7: a549 cells were pretreated with ifn-β (500 ng/ml) for 4 h prior to iav infection. lanes 8-11: a549 cells were infected with iav for 4 h and then stimulated with 500 ng/ml ifn-β. after the indicated treatments, the cells were incubated with or without β-sitosterol for 24 h. the cell lysates were analyzed by immunoblotting for the expression of phospho-stat1 and phospho-stat2. i, j the effect of β-sitosterol on the expression of rig-i. a549 cells were pretreated with ifnβ (500 ng/ml) for 4 h and infected with iav in the presence or absence of β-sitosterol (150-450 μg/ml) (columns 3-6, lanes 4-7) . meanwhile, a549 cells were infected with iav for 4 h prior to ifn-β (500 ng/ml) stimulation (columns 7-10, lanes [8] [9] [10] [11] . i the expression of rig-i was determined by quantitative real-time pcr. ## p < 0.01 versus the iav control group (column 2). *p < 0.05, **p < 0.01, ***p < 0.001 versus ifn-βpretreated group (column 3). j the expression of rig-i was determined by immunoblotting at 24 h p.i. the band intensities of rig-i were semiquantified using imagej (normalized to the loading control gapdh). *p < 0.05 versus ifn-β-pretreated group (column 4). § p < 0.05 versus the group infected with iav before being stimulated with ifn-β (500 ng/ml) (column 8). (fig. 5j) (lane 4) . notably, the dephosphorylation of the 5ʹtriphosphate of rna led to the inactivation of p65 nf-κb and p38 mapk but not stat1 (lane 5). moreover, the vrna-triggered activation of p65 nf-κb, p38 mapk, and stat1 was inhibited by βsitosterol (lanes 6-8). the stimulation of rig-i with vrna initiated signaling events that ultimately led to the expression of proinflammatory cytokines. we detected increased expression of proinflammatory mediators after 24 h of vrna stimulation, and observed that β-sitosterol treatment blocked this effect (fig. 5k) . meanwhile, these proinflammatory mediators induced by iav were effectively reduced by specific rig-i sirnas (fig. 5l) . the levels of il-8, mcp-1, rantes, and gm-csf were further reduced by the combination of rig-i sirnas and β-sitosterol (fig. 5l) . furthermore, the further increased levels of il-6, tnf-α, and ip-10 in flag-rig-i overexpression plasmid-transfected cells with iav infection is dose-dependently decreased by β-sitosterol treatment (fig. 5m) . interestingly, the viral rna-induced upregulation of cox-2 expression was decreased following β-sitosterol treatment (fig. 5n) . to further confirm that β-sitosterol suppresses viral rna-mediated cox-2 upregulation, we quantified the level of its downstream product pge2 in the culture medium using elisa. our results showed that treatment with β-sitosterol dosedependently suppressed the viral rna-induced production of pge2 (fig. 5o) . collectively, these data suggest that β-sitosterol inhibits the activation of rig-i signaling and downstream apoptosis in response to vrna. considering that β-sitosterol exhibited immunomodulatory properties in vitro, we next sought to investigate whether β-sitosterol has a protective effect in a mouse model of influenza. balb/c mice were pretreated with β-sitosterol for 2 days prior to intranasal challenge with 5 ld 50 of iav. at 5 days p.i., histological analysis of lung sections showed that infection with iav resulted in extensive inflammation characterized by massive leukocyte recruitment to the lung parenchyma (fig. 6a, upper panel) . pre-treatment with 200 mg·kg −1 ·d −1 β-sitosterol decreased leukocyte infiltration, and as a result, relatively few inflammatory cells were observed surrounding the bronchioles (fig. 6a, upper panel) . we next performed immunohistochemical staining for cd3 (pan t lymphocytes) and observed that the infiltration of cd3 + t lymphocytes was more pronounced in the lungs of mice with iav analysis of active caspase-3 and parp cleavage in a549 cells transfected with vrna at 24 h. c luminex assay for trail and sfas ligand levels in the supernatants of a549 cells transfected with vrna. *p < 0.05, **p < 0.01 versus the vrna transfection group (column 4). d flow cytometry analysis of the effect of β-sitosterol on apoptotic a549 cells infected with iav at 24 h. *p < 0.05, **p < 0.01 versus iav control group. e analysis of active caspase-3 and parp cleavage in a549 cells infected with iav at 24 h. f luminex assay for trail and sfas ligand levels in the supernatants of a549 cells infected with iav at 24 h. *p < 0.05 versus the iav control group. g flow cytometry analysis of the effect of rig-i knockdown by specific sirnas on iav-induced apoptosis. h luminex assay for trail in the supernatants of a549 cells with rig-i knockdown. ***p < 0.001 versus iav control group (column 2). i luminex assay for ifn-β and ifn-λ1 levels in the supernatants of a549 cells transfected with vrna at 24 h. *p < 0.05, **p < 0.01, ***p < 0.001 versus the vrna-transfected group (column 4). j a549 cells were transfected with vrna in the presence or absence of the indicated concentration of β-sitosterol; 24 h later, the cells were lysed and analyzed by immunoblotting with the indicated antibodies. gapdh was used as a control for equal loading. k the effect of β-sitosterol on the protein expression of cytokines and chemokines in the supernatants of vrna-transfected cells. *p < 0.05, **p < 0.01, ***p < 0.001 versus the vrna transfection group (column 4). l luminex assay for proinflammatory mediators in the supernatants of a549 cells with knockdown of rig-i or in combination with β-sitosterol treatment. ***p < 0.001 versus the iav control group (column 2). # p < 0.05, ### p < 0.001 versus the rig-i #1 sirna transfection group (column 3). § p < 0.05, § § p < 0.01, § § § p < 0.001 versus the rig-i #2 sirna transfection group (column 7). m luminex assay for cytokines and chemokines in the culture supernatants of iav-infected a549 cells transfected with flag-rig-i overexpression plasmid with or without β-sitosterol treatment. ## p < 0.01 versus the iav control group (column 2). *p < 0.05, ***p < 0.001 versus the flag-rig-i overexpression plasmid transfection control group (column 3). n the effect of β-sitosterol on the expression of cox-2 in vrna-transfected cells. the cells were transfected with vrna in the presence or absence of the indicated concentration of β-sitosterol; 24 h later, the cells were lysed and analyzed by immunoblotting using a specific antibody to cox-2. o quantification of pge2 in the culture supernatants of vrna-transfected cells in the presence or absence of the indicated concentrations of β-sitosterol at 24 h. ***p < 0.001 versus the vrna-transfected group (column 4). β-sitosterol ameliorates iav-induced inflammation and ali bx zhou infection alone compared with those that received β-sitosterol treatment (fig. 6a, lower panel) . consistent with the immunohistochemical results, a significantly greater percentage of cd3 + cd8 + cytotoxic t lymphocytes (ctls) was detected in balf from iav-infected mice compared with balf from mice treated with β-sitosterol (fig. 6b) . moreover, the quantification of granzyme b, a pro-apoptotic enzyme secreted by ctl, in lung homogenates revealed a significant increase in its expression in iav-infected mice that was significantly reduced by β-sitosterol administration (fig. 6c) . similar results were obtained for the levels β-sitosterol ameliorates iav-induced inflammation and ali bx zhou of active caspase-3 measured by immunoblotting (fig. 6c) . although influenza antigen-specific cd8 + t cells are recruited to the sites of infection and contribute to viral clearance, immunemediated lung injury can be elicited by aberrant t-cell responses. lung index and total protein levels in balf can be used as an assessment of lung damage. our results showed that compared with iav infection alone, β-sitosterol treatment produced a significantly lower lung index and protein levels in balf (fig. 6d , e), suggesting that β-sitosterol treatment alleviated lung injury likely through the suppression of cd8 + t-cell recruitment. last, iav infection resulted in 100% (13/13) mortality by 12 days p.i. and rapid and continuous weight loss (fig. 6f, g) . remarkably, the survival rate of mice that were treated with 50 and 200 mg/kg βsitosterol was significantly increased to 61.5% (8/13) and 84.6% (11/13), respectively. furthermore, β-sitosterol-treated mice exhibited less initial weight loss after viral challenge and a gradual recovery of body weight (fig. 6g) . together, these data reveal that β-sitosterol attenuates iav-induced lung injury and reduces mortality. β-sitosterol protects against iav by abrogating the iav-mediated activation of multiple signaling cascades to investigate the mechanisms underlying the protective effect of β-sitosterol against iav in vivo, we focused on inflammationassociated signal transduction in the lung. the phosphorylation levels of stat1, stat3, p38, and erk1/2 in lung homogenates were significantly increased in mice challenged with iav relative to uninfected mice (figs. 7a), whereas the phosphorylation levels of these molecules were decreased in mice treated with β-sitosterol. to address whether β-sitosterol inhibits the signaling events mediating the iav-induced expression of proinflammatory cytokines, we quantified cytokine and chemokine levels using luminex. the expression of cytokines and chemokines in balf (il-6, tnf-α, rantes, kc, mcp-1 and mip-1α) and lung homogenates (il-6, tnf-α, ip-10, and rantes) was reduced in mice treated with β-sitosterol (fig. 7b, c) . the iav-induced elevation of serum cytokines including ifn-γ, ip-10, and rantes, was blocked in β-sitosterol-treated mice (fig. 7d) . given that β-sitosterol inhibited iav-mediated stat1/3 activation in vivo and decreased the expression of rig-i and ifn in vitro (fig. 3b, f) , it was necessary to examine the impact of β-sitosterol treatment on the expression of rig-i and ifns in vivo. as expected, iav-induced expression of rig-i in lung homogenates was decreased by β-sitosterol treatment (fig. 7e) . similarly, the expression of ifns in balf (ifn-α, ifn-β, and ifn-γ) (fig. 7f) and lung homogenates (ifn-β) (fig. 7g) was also decreased. collectively, these data provide evidence regarding the mechanism by which β-sitosterol modulates dysregulated signaling cascades and proinflammatory responses linked to severe influenza. patients with influenza are frequently afflicted with severe pneumonia characterized by excessive infiltration of leukocytes and proinflammatory cytokine production [58] [59] [60] , leading to a high risk of death. recent studies have revealed that iav-mediated ifns play a disease-promoting role in the pathogenesis of influenza [31] . chinese herbal medicines, including l. japonica [35] , c. morifolium [36] , t. mongolicum [37] , f. suspense [38] , and i. indigotica [39] , have a long history of being used for the treatment of the common cold and for heat-clearing. in the current study, we demonstrated that β-sitosterol derived from these herbal medicines has the ability to block iav-mediated ifn and proinflammatory mediator production through the inhibition of rig-i signaling. furthermore, our data showed that β-sitosterol attenuates the amplification of the iav-mediated proinflammatory response in ifn-sensitized cells by disrupting rig-i-mediated stat1 activation. furthermore, we showed that β-sitosterol abrogates the recruitment of cytotoxic t lymphocytes (ctls) in the lung, thereby significantly improving lung injury and survival in mice challenged with iav (fig. 8) . rig-i detects viral rna (5′ppp-rna) in the cytoplasm, which is then ubiquitinated by trim25 (tripartite motif-containing protein 25) and subsequently interacts with ips-1 (ifn-β promotor stimulator 1) to initiate the activation of nf-κb, p38, and irf3/7 [61, 62] . we asked whether rig-i signaling is affected by βsitosterol during iav infection or in cells transfected with viral rna. we found that rig-i expression was increased following iav infection or prestimulation with ifn-β prior to iav infection, and that this increase in expression was significantly reduced in the presence of β-sitosterol. furthermore, the activation of nf-κb and p38 in both iav-infected and viral rna-transfected cells was inhibited by β-sitosterol treatment. these results suggest that βsitosterol treatment antagonizes the rig-i signaling cascades. the activation of rig-i and its downstream targets nf-κb and p38 contributes to the induction of proinflammatory cytokines in response to influenza virus infection [63] . in previous studies, the upregulation of rig-i during fatal h5n1 infection was shown to cause an amplification of inflammatory responses [18] . the hyperinduction of proinflammatory cytokines via rig-i, nf-κb, and p38 signaling has been suggested to contribute to severity of symptoms in patients with h5n1 infection [64, 65] . we measured nf-κb activation using an nf-κb luciferase reporter system and found that β-sitosterol treatment downregulated the transcriptional activation of nf-κb following the administration of tnf-α and iav infection. consistent with these findings, the inhibitory effects of β-sitosterol on rig-i signaling led to reduced production of cytokines, such as il-6 and tnf-α, and chemokines such as il-8 and ip-10 in iav-infected and viral rna-transfected cells. rig-i or nf-κb and p38 activation in response to viral or bacterial infection, respectively, has been reported to induce cox-2 expression [46, 66] . furthermore, it has been shown that decreased expression of cox-2 and its derivative pge2 has beneficial effects during influenza virus infection that lead to reduced hypothermia and enhanced type i ifn antiviral immunity [51] . we observed that iav infection and viral rna stimulation were associated with increased expression of cox-2 and pge2 and that β-sitosterol treatment reversed the increase in a dosedependent manner. fig. 6 β-sitosterol prevents iav-induced lung pathology in mice. two days prior to infection with 5 ld 50 of a/fm1/h1n1 virus, pbs or β-sitosterol (50 mg·kg −1 ·d −1 or 200 mg·kg −1 ·d −1 ) was intragastrically administered to mice for 7 consecutive days. a on day 5 p.i., the lungs were harvested and subsequently subjected to histological analysis by h&e staining (original magnification, ×100) or cd3 antigen (t-cell marker) staining (original magnification, ×200). b representative flow cytometry quantification of cd3 + cd8 + t cells in balf on day 4 p.i. the histograms represent the percentage of cd3 + cd8 + t cells in balf (right panel). the data are representative of three independent experiments using 5-7 mice per group. *p < 0.05, **p < 0.01 versus the iav-infected group. c on day 5 p.i., the lungs were removed and homogenized. lung homogenates were subjected to immunoblot analysis of granzyme b and active caspase-3. the ratios of the relative band intensities of granzyme b and active caspase-3 normalized to gapdh are shown (n = 3-5 mice per group) (right panel). *p < 0.05, ***p < 0.001 versus the iav-infected mice group. d the lung index (lung/body weight ratios) of mice treated with pbs (n = 6) or β-sitosterol (n = 6) on day 5 p.i. *p < 0.05, **p < 0.01 versus the iav-infected group. e on day 7 p.i., the total protein concentrations in balf was measured by bca assay (n = 3-11 mice per group). *p < 0.05, **p < 0.01 versus the iav-infected group. f, g survival rate (f) and weight curves (g) of iav-infected mice treated with or without β-sitosterol (n = 13 mice per group). β-sitosterol ameliorates iav-induced inflammation and ali bx zhou nf-κb, atf2 (a downstream target of p38), and irf3 form a transcriptional complex that drives the expression of the antiviral factor ifn-β [10] . in addition, the viral-induced expression of type iii ifn requires the involvement of rig-i, ips-1, tbk1, and p38 signaling [67] [68] [69] , suggesting that the expression of type i and iii ifns is promoted via a common mechanism. interestingly, some studies have indicated that nf-κb is crucial for ifn-β production when irf3 activation is weak but not when irf3 activation is strong [70] . although we did not detect significant activation of irf3 at 24 h, we were able to detect an inhibitory effect of β-sitosterol on the expression of ifns, including ifn-β and ifn-λ1, in cells infected with iav or in those subjected to viral rna transfection. these findings may be attributable to the inactivation of rig-i, nf-κb, and p38 signaling. a clear link between rig-i expression and stat1 activation has been established by previous studies. experiments involving rig-i overexpression or knockdown have suggested that rig-i is essential for stat1 activation in leukemia cell lines [54, 71] . in accordance with these findings, our results show that the augmentation of stat1 activation by rig-i overexpression was suppressed by β-sitosterol or the inhibition of iav-mediated isre transcriptional activity by specific rig-i sirnas. in addition, we observed that the activation of jaks was not affected by β-sitosterol. therefore, the inhibition of stat1 phosphorylation in response to ifn-β treatment for 15 min or 24 h can be attributed to the downregulation of rig-i by βsitosterol. moreover, the activation of rig-i, but not of mda-5, has been shown to involve double-stranded rna (dsrna)-induced stat1 phosphorylation [72] . our data showed that β-sitosterol treatment abrogated stat1 phosphorylation in cells stimulated with vrna (5′ppp-rna), which binds to and activates rig-i. however, the dephosphorylation of viral rna with ciap did not reduce stat1 phosphorylation. a possible explanation for these findings is that the dsrna that is generated following viral rna dephosphorylation is also a ligand for rig-i and induces stat1 phosphorylation in an ifn-dependent or ifn-independent manner, as described previously [72] . the important role of ifns in the defense against viral infection is widely recognized [73, 74] . however, ifn receptor deficiency does not lead to a detrimental outcome, which is perhaps due to decreased ifn-induced immune injury [31, 33, 34] . recent studies have clearly revealed the pathogenic potential of ifn-β-and ifn-λ1-mediated immunopathology in viral infectious diseases and autoimmune diseases [31, 33, 34, 75] . here, we have proposed a model in which ifns (including type i and iii ifn) secreted from iav-infected cells bind to their receptors and sensitize uninfected neighboring cells, leading to the amplification of proinflammatory responses driven by isgf3 following infection by progeny viruses. β-sitosterol treatment blocked the amplification of this proinflammatory response and the concomitant expression of proinflammatory cytokines through the inhibition of isgf3 complexes. the inhibitory effect on isgf3 complexes was due to the failure of downregulated rig-i to exert a converse effect on stat1 activation in β-sitosterol-treated cells (fig. 8) . the activation of rig-i-mediated apoptosis via type i ifn-dependent and type i ifnindependent mechanisms has been considered a promising strategy for cancer therapeutics [57, 76] . the ifn-induced activation of isgf3 leads to trail expression, resulting in substantial alveolar epithelial cell (aec) apoptosis and lung injury [32, 77] . aec apoptosis has been found to play a critical role in the pathogenesis of h5n1 and pandemic h1n1 in patients with ards [59, 78] . our data suggest that β-sitosterol prevents iav-induced apoptosis associated with decreased ifn-driven expression of trail. the loss of rig-i signaling has been correlated with a reduction in antigen presentation in bone marrow derived dendritic cells (bmdcs), and in the antiviral function of cd8 + cytotoxic t cells [79] . thus, it is clear that the rig-i pathway is important for mediating the production of ifns during antiviral responses. in contrast, the rig-i-mediated expression of inflammatory mediators has been shown to induce the recruitment of monocyte-derived dcs (modcs) to support viral replication [80] . antiviral effector cd8 + t cells and nk cells eliminate invading pathogens through fig. 7 β-sitosterol effectively abrogates iav-triggered signaling in vivo. a mice were infected with 5 ld 50 of a/fm1/h1n1 virus and treated with pbs or β-sitosterol (intragastrically administered for 7 consecutive days beginning 2 days prior to viral infection). the lungs were harvested and homogenized on day 5 p.i. and the processed for immunoblotting with the indicated antibodies. the relative band intensities of the indicated proteins were normalized to that of gapdh (n = 3-5 mice per group) (right panel). *p < 0.05, **p < 0.01 versus the iav-infected group. b-d detection of cytokine and chemokine production in balf (b), lung homogenates (c), and serum (d) by luminex analysis. *p < 0.05, **p < 0.01 versus iav-infected group. e on day 5 p.i., lung homogenates were subjected to immunoblot analysis of rig-i. the rig-i band intensity normalized to that of gapdh is shown (n = 4-6 mice per group) (right panel). *p < 0.05 versus iav-infected group. f, g detection of ifns (ifn-α, ifn-β, and ifn-γ) production in balf (f) and lung homogenates (g) by luminex analysis. *p < 0.05, **p < 0.01 versus the iavinfected group. β-sitosterol ameliorates iav-induced inflammation and ali bx zhou several mechanisms, including the expression of pro-apoptotic proteins such as trail and fas and the secretion of granzyme b (grb) and perforin. through these mechanisms, the apoptotic caspase cascade is activated in virus-infected cells [81] [82] [83] . studies have reported that ifn-γ plays an important role in modulating cd8 + t-cell recruitment and that the production of granzyme b during the recruitment of cd8 + t cells is dependent on the ifn-βinduced activation of stat1 [81, 84, 85] . accordingly, our data show that β-sitosterol administration decreases the levels of ifn-γ and ifn-β and concomitantly induces a low level of cd8 + t-cell recruitment and granzyme b secretion in the lungs. cytotoxic cd8 + t lymphocytes (ctls) seem to be essential for viral clearance. however, severe pneumonia in patients with pandemic influenza a (h1n1) virus infection is apparently related to high levels of cd8 + t cells [86] . interestingly, one study showed that ha-transgenic mice develop lethal lung injury following treatment with influenza ha-specific cd8 + cytotoxic t cells [87] . deficiency of a20 (tnf alpha-induced protein 3, tnfaip3), which is a negative feedback ubiquitin-editing protein that inhibits nf-κb signaling, protects mice against viral challenge by reducing the population of grb + cd8 + t cells [88] . consistent with these studies, our data showed that iav-induced acute lung injury and mortality are attenuated in mice treated with β-sitosterol and that this attenuation is associated with decreased cd8 + t-cell recruitment and granzyme b secretion in the lung. furthermore, we observed the inhibition of stat1/3 and p38 phosphorylation by β-sitosterol in mouse lung tissue and made a similar observation in iavinfected a549 cells. the expression of cytokines driven by stat1/3 and p38, which exacerbate iav-induced immunopathology, in balf and lung tissue was decreased following β-sitosterol administration in balf and lung tissue. in contrast to our in vitro results, the phosphorylation of erk1/2 was attenuated in the lung tissues of β-sitosterol-treated mice. the inhibition of erk1/2 signaling is involved in the retention of viral rnp in the nucleus, but its activation also correlates with cytokine expression [89] . it is likely that the decrease in phosphorylated erk1/2 expression in vivo was a result of the immunoregulatory effects of β-sitosterol. fig. 8 schematic diagram showing the mechanism by which βsitosterol attenuates iav-induced proinflammatory responses and injury. invading viruses are sensed by rig-i, leading to the activation and that of rig-i, nf-κb, and p38, which initiates the expression of proinflammatory mediators and ifns. secreted ifns, including type i and iii ifns, bind to their receptors via an autocrine or paracrine mechanism and then exert their antiviral effects. subsequently, previously uninfected ifn-sensitized neighboring cells become infected by progeny viruses, which triggers the amplification of the inflammatory response. the inhibition of rig-i signaling by βsitosterol attenuates rig-i-linked proinflammatory ifn production, which results in a reduction in stat1 activation and thus a decrease in the amplification of proinflammatory responses driven by isg complexes in ifn-sensitized cells. furthermore, the inhibition of rig-i signaling by β-sitosterol also suppresses the recruitment of cd8 + t cells and granzyme b release in vivo, thereby blocking lung immune injury during influenza virus infection. influenza a(h7n9) virus gains neuraminidase inhibitor resistance without loss of in vivo virulence or transmissibility an ns-segment exonic splicing enhancer regulates influenza a virus replication in mammalian cells clinical aspects and cytokine response in severe h1n1 influenza a virus infection clinical aspects and cytokine response in adults with seasonal influenza infection fatal outcome of human influenza a (h5n1) is associated with high viral load and hypercytokinemia cytokine and chemokine levels in patients infected with the novel avian influenza a (h7n9) virus in china pathogen recognition and inflammatory signaling in innate immune defenses de novo replication of the influenza virus rna genome is regulated by dna replicative helicase recognition of viruses by cytoplasmic sensors an atomic model of the interferon-beta enhanceosome inhibition of p38 mitogen-activated protein kinase impairs influenza virus-induced primary and secondary host gene responses and protects mice from lethal h5n1 infection the kinetic mechanism of the dual phosphorylation of the atf2 transcription factor by p38 mitogen-activated protein (map) kinase alpha. implications for signal/response profiles of map kinase pathways early control of h5n1 influenza virus replication by the type i interferon response in mice protective role of beta interferon in host defense against influenza a virus differential roles of mda5 and rig-i helicases in the recognition of rna viruses distinct rig-i and mda5 signaling by rna viruses in innate immunity cell typespecific involvement of rig-i in antiviral response h5n1 influenza virus-induced mediators upregulate rig-i in uninfected cells by paracrine effects contributing to amplified cytokine cascades inflammatory cytokines in the bal of patients with ards. persistent elevation over time predicts poor outcome bronchoalveolar and systemic cytokine profiles in patients with ards, severe pneumonia and cardiogenic pulmonary oedema ifnlambdas mediate antiviral protection through a distinct class ii cytokine receptor complex cloning and expression of a long form of the beta subunit of the interferon alpha beta receptor that is required for signaling signaling pathways activated by interferons innate immune modulation by rna viruses: emerging insights from functional genomics the human interferon-induced mxa protein inhibits early stages of influenza a virus infection by retaining the incoming viral genome in the cytoplasm binding of the influenza a virus ns1 protein to pkr mediates the inhibition of its activation by either pact or double-stranded rna the primary function of rna binding by the influenza a virus ns1 protein in infected cells: inhibiting the 2'-5' oligo (a) synthetase/rnase l pathway influenza a virus inhibits type i ifn signaling via nf-kappab-dependent induction of socs-3 expression suppression of interferon lambda signaling by socs-1 results in their excessive production during influenza virus infection influenza-induced type i interferon enhances susceptibility to gram-negative and gram-positive bacterial pneumonia in mice pathogenic potential of interferon alphabeta in acute influenza infection macrophageexpressed ifn-beta contributes to apoptotic alveolar epithelial cell injury in severe influenza virus pneumonia alpha/beta interferon receptor signaling amplifies early proinflammatory cytokine production in the lung during respiratory syncytial virus infection dysregulated type i interferon and inflammatory monocyte-macrophage responses cause lethal pneumonia in sars-cov-infected mice honeysuckle-encoded atypical microrna2911 directly targets influenza a viruses antioxidant and anti-inflammatory flavonoids from the flowers of chuju, a medical cultivar of chrysanthemum morifolim ramat taraxacum mongolicum extract exhibits antimicrobial activity against respiratory tract bacterial strains in vitro and in neonatal rats by enhancing systemic th1 immunity antiviral effect of forsythoside a from forsythia suspensa (thunb.) vahl fruit against influenza a virus through reduction of viral m1 protein lariciresinol-4-o-beta-d-glucopyranoside from the root of isatis indigotica inhibits influenza a virus-induced proinflammatory response sterol content of foods of plant origin natural sources of dietary plant sterols antioxidant effects of phytosterol and its components protective role of plant sterol and stanol esters in liver inflammation: insights from mice and humans beta-sitosterol attenuates high-fat dietinduced intestinal inflammation in mice by inhibiting the binding of lipopolysaccharide to toll-like receptor 4 in the nf-kappab pathway beta-sitosterol-induced-apoptosis is mediated by the activation of erk and the downregulation of akt in mca-102 murine fibrosarcoma cells influenza a viruses suppress cyclooxygenase-2 expression by affecting its mrna stability analysis of relative gene expression data using real-time quantitative pcr and the 2(-delta delta c(t)) method influenza virus and cell signaling pathways induction of proinflammatory cytokines in primary human macrophages by influenza a virus (h5n1) is selectively regulated by ifn regulatory factor 3 and p38 mapk role of hypercytokinemia in nf-kappab p50-deficient mice after h5n1 influenza a virus infection targeted prostaglandin e2 inhibition enhances antiviral immunity through induction of type i interferon and apoptosis in macrophages hyperinduction of cyclooxygenase-2-mediated proinflammatory cascade: a mechanism for the pathogenesis of avian influenza h5n1 infection rig-i detects viral genomic rna during negative-strand rna virus infection ra-inducible gene-i induction augments stat1 activation to inhibit leukemia cell proliferation interferons at age 50: past, current and future impact on biomedicine disease-promoting effects of type i interferons in viral, bacterial, and coinfections proapoptotic signaling induced by rig-i and mda-5 results in type i interferon-independent apoptosis in human melanoma cells cytokine response patterns in severe pandemic 2009 h1n1 and seasonal influenza among hospitalized adults pathological and ultrastructural analysis of surgical lung biopsies in patients with swine-origin influenza type a/h1n1 and acute respiratory failure a question of self-preservation: immunopathology in influenza virus infection trim25 ring-finger e3 ubiquitin ligase is essential for rig-i-mediated antiviral activity type i inteferon gene induction by the interferon regulatory factor family of transcription factors antiviral innate immunity and stress granule responses p38 mitogen-activated protein kinase-dependent hyperinduction of tumor necrosis factor alpha expression in response to avian influenza virus h5n1 essential impact of nf-kappab signaling on the h5n1 influenza a virus-induced transcriptome streptococcus pneumoniae induced p38 mapk-and nf-kappab-dependent cox-2 expression in human lung epithelium viral infections activate types i and iii interferon genes through a common mechanism map kinase p38alpha regulates type iii interferon (ifn-lambda1) gene expression in human monocyte-derived dendritic cells in response to rna stimulation rig-i-mediated activation of p38 mapk is essential for viral induction of interferon and activation of dendritic cells: dependence on traf2 and tak1 nf-kappa b rela subunit is crucial for early ifn-beta expression and resistance to rna virus replication involvement of retinoic acid-inducible gene-i in the ifn-{gamma}/stat1 signalling pathway in beas-2b cells doublestranded rna induces biphasic stat1 phosphorylation by both type i interferon (ifn)-dependent and type i ifn-independent pathways interferons and viruses: an interplay between induction, signalling, antiviral responses and virus countermeasures interferon-inducible antiviral effectors interleukin-29 modulates proinflammatory cytokine production in synovial inflammation of rheumatoid arthritis synergy between ra and tlr3 promotes type i ifn-dependent apoptosis through upregulation of trail pathway in breast cancer cells antiviral response by natural killer cells through trail gene induction by ifn-alpha/beta molecular biology, and pathogenesis of avian influenza a (h5n1) infection in humans rig-i signaling is critical for efficient polyfunctional t cell responses during influenza virus infection efficient influenza a virus replication in the respiratory tract requires signals from tlr7 and rig-i type i interferons regulate cytolytic activity of memory cd8 + t cells in the lung airways during respiratory virus challenge role of tumor necrosis factorrelated apoptosis-inducing ligand in immune response to influenza virus infection in mice distinct roles of nk cells in viral immunity during different phases of acute friend retrovirus infection intracellular ifn-gamma expression in natural killer cells precedes lung cd8 + t cell recruitment during respiratory syncytial virus infection production of interferon-gamma by influenza hemagglutinin-specific cd8 effector t cells influences the development of pulmonary immunopathology cd4 + /cd8 + t lymphocytes imbalance in children with severe 2009 pandemic influenza a (h1n1) pneumonia structural and functional consequences of alveolar cell recognition by cd8 + t lymphocytes in experimental lung disease a20 deficiency in lung epithelial cells protects against influenza a virus infection inhibition of influenza virus-induced nf-kappab and raf/mek/erk activation can reduce both virus titers and cytokine expression simultaneously in vitro and in vivo zfy and nsz conceived the study; bxz, zfy, and nsz designed the study; bxz and xll conducted the in vitro experiments; jl and xpp isolated and analyzed the compound β-sitosterol; bxz, xll, hmj, ybh, and pfx performed animal experiments; and bxz and jl wrote the paper. the online version of this article (https://doi.org/10.1038/s41401-020-0403-9) contains supplementary material, which is available to authorized users.competing interests: the authors declare no competing interests. key: cord-313957-hviv5zar authors: masucci, maria grazia title: viral ubiquitin and ubiquitin-like deconjugases—swiss army knives for infection date: 2020-08-01 journal: biomolecules doi: 10.3390/biom10081137 sha: doc_id: 313957 cord_uid: hviv5zar posttranslational modifications of cellular proteins by covalent conjugation of ubiquitin and ubiquitin-like polypeptides regulate numerous cellular processes that are captured by viruses to promote infection, replication, and spreading. the importance of these protein modifications for the viral life cycle is underscored by the discovery that many viruses encode deconjugases that reverse their functions. the structural and functional characterization of these viral enzymes and the identification of their viral and cellular substrates is providing valuable insights into the biology of viral infections and the host’s antiviral defense. given the growing body of evidence demonstrating their key contribution to pathogenesis, the viral deconjugases are now recognized as attractive targets for the design of novel antiviral therapeutics. viruses have shaped the fate of human societies throughout history. understanding how these potentially life-threatening pathogens establish infection and how they interact with their hosts is our best strategy for acquiring the means to control the diseases they cause. being obligatory intracellular parasites, viruses face a double challenge. on one side, they need to commandeer the molecular machinery of the host cell to support the production of new virus particles, while on the other side, they must hold back the multifaceted cellular and organismal defenses that are triggered by infection. these challenges are met by the expression of specialized viral products that hijack or manipulate critical cellular functions. many of these viral pathogenicity factors are multifunctional proteins that mimic the activity of cellular counterparts whose activity controls key aspects of normal cell physiology. virtually all cellular processes are regulated by posttranslational modifications that dictate the function, subcellular localization, and interactions of effector proteins. among those, the covalent attachment of small polypeptides of the ubiquitin family (henceforth collectively referred to as ubiquitin-like polypeptides, ubls) provides a flexible means to control the activity and fate of the modified substrate. cell functions that orchestrate the outcome of infection such as the cell cycle, cell survival and programmed cell death, gene expression, protein trafficking and degradation, autophagy, and the immune response are all dependent on ubl modifications [1] . it is therefore not surprising that viruses have evolved means to interfere with the ubl signaling networks in order to secure a cellular environment conducive to their own replication and spread. the ubls are a family of structurally related small polypeptides that share a β-grasp fold organization consisting of a mixed β-sheet structure with a central α-helix [2] . to date, seventeen human ubls have been reported to be conjugated to other molecules. in addition to ubiquitin (ub), the ubls include the small ubiquitin-related modifier (sumo)-1, -2, and -3; nedd8 (neural precursor cell expressed, developmentally downreagulated-8); isg15 (interferon stimulated gene-15); ufm1 (ubiquitin-fold modifier-1); urm1 (ubiquitin-related modifier-1); fat10 (hla-f adjacent transcript cell expressed, developmentally downreagulated-8); isg15 (interferon stimulated gene-15); ufm1 (ubiquitin-fold modifier-1); urm1 (ubiquitin-related modifier-1); fat10 (hla-f adjacent transcript 10); mnsfβ (monoclonal nonspecific suppressor factor β); and the lc3 (microtubule-associated light chain-3) and gabarap (γ-aminobutyric acid receptor associated protein) family of modifiers [3] . the attachment of ubls to their protein or, in the case of the lc3/gabarap family, lipid substrates is mediated by an enzymatic cascade that starts with processing of the ubl precursor by a specific protease, which generates a c-terminal gly residue required for conjugation. the mature ubl becomes the substrate of an activating enzyme (e1) that forms a high-energy thiolester bond with the c-terminal gly and then loads the activated ubl on the catalytic cys residue of a conjugating enzyme (e2). the e2 transfers the ubl to the substrate with the help of a ligase (e3) that promotes the transfer [4, 5] (figure 1 ). as a rule, each ubl conjugation system involves distinct sets of dedicated e1, e2, and e3, enzymes but enzyme sharing is not uncommon and e3 ligases with mixed specificity for ub and isg15, e.g., trim25 (tripartite motif-25) [6] ; ubiquitin and nedd8, e.g., mdm2 (mouse double minie-2) [7] ; or ub and sumo, e.g., topors (top1 binding arginine/serine rich protein), traf7 (tnf receptor associated factor-7), uhrf2 (ubiquitin like with phd and ring finger domain-2), and trim27 [8] [9] [10] [11] have been described. figure 1 . schematic illustration of the ubl activation, conjugation and deconjugation cycle. the covalent attachment of ubls to their substrates involves sequential catalytic reactions that initiate with processing of the ubl precursor by a specific ubl protease. the mature ubl is activated by an activating enzyme (e1) and then transferred to a conjugating enzyme (e2) that, with the help of a substrate-specific ligase (e3), transfer the activated ubl to the ε-amino residue of a lys on the target protein via a covalent isopeptide bond. additional ubls can be linked to the previous one to form chains. ubl-specific proteases can reverse the modification, supplementing the cellular pools of free ubls. the attachment of a ub moiety to the n-terminal met1 or to an internal lys residue of the previous ub (k6, k11, k27,k29,k33,k48 or k63) results in the formation of topologically different poly-ub chains that, upon recognition by signal transducers contain dedicated binding domains, target the substrates various fates and cellular functions ubiquitin is the first recognized and best-known member of the family. the covalent attachment of ub, ubiquitination, is mediated by specific combinations of e2s and e3s that promote the formation of a peptide bond between the c-terminal gly and the n-terminal met or the ε-amino group of a lys residue in the substrate. in addition to the attachment of a single ub to one (mono-ub) or several figure 1. schematic illustration of the ubl activation, conjugation and deconjugation cycle. the covalent attachment of ubls to their substrates involves sequential catalytic reactions that initiate with processing of the ubl precursor by a specific ubl protease. the mature ubl is activated by an activating enzyme (e1) and then transferred to a conjugating enzyme (e2) that, with the help of a substrate-specific ligase (e3), transfer the activated ubl to the ε-amino residue of a lys on the target protein via a covalent isopeptide bond. additional ubls can be linked to the previous one to form chains. ubl-specific proteases can reverse the modification, supplementing the cellular pools of free ubls. the attachment of a ub moiety to the n-terminal met1 or to an internal lys residue of the previous ub (k6, k11, k27,k29,k33,k48 or k63) results in the formation of topologically different poly-ub chains that, upon recognition by signal transducers contain dedicated binding domains, target the substrates various fates and cellular functions ubiquitin is the first recognized and best-known member of the family. the covalent attachment of ub, ubiquitination, is mediated by specific combinations of e2s and e3s that promote the formation of a peptide bond between the c-terminal gly and the n-terminal met or the ε-amino group of a lys residue in the substrate. in addition to the attachment of a single ub to one (mono-ub) or several (multi-ub) lys residues, poly-ub chains can be formed upon attachment of a new ub moiety to met1 or lys6, 11, 27, 29, 33, 48 , or 63 of the first conjugated ub. in the poly-ub chain, ub is usually attached to the same lys residue on each ub in the chain but mixed-linkage and branched poly-ub chains may be more common than originally thought [12] (figure 1 ). the attachment of ub or poly-ub chains generates new interaction surfaces in the modified substrate that are recognized by signal transducers via dedicated ubiquitin binding domains [13] . signal transducers that recognize other ubls have their own specific binding motifs resulting in a broad spectrum of distinct signals that engage the modified substrate in specialized functions ( figure 2 ). for example, lys48-linked poly-ub chains (k48poly-ub) usually target the substrate for degradation by the proteasome whereas k63poly-ub have non-proteolytic functions, often related to protein localization and protein-protein interactions [14] . mono-or poly-sumoylation regulates protein localization and the formation of protein complexes involved in dna replication and stress responses [15] . poly-sumo chains may also serve as a signal for ub-dependent proteasomal degradation following recognition by specialized e3 that carry multiple sumo-interacting motifs [16] . the activation of cullin-ring ligases (crls) by neddylation of the cullin scaffold provides another example of cross-talk between different types of ubl modification [17] . other ubls play important roles in different types of stress responses as illustrated by involvement of urm1 and ufm1 in the regulation of oxidative stress [18, 19] and er stress [20, 21] , respectively, and by the involvement of fat-10 [22] and isg15 [23, 24] in the cellular and immune response to infection. the conjugation of ubls of the lc3/gabarap family to the membrane lipid phosphatidylethanolamine underlies their involvement in the expansion and fusion of autophagic membranes [25] . biomolecules 2020, 10, 1137 3 of 24 (multi-ub) lys residues, poly-ub chains can be formed upon attachment of a new ub moiety to met1 or lys6, 11, 27, 29, 33, 48 , or 63 of the first conjugated ub. in the poly-ub chain, ub is usually attached to the same lys residue on each ub in the chain but mixed-linkage and branched poly-ub chains may be more common than originally thought [12] ( figure 1 ). the attachment of ub or poly-ub chains generates new interaction surfaces in the modified substrate that are recognized by signal transducers via dedicated ubiquitin binding domains [13] . signal transducers that recognize other ubls have their own specific binding motifs resulting in a broad spectrum of distinct signals that engage the modified substrate in specialized functions ( figure 2 ). for example, lys48-linked poly-ub chains (k48poly-ub) usually target the substrate for degradation by the proteasome whereas k63poly-ub have non-proteolytic functions, often related to protein localization and protein-protein interactions [14] . mono-or poly-sumoylation regulates protein localization and the formation of protein complexes involved in dna replication and stress responses [15] . poly-sumo chains may also serve as a signal for ub-dependent proteasomal degradation following recognition by specialized e3 that carry multiple sumo-interacting motifs [16] . the activation of cullin-ring ligases (crls) by neddylation of the cullin scaffold provides another example of cross-talk between different types of ubl modification [17] . other ubls play important roles in different types of stress responses as illustrated by involvement of urm1 and ufm1 in the regulation of oxidative stress [18, 19] and er stress [20, 21] , respectively, and by the involvement of fat-10 [22] and isg15 [23, 24] in the cellular and immune response to infection. the conjugation of ubls of the lc3/gabarap family to the membrane lipid phosphatidylethanolamine underlies their involvement in the expansion and fusion of autophagic membranes [25] . the best-known function of ubiquitin is the marking of substrates for degradation by the proteasome, but different types of ubiquitination regulate endocytosis protein trafficking, transcription and translation, cell signaling, histone modification and dna repair. other ubl have similar but usually more restricted roles in the regulation of cellular functions. sumoylation is involved in the formation of protein complexes that regulate transcription, dna repair different stress responses and can also mark proteins for ubiquitin-dependent degradation. nedd8 is best known for its role in regulating the activity of cullin-ring ligases, which in turn regulates substrate degradation. ubls, of the lc3/gabarap family are involved in the process of autophagy. the best-known function of ubiquitin is the marking of substrates for degradation by the proteasome, but different types of ubiquitination regulate endocytosis protein trafficking, transcription and translation, cell signaling, histone modification and dna repair. other ubl have similar but usually more restricted roles in the regulation of cellular functions. sumoylation is involved in the formation of protein complexes that regulate transcription, dna repair different stress responses and can also mark proteins for ubiquitin-dependent degradation. nedd8 is best known for its role in regulating the activity of cullin-ring ligases, which in turn regulates substrate degradation. ubls, of the lc3/gabarap family are involved in the process of autophagy. the conjugation of ubls is highly dynamic and reversible, allowing for the fine-tuning and rapid remodeling of signal transduction pathways in response to different stimuli. deconjugating enzymes catalyze the removal of the ubls from their substrates, resulting in either complete loss or editing/trimming of the ubl chain. in humans, around hundred ub-specific deconjugases (also called deubiquitinases, dubs), belong to families that differ in structure and catalytic mechanisms [26, 27] . the majority are cysteine proteases containing an active-site catalytic triad composed of a cys nucleophile and closely situated his and asp/asn residues. the cys protease families include ub-specific proteases (usps), ubiquitin c-terminal hydrolases (uch), ovarian tumor domain proteases (otus), machado-josephin disease proteases (mjd), motif interacting with ubiquitin novel dub family (mindy), and zinc finger with ufm1-specific peptidase domain protein (zufsp), whereas the jab1/mpn/mov34 (jamm) deconjugases are zinc-dependent metalloproteases. members of the usp family generally cleave all ub linkage types without a clear preference [28] , while the members of the otu enzymes are often linkage specific [29] . as a rule, different ubls have their own sets of specific deconjugases. however, several ub deconjugases exhibit promiscuous activity against isg15 or nedd8 [30, 31] , probably due to the closely similar c-terminal tail region of these ubls. in view of their limited genome size, viruses need to co-opt the host-cell machineries for virtually all aspects of their life cycle. hence, ubl-regulated cellular functions are exploited for viral entry, transcription and replication of the viral genomes, synthesis of viral proteins, and assembly of new virions and for the maturation and exit of viral particles from the infected cell [32] [33] [34] [35] [36] . in addition, ubls play key roles in the regulation of the innate and adaptive immune defense that counteract infection. a large number of ubl ligases and deconjugases participate in different aspects of the antiviral immune response, ranging from the signaling of viral nucleic acid sensors in innate immunity and inflammation to the maturation of antigen presenting cells and the activation of antigen-specific t-cell responses [37, 38] . the pleiotropic role of ubls in orchestrating the antiviral defense is well illustrated by their contribution to the activation and fine-tuning of the early response to infection [39] (figure 3 ). the recognition of incoming viral genomes by dna or rna sensors located in endosomes (toll-like receptors, tlrs) or in the cytosol of the infected cells (including the retinoic acid inducible gene, rig-i-like receptors, rlrs, and cytoplasmic dna sensors such as cyclic gmp-amp synthase, cgas) leads via a cascade of signal transduction events to the activation of executor transcription factors that drive the synthesis of antiviral molecules such as type i and ii interferons (ifns) and pro-inflammatory cytokines [40, 41] . in turn, binding of ifns to their specific receptors activates a new signaling cascade that leads to transcription of ifn-sensitive genes for which the products mediate establishment of an antiviral state [42] . e3 ligases regulate these signaling pathways via the attachment of different types of polyubiquitin chains and ubl polypeptides [43, 44] . for example, the viral nucleic acid sensor rig-i is activated by k63-polyubiquitination mediated by the ligases trim4 [45] , riplet [46] , and trim25 [47] , while signaling is terminated by k48-polyubiquitination mediated by the ring finger protein 125 (rnf125) ligase [48] , which promotes proteasomal degradation. the attachment of m1-or k63-polyubiquitin chains to signaling mediators such as irak1 (interleukin 1 associated kinase-1) [49, 50] , traf6 [51] , rip1 (receptor interacting protein-1) [52] , traf3 (tnf receptor associated factor-3) [53] , mavs (mitochondrial antiviral signaling protein) [54] , nemo (nf-κb essential modulator) [55] , and sting (stimulator of ifn genes) [56] promotes activation of the kinases ikk (iκb kinase), tak1 (transforming growth factor beta activated kinase-1), and tbk1 (tank binding kinase-1) [57, 58] that phosphorylate the executor transcription factors nf-κb (nuclear factor-κb, irf3 (interferon regulatory factor-3), and irf7, leading to their activation and nuclear translocation. phosphorylation may also serve as a signal for ubiquitination as illustrated by the phosphorylation-dependent k48-polyubiquitination of iκbα by the βtrcp e3 ligase, which leads to degradation of the inhibitor and activation of nf-κb [59] . other types of ubl modifications exert biomolecules 2020, 10, 1137 5 of 24 similar regulatory functions. thus, isgylation targets rig-i for degradation by autophagy, reducing the levels of both basal and virus-induced ifn promoter activity [60] , while isgylation of irf3 by the herc5 (hect and rld domain containing-5) ligase was shown to promote sustained signaling by protecting irf3 for ubiquitination and proteasomal degradation [61] . fat10 was shown to form an inhibitory complex with rig-i, leading to the formation of insoluble rig-i aggregates, which prevented the translocation of rig-i to mavs and halted signaling [62] . neddylation of myd88 was shown to negatively regulate nf-κb signaling by antagonizing its ubiquitination [63] , while sumoylation of rig-i, irf3, and irf7 was shown to affect both their stability and signaling properties [64] . biomolecules 2020, 10, 1137 5 of 24 containing-5) ligase was shown to promote sustained signaling by protecting irf3 for ubiquitination and proteasomal degradation [61] . fat10 was shown to form an inhibitory complex with rig-i, leading to the formation of insoluble rig-i aggregates, which prevented the translocation of rig-i to mavs and halted signaling [62] . neddylation of myd88 was shown to negatively regulate nf-κb signaling by antagonizing its ubiquitination [63] , while sumoylation of rig-i, irf3, and irf7 was shown to affect both their stability and signaling properties [64] . the production of type i ifn leads to transcriptional activation of numerous ifn stimulated genes (isgs) whose products cooperate in the establishment of an antiviral state in both the infected and adjacent cells [65] . isg15 and its conjugation enzymes are strongly upregulated by ifn, and hundreds of putative targets of isgylation have been identified by mass spectrometry analysis although only a few have been experimentally validated [66] . the conjugation of isg15 to both viral and cellular proteins was shown to impair virus replication and spread [67, 68] . thus, the isgylation of de novo synthesized viral proteins may hinder their interaction with host proteins that are required for replication, may disrupt their catalytic function, or may alter the oligomerization of capsid proteins leading to a decrease in the number and infectivity of virus particles [69] [70] [71] . in addition, isgylation inhibits the function of cellular proteins that regulate vesicular trafficking and are required for virus budding and release, including components of the endosomal sorting complex required for transport (escrt) [72, 73] . recent studies suggest that isg15 may participate in the regulation of herpesvirus latency. numerous isgs were strongly upregulated in primary human oral fibroblasts latently infected with kaposi's sarcoma-associated herpesvirus (kshv) and in kshvpositive primary effusion lymphoma cells, while knockdown of isg15 or the isg15 ligase herc5 induced virus reactivation and the release of infectious virus [74, 75] . it should be noted that conjugation-independent functions of isg15 may also contribute to the control of infection. high serum levels of unconjugated isg15 have been detected in patients treated with interferon and in mice infected with different viruses [76] . furthermore, extracellular isg15 was shown to function as a cytokine with immune modulatory activity [77] and as a chemotactic factor for neutrophils [78] , pointing to a possible function of soluble isg15 in the modulation of inflammatory responses. the production of type i ifn leads to transcriptional activation of numerous ifn stimulated genes (isgs) whose products cooperate in the establishment of an antiviral state in both the infected and adjacent cells [65] . isg15 and its conjugation enzymes are strongly upregulated by ifn, and hundreds of putative targets of isgylation have been identified by mass spectrometry analysis although only a few have been experimentally validated [66] . the conjugation of isg15 to both viral and cellular proteins was shown to impair virus replication and spread [67, 68] . thus, the isgylation of de novo synthesized viral proteins may hinder their interaction with host proteins that are required for replication, may disrupt their catalytic function, or may alter the oligomerization of capsid proteins leading to a decrease in the number and infectivity of virus particles [69] [70] [71] . in addition, isgylation inhibits the function of cellular proteins that regulate vesicular trafficking and are required for virus budding and release, including components of the endosomal sorting complex required for transport (escrt) [72, 73] . recent studies suggest that isg15 may participate in the regulation of herpesvirus latency. numerous isgs were strongly upregulated in primary human oral fibroblasts latently infected with kaposi's sarcoma-associated herpesvirus (kshv) and in kshv-positive primary effusion lymphoma cells, while knockdown of isg15 or the isg15 ligase herc5 induced virus reactivation and the release of infectious virus [74, 75] . it should be noted that conjugation-independent functions of isg15 may also contribute to the control of infection. high serum levels of unconjugated isg15 have been detected in patients treated with interferon and in mice infected with different viruses [76] . furthermore, extracellular isg15 was shown to function as a cytokine with immune modulatory activity [77] and as a chemotactic factor for neutrophils [78] , pointing to a possible function of soluble isg15 in the modulation of inflammatory responses. interfering with ubl-dependent processes through deconjugation is a powerful strategy used by viruses to regulate many cellular functions that contribute to or counteract infection. common means of regulation involve altering the expression of host deconjugases or redirecting the activity of the cellular enzymes towards new cellular or viral substrates [79, 80] . in addition, many viruses encode their own deconjugases and increasing evidence supports the involvement of these viral enzymes in the control of infection [81] [82] [83] . significant effort has been devoted to uncovering the substrates and cellular functions targeted by viral deconjugases. however, caution should be used in the interpretation of the increasing body of data since many experiments have relied on the overexpression of recombinant viral proteins or isolated enzymatic domains that, outside of the physiological context of infection, often exhibit very potent and broad deconjugase activity. indeed, temporal and spatial constraints operating in the infected cells are likely to determine the accessibility of a given substrate, while other viral factors expressed during infection may influence substrate specificity. further complications arise when the viral enzyme exerts both deconjugase and protease activity, as observed for the enzymes encoded by rna viruses. while some of these caveats can be addressed by sophisticated technologies, including structure determination and powerful mass spectrometry, the use of recombinant viruses expressing catalytically dead mutants of the enzymes has in several cases provided conclusive evidence on the cellular functions targeted during infection and reliable information on the putative substrates. both dna and rna viruses were shown to encode proteins with ubl deconjugase activity (table 1) , and bioinformatics analysis coupled with in vitro enzymatic assays suggest that the largest viruses may even contain more than one deconjugase, as exemplified by the identification of three bona-fide dubs in the genome of epstein-barr virus (ebv) [84, 85] . sequence-and structure-based comparisons with eukaryotic ubl-specific protease families have provided interesting clues on the origin and biology of the viral enzymes. in contrast to the eukaryotic enzymes, the deconjugases encoded by viruses usually target more than one ubl. for example, the adenovirus encoded adenain cleaves ub and isg15 conjugates but shares structural similarities with the ubiquitin c-terminal hydrolase uchl3 and with ulp/senp-like proteases that cleave sumo and nedd8 [86] ; the papain-like proteases encoded by coronaviruses (cov) that are structurally related to mammalian dubs such as usp7 and usp14 show specificity for ub and isg15 and possibly nedd8 [110] [111] [112] . ub and isg15 conjugates are also recognized by the otu-like proteases encoded by several animal rna viruses although this double specificity is not observed in their mammalian counterparts [113] . while the structural similarities point to common ancestry, adaptation mechanisms operating in the context of infection may have selected for variants with broader specificity, which could counterbalance the limited coding capacity of the viral genomes. based on tertiary fold and architecture of the catalytic triad, the deconjugases encoded by herpesviruses constitute a unique family of enzymes that belong to the papain protease superfamily but are only distantly related to known cellular dubs [114] . these enzymes cleave with comparable efficiency ub and nedd8 conjugates but fail to recognize isg15 [85, 90] , pointing to a distinct set of substrates and targeted cellular functions. a common feature of the viral deconjugases is the embedding of catalytic domains in large multidomain proteins that play pleiotropic roles in infection. for example, the cov-encoded deconjugases are contained within a relatively well-conserved approximately 20 kd region of the membrane anchored nonstructural protein-3 (nsp3) [115] . the multidomain nsp3 is the largest protein encoded by the cov genome, with an average molecular mass of about 200 kd and an essential component of the replication/transcription complex. although the domain organization differs between cov genera due to duplication or absence of some domains, eight domains are found in all known nsp3. these include, in addition to the deconjugase domain, two ubiquitin-like domains, a catalytically active adp-ribose-1-phosphatase domain that may play a role during synthesis of viral sub-genomic rnas, a nucleic acid binding domain with chaperone function, and other less characterized domains including an er luminal zn finger domain [116] . via interaction with other nsps, nsp3 scaffolds the assembly of the replicase complex that utilizes er membranes to organize a microenvironment where the genome replication and transcription machinery is localized. in a similar fashion, the deconjugases encoded by different herpesviruses are located in an approximately 25 kd n-terminal domain of the 250-350 kd large tegument proteins [90] . the function of the large tegument proteins is only partially understood. studies on the herpes simplex virus (hsv) encoded member of the family, ul36/vp1-2, show that, via binding to the viral capsid protein ul25 [117] and inner tegument protein ul37 [118] , ul36 promotes the transport of a viral dna loaded capsid along microtubules to the sites of secondary envelopment in the trans-golgi network [119] . deletion of ul36 results in failure to fully assemble infectious virus particles [120] . this role in virus assembly is shared by all members of this protein family [101, 121] and is independent on the deconjugase function [118] . in addition, upon de novo infection, ul36 guides the transport of incoming semi-uncoated capsids to the nuclear pore, where the viral genome is discharged into the nucleoplasm for viral transcription and replication [122] . interestingly, cleavage of the n-terminal domain of ul36 that contains the deconjugase activity is required for the release of viral dna into the nucleus [123] . a feature that distinguishes viral enzymes from their eukaryotic counterparts is the double function as ubl deconjugases and endopeptidases that play key roles in the virus cycle by processing viral proteins. the adenovirus protease (avp), adenain, is incorporated into immature virus particles, where it becomes activated by forming a thiol bond with an eleven-residue cleavage product of the capsid protein pvi (pvic) [124] . the activated enzyme cleaves several viral capsid precursor proteins via recognition of the (m/i/l)xgx-g and (m/i/l)xgg-x sequence motifs [87, 109] ; proteolytic maturation promotes the assembly of entry-competent viruses and primes the incoming virus particles for uncoating, which is essential for infectivity [125] . interestingly, binding to the activating peptide induces preferential cleavage at the gx-g site that is overrepresented in the viral proteins [126] , suggesting that the substrate repertoire of adenain may change during different phases of infection depending on availability of the activating peptide. in a similar fashion, the cov papain-like protease (plpro) contained in nsp3 cooperates with the major chymotrypsin-like protease (3clpro) in processing of the viral polyprotein to give rise to the sixteen nsps that form the viral replicase complex [127] . plpro recognizes the sequence lxgg at the nsp1/2, nsp2/3, and nsp3/4 boundaries that is identical to the c-terminal sequence of ub, isg15, and nedd8 [110, 128] . processing of the polyprotein by plpro is required for virus replication, which highlights the essential role of the enzyme in the virus life cycle. analysis of the crystal structure of plpro bound to ub-aldehyde and models of the interaction with ub-chains and isg15 revealed a likely mechanism for discrimination between the ubl-conjugates and viral substrates. the recognition of poly-ub chains and isg15 was shown to be dependent on simultaneous engagement of two binding domains, s1 and s2, on the surface of plpro [112] . mutation of the distal s2 domain significantly impaired the processing of isg15 and poly-ub conjugates but did not affect the activity of the protease against the viral polyprotein [129] . given the importance of coronaviruses, adenoviruses, and herpesviruses for human diseases, the next sections will be focused on the deconjugases encoded by these viruses, with particular emphasis on the affected cellular functions and validated substrates. comprehensive reviews on the ubl deconjugases of animal viruses, including the very interesting family of out-domain containing proteases encoded by nairoviruses, were recently published [82] . coronaviruses are enveloped viruses with positive-sense, single-stranded rna (ssrna) genomes [130] . the first human cov was identified in the 1960s and was recognized as a causative agent of the common cold [131] . since then, highly pathogenic covs causing severe acute respiratory syndrome (sars-cov) [132, 133] , middle east respiratory syndrome (mers-cov) [134] , and the ongoing cov-disease-19 pandemics (sars-cov-2) [135] have emerged in humans via zoonotic transmission. the three highly pathogenic human viruses cause similar forms of atypical pneumonia with bilateral parenchymal "ground-glass" consolidative lesions that may progress to acute respiratory distress syndrome (ards) [136] . the frequency of such complication varies, being highest for mers-cov and lowest for sars-cov-2, suggesting important differences in disease pathogenesis. the severe cases show progressive lymphopenia with loss of both cd4+ and cd8+ t lymphocytes; massive infiltration of the alveolar walls by neutrophil and eosinophil granulocytes; and significantly elevated levels of il-6, il-10, il-2, and ifn-γ, pointing to a direct correlation between the magnitude of the inflammatory response and the severity of the disease [137] . importantly, all three viruses induce very little, if any, type i ifn [138] [139] [140] . work in a mouse model of sars suggests that the impaired ifn production is responsible for the recruitment of monocyte-macrophages and production of proinflammatory cytokines in the lung, resulting in vascular leakage and impairment of the immune response [141] . based on available crystal structures, the plpro encoded by sars-cov, mers-cov, and sars-cov-2 share a similar domain organization with "thumb", "palm", and "fingers" subdomains arranged together to resemble an extended right hand [107] [108] [109] 112, 142] . the cys-his-asp catalytic triad is located at the interface of the thumb and palm domain, with a topology similar to that found in papain. in addition to the core catalytic domain, the enzymes contain an n-terminal ub-fold domain that is not required for catalysis but may play a role in the immunomodulatory activity of plpro [143] . the three enzymes cleave ub-and isg15-reporter substrates and synthetic poly-ub chains, and both sars-cov and sars-cov-2 exhibit weak deneddylase activity [108] . differences in specificity and efficiency of catalysis are likely explained by subtle changes in the surface binding pockets that mediate interaction with the substrates. as a general rule, dubs that disassemble ub chains have surface pockets that bind the ub moiety preceding (s1) and following (s1 ) the scissile bond, whereas dubs that recognize mono-or-polyubiquitinated substrates lack the s1 pocket but contain one (s2) or more (s3, s4, and s5) additional pockets that can accommodate distal ub moieties. based on structure and mutation analysis, the preferential recognition of k48poly-ub by sars-cov plpro relies on interaction with both s1 and s2 binding pockets, where solvent-exposed hydrophobic residues within the thumb domain interact with the ile44 patch of the distal ub [104] . the s2 site is also involved in the recognition of the distal ub-fold domain of isg15 [144] , although interaction with the proximal s1 pocket was shown to play a dominant role [104] . important differences in the architecture of both the catalytic core and binding pockets of mers-cov plpro correlate with overall decreased catalytic activity and capacity to recognize all types of poly-ub chain linkages [142] . in line with the high degree of homology (83% identity), the plpro encoded by sars-cov-2 resembles the sars-cov enzyme in the capacity to target both k48poly-ub and isg15 conjugates. however, changes in the palm domain were shown to improve the interaction with isg15 [107] , while mutation of a key leu residue that engages ub in the s2 pocket of sars-cov to thr diminishes the ability to process k48poly-ub chains [107] [108] [109] , which results in the preference of sars-cov-2 for isg15 conjugates. the deconjugase activity of cov plpro has been implicated in the downregulation of innate immune responses [145, 146] . type i ifns and pro-inflammatory cytokines are hardly expressed or appear late in cell-culture based models of cov infection [147] , and dysregulation of the ifn response is associated with severe lung immunopathology, influx of inflammatory monocyte-macrophages, and elevated levels of cytokines and chemokines in mouse models of sars-cov [141] and mers-cov [148] infection. a similar imbalance of the host response was recently observed in sars-cov-2-infected patients [140] . two lines of evidence support a key role of the viral deconjugase in these effects. first, ectopic expression of both sars-cov [145, 149] and mers-cov [145, 149] and more recently sars-cov-2 plpro [108] was shown to inhibit innate immune signaling pathways. second, direct evidence for the contribution of the deconjugase activity to immune evasion was obtained using recombinant mers-cov viruses encoding for plpro mutants with selective loss of the deconjugase activity but preserved polyprotein cleavage. the transcription of type i ifn and ifn-stimulated genes was markedly increased in cells infected with the mutant viruses, and infected mice showed significantly increased survival rates and faster virus clearance in spite of comparable virus replication rates in the lungs [150] . although the capacity of plpro to regulate innate immune responses appears to be firmly established, the mechanism and cellular substrates involved in this effect are not well understood. the inhibition of the type i ifn by sars-cov plpro was shown to correlate with impaired phosphorylation and nuclear translocation of irf3, suggesting that signaling through tlr3 or rig-i may be affected [145] . surprisingly, while plpro interacted with irf3 both in transfected cells and in cells infected with sars-cov, this was dependent on the presence of the nsp3 transmembrane domain (plpro-tm) and neither binding nor inhibition of irf3 phosphorylation were affected by mutation of the catalytic cys to ala. a possible explanation may be found in the demonstration that plpro-tm interacts with the sting-traf3-tbk1 complex via binding to the sting transmembrane domain, which promotes disruption of the complex and is associated with reduced ubiquitination of rig-i, traf-3, sting, tbk1, and irf3 [105] . in addition, expression of a plpro construct lacking the tm domain but containing the n-terminal ubiquitin-fold domain was shown to block nf-κb signaling by stabilizing phosphorylated iκbα [116] . stabilization of iκbα may be due to deubiquitination and the consequent inhibition of proteasomal degradation, but this was not formally proven. a recent study comparing the plpro of sars-cov and sars-cov-2 suggests that their different activity towards k48poly-ub and isg15 parallels the extent of inhibition of nf-kb versus ifn signaling, with the dominant de-isgylase activity of sars-cov-2 plpro being associated with a stronger decrease of isgylated irf3 and stronger inhibition of the ifn response [108] . however, the multiple roles of isgylation in the inhibition or enhancement of the ifn response via targeting of rig-i [151] or rnf3 [151] call for some caution in assessing the significance of this finding in the context of infection. it is noteworthy that silencing of isg15 did not affect or even enhance the type i ifn response in mice infected with different rna viruses [152, 153] . it is also important to remember that isg15 is itself an ifn target gene and that secreted isg15 regulates the activity of various types of immune cells, including natural killer (nk) cells, dendritic cells (dc), and neutrophils [76, 78] . thus, much remains to be done to achieve a precise molecular understanding of the mechanisms by which the plpro encoded by different coronaviruses modulate innate immunity and how this reflects in the severity of the disease. adenoviruses are double-stranded dna, non-enveloped viruses with a ≈35 kd genome that codes for at least 50 different proteins expressed during the early and late phases of infection. more than forty adenovirus serotypes infect humans, causing a range of pathologies including respiratory, ocular, and gastrointestinal infections [154] . the adenovirus protease (avp) adenain is a 25 kd protein encoded by the conserved late gene l3 [124] . the protease activity plays an essential role in the maturation of virion-associated precursor proteins, which is required for assembly of infectious virus particles and is also involved in the uncoating of incoming virions during primary infection [155] . adenain exhibits a papain-like fold and is structurally related to the ub-specific protease uch-l3 and sumo deconjugase ulp1, with topology of the active site cys and his residues resembling that of ulp1 and organization of s1-s4 substrate binding pocket similar to that of ubiquitin hydrolases [86] . recombinant adenain was shown to cleave k48tetra-ub and isg15 precursor peptides in vitro, and expression of the active enzymes correlated with a global decrease of poly-ubiquitinated proteins in adenovirus-infected cells. however, the cellular substrates of these activities have not been characterized. interestingly, overexpression was associated with decreased levels of mono-ubiquitinated histone h2a in transfected hela cells, suggesting a possible role of the protease in chromatin-related events or in the regulation of the dna damage response. herpesviruses are large dna viruses with double-stranded dna genomes ranging from 124 to 295 kb. a characteristic property of herpesviruses is their capacity to establish latent infections in certain cell types, which allows life-long persistence in the infected hosts [156] . seven herpesviruses are important human pathogens. herpes simplex virus-1 and 2 (hsv-1 and -2; hhv-1 and -2) and varicella zoster virus (vzv; hhv-3) infect epithelial cells, causing cold sores or shingles, and establish latency in sensory neurons [157, 158] . human cytomegalovirus (hcmv; hhv-5) and human herpesvirus-6 and -7 (hhv-6 and -7) infect myelomonocytic and lymphoid cells and cause mononucleosis-like syndromes in immunosuppressed patients and roseola in children [159, 160] ; epstein-barr virus (ebv; hhv-4) and kaposi sarcoma herpesvirus (kshv; hhv-8) establish latency in b-lymphocytes and are associated with the pathogenesis of lymphoid, endothelial, and epithelial cell malignancies [161, 162] . the establishment of latency poses a particular challenge to these viruses since it entails adaptation to different cellular environments and the consequent establishment of cell type-dependent programs of viral gene expression. in addition, transmission to new hosts is dependent on the reactivation of virus production in the face of specific and highly effective immune responses, which requires sophisticated immune evasion strategies [163] . the development of activity-based ubiquitin probes capable of forming covalent adducts with the catalytic cys of dubs [164] was instrumental for the discovery of deconjugase activity in the n-terminal fragment of the hsv-1 large tegument protein ul36 [90] . the activity is conserved in all ul36 homologs encoded by human and animal herpesviruses investigated to date [87, [128] [129] [130] [131] [132] , supporting the notion that the enzymes play important roles in the biology of these viruses. in spite of very low amino acid sequence similarity, sequence alignment identified relatively well-conserved cys and his boxes, and crystal structure of the homolog encoded by the mouse cytomegalovirus (mcmv) m48 revealed a unique organization of the catalytic core, suggesting that the viral enzymes may represent a new family of deconjugases [114] . the in vitro cleavage of fluorogenic substrates and in vivo assays in cells transfected with tagged ubls showed that the enzymes recognize comparable efficiency of k48and k63poly-ub [85] . in addition, a bacterial screen based on co-expression of the ebv encoded homolog, bplf1, with ubl-gfp (green fluorescent protein) reporters revealed specificity for both ub and nedd8 [85] .the deneddylase activity was shown to be conserved in the homologs encoded by hsv-1, hcmv, kshv, and mouse herpesvirus mhv-68 [85] , and sirna knockdown confirmed the involvement of bplf1 in the progressive decrease of neddylated substrates in ebv-positive cells entering the productive virus cycle [165] , corroborating the notion that the deneddylase operates in infected cells under physiological levels of expression. the strict host-specificity of herpesviruses hampers direct testing of the contribution of the deconjugases to viral pathogenesis in humans, but compelling evidence from cell culture and animal models of herpesvirus infection supports an important role of the enzymes in virus replication and pathogenesis. while deletion of the entire or large fragments of the large tegument proteins severely impaired the release of infectious virus [121] , as may be expected given their essential role in the architecture of the mature virions, decreased virus yields were also observed upon infection with recombinant viruses carrying mutation of the active site cys residue [93, 95, 166] . ultrastructural analysis of cells infected with a mouse pseudorabies virus (prv) carrying an inactivating mutation of the pul36 deconjugase revealed accumulation of naked nucleocapsids in the cytoplasm [166] , suggesting that enzymatic activity is required for virus assembly and egress. the contribution of the deconjugase to viral pathogenesis in vivo is clearly illustrated by the strongly reduced formation of t cell lymphomas in chicken infected with mutant marek's disease virus (mdv) [167] and decreased neuro-invasion and longer survival of mice infected with mutant prv [166] . interestingly, in line with the known role of the large tegument protein in the early phases of herpesvirus infection, abrogation of the deconjugase activity was shown to cause the accumulation of incoming viral genomes in the cytoplasm of mhv-68-infected cells, which correlated with strongly enhanced activation of the type i ifn response and hampered the establishment of latent infection [168] . the capacity to interfere with the ifn response is conserved in the deconjugase encoded by human herpesviruses, as confirmed by comparing type i ifn production in cells infected with wild type and mutant viruses [89, 96, 102, 169] . collectively, the findings illustrate a pleiotropic role of the herpesvirus deconjugases in the regulation of multiple steps of the virus life cycle from virus entry, uncoating, and viral genome replication to the assembly and release of infectious virus particles. in addition, by halting the innate immune response, the deconjugase may promote establishment of a cellular and host environment conducive to latency and permissive for virus reactivation. in line with the broad effect of the herpesvirus deconjugases on different cellular functions, several putative substrates have been identified, often based on candidate approaches where the capacity of the isolated enzymatic domains to deconjugate known ubl substrates was tested in co-transfection assays. while the very potent and broad deconjugase activity of the overexpressed enzymes calls for some caution in the interpretation of the data, at least some of the candidate substrates could be validated by mapping the sites of interaction and by comparing their fate in cells infected with wild type and mutant viruses. cullins are the main cellular targets of neddylation and an obvious candidate substrate for the deneddylase activity of the ebv-encoded bplf1 in productively infected cells [165] . bplf1 was shown to interact with a conserved region in the c-terminal domain of the cullins scaffolds, close to the site of neddylation, and to promote cullin deneddylation and their degradation by the proteasome [85, 170] . the phenotype of cells expressing catalytically active bplf1 is similar to that induced by chemical blockade of the neddylation cascade, with accumulation of several substrates of nuclear cullin ligases and arrest in the s/g2 phase of the cell cycle [171] , pointing to a role of the deconjugase in the induction of a pseudo s-phase environment that is required for efficient replication of the herpesvirus genomes [172] . in line with this possibility, viral dna replication was strongly decreased upon sirna-mediated knockdown of bplf1 in cells entering the productive virus cycles, which correlated with failure to accumulate several substrates of cullin ligases, including the cellular dna polymerase licensing factor cdt1 (chromatin licensing and dna replication factor-1) [85] . viral dna replication was restored following reconstitution of cdt1 expression in bplf1 knockdown cell, supporting the involvement of the cellular licensing factors in viral dna replication. the dub activity of the viral enzymes is likely to synergize with the deneddylase activity in promoting the efficiency of virus replication. reactivation of the productive virus cycle triggers the dna damage response [173] . in response to dna damage, pcna (proliferating cell nuclear antigen) is monoubiquitinated by rad18 (ring type ubiquitin ligase-18) to activate the translesion synthesis pathway of post-replication repair. pcna accumulates at the replication sites of many dna viruses, although its function in viral replication is not fully understood. the ebv-encoded bplf1 [97] and hsv-1-encoded ul36 [92] were shown to de-ubiquitinate pcna and to prevent the formation of pcna foci. in addition, bplf1 was shown to interact with and to promote the accumulation of the pcna ligase rad18 [100] and translesion synthesis polymerase polη [174] . taken together, these findings point to an important role of the viral deconjugase in regulating the stability, localization and activity of a variety of cellular factors that are recruited at the site of viral replication to assist or counteract the production of infectious virus. several members of the type i ifn and nf-κb signaling pathways have been proposed as putative targets of the inhibitory effect of the herpesvirus deconjugases on the innate immune responses. in different experimental setups, expression of the catalytically active enzymes was accompanied by impaired ubiquitination of rig-i [102, 169] traf6 [98, 175, 176] , traf3 [89, 176] , irak1 [176] , irf7 [176] , sting [176, 177] and iκbα [88] . however, since the activity of these signaling molecules is interconnected via different ubiquitination and deubiquitination events, the identity of the viral substrates and the molecular interactions leading to failure to activate the key executor transcription factors remain in many cases unknown. in addition, since the poor amino acid sequence conservation of the n-terminal domains is likely to influence the binding properties of the viral enzymes, it is unclear whether the same or different ubiquitination events are targeted by the various member of the family. an alternative unbiased approach to the identification of putative substrates and targeted signaling pathways relies on the identification of binding partners by co-immunoprecipitation and mass spectrometry. while the huge size renders this approach technically challenging for the entire large tegument proteins, the physiological relevance of the much shorter n-terminal domain is supported by the finding that processing by caspase-1 [165] , or by a yet unidentified protease [123] , releases the catalytic domain of bplf1 and ul36 to promote localization of the enzymatic activity to the nucleus. gene ontology analysis of protein interacting with the catalytic domain of bplf1 showed enrichment in proteins involved in numerous cellular functions including rna transcription and metabolism, nuclear transport, intracellular trafficking, cell cycle, and apoptosis, with major interacting hubs centering around proteasome subunits, nuclear transport proteins, and several members of the 14-3-3 family of adaptor proteins [169] . the 14-3-3 proteins are conserved molecular scaffolds expressed in all eukaryotic cells that bind as homo-or heterodimers to a multitude of functionally diverse proteins, including kinases, phosphatases, and transmembrane receptors [178, 179] . network analysis revealed that bplf1 and 14-3-3 share a high number of interacting partners, in addition to cullins, the trim25 ligase that regulates the ifn response via ubiquitination of rig-i [47] . binding of 14-3-3 was shown to stabilize the interaction of trim25 with rig-i [180] , which is essential for targeting the ubiquitination rig-i to mavs for downstream signaling [181] . the recruitment of bplf1 to the 14-3-3:trim25 complex was shown to promote activation and autoubiquitination of the ligase and was associated with failure to ubiquitinate rig-i. interestingly, while catalytically inactive bplf1 induced the k48-linked autoubiquitination and degradation of trim25, the active viral enzyme trimmed the polyubiquitin chain to mono-or di-ubiquitin and promoted the formation of trim25 cytosolic aggregates decorated by the autophagy receptor p62/sqstm1 [182] . aggregate formation and the inhibition of ifn response were abolished by mutation of solvent exposed residues in helix-2 of bplf1 that are also involved in the interaction with cullins [170, 182] , pointing to a critical role of the 14-3-3:bplf1 interaction in the assembly of the inactivating trimolecular complex. mapping of the interacting domains provided interesting insights on the possible mechanism of inhibition. the formation of 14-3-3 homo-or heterodimers builds a groove with two symmetrically oriented binding pockets [179] . in vitro binding assays using isolated trim25 domains and bacterially expressed wild type and mutant 14-3-3 and bplf1 suggest that dimeric 14-3-3 could stabilize the trimolecular complex by simultaneously accommodating in each of the two binding pockets acidic residues located in bplf1 helix-2 and on the tip of the trim25 coiled-coil domain. docking models predict that bplf1 would not prevent the recruitment of rig-i but that the presence of one or two conjugated ubiquitins may hinder correct positioning of the substrate towards the e2 and may contribute to functional inactivation of the ligase [182] . the finding that the bplf1-mediated inhibition of ifn signaling is dependent on the formation of a tri-molecular complex with 14-3-3 and trim25 points to trim25 as the primary target of the viral deconjugase. while the capacity to inhibit the ubiquitination of rig-i and other components of the signaling cascade was previously reported [89, 98, 102, 175] , the homologs encoded by hcmv and kshv shared with bplf1 the capacity to bind to 14-3-3 and trim25 and to promote the functional inactivation of trim25, suggesting that this early step of the signaling cascade is a common target of the viral enzymes. interestingly, this property was not shared by the hsv1 ul36 homolog where changes in the exposed residues of helix-2 prevent efficient binding to 14-3-3 [183] . it remains to be seen whether and how the targeting of different steps of the ifn and nf-κb signaling pathways impacts the life cycle of these viruses. given the growing body of evidence demonstrating the contribution of viral deconjugases to virus replication and pathogenesis, the enzymes are now recognized as attractive targets for the design of antiviral therapeutics. high-throughput screening of small-molecule libraries and rational structure-guided design have led to the identification and development of several lead compounds capable of inhibiting the activities of the plpro of coronaviruses [116] . among those, naphthalene inhibitors are particularly interesting due to their capacity to act as competitive, non-covalent inhibitors of sars-cov plpro via interaction with a mobile loop in the fingers domain that, upon binding of the inhibitor, closes towards the catalytic cleft and shuts down the active site [103] . the compounds were shown to inhibit the deconjugase activity against model substrates and to block ifn production and virus replication in infected cells. while structural difference in the mobile loop are likely to explain the failure of the compounds to inhibit the plpro of mers-cov [107, 184] , recent findings demonstrate potent inhibitory activity against the plpro of sars-cov-2 [108] . of note, the naphthalene compounds did not inhibit the activity of human deconjugases, caspases-3, and cathepsin-k in biochemical assays [185] , which may explain their lack of toxicity in cell cultures and makes them promising candidates for the development of specific antiviral drugs. both covalent and non-covalent inhibitors of the adenovirus protease working at nanomolar concentrations in biochemical assays have been described [186] . however, the compounds have either weak antiviral activity, suggesting poor cell permeability, or are highly cytotoxic for cultured cells. thus, additional medicinal chemistry optimization will be required to harness their therapeutic potential. the involvement of the herpesvirus deconjugases in the regulation of both virus production and antiviral responses makes them interesting candidates for the development of much needed drugs for the treatment of many diseases caused by herpesvirus infection and reactivation. unfortunately, research in this area is still scarce. recently, the antiparasitic drug suramin was identified as a possible candidate in a high-throughput screen for small molecule inhibitors of bplf1 [187] . suramin inhibited the cleavage of k63poly-ub at sub-micromolar concentrations and decreased in a dose-dependent manner the production of infectious virus without apparent cell toxicity. however, comparable levels of inhibition were observed against a panel of ten human dubs, indicating that the compound has relatively poor selectivity and may be acting via a nonspecific mechanism. as regulators of both the virus life cycle and the host innate immunity, viral ubl deconjugases serve as multifunctional swiss army knives to facilitate viral infection and pathogenesis (figure 4 ). herpesviruses provide a particularly enticing example of the involvement of ubl deconjugases in the regulation of a multitude of nuclear and cytoplasmic events that are critical for the establishment of both latent and productive infection in different cell types. since the first report on the ubiquitin deconjugase activity of adenain some twenty years ago, significant progress has been made in the identification, functional characterization, and structure determination of this fascinating class of viral enzymes. however, a precise understanding of the mechanism of action and the identification of viral and cellular substrates remain in many cases a challenge. the huge societal impact of the coronavirus pandemics is providing a strong stimulus towards the characterization of the deconjugases encoded by these viruses and the development of therapeutic inhibitors. regrettably, work on the adenovirus and herpesvirus deconjugases is lagging behind. while the similarity to cellular enzymes is likely to be a major hinder towards the development of specific inhibitors targeting the catalytic core, the mapping of binding domains involved in substrate interaction may offer new opportunities for regulating the function of the viral deconjugases. funding: the work of the author was funded by grants awarded by the swedish research council, the swedishcancer foundation, and the karolinska institutet. acknowledgments: i sincerely thank t frisan, umeå university, n dantuma, karolinska institutet and all members of the masucci group for their critical reading of the manuscript and apologize to many contributors to the field whose work was not cited due to space limitations. the author declares no conflict of interest. the funders had no role in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the manuscript. the ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction origin and function of ubiquitin-like proteins ubiquitin-like proteins the ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction origin and function of ubiquitin-like proteins ubiquitin-like proteins the ubiquitin system modification of proteins by ubiquitin and ubiquitin-like proteins the interferon-inducible ubiquitin-protein isopeptide ligase (e3) efp also functions as an isg15 e3 ligase mdm2-mediated nedd8 conjugation of p53 inhibits its transcriptional activity uhrf2, a ubiquitin e3 ligase, acts as a small ubiquitin-like modifier e3 ligase for zinc finger protein 131 topors acts as a sumo-1 e3 ligase for p53 in vitro and in vivo sumo e3 ligase activity of trim proteins traf7 sequesters c-myb to the cytoplasm by stimulating its sumoylation the emerging complexity of protein ubiquitination ubiquitin-binding domains the increasing complexity of the ubiquitin code mediated regulation of nuclear functions and signaling processes an additional role for sumo in ubiquitin-mediated proteolysis cullin-based ubiquitin ligase and its control by nedd8-conjugating system urm1 couples sulfur transfer to ubiquitin-like protein function in oxidative stress ubiquitin-related modifier urm1 acts as a sulphur carrier in thiolation of eukaryotic transfer rna the ufmylation system in proteostasis and beyond ribosomal protein rpl26 is the principal target of ufmylation regulation of interferon induction by the ubiquitin-like modifier fat10 isg15: in sickness and in health interferon-stimulated gene 15 and the protein isgylation system atg8: an autophagy-related ubiquitin-like protein family breaking the chains: structure and function of the deubiquitinases cellular functions of the dubs the differential modulation of usp activity by internal regulatory domains, interactors and eight ubiquitin chain types otu deubiquitinases reveal mechanisms of linkage specificity and enable ubiquitin chain restriction analysis screen for isg15-crossreactive deubiquitinases identification and characterization of den1, a deneddylase of the ulp family viral takeover of the host ubiquitin system viral interplay with the host sumoylation system the role of ubiquitin and ubiquitin-like modification systems in papillomavirus biology emerging roles for ubiquitin in adenovirus cell entry the ubiquitin-conjugating system: multiple roles in viral replication and infection antigen presentation and the ubiquitin-proteasome system in host-pathogen interactions ubiquitin signaling in immune responses ubiquitination in the antiviral immune response innate immune sensing and signaling of cytosolic nucleic acids pathogen recognition in the innate immune response transcriptional regulation of antiviral interferon-stimulated genes ubiquitin in the immune system ubiquitin ligases and the immune response trim4 modulates type i interferon induction and cellular antiviral response by targeting rig-i for k63-linked ubiquitination a distinct role of riplet-mediated k63-linked polyubiquitination of the rig-i repressor domain in human antiviral innate immune responses trim25 ring-finger e3 ubiquitin ligase is essential for rig-i-mediated antiviral activity negative regulation of the rig-i signaling by the ubiquitin ligase rnf125 the e3 ligase hoil-1 catalyses ester bond formation between ubiquitin and components of the myddosome in mammalian cells lys63-linked polyubiquitination of irak-1 is required for interleukin-1 receptor-and toll-like receptor-mediated nf-kappab activation ubl4a augments innate immunity by promoting the k63-linked ubiquitination of traf6 cellular iap proteins and lubac differentially regulate necrosome-associated rip1 ubiquitination trim24 facilitates antiviral immunity through mediating k63-linked traf3 ubiquitination the ubiquitin e3 ligase trim31 promotes aggregation and activation of the signaling adaptor mavs through lys63-linked polyubiquitination linear ubiquitination of nemo negatively regulates the interferon antiviral response through disruption of the mavs-traf3 complex ubiquitination of sting at lysine 224 controls irf3 activation ubiquitination in signaling to and activation of ikk ubiquitin-mediated activation of tak1 and ikk ben-neriah, y. identification of the receptor component of the ikappabalpha-ubiquitin ligase lrrc25 inhibits type i ifn signaling by targeting isg15-associated rig-i for autophagic degradation positive regulation of interferon regulatory factor 3 activation by herc5 via isg15 modification ubiquitin-like modifier fat10 attenuates rig-i mediated antiviral signaling by segregating activated rig-i from its signaling platform myd88 neddylation negatively regulates myd88-dependent nf-kappab signaling through antagonizing its ubiquitination the implication of sumo in intrinsic and innate immunity regulation of type i interferon responses proteomic identification of proteins conjugated to isg15 in mouse and human cells how isg15 combats viral infection isg15 in antiviral immunity and beyond the isg15 conjugation system broadly targets newly synthesized proteins: implications for the antiviral function of isg15 influenza b virus non-structural protein 1 counteracts isg15 antiviral activity by sequestering isgylated viral proteins inhibition of hepatitis c virus replication by ifn-mediated isgylation of hcv-ns5a type i interferon imposes a tsg101/isg15 checkpoint at the golgi for glycoprotein trafficking during influenza virus infection isg15 inhibits ebola vp40 vlp budding in an l-domain-dependent manner by blocking nedd4 ligase activity transcriptomic analysis of kshv-infected primary oral fibroblasts: the role of interferon-induced genes in the latency of oncogenic virus kaposi's sarcoma-associated herpesvirus viral interferon regulatory factor 1 interacts with a member of the interferon-stimulated gene 15 pathway in vitro and in vivo secretion of human isg15, an ifn-induced immunomodulatory cytokine immunoregulatory properties of isg15, an interferon-induced cytokine identification of a ubiquitin family protein as a novel neutrophil chemotactic factor influenza a virus ns1 protein induced a20 contributes to viral replication by suppressing interferon-induced antiviral response human papillomavirus (hpv) upregulates the cellular deubiquitinase uchl1 to suppress the keratinocyte's innate immune response viral deubiquitinases: role in evasion of anti-viral innate immunity structure and function of viral deubiquitinating enzymes manipulation of viral infection by deubiquitinating enzymes: new players in host-virus interactions epstein-barr virus encodes three bona fide ubiquitin-specific proteases a deneddylase encoded by epstein-barr virus promotes viral dna replication by regulating the activity of cullin-ring ligases deubiquitinating function of adenovirus proteinase adenovirus endopeptidases herpes simplex virus 1 ubiquitin-specific protease ul36 abrogates nf-kappab activation in dna sensing signal pathway herpes simplex virus 1 ubiquitin-specific protease ul36 inhibits beta interferon production by deubiquitinating traf3 a deubiquitinating enzyme encoded by hsv-1 belongs to a family of cysteine proteases that is conserved across the family herpesviridae autocatalytic activity of the ubiquitin-specific protease domain of herpes simplex virus 1 vp1-2 the herpes simplex virus 1 ul36usp deubiquitinase suppresses dna repair in host cells via deubiquitination of proliferating cell nuclear antigen cleavage specificity of the ul48 deubiquitinating protease activity of human cytomegalovirus and the growth of an active-site mutant virus in cultured cells involvement of the n-terminal deubiquitinating protease domain of human cytomegalovirus ul48 tegument protein in autoubiquitination, virion stability, and virus entry high-molecular-weight protein (pul48) of human cytomegalovirus is a competent deubiquitinating protease: mutant viruses altered in its active-site cysteine or histidine are viable cooperative inhibition of rip1-mediated nf-kappab signaling by cytomegalovirus-encoded deubiquitinase and inactive homolog of cellular ribonucleotide reductase large subunit epstein-barr virus bplf1 deubiquitinates pcna and attenuates polymerase eta recruitment to dna damage sites epstein-barr virus large tegument protein bplf1 contributes to innate immune evasion through interference with toll-like receptor signaling the epstein-barr virus (ebv) deubiquitinating enzyme bplf1 reduces ebv ribonucleotide reductase activity the rad6/18 ubiquitin complex interacts with the epstein-barr virus deubiquitinating enzyme, bplf1, and contributes to virus infectivity kaposi's sarcoma-associated herpesvirus encodes a viral deubiquitinase inhibition of rig-i-mediated signaling by kaposi's sarcoma-associated herpesvirus-encoded deubiquitinase orf64 a noncovalent class of papain-like protease/deubiquitinase inhibitors blocks sars virus replication recognition of lys48-linked di-ubiquitin and deubiquitinating activities of the sars coronavirus papain-like protease sars coronavirus papain-like protease inhibits the type i interferon signaling pathway through interaction with the sting-traf3-tbk1 complex mers-cov papain-like protease has deisgylating and deubiquitinating activities mechanisms of inhibition of sars-cov-2 plpro inhibition of papain-like protease plpro blocks sars-cov-2 spread and promotes anti-viral immunity activity profiling and structures of inhibitor-bound sars-cov-2-plpro protease provides a framework for anti-covid-19 drug design the papain-like protease from the severe acute respiratory syndrome coronavirus is a deubiquitinating enzyme biomolecules 2020 deubiquitination, a new function of the severe acute respiratory syndrome coronavirus papain-like protease? severe acute respiratory syndrome coronavirus papain-like protease: structure of a viral deubiquitinating enzyme ovarian tumor domain-containing viral proteases evade ubiquitin-and isg15-dependent innate immune responses structure of a herpesvirus-encoded cysteine protease reveals a unique class of deubiquitinating enzymes nsp3 of coronaviruses: structures and functions of a large multi-domain protein the sars-coronavirus papain-like protease: structure, function and inhibition by designed antiviral compounds the c terminus of the large tegument protein pul36 contains multiple capsid binding sites that function differently during assembly and cell entry of herpes simplex virus cytosolic herpes simplex virus capsids not only require binding inner tegument protein pul36 but also pul37 for active transport prior to secondary envelopment herpes simplex virus type 1 capsids transit by the trans-golgi network, where viral glycoproteins accumulate independently of capsid egress a null mutation in the ul36 gene of herpes simplex virus type 1 results in accumulation of unenveloped dna-filled capsids in the cytoplasm of infected cells knockout of epstein-barr virus bplf1 retards b-cell transformation and lymphoma formation in humanized mice herpes simplex virus replication: roles of viral proteins and nucleoporins in capsid-nucleus attachment proteolytic cleavage of vp1-2 is required for release of herpes simplex virus 1 dna into the nucleus adenain, the adenovirus endoprotease (a review) the role of the adenovirus protease on virus entry into cells cleavage efficiency by adenovirus protease is site-dependent mechanisms and enzymes involved in sars coronavirus genome expression the papain-like protease of severe acute respiratory syndrome coronavirus has deubiquitinating activity structural basis for the ubiquitin-linkage specificity and deisgylating activity of sars-cov papain-like protease the viruses and their replication a new virus isolated from the human respiratory tract identification of a novel coronavirus in patients with severe acute respiratory syndrome a novel coronavirus associated with severe acute respiratory syndrome isolation of a novel coronavirus from a man with pneumonia in saudi arabia a novel coronavirus from patients with pneumonia in china acute respiratory distress syndrome: advances in diagnosis and treatment altered cytokine levels and immune responses in patients with sars-cov-2 infection and related conditions severe acute respiratory syndrome coronavirus fails to activate cytokine-mediated innate immune responses in cultured human monocyte-derived dendritic cells ifn-i response timing relative to virus replication determines mers coronavirus infection outcomes imbalanced host response to sars-cov-2 drives development of covid-19 dysregulated type i interferon and inflammatory monocyte-macrophage responses cause lethal pneumonia in sars-cov-infected mice crystal structure of the papain-like protease of mers coronavirus reveals unusual, potentially druggable active-site features severe acute respiratory syndrome coronavirus papain-like protease ubiquitin-like domain and catalytic domain regulate antagonism of irf3 and nf-kappab signaling selectivity in isg15 and ubiquitin recognition by the sars coronavirus papain-like protease regulation of irf-3-dependent innate immunity by the papain-like protease domain of the severe acute respiratory syndrome coronavirus crystal structure of the middle east respiratory syndrome coronavirus (mers-cov) papain-like protease bound to ubiquitin facilitates targeted disruption of deubiquitinating activity to demonstrate its role in innate immune suppression sars coronavirus and innate immunity mouse-adapted mers coronavirus causes lethal lung disease in human dpp4 knockin mice proteolytic processing, deubiquitinase and interferon antagonist activities of middle east respiratory syndrome coronavirus papain-like protease the deubiquitinating activity of middle east respiratory syndrome coronavirus papain-like protease delays the innate immune response and enhances virulence in a mouse model negative feedback regulation of rig-i-mediated antiviral signaling by interferon-induced isg15 conjugation isg15 modulates type i interferon signaling and the antiviral response during hepatitis e virus replication isg15, an interferon-stimulated ubiquitin-like protein, is not essential for stat1 signaling and responses against vesicular stomatitis and lymphocytic choriomeningitis virus genome analysis of adenovirus type 31 strains from immunocompromised and immunocompetent patients structure, function and dynamics in adenovirus maturation herpesvirus latency herpes simplex: insights on pathogenesis and possible vaccines pathogenesis and current approaches to control of varicella-zoster virus infections the pathogenesis of human cytomegalovirus pathogenesis of human herpesvirus 6 (hhv-6) ebv-induced oncogenesis kshv-induced oncogenesis herpesvirus exploitation of host immune inhibitory pathways chemistry-based functional proteomics reveals novel members of the deubiquitinating enzyme family caspase-1 promotes epstein-barr virus replication by targeting the large tegument protein deneddylase to the nucleus of productively infected cells mutagenesis of the active-site cysteine in the ubiquitin-specific protease contained in large tegument protein pul36 of pseudorabies virus impairs viral replication in vitro and neuroinvasion in vivo a herpesvirus ubiquitin-specific protease is critical for efficient t cell lymphoma formation evasion of innate cytosolic dna sensing by a gammaherpesvirus facilitates establishment of latent infection herpesvirus deconjugases inhibit the ifn response by promoting trim25 autoubiquitination and functional inactivation of the rig-i signalosome herpes virus deneddylases interrupt the cullin-ring ligase neddylation cycle by inhibiting the binding of cand1 an inhibitor of nedd8-activating enzyme as a new approach to treat cancer epstein-barr virus lytic replication elicits atm checkpoint signal transduction while providing an s-phase-like cellular environment virus dna replication and the host dna damage response the translesion polymerase pol eta is required for efficient epstein-barr virus infectivity and is regulated by the viral deubiquitinating enzyme bplf1 epstein-barr virus deubiquitinase downregulates traf6-mediated nf-kappab signaling during productive replication essential role of hcmv deubiquitinase in promoting oncogenesis by targeting anti-viral innate immune signaling pathways hsv1 vp1-2 deubiquitinates sting to block type i interferon expression and promote brain infection the 14-3-3 proteins: integrators of diverse signaling cues that impact cell fate and cancer development 14-3-3 proteins: structure, function, and regulation the mitochondrial targeting chaperone 14-3-3epsilon regulates a rig-i translocon that mediates membrane association and innate antiviral immunity roles of rig-i n-terminal tandem card and splice variant in trim25-mediated antiviral signal transduction 14-3-3 scaffold proteins mediate the inactivation of trim25 and inhibition of the type i interferon response by herpesvirus deconjugases interaction with 14-3-3 correlates with inactivation of the rig-i signalosome by herpesvirus ubiquitin deconjugases catalytic function and substrate specificity of the papain-like protease domain of nsp3 from the middle east respiratory syndrome coronavirus x-ray structural and biological evaluation of a series of potent and highly selective inhibitors of human coronavirus papain-like proteases structure-based design and optimization of potent inhibitors of the adenoviral protease small molecule screening identifies inhibitors of the epstein-barr virus deubiquitinating enzyme, bplf1 this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license acknowledgments: i sincerely thank t frisan, umeå university, n dantuma, karolinska institutet and all members of the masucci group for their critical reading of the manuscript and apologize to many contributors to the field whose work was not cited due to space limitations. the author declares no conflict of interest. the funders had no role in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the manuscript. key: cord-319501-a2x1hvkk authors: wong, lok-yin roy; lui, pak-yin; jin, dong-yan title: a molecular arms race between host innate antiviral response and emerging human coronaviruses date: 2016-01-15 journal: virol sin doi: 10.1007/s12250-015-3683-3 sha: doc_id: 319501 cord_uid: a2x1hvkk coronaviruses have been closely related with mankind for thousands of years. communityacquired human coronaviruses have long been recognized to cause common cold. however, zoonotic coronaviruses are now becoming more a global concern with the discovery of highly pathogenic severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) coronaviruses causing severe respiratory diseases. infections by these emerging human coronaviruses are characterized by less robust interferon production. treatment of patients with recombinant interferon regimen promises beneficial outcomes, suggesting that compromised interferon expression might contribute at least partially to the severity of disease. the mechanisms by which coronaviruses evade host innate antiviral response are under intense investigations. this review focuses on the fierce arms race between host innate antiviral immunity and emerging human coronaviruses. particularly, the host pathogen recognition receptors and the signal transduction pathways to mount an effective antiviral response against sars and mers coronavirus infection are discussed. on the other hand, the counter-measures evolved by sars and mers coronaviruses to circumvent host defense are also dissected. with a better understanding of the dynamic interaction between host and coronaviruses, it is hoped that insights on the pathogenesis of newly-identified highly pathogenic human coronaviruses and new strategies in antiviral development can be derived. [image: see text] coronaviruses (covs) are classified into four genera, namely alpha-, beta-, gamma-and deltacoronavirus, under the family of coronaviridae and the order of nidovirales (woo et al., 2012) . the first three genera were previously known as groups i, ii and iii, respectively (lau et al., 2006; zhong et al., 2012) . covs have been shown to infect many different hosts including bats, birds, dogs, mice and human (woo et al., 2009; de groot et al., 2013) . the infections are commonly zoonotic in nature (chan et al., 2013) . in the past 50 years, several human covs (hcovs) were identified. hcov-229e and hcov-oc43, belonging to alpha-and betacoronavirus respectively, were the first two hcovs identified in the mid-1960s (tyrrell and bynoe, 1965; hamre and procknow, 1966; mclntosh et al., 1967) . healthy individuals infected with either hcov-oc43 or hcov-229e develop illnesses within the range of typical common colds with good prognosis (bradburne et al., 1967) . since the identification of these two hcovs, extensive studies were conducted to understand their pathogenicity. however, almost all studies showed that hcov-oc43 and hcov-229e caused mild illnesses with high titers of neutralizing antibodies (bradburne et al., 1967) . the idea of hcov being a relatively weak respiratory diseasecausing agent was therefore presented to the field. this idea was generally accepted until the outbreak of sars in 2003. sars-cov was the first hcov identified to cause acute respiratory distress syndrome (ar-ds) (cheng et al., 2007; graham et al., 2013) . according to world health organization (who), a total of 8096 cases from 29 countries were reported with a case mortality rate of 9.6%. the sars outbreak changed the landscape of cov studies entirely and marked the new era of combating infectious diseases. tremendous efforts have been put into understanding sars-cov pathogenicity, opening a new page of cov biology. despite advances in infection control and quarantine measures in the past decade, another hcov causing ards was identified in saudi arabia as a novel lineage c betacoronavirus in september 2012 (zaki et al., 2012) . the newly identified hcov was later named mers-cov. up to october 2015, 1611 laboratory-confirmed cases were reported to who with 575 related deaths in 26 countries, including a recent outbreak involving 186 cases and 37 deaths in south korea. mers-cov is closely related phylogenetically to two bat covs, hku4 and hku5, shedding light on the possible zoonotic reservoir of mers-cov (zaki et al., 2012; . together with hcov-hku1 identified in 2005 (woo et al., 2005) and hcov-nl63 discovered in 2004 (fouchier et al., 2004; van der hoek et al., 2004) , six hcovs have been documented up to date. these 6 hcovs present diseases with a range of clinical severity from typical common cold in hcov-oc43, hcov-229e, hcov-hku1 and hcov-nl63 to ards in sars-cov and mers-cov. why these covs show dramatically different pathogenicity in human is an important but unanswered question in the field. one model to explain this difference is based on adaptation and host immunity. according to this model, bats are reservoir of various covs. bat covs constantly emerge in human via intermediate hosts such as civets and dromedaries. exposure of immunologically naïve human populations to these covs commonly causes severe diseases plausibly due to aberrant activation of innate immunity and lack of immune memory. when some covs become better adapted in human by acquiring the ability to transmit from human to human readily, pandemics could arise. meanwhile, as they become fully adapted, the covs might only cause mild diseases in human. existing evidence supports the origin of hcov-oc43, hcov-229e, hcov-hku1 and hcov-nl63 from bats and other animals (woo et al., 2009; huynh et al., 2012; corman et al., 2015) . adaptation and virus-host interaction are also known to be major determinants in cov pathogenesis (pepin et al., 2010; chan et al., 2013) . it will therefore be of great interest to see whether emerging human covs might be particularly capable of evading innate antiviral response while activating pathological inflammation. in other words, we need to determine whether the more severe clinical presentations might be accounted for by the specific interaction between host and emerging human covs, namely sars-cov and mers-cov. in this review, the host innate antiviral response to cov infection is particularly focused. in addition, the viral strategies adopted by sars-cov and mers-cov to subvert innate immunity are also summarized to provide inspiring insights that may explain the discrepancies in virulence (figure 1 ). covs are polycistronic positive-sense single-stranded rna (ssrna) viruses with genomes of about 30 kb in size. the 5′ most two-thirds of cov genome encodes polyprotein 1a (pp1a) and pp1ab replicase polyproteins, which are further cleaved by viral proteases to yield nonstructural proteins (nsps), while the 3′ end of the genome encodes structural and lineage-specific proteins (durai et al., 2015) . the cov life cycle begins with the binding to cellular receptor followed by membrane fusion as well as viral rna and protein synthesis in the cytoplasm. the pp1a and pp1ab polyproteins are co-translationally processed resulting in the formation of the replicase complex. a set of nested subgenomic mrnas and genomic rna, which possess both the same 3′ end and a common 5′ leader sequence derived from the 5′ end of the genome, is then transcribed. normally, only the 5′ end of each mrna is translated. virion assembly is achieved by budding into intracellular membranes and virion release is accomplished through the secretory pathway (cheng et al., 2007; durai et al., 2015) . the coronaviral spike (s) protein is responsible for binding to specific host receptor on cell surface and fusing viral envelope with lipid membrane of host upon infection (bosch et al., 2003; rota et al., 2003; chen et al., 2013) . hcov-nl63 and sars-cov from α-and β-genera respectively recognize angiotensin-converting enzyme 2 (ace2) (li et al., 2003; pyrc et al., 2007; frieman et al., 2008; chen et al., 2013) while mers-cov infects cells through another cell surface enzyme dipetidyl peptidase 4 (dpp4) (chen et al., 2013; raj et al., 2013) . aminopeptidase n (apn) has also been found to be recognized by some α-genus covs like hcov-229e (yeager et al., 1992) . cell surface receptor binding dictates species-specific viral entry as well as tropism. this also confines the direction of cellular antiviral response. we and others have shown the ability of cov s proteins to activate unfolded protein response and endoplasmic reticulum stress fung et al., 2014; siu et al., 2014b) . the activity of s might also be functionally related to coronaviral perturbation of innate antivir-al response including ifn and cytokine production. pattern recognition receptors (prrs) constitute an indispensable part of the host innate immune defense mechan-ism by the detection of foreign, non-self patterns from invading microbes distinct from host. these pathogenassociated molecular patterns (pamps) are usually biomolecules derived from the surface or generated during the life cycle of the microbes. the detection of pamps by host prrs activates innate immune response including the expression of type i ifns and cytokines for clearcycle are exposed to host innate immune sensors, rig-i/mda5 in cytoplasm or toll-like receptors tlr3/7/8 in endosome. activation of these immune sensors initiates a downstream signaling cascade that leads to ifn-β gene expression. rig-i/mda5 conveys signal through a mitochondrial adaptor mavs while tlr signals through trif/myd88. both pathways share the common traf adaptor to activate transcription factors. traf3 serves as an adaptor which activates tank × tbk1/ikkε complex for irf3 phosphorylation and subsequent dimerization, while traf6 is responsible for the activation of ikk complex which phosphorylates the canonical inhibitor of nf-κb (iκb). activated transcription factors are translocated into the nucleus to drive ifn-β expression. (right) ifn-β are secreted into extracellular space and bound to its cognate receptors ifnar to activate downstream jak-stat signaling. receptor-associated tyrosine kinases jak1 and tyk2 are brought to juxtaposition for self-phosphorylation and activation. stats are recruited to and phosphorylated by the tyrosine kinases. phosphorylated stat1/2 with irf9 forms a ternary complex isgf3 which translocates into the nucleus and binds to isre in the promoter region upstream of isg genes. isg genes are expressed consequently to establish an antiviral state in cells. oas is an example of isg which produces 2′, 5′-oligoadenylate (2′, 5′-a) upon detection of dsrna and activates rnase l to cleave viral rna to yield more rlr ligand as a positive-feedback mechanism of ifn production. the cov-encoded proteins shown in red are known to intervene the host innate immune signaling at various action points as evasion mechanisms to sustain viral replication and propagation. the action points at which viral proteins function marked with a question mark (?) represent controversial and inconclusive findings in the field or molecular mechanisms not well studied. mhv: mouse hepatitis virus. arms race between innate immunity and human coronaviruses ance of invading microbes. during cov infection, retinoic acid-inducible gene i (rig-i)-like receptors (rlrs) and toll-like receptors (tlrs) are believed to bear pivotal importance in stimulating host type i ifn induction. it is therefore essential to review the sensing mechanism of the prrs to understand viral evasion mechanisms and provide insights on the development of potential viral antagonists. after viral entry, cov genomes are exposed in the cytoplasm for expression of viral proteins, providing an opportunity for viral rna sensing by host. rlrs are ubiquitously expressed cytoplasmic rna helicases of dexd/h box family responsible for sensing doublestranded rna (dsrna) (yoneyama et al., 2005) . three types of rlrs have been identified up to now, including rig-i, melanoma differentiation-associated gene 5 (mda5) and laboratory of genetics and physiology 2 (lgp2) (loo and gale, 2011) . rig-i and mda5 consist of n-terminal caspase activation and recruitment domain (card) in two tandem copies, a central dexd/h box helicase domain and a c-terminal domain (ctd) (yoneyama et al., 2004 (yoneyama et al., , 2005 . the n-terminal cards are the effector domain of rlrs to mediate downstream transduction, which is held by the ctd when unstimulated (jiang et al., 2011; kowalinski et al., 2011; luo et al., 2011) . however, in the presence of residual amount of cytoplasmic dsrna, rlrs bind to dsrna through the central dexd/h box helicase domain and ctd with atp, causing a conformational change that exposes the n-terminal cards for signal transduction (yoneyama et al., 2004; jiang et al., 2011). lgp2 lacking the n-terminal cards is thought to act as co-factor that augments the function of rig-i and mda5 (satoh et al., 2010; bruns et al., 2014) . exposure of cards leads to oligomerization of rig-i or mda5 to form filamentous structure (berke et al., 2012; peisley et al., 2013; wu et al., 2013) . the card filament recruits and further initiates similar filamentous structure formation of card on mavs, an adaptor protein which further recruits downstream effectors tumor necrosis factor receptor-associated factor 3 (traf3), tank-binding kinase 1 (tbk1) and iκb kinase ε (ikkε) (loo and gale, 2011; wu et al., 2014) . tbk1 and ikkε form a complex of activated protein kinase for phosphorylation and activation of not only mavs adaptor , but also irf3 transcription factor (loo and gale, 2011) . activated irf3 are phosphorylated, dimerized and eventually translocated to the nucleus. on the other hand, traf2/6 is also recruited to mavs for nf-κb activation. specifically, canonical nf-κb inhibitor iκb is phosphorylated and then degraded through proteasomes in a ubiquitinationdependent fashion (loo and gale, 2011) . iκb degrada-tion exposes nuclear localization signal on nf-κb dimer for nuclear translocation. activated irf3 and nf-κb together with other transcription factors including c-jun assemble the enhanceosome that binds to ifn-β promoter for ifn-β expression (ford et al., 2010; loo and gale, 2011) . infection with mouse hepatitis virus induces rig-i expression. in addition, the activation of type i ifn production by this cov in oligodendrocytes requires both rig-i and mda5 . thus, rlrs might play an important role in the sensing of cov infection. several critical questions concerning rlr recognition of covs merit further investigations. first, the role of rlrs in cov sensing should be studied in rlr-null and cov-susceptible cells and animals. when necessary cr-ispr/cas9 technology might be used to disrupt rlr genes in target cells (hsu et al., 2014; yuen et al., 2015) . second, the cov pamps recognized by rlrs should be identified and characterized. particularly, it will be of interest to see whether and how common and highly structured regions in coronaviral genome, such as the aforementioned 5′ leader sequence, might be recognized by rlrs. for example, a polyuridine motif in the 3′ untranslated region of hepatitis c virus genome and the panhandle structure in rna viruses such as influenza a virus have previously been shown to be rig-i agonists (saito et al., 2008; weber et al., 2013; kell et al., 2015; liu et al., 2015b) . in addition, possible involvement of viral proteins such as nucleocapsid (n) in this recognition as in the case of other rna viruses (saito et al., 2008; weber et al., 2013) should also be clarified. finally, comparative analysis of sars-cov, mers-cov and other hcovs for their ability to activate rlrs will shed light on whether rlr activation would be a critical determinant in cov virulence. covs have been observed to infect host cells through more than one pathway. while cov entry by the fusion of viral envelope and host membrane has been described, the endosomal pathway is still considered the classical entry pathway for covs. in this pathway the activation of s protein cleavage by cathepsin l and transmembrane serine protease tmprss2 occurs in the absence of cell surface proteases in certain cell types (shirato et al., 2013; burkard et al., 2014) . in this regard, tlr family may play an essential role in sensing cov infection through the endosomal pathway. tlr family was identified as another prr homologous to drosophila toll receptor (boehme and compton, 2004) , sensing various pamps within the endosome which leads to induction of cytokines and ifns. in human, each of the 11 tlrs is known to specifically recognize a particular pamp and preferentially resides in either plasma or endosomal membrane. the cellular localization of tlrs defines their functions in detecting different pamps. for example, tlrs critically involved in viral nucleic acid sensing, including tlr3 for dsrna, tlr7 and tlr8 for ssrna, and tlr9 for unmethylated cpg island of dsdna viruses, are mainly localized in endosomal membrane while other members having a role in sensing other biomolecules derived from microbial surface components localized to plasma membrane of infected cells (xagorari and chlichlia, 2008; kawai and akira, 2010) . tlr family members being type 1 transmembrane proteins share a similar structure with a single transmembrane domain. tlr specificity is determined by the ectodomain made up of various number of leucine-rich repeats (lrrs) that bind the corresponding pamp directly (boehme and compton, 2004) . signal transduction begins with ligand binding to lrrs in the ectodomain, thus recruiting cytosolic adaptor protein myd88 with cytoplasmic toll/il-1 receptor (tir) domain by homotypic tir-tir domain interaction (xagorari and chlichlia, 2008) . the tlr-myd88 complex then recruits and activates interleukin 1r-associated kinase (irak) by phosphorylation. the activated irak then in turn associates with traf6 and activates a series of downstream effectors leading to the activation of a range of cytokines and ifn-stimulated genes (isgs), while activation of type i ifn expression by tlr3 is independent of myd88 but dependent on trif (boehme and compton, 2004; xagorari and chlichlia, 2008) . tlr pathway is significantly involved in the suppression of cov replication and induction of type i ifn expression. mice deficient of either tlr3 or tlr4 were more prone to sars-cov pathogenesis (mazaleuskaya et al., 2012; totura et al., 2015) . notably, disruption of either myd88 or trif arm of the tlr signaling pathway causes lethal sars-cov disease, indicating the importance of both arms in host innate immunity against sars-cov (totura et al., 2015) . full characterization of the role of tlrs in host innate antiviral response against sars-cov and mers-cov versus other hcovs will not only provide new knowledge about how tlr activation might impact cov pathogenesis, but might also identify new strategies for antiviral and vaccine development. for example, synthetic tlr agonists could potentially serve as antivirals and vaccine adjuvants in the prevention and control of covs. innate antiviral response is the first line of defense against cov infection. type i ifns are important antiviral and immunomodulatory agents. type i ifns function by binding to ifn-α receptor-1 (ifnar-1) and ifnar-2 receptor complex, thus activating janus family tyrosine kinase (jak), leading to the phosphorylation of signal transducer and activator of transcription (stat), a family of transcription factors regulating the expression of isgs. activated stat and irf9 form ifn-stimulated gene factor 3 (isgf3), stimulating expression of isgs by binding to ifn-stimulated response element (isre) in promoters of isgs (levy et al., 2001; samuel, 2001) . viral induction of isgs was abrogated in stat1 -/mice infected with sars-cov. the viral infection could not be cleared resulting in severe disease, extensive lung injury and 100% mortality zornetzer et al., 2010) . this indicates the importance of stat1 in sars-cov pathogenesis. isgs are the workhorses of the innate antiviral response with diverse functions including direct antiviral activities and regulation of adaptive immune system (schneider et al., 2014) . for example, ifn-inducible gene p53 evokes apoptosis in virus-infected cells (takaoka et al., 2003) . ifn-inducible protein kinase pkr, 2′, 5′-oligoadenylate synthetase (oas) and rnase l are important modulators involved in dsrna sensing, viral gene expression and replication. they act sequentially to trigger viral rna degradation and suppression of viral activities (samuel, 2001) . other isgs encoding antiviral effectors such as mx proteins, cholesterol-25-hydrolse, ifitm proteins, trim proteins, viperin, tetherin, cgamp synthase and sting could also be highly relevant to cov infection (schneider et al., 2014; schoggins et al., 2014; ma et al., 2015a; ma et al., 2015b) . inflammatory responses triggered by inflammatory cytokines like tumor necrosis factor α (tnf-α) and ifn-γ are also found to be ifn-dependent (samuel, 2001) . ifns do not only exert antiviral effects through activation of innate immunity but also act as modulators of adaptive immunity. adaptive immune response is activated by increased level of ifns. the levels of major histocompatibility complex (mhc) proteins class i and ii are found up-regulated by ifns. this facilitates efficient antigen presentation and hence cellular immune response to cov infection (samuel, 1991 (samuel, , 2001 ivashkiv and donlin, 2014) . in addition, the roles of non-conventional isgs including micrornas, long non-coding rnas and alternatively spliced isoforms have been increasingly recognized in recent years (schneider et al., 2014) . it will be of importance to determine whether sars-cov and mers-cov might be unique in isg activation as suggested in a recent study, which demonstrated that mers-cov induces repressive histone modifications to down-regulate specific subsets of isgs (menanchery et al., 2014b) . in relation to this, two areas concerning isg activation by covs might require more attention and research efforts. first, unbiased and large-scale screening of antiviral isgs using rna interference or crispr/cas9 technology might be carried out to identify key cellular factors that restrict sars-cov and mers-cov replication and infection. second, small-molecule compounds that activate antiviral isgs could be identified and tested for inhibition of sars-cov and mers-cov replication and infection. for example, establishing the significance of cgas and sting in cov infection might lead to the development of cyclic dinucleotides such as c-di-gmp and cgamp as novel anti-cov agents. covs have been reported to directly or indirectly suppress ifn production and signaling pathways by a subset of viral proteins via various mechanisms. in many cases, infected patients have shown diminished levels of type i ifns. this is especially true for sars and mers patients with severe diseases (faure et al., 2014) . it was also shown that sars-cov and mers-cov were capable of evading type i ifn production and signaling to different extents in cultured cells (kindler et al., 2013) . when the deficiency in type i ifn production in cov-infected cells was remedied by ifn-α treatment, cov replication was inhibited (falzarano et al., 2013) . combination of ifn-α with other antiviral drugs further improves the survival of infected patients (omrani et al., 2014) . this evidence suggests an essential role of type i ifns in the antiviral effect against cov infection. covs have evolved strategies to counter host antiviral response by antagonizing type i ifn production and signaling. cov proteins have been characterized to exhibit innate immunosuppressive effects in cellular models. below we will discuss them in three categories: structural, lineagespecific and non-structural proteins (nsps) (de groot et al., 2013) . nsps of covs are involved in the assembly of the replicase complex for viral rna synthesis (sevajol et al., 2014) . certain nsps have also been reported to possess innate immunosuppressive effect that facilitates viral replication and propagation, although these proteins per se are not required for viral life cycle (narayanan et al., 2008b; lokugamage et al., 2015) . nsps of different covs are more or less evolutionarily conserved suggesting their functional significance, with the exception of nsp1 and nsp2, which are thought to contribute to virulence of certain covs (neuman et al., 2014) . four structural proteins are found in covs, namely s, membrane (m), envelope (e) and n proteins. structural proteins contribute the architecture for virion assembly. accessory proteins are lineage-specific with diverse behaviors in different covs but are not essential for viral replication and propagation (de groot et al., 2013) . cov nsps have shown suppressive effects in various immune pathways including type i ifn production and signaling. sars-cov and mers-cov nsp1 proteins have been shown to selectively induce degradation of host mrna by inducing endonucleolytic cleavage while leaving viral rnas intact (huang et al., 2011; lokugamage et al., 2015) . in addition to the induction of endonucleolytic cleavage of host mrna, general inhibition of host mrna translation is achieved by binding of 40s subunit of ribosome with sars-cov nsp1 (huang et al., 2011) . particularly, sars-cov nsp1 inhibits innate immune response by translational repression of ifn mrna transcripts, hence altering ifn production and signaling (narayanan et al., 2008a; tanaka et al., 2012) . mers-cov nsp1 has also been characterized to specifically induce endonucleolytic cleavage of nuclear transcribed mrna while sparing cytoplasmic host mrna and viral rna (lokugamage et al., 2015) . this suggests a novel mechanism for evading host immune response. cov nsp3 protein has been characterized with a papain-like protease (plpro) domain for enzymatic cleavage of pp1a and pp1ab as well as a plp2 domain with deubiquitinating and deisgylating activity (clementz et al., 2010; mielech et al., 2014) . mers-cov plpro is able to antagonize ifn production induced by rig-i and mda5 as well as nf-κb activation . mers-cov plpro is catalytically more efficient (báez-santos et al., 2014) and its catalytic activity is indispensable for the suppressive effect on rig-i, mda5 and nf-κb (mielesh et al., 2014) . in contrast, sars-cov plpro does not require enzymatic activity for ifn antagonism (clementz et al., 2010) . hcov-nl63 and sars-cov plp2 transmembrane domain can also act as potent ifn antagonists to suppress ifn production induced by rig-in, a dominant active form of rig-i (clementz et al., 2010) . in another view of direct inhibition of ifn induction, nsp3 with deubiquitinating and deisgylating activity may also influence the ubiquitination and isgylation pattern and dynamics thus indirectly hindering innate immune response against cov infections (clementz et al., 2010) . for example, isgylation and ubiquitination of irf3 required for optimal activation is probably altered by plp domain of nsp3. apart from directly manipulating the signaling pathway involved in ifn production, several cov nsps were identified to act on viral rna to minimize ifn stimulation. n7-methylguanosine is the fundamental moiety of eukaryotic mrna cap structure and 2′-o-methylation on this moiety is a representative host signature to avoid prr activation as well as isg action. particularly, viral rna with this modification evades recognition by mda5 or ifit family antiviral factors (züst et al., 2011; daffis et al., 2010) . this is a common immunoevasive mechanism adopted by not only different covs but also other rna viruses. functional screening in yeasts suggested a novel function of sars-cov nsp14 as a guanine-n7-methyltransferase, the activity of which is required for viral replication and transcription (chen et al., 2009) . another nsp of sars-cov, nsp16, also possesses 2′-o-methyltransferase activity (menachery et al., 2014a; menachery et al., 2014c) . structural modeling suggested that sars-cov nsp16 associates with nsp10 in 1:1 ratio to form a complex of mature 2′-o-methyltransferase for viral cap methylation (chen et al., 2011; decroly et al., 2011) . a short peptide derived from nsp10 conserved region has been shown to be a promising nsp16 antagonist which outcompetes native nsp10 to blunt 2′-o-methyltransferase activity and restrict viral replication . plausibly, cov nsps might execute their innate immunosuppressive roles by targeting type i ifn production and signaling. further investigations are required to clarify whether and how far the sensing of cov rna and the induction of innate antiviral response are involved in the inhibitory activity of the nsp antagonists on cov replication. cov structural proteins have been shown to inhibit ifn production and signaling at multiple levels. sars-cov n protein showed inhibitory effects on ifn production induced by sendai virus and dsrna analogue poly(i:c) but no inhibition could be observed when downstream signaling molecules of tlr and rlr pathway were overexpressed. truncation mutant of n protein shows that the c-terminal domain is critical for rna-binding and ifn-antagonizing effect (lu et al., 2011) . this suggests sars-cov n may interfere with rna recognition by host immune sensors such as rig-i and mda5 thus achieving suppressive role in ifn production. other than n protein, sars-cov m protein has been characterized to potently down-regulate ifn production by impeding the formation of traf3·tank· tbk1/ikkε complex through the first transmembrane domain (siu et al., 2009 (siu et al., , 2014a . sars-cov m protein inhibits ifn production possibly through a sequestration model in which components of traf3·tank·tbk1/ikkε complex, an active complex for irf3 phosphorylation, are sequestered to specific locations in the cell (siu et al., 2009) . sars-cov m protein therefore exerts its inhibitory effects by impeding the formation of traf3·tank· tbk1/ikkε complex but not by modulating the catalytic activity of the complex. mers-cov m protein also exhibits ifn-antagonizing effects similar to its counterpart in sars-cov. in a previous study, mers-cov m is shown to impede ifn production by preventing irf3 translocation into the nucleus (yang et al., 2013) . however, the detailed mech-anism of inhibition remains unknown. recently, our group has characterized the mode of inhibition of ifn production by mers-cov m. consistently with previous report, we show that mers-cov m suppresses ifn production by preventing irf3 activation. we showed that mers-cov m interacts with traf3 which impedes the recruitment of tbk1 to traf3 complex. irf3 activation and dimerization have also been hampered as a result. the inhibitory effect is at least in part accounted for by the n-terminal transmembrane domains. despite of the similar behaviors, mers-cov m can only moderately suppress ifn expression when compared to sars-cov m. interestingly, hcov-hku1 m protein does not exert any inhibitory effects on ifn production (siu et al., 2014a) , suggesting that the ifn-antagonizing activity of structural proteins is unique to each cov but not universal. it will be of great interest to see whether this may correlate with the pathogenicity of different hcovs. eight accessory proteins have been identified in sars-cov and five are found in mers-cov (narayanan et al., 2008b) . sars-cov genome encodes orf3a, orf3b, orf6, orf7a, orf7b, orf8a, orf8b and orf9b as accessory proteins (narayanan et al., 2008b) . sars-cov orf3b and orf6 have been found to antagonize type i ifn production and signaling. particularly, sars-cov orf3b and orf6 suppress ifn-β production by perturbing irf3 activation induced by sendai virus infection. sars-cov orf3b and orf6 also suppress ifn-β-induced activation of isre in isg promoters (kopecky-bromberg et al., 2007) , although they are not able to reduce the level of phosphorylation of stat1, a transcription factor that activates isre activity once phosphorylated. however, sars-cov orf6 has been shown to inhibit stat1 translocation for isre activation (kopecky-bromberg et al., 2007) . the findings suggest a mode of inhibition of ifn-β signaling by sars-cov. ifn antagonism of accessory proteins has also been observed in another deadly hcov. mers-cov genome encodes orf3, orf4a, orf4b, orf5 and orf8b (de groot et al., 2013) . among the five accessory proteins, orf4a, orf4b and orf5 show the ability to dampen ifn production (yang et al., 2013) . suppression of ifnβ promoter-driven luciferase activity has been observed in cells transfected with orf4a, orf4b and orf5 plasmids. all these 3 accessory proteins are able to block irf3 translocation to the nucleus to activate ifn promoter (yang et al., 2013) . mers-cov orf4a shows an additional level of inhibition of innate immunity by intervening nf-κb activation. in another study, orf4a has been shown as an antagonist of ifn production by inhib-iting irf3 translocation but has no effect on ifn signaling (niemeyer et al., 2013) . our group demonstrated that mers-cov orf4a interacts with pact, a cellular dsrna-binding protein that optimally activates rig-iand mda5-induced type i ifn production, in an rnadependent manner (siu et al., 2014c) . this suggests that orf4a may compete with rig-i and mda5 for rna, rendering the inactivation of rig-i and mda5. direct interaction of orf4a with pact may also prevent interaction of pact with rig-i and mda5, thus compromising pact-dependent activation of rig-i and mda5 required for optimal induction of ifn production. although we and others have observed the ifn-antagonizing activity of mers-cov orf4b, different activity profiles and mechanisms have been suggested (yang et al., 2013; matthews et al., 2014) . one recent report suggested that orf4b directly interacts with and inhibits tbk1/ikke in the cytoplasm but might also perturb type i ifn production in the nucleus through an unknown mechanism (yang et al., 2015) . mouse hepatitis virus, another betacoronavirus closely related to hcov-oc43 and hcov-hku1, encodes a lineage-specific accessory protein named ns2 with innate immunosuppressive property (zhao et al., 2012) . biochemical assays indicate that ns2 protein has phosphodiesterase activity against 2′, 5′-a, the product of oas . thus, ns2 is a potent inhibitor of an ifn effector molecule and it might represent a new family of viral and cellular proteins with innate immunosuppressive activity gusho et al., 2014) . whether distantly related proteins in hcov-oc43 and hcov-hku1 might have similar activity remains to be determined. more importantly, it will be of interest to see whether sars-cov and mers-cov might encode proteins with similar enzymatic activity. multiple ifn antagonists have been identified and characterized in sars-cov and mers-cov. some differences between these ifn-antagonizing viral proteins and their counterparts in other covs such as the parental bat viruses of mers-cov have also been noticed (siu et al., 2014c) . existing evidence supports several important notions. first, although sars-cov and mers-cov share some features in common, they are distinct and use unique mechanisms for innate immune evasion (perlman and zhao, 2013) . second, both sars-cov and mers-cov are bat-origin covs that are well adapted in bats but newly emerge in human. this provides a golden opportunity for the study of cov-host interaction, cov adaptation as well as the arms race between host innate antiviral immunity and covs. observing how the arms race between the host and sars-cov or mers-cov might evolve when the viruses become adapted to human will be most revealing and could provide important clues as to how a balance of power in this arms race might result in attenuation with increased transmissibility. finally, studies on sars-cov and mers-cov have overturned existing concepts and derived new principles and thoughts to cov biology. particularly, mechanisms by which sars-cov and mers-cov evade innate immunity have attracted increasing attention. however, many key issues remain obscure. particularly, better in vivo evidence should be obtained to clarify whether more potent inhibition of innate ifn production and signaling by sars-cov and mers-cov is a key determinant in virulence and disease severity. covs have drawn a lot of interests in the light of the recent emergence of mers-cov. it remains to be understood whether the emerging deadly covs causing ards might ultimately be established and adapted in human resulting in significant attenuation of virulence. from the identification of the first two hcovs, hcov-229e and hcov-oc43 in the mid-1960s, we learned that hcov was able to cause only common cold. however, the outbreaks of sars and mers that have claimed hundreds of lives revealed the other extreme of cov pathogenicity and raised new questions in cov biology. so far no vaccines have been developed against sars-cov and mers-cov. infection with sars-cov and mers-cov has been accompanied with suppression of innate immune response, most notably with the suppression of type i ifn production and signaling pathways. as the first-line defense in the immune system, suppression of innate immune response by these covs has impeded the host ability to restrict infection, causing significant casualties. although many reports have shed light on the molecular mechanism by which various cov proteins antagonize type i ifn production and signaling, most of the studies were performed with overexpression experiments in cellular models. future emphasis should be put on the characterization of knock-out viruses with which the function of a particular viral gene could be studied in a more physiologically relevant context. infectious clones and replicons for sars-cov and mers-cov have been generated for this reverse genetic approach (yount et al., 2003; almazán et al., 2006 almazán et al., , 2013 almazán et al., , 2014 scobey et al., 2013) . ifn and cytokine profiles of deadly hcovs such as sars-cov and mers-cov can be compared with hcov-229e and hcov-oc43 causing mild diseases. the pivotal significance of type i ifns in innate immune activation and modulation has been discussed in this review. suppression pattern of ifn may provide insights on the high pathogenicity of deadly hcovs. the arms race between host innate antiviral response and emerging human covs might evolve after their introduc-tion and establishment in human populations, with significant impact on virulence, transmissibility and disease severity. emerging human covs remain a potential threat to global public health. new knowledge about the host-cov arms race will provide new ideas, targets and attenuated strains for the design and development of antivirals and vaccines for prevention and control of deadly cov infections. construction of a severe acute respiratory syndrome coronavirus infectious cdna clone and a replicon to study coronavirus rna synthesis engineering a replication-competent, propagation-defective middle east respiratory syndrome coronavirus as a vaccine candidate coronavirus reverse genetic systems: infectious clones and replicons catalytic function and substrate specificity of the papainlike protease domain of nsp3 from the middle east respiratory syndrome coronavirus mda5 assembles into a polar helical filament on dsrna innate sensing of viruses by tolllike receptors the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex effects of a "new" human respiratory virus in volunteers the innate immune sensor lgp2 activates antiviral signaling by regulating mda5-rna interaction and filament assembly coronavirus cell entry occurs through the endo-lysosomal pathway in a proteolysis-dependent manner modulation of the unfolded protein response by the severe acute respiratory syndrome coronavirus spike protein interspecies transmission and emergence of novel viruses: lessons from bats and birds functional screen reveals sars coronavirus nonstructural protein nsp14 as a novel cap n7 methyltransferase crystal structure of the receptor-binding domain from newly emerged middle east respiratory syndrome coronavirus biochemical and structural insights into the mechanisms of sars coronavirus rna ribose 2′-o-methylation by nsp16/nsp10 protein complex severe acute respiratory syndrome coronavirus as an agent of emerging and reemerging infection deubiquitinating and interferon antagonism activities of coronavirus papain-like proteases evidence for an ancestral association of human coronavirus 229e with bats 2′-o methylation of the viral mrna cap evades host restriction by ifit family members crystal structure and functional analysis of the sars-coronavirus rna cap 2′-o-methyltransferase nsp10/nsp16 complex middle east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus study group middle east respiratory syndrome coronavirus: transmission, virology and therapeutic targeting to aid in outbreak control inhibition of novel β coronavirus replication by a combination of interferon-α2b and ribavirin distinct immune response in two mers-cov-infected patients: can we go from bench to bedside? the transcriptional code of human ifn-β gene expression a previously undescribed coronavirus associated with respiratory disease in humans sars coronavirus and innate immunity sars-cov pathogenesis is regulated by a stat1 dependent but a type i, ii and iii interferon receptor independent mechanism coronavirus-induced er stress response and its involvement in regulation of coronavirus-host interactions a decade after sars: strategies for controlling emerging coronaviruses murine akap7 has a 2′, 5′-phosphodiesterase domain that can complement an inactive murine coronavirus ns2 gene a new virus isolated from the human respiratory tract development and applications of crispr-cas9 for genome engineering sars coronavirus nsp1 protein induces template-dependent endonucleolytic cleavage of mrnas: viral mrnas are resistant to nsp1-induced rna cleavage evidence supporting a zoonotic origin of human coronavirus strain nl63 regulation of type i interferon responses structural basis of rna recognition and activation by innate immune receptor rig-i the role of pattern-recognition receptors in innate immunity: update on toll-like receptors pathogen-associated molecular pattern recognition of hepatitis c virus transmitted/founder variants by rig-i is dependent on u-core length efficient replication of the novel human betacoronavirus emc on primary human epithelium highlights its zoonotic potential severe acute respiratory syndrome coronavirus open reading frame (orf) 3b, orf 6, and nucleocapsid proteins function as interferon antagonists structural basis for the activation of innate immune pattern-recognition receptor rig-i by viral rna coronavirus hku1 and other coronavirus infections in hong kong the virus battles: ifn induction of the antiviral state and mechanisms of viral evasion murine coronavirus induces type i interferon in oligodendrocytes through recognition by rig-i and mda5 angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus influenza a virus panhandle structure is directly involved in rig-i activation and interferon induction phosphorylation of innate immune adaptor proteins mavs, sting, and trif induces irf3 activation middle east respiratory syndrome coronavirus nsp1 inhibits host gene expression by selectively targeting mrnas transcribed in the nucleus while sparing mrnas of cytoplasmic origin immune signaling by rig-i-like receptors sars-cov nucleocapsid protein antagonizes ifn-β response by targeting initial step of ifnβ induction pathway, and its c-terminal region is critical for the antagonism structural insights into rna recognition by rig-i positive feedback regulation of type i ifn production by the ifn-inducible dna sensor cgas positive feedback regulation of type i interferon by the interferonstimulated gene sting the orf4b-encoded accessory proteins of middle east respiratory syndrome coronavirus and two related bat coronaviruses localize to the nucleus and inhibit innate immune signalling protective role of toll-like receptor 3-induced type i interferon in murine coronavirus infection of macrophages recovery in tracheal organ cultures of novel viruses from patients with respiratory disease middle east respiratory syndrome coronavirus in bats, saudi arabia coronavirus nonstructural protein 16: evasion, attenuation, and possible treatments pathogenic influenza viruses and coronaviruses utilize similar and contrasting approaches to control interferon-stimulated gene responses attenuation and restoration of severe acute respiratory syndrome coronavirus mutant lacking 2′-o-methyltransferase activity mers-cov papain-like protease has deisgylating and deubiquitinating activities severe acute respiratory syndrome coronavirus nsp1 suppresses host gene expression, including that of type i interferon, in infected cells sars coronavirus accessory proteins atlas of coronavirus replicase structure middle east respiratory syndrome coronavirus accessory protein 4a is a type i interferon antagonist ribavirin and interferon alfa-2a for severe middle east respiratory syndrome coronavirus infection: a retrospective cohort study rig-i forms signaling-competent filaments in an atp-dependent, ubiquitin-independent manner identifying genetic markers of adaptation for surveillance of viral host jumps human coronavirus emc is not the same as severe acute respiratory syndrome coronavirus the novel human coronaviruses nl63 and hku1 dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc characterization of a novel coronavirus associated with severe acute respiratory syndrome antiviral actions of interferon interferon-regulated cellular proteins and their surprisingly selective antiviral activities antiviral actions of interferons lgp2 is a positive regulator of rig-i-and mda5-mediated antiviral responses innate immunity induced by composition-dependent rig-i recognition of hepatitis c virus rna interferon-stimulated genes: a complex web of host defenses pan-viral specificity of ifn-induced genes reveals new roles for cgas in innate immunity reverse genetics with a full-length infectious cdna of the middle east respiratory syndrome coronavirus insights into rna synthesis, capping, and proofreading mechanisms of sars-coronavirus middle east respiratory syndrome coronavirus infection mediated by the transmembrane serine protease tmprss2 suppression of innate antiviral response by severe acute respiratory syndrome coronavirus m protein is mediated through the first transmembrane domain comparative analysis of the activation of unfolded protein response by spike proteins of severe acute respiratory syndrome coronavirus and human coronavirus hku1 severe acute respiratory syndrome coronavirus m protein inhibits type i interferon production by impeding the formation of traf3·tank·tbk1/ikkϵ complex middle east respiratory syndrome coronavirus 4a protein is a double-stranded rna-binding protein that suppresses pact-induced activation of rig-i and mda5 in the innate antiviral response integration of interferon-α/β signalling to p53 responses in tumour suppression and antiviral defence severe acute respiratory syndrome coronavirus nsp1 facilitates efficient propagation in cells through a specific translational shutoff of host mrna toll-like receptor 3 signaling via trif contributes to a protective innate immune response to severe acute respiratory syndrome coronavirus infection cultivation of a novel type of common-cold virus in organ cultures coronavirus nsp10/nsp16 methyltransferase can be targeted by nsp10-derived peptide in vitro and in vivo to reduce replication and pathogenesis incoming rna virus nucleocapsids containing a 5′-triphosphorylated genome activate rig-i and antiviral signaling characterization and complete genome sequence of a novel coronavirus, coronavirus hku1, from patients with pneumonia coronavirus diversity, phylogeny and interspecies jumping discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus structural basis for dsrna recognition, filament formation, and antiviral signal activation by mda5 molecular imprinting as a signalactivation mechanism of the viral rna sensor rig-i toll-like receptors and viruses: induction of innate antiviral immune responses middle east respiratory syndrome coronavirus orf4b protein inhibits type i interferon production through both cytoplasmic and nuclear targets the structural and accessory proteins m, orf 4a, orf 4b, and orf 5 of middle east respiratory syndrome coronavirus (mers-cov) are potent interferon antagonists human aminopeptidase n is a receptor for human coronavirus 229e shared and unique functions of the dexd/h-box helicases rig-i, mda5, and lgp2 in antiviral innate immunity the rna helicase rig-i has an essential function in double-stranded rnainduced innate antiviral responses reverse genetics with a full-length infectious cdna of severe acute respiratory syndrome coronavirus crispr/cas9-mediated genome editing of epstein-barr virus in human cells isolation of a novel coronavirus from a man with pneumonia in saudi arabia homologous 2′, 5′-phosphodiesterases from disparate rna viruses antagonize antiviral innate immunity antagonism of the interferon-induced oas-rnase l pathway by murine coronavirus ns2 protein is required for virus replication and liver pathology recent progress in studies of arterivirus-and coronavirus-host interactions transcriptomic analysis reveals a mechanism for a prefibrotic phenotype in stat1 knockout mice during severe acute respiratory syndrome coronavirus infection ribose 2′-o-methylation provides a molecular signature for the distinction of self and non-self mrna dependent on the rna sensor mda5 we thank hinson cheung, kitty fung, edwin kong and sam yuen for reading the manuscript critically. coronavirus research in our laboratory was supported by hong kong health and medical research fund (13121032, 14130822 and hkm-15-m01) and hong kong research grants council (hku1/crf/11g, c7011-15r and t11-707/15-r). the authors declare that they have no conflict of interest. this article does not contain any studies with human or animal subjects performed by any of the authors. key: cord-323756-atnrw9ew authors: vabret, nicolas; blander, j. magarian title: sensing microbial rna in the cytosol date: 2013-12-25 journal: front immunol doi: 10.3389/fimmu.2013.00468 sha: doc_id: 323756 cord_uid: atnrw9ew the innate immune system faces the difficult task of keeping a fine balance between sensitive detection of microbial presence and avoidance of autoimmunity. to this aim, key mechanisms of innate responses rely on isolation of pathogens in specialized subcellular compartments, or sensing of specific microbial patterns absent from the host. efficient detection of foreign rna in the cytosol requires an additional layer of complexity from the immune system. in this particular case, innate sensors should be able to distinguish self and non-self molecules that share several similar properties. in this review, we discuss this interplay between cytosolic pattern recognition receptors and the microbial rna they detect. we describe how microbial rnas gain access to the cytosol, which receptors they activate and counter-strategies developed by microorganisms to avoid this response. when janeway formulated the theory of pattern recognition in 1989, he proposed that host cells could sense microbial infection owing to receptors able to recognize invariant molecular structures defined as pathogen-associated molecular patterns (pamps). these patterns would be present in groups of pathogens, but absent in the host (1) . years later, janeway and medzhitov described the activity of the first mammalian member of the toll-like receptor (tlr) family, toll-like receptor 4 (2) . tlrs comprise a family of transmembrane proteins able to recognize conserved microbial features and activate the immune response (3) . once activated, tlrs and others pattern recognition receptors (prrs) initiate several intracellular pathways, including those mediated by nuclear factor-κb (nf-κb), mitogen-activated protein kinases (mapks), and interferon regulatory factors (irfs). another outcome of activation of distinct members of cytosolic prrs is their oligomerization into multimeric cytosolic structures called inflammasomes, which activate the cysteine protease caspase-1, subsequently leading to the production of biologically active forms of pro-inflammatory cytokines (4) . initially thought to detect exclusively microbial derived ligands, prrs were later shown to recognize host derived danger signals, which are released in response to stress conditions such as cellular damage or tissue injury (3) . under normal physiological conditions, these ligands are not accessible to their respective prrs and do not activate the immune system. conversely, it was first suggested that self-dna artificially introduced into the cytoplasm by transfection could activate nf-κb and the mapk pathway (5) . evidence that any dna, regardless of its origin, can engage innate immune receptors when localized outside of the nucleus was further confirmed by the identification of several endosomal and cytosolic dna sensors [reviewed in ref. (6) ]. in contrast to cytosolic dna, rna sensing in the cytoplasm raises many questions on the mechanisms used by the innate system to specifically distinguish non-self-rna from self-rna. during infection, microbial rnas share the cytosolic cellular compartment with several host rna species, including messenger rna (mrna), transfer rna (trna), ribosomal rna (rrna), microrna, and other small regulatory rnas. as a consequence, cytosolic sensors must display a high affinity for specific microbial features to avoid activation by host molecules that would otherwise elicit autoimmune responses. despite this apparent challenge, efficient detection of foreign rna in the cytosol is essential for innate immunity. during certain viral infections, rna may be the only microbial pamp produced throughout most of the replication cycle. additionally, our laboratory previously showed that recognition of bacterial mrna in the cytosol was critical to elicit a robust innate response against bacterial infection (7) . finally, cytosolic sensing of pathogen invasion by non-immune infected cells provides the very first steps of innate response against infection, before phagocytosis-competent immune cells are recruited to the site of infection. in this review, we summarize the current understanding of cytosolic rna sensing. we describe instances in which microbial rnas gain access to the cytosol, the prrs they activate, their corresponding ligands and strategies developed by microorganisms to conceal their rnas. rna entry into host cells generally takes place during the first steps of a microbial infection. we distinguish four processes leading to the presence of microbial rna in the cytosol of eukaryotic cells, where it can engage host prrs (figure 1 ). figure 1 | cytosolic recognition of microbial rna. genomic rna from rna viruses access the cytosol immediately after the cell entry step of the replication cycle, where it may be amplified by viral rna-dependent rna polymerase (rdrp). genomic dna from dna viruses is transcribed by viral or cellular rna polymerase, including the cytosolic rna polymerase iii. bacterial rna can access the cytosol through the activity of auxiliary secretion systems or during passive leakage of phagosomal products. once in the cytosol, microbial rna binds different families of prrs classified as rlrs, non-rlr helicases, and other receptors. downstream signaling pathways include activation of mavs, trif, and the nlrp3 inflammasome. black arrows, rna entry; red arrows, signaling pathways. step of the replication cycle. viruses can directly release their genome at the plasma membrane after binding to a receptor. alternatively, they can be first internalized through endocytosis or macropinocytosis. endocytosed virus particles will typically traffic through endosomal vesicles by actin-dependent and/or microtubule-dependent transport (8) . specific environmental triggers like endosomal ph acidification induce either fusion of enveloped virus with the endosome, or membrane penetration of viral proteins, allowing viral genetic material to be released into the cytoplasm (8) . alternatively, viruses can spread by direct cell-cell contact (9) . cell-to-cell transmission of viral material can activate cytoplasmic innate pathways, as exemplified with hepatitis c virus (10), lymphocytic choriomeningitis virus (11) , or human immunodeficiency virus transmission (12) . other viral rna pamps can be produced during viral replication. david baltimore has defined a classification of viruses based on the mechanism of mrna production (13) . viruses are clustered in seven groups depending on their genomes (dna, rna), strandedness (single or double), sense or antisense, and method of replication. the type of rna ligands produced during viral replication will depend on the type of viral genome and the strategy used to generate mrna. rna ligands can be generated by dna viruses and retroviruses via genome transcription, or by synthesis of mrna and replication intermediates by rna-dependent rna polymerases (rdrps) of rna viruses (8) . it has been shown that ligands generated in phagolysosomes after phagocytosis of bacteria by immune cells can engage cytosolic innate immune receptors (14) . similarly, we showed that rna from escherichia coli could activate receptors in the cytosol after phagocytosis by macrophages (7) . we demonstrated that phagosomes carrying e. coli exhibit intrinsic leakiness, suggesting a mechanism by which bacterial rna, irrespective of the activity of virulence factors, can gain access to the cytoplasm (7) . alternatively, bacteria express secretion systems to translocate products outside of the bacterial cell wall. in the case of intracellular bacteria, auxiliary secretion systems like seca2 in listeria monocytogenes have been shown to actively translocate listeria rna into the cytoplasm, resulting in activation of cytosolic sensors (15, 16) . similarly, another study proved that cytosolic rna sensors participate in the type 1 interferon (ifn-i) response to legionella pneumophila. although the authors did not demonstrate the translocation of legionella rna into the cytosol of infected cells, they discuss their data through a model where it would be the case (17) . future studies looking for additional secreted rna will likely provide additional insights on their interaction with the innate immune system. two independent groups have demonstrated that cytoplasmic dsdna triggers ifn-i production via rna polymerase iii, which transcribes dna into 5'-triphosphate (5 -ppp) rna, subsequently recognized by cytosolic rna receptors (18, 19) . this pathway has been involved in the sensing of dna viruses, like epstein-barr virus, or intracellular bacteria, like l. pneumophila (18, 19) . although the rna intermediate produced is not sensu stricto microbial, its generation is due to the activity of a microbial invader. the best-studied cytosolic rna sensors are the three members of rig-i-like receptors (rlrs), a subfamily of the dexd/hbox family of helicases. they consist of retinoic acid-inducible gene i (rig-i), melanoma differentiation factor 5 (mda5), and laboratory of genetics and physiology 2 (lgp2). they share a similar organization with three distinct domains: (i) a c-terminal repressor domain (rd) embedded within the c-terminal domain (ctd); (ii) a central atpase containing dexd/h-box helicase domain able to bind rna; and (iii) a n-terminal tandem card domain that mediates downstream signaling, and which is present in rig-i and mda5 but absent in lgp2. upon activation by rna ligands, rig-i and mda5 are subsequently recruited to the adaptor protein mitochondrial antiviral signaling (mavs) via a card-card interaction and lead to activation of nf-κb and irfs (20) (21) (22) (23) . in contrast to tlr expression that is predominantly expressed in specialized immune cells such as macrophages and dendritic cells (dcs), rlrs are found in the cytosol of most cell types and are strongly induced in response to ifn-i (24, 25) . the rig-i ligand has been characterized as an rna molecule with two distinct features: (i) a 5 -ppp moiety (26, 27) and (ii) bluntend base pair at the 5'-end (28, 29) . blunt-end base pairs can be found in double-stranded rna (dsrna) and secondary rna structures such as hairpin or panhandle conformations (28, 29) . recent structural studies have contributed toward a better understanding of ligand binding and activation of rig-i. specificity of 5 -ppp binding is conferred by the ctd, and the helicase domain binds the double-stranded part of the rna. rig-i is normally held in an auto-repressed conformation, and ligand binding results in a conformational change, releasing the card domain which can subsequently initiate signaling by association with mavs (30) (31) (32) . despite the increasing amount of high-resolution crystal data, the consensus definition of rig-i ligand remains controversial. other rig-i ligands have been indeed described in the literature including long (33) or short dsrna (34) (35) (36) lacking the 5 -ppp. however, thermodynamic analysis have shown that the full-length human rig-i protein binds 5'-ppp dsrna with 126-fold higher affinity than 5'-oh dsrna, and dsrna with a 361-fold higher affinity than short single stranded rna (ssrna) lacking secondary structure (37) . many viral families display blunt-end base-paired rna with a 5 -ppp, directly in their genomic rna or in replication intermediates. consistent with this notion, rig-i has been shown to be involved in the recognition of many viruses, either antisense (−)ssrna viruses (38, 39) or sense (+)ssrna/dsrna viruses (40, 41) . notably rig-i can detect panhandle structures found in lacrosse viral particles (39) or in influenza genomic rna (28, 38) . sendai virus (sev) and other mononegavirales produce defective interfering (di) viral genomes containing panhandle structures that activate rig-i in infected cells (42) . retinoic acid-inducible gene i recognition has not been limited to rna virus since rig-i is involved in recognition of dna viruses, such as epstein-barr virus or adenovirus through the rna polymerase iii pathway (18, 43, 44) . moreover, rig-i is also able to detect bacterial infections. bacterial mrna are not capped and it has been estimated that approximately 40% of rna oligonucleotides in e. coli have a 5 -ppp (45) . reports in the literature describe sensing of l. monocytogenes secreted rna (15, 16) or purified legionella (17) and helicobacter pylori rna (46) by rig-i. finally, rig-i can also sense shigella flexneri infection in macrophages through the rna polymerase iii pathway (47) . melanoma differentiation factor 5 ligand is less characterized than rig-i. using poly(i:c) as a synthetic dsrna mimic, studies have shown that mda5 binds long, but not short dsrna (35, 40, 48) . structural analyses have demonstrated that mda5 specifically recognizes the internal duplex structure of dsrna and uses it as a platform to stack along dsrna in a head-to-tail arrangement. this mechanism promotes stochastic assembly of the tandem card oligomers that activates the signaling adaptor mavs (49) . melanoma differentiation factor 5 detects infection by viral families known to produce long dsrna structures during their replication cycle, including (+)ssrna viruses like picornaviruses, dsrna viruses like reoviruses, or dna viruses like poxviruses (35, (50) (51) (52) (53) . in the case of (+)ssrna virus infection, fluorescent imaging studies have confirmed that mda5 recognizes preferentially the dsrna generated during the replication of these viruses, but not the genomic ssrna (54) . prior to the structural study mentioned above, multiple observations raised the possibility that there may exist additional mda5 ligands, different from the consensus long dsrna. thus, a study has shown that mda5 cooperates with the ribonuclease rnase l to induce ifn-i in response to a viral mrna from parainfluenza 5 virus (55) . interestingly, rnase l converts rna into small rna products, with shorter length than the current mda5 www.frontiersin.org ligand definition (56) . another work published the same year has shown that mrna lacking 2'-o-methylation at their 5' cap structure induces production of ifn-i through mda5 activation (57) . however, the data published, which focus on coronavirus infection, did not elucidate whether the absence of methylation was directly recognized by mda5 or via another intermediate (57) . after binding to their specific ligands, both rig-i and mda5 activate mavs to trigger a common signaling pathway. the majority of mavs is localized on the mitochondrial membrane and its engagement by rlrs causes a conformational change that propagates to adjacent un-activated mavs in a prion-like behavior (58) . the formation of these very large mavs aggregates results in a largescale amplification of the signaling cascade. this cascade involves the recruitment of cytosolic adaptor molecules, followed by the activation of the canonical ikks, ikk-α, ikk-β, and ikk-γ, the mapk and the non-canonical ikk-related kinase, tbk1 and ikki/ε. ultimately, specific transcription factors, such as irf3, nf-κb, and depending on the cell type irf5 and irf7, are translocated to the nucleus where they promote the expression of ifn-i genes and pro-inflammatory cytokines [reviewed in ref. (59) ]. finally, mavs has been recently shown to interact with nodlike receptor family, pyrin domain containing 3 (nlrp3) and promote its recruitment to the mitochondria. the authors emphasize the central role of mavs in innate immune signaling events by showing its importance in the functioning of nlrp3 inflammasome and the production of il-1β (60) . of note, mavs independent activation of the nlrp3 inflammasome by rig-i has also been reported (61, 62) . the third member of rlrs, lgp2, is able to bind dsrna (63, 64) , however, its role in immune activation is poorly understood. lgp2 was proposed to be a modulator of rlr signaling. studies showed that lgp2 was required for rig-i and mda5 activity, in particular during picornaviral infection (65) (66) (67) . another work proposed that lgp2 would inhibit rig-i through competition with its ligand (64) . it is however unclear whether lgp2 binds microbial rna in an infectious context, and what specific features of the rna it would recognize. further studies will be required to clarify the precise role of lgp2. apart from rlr, several recent studies have highlighted the importance of other dexd/h-box helicases in microbial rna sensing. rna helicases of the dead box family are involved in various different steps of rna metabolism [reviewed in ref. (68) ]. they share eight conserved motifs that are involved in atp binding, atp hydrolysis, nucleic acid binding, and rna unwinding activity. additionally, most dexd/h-box helicases contain auxiliary nand c-terminal domains that confer on them functional specificities, such as an ability to induce downstream signaling or to bind specific rna targets (69) . ddx3 (ddx3x) can bind poly(i:c) or vesicular stomatitis virus (vsv) rna and was shown to enhance the ifn-i response to vsv infection by interaction with the rlr-mavs complex. overexpression assays suggest that ddx3 precipitates with rig-i and mda5 (70) . since ddx3 is easily detected in resting cells, the authors propose a sentinel role for this helicase, the activity of which would be required during the initial steps of viral infection. another study showed that upon sev infection, ddx3 interacts with ikkε, an essential component of the irf3 signaling pathway, increasing the induction of the ifn-β promoter (71) . moreover, ddx3 is targeted by vaccinia virus protein k7 (71) , an inhibitor of ifn-β production, and by hcv core protein, which can disrupt its interaction with mavs (72) . these observations highlight the importance of ddx3 in efficient viral sensing. using overexpression and knock-down experiments, dhx9 was shown to be required for the production of ifn-i and proinflammatory cytokines in response to poly(i:c), influenza virus, and reovirus by a murine splenic dc line and bone-marrow derived dcs. dhx9 can bind dsrna via its dsrna-binding motif and interact with mavs through both its helicase c-terminal domain and ha2-duf (73). myeloid dcs have also been shown to express a complex composed of ddx1, ddx21, and dhx36 that triggers an antiviral program in response to poly(i:c), in a pathway dependent of the adapter molecule tir-domain containing adapter-inducing interferon-β (trif). ddx1 binds to poly(i:c) via its helicase a domain, while dhx36 and ddx21 bind the tir domain of trif via their ha2-duf and prk domains, respectively. this complex seems to be required for the innate response against influenza or reovirus infection (74) . notably, a separate study also characterized dhx36 and dhx9 as a sensor for the dsdna species cpg-a and -b, respectively. in this case, both dhx36 and dhx9 activate the cytosolic adapter protein myeloid differentiation primary response gene 88 (myd88) by binding to its tir domain (75) . another recent study by yong-jun liu's group identified another helicase, dhx33, as a cytosolic rna receptor able to activate the nlrp3 inflammasome (76) . dhx33 is involved in inflammasome activation after sensing cytosolic rna such as poly(i:c) or reoviral rna when directly delivered by lipofection to the cytoplasm of a macrophage cell line or human monocyte-derived macrophages. additional experiments suggested that dhx33 could also possibly be involved in detection of cytosolic bacterial rna. the authors showed that dhx33 can bind to dsrna through its helicase c domain and to nlpr3 through its dead domain (76) . a few months later, another study performed on myeloid dcs confirmed the role of dhx33 in the sensing of cytosolic poly(i:c) and reoviral rna. surprisingly, in this case, poly(i:c)-induced activation of mapk, nf-κb, and irf3 was mediated by mavs, which binds the helicase c domain of dhx33 (77) . ddx60 has also been shown to enhance the ifn-i response to rna and dna stimulation through formation of complexes with frontiers in immunology | molecular innate immunity rig-i, mda5, and lgp2 but not with mavs. this complex formation has been deciphered with overexpression assays in the case of mda5 and lgp2, and with endogenous rig-i during vsv infection. ddx60 expression is induced by viral infection and its helicase domain can bind ds-or ss-vsv rna generated in vitro, independently of the 5 -ppp (78) . interestingly, ddx60 can also bind dsdna, and was shown to play role in ifn-i expression after infection with herpes simplex virus-1, a dna virus. this ability to bind both dsrna and dna raises the question of the feature ddx60 recognizes. it should be finally noted that the role of ddx60 in the ifn-i pathway has been questioned (79) . several other cytoplasmic receptors have been shown to play a role in microbial rna recognition. this is the case for the cytoplasmic protein kinase r (pkr), which is important for antiviral activity. pkr is activated by dsrna from viruses and is a component of mapk and nf-κb signaling pathways [reviewed in ref. (80) ]. activation of pkr can also be mediated by short 5 -ppp rnas containing limited secondary structures (81) . proteins from the interferon-induced protein with tetratricopeptide repeats (ifits) family, such as ifit1 and 5, bind 5 -ppp of viral rna (82) . using short in vitro transcribed oligonucleotides, crystal structure studies have demonstrated that ifit proteins contain a positively charged cavity designed to engage, without any particular sequence specificity, ssrna with a 5 -ppp end. contrary to rig-i, ifit proteins cannot bind blunt-ended 5 -ppp dsrna, and owing to the limitations imposed by their rna-binding pockets, ifit1 and ifit5 require 5'-overhangs of at least 5 or 3 nt, respectively (83) . using a 2 -o-methyltransferase mutant of japanese encephalitis virus, another study showed that ifit1 preferentially binds to 5 capped 2 -o-unmethylated mrna (84) , confirming previous findings showing that 2 -o-methylation of viral mrna caps promotes ifit1 evasion (85, 86) . the mechanism of ifit1 antiviral action is not completely understood, and it has been proposed that ifit might sequester viral rnas (82) or inhibit viral mrna translation (84) . the crystal structure of ifit2 (known as isg54) was also described. ifit2 specifically binds adenylate uridylate (au)rich rnas in vitro, independently of the presence of a 5 -ppp (87) . the authors showed that rna-binding capacity of this protein mediates its antiviral properties, using a model of hek293t cells infected by newcastle disease virus or sev (87) . nucleotide-binding oligomerization domain containing protein 2 (nod2) is a member of the nod1/apaf-1 family and encodes a protein with two card domains and six leucinerich repeats (lrrs). nod2 is primarily known for its ability to recognize bacterial peptidoglycan, but it also plays a role in the antiviral response. nod2 has been shown to activate mavs after stimulation with viral ssrna or human respiratory syncytial virus infection (88) . nlrp3 is involved in cytosolic rna sensing. caspase-1 cleavage triggered by influenza virus, sev, or bacterial mrna is dependent on nlrp3 inflammasome activation (7, 89, 90) . however, direct binding of nod2 or nlrp3 to microbial rna has not been established. leucine-rich repeat flightless-interacting protein 1 (lrrfip1) contributes to the production of ifn-β induced by vsv and l. monocytogenes in macrophages (91) . mostly located in the cytosol, lrrfip1 can also be found in rna-containing lysosomes (92) . lrrfip1 can bind both dsrna and dsdna and subsequently induce ifn-i expression through β-catenin phosphorylation. activated β-catenin is translocated to the nucleus and increases ifn-β expression by binding to the c-terminal domain of the transcription factor irf3 and promoting the recruitment of the acetyltransferase p300 to the ifnb1 promoter. several other microbial rna features have been suspected or proposed to act as potential signals for cytosolic sensing, suggesting the existence of receptors detecting these characteristics. a computational analysis identified cpg motifs in an au-rich rna as an immunostimulatory feature. this sequence motif is underrepresented in both ssrna viruses and host innate immune gene mrna, and its frequency in influenza virus genomes has decreased throughout evolution (93) . since this evolutionary pressure seems to also be applied on host mrna, the implication of a cytosolic receptor is possible, although experimental studies identified endosomal tlr7 as a potential prr (94) . another study identified the nucleotide bias of a-rich hiv-1 genome as a strong inducer of ifn-i and potent mediator of lentiviral pathogenicity. the authors showed that the ability of rna sequences derived from the hiv-1 genome to induce an interferon response correlated with their nucleotide bias and that codon-optimized sequences lost their stimulatory activity (95) . the experimental procedure used in this study consisted of direct delivery via lipofection of in vitro transcribed rna sequences into the cytosol of a reporter cell line, suggesting a potential role for a cytoplasmic rna sensor (95) . recently, our group identified bacterial mrnas as an activator of the nlrp3 inflammasome. polyadenylation of these rnas abrogated their immunostimulatory activities, suggesting that features at the 3 end of mrna, rather than the 5 end, could engage cytoplasmic cellular sensors (7) . philip bevilacqua's group has shown that different nucleoside modifications on rna, such as base or sugar internal modifications, suppress their intrinsic ability to activate immune sensors, notably pkr. the authors propose that self-rna editing could be a mechanism used by the innate immune system to discriminate self-transcripts from "unmodified" microbial rnas (96, 97) . conversely, microbial rna editing by cellular deaminase enzymes such as dsrna-specific adenosine deaminase (adar) have been shown to enhance its recognition by cytosolic sensors (98) . other host transcript specificities, like association to cellular components that prevent prr binding, or specific tertiary structure such as the eukaryotic mrna closed loop conformation (99) , could be determinants for the differentiation of host mrnas from microbial rnas. identification of receptors able to recognize such features are lacking so far. infectious microorganisms have developed several strategies to evade cytosolic sensing. one of these strategies, which we only mention briefly here, is the direct targeting by microbial proteins www.frontiersin.org some (−)ssrna viruses edit the 5 -ppp moieties in their genomes as well as replication intermediates into 5 mono-phosphates to avoid recognition by rlrs (101) . arenaviruses produce rna panhandle structures with a 5 -ppp containing a gtp overhanging nucleotide. this viral structure is suggested to act as a rig-i ligand decoy, by trapping rig-i but not activating it (102) . we are beginning to understand how eukaryotic cells use nucleoside modifications in order to protect self-rnas from innate sensing. for example, higher eukaryotes have acquired the ability to 2 -omethylate their mrnas, allowing cellular receptors to distinguish self from unmethylated non-self mrna through specific types of antiviral sensors such as mda5 and ifits (57, 85) . consistent with the red queen hypothesis (103) , which postulates that parasites have to constantly evolve in order to adapt to their host species, the same immune escape strategy has been mimicked by several pathogens, like flaviviruses (84, 86) . similarly, 2 -o-methylation of g18 (gm18) on bacterial trna suppresses activation of the immune response in plasmacytoid dcs (104, 105) . flaviviruses and other viruses are also known to induce cellular membrane reorganization that allows them to replicate in subcellular compartments, creating new replication-dependent organelles (106) . thus, tick-borne encephalitis virus or japanese encephalitis virus have been shown to rearrange endoplasmic reticulum membranes to provide a compartment where viral dsrna is concealed from prr recognition. this hijacking of internal cell membrane induces a delayed cytosolic exposure of viral rna to innate receptors and accordingly, ifn-i responses are only measured late in the replication cycle (107) (108) (109) . the ns1 protein from influenza virus can prevent rna sensing through the formation of a chain of ns1 molecules along the influenza dsrna backbone (110) . picornaviruses mask their 5ppp with a viral encoded protein, vpg, which functions as a 5 cap and as a primer during rna synthesis. interestingly, studies have shown that vpg could be used to evade rig-i recognition (111) . similarly, ebola virus vp35 assembles into dimmers to cap the ends of viral dsrna and hide the specific rig-i recognition site (112) . while one vp35 monomer binds the terminus and backbone of dsrna, the other vp35 monomer binds only the phosphate backbone of the dsrna, displaying a unique mode of dsrna concealing from prr (112) . another hemorrhagic fever virus, lassa fever virus, uses the 3'-5' exonuclease activity of its nucleoprotein (np) to degrade stimulatory dsrna (113) . this activity seems to be shared by other arenaviruses (114) . finally, the protein c from human parainfluenza virus type 1 (hpiv1), a paramyxoviridae, has been shown to limit the accumulation of dsrna. cell infection by a virus mutant defective for the c protein displays higher accumulation of several viral rnas, including viral genome, antigenome, and mrna, eventually leading to the accumulation of dsrna. thus, by limiting intracytosolic quantities of viral dsrna, the c protein of hpiv1 avoids dsrna triggering of mda5 and pkr in infected cells (115) . the multiplicity of prr pathways is an essential determinant of the immune system's ability to sense with precision the level of frontiers in immunology | molecular innate immunity microbial threat and to respond accordingly (4) . however, as far as cytosolic rna sensors are concerned, it is striking to observe the contrast between the high number of prrs that have been isolated and the similarities of the pamps they recognize ( table 1) . while 5 -ppp and dsrna are undoubtedly powerful triggers of the innate immunity, they cannot account for the diversity of responses that the organism is able to elicit against a wide range of pathogens. our understanding of how the immune system distinguishes between foreign and self-nucleic acids will continue to improve over time. this will help us better define the precise role played by cytosolic rna sensors in the global immune response against pathogens. approaching the asymptote? evolution and revolution in immunology a human homologue of the drosophila toll protein signals activation of adaptive immunity approaching the asymptote: 20 years later beyond pattern recognition: five immune checkpoints for scaling the microbial threat activation of target-tissue immune-recognition molecules by double-stranded polynucleotides immune sensing of dna detection of prokaryotic mrna signifies microbial viability and promotes immunity principles of virology cell-to-cell transmission of viruses plasmacytoid dendritic cells sense hepatitis c virus-infected cells, produce interferon, and inhibit infection human pdcs sense lcmv infected cells in vitro innate sensing of hiv-infected cells expression of animal virus genomes bacterial ligands generated in a phagosome are targets of the cytosolic innate immune system rig-i detects infection with live listeria by sensing secreted bacterial nucleic acids rig-i detects triphosphorylated rna of listeria monocytogenes during infection in non-immune cells identification of host cytosolic sensors and bacterial factors regulating the type i interferon response to legionella pneumophila rig-i-dependent sensing of poly(da:dt) through the induction of an rna polymerase iii-transcribed rna intermediate rna polymerase iii detects cytosolic dna and induces type i interferons through the rig-i pathway ips-1, an adaptor triggering rig-i-and mda5-mediated type i interferon induction cardif is an adaptor protein in the rig-i antiviral pathway and is targeted by hepatitis c virus identification and characterization of mavs, a mitochondrial antiviral signaling protein that activates nf-kappab and irf 3 visa is an adapter protein required for virus-triggered ifn-beta signaling expression analysis and genomic characterization of human melanoma differentiation associated gene-5, mda-5: a novel type i interferon-responsive apoptosis-inducing gene the rna helicase rig-i has an essential function in double-stranded rna-induced innate antiviral responses 5'-triphosphate rna is the ligand for rig-i rig-i-mediated antiviral responses to single-stranded rna bearing 5'-phosphates recognition of 5' triphosphate by rig-i helicase requires short blunt doublestranded rna as contained in panhandle of negative-strand virus 5'-triphosphate rna requires base-paired structures to activate antiviral signaling via rig-i structural basis of rna recognition and activation by innate immune receptor rig-i structural basis for the activation of innate immune pattern-recognition receptor rig-i by viral rna structural insights into rna recognition by rig-i molecular mechanism of signal perception and integration by the innate immune sensor retinoic acid-inducible gene-i (rig-i) a structural basis for discriminating between self and nonself double-stranded rnas in mammalian cells length-dependent recognition of double-stranded ribonucleic acids by retinoic acid-inducible gene-i and melanoma differentiation-associated gene nonself rna-sensing mechanism of rig-i helicase and activation of antiviral immune responses the thermodynamic basis for viral rna detection by the rig-i innate immune sensor rig-i detects viral genomic rna during negative-strand rna virus infection incoming rna virus nucleocapsids containing a 5'-triphosphorylated genome activate rig-i and antiviral signaling differential roles of mda5 and rig-i helicases in the recognition of rna viruses innate immunity induced by composition-dependent rig-i recognition of hepatitis c virus rna preference of rig-i for short viral rna molecules in infected cells revealed by next-generation sequencing epstein-barr virus-encoded small rna induces il-10 through rig-i-mediated irf-3 signaling adenovirus virus-associated rnas induce type i interferon expression through a rig-i-mediated pathway the 5' ends of rna oligonucleotides in escherichia coli and mrna degradation extracellular and intracellular pattern recognition receptors cooperate in the recognition of helicobacter pylori ifngamma inhibits the cytosolic replication of shigella flexneri via the cytoplasmic rna sensor rig-i essential role of mda-5 in type i ifn responses to polyriboinosinic:polyribocytidylic acid and encephalomyocarditis picornavirus structural basis for dsrna recognition, filament formation, and antiviral signal activation by mda5 double-stranded rna is produced by positive-strand rna viruses and dna viruses but not in detectable amounts by negative-strand rna viruses activation of mda5 requires higher-order rna structures generated during virus infection mda5 detects the double-stranded rna replicative form in picornavirus-infected cells innate immune response after adenoviral gene delivery into skin is mediated by aim2, nalp3, dai and mda5 visualisation of direct interaction of mda5 and the dsrna replicative intermediate form of positive strand rna viruses activation of ifn-&#946; expression by a viral mrna through rnase l and mda5 new insights into the role of rnase l in innate immunity ribose 2'-o-methylation provides a molecular signature for the distinction of self and non-self mrna dependent on the rna sensor mda5 mavs forms functional prion-like aggregates to activate and propagate antiviral innate immune response intracellular pathogen detection by rig-i-like receptors the adaptor mavs promotes nlrp3 mitochondrial localization and inflammasome activation recognition of rna virus by rig-i results in activation of card9 and inflammasome signaling for interleukin 1 beta production type i ifn triggers rig-i/tlr3/nlrp3-dependent inflammasome activation in influenza a virus infected cells the rig-i-like receptor lgp2 recognizes the termini of double-stranded rna the regulatory domain of the rig-i family atpase lgp2 senses doublestranded rna lgp2 is a positive regulator of rig-i-and mda5-mediated antiviral responses atp hydrolysis enhances rna recognition and antiviral signal transduction by the innate immune sensor, laboratory of genetics and physiology 2 (lgp2) lgp2 plays a critical role in sensitizing mda-5 to activation by double-stranded rna from unwinding to clamping -the dead box rna helicase family dexd/h-box rna helicases as mediators of anti-viral innate immunity and essential host factors for viral replication dead/h box 3 (ddx3) helicase binds the rig-i adaptor ips-1 to up-regulate ifn-beta-inducing potential viral targeting of dead box protein 3 reveals its role in tbk1/ikkepsilon-mediated irf activation hepatitis c virus core protein abrogates the ddx3 function that enhances ips-1-mediated ifn-beta induction dhx9 pairs with ips-1 to sense double-stranded rna in myeloid dendritic cells ddx1, ddx21, and dhx36 helicases form a complex with the adaptor molecule trif to sense dsrna in dendritic cells aspartateglutamate-alanine-histidine box motif (deah)/rna helicase a helicases sense microbial dna in human plasmacytoid dendritic cells the dhx33 rna helicase senses cytosolic rna and activates the nlrp3 inflammasome the interaction between the helicase dhx33 and ips-1 as a novel pathway to sense double-stranded rna and rna viruses in myeloid dendritic cells ddx60, a dexd/h box helicase, is a novel antiviral factor promoting rig-i-like receptor-mediated signaling cytosolic sensing of viruses interferon-inducible antiviral effectors 5'-triphosphate-dependent activation of pkr by rnas with short stem-loops ifit1 is an antiviral protein that recognizes 5'-triphosphate rna structural basis for viral 5'-ppp-rna recognition by human ifit proteins ifit1 inhibits japanese encephalitis virus replication through binding to 5' capped 2'-o unmethylated rna 2'-o methylation of the viral mrna cap evades host restriction by ifit family members 2'-o methylation of the viral mrna cap by west nile virus evades ifit1-dependent and -independent mechanisms of host restriction in vivo crystal structure of isg54 reveals a novel rna binding structure and potential functional mechanisms activation of innate immune antiviral responses by nod2 critical role for cryopyrin/nalp3 in activation of caspase-1 in response to viral infection and double-stranded rna the nlrp3 inflammasome mediates in vivo innate immunity to influenza a virus through recognition of viral rna the cytosolic nucleic acid sensor lrrfip1 mediates the production of type i interferon via a betacatenin-dependent pathway characterization of lrrfip1 patterns of oligonucleotide sequences in viral and host cell rna identify mediators of the host innate immune system oligonucleotide motifs that disappear during the evolution of influenza virus in humans increase alpha interferon secretion by plasmacytoid dendritic cells the biased nucleotide composition of hiv-1 triggers type i interferon response and correlates with subtype d increased pathogenicity nucleoside modifications modulate activation of the protein kinase pkr in an rna structure-specific manner native tertiary structure and nucleoside modifications suppress trna's intrinsic ability to activate the innate immune sensor pkr inosine-containing rna is a novel innate immune recognition element and reduces rsv infection the mechanism of eukaryotic translation initiation and principles of its regulation targeting of immune signalling networks by bacterial pathogens processing of genome 5' termini as a strategy of negative-strand rna viruses to avoid rig-i-dependent interferon induction short doublestranded rnas with an overhanging 5' ppp-nucleotide, as found in arenavirus genomes, act as rig-i decoys a new evolutionary law identification of modifications in microbial, native trna that suppress immunostimulatory activity the 2'-o-methylation status of a single guanosine controls transfer rna-mediated toll-like receptor 7 activation or inhibition viral reorganization of the secretory pathway generates distinct organelles for rna replication tick-borne encephalitis virus delays interferon induction and hides its double-stranded rna in intracellular membrane vesicles delayed cytosolic exposure of japanese encephalitis virus double-stranded rna impedes interferon activation and enhances viral dissemination in porcine cells formation of membrane-defined compartments by tick-borne encephalitis virus contributes to the early delay in interferon signaling x-ray structure of ns1 from a highly pathogenic h5n1 influenza virus the genome-linked protein vpg of vertebrate viruses -a multifaceted protein ebolavirus vp35 uses a bimodal strategy to bind dsrna for innate immune suppression structure of the lassa virus nucleoprotein reveals a dsrna-specific 3' to 5' exonuclease activity essential for immune suppression structures of arenaviral nucleoproteins with triphosphate dsrna reveal a unique mechanism of immune suppression the c proteins of human parainfluenza virus type 1 limit double-stranded rna accumulation that would otherwise trigger activation of mda5 and protein kinase r the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. key: cord-328252-dk54w8z9 authors: kikkert, marjolein title: innate immune evasion by human respiratory rna viruses date: 2019-10-14 journal: journal of innate immunity doi: 10.1159/000503030 sha: doc_id: 328252 cord_uid: dk54w8z9 the impact of respiratory virus infections on the health of children and adults can be very significant. yet, in contrast to most other childhood infections as well as other viral and bacterial diseases, prophylactic vaccines or effective antiviral treatments against viral respiratory infections are either still not available, or provide only limited protection. given the widespread prevalence, a general lack of natural sterilizing immunity, and/or high morbidity and lethality rates of diseases caused by influenza, respiratory syncytial virus, coronaviruses, and rhinoviruses, this difficult situation is a genuine societal challenge. a thorough understanding of the virus-host interactions during these respiratory infections will most probably be pivotal to ultimately meet these challenges. this review attempts to provide a comparative overview of the knowledge about an important part of the interaction between respiratory viruses and their host: the arms race between host innate immunity and viral innate immune evasion. many, if not all, viruses, including the respiratory viruses listed above, suppress innate immune responses to gain a window of opportunity for efficient virus replication and setting-up of the infection. the consequences for the host's immune response are that it is often incomplete, delayed or diminished, or displays overly strong induction (after the delay) that may cause tissue damage. the affected innate immune response also impacts subsequent adaptive responses, and therefore viral innate immune evasion often undermines fully protective immunity. in this review, innate immune responses relevant for respiratory viruses with an rna genome will briefly be summarized, and viral innate immune evasion based on shielding viral rna species away from cellular innate immune sensors will be discussed from different angles. subsequently, viral enzymatic activities that suppress innate immune responses will be discussed, including activities causing host shut-off and manipulation of stress granule formation. furthermore, viral protease-mediated immune evasion and viral manipulation of the ubiquitin system will be addressed. finally, perspectives for use of the reviewed knowledge for the development of novel antiviral strategies will be sketched. sion. many, if not all, viruses, including the respiratory viruses listed above, suppress innate immune responses to gain a window of opportunity for efficient virus replication and setting-up of the infection. the consequences for the host's immune response are that it is often incomplete, delayed or diminished, or displays overly strong induction (after the delay) that may cause tissue damage. the affected innate immune response also impacts subsequent adaptive responses, and therefore viral innate immune evasion often undermines fully protective immunity. in this review, innate immune responses relevant for respiratory viruses with an rna genome will briefly be summarized, and viral innate immune evasion based on shielding viral rna species away from cellular innate immune sensors will be discussed from different angles. subsequently, viral enzymatic activities that suppress innate immune responses will be discussed, including activities causing host shut-off and manipulation of stress granule formation. furthermore, viral protease-mediated immune evasion and viral manipulation of the ubiquitin system will be addressed. finally, perspectives for use of the reviewed knowledge for the development of novel antiviral strategies will be sketched. the epithelium of the lungs is the largest surface in the human body that is in contact with our environment. huge amounts of air and aerosols pass these cells each day, whereby the lung tissue, as well as the rest of the respiratory tract is probably almost constantly exposed to viruses and bacteria present in the inhaled air. an elaborate machinery is therefore present at this large surface to defend this tissue against invading pathogens, including mechanical barriers such as a mucus layer. the first line of defense at the entire length of the tract from the nasopharynx to the alveolar membrane is formed by the innate immune system [1, 2] . in this review, the focus will be on the selection of common viruses that invade the lungs: coronaviruses (covs), rhinoviruses, respiratory syncytial virus (rsv), and influenza, which all have an rna genome. this latter feature is of importance to the set of cellular innate immune sensors that recognize these viruses when they enter the cells of the respiratory tract, and the subsequent downstream signaling cascades that are triggered as a result. a myriad of different cell types such as alveolar macrophages, airway epithelial cells, innate lymphoid cells, and dendritic cells (dcs) have a major role in this first defense, while in these and other cells of the respiratory tract the sensing, and several subsequent specific molecular intra-and intercellular signaling cascades ensure the establishment of the so-called antiviral state in the lungs. this state can inhibit the development of a productive infection with each of these invading viruses, thereby preventing or at least mitigating illness, before adaptive immunity kicks in to completely clear these viruses from the lungs. importantly, as a countermeasure against these elaborate defense mechanisms, invading respiratory viruses evolve activities that either circumvent or suppress the innate immune responses to create a window of opportunity for efficient virus replication, thereby often causing disease. ultimately, the balance between the efficacy of the combined innate and adaptive responses on the host's side, and the virulence and its capacity to evade the host's immune responses on the virus' side, together dictate the disease outcome. this review will focus on the evasion of the innate immune system by the array of respiratory viruses as introduced above, to highlight this important aspect of the virus-host interaction that may provide us with possible opportunities for exploration of novel antiviral strategies against these important viruses. particular viral activities will be highlighted and different viruses compared, but the information discussed will not be complete. i therefore apologize to any authors who miss discussion of their interesting work in this review. to facilitate comparison between the respiratory viruses described here, known and arguably important innate immune evasion strategies are listed, and for each strategy it is discussed how each virus group exploits its own mechanism. innate immune evasion obviously links to the innate immune responses that are known to be elicited by respiratory and other (rna) viruses, and while this will be elaborated to a limited extent below, they have also been reviewed comprehensively in recent reviews by others [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] . arguably, the innate immune system is more important in early life, when the adaptive functions are still underdeveloped [14] . yet, the young infant is probably exposed to as many incoming pathogens as older children and adults are, so the innate immune system plays a very important role in the protection from respiratory infection in young children. the fact that respiratory infections are one of the leading causes of mortality in children under 5 years of age [18, 19] suggests that the interactions of the (innate) immune responses in the infant respiratory tract with incoming pathogens is indeed a delicate one, and the balance between severe illness and overcoming an infection may be relatively easily tipping towards the dangerous side. that the innate immune response plays an important role in defense against respiratory infections in early life may be further illustrated by the fact that severe rsv infections in children are linked with polymorphisms in genes encoding innate immune factors (reviewed in [14, 20] ). also later in life, the innate immune system plays an important role in the response against respiratory viruses (reviewed in [1] ), and in the lungs these first responses against incoming viruses are governed primarily by alveolar and interstitial macrophages, dcs, airway epithelial cells, innate lymphocytes, and neutrophils. the innate immune response signaling cascade starts with the recognition of pathogen-associated molecular patterns by pattern recognition receptors (prrs). for rna viruses in the lungs, the toll-like receptors (tlrs) 3, 7 and 8, which are expressed on several of the mentioned cell types, are important prrs. also, intracellular cytosolic prrs such as mda5 and rig-i, which are present in virtually any cell type including those of the lung, have been shown to be relevant for respiratory infections, as will be elaborated below. each of these mentioned receptors, or sensors, recognize forms of rna (e.g., 5′ triphosphate rna, double-stranded rna [dsrna]) that are produced by (respiratory) rna viruses during their infection process, and which are distinguishable from the rna species that are normally present in the cells (such as capped mrna in the cytosol). in this way, the innate immune system senses foreign material that is possibly pathogenic, and this triggers downstream signaling to ultimately induce transcription factors in the nucleus which in turn stimulate expression of types i and iii interferons (ifns) and other proinflammatory cytokines. a second round of autocrine and paracrine signaling subsequently ensures that infected, and the surrounding uninfected cells, express a myriad of interferon stimulated genes (isgs) that establish a so-called antiviral state. this state quite efficiently inhibits further spread of the infection, and simultaneously triggers further adaptive responses that in most cases eventually will clear the virus from the infected individual. during all these signal transduction pathways, regulation of activation and inhibition of signal transduction in the cascades is governed in a strict manner by phosphorylation events as well as ubiquitination of different linkage types (k48, k63, k27, etc.) on numerous factors in the pathways (reviewed in [21] ). these events critically regulate the downstream signaling to ensure a sufficiently strong, but not overly explosive triggering of innate immune responses, and a timely downregulation of these responses to protect the individual from damaging immunopathology. recently, it has become clear that particular type iii ifns (il-28/29), or ifn lambdas, which were discovered in 2003 [22, 23] , play a prominent role in defense of epithelial surfaces such as that in the lung (reviewed in [3, 5, 24] ). they bind to a distinct heterodimeric receptor consisting of ifnlr1 and il10rb (as opposed to type i ifn that binds to ifnar1/2), but seem to trigger downstream signaling that is very similar to the type i ifn-induced pathways, and are also induced by the same prrs as those triggering type i ifns. however, whereas type i ifns are made by many different cell types, ifn lambdas are primarily expressed by epithelial cells and dcs. recent literature suggests that despite the clear similarities between the types i and iii ifn signaling pathways, the type iii ifn machinery seems especially equipped to protect epithelial surfaces from pathogenic attacks, and forms the primary local defense upon invasion of low doses of viruses and bacteria. when this first activation of the type iii ifn machinery is insufficient due to higher doses of pathogens coming in, the more systemic type i ifn machinery forms the second line of defense over broader areas of the tissue (reviewed in [24] ). additionally, it seems that type iii ifn does not trigger inflammation as much as type i ifn, and this probably indicates an important unique aspect of the type iii ifn induction, which may have a role in the protection of, for example, the lung epithelial tissue from immunopathology [25] . recently, it has become clear that the strict distinction between innate and adaptive responses that has been the general view for a long time is probably not accurate. in the respiratory tract, several of the newly identified cell types and mechanisms that integrate aspects from both branches of human immunity are now thought to be very important for the defense against respiratory infections. natural killer t cells, mucosal-associated invariant t cells, and neutrophils, for example, each form a bridge between the innate and adaptive machineries and play very important roles during the clearance of respiratory viruses (reviewed in [1, 6, 10] ). aspects of immunological memory, which were thought to be only present in the adaptive immune system, have now clearly been shown to play a role in the innate immune response as well, also that induced by viruses, and was named "trained innate immunity" [9] . the general idea about the mechanism governing this is that epigenetic changes on innate immune factor genes in specialized immune cells such as macrophages are made after the activation of the innate immune response. this then positively influences the response upon a subsequent pathogen encounter, just as in the adaptive immune system [26] . recently, it also became clear that after respiratory (bacterial) infections this mechanism indeed has a role, and strikingly, signaling from adaptive (cd8+ t cell responses) "back" to innate immune systems (alveolar macrophages) via ifn-gamma plays a role in generating epigenetically triggered innate immune memory to protect from re-infection [27, 28] . besides these different responses, most of which are ifn-mediated, small non-coding (micro, circular, ...) rnas, rnai, and ifn-independent antiviral responses can be regarded as part of the innate immune response package as well [29] [30] [31] . an emerging hot topic is also the interplay of innate immune response with cellular metabolism, so-called immunometabolism, which likely is quite relevant for respiratory viral infections [4, 32, 33] . the general idea is that immune cells such as macrophages and dcs adapt the choice for the use of their metabolic systems to an immune-activated situation that requires increased amounts of energy. this resembles "the warburg effect", as described in tumor cells, and after pathogen sensing innate immune response thus triggers changes in the cell's metabolism from oxidative phosphorylation to glycolysis, thereby optimizing the cell's metabolism for the new situation [34] . since the new insights mentioned above have generally not yet, or only to a limited extent, been investigated in the context of viral evasion, this will not be further elaborated in the subsequent sections for the selected respiratory viruses. viruses with an rna genome, such as the respiratory viruses highlighted in this review, produce several rna species during viral replication, which are normally absent in uninfected cells. for example, dsrna and rna with a 5′-triphosphate are commonly produced by rna viruses during replication, but since the host cells do not normally copy rna from rna templates, these intermediate rna species are recognized by the innate immune sensors discussed above as foreign, resulting in antiviral effector activation. to be able to set up a productive infection in the cell, these viruses therefore need to circumvent and/or suppress these intracellular innate antiviral responses. an obvious primary strategy would be to shield away the replication intermediates with their dangerous, recognizable features, from the innate immune sensors roaming the cytosol. indeed, the viruses that have a +rna genome, which replicate exclusively in the cytosol such as the covs and rhinoviruses that invade the lungs, generally modify intracellular membranes elaborately to form headquarters of viral rna replication, also called "replication organelles" (ros; covs), "replication factories," "double membrane vesicles" (dmvs; covs, enteroviruses), "invaginations," or other (reviewed in [35] [36] [37] ). also, the negative-stranded rsv genome and its replication enzymes are found associated with cytosolic occluded structures, in that case named inclusion bodies [38, 39] . expression of a selection of specific hydrophobic viral proteins can usually mimic the formation of these structures, for example, nsp3 and nsp4 of covs [40] , the n and p proteins of rsv [41] , and 2b,2c and 3a proteins of enterovirus (polio; [42] ). all these structures, while divers in morphology and contents, seem to concentrate the viral replication machinery, intermediates and products inside membrane-bound vesicles or invaginations, seemingly unreachable for the innate immune sensors of the cytosol. it is interesting to note that very little is known about the details of interaction of viral replication organelles with the innate immune system. while the protective function of such organelles in the context of innate immune sensing is assumed by many researchers, hardly any reports present investigation, let alone proof, of this concept. a report by al-mulla and co-workers showed that in cov mutants that produced only half the number of ros during infection or in which the structures were smaller, replication as well as fitness of these viruses was in fact unaffected or even higher than for wt viruses. this was also true in cultures of primary host cells, which presumably have a fully functional intracellular innate immune system [43] . their results indicated that there is no strict correlation between the number of replication organelles and the replication rate of these viruses. it is not clear, however, whether (part of) the viral replication takes place outside the replication organelles in these mutant virus infections, and whether replication organelles do, or do not, protect viral replication from innate immune attack therefore remains elusive after all. importantly, virtually all research investigating the role and structure of viral ros was performed in cell cultures, and little is known about their presence or numbers during infections in animal models or real hosts. investigation of the latter will, therefore, be pivotal for the true understanding of viral ros and their role in protection from innate immune responses. besides the question whether the replication organelles protect from innate immune sensors that recognize viral rna, it is also largely unclear whether the innate immune system possesses sensors or effectors that target viral replication organelles themselves. after all, all +rna viruses produce membranous replication organelles, and since they are probably indeed supporting viral replication, recognizing and attacking them would provide an efficient way for the innate immune system to inhibit viral infection. our recent research revealed that the type i ifn signaling cascade, which is utterly relevant for defense against +rna viruses, indeed includes effectors that influence the integrity of ros induced by equine arteritis virus, a +rna arterivirus and a distant relative of the covs [44] . however, it is not yet clear which type i ifn-inducible factors are responsible. some recent reports (reviewed in [45] [46] [47] ) suggest that intracellular membrane modifications such as viral ros can be recognized and targeted by guanylate-binding proteins (gbps), a family of dynamin-related large gtpases, of which mxa is a member. mxa is a well-known human type i and iii interferon-inducible factor that inhibits influenza virus infections [48] . although the exact mechanism of inhibition is still not clear for several of the viruses inhibited by mx proteins, mx gtpase family members bind to intracellular membranes, and in cytosolic +rna virus infections mx proteins could target the ros [48] . since influenza replicates in the nucleus (see also below), the idea is that mxa attacks influenza while its products are in the cytosol. several reports indicate that gbps other than the mx proteins act against human +rna viruses such as hepatitis c virus, classical swine fever virus, and dengue virus, which are all members of the flavivirus family, possibly by attacking their ros. in pigs, gbps inhibit porcine reproductive and respiratory syndrome virus (an arterivirus, distantly related to the covs). in mice, encephalomyocarditis virus and murine norovirus, which are both +rna viruses, are suppressed by interferon (-gamma)induced gbps [45] . for murine norovirus, it has now become clear that gbps are indeed targeted to viral ros and that this depends on part of the autophagy machinery, namely the lc3 conjugation system [49] . lipidated lc3 associates with viral ros, and while this does not depend on ifn-gamma induction it is clearly stimulated by it. the authors of this paper also mention in their discussion that similar mechanisms can be shown for encephalomyocarditis virus, suggesting that at least several +rna virus induced ros can be targeted by the innate immune system via gbps [49] . ultimately, the idea is that once gbps associate with the viral ro membranes, they cause disruption and/or modification of these structures, resulting in less efficient viral replication [49, 50] . mechanistically, this effect on viral replication could link to the viral rna species and intermediates becoming exposed upon disruption of ro membranes by gbps to the cytosolic innate immune rna sensors such as rig-i and mda5, which subsequently triggers antiviral innate and adaptive immune responses to suppress further replication. further research is needed to confirm such a hypothesis. interestingly, while covs, rhinoviruses, and rsv replicate in the cytosol of respiratory epithelial cells and shield their replicating rnas as discussed above, influenza virus apparently takes another route, and as the only known exception to the rule this rna virus replicates in the nucleus. rna sensors like the rig-i-like sensors or tlrs were thought to be absent there, and therefore replication inside the nucleus may have been an alternative solution to avoid innate immune recognition of viral rna intermediates during replication. however, recent data indicated that rig-i can be active in the nucleus against influenza rna [51] . the viral genome, packaged in nucleocapsid proteins and bearing a panhandle-and 5′-triphosphate structure is recognized by rig-i, presumably in the cytosol while on its way to the nucleus, or when being incorporated into new virus particles [52] [53] [54] . the recognition by rig-i is the major trigger to the production of type i ifn during influenza infection, while also tlr3 plays a role [55] . additionally, the cell has evolved multiple ways to attack influenza replication, for example, by gbps that are localized in the nucleus and the cytosol [56] . in summary, the formation of membranous headquarters may be a major strategy for respiratory viruses to avoid innate immune recognition of viral nucleic acid products in the cytosol. whether the cell can in turn recognize and attack these structures is still relatively unknown, along with viral countermeasures against these attacks. this kind of interactions illustrates the arms race between the cellular immune responses and viral evasion, which due to continuous evolution often has multiple levels. protection of the 5′ terminus of viral rnas apparently, the shielding of viral replication products by ros is not a watertight system, and to further avoid recognition of their foreign rna species, respiratory viruses have evolved several means of directly modifying these rnas to avoid recognition by the innate immune rna sensors. adding a cap-structure or a mimic of this structure to the 5′-end is an effective way, since in this way the cell's own mrnas are protected from recognition by the innate immune sensors. the respiratory viruses discussed here use quite diverse methods to achieve this kind of protection from recognition, concomitantly making sure their mrnas can be properly recognized by the translation machinery of the cell, which they "chose" to utilize. the rhinoviruses are members of the picornavirus family, and these use a specialized, virally encoded capmimicking peptide, called vpg, and attach this to the viral rna 5′ end to protect it from recognition by the innate rna sensors [57, 58] . these viruses indeed do not need a cap structure for translation of their rnas, since they use cap-independent internal ribosomal entry site-mediated translation [59, 60] . influenza viruses steal mrna cap-structures from host mrnas in the nucleus during transcription in a process called "cap-snatching," in j innate immun 2020;12:4-20 doi: 10.1159/000503030 which the viral nucleoprotein plays a major role [61] . rsv and covs provide their mrnas with cap-structures themselves, using enzymatic functions in their polymerase complexes. interestingly, rsv rnas have capstructures that contain a 7-methyl guanosine; however, these caps are devoid of 2'-o-methylation [62] . both methylations are part of the canonical cap-structures on cellular mrnas, but why this is necessary was actually unknown. a more recent report in which covs and their cap-structures were studied indicated that the latter viruses make sure to add 2'-o methylation to their capstructures using a dedicated viral enzyme called nsp16. this turned out to be important to avoid recognition by the mda5 sensor and subsequent triggering of innate immune responses [63, 64] . rsv apparently does not need this 2'-o-methylation on its caps, and this may be explained by the observation that this virus is able to sequester mda5 (and innate immune adapter mavs) into its inclusion bodies (the rsv replication headquarters as discussed above) using association with its n protein, to avoid mda5-dependent recognition of viral rna species and subsequent innate immune response [38] . yet another activity provides additional means of avoiding recognition, and that is viral endoribonuclease activity. covs encode endonuclease activity in one of their non-structural proteins, and recent reports indicated that this is instrumental to avoid recognition by the mda5, protein kinase r (pkr), and oas/rnase l machineries [65, 66] . the latter 2 systems recognize and destroy foreign rna in the cytosol independently of the rig-i-like sensors to remove microbial products. though it may be counter-intuitive for an rna virus to express an rnase, the virus apparently destroys its own rna at certain locations or in certain stages of the infection to avoid the triggering of the rna sensing and virus-destroying machineries. influenza also encodes one or more endoribonucleases, the primary one in the pa protein, which is part of the viral polymerase complex together with the pb-1 and pb-2 subunits. the pa endonuclease is responsible for cleaving the host mrnas for cap-snatching during transcription of the influenza rna [67, 68] , another mechanism of innate immune evasion that was discussed above. additionally, many influenza strains express shorter forms of this protein encoded by the same gene, overlapping with pa at the n-terminal region, but with an alternative or truncated c-terminal region, added through a ribosomal frame shift or by natural truncation, respec-tively [69] . these alternative products of the pa gene from segment 3 of the influenza genome are called pa-x or paxdeltac20, which were discovered recently to also have an endonuclease activity. these were shown to have a role in innate immune evasion, although the truncated paxdeltac20 seems to have very low endonuclease activity [70] . the immune modulation by these alternative pa proteins is thought to be achieved by stimulating host shut-off, another innate immune evasion strategy further discussed below, whereby host cell mrnas are destroyed to suppress the expression of host proteins, including those involved in the activation of the innate antiviral state. interestingly though, pa-x was shown to cleave dsrna quite efficiently [70] , which may not be very relevant for host shut-off, as the cell does not really produce dsrna. whether pa-x also degrades viral dsrna species to prevent recognition by cytosolic rna sensors is not entirely clear, but mutant viruses in which this pa-x protein was expressed in significantly lower amounts elicited higher levels of innate immune response; for example, ifn-beta production was much higher in these infections [71] . this indeed suggests that pa-x, besides having a role in the degradation of cellular mrnas, may also degrade viral rna to prevent recognition by innate immune sensors and activation of innate immune responses, similar to what was shown for the covs. to my knowledge, an endoribonuclease has not been identified in the rsv genome, so this virus may use alternative innate immune evasion strategies, as discussed elsewhere in this review. the same counts for the rhinoviruses. besides the replication organelles, the viral 5′ end rna capping/protection mechanisms, and the viral endonucleases, other ways of shielding rna from innate immune sensors or protecting it from degradation are exploited by respiratory viruses. influenza non-structural protein ns1, for which many different innate immune evasion strategies have been described, binds and sequesters viral rna to protect it from being sensed by rig-i, and this also protects from the activation of pkr and oas/rnase l-mediated viral rna degradation [72] [73] [74] . recent data hint at the importance of protecting the 3′ ends of viral rnas as well, besides the 5′ ends, as it was shown that tut4 and tut7, 2 cellular terminal uridylyltransferases, can add one or 2 uridines to the 3′ ends of polyadenylated influenza mrnas, as well as rnas of several other viruses, to target these rnas for degradation by cellular machineries [75] [76] [77] . additionally, a recent report indicated that cytosolic coronaviral mrnas are targeted by the cellular nonsense-mediated decay pathway, a pathway that detects aberrant translation termination doi: 10.1159/000503030 features such as premature termination codons in mrna, resulting in the degradation of these mrnas [78] . in the case of cov, the viral n protein plays a role in counteracting this latter effect [79] , presumably by packaging the viral rnas, thereby protecting them from degradation. all these data suggest that viruses likely evolved escape mechanisms to avoid all these different cellular mechanisms for rna degradation to be able to set-up a productive infection in this hostile environment, however, the details of several of these mechanisms for the respiratory viruses discussed here are still unknown. host shut-off general host shut-off, that is viruses halting cellular protein expression, is an effective way to actively suppress all cellular innate immune responses against the virus, and simultaneously provide the virus with the full capacity of the cellular translation machinery for their own use. besides using viral endoribonucleases pa-x and derivatives to attack cellular mrnas, as has been briefly discussed for influenza viruses above, the viral polymerase complex and the viral "immune evasion" ns1 each also contribute importantly to host shut-off during influenza infection. since the polymerase complex takes care of cap-snatching, it will leave considerable amounts of cellular mrnas without a cap, and this in fact triggers the degradation of these molecules by cellular machineries such as xrn2 exonuclease, diminishing the general cellular mrnas available for translation. additionally, the interactions of viral polymerase complex with the cellular translation machinery cause degradation of pol ii, thereby inhibiting cellular mrna production and translation [80] . in 1998, nemerof et al. discovered the role of influenza encoded ns1 in host shut-off [81] . ns1 interacts with an essential component of the 3′ end processing machinery of cellular pre-mrnas, cpsf30, whereby 3′-end cleavage and polyadenylation of cellular mrnas is inhibited, thereby contributing to host shut-off. in the past decades, details of the molecular mechanism in which ns1 influences host shut-off have been investigated, and it is also clear that these mechanisms can be strain-specific [72, 80] . like influenza viruses, covs such as sars-cov and mers-cov also use a combination of ways to achieve host shut-off both at the transcriptional and the translational levels. nsp1, the most 5′-terminal subunit of the replicate polyprotein of these viruses, was shown to cause host shutoff by binding to cellular factors of the translation machin-ery thereby preventing translation of host mrnas. sars-cov nsp1 binds the 40s subunit of ribosomes to halt translation [82] [83] [84] [85] , however, for the mers-cov encoded nsp1 the mechanism of halting translation of cellular mrna seems a bit different [86] . one of the differences is that mers-cov encoded nsp1 distinguishes between cellular mrnas produced in the nucleus, and viral mrnas in the cytosol, and the translation of the latter is not inhibited by mers-cov nsp1. in this way, specificity towards disrupting cellular mrna translation is achieved [86] . this is different from sars-cov nsp1, which inhibits all mrna translations. additionally, the nsp1 protein of both viruses causes host mrna degradation, however, not through intrinsic endoribonuclease activity of nsp1 itself but by activating the cellular mrna degradation machinery and its exonuclease xrn1 [82, 83, 86, 87] . rhinoviruses, like poliovirus and other enteroviruses, cleave translation initiation factor elf4g to shut down cap-dependent translation of cellular mrnas. this does not interfere with the translation of viral mrnas since these viruses depend on internal ribosomal entry site-mediated translation (see above). the 2a protease of these viruses is responsible for this, by directly cleaving this factor [88, 89] . recent work indicated that interaction of rhinovirus a encoded 2a protease with elf4e, another subunit of the cellular translation initiation complex, is required for the cleavage of elf4g during infection [90] . finally, for rsv little is known about possible host shut-off mechanisms. a report by bruce et al. [91] suggested that rsv specifically targets mrna encoding surfactant protein a, an innate immune factor with an important role in the epithelial tissue of the lung, which directly binds to virus particles to cause their destruction by host defense mechanisms. during rsv infection, surfactant protein a mrna translation efficiency seems inhibited, however, the mechanism for this effect has not been elucidated to date. an indirect way for viruses to manipulate host mrna expression besides the classical host shut-off mechanisms discussed for the other respiratory viruses above, may be the induction of stress granules. in these structures, cellular mrnas are accumulated upon induction of cellular stress responses that lead to inhibition of cellular translation. rsv, for example, seems to induce stress granules and this benefits its replication, as will be discussed in the next section. stress granules are structures in which, upon stress responses such as resulting from virus infections, the cell concentrates mrnas that are produced but can no longer j innate immun 2020;12:4-20 doi: 10.1159/000503030 be translated. the triggering of pkr, a viral rna sensor, for example, causes phosphorylation of the eif2alpha translation factor, which halts cellular translation thereby also affecting viral translation. the accumulation of untranslated mrnas and stalled translation and pre-initiation complexes trigger the formation of stress granules. recent insights suggest that stress granules may form a platform for innate immune responses, since the accumulation of (viral) rna species there provides a pool of substrates for cellular sensors such as rig-i and mda5 [92] [93] [94] . indeed, these sensors have been shown to be recruited to stress granules, supporting this view [94] [95] [96] . in the last decade, it has become clear that many viruses manipulate stress granule formation to benefit their replication, for example, rsv, as was also mentioned above. in the later stages of rsv infection in epithelial cells, stress granules are formed, and if expression of g3bp, a factor that is essential for stress granule formation, is knocked-down, replication of rsv is inhibited, suggesting a beneficial role for stress granules [97] . a subsequent report showed that pkr activation is required for the induction of stress granules by rsv, however, this is dispensable for viral replication [98] . several other reports also show how rsv counteracts the formation of stress granules [99] , suggesting a negative effect of stress granule formation on rsv infection. up till today, it therefore remains unclear what the role of stress granules during rsv infection is exactly. covs also manipulate stress granule formation. in collaboration with the group of frank van kuppeveld, our lab showed that mers-cov encoded 4a protein (translated from orf4 in the virus) impedes dsrna-mediated pkr activation, thereby preventing stress granule formation [100] . protein 4a binds viral dsrna, which is essential for its antagonistic function in pkr activation and stress granule formation, suggesting that 4a prevents recognition of viral rna by pkr. recombinant mers-cov in which orf 4 (encoding 4a and 4b proteins) was removed, however, still suppressed stress granule formation in vero cells, suggesting that 4a's activity is not the only way in which the virus inhibits stress granule formation [100] . indeed, cov nsp1 with its host-shut-off activities (see above) is a likely candidate viral protein that could play a role. a later report by nakagawa et al. [101] however showed that the orf4 mers-cov mutant virus did induce stress granules in another cell line (hela/ cd26), and also a virus mutant in which 4a alone was removed was not able to suppress sg formation in these cells. this suggests that the activity of 4a, and possibly other stress granule-inhibiting mers-cov proteins, may differ per cell line, or that cell lines differ in the activity of their antiviral pathways. influenza virus infection is also negatively influenced by the triggers that induce stress granule formation [102, 103] . indeed, this virus also inhibits the formation of stress granules, and influenza virus encoded ns1 seems to play a major role in this [104] . this is not surprising given the role of ns1 in host-shut-off as well as in protecting the viral rna from recognition by rna sensors in the cell (see above), thereby preventing the activation of pkr and concomitant eif2alpha phosphorylation and stress granule formation. interestingly, this innate immune evasion activity of ns1 is counteracted by cellular protein nf90, which partly prevents the suppression of pkr triggered stress granule formation by ns1 by binding both pkr and ns1 [105] . besides ns1, the influenza nucleoprotein np and polymerase subunit pa-x help to prevent stress granule formation, due to their rna protection and host-shut off functions, respectively [103] . for rhinoviruses, nothing is known about their capacity to manipulate stress granule formation, however, for other picornaviruses the 2a and l proteases have recently been shown to interfere by cleaving stress granule factors such as g3bp1 and g3bp2 [106] [107] [108] [109] . a recent report showed that binding of eif4gi translation factor to stress granule-inducing protein g3bp1 is essential for antiviral stress granule formation, and this interaction is disrupted by the 2a or l proteases of picornaviruses [110] . given these data, it may be likely that rhinoviruses also affect stress granule formation using their proteases, which is further supported by data described in the next paragraph, but this needs to be investigated. most, if not all, positive strand rna viruses encode proteases, which they generally use to cleave their viral polyproteins into functional subunits during the viral life cycle. it has lately become apparent that these proteases often have side-functions that support immune evasion by these viruses. among the viruses discussed here, rhinoviruses and covs carry a positive strand rna genome, and each of the members of these virus families encode at least 2 proteases. rhinoviruses use their 2a papain-like protease (plpro) to effectively disable cap-dependent translation by cleaving eif4g to induce host-shut off. this may well also prevent stress granule formation, however as mentioned, this has not been investigated for rhinoviruses yet. addition-doi: 10.1159/000503030 ally, rhinovirus 2a protease cleaves nuclear pore proteins nup62 and nup98, while 3c protease seems to cleave nup153 [111, 112] . these activities are thought to influence host immune response signaling for which cytosolnucleus communication and trafficking is essential. it recently became clear that rhinovirus 2a protease activity also plays a role in targeting rhinovirus 3c protein to the nucleus [113, 114] , however, it is not clear what 3c protease is doing there exactly [114, 115] . as discussed in the beginning of this review, the type i ifn antiviral pathway is very relevant for rna virus infections, and an essential adaptor that enables downstream signaling in this pathway is ips-1 (also called mavs). this factor is cleaved by both 2a and 3c proteases of rhinovirus to halt type i ifn signal transduction [116] . rhinovirus 3c protease can inhibit apoptotic cell death and activation of antiviral protein complexes by cleaving cellular apoptosis factor ripk1 [117, 118] . cov proteases also cleave cellular substrates to benefit the infection. the functions of plpro of covs in manipulating the ubiquitin regulation of the innate immune system will be discussed later. the main, or 3c-like, protease of covs may have side-functions in cleaving innate immune factors as it was shown for 2 porcine covs that their main proteases cleave nemo [119, 120] , however, nothing is known yet in this respect for the human respiratory covs. the ubiquitin system is essential for the correct functioning of virtually all important cellular processes. the central molecule is ubiquitin, a small 76 amino acid protein that can be conjugated with its c-terminus to lysine residues in substrate proteins. three classes of enzymes are needed for the conjugation: activating e1 enzyme, conjugating e2 enzyme, and an e3 ligase. additional ubiquitins can be added to the first via one of 7 lysines in ubiquitin itself, yielding poly-ubiquitin chains. the signal that the ubiquitin chain gives depends on the linkage type(s) of the chain. k48 and k63-linked ubiquitin chains are best studied and are generally the cause of degradation or activation of the substrate, respectively. in antiviral innate immune signaling, ubiquitin is an important regulating factor, and isg15, an interferon-induced ubiquitin-like molecule, is also an important factor in antiviral innate immunity. it is therefore not a surprise that a lot of viruses have evolved ways to manipulate the ubiquitin system and ubiquitin-like molecules such as isg15, which they do in very diverse ways [121] . after the discovery of a structural resemblance between sars-cov expressed plpro and the cellular deubiquitinase hausp/usp7 [122] , it soon became clear that cov plpros had intrinsic deubiquitinating activity and could potentially deconjugate cellular (or viral) substrates to disrupt ubiquitin-mediated signaling, as well as deconjugate isg15 off its substrates [123] . it is still not clear which cellular and viral factors are deconjugated by plpro during infection, but mutant mers-cov in which the deubiquitinating/de-isg15ylating function of plpro was removed clearly showed increased type i ifn innate immune responses (knaap et al., unpublished results) , indicating that plpro's dub activity has an important role in the suppression of innate immunity during infection. for plpro from human common cold virus hcov-nl63 it was shown, although only by over-expression experiments, that it can deubiquitinate mdm2, the e3 ligase that mediates p53 ubiquitination and subsequent degradation, thereby possibly inhibiting apoptosis and innate immune signaling [124] . similarly, sars-cov plpro can deubiquitinate e3 ligase rchy1 to stimulate ubiquitination of p53 by this ligase, and thus also potentially inhibit apoptosis [125] . for influenza, several different interactions with the ubiquitin system have been identified that critically influence the outcome of the infection [126] . generally, the activation of the rig-i -mavs -irf3 signaling axis in type i ifn signaling, which is important for all viruses discussed in this review, is governed by ubiquitin linked through its lysine at position 63 (forming k63-linked chains). about a decade ago, influenza virus ns1 was shown to bind e3 ligase trim25, thereby interfering with k63-linked ubiquitination of rig-i, and therefore uniquely inhibiting innate immune signaling in the type-i ifn pathway [127] . there has been a recent debate as to whether these chains are actually conjugated to rig-i or other factors within the cascade or whether they are free ubiquitin chains that provide a scaffold for activating the aggregation of rig-i and mavs, which in turn enables downstream signaling [128] . influenza b virus-encoded ns1 additionally inhibits isg15 antiviral activity by binding the n-terminus of human isg15 (and not mouse isg15) [129] . furthermore, and similar to what some of the cov plpro's may do (see above in this section), influenza ns1 was recently shown to destabilize mdm2 e3 ligase which somehow benefits the iav infection. according to the authors, this is because mdm2 seems to have a p53-independent antiviral function which is then alleviated [130] . this is, however, in contrast to what was mentioned for nl63 cov, where plpro seems to stabilize innate immune evasion by respiratory viruses mdm2 to also benefit infection [124] . further research is needed to conclude whether these opposite effects indeed benefit the respective infections, or whether either of the results is incorrect. finally, influenza ns1 was shown to mediate the upregulation of a20, a deubiquitinase with a role in the downregulation of rig-i activation, to suppress the activation of rig-i [131] . rsv also manipulates ubiquitin-mediated signaling, mainly directed by its non-structural proteins ns1 and ns2. quite recently it was shown that rsv ns1 targets trim25 to suppress rig-i ubiquitination, very similar to influenza's ns1's strategy [132] . this probably corroborates the importance of trim25-mediated ubiquitination in the innate immune signaling cascade. earlier reports suggested that ns2 of rsv can direct proteasomal degradation of signal transducer and activator of transcription 2 (stat2) in lung epithelial cells [133, 134] . stat2 and stat1 are transcription factors in the second round of innate immune signaling after binding of ifn to its receptor on the original, or surrounding cells. however, the mechanistic details of ns2's action has not become completely clear yet, although it has been claimed that ns2 somehow stimulates (k48-linked) ubiquitination of proteins, which can be alleviated again by a com-bination of mutations in ns2. these mutations when introduced into the virus prevent stat2 from being degraded during infection, providing possibilities for novel vaccines [135] . although it was reported that rsv infection in cell culture and in patients causes induction of isg15 and that isg15 conjugation to proteins has an antiviral effect [136] , it is not clear whether rsv inhibits or evades isg15 antiviral effects or not. for rhinoviruses, it is unclear how it interacts with the cell's ubiquitin system. while picornavirus family member foot-and-mouth disease virus leader protease was shown to have deubiquitinating activity [137] , neither 2a nor 3c protease from rhinovirus has been implicated in ubiquitin-regulated processes to date, and no other reports hinting at manipulation of the ubiquitin system by rhinoviruses have been published to my knowledge. the data summarized and discussed above illustrate that innate immune evasion is a major function of respiratory and other rna viruses (fig. 1) , which probably takes a significant volume of the genetic capacity of these viruses. this also implies that, given the restricted genetic space available to these viruses, the evasive functions must be pivotal for viruses to survive, otherwise they would likely not have evolved. since each virus employs multiple different activities to suppress immune responses, and often evolved multifunctional proteins to do so, it remains difficult to acquire a complete picture of the immune evasive arsenal of a virus and how this is balanced with symptoms and disease outcome in different cell types or situations. nevertheless, in-depth knowledge of this virus-host interaction creates important avenues for novel antiviral strategies, some of which have already been mentioned in the text above, and some more examples will be discussed in the next section. besides the major strategies for innate immune suppression by respiratory viruses discussed, several other mechanisms of innate immune evasion have been described for the 4 respiratory viruses discussed here, and/ or members of their families, of which many may be unique to only one or 2 of the respiratory viruses discussed here. one example is the virus-encoded macrodomain. these domains have been identified in covs (and several other non-respiratory +rna viruses) and have been shown to counteract ifn signaling with a yet unknown mechanism [138] . they are absent in influenza virus, rhinoviruses, and rsv, and therefore has not been discussed in this review. many of these additional evasive activities have been comprehensively reviewed recently by others [13, 17, 72, 74, 99, 108, 123, . undoubtedly, yet other evasive activities are additionally still to be identified. the newly discovered aspects of human innate immunity, such as trained innate immunity and the integrated innate/adaptive cell types, as well as the links between innate immune responses and cellular metabolic changes, as discussed in the first part of this review, due to their recent discovery have not yet been studied extensively in the context of possible viral evasion strategies. this direction of course forms an obvious avenue for new research that should be undertaken, since it is likely that viruses also target these newly discovered mechanisms. an important question is how exactly the viral innate immune evasive functions of respiratory viruses influence disease outcome and ultimate immune responses. it is noticeable that many of the viruses discussed here do not elicit a long-lasting immune protection after infection, and indeed rhino, corona, and rsv can re-infect individuals sometime after earlier infection, again causing symptoms (reviewed in [178, 179] ), which is in sharp contrast to several other childhood-associated viral infections, where lifelong protection is achieved after generally experiencing only one episode of disease. it may well be that, besides their strong genetic variation, the innate immune evasive activities of the mentioned respiratory viruses play a role in this lack of eliciting protective immunity [180] , and to possibly improve our options for effective antiviral strategies, it seems pivotal to further investigate this. for influenza the situation is slightly different, since this virus elicits protective immunity [172] ; however, its genetic drift and shift causes new strains that are not, or inefficiently, recognized by existing influenza immunity which generally means that individuals will experience multiple influenza infections in the course of their lives. besides contributing to the problem of limited immunological protection, viral innate immune evasion may also contribute to often reported immune over-reactions associated with respiratory infections, including cytokine storms, damaging inflammation, and other severe complications [181] [182] [183] [184] . some studies on sars-cov and mers-cov infections in patients suggest that the delayed innate immune response that is the result of temporary suppression by innate immune evasion, contributes to an exacerbated response [144] . how this works exactly is unclear to date. respiratory pathogens are associated with asthma. the exacerbation of asthma symptoms upon infection with rhinoviruses have been associated with defective types i and iii ifn responses [185, 186] . in lung tissue, antiviral defenses may be further compromised by other mechanisms that impair these defenses such as th2 cytokines il-4 and il-13 [187] , and possibly high affinity ige receptor expression and crosslinking [188] . however, suppressed antiviral innate immune response during virus-induced asthma exacerbations is likely also influenced by the innate immune evasive functions of respiratory viruses, as these activities contribute to more severe pathogenicity and slower virus clearance, likely stimulating asthmatic manifestations [182, 189] . understanding (innate) immune evasion by respiratory viruses could, therefore, shed light on the possibilities for the prevention and cure of asthmatic complications associated with respiratory infections. for particularly rsv and influenza, efforts to develop effective and long-lasting vaccines and antivirals have been relatively unsuccessful for decades [179, 190] link to the viruses' capability to manipulate the host's immune responses, thereby breaking through pre-existing natural or vaccine inflicted immunity. detailed knowledge on the mechanisms whereby these viruses deal with and modify the immune responses they encounter is therefore pivotal to genuinely advance this field. some studies have focused on mapping the interactions of respiratory viruses, and their immune evasion proteins, with their host cells to find promising cell-based drug targets [191, 192] , and this could be an effective way to developing novel vaccines and antiviral drugs. effective rhinovirus and cov antivirals and vaccines have also been lacking, and for these viruses causing common colds, an additional hurdle is the cost-effectiveness of these medicines. the general symptoms of these virus infections are mild, and before the public is willing to buy specific and effective medicines against these infections, these should be relatively cheap. given that these viruses are generally difficult to control due to factors of viral immune modulation, the more knowledge we gain on the link between virus infection and (innate) immune responses in the host, the higher the chance that we may be able to develop successful and cost-effective remedies. although the impact of a common cold may not be high, the fact that these infections are extremely widespread in the human population makes controlling these viruses a desirable goal. the cost-effectiveness balance is also a factor for the covs causing severe infections, that is, sars-cov and mers-cov, since infections with these viruses are either not being reported any more (sars-cov), or are quite localized and relatively scarce (mers-cov). still, since 35% of mers-cov-infected patients succumb to the infection, and the lingering threat of larger outbreaks is felt as long as the virus replicates in humans, who has been recommending the development of specific vaccines for both viruses. recently, a number of efforts for mers-cov vaccines have reached the stage of clinical trials, and having these vaccines "on the shelf" will at least ease societal concerns of dangerous outbreaks with this lethal virus [193] . a more or less obvious way of exploiting a virus' innate immune evasive functions for the development of new vaccines is to remove one or more of these from the virus using reverse genetic technology. in this way, the virus may become attenuated and at the same time it may trigger better innate immune responses due to the lack of one or more of its evasive functions. this could yield effective modified live virus vaccines that are attenuated by design, and for influenza there has been many attempts at constructing vaccine viruses lacking (parts of) ns1 or containing mutated ns1. none of these have, however, reached the market yet [73, 150, 194] . in our own group, we have been exploring the removal of viral deubiquitination activity from the viral plpro of mers-cov and are in the process of analyzing the effect on disease outcome and immune responses in a mouse model ( [195] and unpublished results). the knowledge we gained about the innate immune evasive activity of viral deubiquitinases like mers-cov plpro also prompted an innovative antiviral option encompassing the screening for high affinity ubiquitin sequence variants that actually block the entire activity of the viral protease and therefore form promising antiviral molecules [196] . innate immunity in the lungs cellular innate immunity: an old game with new players type iii interferons in viral infection and antiviral immunity interplay between cellular metabolism and cytokine responses during viral infection. viruses type iii interferons in antiviral defenses at barrier surfaces how myeloid cells contribute to the pathogenesis of prominent emerging zoonotic diseases viruses seen by our cells: the role of viral rna sensors infectious agents as stimuli of trained innate immunity neutrophils in viral infection dead-box helicases: the yin and yang roles in viral infections natural killer cell specificity for viral infections recognition of viral rna by pattern recognition receptors in the induction of innate immunity and excessive inflammation during respiratory viral infections innate immunity to respiratory infection in early life. front immunol multifaceted roles of trim38 in innate immune and inflammatory responses sustained ifn-i expression during established persistent viral infection: a "bad seed" for protective immunity strategies of highly pathogenic rna viruses to block dsrna detection by rig-i-like receptors: hide, mask, hit global, regional, and national causes of child mortality in 2000-13, with projections to inform post-2015 priorities: an updated systematic analysis global burden of childhood pneumonia and diarrhoea immunity to rsv in early-life. front immunol ubiquitination in the antiviral immune response ifn-lambdas mediate antiviral protection through a distinct class ii cytokine receptor complex il-28, il-29 and their class ii cytokine receptor il-28r koltsida o. interferon-λs: front-line guardians of immunity and homeostasis in the respiratory tract. front immunol interferon-λ mediates non-redundant front-line antiviral protection against influenza virus infection without compromising host fitness trained immunity: a program of innate immune memory in health and disease induction of autonomous memory alveolar macrophages requires t cell help and is critical to trained immunity trained immunity and local innate immune memory in the lung mini viral rnas act as innate immune agonists during influenza virus infection rnai-mediated antiviral immunity in mammals innate antiviral defenses independent of inducible ifnα/β production the interplay between central metabolism and innate immune responses mtor-and hif-1α-mediated aerobic glycolysis as metabolic basis for trained immunity metabolic reprogramming in macrophages and dendritic cells in innate immunity membranous replication factories induced by plusstrand rna viruses organelle-like membrane compartmentalization of positivestrand rna virus replication factories biogenesis and architecture of arterivirus replication organelles human respiratory syncytial virus nucleoprotein and inclusion bodies antagonize the innate immune response mediated by mda5 and mavs respiratory syncytial virus: virology, reverse genetics, and pathogenesis of disease expression and cleavage of middle east respiratory syndrome coronavirus nsp3-4 polyprotein induce the formation of double-membrane vesicles that mimic those associated with coronaviral rna replication cytoplasmic inclusions of respiratory syncytial virus-infected cells: formation of inclusion bodies in transfected cells that coexpress the nucleoprotein, the phosphoprotein, and the 22k protein remodeling the endoplasmic reticulum by poliovirus infection and by individual viral proteins: an autophagy-like origin for virus-induced vesicles competitive fitness in coronaviruses is not correlated with size or number of double-membrane vesicles under reduced-temperature growth conditions antiviral innate immune response interferes with the formation of replication-associated membrane structures induced by a positive-strand rna virus regulation of innate immune functions by guanylate-binding proteins interaction of the innate immune system with positive-strand rna virus replication organelles. cytokine growth factor rev sensing of invading pathogens by gbps: at the crossroads between cellautonomous and innate immunity mx gtpases: dynamin-like antiviral machines of innate immunity viral replication complexes are targeted by lc3-guided interferon-inducible gtpases. cell host microbe quo vadis? interferon-inducible gtpases go to their target membranes via the lc3-conjugation system of autophagy. small gtpases nuclear-resident rig-i senses viral replication inducing antiviral immunity rig-i-mediated antiviral responses to single-stranded rna bearing 5′-phosphates rig-i detects viral genomic rna during negative-strand rna virus infection influenza virus adaptation pb2-627k modulates nucleocapsid inhibition by the pathogen sensor rig-i. cell host microbe rig-i and tlr3 are both required for maximum interferon induction by influenza virus in human lung alveolar epithelial cells a new splice variant of the human guanylate-binding protein 3 mediates anti-influenza activity through inhibition of viral transcription and replication covalent linkage of a protein to a defined nucleotide sequence at the 5′-terminus of virion and replicative intermediate rnas of poliovirus a protein covalently linked to poliovirus genome rna internal initiation of translation of eukaryotic mrna directed by a sequence derived from poliovirus rna. nature a segment of the 5′ nontranslated region of encephalomyocarditis virus rna directs internal entry of ribosomes during in vitro translation insight into influenza: a virus cap-snatching. viruses the structure of the 5′ terminal cap of the respiratory syncytial virus mrna ribose 2′-o-methylation provides a molecular signature for the distinction of self and nonself mrna dependent on the rna sensor mda5 pathogenic influenza viruses and coronaviruses utilize similar and contrasting approaches to control interferon-stimulated gene responses early endonuclease-mediated evasion of rna sensing ensures efficient coronavirus replication. plos pathog coronavirus nonstructural protein 15 mediates evasion of ds-rna sensors and limits apoptosis in macrophages the cap-snatching endonuclease of influenza virus polymerase resides in the pa subunit crystal structure of an avian influenza polymerase pa(n) reveals an endonuclease active site an overlapping protein-coding region in influenza a virus segment 3 modulates the host response the novel influenza a virus protein pa-x and its naturally deleted variant show different enzymatic properties in comparison to the viral endonuclease pa influenza a virus protein pa-x contributes to viral growth and suppression of the host antiviral and immune responses modulation of innate immune responses by the influenza a ns1 and pa-x proteins. viruses interactions between ns1 of influenza a viruses and interferon-α/β: determinants for vaccine development functions of the influenza a virus ns1 protein in antiviral defense terminal uridylyltransferases target rna viruses as part of the innate immune system uridylation by tut4 and tut7 marks mrna for degradation widespread 3′-end uridylation in eukaryotic rna viruses. sci rep the evolution and diversity of the nonsense-mediated mrna decay pathway. f1000res interplay between coronavirus, a cytoplasmic rna virus, and nonsense-mediated mrna decay pathway host shutoff in influenza a virus: many means to an end. viruses influenza virus ns1 protein interacts with the cellular 30 kda subunit of cpsf and inhibits 3'end formation of cellular pre-mrnas severe acute respiratory syndrome coronavirus nsp1 facilitates efficient propagation in cells through a specific translational shutoff of host mrna severe acute respiratory syndrome coronavirus protein nsp1 is a novel eukaryotic translation inhibitor that represses multiple steps of translation initiation sars coronavirus nsp1 protein induces template-dependent endonucleolytic cleavage of mrnas: viral mrnas are resistant to nsp1-induced rna cleavage a two-pronged strategy to suppress host protein synthesis by sars coronavirus nsp1 protein middle east respiratory syndrome coronavirus nsp1 inhibits host gene expression by selectively targeting mrnas transcribed in the nucleus while sparing mrnas of cytoplasmic origin a common strategy for host rna degradation by divergent viruses mapping the cleavage site in protein synthesis initiation factor eif-4 gamma of the 2a proteases from human coxsackievirus and rhinovirus the structure of the 2a proteinase from a common cold virus: a proteinase responsible for the shut-off of host-cell protein synthesis interaction of 2a proteinase of human rhinovirus genetic group a with eif4e is required for eif4g cleavage during infection respiratory syncytial virus infection alters surfactant protein a expression in human pulmonary epithelial cells by reducing translation efficiency the stress granule protein g3bp1 binds viral dsrna and rig-i to enhance ifn-β response translation inhibition and stress granules in the antiviral immune response critical role of an antiviral stress granule containing rig-i and pkr in viral detection and innate immunity subcellular localizations of rig-i, trim25, and mavs complexes leader-containing uncapped viral transcript activates rig-i in antiviral stress granules respiratory syncytial virus induces host rna stress granules to facilitate viral replication activation of protein kinase r is required for induction of stress granules by respiratory syncytial virus but dispensable for viral replication respiratory syncytial virus and cellular stress responses: impact on replication and physiopathology middle east respiratory coronavirus accessory protein 4a inhibits pkr-mediated antiviral stress responses inhibition of stress granule formation by middle east respiratory syndrome coronavirus 4a accessory protein facilitates viral translation, leading to efficient virus replication stress granule-inducing eukaryotic translation initiation factor 4a inhibitors block influenza a virus replication influenza a virus host shutoff disables antiviral stress-induced translation arrest influenza a virus inhibits cytoplasmic stress granule formation nf90 is a novel influenza a virus ns1-interacting protein that antagonizes the inhibitory role of ns1 on pkr phosphorylation foot-and-mouth disease virus leader protease cleaves g3bp1 and g3bp2 and inhibits stress granule formation picornavirus 2a protease regulates stress granule formation to facilitate viral translation induction and suppression of innate antiviral responses by picornaviruses. cytokine growth factor rev inhibition of cytoplasmic mrna stress granule formation by a viral proteinase. cell host microbe sg formation relies on eif4gi-g3bp interaction which is targeted by picornavirus stress antagonists differential processing of nuclear pore complex proteins by rhinovirus 2a proteases from different species and serotypes rhinovirus 3c protease facilitates specific nucleoporin cleavage and mislocalisation of nuclear proteins in infected host cells rhinovirus 16 2a protease affects nuclear localization of 3cd during infection rhinovirus 3c protease can localize in the nucleus and alter active and passive nucleocytoplasmic transport rhinovirus 3c protease precursors 3cd and 3cd' localize to the nuclei of infected cells cleavage of ips-1 in cells infected with human rhinovirus rhinovirus 3c protease suppresses apoptosis and triggers caspase-independent cell death human rhinovirus 3c protease cleaves ripk1, concurrent with caspase 8 activation porcine deltacoronavirus nsp5 inhibits interferon-β production through the cleavage of nemo porcine epidemic diarrhea virus 3c-like protease regulates its interferon antagonism by cleaving nemo ubiquitin in the activation and attenuation of innate antiviral immunity deubiquitination, a new function of the severe acute respiratory syndrome coronavirus papain-like protease? nidovirus papain-like proteases: multifunctional enzymes with protease, deubiquitinating and deisgylating activities p53 degradation by a coronavirus papain-like protease suppresses type i interferon signaling p53 downregulates sars coronavirus replication and is targeted by the sars-unique domain and plpro via e3 ubiquitin ligase rchy1 ubiquitin in influenza virus entry and innate immunity influenza a virus ns1 targets the ubiquitin ligase trim25 to evade recognition by the host viral rna sensor rig-i. cell host microbe regulation of rig-i activation by k63-linked polyubiquitination species-specific antagonism of host isgylation by the influenza b virus ns1 protein influenza a viruses alter the stability and antiviral contribution of host e3-ubiquitin ligase mdm2 during the time-course of infection influenza a virus ns1 protein induced a20 contributes to viral replication by suppressing interferon-induced antiviral response human respiratory syncytial virus ns 1 targets trim25 to suppress rig-i ubiquitination and subsequent rig-i-mediated antiviral signaling. viruses specific inhibition of type i interferon signal transduction by respiratory syncytial virus respiratory syncytial virus nonstructural protein 2 specifically inhibits type i interferon signal transduction identification of respiratory syncytial virus nonstructural protein 2 residues essential for exploitation of the host ubiquitin system and inhibition of innate immune responses isg15 is upregulated in respiratory syncytial virus infection and reduces virus growth through protein isgylation the leader proteinase of foot-andmouth disease virus negatively regulates the type i interferon pathway by acting as a viral deubiquitinase viral macrodomains: unique mediators of viral replication and pathogenesis modulation of the immune response by middle east respiratory syndrome coronavirus viral evasion strategies in type i ifn signaling -a summary of recent developments. front immunol innate immunity evasion by enteroviruses: insights into virus-host interaction molecular mechanisms of innate immune inhibition by non-segmented negative-sense rna viruses mechanisms of innate immune evasion in re-emerging rna viruses molecular pathology of emerging coronavirus infections evasion of natural killer cells by influenza virus innate immune evasion strategies of influenza viruses segmented negativestrand rna viruses and rig-i: divide (your genome) and rule antiviral innate immunity and stress granule responses coronavirus non-structural protein 16: evasion, attenuation, and possible treatments influenza virus non-structural protein ns1: interferon antagonism and beyond coronavirus virulence genes with main focus on sars-cov envelope gene the battle between influenza and the innate immune response in the human respiratory tract. infect chemother enter at your own risk: how enteroviruses navigate the dangerous world of pattern recognition receptor signaling ubiquitin-mediated modulation of the cytoplasmic viral rna sensor rig-i trim proteins: another class of viral victims viral inhibition of the ifn-induced jak/stat signalling pathway: development of live attenuated vaccines by mutation of viral-encoded ifn-antagonists. vaccines (basel) viral evasion of intracellular dna and rna sensing. nat rev microbiol respiratory syncytial virus infection of airway cells: role of micrornas coronavirus nonstructural protein 1: common and distinct functions in the regulation of host and viral gene expression induction of innate immunity and its perturbation by influenza viruses host detection and the stealthy phenotype in influenza virus infection the sars-coronavirus papain-like protease: structure, function and inhibition by designed antiviral compounds virus escape and manipulation of cellular nonsense-mediated mrna decay. viruses complement evasion strategies of viruses: an overview. front microbiol a molecular arms race between host innate antiviral response and emerging human coronaviruses to conquer the host, influenza virus is packing it in: interferon-antagonistic strategies beyond ns1 host and viral modulation of rig-i-mediated antiviral immunity. front immunol interaction of sars and mers coronaviruses with the antiviral interferon response camouflage and interception: how pathogens evade detection by intracellular nucleic acid sensors nsp3 of coronaviruses: structures and functions of a large multi-domain protein induction and subversion of human protective immunity: contrasting influenza and respiratory syncytial virus. front immunol the innate immune response to rsv: advances in our understanding of critical viral and host factors. vaccine molecular mechanisms of foot-and-mouth disease virus targeting the host antiviral response. front cell infect microbiol influenza virus ns1 protein mutations at position 171 impact innate interferon responses by respiratory epithelial cells modulation of host immunity by human respiratory syncytial virus virulence factors: a synergic inhibition of both innate and adaptive immunity. front cell infect microbiol viral mimicry to usurp ubiquitin and sumo host pathways human coronaviruses: what do they cause? antivir ther progress in understanding and controlling respiratory syncytial virus: still crazy after all these years. virus res human respiratory syncytial virus: role of innate immunity in clearance and disease progression inflammatory damage on respiratory and nervous systems due to hrsv infection. curr opin immunol epithelial injury and repair in airways diseases immune and inflammatory response in bronchiolitis due to respiratory syncytial virus and rhinovirus infections in infants the host immune response in respiratory virus infection: balancing virus clearance and immunopathology role of deficient type iii interferon-lambda production in asthma exacerbations asthmatic bronchial epithelial cells have a deficient innate immune response to infection with rhinovirus th2 cytokines impair innate immune responses to rhinovirus in respiratory epithelial cells innate immune responses to rhinovirus are reduced by the high-affinity ige receptor in allergic asthmatic children advances in the treatment of virus-induced asthma. expert rev respir med challenges in developing a cross-serotype rhinovirus vaccine identification of common biological pathways and drug targets across multiple respiratory viruses based on human host gene expression analysis viral immune modulators perturb the human molecular network by common and unique strategies middle east respiratory syndrome vaccine candidates: cautious optimism. viruses influenza virus: a master tactician in innate immune evasion and novel therapeutic interventions. front immunol crystal structure of the middle east respiratory syndrome coronavirus (mers-cov) papain-like protease bound to ubiquitin facilitates targeted disruption of deubiquitinating activity to demonstrate its role in innate immune suppression potent and selective inhibition of pathogenic viruses by engineered ubiquitin variants the author wishes to thank prof. pieter hiemstra (lumc, leiden, the netherlands) for critically reading the manuscript and for helpful comments and discussions. the author declares no conflicts of interest. the author was funded by a primary institutional appointment as associate professor at leiden university medical center, leiden, the netherlands. key: cord-355839-o0m71kvw authors: sedeyn, koen; schepens, bert; saelens, xavier title: respiratory syncytial virus nonstructural proteins 1 and 2: exceptional disrupters of innate immune responses date: 2019-10-17 journal: plos pathog doi: 10.1371/journal.ppat.1007984 sha: doc_id: 355839 cord_uid: o0m71kvw human respiratory syncytial virus (rsv) is the most important cause of acute lower respiratory tract disease in infants worldwide. as a first line of defense against respiratory infections, innate immune responses, including the production of type i and iii interferons (ifns), play an important role. upon infection with rsv, multiple pattern recognition receptors (prrs) can recognize rsv-derived pathogen-associated molecular patterns (pamps) and mount innate immune responses. retinoic-acid-inducible gene-i (rig-i) and nucleotide-binding oligomerization domain-containing protein 2 (nod2) have been identified as important innate receptors to mount type i ifns during rsv infection. however, type i ifn levels remain surprisingly low during rsv infection despite strong viral replication. the poor induction of type i ifns can be attributed to the cooperative activity of 2 unique, nonstructural (ns) proteins of rsv, i.e., ns1 and ns2. these viral proteins have been shown to suppress both the production and signaling of type i and iii ifns by counteracting a plethora of key host innate signaling proteins. moreover, increasing numbers of ifn-stimulated genes (isgs) are being identified as targets of the ns proteins in recent years, highlighting an underexplored protein family in the identification of ns target proteins. to understand the diverse effector functions of ns1 and ns2, goswami and colleagues proposed the hypothesis of the ns degradasome (nsd) complex, a multiprotein complex made up of, at least, ns1 and ns2. furthermore, the crystal structure of ns1 was resolved recently and, remarkably, identified ns1 as a structural paralogue of the rsv matrix protein. unfortunately, no structural data on ns2 have been published so far. in this review, we briefly describe the prrs that mount innate immune responses upon rsv infection and provide an overview of the various effector functions of ns1 and ns2. furthermore, we discuss the ubiquitination effector functions of ns1 and ns2, which are in line with the hypothesis that the nsd shares features with the canonical 26s proteasome. introduction human respiratory syncytial virus (rsv) is a negative strand rna virus belonging to the family pneumoviridae. rsv is a major cause of acute lower respiratory tract infections in the pediatric population and is increasingly recognized as an important cause of severe respiratory disease in the elderly [1] [2] [3] . despite the major clinical impact of rsv on human health worldwide, no approved vaccine or effective antiviral therapy is available. although rsv infections do evoke humoral and cellular immune responses required to clear infection, these responses do not provide strong protection against a subsequent infection with rsv. early during infection, viral replication is sensed by the host's innate immune system, which leads to the production of type i and iii interferons (ifns), which is followed by the induction of an array of genes that code for antiviral proteins. in addition, ifn will recruit and activate innate leukocytes, including antiviral monocytes and natural killer (nk) cells, and stimulate the adaptive immune response [4] . rsv has evolved a marvelous set of mechanisms to subvert this canonical antiviral response of the mammalian host, most notably by virtue of its nonstructural (ns) 1 and ns2 proteins. the innate immune system is an important early line of defense against pathogens. this system comprises pattern recognition receptors (prrs) that can recognize pathogen-associated molecular patterns (pamps). activated prrs can induce the expression of cytokines, e.g., type i and iii ifns, which mount an antiviral state in an autocrine and paracrine fashion. upon rsv infection, airway epithelial cells, macrophages, and dendritic cells (dcs) are the main inducers of innate immune responses. several toll-like receptors (tlrs) have been identified that can act as prrs for rsv-derived pamps (fig 1) . tlr2, -3, and -6, for example, have been implicated in the induction of cytokines and chemokines upon rsv infection [5, 6] . a role for tlr4, which is well known to respond to lipopolysaccharide, as a prr for rsv is currently debated. although some groups reported an impaired innate immune response in tlr4-deficient mice [7-9], ehl and colleagues could not confirm a role for a tlr4-mediated immune response upon rsv infection [10] . in addition, tlr7 might even play a role in tempering immune responses upon rsv infection [11] . moreover, type i ifn production by macrophages and dcs from wild-type and tlr1, -2, -4, -6, and -7 knockout mice is very similar following rsv infection [5, 11] . finally, alveolar macrophages are the primary source of type i ifn in rsv-infected mice, and tlrs do not play a crucial role in this response [4, 12] . in contrast, retinoic-acid-inducible gene-i (rig-i)-like receptors (rlrs) are important for the induction of type i and possibly type iii ifns upon recognition of rsv (fig 1) . type i ifn expression is strongly hampered in the absence of mitochondrial antiviral-signaling protein (mavs), the adaptor protein for rig-i and melanoma differentiation-associated protein 5 (mda5) [12, 13] . early on, gene expression ablation studies revealed that rig-i is the most important rlr to detect rsv, which is supported by the observation that rsv mrna could be coimmunoprecipitated with rig-i but not with mda5 [14-17]. later, however, both rig-i and mda5 were found to colocalize with rsv genomic rna and the n protein [18] . interestingly, whereas rig-i partially localizes to rsv-induced inclusion bodies, mda5 and mavs were found nearly exclusively in these inclusion bodies, thereby dramatically blunting ifn-β induction. a third class of prrs, the nucleotide-binding oligomerization domain-like receptors (nlrs), is also important for the recognition of rsv. nucleotide-binding oligomerization domain-containing protein 2 (nod2) can be activated by intact genomic single-stranded rna (ssrna) and is involved in the induction of ifn-β in mice in a mavs-dependent way (fig 1) [19]. cytoplasmic dna sensors (cdss), such as z-dna binding protein 1 (zbp1) and cyclic gmp-amp synthase (cgas), are well known to induce innate immune responses upon recognition of pathogen-derived double-stranded dna (dsdna) or even rna [20] [21] [22] . a contribution of cdss in innate sensing of rsv replication has not yet been reported. in contrast to other respiratory viruses, i.e., influenza virus and human respirovirus 1 (formerly named human parainfluenza virus 1), nasal washes from rsv-infected infants hardly firstly, tlr-2, -3, -4, -6, and -7 (marked in green) are involved in the production of cytokines and chemokines upon rsv infection. secondly, rig-i and possibly mda5 (rlrs, marked in red), are important in the induction of type i ifns. thirdly, nod2 (marked in dark blue) also induces type i ifns upon rsv infection. currently, there is no evidence for a role of the cdss (marked in purple), which signal through the er-associated sting protein, as prrs during rsv infection. activation of the rlrs or nod2 induces their association with the mitochondrial-associated mavs, which recruits the adaptor proteins traf3 or traf6. via the traf3 adaptor, the kinases ikkε and tbk1 are subsequently activated, which phosphorylate and activate the transcription factors irf3 and irf7. via the traf6 adaptor, 3 kinases are activated, i.e., the ikk kinase complex, jnk, and p38 mapk, which phosphorylate and activate multiple transcription factors such as nf-κb, c-jun, and atf2, respectively. activation of tlrs leads to the recruitment of adaptor proteins, e.g., myd88, ticam1, tirap, and tram. these adaptors can signal via traf3 or traf6. the transcription factors activated by prr signaling ultimately induce expression of cytokines, chemokines, and ifns. above each prr, the confirmed or likely rsv-derived pamp is depicted. atf2, activating transcription factor 2; cds, cytoplasmic dna sensor; er, endoplasmic reticulum; ifn, interferon; ikk, inhibitor of nuclear factor kappa-b kinase; ikkε, inhibitor of nuclear factor kappa-b kinase subunit epsilon; irf3, interferon regulatory factor 3; irf7, interferon regulatory factor 7; jnk, c-jun n-terminal kinase; mapk, mitogen-activated protein kinase; mavs, mitochondrial antiviral-signaling protein; mda5, melanoma differentiation-associated protein 5; myd88, myeloid differentiation primary response protein myd88; nf-kb, nuclear factor-kappa b; nod2, nucleotide-binding oligomerization domain-containing protein 2; pamp, pathogenassociated molecular pattern; prr, pattern recognition receptor; rig, retinoic-acid-inducible gene-i; rlr, rig-i-like receptor; rsv, respiratory syncytial virus; sting, stimulator of interferon protein; tbk1, tank binding kinase 1; ticam1, toll/interleukin-1 receptor domain-containing adapter molecule 1; tirap, toll/ interleukin-1 receptor domain-containing adapter protein; tlr, toll-like receptor; traf3, tumor necrosis factor receptor-associated factor 3; traf6, tumor necrosis factor receptor-associated factor 6; tram, toll-like receptor adaptor molecule. https://doi.org/10.1371/journal.ppat.1007984.g001 contain ifn-α and -β [23-26]. apparently, this virus has evolved ways to outsmart the canonical mammalian antiviral response. two unique viral proteins, ns1 and ns2, are responsible for the suppression of ifn induction and signaling. human and bovine rsv strains that lack ns1 and/or ns2 have been explored as live-attenuated vaccine candidates. such viruses are strongly attenuated in in vivo rsv infection models (cotton rats, calves, and chimpanzees) as well as in human but, at least in calves and chimpanzees, retain their ability to induce antibody responses [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] . positioned 3 0 proximal on the negativestranded rna genome, ns1 and ns2 are the most abundantly transcribed viral genes. recently, the crystal structure of ns1 was determined, revealing that this protein is composed of a β-sandwich flanked by 3 α-helices [37] . interestingly, the 3d structure of ns1 is very similar to the n-terminal domain of rsv m despite the complete absence of any primary sequence homology. both ns1 and ns2 strongly reduce the induction of type i and iii ifns upon rsv infection [32, [37] [38] [39] [40] [41] . infection with recombinant viruses lacking ns1 or ns2, separately or combined, suggests that these proteins function individually and cooperatively to suppress ifn induction. they do so by targeting multiple proteins of the signaling cascade that starts with the recognition of pamps by prrs and ends with the induction of ifn gene expression by several transcription factors. a widespread strategy of viruses to dampen innate immune responses is to counteract one of the early signaling steps in rlr-mediated ifn induction, i.e., the interaction of rig-i or mda5 with mavs [42] [43] [44] [45] . likewise, rsv prevents the interaction of rig-i with its adaptor mavs. in rsv-infected hep-2 cells and a549 cells overexpressing ns1, ns1 was shown to interact with mavs (fig 2) [46] . by binding to mavs, ns1 could dose-dependently prevent the interaction between rig-i and mavs in a549 cells. furthermore, ban and colleagues demonstrated that ectopically expressed ns1 in hek293t cells interacts with the pry-spry domain of e3 ubiquitin/ifn-stimulated gene 15 (isg15) ligase tripartite motif-containing protein 25 (trim25). this domain is responsible for the interaction of trim25 with rig-i [47] . ns1 binding to the pry-spry domain suppresses k63-linked polyubiquitination of rig-i by trim25, which is essential for its downstream interaction with mavs (fig 2) . proteins of 2 other respiratory viruses also target the pry-spry domain of trim25, which highlights the evolutionary importance of trim25 in mammalian antiviral defense. by binding the pry-spry domain of trim25, the ns1 protein of influenza a virus and the nucleocapsid protein of severe acute respiratory syndrome virus also counteract trim25-mediated rig-i ubiquitination [48, 49] . ns2 can interact with the n-terminal domain of rig-i, both upon overexpression in hek293t cells and during rsv infection in a549 cells (fig 2) [50] . as such, overexpressed ns2 disrupts the binding of rig-i with mavs; however, this has not yet been confirmed for endogenous ns2 expressed during an rsv infection. in addition to counteracting the interaction between rig-i and mavs, ns1 may also influence rig-i expression, although the reported findings seem conflicting. rig-i expression in a549 cells is strongly reduced in the presence of rsv ns1, either expressed separately or in the context of an rsv infection [51] . boyapalle and colleagues, however, reported that rig-i expression in a549 cells is reduced following infection with an ns1-deficient rsv [46] . this is surprising, because rig-i is itself an isg. possibly, this discrepancy is caused by the different multiplicity of infection (moi) used by these 2 groups (moi 3 and 1, respectively). in contrast to rig-i, mavs appears to be resistant to ns1-and/or ns2-mediated down-regulation [51] . all together, these results highlight that the interaction of rig-i with mavs is suppressed by both ns1 and ns2 and that rig-i expression itself might be reduced by ns1. currently, it is not clear whether ns1 and/or ns2 can disturb the interaction of mda5 or nod2 with mavs and whether they impact mda5 or nod2 expression levels. type i and iii ifn responses are inhibited by ns1 and ns2 at multiple levels, both during the induction of type i and iii ifns (left panel) and during ifn-induced signaling (right panel). ns1 and ns2 can form a so-called "ns degradasome" complex that is stabilized by mitochondria via mavs. the nsd complex is thought to contain hps, including the proteasome α2 subunit and other as yet unidentified proteins. ns1 and ns2 prevent the interaction of rig-i with mavs in different ways. ns1 binds to the pry-spry domain of trim25, which is responsible for the interaction of trim25 with rig-i. as such, ns1 prevents the trim25-mediated k63-linked polyubiquitination of rig-i, which is necessary for the subsequent interaction of rig-i with mavs (1). moreover, ns2 directly interacts with rig-i (2) and ns1 interacts with mavs (3) to suppress binding of rig-i to mavs. whether the interaction of ns1 with mavs also prevents the interaction of nod2 with mavs is currently unclear. ns1 reduces protein expression of traf3 and ikkε (4), whereas ns2 modestly reduces traf3 and induces ikkε and tbk1 (5). ns1 subsequently inhibits irf3 and irf7 by different proposed mechanisms (6). ns1 reduces irf3 and irf7 protein expression and prevents the interaction between irf3 and cbp, thereby lowering the binding of the irf3-cbp complex to the ifn-β promoter. ns2, and to a lesser extent ns1, enhances the activation and nuclear translocation of nf-κb (7). these ns1/ns2 effector functions (1-7) synergistically reduce the production of type i and iii ifns. furthermore, ns1 and ns2 also suppress type i and iii ifn receptor-mediated signal transduction. ns1 induces mir-29a expression, which targets the mrna coding for ifnar1, one of the 2 subunits of the type i ifn receptor (8). ns1 and ns2 may induce expression of socs proteins (socs1 and 3), which negatively regulate the tyrosine kinases jak1 and tyk2, which are important to transmit signaling from the type i and iii ifn receptors (9). ns2 inhibits jak1/ tyk2-mediated activation of stat1/2 by reducing stat2 protein levels (10) and by reducing stat1 phosphorylation (11). some groups, however, reported that stat2 expression can also be reduced by ns1 (see text) (10). ns1 and ns2 counteract the anti-inflammatory activity of the gr, although the exact mechanism is debated. in one model, ns1 interacts with the nuclear translocator ipo13, which competes with gr for its nuclear translocation (12). recent evidence suggests that the ns proteins may also counteract antiviral effector functions of isgs, e.g., ns1 degrades the oasl, ifit1, and ifitm3 proteins, whereas ns2 degrades mapk8 (13). full and dashed lines indicate robust and moderate inhibitory or stimulatory effector functions, respectively. cbp, creb binding protein; gr, glucocorticoid receptor; hp, host protein; ifit, interferon-induced protein with tetratricopeptide repeats; ifitm, interferon-induced transmembrane protein; ifn, interferon; ifnar, interferon alpha/beta receptor; ikkε, inhibitor of nuclear factor-kappa b kinase subunit epsilon; ipo13, importin-13; irf3, interferon regulatory factor 3; isg, ifn-stimulated gene; jak, janus kinase; mapk8, mitogen-activated protein kinase 8; mavs, mitochondrial antiviral-signaling protein; ns, nonstructural; oasl, 2 0 -5 0 -oligoadenylate synthase-like protein; rig-i, retinoic-acid-inducible gene-i; rlr, rig-i-like receptor; rsv, respiratory syncytial virus; socs, suppressor of cytokine signaling; stat, signal transducer and activator of transcription; tbk1, tank binding kinase 1; trim25, tripartite motif-containing protein; tyk, tyrosine kinase 25. https://doi.org/10.1371/journal.ppat.1007984.g002 association of rlrs with mavs leads to the recruitment of the adaptors tumor necrosis factor receptor-associated factor 3 (traf3) and -6 (traf6). whereas traf3 activates the downstream serine/threonine kinases inhibitor of nuclear factor-kappa b kinase subunit epsilon (ikkε) and tank binding kinase 1 (tbk1), traf6 activates the downstream kinases ikk, c-jun n-terminal kinase (jnk), and p38 mitogen-activated protein kinase (p38 mapk) (fig 1) . the effect of ns1 and ns2 on traf3, ikkε, and tbk1 is currently inconclusive. some groups reported that ns1, either overexpressed or expressed during an rsv infection in a549 cells, reduces the expression levels of both traf3 and ikkε (fig 2) [51, 52] . ren and colleagues, however, observed no difference in endogenous and recombinant traf3 and ikkε expression in a549 cells in the presence or absence of ns1 [39] . possibly, the different strains (rsv long versus a2) or experimental timing used explain these opposing observations. overexpression of ns2 in a549 cells modestly reduces and slightly enhances traf3 and ikkε expression levels, respectively (fig 2) [51, 52] . these effects of ns2 were, however, not observed with an rsv strain that lacks ns1. ling and colleagues also observed that overexpressed ns2 enhances overexpression of ikkε and tbk1 in hek293t cells (fig 2) [50] . to our knowledge, no data have been published on the possible effect of ns1 on the expression of tbk1. coexpression of ns1 and ns2 in a549 cells reduces recombinant ikkε expression, suggesting that the inhibitory effect of ns1 is dominant over the enhancing effect of ns2 [52] . these results suggest that ns1 suppresses traf3 and ikkε expression, although more evidence is needed to confirm this hypothesis. ectopic expression of ns2 slightly suppresses traf3 expression and enhances ikkε and tbk1 expression, although these effects need to be confirmed during an rsv infection. whether ns1 and/or ns2 affect the traf6 adaptor has not been investigated yet. some evidence indicates that rsv may indeed affect traf6 expression. in different models, traf6 expression has been shown to be down-regulated by the microrna (mirna) mir-146a [53, 54] . interestingly, eilam-frenkel and colleagues found that mir-146a expression was up-regulated in rsv-infected hep-2 cells [55] . future research may elucidate whether rsv can downregulate traf6 expression via up-regulating mir-146a and whether this is mediated by ns1 and/or ns2. activated ikkε and tbk1 phosphorylate the transcription factors interferon regulatory factor (irf)-3 and irf7, which induces conformational changes that allow the formation of homo-and heterodimers of irf3 and -7 that translocate to the nucleus. phosphorylation of inhibitor of kappa b (iκb), e.g., by the canonical ikkα/β/γ complex, initiates its degradation with subsequent release and nuclear translocation of nuclear factor-kappa b (nf-κb). the irf and nf-κb transcription factors are essential for the induction of type i and iii ifns (fig 1) . several observations highlight that ns1 and ns2 can impair the activation and effector functions of irf transcription factors. initial work with recombinant rsv strains lacking one or both ns proteins in a549 cells highlighted that ns1 and ns2 prevent nuclear translocation of irf3, especially late (>9 hours post infection) in the rsv replication cycle [40, 50, 56] . this might, in part, be explained by the ability of ns1 to directly reduce the recombinant expression of irf3 and irf7 (fig 2) [51] . in addition, by preventing the formation of the irf3/creb binding protein (cbp) complex in the nucleus, ns1 may also inhibit irf3-dependent gene expression downstream of the nuclear translocation of irf3 (fig 2) [37, 39] . it is important to note that this ns1 effector function has only been demonstrated upon overexpression in hek293t cells, so additional confirmation in an rsv infection is necessary. in a549 and vero cells, nf-κb activation and nuclear translocation occur early after rsv infection and are clearly enhanced by ns2 and, to some extent, by ns1 (fig 2) [56, 57] . in addition to the canonical iκb phosphorylation and subsequent degradation, rsv-induced nf-κb activation involves p65ser536 phosphorylation via the rig-i/mavs/traf6/ikkβ signaling pathway [16] . although nf-κb contributes to the induction of type i and iii ifns, nf-κb also strongly induces the expression of anti-apoptotic genes. in the context of an rsv infection, the induction of an anti-apoptotic cellular environment by nf-κb likely dominates the contribution of nf-κb over the induction of ifn, which is primarily controlled by irf3/7 activation. taken together, ns1 and ns2 suppress the induction of type i and iii ifns by targeting multiple proteins of the signaling cascade that leads to type i or iii gene activation, i.e., rig-i, mavs, traf3, ikkε, tbk1, irf3, and irf7. although some of these effector functions have been confirmed in rsv-infected cells, others have so far only been identified with overexpression of ns1 or ns2 alone. so, future research is needed to elucidate the biological relevance of these recombinant responses, particularly because ns2 can relocate ns1 to the mitochondria [58] . binding of type i and iii ifns to their heterodimeric receptor complex induces a janus kinase (jak)-signal transducer and activator of transcription (stat) signaling cascade that induces an antiviral state by expression of isgs. compared with wild-type rsv, rsv strains that lack ns1 and/or ns2 are more sensitive to type i ifn treatment [30, 51] . thus, rsv ns proteins also have effector functions downstream of the induction of ifn synthesis to suppress ifninduced antiviral responses. in rsv-infected a549 cells, ns1 induces the expression of the mirna mir-29a, which targets the mrna coding for interferon alpha/beta receptor 1 (ifnar1) by an unknown mechanism (fig 2) [59] . as a result, the ifnar1 protein levels are reduced, which decreases responsiveness to type i ifns of rsv-infected cells and thus favors rsv replication. the induction of an antiviral state by type i and type iii ifns requires the phosphorylation of stat1 and stat2 proteins in the proximity of the type i and iii receptors. two tyrosine kinases, jak1 and tyrosine kinase 2 (tyk2), are responsible for the phosphorylation and activation of stat proteins after activation of the type i and iii ifn receptor ( fig 2) . for now, there is no evidence that ns1 or ns2 may alter the levels and activation status of these 2 kinases. several groups reported that the phosphorylation and total protein levels of tyk2 are not altered by the expression of ns1 and/or ns2 [60] [61] [62] . whether this also accounts for jak1 is currently not clear. infection of airway epithelial cell lines with human metapneumovirus, a respiratory virus closely related to rsv, does reduce both jak1 and tyk2 protein levels [63] . additionally, jak kinase activity is regulated by a negative feedback loop that consists of the suppressor of cytokine signaling (socs) family, with socs1 and 3 being the strongest inhibitors of jak kinases. it has been reported that ns1 and/or ns2 may regulate the expression of socs1 and/or socs3, thereby enhancing rsv replication [64] [65] [66] [67] . heterodimers of phosphorylated stat1 and 2 transcription factors are important for the induction of isgs. consequently, numerous viral proteins counteract stat1 and 2 proteins by using different strategies, e.g., by preventing stat1/2 heterodimer formation (nipah and hendra virus nucleoprotein), by suppressing stat1/2 nuclear translocation (ebolavirus vp24), or by degrading stat1 and/or 2 (paramyxovirus v protein) [68] [69] [70] . both stat1 and 2 are targets for rsv. in human tracheobronchial epithelial cells and a549 cells, infection with rsv slightly increases total stat1 protein levels, likely because stat proteins are themselves isg products [60-62, 71, 72] . stat1 phosphorylation induced by exogenous type i ifn, however, is clearly reduced by ns2 but not by ns1 (fig 2) [ [60] [61] [62] 73] . whether ns2 also reduces stat1 phosphorylation during an rsv infection has not been reported yet. in contrast to stat1, both phosphorylated and nonphosphorylated stat2 protein levels are clearly reduced by rsv infection [60, 62, 73] . whether ns1, ns2, or both reduce stat2 levels is currently debated. based on overexpression experiments and infections with rsv strains lacking ns1 and/or ns2, several groups reported that ns2, but not ns1, reduces stat2 levels in vitro (fig 2) [51, 52, 61, 62] . others, however, also observed reduced stat2 levels after overexpression of ns1 (fig 2) [66, 73] . lo and colleagues reported that recombinant coexpression of ns1 and ns2 reduces stat2 levels stronger than ns2 alone, although expression of ns1 alone did not affect stat2 levels [62] . in addition to reducing total stat2 levels, rsv also seems to suppress nuclear translocation of the residual stat2 proteins, an effect that depends on ns1 and/or ns2 [62] . so far, the possible impact of rsv ns proteins on stat1 and 2 expression levels in vivo has not yet been demonstrated. because mouse stat2 appears resistant to ns2, primary human airway epithelial cell cultures or experiments in nonhuman primates may be required to confirm that ns2-mediated stat2 reduction also occurs in vivo [62] . evidence is rising that the ns proteins also suppress the antiviral activity of at least some isg products. cells that express myxovirus resistance protein a (mxa) or cells pretreated with type i ifn only modestly limit rsv replication [74] . moreover, ectopic expression of ns1 and ns2 in hek293-derived cells has been shown to degrade certain isg products, i.e., 2 0 -5 0 oligoadenylate synthetase-like protein (oasl), interferon-induced protein with tetratricopeptide repeats 1 (ifit1), interferon-induced transmembrane protein 3 (ifitm3), and mapk8, in a selective manner (fig 2) [75, 76] . an important remark, however, is that these effector functions have not yet been validated during an rsv infection. as stated by ribaudo and barik, a comprehensive screen of all known isgs will likely unravel additional substrates of ns1 and/or ns2 [76] . ns1 and ns2 affect the induction of apoptosis and cell shedding. ns1 and ns2 individually and cooperatively delay apoptosis in rsv-infected cells, with ns2 being stronger than ns1 in doing so [57] . the early expression of ns1 and ns2 after infection activates the antiapoptotic 3-phophoinositide-dependent protein kinase (pdk)-rac serine/threonine-protein kinase (akt)-glycogen synthase kinase (gsk) pathway [56, 57, 77] . later in infection (>24 hours), activation of this pathway drops, and the incidence of apoptosis increases [57] . by delaying apoptosis, ns1 and ns2 may facilitate prolonged rsv replication with increased viral yields. a typical hallmark of severe disease following rsv infection is the obstruction of the smaller airways by plugs consisting of infiltrating immune cells, mucus, and shed infected epithelial cells. in primary human airway epithelial cells and in an in vivo hamster model, it has been shown by experiments with a set of elegant recombinant virus constructs that rsv ns2 is necessary and sufficient to induce shedding of infected epithelial cells [78] . interestingly, cell death, associated with nuclear changes that are indicative of apoptosis, of infected epithelial cells only occurred after these cells were detached from the epithelial layer. moreover, shedding of infected epithelial cells coincided with reducing viral titers. all together, these results suggest that expression of ns2 early after infection delays apoptosis and induces changes in cell morphology that ultimately result in shedding of infected cells in the airway lumen. these shed and detached infected epithelial cells die. as shedding (and ultimately clearance by mucociliary transport) of infected epithelial cells reduces rsv viral titers, it seems that the bulk of viral spread precedes cell shedding. possibly, (early) changes in cell morphology that ultimately lead to shedding of infected epithelial cells may facilitate rsv production and spreading. as such, ns2 plays a pivotal role in the production and spreading of rsv virions. ns1 and ns2 counteract the anti-inflammatory activity of the glucocorticoid receptor. although rsv-induced bronchiolitis is characterized by a (severe) inflammatory response, the use of anti-inflammatory glucocorticoids has shown no clinical benefits against (severe) disease [79] [80] [81] . based on experiments with different transformed and primary cell cultures as well as mice, several groups concluded that rsv can inhibit the anti-inflammatory activity of glucocorticoids via the glucocorticoid receptor (gr) [82] [83] [84] [85] [86] [87] [88] . the exact mechanism that accounts for this inhibition, however, is debated. one group demonstrated that rsv blocks glucocorticoid-mediated gr activation in a549, beas-2b, and primary human small airway epithelial cells but not in the monocytic thp-1 cell line, suggesting that the effect is restricted to rsv-infected epithelial cells [83, 85] . further mechanistic analysis in a549 cells suggested that ns1 and ns2 reduce the binding of the gr to gr-responsive promoters, without affecting gr total protein levels and nuclear translocation [83, 84, 86] . interestingly, knockdown of mavs in a549 cells lowered dexamethasone-induced transforming growth factor β (tgf-β)-stimulated clone 22 (tsc22) domain family member 3 (also known as glucocorticoid-induced leucine zipper protein [gilz]) mrna expression to a level similar to that of an rsv infection [86] . these results suggest that mavs plays a role in glucocorticoidinduced gr activation in a549 cells and that ns1 and ns2 may indirectly suppress gr activation through inhibition of mavs (see "ns1 and ns2 interfere with rig-i"). xia and colleagues used beas-2b and primary differentiated human bronchial epithelial cells grown at an air-liquid interface to demonstrate that rsv suppresses glucocorticoidinduced gr activation by a mechanism that involves up-regulation of tgf-β expression [87] . a model was proposed in which viral infection is sensed by tlr3, which induces tgf-β that subsequently activates the type i tgf-β receptor activin receptor-like kinase 5 (alk5). alk5 activation subsequently counteracts glucocorticoid activity through an unknown mechanism. in accordance with the aforementioned study, no difference in total grα protein levels or nuclear translocation were observed upon rsv infection. interestingly, blocking alk5 with the selective inhibitor sb431542 and reducing tgf-β activity by tranilast (a compound used to manage a wide variety of diseases, including inflammatory diseases) could subvert the rsvmediated suppression of glucocorticoid activity. although xia and colleagues did not investigate the role of ns1 or ns2, ns1 may contribute to rsv-induced tgf-β expression by up-regulating the transcription factor kruppel-like factor 6 (discussed in the next paragraph) [89] . another study concluded that rsv ns1 prevents gr nuclear translocation [88] . this finding was based on experiments with a549 cells, mouse lung tissue, but also analysis of nasopharyngeal aspirates from rsv-infected infants. moreover, expression of grα and importin-13, which is important for gr nuclear entry, was reduced at the mrna and protein level in mouse lungs, whereas grβ and ipo13 expression at the mrna level were reduced in the nasopharyngeal aspirates upon rsv infection. mechanistically, ns1 was found to directly interact with ipo13 and can thus compete with gr for ipo13 binding. the capacity of rsv to suppress the anti-inflammatory effect of glucocorticoids through ns1 and ns2 are in line with the ineffectiveness of glucocorticoid treatments off severely ill rsv patients. in addition to mir-29a, ns proteins also regulate the expression of the mirnas mir-24, let-7i, and mir-30b [59, 89, 90] . in rsv-infected a549 cells, ns1 suppresses mir-24 expression by up-regulating the transcription factor kruppel-like factor 6, which drives the expression of tgf-β [89] . remarkably, inhibition of mir-24 was shown to actually repress rsv replication in a549 cells [91] . this apparent discrepancy could be explained by the difference in time points after infection that were analyzed (1 day versus 3 days post infection). possibly, ns1 suppresses the rsv-induced up-regulation of mir-24 within 24 hours post infection, whereas later on, mir-24 expression may be up-regulated to enhance viral replication. investigating mir-24 expression levels beyond 24 hours post infection and the impact of ns1 on these levels may result in a better understanding of the role of mir-24 in rsv replication. in normal human bronchial epithelial cells, let-7i and mir-30b expression is up-regulated during an rsv infection by a type i ifn and nf-κbdependent mechanism, respectively, and further increased in the absence of ns1 or ns2 [90] . this can be expected for let-7i, as the ns proteins suppress the type i ifn response. moreover, the mir-30b promoter can be activated by the nf-κb family member p65, which is activated during an rsv infection through a rig-i/mavs/traf6/ikkβ signaling pathway [16, 92] . by counteracting the interaction between rig-i and mavs (see "ns1 and ns2 interfere with rig-i"), the ns proteins may dampen this pathway, leading to reduced activation of p65 and subsequent expression of mir-30b. to our knowledge, the effect of ns1 and/or ns2 on the rsv-induced activation of p65 has not been investigated yet. this could readily be tested by comparing total levels of activated p65 between cells infected with wild-type rsv and ns deletion strains. whether the ns protein-mediated suppression of let7i and mir-30b favors or counteracts rsv replication is currently unclear. ns1 and ns2 interfere with the adaptive immune response. the ns proteins also play a role in the development of adaptive immune responses during rsv infection, partly as a consequence of their capacity to suppress type i and iii ifn levels. in addition to airway epithelial cells, rsv can infect dcs and activate prrs by pamps [93] . munir and colleagues investigated the activation status of isolated human monocyte-derived myeloid dcs upon rsv infection by quantifying several maturation markers, including cluster of differentiation (cd)38, 54, 80, 83, and 86 [41] . infection with wild-type and ns-deficient rsv strains highlighted that ns1, and to a lesser extent ns2, suppress the maturation of human dcs. by using an ifnar2-blocking antibody, this suppression was shown to partially depend on the capacity of the ns proteins to counteract the production of type i ifns. moreover, in vivo pulmonary conventional dc activation, as measured by the up-regulation of cd86 and cd80, was strongly hampered in rsv-infected mice that are deficient for mavs, an essential signaling protein for rsv-induced type i ifn production [12] . taken together, these results highlight that type i ifns play a role in rsv-induced dc maturation. as such, the pleiotropic effector functions of ns1 and ns2 to counteract the production of type i ifns likely account for the reduced dc maturation by ns1 and ns2. in a follow-up report, munir and colleagues investigated the consequence of reduced dc maturation by ns1 and ns2 on subsequent activation of t-cell responses by co-cultivation of rsv-infected human monocyte-derived dcs and autologous t cells [94] . deletion of ns1 was found to promote the proliferation and activation of cd103 + cd8 + t cells and t-helper 17 cells, 2 cell populations that can counteract rsv, and to suppress the activation of il-4-producing cd4 + t cells. remarkably, none of these effects on t cells appeared to depend on type i ifn. possibly, il-12β and il-23α play a role as these cytokines were suppressed in dcs by ns1 in a type i ifn-independent manner. in contrast, a study by kotelkin and colleagues in mice identified that ns2, and not ns1, suppresses cd8 + t-cell responses in a type i ifndependent manner [95] . remarkably, although rsv-induced activation of dcs in mavs -/-and mavs -/myeloid differentiation primary response protein myd88 (myd88) -/mice was strongly impaired, these mice could still mount comparable cd8 + t-cell responses as wildtype mice [12] . this may in part be explained by the approximately 100-fold higher viral loads in mavs -/mice compared with wild-type mice. in addition, a small residual population of cd86-positive pulmonary dcs was still present in these mice that could migrate to the draining mediastinal lymph node to activate naive cd8 + t cells. as mavs -/-myd88 -/mice are still functional for toll/interleukin-1 receptor domain-containing adapter molecule 1 (ticam1), the adaptor of tlr3, these dcs may have matured after the activation of tlr3 by rsvderived double-stranded rna (dsrna) [96] . live-attenuated rsv vaccines with a targeted deletion in ns2 have been tested for use in infants [35] . whereas a strain with only ns2 deleted was insufficiently attenuated, 2 other ns2 deletion strains with additional mutations were found to be over-attenuated, highlighting the importance of a right balance between attenuation and immunogenicity. currently, 2 phase i clinical trials with other ns2 deletion strains (clinicaltrials.gov identifiers: nct03422237 and nct03387137) and one with an ns1 deletion (clinicaltrials.gov identifier: nct03596801) are ongoing. the primary endpoints of these ongoing phase i trials with the live-attenuated rsv strains that lack either ns1 or ns2 are measures for safety, vaccine virus infectivity, and the induction of rsv-neutralizing titers. to our knowledge, cellular immune responses after immunization with these live-attenuated rsv vaccines have not been investigated. taking into account the small size of ns1 and ns2, it is remarkable how many host functions are affected by these rsv proteins. goswami and colleagues hypothesized that ns1 and ns2 form a large (300 to 750 kda) degradative complex, which they called the ns degradasome (nsd) [51] . in a549 cells, this complex could selectively degrade particular innate immune signaling proteins of which rig-i was also confirmed as a substrate of nsd complexes isolated from rsv-infected cells. in this respect, it would be interesting to assess whether other reported ns target proteins are also substrates of the proposed nsd complexes in rsv-infected cells. the nsd complex appears to be metastable, and its activity is enhanced by mitochondria via mavs, possibly through stabilizing the nsd complex [51] . interestingly, in mavs-deficient cells stimulated with ifn-α, wild-type rsv is almost equally attenuated as rsv lacking ns1 and ns2, confirming the importance of mavs for ns protein-mediated suppression of type i ifn responses. in line with these results, ns2 mainly localizes to mitochondria and seems to recruit ns1 towards the mitochondria [58] . furthermore, ns target proteins, but not other innate proteins, relocate from the cytoplasm to the mitochondria upon expression of ns1 and ns2 [51] . all together, these results support the hypothesis of the formation of a mitochondrialassociated nsd complex to selectively degrade innate immune proteins in rsv-infected cells. although the exact composition of the nsd complex is still unresolved, the presence of the α2 subunit from the 20s core proteasome sparked the idea that the nsd complex might act in a similar way as the host 26s proteasome [51] . in line with this hypothesis, ns protein-induced stat2 and oasl degradation is (partially) blocked by the proteasome inhibitors mg132 and lactacystin [51, 60, 61, 73, 75] . in contrast, ns protein-induced traf3 and ikkε degradation appears insensitive to mg132 [52] . proteasome-like activity of the nsd complex is further supported by the identification of ns1 and ns2 as potential inducers of host protein ubiquitination [73, 97] . whereas ns1 is a putative elongin b/c-cullin 2/5-socs box-type e3 ubiquitin ligase, ns2 does not contain an elongin c binding consensus sequence [73, 97, 98] . interestingly, an rsv strain with 3 mutated ns2 residues that appear essential for ubiquitination activity and ns2-mediated stat2 degradation is nearly equally attenuated as an ns2 deletion strain [97] . these results suggest that ns2 and ns1 may induce ubiquitination of host proteins, which are then degraded by the nsd complex. it is currently not known, however, if ns1 and/or ns2 selectively mark innate immune proteins for degradation, which would explain the selective degradative activity of the nsd complex. recently, the crystal structure of ns1 was determined and revealed a remarkable structural homology with the n-terminal domain of the matrix protein of rsv [37]. one clear difference between ns1 and m is the presence of an α-helix (called α3) at the c-terminus of ns1. a truncated ns1 lacking this helix or mutation of 3 residues that are important for the interaction of helix α3 with the rest of ns1 partially abrogates ns1-mediated suppression of ifn-β induction and attenuates recombinant rsv strains. these results confirm that helix α3 is important for at least some ns1-mediated effector functions. unravelling the crystal structure of ns2 and the composition of the nsd complex would greatly enhance our understanding of the remarkable diverse effector functions of ns1 and ns2. moreover, ns1 and/or ns2 may be attractive targets for antiviral therapy. intranasal administration of nanoparticles carrying a plasmid encoding an ns1-targeting small interfering rna (sirna) in mice can reduce lung viral titers, airway hyperresponsiveness, and pulmonary inflammation, both in a prophylactic as well as therapeutic setting [99] . a recent small compound high-throughput screen revealed 4 candidate inhibitors of ns2, highlighting that ns2 could be targeted by a small compound drug [100] . in humans, rsv infections induce remarkably low levels of ifn-α and -β compared with other respiratory viruses. this is largely the consequence of 2 unique viral proteins, ns1 and ns2, that strongly suppress the induction and signaling of ifn. in recent years, evidence is rising that, in addition, antiviral activities of isgs may be hampered by the ns proteins. ns1 and ns2 exert additional functions, such as delaying cell apoptosis, and loss of ns1 is associated with a stronger adaptive immune response and reduced in vivo viral replication. ns1 and ns2 are thought to form a so-called "ns degradasome" complex, which may function as a proteasome-like complex that selectively degrades a plethora of innate immune proteins. further insight in the composition of the nsd and the resolution of the structure of ns2 will likely help to explain the remarkable diverse effector functions of ns1 and ns2. some effector functions, however, have so far only been identified in artificial cell systems and should be interpreted carefully. large-scale protein-protein interaction screens, preferentially performed using multiple complementary protein-protein interaction techniques in the context of an rsv infection, will generate a more comprehensive list of host proteins that interact with ns1 and/or ns2. such new knowledge, combined with co-crystal structure analysis of rsv ns1 and ns2 in complex with host protein factors, will be instrumental to design antiviral drugs that impact on the rsv-host interface and thereby complement directly acting antivirals. global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis global, regional, and national disease burden estimates of acute lower respiratory infections due to respiratory syncytial virus in young children in 2015: a systematic review and modelling study estimates of the global, regional, and national morbidity, mortality, and aetiologies of lower respiratory infections in 195 countries nonstructural proteins ns1 and ns2 of bovine respiratory syncytial virus block activation of interferon regulatory factor 3 a novel mechanism for the inhibition of interferon regulatory factor-3-dependent gene expression by human respiratory syncytial virus ns1 protein replacement of the respiratory syncytial virus nonstructural proteins ns1 and ns2 by the v protein of parainfluenza virus 5 nonstructural proteins 1 and 2 of respiratory syncytial virus suppress maturation of human dendritic cells hepatitis c virus protease ns3/4a cleaves mitochondrial antiviral signaling protein off the mitochondria to evade innate immunity disruption of innate immunity due to mitochondrial targeting of a picornaviral protease precursor mutual antagonism between the ebola virus vp35 protein and the rig-i activator pact determines infection outcome influenza a virus ns1 targets the ubiquitin ligase trim25 to evade recognition by the host viral rna sensor rig-i respiratory syncytial virus ns1 protein colocalizes with mitochondrial antiviral signaling protein mavs following infection human respiratory syncytial virus ns 1 targets trim25 to suppress rig-i ubiquitination and subsequent rig-i-mediated antiviral signaling. viruses the severe acute respiratory syndrome coronavirus nucleocapsid inhibits type i interferon production by interfering with trim25-mediated rig-i ubiquitination molecular mechanism of influenza a ns1-mediated trim25 recognition and inhibition human respiratory syncytial virus nonstructural protein ns2 antagonizes the activation of beta interferon transcription by interacting with rig-i viral degradasome hijacks mitochondria to suppress innate immunity respiratory syncytial virus nonstructural proteins decrease levels of multiple members of the cellular interferon pathways microrna-146a induction during influenza h3n2 virus infection targets and regulates traf6 levels in human nasal epithelial cells (hnecs) extracellular vesicles containing mir-146a attenuate experimental colitis by targeting traf6 and irak1 microrna 146-5p, mir-let-7c-5p, mir-221 and mir-345-5p are differentially expressed in respiratory syncytial virus (rsv) persistently infected hep-2 cells effects of nonstructural proteins ns1 and ns2 of human respiratory syncytial virus on interferon regulatory factor 3, nf-kappab, and proinflammatory cytokines nonstructural proteins of respiratory syncytial virus suppress premature apoptosis by an nf-kappab-dependent, interferon-independent mechanism and facilitate virus growth multiple functional domains and complexes of the two nonstructural proteins of human respiratory syncytial virus contribute to interferon suppression and cellular location respiratory syncytial virus non-structural protein 1 facilitates virus replication through mir-29a-mediated inhibition of interferon-alpha receptor specific inhibition of type i interferon signal transduction by respiratory syncytial virus respiratory syncytial virus nonstructural protein 2 specifically inhibits type i interferon signal transduction respiratory syncytial virus nonstructural proteins ns1 and ns2 mediate inhibition of stat2 expression and alpha/beta interferon responsiveness human metapneumovirus inhibits ifn-beta signaling by downregulating jak1 and tyk2 cellular levels respiratory syncytial virus (rsv) attachment and nonstructural proteins modify the type i interferon response associated with suppressor of cytokine signaling (socs) proteins and ifn-stimulated gene-15 (isg15) rsv replication is attenuated by counteracting expression of the suppressor of cytokine signaling (socs) molecules respiratory syncytial virus ns1 protein degrades stat2 by inducing socs1 expression respiratory syncytial virus nonstructural proteins upregulate socs1 and socs3 in the different manner from endogenous ifn signaling identification of paramyxovirus v protein residues essential for stat protein degradation and promotion of virus replication ebola virus vp24 targets a unique nls binding site on karyopherin alpha 5 to selectively compete with nuclear import of phosphorylated stat1 nipah and hendra virus nucleoproteins inhibit nuclear accumulation of signal transducer and activator of transcription 1 (stat1) and stat2 by interfering with their complex formation ifnbeta-dependent increases in stat1, stat2, and irf9 mediate resistance to viruses and dna damage interferome v2.0: an updated database of annotated interferon-regulated genes respiratory syncytial virus ns1 protein degrades stat2 by using the elongin-cullin e3 ligase respiratory syncytial virus strain a2 is resistant to the antiviral effects of type i interferons and human mxa 2'-5'-oligoadenylate synthetase-like protein inhibits respiratory syncytial virus replication and is targeted by the viral nonstructural protein 1 the nonstructural proteins of pneumoviruses are remarkably distinct in substrate diversity and specificity role of p53/nf-kappab functional balance in respiratory syncytial virus-induced inflammation response rsv-encoded ns2 promotes epithelial cell shedding and distal airway obstruction a randomized, doubleblind, placebo-controlled trial of dexamethasone in severe respiratory syncytial virus (rsv) infection: effects on rsv quantity and clinical outcome the effect of high dose inhaled corticosteroids on wheeze in infants after respiratory syncytial virus infection: randomised double blind placebo controlled trial effect of dexamethasone on respiratory syncytial virus-induced lung inflammation in children: results of a randomized, placebo controlled clinical trial epithelial cells infected with respiratory syncytial virus are resistant to the anti-inflammatory effects of hydrocortisone respiratory syncytial virus represses glucocorticoid receptor-mediated gene activation respiratory syncytial virus (rsv) suppression of glucocorticoid receptor phosphorylation does not account for repression of transactivation poly i:c and respiratory syncytial virus (rsv) inhibit glucocorticoid receptor (gr)-mediated transactivation in lung epithelial, but not monocytic, cell lines the respiratory syncytial virus (rsv) nonstructural proteins mediate rsv suppression of glucocorticoid receptor transactivation glucocorticoid insensitivity in virally infected airway epithelial cells is dependent on transforming growth factor-beta activity respiratory syncytial virus nonstructural protein 1 blocks glucocorticoid receptor nuclear translocation by targeting ipo13 and may account for glucocorticoid insensitivity human respiratory syncytial virus non-structural protein ns1 modifies mir-24 expression via transforming growth factor-beta respiratory syncytial virus regulates human micrornas by using mechanisms involving beta interferon and nf-kappab respiratory syncytial virus modifies micrornas regulating host genes that affect virus replication nf-kappab p65-dependent transactivation of mirna genes following cryptosporidium parvum infection stimulates epithelial cell immune responses primary human mdc1, mdc2, and pdc dendritic cells are differentially infected and activated by respiratory syncytial virus respiratory syncytial virus interferon antagonist ns1 protein suppresses and skews the human t lymphocyte response the ns2 protein of human respiratory syncytial virus suppresses the cytotoxic t-cell response as a consequence of suppressing the type i interferon response respiratory macrophages and dendritic cells mediate respiratory syncytial virus-induced il-33 production in tlr3-or tlr7-dependent manner. int immunopharmacol identification of respiratory syncytial virus nonstructural protein 2 residues essential for exploitation of the host ubiquitin system and inhibition of innate immune responses mutation of the elongin c binding domain of human respiratory syncytial virus non-structural protein 1 (ns1) results in degradation of ns1 and attenuation of the virus inhibition of respiratory syncytial virus infection with intranasal sirna nanoparticles targeting the viral ns1 gene modular cell-based platform for high throughput identification of compounds that inhibit a viral interferon antagonist of choice key: cord-007382-5kb16qb7 authors: hartmann, g. title: nucleic acid immunity date: 2016-12-15 journal: adv immunol doi: 10.1016/bs.ai.2016.11.001 sha: doc_id: 7382 cord_uid: 5kb16qb7 organisms throughout biology need to maintain the integrity of their genome. from bacteria to vertebrates, life has established sophisticated mechanisms to detect and eliminate foreign genetic material or to restrict its function and replication. tremendous progress has been made in the understanding of these mechanisms which keep foreign or unwanted nucleic acids from viruses or phages in check. mechanisms reach from restriction-modification systems and crispr/cas in bacteria and archaea to rna interference and immune sensing of nucleic acids, altogether integral parts of a system which is now appreciated as nucleic acid immunity. with inherited receptors and acquired sequence information, nucleic acid immunity comprises innate and adaptive components. effector functions include diverse nuclease systems, intrinsic activities to directly restrict the function of foreign nucleic acids (e.g., pkr, adar1, ifit1), and extrinsic pathways to alert the immune system and to elicit cytotoxic immune responses. these effects act in concert to restrict viral replication and to eliminate virus-infected cells. the principles of nucleic acid immunity are highly relevant for human disease. besides its essential contribution to antiviral defense and restriction of endogenous retroelements, dysregulation of nucleic acid immunity can also lead to erroneous detection and response to self nucleic acids then causing sterile inflammation and autoimmunity. even mechanisms of nucleic acid immunity which are not established in vertebrates are relevant for human disease when they are present in pathogens such as bacteria, parasites, or helminths or in pathogen-transmitting organisms such as insects. this review aims to provide an overview of the diverse mechanisms of nucleic acid immunity which mostly have been looked at separately in the past and to integrate them under the framework nucleic acid immunity as a basic principle of life, the understanding of which has great potential to advance medicine. immunology is typically categorized in innate and adaptive immunity. while the term innate is associated with conserved molecular patterns detected by germline-encoded receptors, adaptive immunity refers to t cells and b cells which use recombination and clonal selection to specifically adapt their immune receptors (t cell receptor, b cell receptors, and antibodies) in order to target foreign protein antigens. in this wellestablished concept, the innate immune system in the form of myeloid immune cells (macrophages, dendritic cells) provides information whether new protein antigens are associated with potential pathogens or damage. a limited number of germline-encoded innate immune receptors have been identified in the last two decades which are specialized to detect different classes of pathogen-or damage-associated molecules. among them are several groups of immune receptors which are specialized on the detection of foreign or damage-associated nucleic acids. one of these groups of nucleic acid-sensing immune receptors are the toll-like receptors (tlrs) tlr3, tlr7, tlr8, and tlr9 (tlr13 not existent in humans) which are preferentially located in the endolysosomal compartment of distinct immune cell subsets and certain somatic cells. nucleic acid-detecting immune receptors located in the cytosol include the rig-i family of helicases (rig-i, mda5, lgp2), cgas, and aim2. although these nucleic acid-sensing immune receptors as part of the innate immune system participate in the regulation of protein antigen-directed adaptive immunity, they are now appreciated as part of a larger system of nucleic acid-directed immunity which functions to detect and eliminate foreign nucleic acids, whereas protein-directed adaptive immunity evolved to eliminate foreign proteins. although there are crossregulatory functions of both systems, nucleic acid-directed immunity has a purpose on its own. this is underlined by the fact that protein-directed adaptive immunity developed more recently in evolution, whereas nucleic acid-directed immunity dates back to the earliest forms of life represented by bacteria and archaea. with the additions of rnai and the crispr/cas system, specific nucleases including the restriction-modification (r-m) systems, and antiviral effector proteins partially discovered only recently in the context of rare hereditary inflammatory diseases, the new concept of nucleic acid immunity evolves. with crispr/cas and rnai, biology has established two mechanisms which acquire new sequence information of pathogens and memorize this information for later defense against the same type of pathogen, characteristics which functionally correspond to adaptive immunity of t cells and b cells. the various mechanisms comprising nucleic acid immunity are highly relevant for the understanding of many inflammatory and infectious diseases. this review summarizes the currently known nucleic acid recognition-based antiviral response strategies. antiviral response strategies span from ancient sequence or nucleic acid modificationdependent degradation systems (r-m, crispr, rnai) to modern innate immunity in vertebrates, in which innate nucleic acid-sensing receptors induce a broad spectrum of antiviral alarm and effector mechanisms as well as subsequent adaptive immune responses. foreign nucleic acids can be introduced by viruses or bacteriophages. however, species differ in their arsenal of defense mechanisms against such foreign nucleic acid invaders (fig. 1 ). all species from bacteria to humans have established different types of nucleases which cleave nucleic acids that have identified themselves as foreign by their specific structure, by abundance and localization. one of the earliest forms of nucleases are the r-m systems in bacteria and archaea. in this system, modification enzymes and restriction endonucleases (reases) are directed to certain dna sequence motifs in self dna. since modified dna is not cleaved by the corresponding nucleases, dna sequence motifs without modification are identified as foreign and are degraded (for further details, see later). besides the r-m systems, a variety of rnases and dnases are established in evolution. dna outside the nucleus is degraded by dnases i, ii, and iii. fig. 1 mechanisms of nucleic acid immunity in species relevant for human disease. biology has evolved a number of mechanisms to detect and eliminate foreign nucleic acids as introduced by viruses or bacteriophages. all species from bacteria to humans have established nucleases to directly degrade nucleic acids with structural characteristics or localizations which allow to distinguish them from regular cellular self nucleic acids. other mechanisms are predominant in certain groups of species. restrictionmodification systems in bacteria and archaea apply sequence-specific modification of self nucleic acids which allows the specific detection and degradation of foreign nucleic acids (restriction endonucleases). acquired sequence information is used by the crispr/cas system in which new sequence information about pathogenic nucleic acids is integrated into the genome and thereby memorized in order to sequencespecifically degrade foreign nucleic acids. sequence information is also used by rna interference which serves antiviral nuclease functions (sirna/dicer) as well as regulatory (microrna) functions in higher multicellular organisms. in vertebrates, innate immune-sensing receptors including dicer-related helicases rig-i and mda5 dominate over rnai as antiviral defense mechanism. while innate nucleic acid immune-sensing receptors elicit signaling pathways resulting in antiviral functions, a number of nucleic acid receptors (e.g., pkr, adar1, ifit1) directly detect and restrict nucleic acid function and replication. since the principles of nucleic acid immunity are either established in mammals themselves or in pathogens (bacteria, parasites, helminths) or pathogentransmitting insects (e.g., mosquitoes), nucleic acid immunity as such is highly relevant for human health and disease. the rna in dna-rna hybrids is degraded by rnase h. long doublestranded rna in the cytosol is subject to dicer which cleaves rna down to short double-stranded oligoribonucleotides which enter the rnai pathway. another way to acquire sequence information for antiviral defense is used by the crispr/cas system. in this system, nucleic acid sequences derived from pathogens are integrated into the genome which allows the sequence-specific identification of the same type of pathogen during a subsequent challenge. in vertebrates, a number of highly specialized innate immune-sensing receptors such as tlr9 or rig-i evolved to detect pathogen-associated nucleic acids and to induce appropriate immune responses. while innate nucleic acid immune-sensing receptors elicit antiviral signaling pathways, a number of nucleic acid-detecting effector proteins (viral restriction factors, e.g., pkr, adar1, ifit1) directly detect and restrict nucleic acid function and replication. various principles of nucleic acid immunity apply to different species, with only a subset applying to mammals. however, if it comes to infectious diseases, pathogens (bacteria, parasites, helminths) and pathogen-transmitting insects (e.g., mosquitoes) use additional mechanisms not present in mammals, which contribute to the interaction of the pathogen with transmitting organisms, and thus represent potential prophylactic or therapeutic targets. several fields initially developed as independent lines of research and only recently were appreciated to closely cooperate in a defense system specialized in the detection and elimination of foreign genetic material. fig. 2 provides a rough time line of key discoveries of principles, receptors, and ligands emerging from different areas of research all in the context of nucleic acid immunity. this brief overview cannot be comprehensive or provide the exact timing of each single discovery. the idea rather is to provide a picture how different fields evolved over the years. for more detailed information on the different receptors and pathways, the reader is referred to the respective specific paragraphs of this chapter below (figs. 5 and 6). immune sensing of nucleic acids dates back to the early 1960s with the observation that nucleic acids such as long double-stranded rna and specifically poly(i:c) can induce the antiviral factor type i interferon (isaacs, cox, & rotem, 1963) which was first described in 1957 (isaacs, 1957; lindenmann, burke, & isaacs, 1957 fig. 2 overview of the time line of discoveries in nucleic acid immunity. this graph provides a noncomprehensive overview of the time lines when important principles, receptors, and ligands contributing to nucleic acid immunity have been described. immune sensing of nucleic acids dates back to the early 1960s with the observation that nucleic acids such as long double-stranded rna and specifically poly(i:c) can induce type i interferon. later, it was appreciated that bacterial dna is more active than vertebrate dna. in 1995, the activity of bacterial dna was attributed to a higher frequency of unmethylated cpg motifs in bacterial dna. in 2000, tlr9 was identified as the immune receptor for the detection of unmethylated cpg motifs in dna in the endosomal compartment. sensing of cytoplasmic dna remained unclear until in 2009 aim2 and in 2012 cgas were identified as the cytosolic receptors responsible for dna-induced inflammasome activation and type i ifn induction, respectively. for immune sensing of rna, the story of discoveries continued in 2001 with reports on tlr3-sensing long double-stranded rna and was continued in 2004 with the appreciation of tlr7 and tlr8 as receptors sensing shorter forms of unmodified single and double-stranded rna with great implications for the application of sirna. another milestone was reached with the immune sensing of cytoplasmic forms of rna, specifically the detection of 5 0 -triphosphate short double-stranded forms of rna by the cytosolic receptor rig-i. the rig-i-like receptor mda5 added another cytosolic receptor which explained the induction of type i ifn by long double-stranded forms of rna as observed early on in the 1960s. pkr identified in the late 1970s was the first of the receptors restricting nucleic acid function and replication without activating immunity and cytokines. samhd1 that bacterial dna induces type i ifn much more vigorously than genomic dna of vertebrates (yamamoto, kuramoto, shimada, & tokunaga, 1988; yamamoto, yamamoto, shimada, et al., 1992) . it was speculated that bacterial dna in sir william coley's bacterial lysates was responsible for the antitumor activity seen using bacterial lysates for the treatment of tumor patients around 100 years earlier (wiemann & starnes, 1994) . efforts were undertaken to generate synthetic oligodeoxynucleotides which mimic the type i ifn-inducing activity of bacterial dna. palindromic dna sequences were identified, and oligonucleotides containing such palindromes induced type i ifn in vitro (yamamoto, yamamoto, kataoka, et al., 1992) but only showed weak activity in tumor models in vivo due to rapid degradation by dnases. in 1995, the immunological activity of bacterial dna was attributed to a higher frequency of unmethylated cpg motifs in bacterial dna (krieg et al., 1995) . unmethylated cpg motifs were contained in the former palindromic sequences, but a palindromic sequence was not required for the type i ifn-inducing activity. the introduction of the phosphorothioate modification in dna first described in 1977 (vosberg & eckstein, 1977) was used to stabilized these so-called cpg oligonucleotides which now could be successfully applied for treatment in experimental tumor models in vivo (heckelsmiller et al., 2002) . in 2000, tlr9 was identified as the innate immune receptor required for the detection of unmethylated cpg motifs in dna (hemmi et al., 2000) . notably, tlr9 was the first innate immune receptor reported to detect a specific type of nucleic acid and to induce an immune response. despite intensive research, it took almost a decade to identify aim2 as the next innate immune receptor-detecting dna (hornung & latz, 2010) . (depletion of dntps) and adar1 (a-to-i conversion in dsrna) entered the field more recently in the context of genetic alterations in these genes identified in the context of inherited inflammatory syndromes (e.g., ags). ifit1 and ifit5 are two other examples of more recently described receptors which inhibit the translation of mrna. oas1 was identified early on soon after pkr as a factor restricting viral replication by activating rnase l. other nucleases contributing to nucleic acid immunity include rnase h structurally resolved in 2004, which degrades the rna in dna-rna hybrids; furthermore, extracellular dnase i and endolysosomal dnase ii are known since the mid-1950s. knowledge around the function of the cytoplasmic dnase iii which is also called trex1 accumulated since 1999 and gained great impact on nucleic acid immunity like samhd1 and adar1 more recently in the context of inherited type i ifn-dependent inflammatory syndromes. antiviral rnai and the role of dicer were first described in 2005, while the bacterial version of sequence-specific antiviral immunity, crispr/cas, was identified in 2011. restriction-modification systems are studied since the 1950s. however, since aim2 activates the inflammasome but not type i ifn, the field of dna sensing struggled until end of 2012, when the cytosolic dnabinding enzyme cyclic gmp-amp synthase (cgas) was discovered which generates cgamp as second messenger for the downstream signaling molecule sting, resolving a big question mark in the field. at that time, sting was already known to be required for immune sensing of cytosolic dna resulting in ifn induction (barber, 2014) , but sting was unable to bind dna directly. although rna molecules were the first nucleic acids found to induce type ifn as described earlier, this line of research continued only in 2001 with the identification of double-stranded rna as ligand for tlr3 (alexopoulou, holt, medzhitov, & flavell, 2001) . although the activation of tlr3 induces some type i ifn, it could not explain the massive amounts of type i ifn induced upon cytosolic delivery of the double-stranded rna mimic poly(i:c). in parallel with the discovery of rnai and sirna, techniques of chemical synthesis of rna rapidly progressed and highly pure synthetic rna oligonucleotides at high quantities and reasonable costs became available. with easier access to highly pure synthetic rna oligonucleotides, it was found that not only long double-stranded rna but also single-stranded rna stimulated type i ifn, specifically in immune cells, and the immune receptors involved were found to be tlr7 and tlr8 (tlr8 nonfunctional in mouse) (diebold, kaisho, hemmi, akira, & reis e sousa, 2004; heil et al., 2004; judge et al., 2005) . then it was noted that even sirna induce type i ifn in tlr7 expressing immune cells, a surprising finding at that time since sirna was thought to be short enough to not stimulate interferon responses . this work also reported that the immune stimulatory activity of sirna can be avoided by introducing chemical modifications such as 2 0 -o-methylation or by introducing pseudouridine which is used since then to avoid immunostimulation by sirna applied in cells expressing tlr7 in vitro or in vivo . at that time, a cheap way to generate sirna was the use of in vitro transcription. however, it was soon realized that sirna made by in vitro transcription induces high amounts of type i ifn when transfected even in cells not expressing tlr7, including human myeloid immune cells. this stimulated research on the molecular mechanism responsible for type i ifn induction by in vitro-transcribed sirna in myeloid immune cells. finally, the cytosolic helicase rig-i was identified to detect 5 0 -triphosphate ends in short blunt end double-stranded rna (hornung et al., 2006; pichlmair et al., 2006; schlee, roth, et al., 2009) . rig-i was reported earlier as immune receptor involved in antiviral responses (kato et al., 2005) . notably, in vitro transcription but not chemical synthesis of sirna generates such 5 0 -triphosphate ends. the presence of unmodified 5 0 -triphsophate ends in the cytosol indicates the presence of rna polymerase activity in the cytosol which only occurs in the course of viral replication. another member of the rig-i-like helicase family of receptors is mda5 which was found to be responsible for the long sought after type i ifn-inducing activity of cytosolic long double-stranded rna including poly(i:c) . as of today, most of the type i ifn-inducing activities of nucleic acids can be assigned to specific immune receptors. future may still keep some surprises for the field, for example, in the context of immune sensing in the nucleus or in the context of dna damage repair. while activation of the immune receptors described above results in the induction of immunologically active cytokines and immune responses, there is a group of nucleic acid receptors directly restricting nucleic acid function and replication largely without inducing an immune response. pkr was one of the first of these. binding of long double-stranded rna activates pkr to phosphorylate elf2a leading to the inhibition of ribosomal translation of mrna to proteins (clemens & elia, 1997; sen, taira, & lengyel, 1978; thomis, doohan, & samuel, 1992; . soon after pkr, the 2 0 -5 0 -oligoadenylate synthetase (oas) system was reported (rebouillat & hovanessian, 1999; yang et al., 1981) . in parallel to the oas system, rnase l was identified (baglioni, minks, & maroney, 1978; brennan-laun, ezelle, li, & hassel, 2014) . upon binding of oas1 to long double-stranded rna, oas1 generates 2 0 -5 0 -linked oligoadenylates (2 0 5 0 -oa). 2 0 5 0 -oa activate rnase l which then cleaves cellular rnas thereby restricting viral propagation. samhd1 (sterile alpha motif and histidine-aspartate-domaincontaining protein 1), originally described as ifn-inducible gene in 2000 (li, zhang, & cao, 2000) , has triphosphohydrolase activity that rapidly converts dntps to the corresponding deoxynucleoside and inorganic triphosphate, thereby depleting the supply of dntp for reverse transcriptase activity of retroviruses. in 2009, it was learned that mutations in samhd1 cause inherited inflammatory syndromes with a type i ifn signature (e.g., ags) (rice et al., 2009 ), but the exact mechanism leading to type i ifn induction in this context is not well understood. originally cloned in 1994 (kim, wang, sanford, zeng, & nishikura, 1994) , the rna-editing enzyme adar1 binds to double-stranded rna and converts a to i thereby contributing to self vs nonself recognition of rna (nishikura, 2016) . in 2012, it was realized that mutations in adar1 cause inflammatory syndromes associated with a type i ifn signature (rice et al., 2012) . ifit1 and ifit5 are known for many years as type i ifn-inducible rna-binding proteins which bind to single-stranded rna lacking 2 0 -o-methylation at their 5 0 -end and inhibiting rna translation (hyde & diamond, 2015) . more recent work from 2011 (pichlmair et al., 2011) added the information that ifit1 and ifit5 preferentially bind to viral rna containing a 5 0 -triphosphate group, completing the picture how these proteins distinguish self from nonself single-stranded rna. of the proteins which function primarily as nucleases, extracellular dnase i and endolysosomal dnase ii are known since the mid-1950s. the cytoplasmic exonuclease trex1 has been identified decades later in 1999 (hoss et al., 1999) . only since 2006, we know that loss of function in trex1 causes the type i ifn-associated inflammatory syndrome ags (crow et al., 2006) , suggesting that trex1 is critically involved in the clearance self dna within the cytoplasm of cells. rnases h are widely expressed enzymes that hydrolyze rna in rna/dna hybrids (cerritelli & crouch, 2009 ). while reports on rnase h activity date back to 1969 (stein & hausen, 1969) , the heterotrimeric functional complex in eukaryotes was only described in 2004 (jeong, backlund, chen, karavanov, & crouch, 2004) and human in 2009 (chon et al., 2009 ). in 2007, it was reported that mutations in any of the three subunits of human rnase h2 cause aicardi-gouti eres syndrome (ags) (rice et al., 2007) . antiviral rnai and the role of dicer were first described in 2006 (wang et al., 2006; zambon, vakharia, & wu, 2006) , while the role of rna interference (rnai) in mammalian innate immunity is still poorly understood. a bacterial counterpart of acquired sequence-specific antiviral immunity is the crispr/cas system in prokaryotes first described in 2007 (barrangou et al., 2007; marraffini & sontheimer, 2010) . this prokaryotic immune system confers resistance to foreign genetic elements such as those present within plasmids and phages. information around r-m systems accumulated since the early 1950s (luria & human, 1952) with reases first described in 1975 (nathans & smith, 1975) . the concept of nucleic acid immunity integrates different functional components which have been studied and reviewed separately in the past. only in the last few years and with the elegant genetic work on rare human inflammatory disorders associated with a type i ifn signature (of crow and others (crow & rehwinkel, 2009 )), it became evident that many of the antiviral restriction factors and various nuclease systems are tightly connected with innate immune sensing of nucleic acids inducing type i ifn. altogether, biology has established a broad spectrum of effector functions which cover most of the molecular and mechanistic possibilities to restrict the propagation of foreign genetic material. effector functions reach from direct actions on the detected nucleic acid to the elimination of cells containing foreign genetic material. fig. 3 illustrates the functional components of nucleic acid immunity. central to all components is the detection of foreign nucleic acids by the molecular interaction of a protein (immune-sensing receptor, restriction factor, or effector protein) or a specific nucleic acid (rnai, crispr/cas) with the targeted nucleic acid. the molecular challenge on this level is the specificity of the detection and the distinction of self vs foreign. a specific molecular signature of self nucleic acid (e.g., 2 0 -o-methylation at the n1 position in capped mrna), compartmentalization of self nucleic acids, and the rapid clearance of surplus self nucleic acids are three examples which enable specific detection of foreign nucleic acid. structural differences such as long double-stranded rna not present under physiological circumstances allow the highest confidence level of detection. upon detection, the system generates different types of responses. detection of a foreign nucleic acid can trigger an intrinsic effect which acts directly on the detected nucleic acid. examples are degradation (e.g., trex1, crispr/cas), structural modification (e.g., a-to-i conversion by adar1), or disabling the function (e.g., inhibition of translation of mrna by ifit1 or rnai) (see fig. 3 , middle gray layer). extrinsic effects upon detection of foreign nucleic acids require a signaling cascade finally resulting in an effect on the detected nucleic acid. extrinsic effects can occur solely inside the same cell, or they can involve functions outside the cell. extrinsic effects inside the same cell include degradation of the nucleic acid (e.g., rnase l activated by 2 0 -5 0 -oa generated by oas1 upon binding of long double-stranded rna). extrinsic effect inside the same cell can also impact on the function of the detected nucleic acid. examples for such functional effects are inhibition of translation (e.g., phosphorylation of elf2a by activated pkr) or inhibition of replication (e.g., restricting replication of retroviruses by depleting intracellular dntp pools by ifn-inducible samhd1) (see fig. 3 , light gray layer). extrinsic effects that involve functions outside the cell in which the nucleic acid is primarily detected appear as immune responses. they range from alarming neighboring cells overview of functional components in nucleic acid immunity. the primary detection of specific forms of nucleic acids by highly specialized proteins is the central part of nucleic acid immunity. upon binding of nucleic acids, the participating specialized proteins can either exert intrinsic direct effects on the nucleic acid which they have bound, or they can have indirect extrinsic effects which require the participation of additional signaling. extrinsic effects that restrict viral replication and function can be located inside or outside cells, or both. intrinsic direct effects include degradation or structural modification of the bound nucleic acids, or direct inhibition of translation. extrinsic indirect effects via signaling pathways include mechanisms that restrict translation or replication, or that lead to degradation of nucleic acids. extrinsic effects with activities beyond the infected cell include alarming neighboring cells, activating immune effector cells, and guiding immune cells to the site of infection. together, these intrinsic and extrinsic activities represent the repertoire of nucleic acid immunity to restrict viral infection. (e.g., secretion of type i ifn or release of cgamp via gap junctions) to the guidance of immune cells to the respective cell (e.g., tlr7-induced release of ip-10) and the activation of immune cells at the local site and systemically (e.g., activation of nk cells by rig-i). intrinsic and extrinsic effects act in concert to minimize the danger associated with foreign nucleic acids. in classical immunology, we distinguish innate and adaptive immunity. while innate immunity relies on receptors encoded in the germline, adaptive immunity acquires information about pathogens during the life span and memorizes such information for later use. while adaptive immunity directed against proteins relies on the mechanism of genetic recombination to adapt to novel pathogen-derived proteins, in the adaptive part of nucleic acid immunity information on pathogen-derived nucleic acid sequences is acquired and memorized (crispr/cas and rnai) (see fig. 4 ). although the adaptive part of nucleic acid immunity requires the participation of germline-encoded proteins such as dicer, risc, innate information on structure acquired information on sequence nucleic acid receptors with effector functions crispr/cas rna interference nucleic acid receptors inducing immune functions fig. 4 innate and adaptive components in nucleic acid immunity. in classical immunology, we distinguish innate and adaptive immunity. while innate immunity relies on receptors encoded in the germline, adaptive immunity acquires information about pathogens during the life span and memorizes such information for later usage. while adaptive immunity directed against proteins relies on the mechanism of genetic recombination to adapt to novel pathogen-derived proteins, in the adaptive part of nucleic acid immunity information about pathogen-derived nucleic acid sequences is acquired and memorized (crispr/cas and rna interference). unlike the adaptive components, the innate components of nucleic acid immunity rely on germline-encoded receptors which detect certain structures indicating viral pathogens. therefore, these receptors are identical throughout the life span, but regulation of receptor expression (e.g., by means of epigenetics) still allows adaptation to different environments (e.g., low or high burden of viral pathogens). or crispr and cas, the detection of foreign nucleic acids is mediated via acquired sequence information. in contrast, the innate components of nucleic acid immunity solely rely on germline-encoded receptors which via protein-nucleic acid interaction detect certain structures which are characteristic of foreign genetic materials. innate immune-sensing receptors are identical throughout the life span. however, it is important to note that epigenetic regulation of gene expression still allows to adapt quantitatively to different environments (e.g., low or high burden of viral pathogens). the perpetual arms race between bacteria and phages has resulted in the evolution of efficient resistance systems that protect bacteria from phage infection. such systems include r-m systems and crispr-cas. the prokaryotic dna r-m systems are based on the contrasting enzymatic activities of a sequence-specific rease and a matching sequence-specific host methyltransferase (mtase) (vasu & nagaraja, 2013) . by transferring a methyl group to the c-5 carbon or the n4 amino group of cytosine or to the n6 amino group of adenine host-specific mtases protect potential cleavage sites of host dna from reases, which on the other hand recognize and cleave foreign unmethylated or "inappropriately" methylated dna from invading phages. this r-m systems can be considered as an innate defense system. on the other hand, the crispr-cas system in prokaryotes represents a highly sophisticated adaptive immune system in which short fragments of invading dna are integrated into the crispr loci. after transcription and processing of these loci, short crispr rnas (crrnas) are generated which guide the nuclease activity of cas proteins to complementary dna or rna resulting in target cleavage (goldfarb et al., 2015; van der oost, westra, jackson, & wiedenheft, 2014). this chapter provides an overview of the well-established proteins targeting foreign nucleic acids without involving the classical immune functions such as the induction of cytokines or the activation of immune cells. such proteins can directly act on the foreign nucleic acid, or they can elicit pathways indirectly acting on the foreign nucleic acid (see fig. 5 ). for the family of apobec proteins which detect and modify viral nucleic acids, we refer to detailed reviews by others (harris & dudley, 2015) . this graph provides an overview of the proteins which target foreign nucleic acids without involving the classical immune functions such as the induction of cytokines or the activation of immune cells. such proteins can directly act on the foreign nucleic acid, or they can elicit pathways indirectly acting on the foreign nucleic acid. the endonuclease dnase i is the most abundant dnase in the extracellular space which degrades dna down to tetramers. dnase ii is the predominant endonuclease in the endolysosomal compartment of cells. the cytoplasmic dnase iii (trex1) is a 3 0 -to 5 0 exonuclease which degrades both double-and single-stranded dna. the cytoplasmic rnase h recognizes dna-rna hybrids and cleaves the rna in such hybrids. in contrast, rnase l is indirectly activated by oligoadenylates which are formed by oas1 upon binding to long doublestranded rna. furthermore, adar1 modifies long double-stranded rna by a-to-i conversions destabilizing the double strand resulting in changes in the coding sequence of proteins. samhd1 depletes the pool of dntps which is the prerequisite for dna formation. samhd1 hydrolyzes the triphosphate in dntps resulting in deoxynucleosides. at the same time, samhd1 has been proposed to be a 3 0 -exonuclease for single-stranded dna and rna. pkr and ifit1/5 inhibit mrna translation by phosphorylation of the elf2a and by replacing elf4 in the ribosomal complex, respectively. while pkr is activated by long double-stranded rna, ifit1 and ifit5 bind 5 0 -triphosphate ends of singlestranded rna. adenosine to inosine (a-to-i) rna editing was originally discovered as enzymatic activity unwinding double-stranded rna in xenopus laevis oocytes and embryos (bass & weintraub, 1987) . soon after, it became clear that this activity is carried out by an adenosine deaminase acting on rna (adar) (bass & weintraub, 1988; wagner, smith, cooperman, & nishikura, 1989) . adenosine deaminases perform c6 deamination of adenosine in base-paired rna structures resulting in a-to-i conversions, a process termed a-to-i rna editing (hogg, paro, keegan, & o'connell, 2011) . the type i ifn-inducible isoform of adars, adar1, first cloned in 1994 (kim et al., 1994) contains three repeats of a double-stranded rna-binding motif, and sequences conserved in the catalytic center of other deaminases. transcription from separate promoters generates two isoforms of adar1, a full-length, interferon-inducible adar1p150 and a shorter and constitutively expressed adar1p110. interestingly, both adar1p150 and adar1p110 isoforms shuttle between nucleus and cytoplasm (nishikura, 2016) . a-to-i editing frequently occurs in noncoding regions that contain inverted alu repeats but can also occur in proteincoding regions of mrnas resulting in the expression of altered proteins with sequences that are not encoded in the genome. recent studies indicate that adar1 is also found in complex with dicer to promote mirna processing and rnai efficacy (ota et al., 2013) , suggesting that both rnai and adar are functionally related. viral dsrna formed at different stages of replication of many viruses are substrates for rna editing by adar. it is well established that adar enzymes interfer with the virus-host interaction with adars acting as pro-or antiviral factors. the biological consequences of a-to-i changes during viral infection is not completely understood (tomaselli, galeano, locatelli, & gallo, 2015) . the current concept is that two effects oppose each other: on the one hand, adar1-mediated a-to-i editing of viral double-stranded rna directly restricts the correct function of the edited rna and thus directly inhibits viral replication. on the other hand, a-to-i editing of double-stranded rna destabilizes long double-stranded rna thereby reducing the recognition of long double-stranded rna by double-stranded rna receptors such as mda5. as a consequence, depending on the type of virus, adar1 has the potential to negatively interfer or to support viral replication and thus can act as proviral or antiviral factor (george, john, & samuel, 2014) . independently of the presence of viral infection, the lack of adar1 in a cell results in the activation of mda5 by endogenous rna species. the most likely scenario is that a-to-i editing masks endogenous rnas from detection by mda5. the consequence is that if a virus actively inhibits the function of adar1, endogenous rna ligands will form which activate mda5 and thus induce a type i ifn response. indeed, sequencing studies demonstrated that adar1-deficient cells display stretches of endogenous double-stranded rna (liddicoat et al., 2015) . thus, a-to-i editing of endogenous dsrna is an essential function of adar1 preventing the activation of the cytosolic dsrna response by endogenous transcripts. samhd1 is composed of a sam and a hd domain. while the sam domain of samhd1 mediates protein-protein interactions, the hd domain possesses the triphosphohydrolase activity through which samhd1 hydrolyzes dntps to deoxynucleosides (goldstone et al., 2011; powell, holland, hollis, & perrino, 2011) . samhd1 expression has been demonstrated in monocytes, macrophages, myeloid dendritic cells, plasmacytoid dendritic cells (pdcs), and cd4 t cells (baldauf et al., 2012; gelais et al., 2012; kim, nguyen, daddacha, & hollenbaugh, 2012; laguette et al., 2011; pauls et al., 2014) . the involvement of samhd1 in innate immunity was initially proposed based on its mouse ortholog mg11 which is ifn-inducible in macrophages and dendritic cells (li et al., 2000) , hence the alternative name dendritic cell-derived ifn-î³-induced protein. subsequent studies showed an increased samhd1 expression upon stimulation of macrophages with double-stranded dna (rice et al., 2009) and its upregulation in the context of viral infections (hartman et al., 2007) . mutations in samhd1 have been shown to be responsible for 5% of patients with ags which is characterized by inappropriate and aberrant type i ifn secretion causing symptoms reminiscent of a congenital infection (rice et al., 2009) . a loss of function of samhd1 results in spontaneous type i ifn production in ags patients and samhd1ã�/ã� mice (behrendt et al., 2013; rehwinkel et al., 2013) . samhd1 was identified as a potent restriction factor for hiv (hrecka et al., 2011; laguette et al., 2011; simon, bloch, & landau, 2015) , other nonhuman retroviruses (gramberg et al., 2013) , and herpesviruses, including hsv-1 (hollenbaugh et al., 2013; kim, white, brandariz-nunez, diaz-griffero, & weitzman, 2013) . the current model is that samhd1 through its function as dntp triphosphohydrolase decreases intracellular dntp pools in nondividing cells below the threshold level required for efficient viral reverse transcriptase or viral dna polymerase activity (lahouassa et al., 2012; wu, 2013) . the observation that functional loss of samhd1 leads to a spontaneous type i ifn response suggests that uncontrolled activity of endogenous retroelements may be a source of the ifn-inducing nucleic acids, but the identity of such endogenous ligands is unknown to date. besides dntp triphosphohydrolase activity, a metal-dependent 3 0 -to 5 0 -exonuclease activity of samhd1 for ssdna and ssrna was demonstrated (beloglazova et al., 2013) . the rnase activity was reported to directly degrade hiv-1 rna (ryoo et al., 2014) , but further work will be necessary to confirm and exactly characterize the proposed nuclease activity of samhd1 (rice et al., 2009). the interferon-inducible, double-stranded rna-activated protein kinase (protein kinase rna-activated, pkr; also known as eukaryotic translation initiation factor 2-alpha kinase 2, eif2ak2) was first cloned in 1992 and represents a key mediator of antiviral activities (feng, chong, kumar, & williams, 1992; garcia et al., 2006; . pkr contains an n-terminal dsrna-binding domain (dsrbd) which consists of two tandem copies of a conserved doublestranded rna-binding motif, dsrbm1 and dsrbm2, and a c-terminal kinase domain. binding of pkr to long double-stranded rna (longer 30 bp) activates pkr by inducing dimerization and subsequent autophosphorylation. activated pkr inhibits 5 0 -cap-dependent mrna translation by phosphorylation of the eukaryotic translation initiation factor eif2a thereby preventing viral protein synthesis (farrell et al., 1978; levin & london, 1978) . besides long double-stranded rna, pkr has been shown to recognize rna with limited secondary structures (rna with a length of about 47 nt and weak structure; short stem-loops) containing uncapped 5 0 -triphosphates (nallagatla et al., 2007) . antiviral functions of pkr beyond the mechanism of translation inhibition are still controversial. pkr affects diverse transcriptional factors such as interferon regulatory factor 1, stats, p53, activating transcription factor 3, and nf-îºb. in particular, how pkr triggers a cascade of events involving ikk phosphorylation of ikappab and nf-îºb nuclear translocation has been intensively studied. pkr was also reported to enhance but not being required for nf-îºb-dependent type i ifn induction (chu et al., 1999; kumar, haque, lacoste, hiscott, & williams, 1994; maggi et al., 2000) . involvement of pkr in inflammasome activation is controversial (he, franchi, & nunez, 2013; lu et al., 2012) . pkr-mediated 5 0 -cap-specific inhibition of translation is expected to perturb the proteome. since pkr-activated elf2a is involved in the initiation of the translation from an aug codon, the alternative non-aug initiation takes place instead. an example of mrnas using non-aug initiation are mrnas for the heat shock proteins. another effect is the selected reduction of proteins with short half-life. reduced translation of the nf-îºb inhibitor protein ikappab-alpha is one plausible explanation for the activation of the nf-îºb pathway in response to pkr activation (mcallister, taghavi, & samuel, 2012) . other signaling pathways may be affected in the same way by the removal of an inhibitor with short half-life resulting in the activation of the pathway. interferon-induced proteins with tetratricopeptide repeats (ifits) are among the most abundantly expressed proteins of the group of interferonstimulated genes (isgs). they represent innate immune effector molecules that confer antiviral defense downstream of type i ifn through disruption of the host translation initiation machinery (daffis et al., 2010; pichlmair et al., 2011) . they are evolutionarily conserved from fish to mammals. in humans, there are four well-characterized paralogues, ifit1 (p56/isg56), ifit2 (p54/isg54), ifit3 (p60/isg60), and ifit5 (p58/isg58). like rig-i, productive binding of both ifit1 and ifit5 was shown to depend on the presence of cytosolic 5 0 -triphosphate rna and is nonsequence specific. unlike rig-i, ifit1 and ifit5 preferentially bind to single-stranded rna or to double-stranded rna with a minimum three (ifit5) or five (ifit1)nucleotide overhangs containing an uncapped triphosphate group at the 5 0 -end of rna (abbas, pichlmair, gorna, superti-furga, & nagar, 2013; habjan et al., 2013; kumar et al., 1994) . replacing the triphosphate with a 5 0 -cap, a 5 0 -monophosphate or 5 0 -oh diminishes the binding significantly (abbas et al., 2013) . ifit1 competes with eif4e, the endogenous 5 0 -cap binding and translation factor, in the 48s initiation complex formation. however, while in vitro competition experiments convincingly show that ifit1 can compete with eif4e for binding at completely unmethylated cap0 rna, the out-competing of eif4e from n7 methylated cap (cap0) structures is unclear. binding of eif4e to cap0 structures in lysates of ifn-primed cells is rather enhanced than reduced, suggesting additional mechanisms beyond eif4e competition for 48s disruption (habjan et al., 2013) . a key role for ifit1 in negative-strand rna viruses (vsv, influenza) and positive-strand rna viruses (wnv, mhv) except picornaviruses was reported (daffis et al., 2010; habjan et al., 2013; pichlmair et al., 2011) . the human 2 0 -5 0 -oas family comprise four type i ifn-inducible genes: oas1, oas2, oas3, and oasl (oas-like protein) (melchjorsen et al., 2009) . upon binding to long double-stranded rna, oas1, oas2, and oas3 catalyze the formation of 2 0 -5 0 -oa, whereas oasl has no enzymatic activity but still has potent antiviral activity due to its coactivating role in the rig-i pathway (schoggins et al., 2011; zhu et al., 2014) . the formation of 2 0 -5 0 -oligomers of adenosine (2 0 -5 0 -oa) upon exposure to dsrna and subsequent inhibition of translation has been described early on (clemens & williams, 1978; farrell et al., 1978; hovanessian, brown, & kerr, 1977; zilberstein, kimchi, schmidt, & revel, 1978) . 2 0 -5 0 -oa function as second messenger of oas binding to rnase l leading to dimer formation and subsequent degradation of cellular and viral rna (dong & silverman, 1997) . the structural mechanisms of rnase l activation by 2 0 -5 0 -oa and its dimer formation have recently been described (han et al., 2014; huang et al., 2014) . all three human oas isoforms are activated by dsrna in vitro which is the presumed ligand in vivo as well. the full activation of the oas system in virally infected cells leads to the inhibition of protein synthesis and the induction of apoptosis, thereby interfering with viral replication (castelli et al., 1998) . activation of the oas-rnase l system restricts replication of a variety of viruses, in particular positive-strand viruses (e.g., picornaviruses, flaviviruses, and alphaviruses) which produce high numbers of dsrna during replication (silverman, 2007) . virus-encoded inhibitors of the oas-rnase l system such as the nonstructural protein 2 (ns2) of murine coronavirus or inhibitors expressed by picornaviruses support a key role of this system in the restriction of viruses. it is interesting to note that oas and cgas (see later) share similar structural features and enzymatic function. both oas and cgas catalyze the uncommon 2 0 -5 0 phosphodiester linkage upon binding to a nucleic acid ligand (hornung, hartmann, ablasser, & hopfner, 2014) . rnases h are a family of widely expressed nonsequence-specific endonucleases that hydrolyze solely the rna of rna/dna hybrids resulting in 3 0 -hydroxyl and 5 0 -phosphate terminated products and an intact dna strand (cerritelli & crouch, 2009) . rnases h play crucial roles in the biochemical processes associated with dna replication, gene expression, and dna repair where rna/dna hybrids can occur. furthermore, rnases h degrade rna/dna hybrids generated during viral replication. members of the rnase h family can be found in nearly all organisms, from bacteria to archaea to eukaryotes. unlike in prokaryotes and in single-cell eukaryotes, in higher eukaryotes rnases h are essential for development. the catalytic subunit of eukaryotic rnase h2, rnaseh2a, is well conserved and similar to the monomeric prokaryotic rnase hii. in contrast, the rnaseh2b and rnaseh2c subunits share very little homology between human and saccharomyces cerevisiae or bacteria. rnaseh2b and rnaseh2c serve as a nucleation site for the addition of rnaseh2a to form an active rnase h2. furthermore, they contain interaction sites with other proteins to support functions other than rnase h nuclease activity, but these functions are not well-defined yet. rnase h2 deficiency can cause a number of pathogenetic principles including the occurrence of ribonucleoside monophosphates accumulating in genomic dna and activating the dna damage-response pathway. rnase h2 deficiency leads to abundance of cytosolic rna-dna hybrids and to an increase in retroelements which both represent potential ligands for the cgas-sting signaling pathway (mankan et al., 2014; rigby et al., 2014) . in fact, mutations in any of the three subunits rnaseh2a-, rnaseh2b-, or rnaseh2c of human rnase h2 cause ags, a human neurological disorder with devastating consequences (rice et al., 2007) . mutations that impair rnase h2 are also associated with systemic lupus erythematosus (sle). pathogenicity is supported by mouse models of ags-associated mutations of rnase h which show a spontaneous cgas/sting-dependent type i ifn-driven phenotype (mackenzie et al., 2016; pokatayev et al., 2016) . 7.7 dnases 7.7.1 dnase i deoxyribonuclease i (dnase i) is an endonuclease which is secreted to cleave dna in the extracellular space down to an average of tetranucleotides with 5 0 monophosphate and 3 0 hydroxyl dna ends (baranovskii, buneva, & nevinsky, 2004) . both single-stranded dna and doublestranded dna are degraded by dnase i. this nuclease appears to account for the major nucleolytic activity on dna in serum and is responsible for the degradation of the majority of circulating dna derived from apoptotic and necrotic cell death and from neutrophil extracellular traps. in addition to its role in the serum, it has been proposed as one of the deoxyribonucleases responsible for dna fragmentation in the process of apoptosis (samejima & earnshaw, 2005) . notably, dnase1l3 complements the activity of dnase i. although dnase1l3 harbors nuclear localization signals, its main function appears to be in the serum, where it can degrade proteincomplexed dna (napirei, ludwig, mezrhab, klockl, & mannherz, 2009 ). in the absence of dnase i, degradation of extracellular dna is heavily reduced resulting in the activation of dna-sensing immune receptors. mice deficient in dnase i display a lupus-like phenotype with increased antinuclear antibody titers and glomerulonephritis (napirei et al., 2000) . mutations in the human dnase i gene and factors inactivating dnase i have been associated with sle (hakkim et al., 2010; yasutomo et al., 2001) . in a subset of sle patients, the presence of dnase i inhibitors or autoantibodies was associated with impaired dna clearance and poor prognosis, suggesting that decreased dnase i activity can also be acquired (hakkim et al., 2010) . notably, loss-of-function variants in dnase1l3 have also been associated with familiar sle, supporting an important functional contribution of dnase1l3 in clearing dna (al-mayouf et al., 2011) . dnase ii is a mammalian endonuclease with highest activity at low ph and in the absence of divalent cations. dnase ii cleaves dna between 5 0 -phosphate and 3 0 -hydroxyl resulting in the formation of nucleoside-3 0phosphates. dnase ii is the predominant dnase located in lysosomes of cells in various tissues including macrophages (evans & aguilera, 2003; yasuda et al., 1998) . with its lysosomal localization and ubiquitous tissue distribution, this enzyme plays a pivotal role in the degradation of exogenous dna encountered by endocytosis. it has been demonstrated that digestion of large dna molecules and of cpg-a (hartmann et al., 2003) by dnase ii creates short dna fragments which are sensed by tlr9 (chan et al., 2015; pawaria et al., 2015) . dnase ii deficiency is another example of a cell-intrinsic nuclease defect driving autoimmunity. loss of dnase ii leads to a defect in the disposal of dna within lysosomal compartments (kawane et al., 2006; yoshida, okabe, kawane, fukuyama, & nagata, 2005) . accumulation of undigested dna can result in the translocation of dna into the cytoplasm which is then sensed by the cgas-sting pathway (ahn, gutman, saijo, & barber, 2012; gao et al., 2015) as well as the aim2 inflammasome (baum et al., 2015; jakobs, perner, & hornung, 2015) . mice lacking dnase ii display an inflammatory response that depends on both cgas and aim2. besides cell-extrinsic sources of dna (e.g., nuclei expelled from erythroid precursor cells), recent work in dnase ii-deficient mice suggests that damaged nuclear dna is also subject to dnase ii degradation and might stimulate cytosolic dna immune-sensing receptors if not properly degraded (lan, londono, bouley, rooney, & hacohen, 2014) . in a model of cardiacspecific deletion of dnase ii, severe myocarditis and dilated cardiomyopathy developed which was attenuated if immune sensing of accumulating mitochondrial dna by tlr9 was inhibited (oka et al., 2012) . the cytoplasmic dnase iii (3 0 -repair exonuclease 1, trex1) has been identified decades later than dnase i and ii (hoss et al., 1999) . trex1 is a 3 0 -to 5 0 -exonuclease which degrades both double-and single-stranded dna. most dna reaching the cytosol is promptly removed by trex1. modifications have been reported which render dna resistant to trex1. for example, oxidation of guanine bases to 8-hydroxydeoxyguanine (8-ohdg) protects dna from trex1-dependent degradation leading to accumulation and cgas-mediated recognition of oxidized dna in the cytosol (gehrke et al., 2013) . only since 2006, it is known that loss of function in trex1 causes the type i ifn-associated inflammatory syndrome ags (crow et al., 2006) , suggesting that trex1 is critically involved in the clearance self dna within the cytoplasm of cells which otherwise is recognized by the immune sensor cgas. defects in trex1 have been associated with sle and with familial chilblain lupus ; furthermore, genetic defects in trex1 can cause retinal vasculopathy with cerebral leukodystrophy . trex1-deficient mice develop severe autoimmunity (gall et al., 2012; morita et al., 2004) . the pathology is fully rescued by additional genetic defects in cgas gao et al., 2015; gray, treuting, woodward, & stetson, 2015) or the type i ifn system demonstrating the critical pathogenic role of the ifn response triggered by endogenous dna species. the source and identity of this dna remain controversial but may derive from endogenous retroelements (beck-engeser, eilat, & wabl, 2011; stetson, ko, heidmann, & medzhitov, 2008) . alternatively, dna ligands originating during chromosomal replication have been proposed (yang, lindahl, & barnes, 2007) . rnai is considered one of the major mechanism for sequencespecific detection and elimination of rna genome-based viruses in plants and invertebrates (szittya & burgyan, 2013; zhou & rana, 2013) . besides its antiviral function, rnai regulates gene expression in many organisms. by suppressing transcription or translation or by targeted degradation of mrna, it controls many cellular developmental and physiological processes (burger & gullerova, 2015) . rnai is initiated by rnase iii family nucleases (nuclear drosha and cytosolic dicer) that cleave endogenous or exogenous double-stranded rna to finally yield short 21-23 bp exogenous sirna or endogenous mirna (bernstein, caudy, hammond, & hannon, 2001; elbashir, lendeckel, & tuschl, 2001; lee et al., 2003) . the mirnas/sirnas are then integrated in the rna-induced silencing complex (risc) which target complementary rna for degradation or inhibition of translation (iwakawa & tomari, 2015) . ago family proteins in the risc complex determine its effector function: perfectly matched mi/sirnas mediate direct target cleavage by ago2, while imperfectly matched mi/sirnas inhibit translation of target mrnas by ago1, 3, or 4 and recruit additional effector proteins which in turn can degrade target rna (doench, petersen, & sharp, 2003; meister et al., 2004) . while an important role for rnai for the antiviral responses in helminths, insects, and plants is well established, the contribution to antiviral immunity in vertebrates is under debate. evidence accumulates for an antiviral role of rnai in mammalian cells (gantier, 2014) , specifically if the otherwise dominating dicer-related rig-i-like helicases are inhibited. the major obstacle is that the contribution of a sirna-mediated antiviral response cannot be studied by the knockout of dicer which is lethal at early stages of mouse embryo development (bernstein et al., 2003) . it was reported that type i ifn-dominated innate immune responses suppress rnai and vice versa (seo et al., 2013) . the absence of dicer products from small rna libraries of (+)ssrna virus (yfv, hcv) infections strengthens this assumption (pfeffer et al., 2005) . however, more recent studies using the more sensitive next-generation sequencing indeed provide evidence for the generation of short virus-derived small rnas (vsrnas) in complex with ago proteins and conforming to dicer cleavage fragments of 24-31 bp (sirna and pirna) in vertebrate infection systems (parameswaran et al., 2010) . in a seminal work, suckling mice were infected by nodamura virus (nov) or a mutant virus lacking the nov virus-encoded suppressor of rnai, b2 (li, lu, han, fan, & ding, 2013) . nov is a mosquitotransmissible (+)ssrna virus, which is highly virulent to suckling mice and suckling hamsters. loss of b2 leads to abundant occurrence of viral sirnas and rendered mice completely resistant to nov titers which are lethal in the presence of b2 (li et al., 2013) . since b2 appears not to prevent recognition by rig-i (fan, dong, li, ding, & wang, 2015) , an important innate immune sensor of (+)ssrna-based viruses, the data suggest a strong role of virus rna-specific rnai during nov infection. still, an impact of b2 on endogenous mirna networks or inhibition of other dsrnasensing receptors (mda5, pkr, oas) as a major cause of lethality is not completely excluded in this work and requires further analysis. nevertheless, this study adds to the concept that virus-encoded suppressors of rnai mask the actual role of rnai in antiviral defense of vertebrates (va1 noncoding rna, influenza ns1, vaccinia virus e3l, ebola virus vp35, primate foamy virus tas, hiv-1 tat west nile virus sfrna) (bennasser, le, benkirane, & jeang, 2005; haasnoot et al., 2007; lecellier et al., 2005; li et al., 2004; lu & cullen, 2004; schnettler et al., 2012; svoboda, 2014) . importantly, murine or rat oocytes or embryonic stem cells in rat and mouse express a shortened form of dicer (dicero) with enhanced cleavage activity for long dsrna which complicate the interpretation of results (flemr et al., 2013) . on the other hand, the finding that poly(i:c) stimulation or infection with dna or rna viruses leads to parp13 induced poly-adp-ribosylation of ago2 which induces ago2 degradation in all tested cells strongly indicates a competition rather than a cooperation between antiviral rnai and nucleic acid immune-sensing pathways (seo et al., 2013) . although some recent work propose a role of rnai in the antiviral defense of vertebrates as described earlier, the current concept is that immune-sensing receptors represent the major antiviral defense strategy in vertebrates. immune-sensing receptors recognize characteristic features of foreign and invading nucleic acids such as unusual localization, specific structural elements, and modifications. stimulation of nucleic acid-sensing receptors results in the induction of cytokines (e.g., type i interferons) and chemokines to alarm neighboring cells, in the recruitment of immune cells, in the activation of cell autonomous mechanisms interfering with virus assembly and protein translation, or in the induction of several types of cell death including apoptosis, necroptosis, and pyroptosis. this chapter provides an overview of the most important immune-sensing receptors of nucleic acids for which robust evidence exists regarding the molecular mechanism of detection, the structural aspects of receptor ligand interaction, and the downstream signaling pathways (fig. 6 ). tlr3 detects long double-stranded rna (alexopoulou et al., 2001) , and unlike other nucleic acid-sensing tlrs, besides its endolysosomal localization, it is also expressed on the surface of certain cell types (matsumoto, kikkawa, kohase, miyake, & seya, 2002; pohar, pirher, bencina, mancek-keber, & jerala, 2013) . tlr3 detects dsrna longer than 40 bp. the ectodomains of two tlr3 molecules bind one dsrna molecule in a way that the cytoplasmic c-terminal signaling domains are juxtaposed to each other resulting in downstream signaling (liu et al., 2008) . tlr3 interacts with the ribose-phosphate backbone of dsrna and has no specific sequence requirements. given the absence of long dsrna under physiological conditions, tlr3 should be inactive in the absence of an infection. still, a number of studies proposed recognition of endogenous dsrna by tlr3 in situations of sterile tissue damage, but the specific ligand is not well defined. the identification of ligand specificities of tlr7 and tlr8 has been hampered by their mutually exclusive expression in different cell types and by considerable differences between mouse and human. tlr7 and tlr8 are examples where distinct function of nucleic acid-sensing tlrs is determined by their differential expression in immune cell subsets. while the expression of tlr7 in the human immune system is almost restricted to b cells and pdc, tlr8 is preferentially expressed in myeloid immune cells. consequently, tlr7 ligands drive b cell activation and the production of large amounts of ifn-alpha in pdc, while tlr8 induces the secretion of high amounts of il-12p70 in myeloid immune cells. fig. 6 immune-sensing receptors-detecting foreign nucleic acids and inducing indirect effector responses. this graph provides an overview of immune-sensing receptors of nucleic acids. tlr3 is the only one which besides its endosomal localization is also reported to be expressed on the cell membrane. tlr3 binds long double-stranded rna which is not present in the cytosol of normal cells and is an indicator of foreign. tlr3 is expressed in myeloid immune cells and in a number of somatic cells including fibroblasts and endothelial cells. the other three tlrs expressed in the endolysosomal compartment of distinct immune cell subsets are tlr7, tlr8, and tlr9. tlr7 detects even short rna, preferentially double-stranded and containing g and u. tlr8 detects single-stranded rna. while tlr8 is expressed in human myeloid immune cells, tlr7 and tlr9 are predominantly expressed in human b cells and plasmacytoid dendritic cells. tlr9 detects single-stranded dna containing unmethylated cpg dinucleotides. in the cytoplasm, rig-i specifically detects rna if it contains at least a short double strand with a blunt end and a 5 0 -triphosphate. the rig-i-like receptor mda5 detects long irregular forms of double-stranded rna, but the exact definition of the ligand structure is unclear. both rig-i and mda5 are widely expressed in immune cells and nonimmune cells, and induce a broad array of cell autonomous and extracellular antiviral responses including the production of type i interferon. mda5 ligands also activate multiple other receptor pathways that depend on the detection of long double-stranded rna, including pkr, adar1, and tlr3. the cytosolic receptor aim2 detects long double-stranded dna and activates the inflammasome. the other key receptor for the detection of dna in the cytoplasm is cgas. cgas is activated by long double-stranded dna and short forms of double-stranded dna with single-stranded overhangs containing gs, a structure which was termed y-form dna and which is presented during retroviral infection or by endogenous retroelements. upon activation, cgas catalyzes the formation of 2 0 -5 0 -cgamp from gtp and atp. 2 0 -5 0 -cgamp acts as a second messenger which binds to the downstream signaling protein sting which induces type i interferon via tbk1 and irf3. 2 0 -5 0 -cgamp can travel to and alarm neighboring cells via gap junctions. sting also activates nf-îºb activation and inflammatory cytokines. tlr7 and tlr8 are preferentially activated by polyu or by g-and u-rich sequences (diebold et al., 2004; heil et al., 2004; hornung et al., 2005; judge et al., 2005) . however, confounding factors need to be considered while interpreting these results (forsbach et al., 2008) . furthermore, it has been demonstrated that tlr8 selectively detects ssrna, while tlr7 primarily detects short stretches of dsrna but can also accommodate certain ssrna oligonucleotides sioud, 2006) . however, since neither polyu or g-and u-rich sequences nor ssrna or short dsrna structures are overrepresented in microbial or viral rna, the distinction of self vs nonself by tlr7 and tlr8 requires additional information. in fact, endogenous rnas are posttranscriptionally modified at their nucleobases and backbone. the current concept is that the addition of certain modifications to self rna inside the nucleus provides a signature for self. one example is 2 0 -o-methylation which is a common nuclear modification of rna performed by a specific mtase located in the nucleolus. the mtase adds a methyl group to the 2 0 -hydroxyl group of the ribose. this modification represents a marker of self in higher eukaryotic cells and prevents the recognition of endogenous rna by tlr7 and tlr8 judge et al., 2005; kariko, buckstein, ni, & weissman, 2005) . other modifications of rna molecules which potently inhibit the detection of transfer rna and ribosomal rna by tlr7 and tlr8 are the incorporation of pseudouridine (î¨), 5-methylcytidine (m5c), 2-thio-uridine (s2u), or n6-methyladenosine (m6a) (kariko et al., 2005) . the presence of such modifications in part explains the lack of immunostimulation of host-derived rna vs microbial rna (gehrig et al., 2012; jockel et al., 2012) . however, endogenous rna from apoptotic or dying cells still activates tlr7 and tlr8 once entering the endolysosomal compartment (busconi et al., 2006; ganguly et al., 2009; vollmer et al., 2005) . thus, additional factors such as intracellular localization and degradation by nucleases likely support a faithful discrimination of self from nonself by tlr7 and tlr8. it is interesting to note that there is an obvious need to sense and eliminate certain endogenous rnas as well. in this context, it has been reported that the loss of tlr7 function causes retroviral viremia (yu et al., 2012) indicating that endogenous rnas transcribed from rna polymerase ii promoters are not generally excluded from tlr-mediated recognition. tlr9 senses dna in the endolysosomal compartment of certain immune cells (hemmi et al., 2000) . like tlr7 and tlr8, tlr9 travels to the endolysosomal compartment via the chaperone protein unc93b1 (protein unc-93 homolog b1) (latz et al., 2004; pelka, shibata, miyake, & latz, 2016) . there, tlr9 is proteolytically processed at a defined protruding loop structure without disrupting the horseshoe shape of the protomer (bauer, 2013; onji et al., 2013; peter, kubarenko, weber, & dalpke, 2009) . cleavage is necessary for the activation of tlr signaling (ewald et al., 2008; park et al., 2008) . tlr9 preferentially detects dna with unmethylated (no methylation at the c5 carbon of cytosine) cpg dinucleotides (cpg dna) with a preference for certain sequence contexts (hexamer cpg motifs, in humans 5 0 -gtcgtt-3) which vary between species and which altogether are less frequent in eukaryotic self dna hartmann, weiner, & krieg, 1999; hemmi et al., 2000; krieg et al., 1995) . activation of tlr9 signaling is preceded by dimer formation where two cpg dna molecules symmetrically bind two tlr9 molecules (ohto et al., 2015) . both cpg dna molecules bind to the c-terminal fragment of one protomer and the cpg-binding groove in the n-terminal fragment of the other. inhibitory dna oligonucleotides only bind to the n-terminal fragment. methylated single-stranded cpg dna and double-stranded dna exhibit lower binding to tlr9 and are less potent to induce tlr9 dimer formation. of note, digestion of dna molecules by the lysosomal endonuclease dnase ii creates short tlr9stimulatory dna fragments (chan et al., 2015; pawaria et al., 2015) . notably, specificity for unmethylated cpg motifs is reduced if the cpg motif is within a phosphorothioate-modified dna often used to stabilize oligodeoxynucleotides against dnases. nevertheless, a high degree of specificity is well established for unmethylated cpg motif containing dna within a natural phosphodiester backbone including microbial dna hartmann & krieg, 2000) . it is specifically noteworthy that genomic microbial dna displays a much stronger activity to stimulate tlr9 as compared to genomic dna of vertebrates. although eukaryotic dna presents with a lower frequency of nonmethylated cpg motifs than microbial dna, this difference in frequency of unmethylated cpg motifs does not allow a clear cut distinction of self from nonself on a structural basis. endogenous dna at high concentrations can activate tlr9 once delivered into the endolysosome (marshak-rothstein, 2006) . it is also of great importance to be aware of the differences of tlr9mediated dna recognition between species. in humans, tlr9 is almost exclusively expressed in b cells and pdc (hornung et al., 2002) , while in mice, tlr9 is expressed more widely including myeloid immune cells. in humans, tlr9 predominantly induces type ifn production in pdcs and polyclonal activation in b cells via myd88/irf7-dependent signaling. species-specific expression patterns of tlr9 are responsible for the fundamental functional differences of tlr9 in mouse and man. another important issue is that in preparations of human immune cell subsets, minute amounts of pdc indirectly activate other immune cell subsets such as monocytes, myeloid dendritic cells, or nk cells. this needs to be carefully considered when direct effects of tlr9 activation in immune cells other than b cells and pdc are claimed, such as direct tlr9 effects in human myeloid immune cells and nk cells. three different classes of cpg oligonucleotides have been described, cpg-a, cpg-b, and cpg-c (avalos et al., 2009; hartmann et al., 2003; kerkmann et al., 2003; krug et al., 2003; rothenfusser et al., 2004) . based on the palindromic structure, cpg-a spontaneously forms nanoparticle-like complexes (kerkmann et al., 2005) that explain much higher type i ifn-inducing capacity in pdc as compared to cpg-b which are monomeric. monomeric cpg-b potently activates b cells which do not internalize larger particles of dna as with cpg-a complexes. cpg-c potently stimulates both b cells and pdcs. in cell culture, delayed tlr9 activation due to slower uptake of cpg-a nanoparticles allows a longer self-priming of pdc by minute amounts of spontaneously released type i ifn. priming of pdc results in higher ifn-inducing activity of cpg-a seen in cell culture . rig-i belongs to the cytosolic dexd/h box rna helicases and is one of three members of the so-called family of rig-i-like helicases (others: mda5 and lgp2). rig-i is closely related to the dicer family of helicases of the rnai pathway. rig-i contains a rna helicase domain and a two n-terminal card domains which relay the signal to the downstream signaling adaptor mavs (mitochondrial antiviral-signaling protein). rig-i signaling via mavs not only leads to the induction of type i ifn responses via tbk1 and irf7/8, it also activates caspase-8-dependent apoptosis, preferentially in tumor cells (besch et al., 2009; el maadidi et al., 2014; glas et al., 2013; kumar et al., 2015) . furthermore, rig-i was also found to mediate mavs-independent inflammasome activation (poeck et al., 2010) , specifically in the context of viral infection (poeck et al., 2010; pothlichet et al., 2013) . rig-i detects blunt ends of double-stranded rna containing a 5 0 -triphosphate or a 5 0 -diphosphate (goubau et al., 2014; hornung et al., 2006; marq, hausmann, veillard, kolakofsky, & garcin, 2010; pichlmair et al., 2006; schlee, roth, et al., 2009) . such rna ligands are presented, for example, by negative-strand rna viruses which form panhandle structures with their matching 5 0 -and 3 0 -ends of the single-stranded genomic rna (rehwinkel et al., 2010; schlee, roth, et al., 2009) . while crystal structures confirmed the structural requirements as determined in functional studies (civril et al., 2011; jiang et al., 2011; kowalinski et al., 2011; luo et al., 2011; wang et al., 2010) , the minimum length of the double-strand required for rig-i activation is still controversial. while approaches with synthetic or highly purified enzymatic double-stranded 5 0 -triphosphate rna revealed a minimum length of 18-19 bp (marq, hausmann, et al., 2010; schlee, hartmann, et al., 2009) , 10 bp were demonstrated to be sufficient for a hairpin forming oligonucleotide (kohlway, luo, rawling, ding, & pyle, 2013) . however, alternatively to the predicted hairpin, these oligonucleotides may form 20mer duplexes when entering the cell. although oligomerization of 2card modules of each rig-i protein along the rig-i filament bound to a longer double-stranded rna molecule induces robust rig-i signaling, the minimal signaling unit is sufficient for rig-i to trigger signal transduction. in the latter case, a 2card tetramer is stabilized by ubiquitin chains . furthermore, it is interesting to note that rig-i mutants deficient in atp hydrolysis of their helicase domain cannot detach from suboptimal endogenous rna ligands leading to erroneous signaling which can cause autoimmunity (lassig et al., 2015) . importantly, n1-methylation (2 0 -o-methylation at the first nucleotide of capped rna) serves as a signature of self rna and completely abrogates rig-i sensing of rna, while in the absence of n1 methylation, rig-i binding is hardly impaired by the 5 0 ppp5 0 -linked m7g cap structure itself (schuberth-wagner et al., 2015) . rig-i is ubiquitously expressed in all cell types including tumor cells. however, the type of rig-i induced responses differs between cells. while normal healthy cells such as melanocytes and fibroblasts are quite resistant to rig-i-induced apoptosis, tumor cells are highly susceptible to rig-iinduced cell death (besch et al., 2009; kubler et al., 2010) . based on this tumor selective activity and a favorable toxicity profile, rig-i-specific ligands are currently being developed for immunotherapy of cancer (duewell et al., , 2015 ellermeier et al., 2013; schnurr & duewell, 2013 . part of the potent antitumor activity of rig-i ligands is its ability to promote crosspresentation of antigens to cd8 t cells and to induce cytotoxic activity (hochheiser et al., 2016) . rig-i ligands show strong therapeutic activity in viral infection models such as influenza (weber-gerlach & weber, 2016) . notably, rig-i has also been shown to be involved in the detection of intracellular bacteria (abdullah et al., 2012) . rare genetic gain-of-function variants of rig-i have been associated with an atypical form of singleton merten syndrome (jang et al., 2015) . like rig-i, melanoma differentiation associated gene 5 (mda5) is a cytosolic dexd/h box rna helicase which signals through mavs and irf3/ irf7 . despite its similar structure, mda5 senses a different type of ligand which has been described as higher order rna structures (pichlmair et al., 2009) . so far, a mda5-specific ligand has not been described. double-stranded rna ligands activating mda5 are typically promiscuous ligands, such as poly(i:c) which also activates tlr3 and antiviral effector proteins which inhibit translation upon binding to doublestranded rna, such as pkr and oas. multiple effects of mda5 ligands cause a high degree of toxicity in vivo strictly limiting the clinical application of mda5 ligands. unlike rig-i which primarily binds to the ends of rna, mda5 proteins bind double-stranded rna internally, independently of its terminal structures. additional mda5 molecules then closely stack in a helical headto-tail arrangement around dsrna resulting in the formation of long mda5 filaments which initiate signaling toward activation of mavs (del toro duany, . lgp2 (laboratory of genetics and physiology 2) is a third cytosolic rig-i-like helicase lacking card domains for signaling. lgp2 appears to contribute to the fine tuning of immune responses by inhibition of rig-i and supporting mda5 signaling venkataraman et al., 2007) . while mda5 contains the signaling card domains but has relatively weak binding to double-stranded rna, lgp2 readily detects diverse double-stranded rna species but lacks a signaling domain. the current concept is that lgp2 assists the interaction of mda5 with double-stranded rna and filament formation, thereby enhancing mda5-mediated antiviral signaling (bruns & horvath, 2015; bruns, leser, lamb, & horvath, 2014) . notably, genetic gain-of-function variants of mda5 have been associated with autoimmune disorders (junt & barchet, 2015; kato & fujita, 2015) . the hin-200 (hematopoietic interferon-inducible nuclear proteins with a 200-amino acid repeat) family member aim2 (absent in melanoma 2) binds and oligomerizes on cytoplasmic double-stranded dna through its c-terminal hin domain in a sequence-independent manner (fernandes-alnemri, yu, datta, wu, & alnemri, 2009; hornung et al., 2009; roberts et al., 2009) . dna binding of the hin domain relieves the autoinhibitory conformation of aim2 and allows the n-terminal pyrin domain of multiple aim2 proteins to form a helical structure which nucleates the helical assembly of asc (apoptosis-associated speck-like protein containing a card) filaments (lu, kabaleeswaran, fu, magupalli, & wu, 2014; lu et al., 2015) thereby forming an inflammasome that results in the release of il-1beta. the formation of an aim2 inflammasome requires a minimal length of double-stranded dna of 50-80 bp (jin et al., 2012) . the cytosolic immune-sensing receptor cgas (cai, chiu, & chen, 2014; wu et al., 2012) detects long double-stranded dna (dsdna) or short dsdna with unpaired open ends containing guanosines (y-form dna) as, for example, presented in highly structured single-stranded dna of retroviruses or certain endogenous retroelements . it is important to note that trex1 has a gate keeper function for cgas. usually, cytosolic dna is efficiently degraded by trex1. only in the case of excess cytosolic dna, or dna modifications rendering dna resistant to trex1mediated degradation, dna gains access to cgas resulting in downstream signaling. along these lines, it has been reported that oxidized dna (e.g., 8-hydroxyguanosine, 8-ohg) as occurring in the context of uv radiation or upon exposure to reactive oxygen species resists trex1-mediated degradation (gehrke et al., 2013) . this results in an accumulation of dna in the cytosol. oxidized dna has the same affinity to cgas than nonoxidized dna. cgas is monomeric in its unligated state. however, two cgas molecules bind to two dsdna molecules in a way that each cgas protomer presents an additional interaction site with the dna bound to the other cgas protein. upon activation by cytosolic dna, cgas catalyzes the formation of 2 0 -5 0 -cgamp from gtp and atp (ablasser, goldeck, et al., 2013; gao, ascano, wu, et al., 2013) . 2 0 -5 0 -cgamp acts as a second messenger which binds to the downstream signaling protein sting (gao, ascano, zillinger, et al., 2013) which induces type i interferon via tbk1 and irf3. 2 0 -5 0 -cgamp can travel to and alarm neighboring cells via gap junctions (ablasser, schmid-burgk, et al., 2013) . sting activation is also associated with nf-îºb activation and prominent induction of inflammatory cytokines such as il-6 and tnf-a. the general view is that most of the relevant immune receptors, effector proteins, and pathways participating in nucleic acid immunity have now been identified. these different players have in common that they all serve the function to detect and to disable foreign nucleic acids. altogether they constitute the system of nucleic acid immunity. all of these pathways are relevant for human disease, either as part of the human antiviral defense system, or indirectly by being active in pathogens or pathogen-transmitting organisms. examples are arboviruses (zika, dengue, yellow-fever, west nile) which are transmitted by arthropod vectors. successful arboviruses need to escape both rnai in insects and immunoreceptors such as rig-i in humans. only if they manage to inhibit both pathways, they can establish as pathogens. therefore, it will be interesting to understand the molecular evolution of escape strategies in emerging viruses such as zika, which may lead to the identification of the molecular step that allowed the virus to spread more efficiently (rasmussen & katze, 2016) . another example is the important role of rnai in pathogens such as filariae and other human pathogenic helminths. there, rnai may be useful as a therapeutic target. another example is the presence of endosymbionts such as wolbachia, a genus of bacteria, in filarial nematodes (taylor, bandi, & hoerauf, 2005) . the release of wolbachia nucleic acids may contribute to the pathogenesis of filarial infection. furthermore, crispr/cas is involved in the evolution of pathogenic bacteria. nucleic acid sensing in vertebrates is required for antimicrobial immunity and is involved in the pathogenesis of many inflammatory diseases. although much is known about the structure and the function of the single pathways, the functional interaction of the different pathways is far from being understood. distinct expression patterns of the receptors in different cell types and cell-type-dependent differences in the expression of downstream signaling components and transcription factors contribute to the complexity of nucleic acid immunity. as a result, the same type of ligand can have different functional outcomes in different cell types. furthermore, since different receptor pathways are activated by the same type of ligand, it is currently unclear whether competition of receptor binding or distinct molecular trafficking to the corresponding receptors or both impact on the functional outcome of ligand exposure. for example, long doublestranded dna in principle binds to both aim2 and cgas, and functional cooperation or inhibition of the respective pathways are unclear. now since most of the individual molecular pathways of nucleic acid immunity are on the table, we see ourselves just at the dawn of an exciting new research field which is expected to advance medicine specifically in the areas of infection and inflammation and with broad implication for human diseases. this work was supported by the dfg-funded excellence cluster immunosensation, and the helmholtz-funded german center for infection research (deutsches zentrum fâ�¬ ur infektionsforschung, dzif). structural basis for viral 5'-ppp-rna recognition by human ifit proteins rig-i detects infection with live listeria by sensing secreted bacterial nucleic acids rig-i-dependent sensing of poly(da:dt) through the induction of an rna polymerase iii-transcribed rna intermediate cgas produces a 2'-5'-linked cyclic dinucleotide second messenger that activates sting trex1 deficiency triggers cell-autonomous immunity in a cgas-dependent manner cell intrinsic immunity spreads to bystander cells via the intercellular transfer of cgamp sting manifests self dnadependent inflammatory disease recognition of double-stranded rna and activation of nf-kappab by toll-like receptor 3 loss-of-function variant in dnase1l3 causes a familial form of systemic lupus erythematosus differential cytokine production and bystander activation of autoreactive b cells in response to cpg-a and cpg-b oligonucleotides interferon action may be mediated by activation of a nuclease by pppa2'p5'a2'p5'a samhd1 restricts hiv-1 infection in resting cd4(+) t cells human deoxyribonucleases sting-dependent cytosolic dna sensing pathways crispr provides acquired resistance against viruses in prokaryotes a developmentally regulated activity that unwinds rna duplexes an unwinding activity that covalently modifies its double-stranded rna substrate toll-like receptor 9 processing: the key event in toll-like receptor 9 activation? cutting edge: aim2 and endosomal tlrs differentially regulate arthritis and autoantibody production in dnase ii-deficient mice an autoimmune disease prevented by anti-retroviral drugs mouse samhd1 has antiretroviral activity and suppresses a spontaneous cell-intrinsic antiviral response nuclease activity of the human samhd1 protein implicated in the aicardi-goutieres syndrome and hiv-1 restriction evidence that hiv-1 encodes an sirna and a suppressor of rna silencing role for a bidentate ribonuclease in the initiation step of rna interference dicer is essential for mouse development proapoptotic signaling induced by rig-i and mda-5 results in type i interferonindependent apoptosis in human melanoma cells rnase-l control of cellular mrnas: roles in biologic functions and mechanisms of substrate targeting lgp2 synergy with mda5 in rlr-mediated rna recognition and antiviral signaling the innate immune sensor lgp2 activates antiviral signaling by regulating mda5-rna interaction and filament assembly swiss army knives: non-canonical functions of nuclear drosha and dicer dna and rna autoantigens as autoadjuvants the cgas-cgamp-sting pathway of cytosolic dna sensing and signaling the role of 2'-5' oligoadenylate-activated ribonuclease l in apoptosis ribonuclease h: the enzymes in eukaryotes dnase ii-dependent dna digestion is required for dna sensing by tlr9 contributions of the two accessory subunits, rnaseh2b and rnaseh2c, to the activity and properties of the human rnase h2 complex jnk2 and ikkbeta are required for activating the innate response to viral infection the rig-i atpase domain structure reveals insights into atp-dependent antiviral signalling the double-stranded rna-dependent protein kinase pkr: structure and function inhibition of cell-free protein synthesis by pppa2'p5'a2'p5'a: a novel oligonucleotide synthesized by interferon-treated l cell extracts higher activation of tlr9 in plasmacytoid dendritic cells by microbial dna compared with self-dna based on cpg-specific recognition of phosphodiester dna mutations in the gene encoding the 3'-5' dna exonuclease trex1 cause aicardi-goutieres syndrome at the ags1 locus aicardi-goutieres syndrome and related phenotypes: linking nucleic acid metabolism with autoimmunity 2'-o methylation of the viral mrna cap evades host restriction by ifit family members mda5-filament, dynamics and disease. current opinion in virology innate antiviral responses by means of tlr7-mediated recognition of single-stranded rna sirnas can function as mirnas a bipartite model of 2-5a-dependent rnase l targeted activation of melanoma differentiation-associated protein 5 (mda5) for immunotherapy of pancreatic carcinoma rig-i-like helicases induce immunogenic cell death of pancreatic cancer cells and sensitize tumors toward killing by cd8(+) t cells a novel mitochondrial mavs/caspase-8 platform links rna virus-induced innate antiviral signaling to bax/bak-independent apoptosis rna interference is mediated by 21-and 22-nucleotide rnas therapeutic efficacy of bifunctional sirna combining tgf-beta1 silencing with rig-i activation in pancreatic cancer dnase ii: genes, enzymes and function the ectodomain of toll-like receptor 9 is cleaved to generate a functional receptor rig-i-dependent antiviral immunity is effective against an rna virus encoding a potent suppressor of rnai interferon action: two distinct pathways for inhibition of protein synthesis by doublestranded rna identification of doublestranded rna-binding domains in the interferon-induced double-stranded rnaactivated p68 kinase aim2 activates the inflammasome and cell death in response to cytoplasmic dna a retrotransposon-driven dicer isoform directs endogenous small interfering rna production in mouse oocytes identification of rna sequence motifs stimulating sequence-specific tlr8-dependent immune responses autoimmunity initiates in nonhematopoietic cells and progresses via lymphocytes in an interferon-dependent autoimmune disease self-rna-antimicrobial peptide complexes activate human dendritic cells through tlr7 and tlr8 processing of double-stranded rna in mammalian cells: a direct antiviral role cyclic [g(2',5')pa(3',5')p] is the metazoan second messenger produced by dna-activated cyclic gmp-amp synthase structure-function analysis of sting activation by c[g(2',5')pa(3',5')p] and targeting by antiviral dmxaa activation of cyclic gmp-amp synthase by self-dna causes autoimmune diseases impact of protein kinase pkr in cell biology: from antiviral to antiproliferative action identification of modifications in microbial, native trna that suppress immunostimulatory activity oxidative damage of dna confers resistance to cytosolic nuclease trex1 degradation and potentiates sting-dependent immune sensing samhd1 restricts hiv-1 infection in dendritic cells (dcs) by dntp depletion, but its expression in dcs and primary cd4+ t-lymphocytes cannot be upregulated by interferons an rna editor, adenosine deaminase acting on double-stranded rna (adar1) targeting the cytosolic innate immune receptors rig-i and mda5 effectively counteracts cancer cell heterogeneity in glioblastoma brex is a novel phage resistance system widespread in microbial genomes hiv-1 restriction factor samhd1 is a deoxynucleoside triphosphate triphosphohydrolase antiviral immunity via rig-i-mediated recognition of rna bearing 5'-diphosphates restriction of diverse retroviruses by samhd1 cutting edge: cgas is required for lethal autoimmune disease in the trex1-deficient mouse model of aicardigoutieres syndrome the ebola virus vp35 protein is a suppressor of rna silencing sequestration by ifit1 impairs translation of 2'o-unmethylated capped rna impairment of neutrophil extracellular trap degradation is associated with lupus nephritis structure of human rnase l reveals the basis for regulated rna decay in the ifn response apobecs and virus restriction adenovirus infection triggers a rapid, myd88-regulated transcriptome response critical to acute-phase and adaptive immune responses in vivo rational design of new cpg oligonucleotides that combine b cell activation with high ifn-alpha induction in plasmacytoid dendritic cells cpg dna and lps induce distinct patterns of activation in human monocytes mechanism and function of a newly identified cpg dna motif in human primary b cells delineation of a cpg phosphorothioate oligodeoxynucleotide for activating primate immune responses in vitro and in vivo cpg dna: a potent signal for growth, activation, and maturation of human dendritic cells the protein kinase pkr is critical for lps-induced inos production but dispensable for inflammasome activation in macrophages peritumoral cpg dna elicits a coordinated response of cd8 t cells and innate effectors to cure established tumors in a murine colon carcinoma model species-specific recognition of single-stranded rna via toll-like receptor 7 and 8 a tolllike receptor recognizes bacterial dna sequence-specific activation of the dna sensor cgas by y-form dna structures as found in primary hiv-1 cdna cutting edge: the rig-i ligand 3prna potently improves ctl cross-priming and facilitates antiviral vaccination rna editing by mammalian adars host factor samhd1 restricts dna viruses in non-dividing myeloid cells aim2 recognizes cytosolic dsdna and forms a caspase-1-activating inflammasome with asc 5'-triphosphate rna is the ligand for rig-i sequence-specific potent induction of ifn-alpha by short interfering rna in plasmacytoid dendritic cells through tlr7 oas proteins and cgas: unifying concepts in sensing and responding to cytosolic nucleic acids intracellular dna recognition quantitative expression of toll-like receptor 1-10 mrna in cellular subsets of human peripheral blood mononuclear cells and sensitivity to cpg oligodeoxynucleotides a human dna editing enzyme homologous to the escherichia coli dnaq/mutd protein synthesis of low molecular weight inhibitor of protein synthesis with enzyme from interferon-treated cells vpx relieves inhibition of hiv-1 infection of macrophages mediated by the samhd1 protein dimeric structure of pseudokinase rnase l bound to 2-5a reveals a basis for interferon-induced antiviral activity innate immune restriction and antagonism of viral rna lacking 2-o methylation particle counts and infectivity titrations for animal viruses foreign nucleic acids as the stimulus to make interferon the functions of micrornas: mrna decay and translational repression aim2 drives joint inflammation in a self-dna triggered model of chronic polyarthritis mutations in ddx58, which encodes rig-i, cause atypical singleton-merten syndrome rnase h2 of saccharomyces cerevisiae is a complex of three proteins structural basis of rna recognition and activation by innate immune receptor rig-i structures of the hin domain:dna complexes reveal ligand binding and activation mechanisms of the aim2 inflammasome and ifi16 receptor the 2'-o-methylation status of a single guanosine controls transfer rna-mediated tolllike receptor 7 activation or inhibition sequence-dependent stimulation of the mammalian innate immune response by synthetic sirna translating nucleic acid-sensing pathways into therapies suppression of rna recognition by toll-like receptors: the impact of nucleoside modification and the evolutionary origin of rna rig-i-like receptors and autoimmune diseases cell type-specific involvement of rig-i in antiviral response chronic polyarthritis caused by mammalian dna that escapes from degradation in macrophages spontaneous formation of nucleic acid-based nanoparticles is responsible for high interferon-alpha induction by cpg-a in plasmacytoid dendritic cells activation with cpg-a and cpg-b oligonucleotides reveals two distinct regulatory pathways of type i ifn synthesis in human plasmacytoid dendritic cells self-priming determines high type i ifn production by plasmacytoid dendritic cells tight interplay among samhd1 protein level, cellular dntp levels, and hiv-1 proviral dna synthesis kinetics in human primary monocyte-derived macrophages molecular cloning of cdna for double-stranded rna adenosine deaminase, a candidate enzyme for nuclear rna editing samhd1 restricts herpes simplex virus 1 in macrophages by limiting dna replication defining the functional determinants for rna surveillance by rig-i structural basis for the activation of innate immune pattern-recognition receptor rig-i by viral rna cpg motifs in bacterial dna trigger direct b-cell activation cpg-a oligonucleotides induce a monocyte-derived dendritic cell-like phenotype that preferentially activates cd8 t cells targeted activation of rna helicase retinoic acid-inducible gene-i induces proimmunogenic apoptosis of human ovarian cancer cells double-stranded rna-dependent protein kinase activates transcription factor nf-kappa b by phosphorylating i kappa b ips-1 differentially induces trail, bcl2, birc3 and prkce in type i interferonsdependent and -independent anticancer activity samhd1 is the dendritic-and myeloid-cell-specific hiv-1 restriction factor counteracted by vpx samhd1 restricts the replication of human immunodeficiency virus type 1 by depleting the intracellular pool of deoxynucleoside triphosphates dnase2a deficiency uncovers lysosomal clearance of damaged nuclear dna via autophagy atp hydrolysis by the viral rna sensor rig-i prevents unintentional recognition of self-rna tlr9 signals after translocating from the er to cpg dna in the lysosome a cellular microrna mediates antiviral defense in human cells the nuclear rnase iii drosha initiates microrna processing a mutation in trex1 that impairs susceptibility to granzyme a-mediated cell death underlies familial chilblain lupus mutations in the gene encoding the 3'-5' dna exonuclease trex1 are associated with systemic lupus erythematosus regulation of protein synthesis: activation by doublestranded rna of a protein kinase that phosphorylates eukaryotic initiation factor 2. proceedings of the national academy of sciences of the united states of america interferon antagonist proteins of influenza and vaccinia viruses are suppressors of rna silencing rna interference functions as an antiviral immunity mechanism in mammals identification of human homologue of mouse ifngamma induced protein from human dendritic cells rna editing by adar1 prevents mda5 sensing of endogenous dsrna as nonself studies on the production, mode of action and properties of interferon structural basis of toll-like receptor 3 signaling with double-stranded rna adenovirus va1 noncoding rna can inhibit small interfering rna and microrna biogenesis crystal structure of the f27g aim2 pyd mutant and similarities of its self-association to ded/ded interactions plasticity in pyd assembly revealed by cryo-em structure of the pyd filament of aim2 unified polymerization mechanism for the assembly of asc-dependent inflammasomes novel role of pkr in inflammasome activation and hmgb1 release structural insights into rna recognition by rig-i a nonhereditary, host-induced variation of bacterial viruses ribonuclease h2 mutations induce a cgas/sting-dependent innate immune response potential role of pkr in double-stranded rna-induced macrophage activation cytosolic rna:dna hybrids activate the cgas-sting axis short doublestranded rnas with an overhanging 5' ppp-nucleotide, as found in arenavirus genomes, act as rig-i decoys unpaired 5' ppp-nucleotides, as found in arenavirus double-stranded rna panhandles, are not recognized by rig-i crispr interference: rna-directed adaptive immunity in bacteria and archaea tolling for autoimmunity-prime time for 7 establishment of a monoclonal antibody against human toll-like receptor 3 that blocks double-stranded rna-mediated signaling protein kinase pkr amplification of interferon beta induction occurs through initiation factor eif-2alpha-mediated translational control human argonaute2 mediates rna cleavage targeted by mirnas and sirnas differential regulation of the oasl and oas1 genes in response to viral infections genetargeted mice lacking the trex1 (dnase iii) 3'-> 5' dna exonuclease develop inflammatory myocarditis 5'-triphosphate-dependent activation of pkr by rnas with short stem-loops features of systemic lupus erythematosus in dnase1-deficient mice murine serum nucleases-contrasting effects of plasmin and heparin on the activities of dnase1 and dnase1-like 3 (dnase1l3) restriction endonucleases in the analysis and restructuring of dna molecules a-to-i editing of coding and non-coding rnas by adars structural basis of cpg and inhibitory dna recognition by toll-like receptor 9 mitochondrial dna that escapes from autophagy causes inflammation and heart failure an essential role for the n-terminal fragment of toll-like receptor 9 in dna sensing adar1 forms a complex with dicer to promote microrna processing and rnainduced gene silencing six rna viruses and forty-one hosts: viral small rnas and modulation of small rna repertoires in vertebrate and invertebrate systems proteolytic cleavage in an endolysosomal compartment is required for activation of tolllike receptor 9 p21 regulates the hiv-1 restriction factor samhd1. proceedings of the national academy of sciences of the united states of america cutting edge: dnase ii deficiency prevents activation of autoreactive b cells by doublestranded dna endogenous ligands nucleic acid-sensing tlrs and autoimmunity: novel insights from structural and cell biology identification of an n-terminal recognition site in tlr9 that contributes to cpg-dna-mediated receptor activation identification of micrornas of the herpesvirus family ifit1 is an antiviral protein that recognizes 5'-triphosphate rna rig-i-mediated antiviral responses to single-stranded rna bearing 5'-phosphates activation of mda5 requires higher-order rna structures generated during virus infection recognition of rna virus by rig-i results in activation of card9 and inflammasome signaling for interleukin 1 beta production the role of unc93b1 protein in surface localization of tlr3 receptor and in cell priming to nucleic acid agonists rnase h2 catalytic core aicardi-goutieres syndrome-related mutant invokes cgas-sting innate immune-sensing pathway in mice type i ifn triggers rig-i/tlr3/nlrp3-dependent inflammasome activation in influenza a virus infected cells aicardi-goutieres syndrome gene and hiv-1 restriction factor samhd1 is a dgtp-regulated deoxynucleotide triphosphohydrolase genomic signatures of emerging viruses: a new era of systems epidemiology the human 2',5'-oligoadenylate synthetase family: interferon-induced proteins with unique enzymatic properties samhd1-dependent retroviral control and escape in mice rig-i detects viral genomic rna during negative-strand rna virus infection mutations involved in aicardi-goutieres syndrome implicate samhd1 as regulator of the innate immune response mutations in adar1 cause aicardi-goutieres syndrome associated with a type i interferon signature clinical and molecular phenotype of aicardi-goutieres syndrome c-terminal truncations in human 3'-5' dna exonuclease trex1 cause autosomal dominant retinal vasculopathy with cerebral leukodystrophy rna:dna hybrids are a novel molecular pattern sensed by tlr9 hin-200 proteins regulate caspase activation in response to foreign cytoplasmic dna the rna helicase lgp2 inhibits tlr-independent sensing of viral replication by retinoic acid-inducible gene-i cpg-a and cpg-b oligonucleotides differentially enhance human peptidespecific primary and memory cd8 + t-cell responses in vitro the ribonuclease activity of samhd1 is required for hiv-1 restriction trashing the genome: the role of nucleases during apoptosis approaching the rna ligand for rig-i? recognition of 5' triphosphate by rig-i helicase requires short blunt doublestranded rna as contained in panhandle of negative-strand virus noncoding flavivirus rna displays rna interference suppressor activity in insect and mammalian cells breaking tumor-induced immunosuppression with 5'-triphosphate sirna silencing tgfbeta and activating rig-i. oncoimmunology, 2. e24170 induction of immunogenic cell death by targeting rig-i-like helicases in pancreatic cancer a diverse range of gene products are effectors of the type i interferon antiviral response a conserved histidine in the rna sensor rig-i controls immune tolerance to n1-2'o-methylated self rna characteristics of a double-stranded rna-activated protein kinase system partially purified from interferon treated ehrlich ascites tumor cells reciprocal inhibition between intracellular antiviral signaling and the rnai machinery in mammalian cells viral encounters with 2',5'-oligoadenylate synthetase and rnase l during the interferon antiviral response intrinsic host restrictions to hiv-1 and mechanisms of viral escape single-stranded small interfering rna are more immunostimulatory than their double-stranded counterparts: a central role for 2'-hydroxyl uridines in immune responses enzyme from calf thymus degrading the rna moiety of dna-rna hybrids: effect on dna-dependent rna polymerase trex1 prevents cellintrinsic initiation of autoimmunity renaissance of mammalian endogenous rnai rna interference-mediated intrinsic antiviral immunity in plants wolbachia bacterial endosymbionts of filarial nematodes mechanism of interferon action: cdna structure, expression, and regulation of the interferon-induced, rna-dependent p1/eif-2 alpha protein kinase from human cells mechanism of interferon action: autoregulation of rna-dependent p1/eif-2 alpha protein kinase (pkr) expression in transfected mammalian cells adars and the balance game between virus infection and innate immune cell response unravelling the structural and mechanistic basis of crispr-cas systems diverse functions of restriction-modification systems in addition to cellular defense loss of dexd/h box rna helicase lgp2 manifests disparate antiviral responses immune stimulation mediated by autoantigen binding sites within small nuclear rnas involves toll-like receptors 7 and 8 incorporation of phosphorothioate groups into fd and phi x174 dna a double-stranded rna unwinding activity introduces structural alterations by means of adenosine to inosine conversions in mammalian cells and xenopus eggs rna interference directs innate immunity against viruses in adult drosophila structural and functional insights into 5'-ppp rna pattern recognition by the innate immune receptor rig-i standing on three legs: antiviral activities of rig-i against influenza viruses coley's toxins, tumor necrosis factor and cancer research: a historical perspective cellular and biochemical mechanisms of the retroviral restriction factor samhd1 how rig-i like receptors activate mavs cyclic gmp-amp is an endogenous second messenger in innate immune signaling by cytosolic dna in vitro augmentation of natural killer cell activity and production of interferon-alpha/beta and -gamma with deoxyribonucleic acid fraction from mycobacterium bovis bcg unique palindromic sequences in synthetic oligonucleotides are required to induce ifn [correction of inf] and augment ifn-mediated [correction of inf] natural killer activity dna from bacteria, but not from vertebrates, induces interferons, activates natural killer cells and inhibits tumor growth trex1 exonuclease degrades ssdna to prevent chronic checkpoint activation and autoimmune disease interferons, double-stranded rna, and rna degradation. isolation and characterization of homogeneous human (2'-5')(a)n synthetase molecular cloning of the cdna encoding human deoxyribonuclease ii mutation of dnase1 in people with systemic lupus erythematosus shared and unique functions of the dexd/h-box helicases rig-i, mda5, and lgp2 in antiviral innate immunity lethal anemia caused by interferon-beta produced in mouse embryos carrying undigested dna nucleic acidsensing toll-like receptors are essential for the control of endogenous retrovirus viremia and erv-induced tumors rnai is an antiviral immune response against a dsrna virus in drosophila melanogaster rna-based mechanisms regulating host-virus interactions antiviral activity of human oasl protein is mediated by enhancing signaling of the rig-i rna sensor isolation of two interferoninduced translational inhibitors: a protein kinase and an oligo-isoadenylate synthetase key: cord-321607-3r736dnk authors: ezelle, heather j.; malathi, krishnamurthy; hassel, bret a. title: the roles of rnase-l in antimicrobial immunity and the cytoskeleton-associated innate response date: 2016-01-08 journal: int j mol sci doi: 10.3390/ijms17010074 sha: doc_id: 321607 cord_uid: 3r736dnk the interferon (ifn)-regulated endoribonuclease rnase-l is involved in multiple aspects of the antimicrobial innate immune response. it is the terminal component of an rna cleavage pathway in which dsrna induces the production of rnase-l-activating 2-5a by the 2′-5′-oligoadenylate synthetase. the active nuclease then cleaves ssrnas, both cellular and viral, leading to downregulation of their expression and the generation of small rnas capable of activating retinoic acid-inducible gene-i (rig-i)-like receptors or the nucleotide-binding oligomerization domain-like receptor 3 (nlrp3) inflammasome. this leads to ifnβ expression and il-1β activation respectively, in addition to broader effects on immune cell function. rnase-l is also one of a growing number of innate immune components that interact with the cell cytoskeleton. it can bind to several cytoskeletal proteins, including filamin a, an actin-binding protein that collaborates with rnase-l to maintain the cellular barrier to viral entry. this antiviral activity is independent of catalytic function, a unique mechanism for rnase-l. we also describe here the interaction of rnase-l with the e3 ubiquitin ligase and scaffolding protein, ligand of nump protein x (lnx), a regulator of tight junction proteins. in order to better understand the significance and context of these novel binding partners in the antimicrobial response, other innate immune protein interactions with the cytoskeleton are also discussed. rnase-l is an endoribonuclease that cleaves single-stranded rna as the effector portion of a two-component system regulated by the antiviral type i and iii interferons (ifn-α/β and ifn-λ, respectively) [1, 2] . ifn signaling leads to the induction of oligoadenylate synthetases (oas) which, following activation by double stranded rna (dsrna), generate 2 1 -5 1 -linked oligoadenylates (2-5a) [3, 4] . 2-5a then binds to rnase-l, leading to its dimerization and enabling its nuclease activity [4] . although originally characterized as an antiviral agent, almost 40 years of research has unveiled many additional and diverse functions for this endoribonuclease, as well as novel mechanisms for both these new and its more established antimicrobial activities. in the late 1970 1 s, the 2-5a-dependence and endonucleolytic properties of rnase-l were being investigated by multiple laboratories around the world [5] [6] [7] [8] [9] . the cloning of rnase-l by the silverman lab in 1993 then opened the door for genetic manipulation and more definitive characterization of is normally repressed due to interactions between the ankyrin repeat domain and the enzymatic domain, however upon binding to 2-5a, a conformational change in the ken domain occurs to expose the pseudokinase and ribonuclease domains [43] . recent crystal structure data indicate that 2-5a binds to the second and fourth ankyrin repeats and the pseudokinase domain. these interactions, in conjunction with binding between the pseudokinase domains of the two protomers, mediate dimerization and enzymatic activation within minutes [48] [49] [50] . the crystallization studies have also cast a new light on other canonical aspects of rnase-l activation. previously, active rnase-l was generally believed to be a homodimer bound to 2-5a. in vitro activation analysis indicates that rnase-l can in fact oligomerize into complexes of two or more monomers and that at micromolar concentrations of rnase-l, 2-5a is dispensable for this oligomerization [49] . whether physiologic concentrations of rnase-l reach these levels is unknown. once active, rnase-l cleaves ssrna, including cellular mrna and rrna as well as microbial rnas [51, 52] . the targeting of microbial rnas has a significant impact on the ability of the virus or bacterium to replicate, as does the inhibition of cellular translation resulting from the cleavage of rrna and ribosomal protein transcripts [6, 26, 41, 53] . mrna targeting can facilitate rnase-l-dependent regulation of gene expression through direct cleavage of transcripts or indirectly through the destabilization of transcriptional regulators [54] . historically, this ribonuclease activity has been considered the major mediator of the biological activities of rnase-l. nucleolytic activity is normally repressed due to interactions between the ankyrin repeat domain and the enzymatic domain, however upon binding to 2-5a, a conformational change in the ken domain occurs to expose the pseudokinase and ribonuclease domains [43] . recent crystal structure data indicate that 2-5a binds to the second and fourth ankyrin repeats and the pseudokinase domain. these interactions, in conjunction with binding between the pseudokinase domains of the two protomers, mediate dimerization and enzymatic activation within minutes [48] [49] [50] . the crystallization studies have also cast a new light on other canonical aspects of rnase-l activation. previously, active rnase-l was generally believed to be a homodimer bound to 2-5a. in vitro activation analysis indicates that rnase-l can in fact oligomerize into complexes of two or more monomers and that at micromolar concentrations of rnase-l, 2-5a is dispensable for this oligomerization [49] . whether physiologic concentrations of rnase-l reach these levels is unknown. once active, rnase-l cleaves ssrna, including cellular mrna and rrna as well as microbial rnas [51, 52] . the targeting of microbial rnas has a significant impact on the ability of the virus or bacterium to replicate, as does the inhibition of cellular translation resulting from the cleavage of rrna and ribosomal protein transcripts [6, 26, 41, 53] . mrna targeting can facilitate rnase-l-dependent regulation of gene expression through direct cleavage of transcripts or indirectly through the destabilization of transcriptional regulators [54] . historically, this ribonuclease activity has been considered the major mediator of the biological activities of rnase-l. in the event of pathogen infection, rapid activation of rnase-l increases the chance for clearance and host survival; however, uncontrolled activation of rnase-l could lead to cell death or dysregulated gene expression [13, 15, 26, 55] . therefore, rnase-l is tightly regulated by multiple mechanisms in order to maintain cell homeostasis. rnase-l is typically expressed at low levels in most mammalian cell types. it is marginally induced by ifnα/β and ifnγ, and sequence analysis primarily indicates tissue-specific and ubiquitous elements in the promoter [1, 2, 56] . amongst these were sites for the specificity protein 1 (sp1) transcription factor, which are present in many housekeeping genes. recent findings show that overexpression of sp1, which is associated with tumorigenesis, can induce rnase-l and many other rig-i pathway members [57] . although this may appear to contradict the role of rnase-l as a tumor suppressor, our lab has shown that rnase-l can have oncogenic activity in chronic myelogenous leukemia and this may contribute to that novel function in certain cell types [58] . in addition to transcriptional control, rnase-l mrna is also regulated at the post-transcriptional level by the rna-binding protein (rbp) human antigen r (hur) [59] . hur binds to au-rich elements (are) typically found in the 3 1 utrs of post-transcriptionally regulated mrnas, and stabilizes them [60] . rnase-l has eight ares, and deletion mapping indicates that there are both positive and negative regulatory elements in the 3 1 utr. the two ares closest to the 3 1 terminus are stabilized by hur and result in a several-fold increase in rnase-l expression [59] . even low-to-moderate changes in rnase-l expression yield significant functional impact as rna cleavage activity is disproportionately enhanced with even two-fold increases in expression [49] . once the protein is made, rnase-l is susceptible to two forms of post-translational modification: ubiquitination and hydroxylation. chase et al. [61] found that treatment of l929 cells with the phorbol ester phorbol-12-myristate-13 acetate (pma) resulted in the rapid and almost complete reduction of rnase-l protein levels. this degradation was ablated by treatment with proteasome inhibitors. similarly, ubiquitin modification of rnase-l was seen in the muscle tissue of a transgenic β5t mouse model that inhibited proteasomal chymotrypsin-like activity [62] . hydroxylation of rnase-l was discovered in a screen for novel substrate interactors for the asparaginyl hydroxylase factor inhibiting hypoxia-inducible factor (hif). hydroxylation typically adds an -oh group to either pro or asn residues and has been shown to increase stability, inhibit signaling, or disrupt protein complex formations. the hydroxylation of rnase-l occurs at asn-196 in the fifth ankyrin repeat domain; however, the effect of this modification is unknown [63] . rnase-l can also be regulated through several other protein-protein interactions. the best characterized rnase-l binding protein is the rnase-l inhibitor (rli). rli inhibits rnase-l as part of a complex containing rli, rnase-l, and the translation termination-associated gtpase, erf3 [64] . rli, also known as abce1, is a member of the atp binding cassette superfamily and, aside from its role as an rnase-l inhibitor, is believed to be involved in translation termination and ribosome recycling [65] [66] [67] . the mechanism for rnase-l inhibition is believed to be through direct binding with rli, as rli is not known to bind competitively with 2-5a. regardless of the mechanism, however, overexpression or knockdown of rli has inhibitory or promoting effects on the binding of rnase-l to 2-5a, rrna cleavage, destabilization of mitochondrial mrna, antiviral activity, and antitumor activity [68] [69] [70] [71] . three other interacting proteins are believed to possibly help target rnase-l to ssrna substrates: the eukaryotic translation termination release factor erf3, mitochondrial translation initiation factor (if2mt), and tristetraprolin (ttp). erf3 was discovered as an rnase-l interacting partner in 1993 but was originally termed rnabp (rna binding protein) until its proper identification in 2005 by le roy et al. [72] . it binds to rnase-l as part of a complex and is believed to help facilitate its activity, since a presumed erf3-inhibitory antibody, mab3, blocks rnase-l-mediated rrna cleavage and antiviral activity [73] . although erf3 does not bind 2-5a directly, the presence of 2-5a enhances the erf3-rnase-l interaction, again suggesting that erf3 is important to rnase-l function. erf3, in conjunction with erf1, mediates the release of the polypeptide chain from the ribosome during translation termination. it also interacts with rli and the poly(a)-binding protein (pabp), which facilitates ribosome recycling by reinitiating translation after termination [74] . rnase-l localization with this translation termination complex places it in close proximity to potential mrna targets as they are translated on polysomes. actively translating rnas adopt a closed-loop conformation in which the 3 1 utr forms an exposed loop that may be accessible to regulatory factors. when active, rnase-l can outcompete pabp for erf3 interaction in the termination complex, displacing it and providing rnase-l with access to mrna substrates [25, 72] . one caveat to this targeting model is that while ribosomes translate all mrnas, rnase-l is selective, indicating that other factors are needed for specific targeting. similar to the accessibility that erf3 may provide rnase-l to its targets, another binding partner may serve the same function in the mitochondrion. although rnase-l is generally considered to be cytosolic, it is also found in the nucleus and mitochondria, where it has been shown to downregulate the expression of several mitochondrial rnas (mtrna), such as cytochrome b, atpase6, and cytochrome oxidase subunit ii [68, 75, 76] . in support of a role for rnase-l regulation of mitochondrial rnas is the observation that the rnase-l regulators oas, rli, and 2 1 pde have also been observed in mitochondria, and their modulation impacts the expression of rnase-l mitochondrial targets correspondingly [68, 77, 78] . in a yeast-two hybrid screen using rnase-l as bait, mitochondrial translation initiation factor (if2mt) was isolated as a potential interactor and confirmed in rabbit reticulocyte lysate. if2mt is a nuclear-encoded translation factor that delivers n-formyl methionyl-trna to the p-site of the mitochondrial ribosome during initiation. using a human t cell lymphoma cell system in which ifnα induces rnase-l-dependent mtrna degradation and cell death, it was shown that rnase-l activity required active translation. in addition, if excess if2mt was introduced to the system so that rnase-l was competed away from translation initiation complex-bound if2mt, the degradation of mtrnas was suppressed, resulting in an increase in proliferation and a decrease in apoptotic signaling [76] . these data indicate that proximity to ribosome-bound mtrnas is necessary for their rnase-l-dependent degradation. therefore, like erf3, if2mt may facilitate accessibility, while other mechanisms must exist to provide specificity. a third interacting protein may aid in providing this rnase-l target specificity. our lab has shown that ttp can bind to rnase-l and coordinately regulate serum response factor (srf) mrna destabilization [54] . ttp is an are-binding protein that controls inflammation and suppresses tumorigenicity through the decay of target mrnas. it binds these mrnas using two zinc finger domains, often recognizing a uuauuuauu nonamer sequence, but utilizes other enzymes to mediate degradation [79, 80] . our data indicate that ttp may guide rnase-l to specific targets like srf mrna, facilitating rna cleavage by the nuclease, possibly at the uu and ua dinucleotide sites within the ttp nonamer. ttp and rnase-l share a subset of targeted rnas, including il-8, hmga2, and ttp itself, indicating that this mechanism may not be unique to srf [54] . also lending support to this cooperative function is the identification of the nuclease regnase-1. regnase-1 contains both endoribonucleolytic activity like rnase-l, and a ccch-type zinc finger that mediates mrna binding similar to ttp [81, 82] . it essentially combines the activities of rnase-l and ttp into a single enzyme, setting precedent for this cooperative function. many rnase-l substrates are not shared with ttp, indicating that additional mechanisms are likely involved and other are-binding proteins (arebp) may serve as potential targeting candidates. arebp post-transcriptional regulators are strong contenders because although no definitive consensus recognition site exists for rnase-l, its preference for uu and ua dinucleotides is identical to those found in ares. rnase-l was initially discovered and characterized as a mediator of type i ifn antiviral activity. although cleavage of rna virus genomes appeared as the most direct mechanism of action, other important pathways have become evident, such as the regulation of host gene expression, stimulation of ifnβ production, activation of the nacht, lrr, and pyd-containing protein-3 (nlrp3) inflammasome, and maintenance of the cell's structural barrier to infection [27, 55, 83, 84] . these will be discussed in further detail below. here we highlight some of the decades of work demonstrating the importance of rnase-l to the replication and survival of specific viruses; a full examination of this subject is provided by r.h. silverman [85] . rnase-l has been shown to play a role in antiviral activity against both rna viruses (picornaviridae, reoviridae, togaviridae, paramyxoviridae, orthomyxoviridae, flaviviridae, coronaviridae and retroviridae) and dna viruses (poxviridae, herpesviridae, and polyomaviridae). the prototypical virus for demonstrating rnase-l activity is the picornavirus, encephalomyocarditis virus (emcv). oas has been isolated while bound to both the positive and negative strands of the emcv genome, indicating that it can be activated by the double-stranded replicative intermediate of the positive-stranded rna genome [86, 87] . following 2-5a production, activated rnase-l then cleaves both cellular and viral rnas, which not only directly inhibits virus replication, but produces small rnas capable of stimulating rig-i activation and subsequent downstream ifnβ transcription to protect surrounding tissue by creating an antiviral state [27, 53] . overexpression of rnase-l has been shown to suppress emcv replication whereas dominant-negative rnase-l inhibits ifn-induced protection against infection, indicating the effectiveness of rnase-l antiviral activity [14, 53] . in order to determine whether these effects were physiologic in vivo, rnase-l knock-out (ko) and rnase-l wild type (wt) mice were infected with emcv, and wild type mice exhibited both a better rate of survival and delayed onset of death as compared to knockout mice [15] . though emcv is sensitive to rnase-l activity, other members of the picornaviridae family, such as theiler's virus and poliovirus, have adapted mechanisms for evading rnase-l activity [88] [89] [90] [91] . the evolution of these and other inhibitors are a testament to the antiviral potency of the oas/rnase-l pathway. in an in vitro hepatitis c virus (hcv) infection model, rnase-l was demonstrated to cleave the genome at uu and ua dinucleotides into 200-500 bp fragments [92] . sequence analysis indicated that uu and ua dinucleotides are underrepresented in the hcv genome and that ifn-sensitive hcv genotypes contain more uu and ua dinucleotides than ifn-resistant genotypes [93] . this minimizing of potential rnase-l cleavage sites could be an effective evasion strategy by this chronic pathogen [94] . influenza a virus (iav) has also been shown to be cleaved at discrete locations in both the positive and negative rna strands of the replicative intermediate genome, but only in the absence of the ifn inhibitor ns1 [95, 96] . ns1 binds to and sequesters dsrna in order to prevent the activation of the viral sensors rig-i, oas, and the ifn-induced protein kinase pkr [97] . since this mechanism not only inhibits the oas/rnase-l pathway, but other potent antiviral actions including the induction of ifnβ, other viruses such as vaccinia virus, human immunodeficiency virus (hiv), and reovirus, have also adopted this strategy [85] . this sequestration of activating rnas may not be completely efficient throughout the course of infection, potentially resulting in low levels of free 2-5a. therefore, hiv also induces the expression of rli to prevent the binding of any free 2-5a to rnase-l, further safeguarding against activation [70, 98] . a relatively new mechanism for inhibiting rnase-l activity was identified in the coronavirus mouse hepatitis virus (mhv). the mhv ns2 gene encodes its own 2 1 phosphodiesterase to degrade 2-5a. when compared to wild type virus, the mutant virus ns2-h126r that expresses catalytically inactive ns2, was unable to inactivate 2-5a and inhibit rnase-l activation, thus the development of hepatitis and replication in the liver was unimpaired. this demonstrates that mhv-ns2 is a potent antagonist of the oas/rnase-l pathway [99] . a more detailed analysis of these and other viral evasion mechanisms are reviewed by drappier et al. [100] , highlighting the importance of rnase-l as an antiviral effector. although the type i ifns were originally discovered as agents of antiviral activity, their role in defense against other pathogens has been a continually expanding field, particularly since the discovery of the tlrs. in 2008, our lab provided the first evidence of antibacterial activity by rnase-l. we demonstrated that rnase-l ko mice are more susceptible to sublethal infection by both escherichia coli (e. coli) and bacillus anthracis (ba) than wt mice. multiple mechanisms were found to contribute to this sensitivity, including changes in gene regulation that likely contributed to immune cell dysfunction. specifically, ifnβ, the proinflammatory cytokines tumor necrosis factor-α (tnfα) and interleukin-1β (il-1β), and a protease involved in endolysosomal maturation, cathepsin e, were all dysregulated in rnase-l ko mice and purified peritoneal macrophages. this likely contributed to the decreased bactericidal activity observed in infected macrophages, altered macrophage maturation, and differences in neutrophil and lymphocyte recruitment. these changes in proinflammatory gene expression were also seen in response to dsrna, lipopolysaccharide, and cpg dna stimulation of tlrs 3, 4, and 9, respectively [26] . on the heels of this work, it was discovered that e. coli rna could activate rnase-l induction of ifnβ and that rnase-l also plays an important part in gut immunity [23] . therefore, our lab investigated the role of rnase-l in the pathogenesis of the intestinal pathogen enteropathogenic e. coli (epec). in a novel antimicrobial role for rnase-l, we found that it helps maintain the intercellular barrier formed by intestinal epithelial cell (iec) monolayers, preventing pathogen translocation from the gut into the host. similar to our previous studies, we also saw dysregulation of ifnβ and tnfα as well as novel regulation of the tight junction proteins occludin and claudin-1, which undoubtedly contributed to the defects in barrier function [84] . many types of bacteria may activate rnase-l through endocytosis and either escape of the bacterium or leakage of rna into the cytosol during infection. however, since rna in actively replicating bacteria is not exposed to cytosolic rnase-l, it is likely resistant to direct cleavage as an effector mechanism. therefore, as touched on above, many of the other antimicrobial functions of rnase-l may be more prominent in these types of infections. as exemplified by the examples of antibacterial activity, rnase-l utilizes a variety of mechanisms to combat microbial infections. early investigations into antiviral activity focused on direct cause-and-effect events such as rnase-l activation and virus genome cleavage to inhibit replication. the expansion of studies into dna viruses and then bacteria have corresponded with broadening ideas in the field about rnase-l functions via more indirect antimicrobial activities, including apoptosis, immune modulation, gene regulation, autophagy, and structural integrity. one of the first alternative mechanisms was elucidated by castelli et al. [12, 13] , who demonstrated that rnase-l mediated apoptosis in response to activation by virus infection or dsrna. zhou et al. [15] confirmed this role for rnase-l in fas, tnfα, and α-cd3 antibody-induced apoptosis. further characterization showed that rnase-l-dependent cell death resulted in the release of cytochrome c from the mitochondria and required caspase-3 activation [101] . this early described role for rnase-l may be partially coordinated with a newly discovered function in the induction of autophagy. autophagy is a stress response that induces the recycling of proteins, damaged organelles, and pathogens by isolating them within a double-membraned autophagosome that then fuses with a lysosome in order to degrade its contents into reusable components [102] . rnase-l induces autophagy in response to viral infection, or more directly by 2-5a. the signaling involved in activating this process involves both c-jun n-terminal kinase and pkr, resulting in the degradation of p62 (sqstm1), lcsbi/lcs3bii conversion, and the accumulation of autophagosomes, all hallmarks of autophagy. rnase-l-induced autophagy was shown to be detrimental to virus replication in the early stages of infection and then advantageous in later stages, or under high infectious doses of virus [29, 103] . it has previously been shown that autophagy suppresses apoptosis at early timepoints in order to give the cell time to recover. if the activating stressor continues or autophagy is excessive, the cell can then switch in favor of a cell death response [104] . recently, siddiqui et al. [30] , have shown that rnase-l cleavage products can promote this switch to apoptosis by activating caspase-3 to cleave beclin-1, which then translocates to the mitochondria to induce cytochrome c release. like the induction of autophagy, the exact mechanisms for many of the cellular functions of rnase-l are unknown. it is broadly believed that the regulation of gene expression plays a major role in facilitating these activities, such as the modulation of the immune response. the gene most notably regulated by rnase-l is ifnβ. in 2007, malathi et al. [27] showed that activated rnase-l cleaved cellular and viral rnas into small rnas (<200 bp) that often form a duplex. these small rnas are capable of stimulating rig-i and mda5 (melanoma differentiation associated gene-5) to activate mitochondrial antiviral signaling protein (mavs) and induce the subsequent translocation of interferon regulatory factor 3 (irf3) to the nucleus to drive transcription of ifnβ. once secreted, ifnβ can not only create an antiviral state in surrounding cells, but it can affect immunoglobulin class switching in b cells as well as t and nk cell activation, affecting the adaptive immune response to pathogens [105] [106] [107] . skin allograft rejection and contact hypersensitivity experiments conducted in rnase-l ko and wt mice revealed a five day delay in allograft rejection in the ko mice and a reduction of inflammatory infiltrates [31] . this may indicate a defect in t-cell priming or immune cell trafficking; however, further studies are required to determine the exact mechanism for rnase-l activity. the possible involvement of rnase-l in t cell function is not surprising, as rnase-l ko mice have enlarged thymuses, likely due to defects in apoptosis. other investigations of immune cell functions have shown that, in addition to our work with e. coli and ba infections above, macrophages deficient in rnase-l exhibit decreased migration, endocytic activity, and proinflammatory gene regulation [26, 32] . these data clearly indicate that rnase-l has an important role in immune cell function, including the adaptive response. in addition to type i ifn and tlr signaling, which were shown to be regulated by rnase-l during bacterial infection, another family of innate immune regulators also interacts with the oas/rnase-l pathway. nucleotide-binding and oligomerization domain (nod)-like receptors (nlr) detect microbial products and are divided into two subsets, those that drive mitogen-activated protein kinase (mapk) and nuclear factor-κb (nf-κb) signaling (such as nod1 and nod2), and those that activate the inflammasome, leading to caspase-1, il-18, and il-1β processing and secretion (including nlrps 1, 3, and 4 and aim2) [108] . recently, rnase-l has been shown to play a role in the activation of the nlrp3 inflammasome. in this process, rnase-l is activated following virus infection to generate small rna cleavage products that bind to the dexd/h helicase dhx33. this leads to the formation of a dhx33, nlrp3, and mavs complex and increased il-1β production [83] . association with the other subset of nlrs occurs when oas binds to nod2. nod2 is activated by the peptidoglycan breakdown product muramyl dipeptide as well as viral ssrna. overexpression of nod2 when oas is active leads to increased rnase-l activity, suggesting a connection between the pathways [109] . any effects of oas interaction on nod2 activation of mapk or nf-κb signaling are unknown. the long-standing model of rnase-l activity involves low levels of expression in the cytosol in an inactive conformation, activation by 2-5a, and cleavage of ssrna in order to exert its biological functions. though all of this is well-documented, it does not address whether constitutively expressed rnase-l serves a physiologic role in the absence of 2-5a. in addition, while rnase-l is widely considered to be a cytosolic protein, it is also associated with the nucleus, mitochondria, and cytoskeleton [76, [110] [111] [112] . although rnas are accessible in all of these cellular compartments, it prompts the question whether there are alternative functions for this localization. early insights into this were provided by the 1998 study by tnani et al. [112] , in which they used biochemical methods to detect rnase-l in the cytoskeletal fraction of cell extracts. they demonstrated that rnase-l associated with the cytoskeleton in a conformation that did not allow it to bind 2-5a, which supports a model in which cytoskeletal association is incompatible with rnase-l activity. indeed, it is not surprising that the dramatic structural changes that occur upon rnase-l activation would impact its interactions with cytoskeletal components. treatment with pma, a known inducer of cytoskeletal alterations, dissociated rnase-l from the cytoskeleton and corresponded with its phosphorylation by protein kinase c (pkc) [112] [113] [114] . thus, changes in cell structure may induce the phosphorylation of rnase-l as a mechanism to promote its release from the cytoskeleton or induce conformational changes that enable activation [61, 112] . this would suggest a model in which rnase-l bound to the cytoskeleton is inactive, however, pma or the subsequent actin reorganization may disrupt this association so that it can bind 2-5a and initiate downstream signaling and effector functions. more recently, gupta et al. [111] performed a proteomic analysis of rnase-l binding proteins and found that over 50% of those identified were involved in cytoskeletal and motor assembly. these included β-actin, troponin i, myosin heavy chain 9, fibronectin precursor and an uncharacterized protein with homology to myosin binding protein c, slow type isoform cra_j. in addition to this screen, three additional interacting proteins have been more fully characterized and contribute towards a new model of cytoskeleton-associated rnase-l activity. the first of these interacting partners is a scaffold protein involved in actin reorganization, identified as the isoleucine-glutamine (iq) motif-containing ras gtpase-activating-like protein 1, or iqgap1. iqgap1 is a 190 kda, ubiquitously expressed protein that shares a number of biological functions with rnase-l, including the regulation of migration, proliferation, and differentiation. in addition, both proteins regulate the formation of tight junctions (tj). iqgap1 alters tj assembly by reducing the recruitment of claudin 2 and increasing the assembly of claudin 4 into tj complexes. it also diminishes barrier function by inhibiting the cdc42-jnk signaling pathway [115] . conversely, rnase-l promotes the expression of tj proteins claudin 1 and occludin, enhances barrier function, and prevents the transepithelial migration of pathogens such as epec [84] . iqgap1 functions as a multifunctional scaffolding protein, involving interactions with over 50 binding partners thus far [116, 117] . one of the main roles for iqgap1 is in actin polymerization. it can help maintain cdc42 and rac1 in their active, gtp-bound state by inhibiting gtp hydrolysis. in the case of cdc42, this activity keeps wasp (wiscott-aldrich syndrome protein) bound to cdc42, stabilizing it and maintaining its interactions with actin-related protein 2 and 3 (arp2/3) and globular actin. this preservation of the interactions enhances actin polymerization and branching [118] . iqgap1 was originally identified as an rnase-l interacting protein in screens searching for a mechanism for rnase-l-mediated cell death induced by the drug 1-(3-c-thynyl-β-d-ribo-pentofuanosyl) cytosine (ecyd). ecyd is a cytotoxic nucleoside analogue that is active against cancer cells, and both rnase-l and iqgap1 were necessary for ecyd-induced apoptosis in human fibrosarcoma ht1080 cells. ecyd treatment enhanced iqgap1 interaction with rnase-l, however, no significant function for this interaction was determined [119, 120] . we recently identified filamin a (flna), an actin-binding protein that functions as a scaffold linking many diverse proteins to the cytoskeleton, as a second rnase-l interacting protein. this interaction revealed a novel biological function for rnase-l in membrane integrity and was abrogated upon rnase-l activation by either 2-5a or emcv infection. complex formation did not require rnase-l nuclease or dimerization functions, but did require the n-terminal ankyrin repeat domains. other rnase-l interacting proteins have, somewhat surprisingly, bound to the c-terminus, possibly indicating that rnase-l may bind multiple proteins simultaneously. in addition, binding to flna did not inhibit or enhance the activation or nucleolytic activities of rnase-l. examination of the potential reciprocal consequences of the interaction on actin dynamics found that rnase-l ko primary murine embryonic fibroblasts (mefs) had a 30% reduction in actin filaments compared to wt mefs. further probing into this role for rnase-l in actin cytoskeleton function demonstrated that rnase-l inhibited the endocytosis of both an inert synthetic agent and viral particles. significantly, inhibition of virus entry was independent of endoribonuclease activity, providing the first evidence of a non-catalytic mechanism of rnase-l action. all other functions for rnase-l have been shown, or presumed to be the result of rna cleavage leading to altered regulation of gene expression via direct or indirect degradation mechanisms [25] . the diminished uptake of virus in rnase-l-expressing cells was sufficient to inhibit overall virus production and this was complemented by similar results in the presence or absence of flna [28] . these data provide a new model for rnase-l activity as a structural component of the cytoskeletal barrier that refracts the actin reorganization involved in the endocytosis of viral particles. this localization may also place rnase-l in immediate proximity to virus that is able to bypass this first layer of host defense in order to hasten the antiviral response via its canonical nuclease-dependent functions. activation of rnase-l may then cause a conformational change that induces its release from flna. although flna does not alter the capacity of virus to activate rnase-l, the interaction may aid in preventing incidental oligomerization of latent rnase-l monomer, adding to the many layers of regulation needed to keep rnase-l activity in check. these findings were the first to truly characterize a function for rnase-l in the cytoskeleton. our lab has also identified a third interacting protein that links rnase-l to the cytoskeleton, the ligand of numb protein x-1 (lnx). lnx is a cytoskeleton/tj-associated protein that was isolated through a yeast two-hybrid screen using rnase-l as bait. lnx is an e3 ubiquitin ligase that contains a ring finger in its n-terminus, which mediates ubiquitin ligase activity, and four pdz domains (named after the proteins postsynaptic density 95, drosophila disc large tumor suppressor, and zonula occludens-1) that facilitate protein-protein interactions and are present in many tj/cytoskeletal proteins [121, 122] . the interaction between lnx and rnase-l was confirmed in 293t cells and mapped to the c-terminus of rnase-l and the pdz domain-containing c-terminus of lnx ( figure 2 ). lnx also functions as a scaffold protein. in addition to several characterized interactions, a proteomic screen for potential lnx ubiquitylation substrates identified 64 candidate interactors, and yeast data suggest that the lnxp80 and p70 isoforms can form homo-or hetero-oligomers, potentially forming large complexes [123] . together, these e3 ligase and scaffolding functions are thought to mediate the physiologic activities of lnx, including cell signaling, cell cycle progression, tight junction reorganization, epithelial-to-mesenchymal transition, and its association with glioblastoma [124] [125] [126] [127] [128] [129] . our lab has also identified a third interacting protein that links rnase-l to the cytoskeleton, the ligand of numb protein x-1 (lnx). lnx is a cytoskeleton/tj-associated protein that was isolated through a yeast two-hybrid screen using rnase-l as bait. lnx is an e3 ubiquitin ligase that contains a ring finger in its n-terminus, which mediates ubiquitin ligase activity, and four pdz domains (named after the proteins postsynaptic density 95, drosophila disc large tumor suppressor, and zonula occludens-1) that facilitate protein-protein interactions and are present in many tj/cytoskeletal proteins [121, 122] . the interaction between lnx and rnase-l was confirmed in 293t cells and mapped to the c-terminus of rnase-l and the pdz domain-containing c-terminus of lnx ( figure 2 ). lnx also functions as a scaffold protein. in addition to several characterized interactions, a proteomic screen for potential lnx ubiquitylation substrates identified 64 candidate interactors, and yeast data suggest that the lnxp80 and p70 isoforms can form homo-or hetero-oligomers, potentially forming large complexes [123] . together, these e3 ligase and scaffolding functions are thought to mediate the physiologic activities of lnx, including cell signaling, cell cycle progression, tight junction reorganization, epithelial-to-mesenchymal transition, and its association with glioblastoma [124] [125] [126] [127] [128] [129] . [43, 127] . rnase-l deletions were subcloned from the pgex4t3 plasmid (kindly provided by r.h. silverman) into the eukaryotic expression plasmid pcmv3b using the bamhi and xhoi restriction sites. interaction with lnx validated our model for a role for rnase-l in the cytoskeleton. although a logical function for this relationship, lnx does not ubiquitylate rnase-l, a known target of modification, and like filamin a (flna), it also does not alter ifnβ induction by rnase-l cleavage products in response to activating conditions ( figure 3a,b) . contrary to the relationship with flna, however, lnx interaction with rnase-l is not abrogated under activating conditions ( figure 3c ). this lack of regulation or modification may indicate that lnx is functioning as a . cell lysates were immunoprecipitated (ip) using anti-myc-tag or igg control antibodies bound to protein a/g agarose beads. bound protein complexes were detected by western blot analysis using the antibodies as indicated; (b) rnase-l and (c) lnx deletions were used in ip studies as in (a) to determine their respective interaction domains [43, 127] . rnase-l deletions were subcloned from the pgex4t3 plasmid (kindly provided by r.h. silverman) into the eukaryotic expression plasmid pcmv3b using the bamhi and xhoi restriction sites. interaction with lnx validated our model for a role for rnase-l in the cytoskeleton. although a logical function for this relationship, lnx does not ubiquitylate rnase-l, a known target of modification, and like filamin a (flna), it also does not alter ifnβ induction by rnase-l cleavage products in response to activating conditions ( figure 3a,b) . contrary to the relationship with flna, however, lnx interaction with rnase-l is not abrogated under activating conditions ( figure 3c ). this lack of regulation or modification may indicate that lnx is functioning as a scaffold when binding to rnase-l or that rnase-l is regulating lnx activity. like rnase-l and iqgap1, lnx regulates multiple tight junction (tj) proteins [126, [130] [131] [132] . claudin-1 is ubiquitylated by lnx, leading to its lysosomal degradation and a reduction in tj strands. it also colocalizes with claudin-2 in late endosomes and lysosomes, suggesting that it may mediate the endocytosis of claudins to lysosomes for degradation [126] . given our previous findings that rnase-l upregulates claudin-1 and occludin following enteropathogenic e. coli (epec) infection, it is plausible that rnase-l may normally bind to lnx and prevent its ubiquitylation and degradation of claudin-1. whether this is sufficient to maintain the barrier function observed in rnase-l-expressing cells compared to deficient cells, or if other factors including flna and iqgap1 also contribute, has yet to be determined. in support of a cooperative complex however, is data indicating that flna and lnx are capable of interacting with each other in a manner that is not altered by rnase-l overexpression ( figure 3d ). given that flna binds to the n-terminus of rnase-l and lnx binds to the c-terminus ( figure 2b) , it is plausible that rnase-l could bind to both proteins simultaneously in a complex at the interface of tjs and the actin cytoskeleton. scaffold when binding to rnase-l or that rnase-l is regulating lnx activity. like rnase-l and iqgap1, lnx regulates multiple tight junction (tj) proteins [126, [130] [131] [132] . claudin-1 is ubiquitylated by lnx, leading to its lysosomal degradation and a reduction in tj strands. it also colocalizes with claudin-2 in late endosomes and lysosomes, suggesting that it may mediate the endocytosis of claudins to lysosomes for degradation [126] . given our previous findings that rnase-l upregulates claudin-1 and occludin following enteropathogenic e. coli (epec) infection, it is plausible that rnase-l may normally bind to lnx and prevent its ubiquitylation and degradation of claudin-1. whether this is sufficient to maintain the barrier function observed in rnase-l-expressing cells compared to deficient cells, or if other factors including flna and iqgap1 also contribute, has yet to be determined. in support of a cooperative complex however, is data indicating that flna and lnx are capable of interacting with each other in a manner that is not altered by rnase-l overexpression ( figure 3d ). given that flna binds to the n-terminus of rnase-l and lnx binds to the c-terminus ( figure 2b ), it is plausible that rnase-l could bind to both proteins simultaneously in a complex at the interface of tjs and the actin cytoskeleton. collectively, the data suggests a model in which flna, lnx, and rnase-l form a complex associated with the actin cytoskeleton. during resting conditions, this complex supports membrane integrity to inhibit endocytosis or viral entry into the cell. if the virus breaches the membrane barrier and activates oas to produce 2-5a, then the rnase-l-flna interaction is abolished and rnase-l is released from the cytoskeleton ( [23] and figure 4) . since the n-terminus of rnase-l is bound and then released by flna and the c-terminus of rnase-l is still capable of binding to lnx, it is likely that 2-5a binding to rnase-l causes a conformational change to lnx-bound rnase-l that induces collectively, the data suggests a model in which flna, lnx, and rnase-l form a complex associated with the actin cytoskeleton. during resting conditions, this complex supports membrane integrity to inhibit endocytosis or viral entry into the cell. if the virus breaches the membrane barrier and activates oas to produce 2-5a, then the rnase-l-flna interaction is abolished and rnase-l is released from the cytoskeleton ( [23] and figure 4) . since the n-terminus of rnase-l is bound and then released by flna and the c-terminus of rnase-l is still capable of binding to lnx, it is likely that 2-5a binding to rnase-l causes a conformational change to lnx-bound rnase-l that induces the dissociation of flna from lnx. this may not only release rnase-l from flna but also alter lnx localization. rnase-l is then free to oligomerize into an active endoribonuclease, cleave viral and cellular rnas into rig-i activators, and induce ifnβ expression to create a broader antiviral state ( figure 5 ). the dissociation of flna from lnx. this may not only release rnase-l from flna but also alter lnx localization. rnase-l is then free to oligomerize into an active endoribonuclease, cleave viral and cellular rnas into rig-i activators, and induce ifnβ expression to create a broader antiviral state ( figure 5 ). this model of rnase-l function parallels an emerging network of innate immune proteins that are directly associated with cytoskeletal components to serve roles both as a barrier to pathogen infection and to initiate signaling and effector functions upon infection. although each protein has been identified and characterized singularly, this growing trend may indicate broader relationships the dissociation of flna from lnx. this may not only release rnase-l from flna but also alter lnx localization. rnase-l is then free to oligomerize into an active endoribonuclease, cleave viral and cellular rnas into rig-i activators, and induce ifnβ expression to create a broader antiviral state ( figure 5 ). this model of rnase-l function parallels an emerging network of innate immune proteins that are directly associated with cytoskeletal components to serve roles both as a barrier to pathogen infection and to initiate signaling and effector functions upon infection. although each protein has been identified and characterized singularly, this growing trend may indicate broader relationships this model of rnase-l function parallels an emerging network of innate immune proteins that are directly associated with cytoskeletal components to serve roles both as a barrier to pathogen infection and to initiate signaling and effector functions upon infection. although each protein has been identified and characterized singularly, this growing trend may indicate broader relationships between cytoskeletal functions and innate immune regulation and activity. consistent with this view, bacteria and viruses both modulate actin polymerization as central components of infection and replication [133, 134] . furthermore, perturbation of the actin cytoskeleton activates cytoskeleton-associated prrs [135] [136] [137] . these observations suggest that the cytoskeleton may serve as a sensor for infection and that pathogen-induced disruption of this structural matrix serves as a signal to activate an innate immune response. here we describe specific innate immune mediators and their proposed roles in this model (summarized in table 1 ). understanding these interactions will likely reveal a novel and coordinated mechanism of cellular host defense. rig-i: perhaps the first prr discovered to be activated by the actin cytoskeleton was rig-i. rig-i was found to localize to the apico-lateral cell junctions and tight junctions of polarized intestinal epithelial cells as well as actin-rich membrane ruffles in non-polarized cells. physiologically, rig-i expression positively correlated with cell migration, which requires both actin polymerization and depolymerization, indicating that rig-i may contribute to cytoskeletal function as well [137] . in fact, lps-activated, rig-i-deficient macrophages have impaired phagocytosis and actin polymerization [138] . when cells were treated with the actin depolymerizing drug cytocholasin-d (cytd), rig-i relocalized to punctate domains and induced both ifnβ and nf-κb signaling [137] . although the use of cytd may mimic the actin depolymerization that can occur during virus infection, cytd activation of rig-i and induction of ifnβ expression in the absence of known viral agonists supports the role of the cytoskeleton as an innate immune sensor. mavs: a second mechanism of actin-induced ifnβ involves the interaction of mavs (mitochondrial antiviral signaling protein) and focal adhesion kinase (fak). mavs is an adaptor protein that binds to rig-i or mda-5 after they interact with their corresponding rna activators. this interaction then leads to the activation of the kinases tank binding kinase-1 (tbk-1) and inhibitor of κb kinase-ε (ikkε), which phosphorylate irf3 and induce ifnβ expression. as its name denotes, fak is a protein tyrosine kinase localized to focal adhesion (fa) contacts with the extracellular matrix (ecm). the fas relay signals from the ecm to the cytoplasm and are therefore extremely sensitive to activation by alterations in the actin cytoskeleton. as a result, virus infection was shown to relocate fak from fas to the mitochondrion where it binds to mavs to induce ifnβ. fak-deficient cells are defective in ifnβ expression and more susceptible to virus infections, indicating that fak may serve as an important link between virus-induced cytoskeletal disruption and irf3 signaling [139] . tbk1: gef-h1 is a rhoa guanine nucleotide exchange factor that binds to the dynein motor complex on microtubules. past work demonstrated that it activates nod1, nod2, and phosphorylates rip2 in response to bacterial infection [147, 148] . recently, it has also been shown to be critical for ifnβ expression in response to nucleic acid pamps. upon activation of rlrs, gef-h1 is dephosphorylated and released from microtubules, freeing it to bind to tbk1 and thus activating irf3 to express ifnβ. this interaction between gef-h1 and tbk1 is dependent on functional microtubules. for undetermined reasons, this regulation is only seen in response to rig-i, mda-5, and sting, but not tlr activation [140] . nod2: nod2 can be activated by bacterial pamps as well as viral ssrna to induce nf-κb and irf3 signaling [108] . legrand-poels et al. [136] , have shown that nod2 binds to rac1, a gtp-binding rho family member that regulates actin polymerization, and recruits it to actin-rich membrane ruffles. there it is sequestered in a latent state until actin disruption, potentially by infection, releases it to begin signaling. treatment of cells with cytd or the actin polymerization inhibitor latrunculin b significantly increased nf-κb signaling and caused nod2 to shift from the insoluble triton-x-100 cell fraction to the soluble fraction. as previously described, oas also binds to nod2, which could potentially localize it to the cytoskeleton as well. this would place it in proximity to cytoskeleton-associated rnase-l for rapid, and perhaps localized, activation. in addition, the presence of nod2 was shown to enhance rnase-l activation in response to dsrna, possibly generating a more robust response to viral infection [109] . asc: inflammasome activation is induced by either pamps or damps which typically stimulate the oligomerization of one of the nlrs, asc (apoptosis-associated speck-like protein containing card; caspase recruitment domain), and caspase-1. this leads to caspase-1 activation, enabling it to cleave il-1β and il-18 into their mature forms [108] . asc ko cells have been shown to be deficient in antigen uptake and presentation, independent of its inflammasome activity. gene expression comparisons between wt and asc ko cells identified a novel role for asc in the stabilization of dock2 mrna. dock2 is a guanine nucleotide exchange factor that mediates rac-dependent actin polymerization; thus, in the absence of asc, dock2 levels are low, resulting in inactive rac and deficient filamentous actin production [142] . this inability to polymerize actin would severely diminish active processes like endocytosis and cell migration. this asc-mediated indirect regulation of actin polymerization may serve to stabilize the cytoskeleton and enhance barrier function. pyrin: pyrin is an inflammasome receptor that interacts with microtubules and actin filaments. utilizing a mouse model in which the actin depolymerizing factor wdr1 was mutated, kim et al. [135] demonstrated that the perturbation of actin resulted in autoinflammation that is dependent on the pyrin inflammasome and activation of il-18. the wdr1 mutation implies that in this system, excessive actin polymerization triggers this inflammasome activation; however, another group found that decreased polymerization resulting from bacterial toxins targeting rho gtpases also activated the pyrin inflammasome [149] . this discrepancy may be a consequence of numerous differences in the two model systems, however both reports demonstrate a link between cytoskeletal and inflammasome components that requires further investigation. pkr: pkr was found to interact with the actin-binding protein gelsolin, an enzyme that catalyzes actin cleavage and nucleation. activation of pkr by dsrna diminishes the interaction with gelsolin, as gelsolin is only capable of binding to pkr monomer and not the active dimeric form. in its latent state, pkr inhibits the ability of gelsolin to sever actin filaments to form lammelopodia. in fact, actin staining of pkr ko and wt mefs indicate that ko cells contain less than one third of the amount of filamentous actin as wt cells. given that knockdown of gelsolin inhibits virus uptake, the suppression of gelsolin activity by pkr represents a novel antiviral mechanism [143] . caspase-11 and caspase-1: the yuan lab has identified two actin-associated caspase-11 interacting proteins, flightless and actin interacting protein 1 (aip1). flightless is an actin-capping protein and a member of the gelsolin superfamily. it localizes caspase-11 to the leading edge of migrating cells and to insoluble cell fractions. it also binds to and inhibits caspase-1 activation and subsequent il-1β maturation. analysis of flightless cleavage products indicates that it may inhibit caspase-1 activity by serving as a pseudosubstrate for its catalytic activity. this inhibition of caspase-1 and relocalization of caspase-11 by flightless could suppress pro-il-1β processing to the secreted form, thus dysregulating the inflammatory response [141, 150, 151] . aip1 activates cofilin-mediated actin depolymerization. binding to caspase-11 enhances this polymerization by a mechanism independent of caspase activity and leads to enhanced migration during inflammation [144] . ifitm1: interferon-induced transmembrane protein 1 (ifitm1) is one of hundreds of ifn-stimulated genes. in hepatocytes, it was found to accumulate in tight junctions in hcv-infected patients undergoing ifn therapy. it bound to the tight junction proteins occludin, claudin-1, and zo-1, and appeared to enhance the interaction between the hcv coreceptors occludin and cd81, yet inhibited virus entry. it is proposed that this disruption in entry results from the altered coordination of coreceptor interactions and complex formation, however this mechanism requires further investigation [146] . adap2: adap2 (adp-ribosylation factor (arf) gtpase-activating protein (gap) with dual pleckstrin homology domains 2) is an ifn-inducible, gtpase-activating protein for arf6. it associates with and can control actin dynamics, as well as induce membrane ruffling and macropinocytosis, leading to the formation of adap2-associated vesicles. these vesicles are also positive for rab8a (recycling endosomes) and lysosomal-associated membrane protein 1 (lamp1; lysosomes). adap2 expression shifts the endocytic association of dengue virus and vesicular stomatitis virus (vsv) into these adap2-positive vesicles instead of rab5 and rab7 endosomes. this not only inhibits their entry into the cell but also likely delivers the viruses directly to degradative lysosomes [145] . the emerging model of pathogen-induced actin alteration as a sensor and regulator of immune activation ( figure 5) involves the contribution of a diverse profile of cytoskeletal and innate immune components (table 1) . interestingly, functional roles for cytoskeletal proteins in the innate immune response appears to occur primarily through an intersection with rlr and inflammasome pathways, whereas few tlr signaling components have been identified. whether this is a fundamental difference between cytosolic receptors and membrane receptors, or if tlr interactors simply have yet to be found, is still to be determined. if these interactions are truly beginning to define a regulatory mechanism for innate immune activity, it is interesting to speculate as to why it may have developed. viruses and bacteria manipulate the cytoskeleton throughout the course of infection. bacteria utilize actin polymerization for many purposes such as forming pedestals to mediate attachment and invasion, generating actin tails for disseminating throughout the cytosol, or to mediate intercellular spread [133] . viruses remodel actin for almost every step of replication including invasion, migration to the nucleus, assembly, exocytosis, and budding [134] . these are all conserved and universal activities that are unlikely to be lost through adaptation, and therefore make pathogen-induced cytoskeletal perturbations good targets for innate immune activation. most of these interactions localize normally cytosolic innate signaling molecules to the cytoskeleton, particularly to the cell membrane or tight junctions. several viruses, such as hcv and coxsackievirus, bind to receptors located at tight junctions, making this site an ideal location for innate immune activators [152, 153] . similarly, rnase-l, pkr, rig-i, and nod2 are all viral rna sensors and proximity to viral entry may provide a kinetic advantage to the cell. in addition, rig-i, rnase-l, and pkr have been found to associate with antiviral stress granules (avsg) that form after virus infection and contain viral rna. formation of these complexes is necessary for ifnβ induction in response to multiple stimuli [154, 155] . avsgs are related to stress granules which form during cellular stress from inhibited translation and are linked to the cytoskeleton. if avsgs are also associated with the cellular structure, this may mean that rig-i, pkr, and rnase-l either require their interaction with the cytoskeleton in order to be incorporated into avsgs or to control them. indeed, rig-i forms punctate domains when cells are treated with cytd to inhibit actin polymerization, however it is unknown whether these are avsgs. an alternative function of this association between members of the innate immune response and the cytoskeleton may be to sequester these proteins to prevent unintended activation. incidental overexpression of ifn would lead to the needless upregulation of hundreds of genes and create an unnecessary heightened antiviral state in surrounding tissue. similarly, rnase-l and pkr are both proapoptotic and require tight regulation to prevent accidental cell death [3] . the question that ultimately arises, however, is how does the cell distinguish between pathogen-induced cytoskeletal remodeling and that due to physiologic cellular processes? inflammasome activation requires a priming step to transcriptionally induce il-1β and il-18 which indicates that an authentic pamp is required to avoid spurious activation. the priming signal is typically initiated through tlr engagement. this upstream role may explain why tlr signaling molecules that are activated by cytoskeletal perturbations have not yet been identified [156] . rlr activation does not need a secondary stimulus and therefore may require an alternative mechanism for differentiating between friendly and foreign actin rearrangement. collectively, this emerging area of study still has many discoveries to be made and questions to answer. recent findings have shown that rnase-l is not only a member of this new class of host defense mechanisms, but may do so on multiple levels through its interactions with filamin a, iqgap, and lnx. despite over 40 years of research, this endoribonuclease still has many lessons to offer and may be a key to helping elucidate the link between cytoskeletal integrity and host defense. socs-1 inhibits expression of the antiviral proteins 2 1 ,5 1 -oas and mxa induced by the novel interferon-λs il-28a and il-29 identification of genes differentially regulated by interferon α,β, or γ using oligonucleotide arrays on the discovery of interferon-inducible, double-stranded rna activated enzymes: the 2 1 -5 1 oligoadenylate synthetases and the protein kinase pkr. cytokine growth factor rev structural basis for recognition of 2 1 ,5 1 -linked oligoadenylates by human ribonuclease l interferon action may be mediated by activation of a nuclease by pppa2 1 p5 1 a2 1 p5 1 a inhibition of cell-free protein synthesis by pppa2 1 p5 1 a2 1 p5 1 a: a novel oligonucleotide synthesized by interferon-treated l cell extracts mechanism of interferon action. properties of an interferon-mediated ribonucleolytic activity from mouse l929 cells synthesis of low molecular weight inhibitor of protein synthesis with enzyme from interferon-treated cells interferon, double-stranded rna and rna degradation. fractionation of the endonucleaseint system into two macromolecular components; role of a small molecule in nuclease activation expression cloning of 2-5a-dependent rnaase: a uniquely regulated mediator of interferon action germline mutations in the ribonuclease l gene in families showing linkage with hpc1 the role of 2 1 -5 1 oligoadenylate-activated ribonuclease l in apoptosis a study of the interferon antiviral mechanism: apoptosis activation by the 2-5a system a dominant negative mutant of 2-5a-dependent rnase suppresses antiproliferative and antiviral effects of interferon interferon action and apoptosis are defective in mice devoid of 2 1 ,5 1 -oligoadenylate-dependent rnase l ribonuclease l proteolysis in peripheral blood mononuclear cells of chronic fatigue syndrome patients characterization of a 2 1 ,5 1 -oligoadenylate (2-5a)-dependent 37-kda rnase l: azido photoaffinity labeling and 2-5a-dependent activation biochemical evidence for a novel low molecular weight 2-5a-dependent rnase l in chronic fatigue syndrome role of 2-5a-dependent rnase-l in senescence and longevity defects in tlr3 expression and rnase l activation lead to decreased mnsod expression and insulin resistance in muscle cells of obese people rnase l controls terminal adipocyte differentiation, lipids storage and insulin sensitivity via chop10 mrna regulation rnase l mediated protection from virus induced demyelination rnase-l deficiency exacerbates experimental colitis and colitis-associated cancer rnase l contributes to experimentally induced type 1 diabetes onset in mice rnase-l control of cellular mrnas: roles in biologic functions and mechanisms of substrate targeting an essential role for the antiviral endoribonuclease, rnase-l, in antibacterial immunity small self-rna generated by rnase l amplifies antiviral innate immunity rnase l interacts with filamin a to regulate actin dynamics and barrier function for viral entry rnase l induces autophagy via c-jun n-terminal kinase and double-stranded rna-dependent protein kinase signaling pathways rnase l cleavage products promote switch from autophagy to apoptosis by caspase-mediated cleavage of beclin-1 skin allograft rejection is suppressed in mice lacking the antiviral enzyme, 2 1 ,5 1 -oligoadenylate-dependent rnase l lack of rnase l attenuates macrophage functions interferons at age 50: past, current and future impact on biomedicine interferon-inducible antiviral effectors the human 2 1 ,5 1 -oligoadenylate synthetase family: interferon-induced proteins with unique enzymatic properties the human 2 1 -5 1 oligoadenylate synthetase family: unique bond formation antiviral activity of human oasl protein is mediated by enhancing signaling of the rig-i rna sensor activation of 2 1 -5 1 oligoadenylate synthetase by single-stranded and double-stranded rna aptamers selection and cloning of poly(rc)-binding protein 2 and raf kinase inhibitor protein rna activators of 2 1 ,5 1 -oligoadenylate synthetase from prostate cancer cells heterogeneous nuclear rna from hairy cell leukemia patients activates 2 1 ,5 1 -oligoadenylate synthetase ribosomal protein mrnas are primary targets of regulation in rnase-l-induced senescence germline mutation in rnase l predicts increased risk of head and neck, uterine cervix and breast cancer a bipartite model of 2-5a-dependent rnase l radiobinding and hplc analysis of 2-5a and related oligonucleotides from intact cells identification of 2 1 -phosphodiesterase, which plays a role in the 2-5a system regulated by interferon synthesis, characterization and properties of ppp(a2 1 p)napcp and related high-specific-activity 32p-labelled derivatives of ppp(a2 1 p)na the role of phosphodiesterase 12 (pde12) as a negative regulator of the innate immune response and the discovery of antiviral inhibitors structure of human rnase l reveals the basis for regulated rna decay in the ifn response innate immune messenger 2-5a tethers human rnase l into active high-order complexes dimeric structure of pseudokinase rnase l bound to 2-5a reveals a basis for interferon-induced antiviral activity interferon action: rna cleavage pattern of a (2 1 -5 1 )oligoadenylate-dependent endonuclease interferon action-sequence specificity of the ppp(a2 1 p)na-dependent ribonuclease rnase l mediates the antiviral effect of interferon through a selective reduction in viral rna during encephalomyocarditis virus infection rnase l attenuates mitogen-stimulated gene expression via transcriptional and post-transcriptional mechanisms to limit the proliferative response a transcriptional signaling pathway in the ifn system mediated by 2 1 -5 1 -oligoadenylate activation of rnase l mapping of the human rnase l promoter and expression in cancer and normal cells overexpression of the transcription factor sp1 activates the oas-rnase l-rig-i pathway regulation of human rnase-l by the mir-29 family reveals a novel oncogenic role in chronic myelogenous leukemia post-transcriptional regulation of rnase-l expression is mediated by the 3 1 -untranslated region of its mrna posttranscriptional regulation of cancer traits by hur proteasome-mediated degradation of rnase l in response to phorbol-12-myristate-13-acetate (pma) treatment of mouse l929 cells decreased proteasomal activity causes age-related phenotypes and promotes the development of metabolic abnormalities proteomics-based identification of novel factor inhibiting hypoxia-inducible factor (fih) substrates indicates widespread asparaginyl hydroxylation of ankyrin repeat domain-containing proteins cloning and characterization of a rnase l inhibitor. a new component of the interferon-regulated 2-5a pathway the essential atp-binding cassette protein rli1 functions in translation by promoting preinitiation complex assembly the iron-sulphur protein rnase linhibitor functions in translation termination biogenesis of cytosolic ribosomes requires the essential iron-sulphur protein rli1p and mitochondria the 2-5a/rnase l/rnase l inhibitor (rli) [correction of (rni)] pathway regulates mitochondrial mrnas stability in interferon α-treated h9 cells hpc1/rnase l mediates apoptosis of prostate cancer cells treated with 2 1 ,5 1 -oligoadenylates, topoisomerase i inhibitors, and tumor necrosis factor-related apoptosis-inducing ligand rnase l inhibitor is induced during human immunodeficiency virus type 1 infection and down regulates the 2-5a/rnase l pathway in human t cells rnase l inhibitor (rli) antisense constructions block partially the down regulation of the 2-5a/rnase l pathway in encephalomyocarditisvirus-(emcv)-infected cells a newly discovered function for rnase l in regulating translation termination 2 1 ,5 1 -oligoadenylate-dependent rnase l is a dimer of regulatory and catalytic subunits eukaryotic release factors (erfs) history rnase lregulates the stability of mitochondrial dna-encoded mrnas in mouse embryo fibroblasts regulation of mitochondrial mrna stability by rnase l is translation-dependent and controls ifnα-induced apoptosis ultrastructural localization of interferon-inducible double-stranded rna-activated enzymes in human cells human 2 1 -phosphodiesterase localizes to the mitochondrial matrix with a putative function in mitochondrial rna turnover the mrna-destabilizing protein tristetraprolin is suppressed in many cancers, altering tumorigenic phenotypes and patient prognosis ttp): interactions with mrna and proteins, and current thoughts on mechanisms of action zinc-finger antiviral protein mediates retinoic acid inducible gene i-like receptor-independent antiviral response to murine leukemia virus mrna degradation by the endoribonuclease regnase-1/zc3h12a/mcpip-1 rnase l activates the nlrp3 inflammasome during viral infections enteropathogenic escherichia coli inhibits type i interferon-and rnase l-mediated host defense to disrupt intestinal epithelial cell barrier function viral encounters with 2 1 ,5 1 -oligoadenylate synthetase and rnase l during the interferon antiviral response interferon action: binding of viral rna to the 40-kilodalton 2 1 -5 1 -oligoadenylate synthetase in interferon-treated hela cells infected with encephalomyocarditis virus natural occurrence of 2-5a in interferon-treated emc virus-infected l cells a phylogenetically conserved rna structure in the poliovirus open reading frame inhibits the antiviral endoribonuclease rnase l evasion of antiviral innate immunity by theiler's virus l* protein through direct inhibition of rnase l a viral rna competitively inhibits the antiviral endoribonuclease domain of rnase l a putative loop e motif and an h-h kissing loop interaction are conserved and functional features in a group c enterovirus rna that inhibits ribonuclease l activation and evasion of the antiviral 2 1 -5 1 oligoadenylate synthetase/ribonuclease l pathway by hepatitis c virus mrna hepatitis c virus rna: dinucleotide frequencies and cleavage by rnase l clinical relevance of the 2 1 -5 1 -oligoadenylate synthetase/rnase l system for treatment response in chronic hepatitis c rnase l targets distinct sites in influenza a virus rnas the primary function of rna binding by the influenza a virus ns1 protein in infected cells: inhibiting the 2 1 -5 1 oligo (a) synthetase/rnase l pathway mechanisms of ifn resistance by influenza virus diagnostic value of the determination of an interferon-induced enzyme activity: decreased 2 1 ,5 1 -oligoadenylate dependent binding protein activity in aids patient lymphocytes antagonism of the interferon-induced oas-rnase l pathway by murine coronavirus ns2 protein is required for virus replication and liver pathology inhibition of the oas/rnase l pathway by viruses caspase-dependent apoptosis by 2 1 ,5 1 -oligoadenylate activation of rnase l is enhanced by ifn-β viruses and the autophagy pathway rnase l triggers autophagy in response to viral infections the crosstalk between autophagy and apoptosis: where does this lead regulation of antiviral t cell responses by type i interferons transcription factor stat3 and type i interferons are corepressive insulators for differentiation of follicular helper and t helper 1 cells type i ifn enhances follicular b cell contribution to the t cell-independent antibody response nod-like receptors: guardians of intestinal mucosal barriers nucleotide oligomerization domain-2 interacts with 2 1 -5 1 -oligoadenylate synthetase type 2 and enhances rnase-l function in thp-1 cells 2 1 ,5 1 -oligoadenylate-dependent rnase located in nuclei: biochemical characterization and subcellular distribution of the nuclease in human and murine cells expression of mrna and protein-protein interaction of the antiviral endoribonuclease rnase l in mouse spleen localization of a molecular form of interferon-regulated rnase l in the cytoskeleton arf1 and arf6 promote ventral actin structures formed by acute activation of protein kinase c and src activation of pkcβii by pma facilitates enhanced epithelial wound repair through increased cell spreading and migration rodriguez-boulan, e. iqgap1 controls tight junction formation through differential regulation of claudin recruitment iqgap1: insights into the function of a molecular puppeteer pathologic effects of rnase-l dysregulation in immunity and proliferative control iqgap1 in microbial pathogenesis: targeting the actin cytoskeleton role of rnase l in apoptosis induced by 1-(3-c-ethynyl-β-d-ribopentofuranosyl)cytosine association of rnase l with a ras gtpase-activating-like protein iqgap1 in mediating the apoptosis of a human cancer cell-line the mammalian numb phosphotyrosine-binding domain. characterization of binding specificity and identification of a novel pdz domain-containing numb binding protein, lnx lnx functions as a ring type e3 ubiquitin ligase that targets the cell fate determinant numb for ubiquitin-dependent degradation proteomics strategy to identify substrates of lnx, a pdz domain-containing e3 ubiquitin ligase characterization of human lnx, a novel ligand of numb protein x that is downregulated in human gliomas ectopic expression of ligand-of-numb protein x promoted tgf-β induced epithelial to mesenchymal transition of proximal tubular epithelial cells the e3 ubiquitin ligase lnx1p80 promotes the removal of claudins from tight junctions in mdck cells moelling, k. c-src is a pdz interaction partner and substrate of the e3 ubiquitin ligase ligand-of-numb protein x1 a global genomic view on lnx sirna-mediated cell cycle arrest lnx (ligand of numb-protein x) interacts with rhoc, both of which regulate ap-1-mediated transcriptional activation ligand-of-numb protein x is an endocytic scaffold for junctional adhesion molecule 4 the cell surface protein coxsackie-and adenovirus receptor (car) directly associates with the ligand-of the coxsackievirus and adenovirus receptor (car) forms a complex with the pdz domain-containing protein ligand-of-numb protein-x (lnx) pathogens and polymers: microbe-host interactions illuminate the cytoskeleton viral exploitation of actin: force-generation and scaffolding functions in viral infection aberrant actin depolymerization triggers the pyrin inflammasome and autoinflammatory disease that is dependent on il-18, not il-1β modulation of nod2-dependent nf-κb signaling by the actin cytoskeleton retinoic acid-induced gene-1 (rig-i) associates with the actin cytoskeleton via caspase activation and recruitment domain-dependent interactions an essential role for rig-i in toll-like receptor-stimulated phagocytosis focal adhesion kinase is a component of antiviral rig-i-like receptor signaling gef-h1 controls microtubule-dependent sensing of nucleic acids for antiviral host defenses flightless-i regulates proinflammatory caspases by selectively modulating intracellular localization and caspase activity the inflammasome adaptor asc regulates the function of adaptive immune cells by controlling dock2-mediated rac activation and actin polymerization regulation of actin dynamics by protein kinase r control of gelsolin enforces basal innate immune defense caspase-11 regulates cell migration by promoting aip1-cofilin-mediated actin depolymerization adap2 is an interferon stimulated gene that restricts rna virus entry ifitm1 is a tight junction protein that inhibits hepatitis c virus entry gef-h1 mediated control of nod1 dependent nf-κb activation by shigella effectors control of nod2 and rip2-dependent innate immune activation by gef-h1 innate immune sensing of bacterial modifications of rho gtpases by the pyrin inflammasome caspase-11 cleaves gasdermin d for non-canonical inflammasome signalling non-canonical inflammasome activation targets caspase-11 picornavirus entry how hepatitis c virus invades hepatocytes: the mystery of viral entry critical role of an antiviral stress granule containing rig-i and pkr in viral detection and innate immunity dhx36 enhances rig-i signaling by facilitating pkr-mediated antiviral stress granule formation new insights into the regulation of signalling by toll-like receptors and nod-like receptors acknowledgments: this work was supported by the veterans affairs visn5 pilot award (heather j ezelle), national institutes of health grant ai089518 (krishnamurthy malathi) and startup funds from university of toledo (krishnamurthy malathi, toledo, oh, usa). we thank robert silverman and gerald radziwill for generously providing rnase-l and lnx deletion constructs, respectively. the authors declare no conflict of interest. key: cord-319729-6lzjhn8j authors: tian, bin; zhou, ming; yang, yu; yu, lan; luo, zhaochen; tian, dayong; wang, ke; cui, min; chen, huanchun; fu, zhen f.; zhao, ling title: lab-attenuated rabies virus causes abortive infection and induces cytokine expression in astrocytes by activating mitochondrial antiviral-signaling protein signaling pathway date: 2018-01-19 journal: front immunol doi: 10.3389/fimmu.2017.02011 sha: doc_id: 319729 cord_uid: 6lzjhn8j rabies is an ancient disease but remains endemic in most parts of the world and causes approximately 59,000 deaths annually. the mechanism through which the causative agent, rabies virus (rabv), evades the host immune response and infects the host central nervous system (cns) has not been completely elucidated thus far. our previous studies have shown that lab-attenuated, but not wild-type (wt), rabv activates the innate immune response in the mouse and dog models. in this present study, we demonstrate that lab-attenuated rabv causes abortive infection in astrocytes, the most abundant glial cells in the cns. furthermore, we found that lab-attenuated rabv produces more double-stranded rna (dsrna) than wt rabv, which is recognized by retinoic acid-inducible gene i (rig-i) or melanoma differentiation-associated protein 5 (mda5). activation of mitochondrial antiviral-signaling protein (mavs), the common adaptor molecule for rig-i and mda5, results in the production of type i interferon (ifn) and the expression of hundreds of ifn-stimulated genes, which suppress rabv replication and spread in astrocytes. notably, lab-attenuated rabv replicates in a manner identical to that of wt rabv in mavs−/− astrocytes. it was also found that lab-attenuated, but not wt, rabv induces the expression of inflammatory cytokines via the mavsp38/nf-κb signaling pathway. these inflammatory cytokines increase the blood–brain barrier permeability and thus enable immune cells and antibodies infiltrate the cns parenchyma, resulting in rabv control and elimination. in contrast, wt rabv restricts dsrna production and thus evades innate recognition by rig-i/mda5 in astrocytes, which could be one of the mechanisms by which wt rabv evades the host immune response in resident cns cells. our findings suggest that astrocytes play a critical role in limiting the replication of lab-attenuated rabv in the cns. rabies is an ancient disease but remains endemic in most parts of the world and causes approximately 59,000 deaths annually. the mechanism through which the causative agent, rabies virus (rabv), evades the host immune response and infects the host central nervous system (cns) has not been completely elucidated thus far. our previous studies have shown that lab-attenuated, but not wild-type (wt), rabv activates the innate immune response in the mouse and dog models. in this present study, we demonstrate that lab-attenuated rabv causes abortive infection in astrocytes, the most abundant glial cells in the cns. furthermore, we found that lab-attenuated rabv produces more double-stranded rna (dsrna) than wt rabv, which is recognized by retinoic acidinducible gene i (rig-i) or melanoma differentiation-associated protein 5 (mda5). activation of mitochondrial antiviral-signaling protein (mavs), the common adaptor molecule for rig-i and mda5, results in the production of type i interferon (ifn) and the expression of hundreds of ifn-stimulated genes, which suppress rabv replication and spread in astrocytes. notably, lab-attenuated rabv replicates in a manner identical to that of wt rabv in mavs−/− astrocytes. it was also found that lab-attenuated, but not wt, rabv induces the expression of inflammatory cytokines via the mavs-p38/nf-κb signaling pathway. these inflammatory cytokines increase the blood-brain barrier permeability and thus enable immune cells and antibodies infiltrate the cns parenchyma, resulting in rabv control and elimination. in contrast, wt rabv restricts dsrna production and thus evades innate recognition by rig-i/mda5 in astrocytes, which could be one of the mechanisms by which wt rabv evades the host immune response in resident cns cells. our findings suggest that astrocytes play a critical role in limiting the replication of lab-attenuated rabv in the cns. introduction rabies is an acute encephalomyelitis. the hallmark of rabies is that the disease is almost always fatal once clinical signs develop (1, 2) . the causative agent, rabies virus (rabv), is a negative-strand rna virus belonging to the genus lyssavirus in the family rhabdoviridae (3) . rabv enters neurons from the neuromuscular junction closest to the site of infection. after a short incubation, rabv travels to the central nervous system (cns) through sensory or motor neurons. only slight tissue damage and neuroinflammation can be observed in the brains of rabid patients (4) . in contrast, lab-attenuated rabv induces extensive inflammation and apoptosis, as well as increases in the expression levels of innate immunity-related genes in the cns of infected mice (5) (6) (7) (8) (9) (10) . these findings suggest that wt, but not lab-attenuated, rabv evades the host immune responses. the innate immune system is the first line of defense against viral invasion. viruses are usually confronted by various pattern recognition receptors (prrs), including toll-like receptors (tlrs) and retinoic acid-inducible gene i (rig-i) like helicases (rlrs) (11) . tlr family members, such as tlr3 and tlr7, are generally involved in recognizing negative-strand rna viruses (12) . tlr3 binds double-stranded rna (dsrna), whereas tlr7 recognizes single-strand rna. the main sources of dsrna in infections with single-strand rna viruses are the replicative intermediates generated by viral rna-dependent rna polymerase (13) . tlr3 and tlr7 initiate signaling though the adaptor molecules trif and myd88, respectively. both signaling cascades triggered by these proteins lead to irf3 phosphorylation in the c terminal region at serine 386, which is critical for irf3 activation by two iκb kinases (tbk-1 and ikkε) (14, 15) . activated irf3 homo-or hetero-dimerizes with irf7 and then translocates into the nucleus, to interacts with the creb binding protein cbp/p300 and stimulates the transcription of interferon (ifn)-β, as well as some ifn-stimulated genes (isgs) (15, 16) . alternatively, rna viruses can be recognized by two rlrs, rig-i, and melanoma differentiation-associated protein 5 (mda5), located in the cytoplasm (17, 18) . rig-i solely senses short and blunt dsrna of negative-strand rna viruses containing 5′ triphosphate rna in the panhandle region of their single-stranded genome (17) . unlike rig-i, mda5 preferentially binds blunt-ended dsrna, with or without 5′ triphosphate (18) . rig-i and mda5 signaling is mediated through mitochondrial antiviral-signaling protein (mavs), which is also known as ips1, visa or cardif (19) . similar to tlr signaling, rlr signaling results in irf3 activation and nuclear translocation (20) . several groups have attempted to identify the prrs that recognize rabv. prehaud et al. found that tlr3 mrna expression is upregulated following rabv infection in postmitotic human neurons (21) . furthermore, enhanced tlr3 expression has also been observed in the cerebellar cortical tissues of rabies patients (22) . the observation that tlr3 is upregulated following infection suggests that tlr3 plays a role in the innate recognition of rabv. faul et al. found that both rig-i and mda5 were responsible for inducing dendritic cell (dc) activation and type i ifn production upon rabv infection (23) . mavs, the adaptor protein for both rig-i and mda5, is essential for inducing innate immune responses in dcs. in another study, li et al. found that mice lacking tlr7 exhibited a phenotype associated with intermediate mortality rates between those of myd88−/− and control mice, indicating that tlr7 may play an important role in controlling rabv infections. however, the role of tlr7 in rabv infection is not entirely understood (24) . astrocytes are the most abundant glial cells in the cns and constitute the blood-brain barrier (bbb) along with endothelial cells and pericytes (25) . astrocytes are generally involved in regulating the cns microenvironment and also play roles in neuronal metabolic support, synaptic transmission, and neurotropism. moreover, astrocytes participate in developing and maintaining the bbb and guiding neuronal migration during development. the roles of astrocytes in innate immunity and inflammation have been reported recently (26) . astrocytes are the reservoir for many neuroinvasive viruses, such as human immunodeficiency virus, theiler's murine encephalomyelitis virus (tmev), john cunningham virus, and herpes simplex virus (hsv) (27) (28) (29) (30) . astrocytes have also been shown to play multiple roles in viral infections. specifically, they can increase bbb permeability (31) by producing cytokines and degrading tight junction proteins (32) . moreover, astrocytes have been shown to induce the innate immune response to produce ifn (33) . an early report showed that both wt and lab-attenuated rabv could successfully infect primary astrocytes in the early stages of rabv infection (34) . a recent study demonstrated that a rabv vaccine strain sad-l16 caused an abortive infection in astrocytes, but the detailed mechanism was not revealed (35) . in this study, it was found that lab-attenuated rabv produces higher level of dsrna than wt rabv, which is recognized by rig-i/mda5 and results in the activation of mavs signaling pathway. following mavs activation, the production of isg and inflammatory cytokines helps to clear the lab-attenuated rabv from the cns. in contrast, wt rabv can maintain a persistent infection in astrocytes by evading the innate recognition. mouse bend.3 cells and vero cells were obtained from the american type culture collection (atcc; manassas, va, usa) and were maintained in dulbecco's modified eagle medium (dmem) supplemented with fetal bovine serum (fbs; gibco, carlsbad, ca, usa). mouse neuroblastoma (na) cells were maintained in rpmi-1640 medium (thermo-fisher, usa) supplemented with 10% fbs. the drv-ah08 (drv) was isolated from a rabid dog in anhui province, china (36, 37) . cvs-b2c (b2c), originated from cvs-24 virus by passage in bhk-21 cells (6), has been used as a lab-attenuated rabv (38) (39) (40) . both drv and cvs-b2c were propagated in suckling icr mouse brains. all the viruses were manipulated under the standard biosecurity procedures made by the ministry of agriculture of china. a rabbit anti-ubiquitin protein c (ubc) polyconal antibody was purchased from abclonal technology (woburn, ma, usa). a rabbit anti-rig-i monoclonal antibody was purchased from enzo life technology (farmingdale, ny, usa), and rabbit polyclonal antibodies against irf7, stat1 and occludin were obtained from santa cruz biotechnology (santa cruz, ca, usa). a rabbit antiphospho-irf7 monoclonal antibody was purchased from cell signaling technology (washington, dc, usa), and rabbit anti-ifit1 and irf3 monoclonal antibodies were purchased from abcam (cambridge, ma, usa). a rabbit polyclonal anti-claudin-5 antibody, a biotinylated goat anti-rabbit or mouse 594 antibody, and an alexa fluor 488-conjugated goat antimouse or rabbit antibody were purchased from invitrogen (grand island, ny, usa). mouse monoclonal anti-zoluna occludens-1 (anti-zo-1) antibodies were obtained from sigma (st. louis, mo, usa), and mouse monoclonal anti-gapdh antibody was purchased from proteintech (wuhan, china). the mouse anti-rabv n and p monoclonal antibodies were prepared in our laboratory, and the fluorescein isothiocyanate (fitc)-conjugated anti-rabv nucleoprotein antibody used herein was obtained from female wt or mavs-knockout (mavs−/−) c57bl/6 mice (6-8 weeks old) were infected intracerebally (i.c.) with 20 µl of drv-ah08 (200 ffu), b2c (20 ffu), or mock infected with the same volume of dmem. at 7 days postinfection (d.p.i.), the mice were euthanized with co2 when moribund, and their brains were collected for immunohistochemistry analysis. blood-brain barrier permeability was determined by measuring sodium fluorescein (naf) uptake as described previously with minor modifications (41) . briefly, 100 µl of naf (100 mg/ml) was injected intraperitoneally (i.p.) into each mouse. after anesthetization, peripheral blood and brains of each mouse were collected. the fluorescence in serum and brain homogenate samples was analyzed by a spectrophotometer (biotek instruments, vt, usa) with excitation at 485 nm and emission at 530 nm. standards (125-4,000 g/ml) were prepared to calculate the naf content. naf uptake into tissue is calculated as (μg of fluorescence spinal cord/mg of tissue)/(μg of fluorescence sera/ml of blood) to normalize values for blood levels of the dye at the time of tissue collection. to detect cd45 + cell in the brain, infected mice were anesthetized with ketamine-xylazine (0.1 ml/10 g body weight), perfused with 50 ml pbs, and then the brains were transferred into 4% neutral buffered paraformaldehyde (pfa) for at least 24 h (39) . briefly, the brain sections were antigenic recovered and blocked with donkey serum, incubated with primary antibodies overnight at 4°c, and then secondary antibodies was applied. pbs was treated as a negative control by replacing primary antibodies. sections were photographed and analyzed using an olympus bx41 microscope (tokyo, japan). primary mixed glial cell cultures were established as described previously (42) . briefly, the brain cells of 1-to 3-day-old neonatal c57bl/6 mice were dissociated by repeated pipetting and then passed through a 75-nm nylon mesh (corning, ny, usa). the cells were subsequently washed once in cold pbs and cultured in dmem (with high glucose) supplemented with 10% fbs and 1% penicillin-streptomycin. the medium was changed on days 3, 5, and 7 for the astrocytes and on day 3 only for the microglia. on day 10, the flasks were shaken at 260 rpm for 2 h to remove any non-adherent cells (mainly microglia). the remaining adherent astrocytes were detached with trypsin-edta and then plated again for further experiments. the purity of the astrocyte cultures was greater than 95%. mouse neurons were obtained from embryonic mouse brains as previously described (42) and then dissociated by repeated pipetting (approximately 20 times) before being passed through a 75-nm nylon mesh. the cells were then washed once in cold pbs and cultured in dmem (with high glucose) supplemented with 5% fbs and 1% penicillin-streptomycin for 6 h, after which the medium was replaced with serum-free neural-basal medium (invitrogen, carlsbad, ca, usa) supplemented with 2% b-27 (invitrogen, carlsbad, ca, usa). viral titers were determined by direct fluorescent antibody assay (40) . na cells cultured in 96-well plates were inoculated with viruses diluted serial 10-fold and then incubated at 37°c for 48 h. then the culture supernatant was subsequently removed, and the cells were fixed and stained with fitc-conjugated anti-rabv n antibodies. the antigen-positive foci were counted under a fluorescence microscope (zeiss, germany), and the viral titers were calculated as ffu per milliliter. all titrations were conducted in quadruplicate. confocal microscopy table 1 | primers used for quantification of viral mrna, ifn-stimulated genes, chemokines, and cytokines. retinoic acid-inducible gene i (rig-i) f gcgtctcagtgcagcacatcatt qrt-pcr antibodies against dsrna, rabv n, rabv p, claudin-5, occludin, zo-1, or dapi. infected wt or mavs−/− mice were anesthetized with ketamine-xylazine (0.1 ml/10 g body weight) and then perfused with pbs followed by 10% neutral buffered formalin, as described previously (39) . three independent mouse brain samples were collected from each group and embedded in paraffin for coronal sectioning. the sections were subsequently stained with antibodies against gfap, map2, rabv p, rabv n, or dapi. after being washed, the cells or sections were incubated with an alexa fluor 488-conjugated goat antirabbit or mouse secondary antibodies or an alexa fluor 594-conjugated goat antirabbit or mouse secondary antibodies for 1 h at room temperature. staining was visualized with a nikon a1 confocal laser microscope system equipped with nis-elements imaging software (nikon, melville, ny, usa) and was quantified using fiji, an imagej distribution package manufactured by nih (http://imagej.net/introduction). mean fluorescence intensity (mfi) was quantified using the region of interest, which encompassed the entire cell to include the membrane, and background staining was quantified using three negatively stained regions per cell. these regions were subtracted from the total mfi. for the protein synthesis inhibition tests, primary astrocytes were pretreated with cycloheximide (chx) (invivogen, san diego, ca, usa) for 1 h at a dose of 50 µg/ml, infected with drv or b2c at an moi of 0.1 and then continued to be incubated with chx for another 24 h. for the tbk1 activation blockage assays, bx795 (invivogen, san diego, ca, usa) at a dose of 1 µm was used to treat primary astrocytes. for the inflammatory pathway blockage assays, the primary astrocytes were pretreated with a p38 inhibitor (skepinone-l; sellek, houston, tx, usa), a jnk inhibitor (jnk inhibitor ix; sellek, houston, tx, usa), an nf-κb inhibitor (sc75741; sellek, houston, tx, usa), or dmso as control at a dose of 5 µg/ml for 1 h. then the cells were infected with drv or b2c at moi 1 and incubated with the above inhibitors for 48 h. the concentrations of tnf-α and il-6 in the supernatant of astrocytes were measured by the commercial elisa kits according to the manufacturer's instruction (abclonal technology, woburn, ma, usa). rna was isolated with trizol ® reagent (invitrogen), according to the manufacturer's instructions, and qrt-pcr was performed as described previously (39) . briefly, 800 ng of total rna (from either cells or tissue) was transcribed into cdna in a reaction mixture with a total of volume of 20 µl using a superscript iii reverse transcription kit (toyobo). the reaction mixture comprised 1 μl of cdna combined with 5 µl of iq5 sybr green mix (biorad, hercules, ca, usa), 3 µl of diethyl pyrocarbonatetreated water, and 0.5 µl of primer mix (the concentration of each primer was 10 mm). the cdna was amplified using an iq5 icycler (bio-rad), and the cycle threshold (ct) values were recorded. the ct value was inversely correlated with the mrna concentration, and each ct unit represented a twofold change in the mrna concentration. basal mrna expression levels were expressed as δ ct values and were normalized to β-actin mrna expression levels [ δ ct ct (isg)/ δ ct (β-actin)]. induced mrna expression levels were expressed as fold changes relative to mock-infection levels using the 2 δδct method. all the primer sequences are listed in (table 1) . to quantify cellular rabv n rna levels, we transcribed the total rna using avian myeloblastosis virus reverse transcriptase xl (takara, kusatsu, japan) and a primer specific for the rabv n genomic sequence. a standard curve was generated from serially diluted plasmids carrying a rabv n gene and the copy numbers of n mrna were normalized to 1 mg of total rna. the cells were lysed with ripa buffer containing protease inhibitors (roche), and the protein concentrations were measured using a dc protein assay kit (bio-rad). equal quantities of protein were resolved by 12 or 15% sds-page and then transferred to polyvinylidene difluoride membranes (bio-rad), which were blocked with 5% nonfat milk before being incubated with primary antibodies against rig-i, phosphorylated irf7 (p-irf7), stat1, ifit1, rabv n, claudin-5, occludin, zo-1, or gapdh and then probed with the appropriate secondary antibodies. the blots were then visualized using ecl reagent (ge, pittsburgh, pa, usa) and detected under an intelligent dark box ii (ge, pittsburgh, pa, usa). the 590-gain pmt scan generated the optimal standard curve, and the results of this scan were analyzed using q-analyzer software for qam-cyt-1 (raybiotech). transendothelial permeability assay was performed according to the methods described previously (43), with some modifications. b.end3 cells were grown on 3-μm-pore transwell filter inserts until they reached 100% confluency. the medium was then treated with uv-inactivated cell culture supernatants collected from rabv-infected astrocytes. after the cells had incubated for 48 h, they were treated apically with dextran-fitc at a dose of 0.1 µg/ml for 30 min. the samples were then removed from the lower chamber and subjected to fluorescence measurement, which were performed using a fluorimeter (biotek, winooski, vt, usa; the excitation wavelength was 492 nm, and the emission wavelength was 520 nm). the fluorescence values for the experimental cells were subsequently compared to corresponding values for a control cell monolayer. data are expressed as the mean and standard error of the mean (sem), and the significance of the differences between groups was evaluated by student's t test or one-way analysis of variance followed by tukey's post hoc test. the survival ratios were analyzed by log-rank (mantel-cox) test. the asterisks indicated statistical significance (*, p < 0.05; **, p < 0.01; ***, p < 0.001). graphs were plotted and analyzed using graphpad prism software, version 6.0 (graphpad software, la jolla, ca, usa). results comparison of the pathogenicity of drv-ah08 and cvs-b2c in mice first, the pathogenicity of the wt rabv strain drv-ah08 (drv) and lab-attenuated strain cvs-b2c (b2c) used in this study were compared in a mouse model. c57/bl6 mice were i.c. inoculated with 20 ffu b2c or drv and the development of rabies was observed. as expected, drv-infected mice displayed development of the diseases at 5 d.p.i. and all moribund at 9 d.p.i., earlier than b2c-infected mice which all succumb to rabies at 10 d.p.i. (figure 1a) . we found that b2c replicated faster than drv at the early stage of the infection in the cns. to ensure that the viral load in the brains is similar, c57/bl6 mice were i.c. inoculated with 20 ffu b2c or 200 ffu drv. then the viral load in the mice brain were determined at 3, 5, 6, 7, 8, and 9 d.p.i. the results showed that the genomic rna of drv was lower than that of b2c at 3 and 5 d.p.i., but reached the same level as that of b2c at 6 and 7 d.p.i. (figure 1b) . previous studies have shown that the lab-attenuated rabv infection enhances the bbb permeability and induces inflammation in the brain (8, 38) . thus, the na-fluorescence (na-f) uptake of both strains at 7 d.p.i. was measured, and the data showed that na-f uptake in b2c-infected brain was significantly higher than that of drv or mock-infected brains ( figure 1c) . consistently, there were more cd45 + lymphocytes in b2c-infected brains than drv-infected mouse brains (figures 1d,e) . all these results are consistent with the previous findings related to the cns inflammation and bbb permeability change during comparison of the pathogenicity between wt and lab-attenuated rabv (6, 8, 39) . astrocytes play an important role in the induction of innate immunity in the cns. to investigate the role of astrocytes in the pathogenesis of rabv, we isolated primary astrocytes and neurons from suckling mice and infected them with drv or b2c. the growth kinetics of both viruses in astrocytes was assessed by virus titration and immunofluorescence assay (ifa). the viral loads in the cell culture supernatants infected with b2c quickly reached peak at 1 d.p.i., and then gradually decreased until 15 d.p.i. in contrast, the virus titer of drv was initially relatively low but subsequently increased steadily until the endpoint (figure 2a) . to be noted, the viral titer of drv in the cell supernatant maintained at a relatively low level than that of b2c in astrocyte at 3 and 5 d.p.i., but n mrna transcription levels of drv was significantly higher than that of b2c at 3 and 5 d.p.i. (figure 2c) . the ifa results showed that drv persistently replicated in astrocytes and large immunofluorescence foci could be observed at 7 d.p.i., while no obvious immunofluorescence foci could be found in b2c-infected cells ( figure 2d) . as a control, virus replication kinetics in primary neurons was also compared. it was found that viral titers of b2c were always higher than those of wt rabv at the indicated time points (figure 2b) . the ifa results also showed that both drv and b2c could efficiently replicate in neuron ( figure 2d) . to confirm these observations in vivo, c57bl/6 mice were i.c. infected with 20 ffu b2c or 200 ffu drv. infected mice were euthanized when moribund and the brains were harvested for fluorescence ihc analysis. gfap was a well-known surface marker for astrocytes (44) , and the gfap staining demonstrated drv could effectively infect astrocytes in the brains ( figure 2e) . however, few infected astrocytes could be observed in b2cinfected mouse brains (figure 2e ). in contrast, neuron was intensively infected by both drv and b2c ( figure 2f) . together, these results show that the b2c causes abortive infection in astrocytes both in vitro and vivo. previous studies have demonstrated that rabv can be recognized by rig-i and mda5, which share a common adaptor mavs in dcs (23) . to assess innate immune responses in astrocytes, cells were infected with drv or b2c at an moi of 0.1 and the expression of several proteins involved in the mavs signaling pathway, namely, rig-i, p-irf7, stat1 and ifit1 (isg56), was measured by western blot. the ubiquitin ligase trim25 mediates lysine 63-linked ubiquitination of rig-i's n-terminal card domains is indispensable to induce type i ifn production and antiviral immunity (45) . thus, the immunoprecipitation of rig-i was carried out and then resolved by western blot by using an anti-ubiquitin antibody. as expected, rig-i was much more robustly ubiquitinated in astrocytes infected with b2c than that in astrocytes infected with drv. consistently, the expression levels of rig-i, p-irf7, stat1, and ifit1 in b2c-infected astrocytes were higher than those in drv-infected cells ( figure 3a) . next, we attempted to determine which viral product (rna or protein) activates mavs signaling pathway in astrocytes. primary astrocytes were treated with chx, a protein synthesis inhibitor, to inhibit viral protein synthesis. it was found that b2c and drv n transcription levels were similar between chxtreated and mock-treated astrocytes at 24 h p.i. (figure 3b) . however, the expression levels of the genes involved in the mavs signaling pathway, namely, rig-i, mda5, mavs, irf7, ifn-β, stat1, and ifit1, were significantly upregulated by b2c compared with drv in both chx-treated and mock-treated astrocytes, indicating that viral rna rather than proteins activates ifn pathway depending on mavs (figures 3c-i) . taken together, these data demonstrate that the lab-attenuated rabv, but not wt rabv, activates the mavs signaling pathway by viral rna, resulting in the production of ifn as well as isgs in astrocytes. retinoic acid-inducible gene i and mda5 activation is induced mostly by dsrna, which is produced during viral replication (13) . to compare the amount of dsrna produced by wt and labattenuated rabv, primary astrocytes were infected with drv or b2c at an moi of 0.1. at 1 and 3 d.p.i., dsrna was stained with specific antibody and observed by a confocal fluorescence microscope. the results demonstrate that more dsrna is synthesized in b2c-infected astrocytes than those in drv-infected cells ( figure 4a) . the dsrna intensity per cell in b2c-infected cell was significantly higher than that in drv-infected cells (figure 4b) , considering the similar rabv intensity per cell (figure 4c) . the ratio of dsrna intensity to rabv intensity in b2c-infected astrocyte was significantly higher than that in drv-infected cells ( figure 4d) . these results suggest that lab-attenuated rabv produces more viral dsrna than wt rabv during viral replication, (20 ffu) . (e) at 7 d.p.i., mice were euthanized, perfused with pbs and then fixed with 4% pfa. the brains were subsequently coated with paraffin, and the brain sections were stained with antibodies against gfap (red), rabv p protein (green), or dapi (blue). the white arrows indicate rabv-infected astrocytes. the scale bars represent 50 µm. (f) the same brain sections as (e) were stained with antibodies against map2 (green), rabv p protein (red), or dapi (blue), then visualized under a confocal microscope. the scale bars represent 50 µm. notably, b2c titers were significantly increased in mavs−/− astrocytes compared with wt astrocytes (figures 5a,b) . moreover, the cell numbers of immunofluorescence plaques in mavs−/− astrocytes caused by b2c infection were significantly more than those in wt astrocytes (figures 5e,f) . in contrast, mavs deficiency had no significant influence on drv replication and spread in astrocytes (figures 5a,e) . tbk1 is an iκb kinase downstream of the mavs signaling pathway and is critical for irf3 phosphorylation. treatment with the tbk1 specific inhibitor bx795 significantly increased b2c titers in astrocytes (figures 5c,d) . to verify these observations in vivo, mavs−/− mice were i.c. infected with drv or b2c, and virus infection of astrocytes was determined by ifa. we found that both drv and b2c could efficiently infected mavs−/− astrocytes (figures 5g,h) . taken together, these findings suggest that mavs signaling significantly restricts the replication and spread of lab-attenuated but not wt rabv in astrocytes. recent studies have shown that tlr7 may be another innate immune molecule that recognizes rabv (24) . thus, the role of tlr7 in rabv replication was investigated in astrocytes. astrocytes from tlr7−/− and control mice were isolated and then infected with drv or b2c at an moi of 0.01. at different time points p.i., viral titers (figures 5a,b ) and the formation of viral immunofluorescence foci was analyzed (figures 5e,f) . the results demonstrated that tlr7 deficiency did not significantly affect the replication and spread of either drv or b2c in astrocytes, suggesting that tlr7 does not play a role in containing rabv replication and spread in astrocytes. astrocytes are one of the major sources of inflammatory cytokines in the cns post viral infection, and these cytokines play an important role in regulating bbb permeability. to investigate rabv-induced cytokine production, primary astrocytes from wt and mavs−/− mice were prepared and infected with drv or b2c at an moi of 0.01. at indicated time points, the cell culture supernatants were harvested and analyzed with a cytokine array kit. the results showed that b2c induced significantly higher levels of cytokine expression, namely, tnf-α, il-6, il-1β, ifn-γ, il-17, and vegf expression, than drv in wt and mavs−/− astrocytes. the levels of cytokine expression in mavs−/− astrocytes were significantly lower than those in wt astrocytes, indicating that rabv-induced inflammatory cytokine production in astrocytes is dependent on the mavs signaling pathway (figures 6a-f) . the specific pathway through which rabv induces cytokine production was subsequently identified in astrocytes. a previous study demonstrated that rabv induces cytokine production in macrophages mainly through p38, jnk, and nf-κb pathways (46, 47) . thus, primary astrocytes were treated with p38, jnk, and nf-κb pathway inhibitors skepinone-l, jnk ix, and sc75741, respectively, and the concentrations of tnf-α and il-6 in the cell supernatant were measured by elisa. the results showed that skepinone-l and sc75741 caused greater reductions in tnf-α ( figure 6g ) and il-6 ( figure 6h ) protein levels than jnk ix in b2c-infected astrocytes. none of these inhibitors significantly altered the expression levels of tnf-α and il-6 in drv-infected astrocytes. these findings suggest that lab-attenuated rabv induces cytokine expression in astrocytes mainly through the p38 and nf-κb pathways and that wt rabv suppresses cytokine production in astrocytes. our previous studies demonstrated that the chemokines/ cytokines induced by rabv infection are responsible for reducing tj protein expression and enhancing bbb permeability (39) . to investigate the effect of these cytokines on bbb permeability, a mouse brain microvascular endothelial cell line b.end3, was cocultured with uv-inactivated supernatants infected with drv or b2c and collected them at different time points after infection. treatment with the supernatants from b2c-infected astrocytes for 48 h (figure 7a ) induced significant increase in dextran-fitc infiltration from 48 to 96 h p.i. notably, treatment with the supernatants from b2c-infected mavs−/− astrocytes elicited significant increases in dextran-fitc permeability at 48 h p.i. (figure 7b) . no significant increases in dextran-fitc permeability were observed after treatment with the supernatants from wt or mavs−/− astrocytes infected with drv. to gain an insight into the mechanisms by which labattenuated rabv enhances bbb permeability, the expression levels of the indicated tj proteins (claudin-5, occludin, and zo-1) were assessed in astrocytes by western blot. it was found that the expression levels of claudin-5 and occludin were unchanged, while zo-1 was significantly reduced after the cells were treated with the supernatants from b2c-infected wt astrocyte ( figure 7c) . however, this reduction was attenuated in cells treated with the supernatants from mavs−/− astrocytes infected with b2c. no significant changes in zo-1 expression were observed in b.end3 cells treated with supernatants from wt or mavs−/− astrocyte infected with drv ( figure 7d) . similarly, ifa results showed that zo-1 expression was significantly decreased in b.end3 cells cocultured with the supernatants from b2c-infected astrocytes collected at 48 h p.i. (figure 7e) . zo-1 fluorescence intensity was only slightly decreased in b.end3 cells cocultured with the supernatants from b2c-infected mavs−/− astrocytes collected at 48 h p.i., indicating that cell-to-cell contact was not significantly decreased in these cells. moreover, no zo-1 degradation was observed in b.end3 cells cocultured with the supernatants from wt or mavs−/− astrocytes infected with drv. to be noted, no significant changes in claudin-5 and occludin expression were detected in either wt or mavs−/− astrocytes upon rabv infection (figures 7c,e) . together, these results illustrate that lab-attenuated, but not wt, rabv upregulates the production of inflammatory cytokines in astrocytes, resulting in zo-1 degradation in bmecs and subsequent increases in bbb permeability. furthermore, these results indicate that cytokine production is dependent on mavs signaling pathway. astrocytes play critical roles in host defense during viral infections of the cns (7) . prr activation in astrocytes results in the expression of many immune mediators, including type i ifns and inflammatory cytokines (6, 7) . during infections caused by pathogens for which glia are not susceptible targets, activation of the innate immune system caused by pathogen recognition in astrocytes may promote antiviral immune responses in susceptible neurons, as well as cns leukocyte trafficking (3, 5, 7) . in an early report, primary murine, feline, and human astrocytes were infected with wt (srv) and lab-attenuated rabv (era) (34), after which viral loads and replication were assessed by infectivity assay and immunofluorescence. the results showed that astrocytes can be infected by rabv, suggesting that astrocytes may play a role in viral spread and persistence and/or neuronal dysfunction (34) . however, in that study, viral loads and replication were evaluated only at the early time point after infection (3 d.p.i.) . in this present study, the growth of drv with that of b2c, which were used as a pair of wt and lab-attenuated rabv in previous studies (38) (39) (40) , was compared in a long-term experiment. surprisingly, we found that lab-attenuated rabv, but not wt rabv, caused abortive replication in astrocytes, a feature that may be associated with the ability of the virus to evade the innate immune response. productive infection of the astrocyte is critical for neurotropic pathogens to induce encephalitis. astrocytes sensed viral entry into the cns and mounted a type i ifn response, which rapidly restricts the virus after neuronal transport into the cns. previous studies demonstrated that tlr3−/− astrocytes were more permissive to hsv infection and caused severe symptom of encephalitis and tissue damage, which was due to impaired type i ifn production in the absence of tlr3 (30) . a recently work found that abortively infected astrocytes are the major producers of ifn-β after infection of the brain with diverse neurotropic viruses, including tmev, rabv, and vesicular stomatitis virus (vsv) (35) . consistent with these studies, we also found that the abortive infection of lab-attenuated rabv in astrocytes was related to its ability to activate ifn signaling pathway. basal isg expression levels are an important determinant of susceptibility to viral infection. we have found that astrocytes have higher basal expression levels of the mrnas encoding isg proteins, such mda5 and stat1, and other molecules crucial for recognizing viral invasion and creating an more antiviral environment than neurons (9) , which may explain why lab-attenuated rabv activates the innate immune response to a degree sufficient to restrict viral replication in astrocytes but not in neurons. double-stranded rna is a viral product that plays an essential role in inducing innate immunity, which leads to the production of type i ifns and the activation of hundreds of isgs. early biochemical studies of viral replication suggested that most viruses produce dsrnas (13) . however, in 2006, weber et al. reported that dsrna could be detected by ifa in cells infected with positive-stranded rna viruses, but not with negative-stranded rna viruses (48) . this notion was challenged by two other studies on dsrna production in cells infected with negative-stranded rna viruses (13, 48) . moreover, a recent study demonstrated the dsrna formation in cells infected with several negative-stranded rna viruses, such as vsv, measles virus (mev), and influenza a virus, although the intensity of the staining of dsrna tended to be weaker in cells infected with negative-stranded rna viruses when compared with those infected with positive-stranded rna viruses (13) . consistent with this finding, the production of dsrna was detected in both lab-attenuated and wt rabv-infected astrocytes, although lab-attenuated rabv produced significantly more dsrna in the cytoplasm than wt rabv. we attempted to investigate why lab-attenuated rabv produces more dsrna than wt rabv. pfaller et al. found that dsrna expression was much lower in cells infected with wt mev than in cells infected with a mutant mev whose c protein was knocked out which is known to control genome replication and transcription (49) . similarly, takeuchi et al. observed dsrna formation in cells infected with sendai virus (50) with c protein knocked out but not in cells infected with wt sev, indicating that the c protein limits or masks dsrna production (51) . both mev and sev are within the family paramyxoviridae, thus it is possible that their c protein possess the similar function to subvert the production of dsrna. ebola virus protein vp35 adopts a unique strategy to mask key cellular recognition sites on dsrna (52) . a recently study showed that the coronavirus endonuclease (endou) activity is the key to prevent early induction of dsrna. replication of endou-deficient coronaviruses is greatly attenuated in vivo and severely restricted in primary cells even during the early phase of virus infection (53) . in this present study, by exchanging viral genes between wt and lab-attenuated rabv, we found that single n, p, and g of wt rabv could not suppress dsrna formation (data not shown). however, multiple viral proteins of rabv may work together to limit the production of dsrna. further studies are needed to elucidate the mechanism through which wt rabv restricts dsrna formation and thus evades recognition by the innate immune system in infected cells. the bbb, which is composed of specialized bmecs joined by tjs and ensheathed by astrocytes and pericytes, plays an important role in protecting the cns. our previous studies have shown that rabv does not infect bmecs, nor does it modulate tj protein expression in bmecs (39) . however, brain extracts prepared from mice infected with lab-attenuated rabv but not wt rabv reduced tj protein expression in bmecs, indicating that the above enhancements of bbb permeability and reductions in tj protein expression are not caused by rabv infection. rather, they are caused by virus-induced inflammatory chemokines/ cytokines. the innate immune mechanisms that regulate bbb function in the setting of infectious diseases have been appreciated only recently. multiple inflammatory cytokines, including tnf-α, il-6, il-1β, ifn-γ, il-17, and vegf, disrupt bbb and tj integrity in bmecs (46, 50, (54) (55) (56) , and inflammatory cytokine signaling at the bbb during infection facilitates leukocyte trafficking into the cns, which is essential for the clearance of many pathogens (39, 41) . our present study demonstrates that lab-attenuated rabv induces production of several inflammatory cytokines in astrocytes, especially tnf-α, il-6, il-1β, ifn-γ, il-17, and vegf, which cause disruption of the bbb and tj integrity. our findings suggest that astrocytes play a critical role in regulating bbb permeability as a major source of cytokines during viral infection. furthermore, we found that the production of inflammatory cytokines in astrocytes by lab-attenuated rabv was dependent on mavs signaling pathway, underscoring the critical role of mavs signaling in defensing against rabv infection in cns. 8 | the proposed model for the mechanism through which wild-type (wt) and lab-attenuated rabies virus (rabv) infect astrocytes. during infection, lab-attenuated rabv produces double-stranded rna (dsrna), which is recognized by retinoic acid-inducible gene i (rig-i)/melanoma differentiation-associated protein 5 (mda5). activation of mitochondrial antiviral-signaling protein (mavs), the common adaptor protein for rig-i and mda5, leads to enhanced production of interferon, as well as some ifn-stimulated genes, which limit rabv replication and spread. mavs activation stimulates the p38 and nf-κb signaling pathways and induces cytokine production in astrocytes. inflammatory cytokines promote blood-brain barrier (bbb) permeability, enabling peripheral inflammatory cells and antibodies to infiltrate into the central nervous system, thereby facilitating rabv clearance. in contrast, wt rabv prevents activation of the mavs signaling pathway by restricting dsrna production. conclusion based on the results, we propose the following model: labattenuated rabv produces dsrna recognized by rig-i, mda5, or both, resulting in the activation of the mavs signaling pathway in astrocytes. ifn expression induces the transcription of hundreds of isgs to inhibit rabv replication in astrocytes and causes the abortive infection by lab-attenuated rabv. the inflammatory cytokines induced by lab-attenuated rabv enhance bbb permeability, enabling immune cells and antibodies to infiltrate the cns and facilitate rabv clearance. conversely, wt rabv restricts dsrna production and then evades recognition by the innate immune system, resulting in persistent viral replication in astrocytes (figure 8 ). current status of rabies and prospects for elimination the cell biology of rabies virus: using stealth to reach the brain rhabdoviruses: rabies virus role of apoptosis in rabies viral encephalitis: a comparative study in mice, canine, and human brain with a review of literature silver-haired bat rabies virus variant does not induce apoptosis in the brain of experimentally infected mice attenuated rabies virus activates, while pathogenic rabies virus evades, the host innate immune responses in the central nervous system rabies virus-induced apoptosis involves caspase-dependent and caspase-independent pathways failure to open the bloodbrain barrier and deliver immune effectors to central nervous system tissues leads to the lethal outcome of silver-haired bat rabies virus infection the roles of chemokines in rabies virus infection: overexpression may not always be beneficial neuronal apoptosis does not play an important role in human rabies encephalitis toll-like receptors in innate immunity toll-like receptor function and signaling double-strand rna is detected by immunofluorescence analysis in rna and dna virus infections including those by negative-strand rna viruses myd88 functions as a negative regulator of tlr3/trif-induced corneal inflammation by inhibiting activation of c-jun n-terminal kinase regulation of innate antiviral defenses through a shared repressor domain in rig-i and lgp2 involvement of the ubiquitin-like domain of tbk1/ikk-i kinases in regulation of ifn-inducible genes recognition of 5' triphosphate by rig-i helicase requires short blunt double-stranded rna as contained in panhandle of negative-strand virus structural basis of double-stranded rna recognition by the rig-i like receptor mda5 mavs forms functional prion-like aggregates to activate and propagate antiviral innate immune response induction of irf-3 and irf-7 phosphorylation following activation of the rig-i pathway virus infection switches tlr-3-positive human neurons to become strong producers of beta interferon toll-like receptor 3 (tlr3) plays a major role in the formation of rabies virus negri bodies rabies virus infection induces type i interferon production in an ips-1 dependent manner while dendritic cell activation relies on ifnar signaling the role of toll-like receptors in the induction of immune responses during rabies virus infection blood-brain barrier biology and methodology immune players in the cns: the astrocyte susceptibility to theiler's virus-induced demyelinating disease correlates with astrocyte class ii induction and antigen presentation longlasting astrocyte reaction to persistent junin virus infection of rat cortical neurons astrocyte infection by hiv-1: mechanisms of restricted virus replication, and role in the pathogenesis of hiv-1-associated dementia tlr3 deficiency renders astrocytes permissive to herpes simplex virus infection and facilitates establishment of cns infection in mice potential mechanisms for astrocyte-timp-1 downregulation in chronic inflammatory diseases viral disruption of the blood-brain barrier astrocyte response to junin virus infection rabies viruses infect primary cultures of murine, feline, and human microglia and astrocytes abortively infected astrocytes appear to represent the main source of interferon beta in the virus-infected brain complete genome sequence of a street rabies virus isolated from a rabid dog in china rabies viruses leader rna interacts with host hsc70 and inhibits virus replication role of chemokines in the enhancement of bbb permeability and inflammatory infiltration after rabies virus infection enhancement of blood-brain barrier permeability and reduction of tight junction protein expression are modulated by chemokines/cytokines induced by rabies virus infection the inability of wild-type rabies virus to activate dendritic cells is dependent on the glycoprotein and correlates with its low level of the de novo-synthesized leader rna expression of neuronal cxcl10 induced by rabies virus infection initiates infiltration of inflammatory cells, production of chemokines and cytokines, and enhancement of blood-brain barrier permeability cell-type-specific type i interferon antagonism influences organ tropism of murine coronavirus infection of pericytes in vitro by japanese encephalitis virus disrupts the integrity of the endothelial barrier regional astrocyte ifn signaling restricts pathogenesis during neurotropic viral infection influenza a virus ns1 targets the ubiquitin ligase trim25 to evade recognition by the host viral rna sensor rig-i overexpression of tumor necrosis factor alpha by a recombinant rabies virus attenuates replication in neurons and prevents lethal infection in mice rabies virus-induced activation of mitogen-activated protein kinase and nf-kappab signaling pathways regulates expression of cxc and cc chemokine ligands in microglia doublestranded rna is produced by positive-strand rna viruses and dna viruses but not in detectable amounts by negative-strand rna viruses measles virus c protein impairs production of defective copyback double-stranded viral rna and activation of protein kinase r production of il-8, il-17, ifn-gamma and ip-10 in human astrocytes correlates with alphavirus attenuation sendai virus c protein plays a role in restricting pkr activation by limiting the generation of intracellular double-stranded rna ebolavirus vp35 coats the backbone of double-stranded rna for interferon antagonism early endonuclease-mediated evasion of rna sensing ensures efficient coronavirus replication astrocytes produce and release interleukin-1, interleukin-6, tumor necrosis factor alpha and interferon-gamma following traumatic and metabolic injury cellular mechanisms of il-17-induced blood-brain barrier disruption expression of interferon gamma by a recombinant rabies virus strongly attenuates the pathogenicity of the virus via induction of type i interferon the authors wish to thank the staff members of the animal facility at huazhong agricultural university for caring for the mice. this work was partially supported by the national natural science foundation of china (31720103917 and 31330078 to zf and 31372419 and 31522057 to lz); the national program on key research project of china (2016yfd0500400 to lz). key: cord-351520-c5fi2uoh authors: zhong, bo; wang, yan-yi; shu, hong-bing title: regulation of virus-triggered type i interferon signaling by cellular and viral proteins date: 2010-02-01 journal: front biol (beijing) doi: 10.1007/s11515-010-0013-x sha: doc_id: 351520 cord_uid: c5fi2uoh host pattern recognition receptors (prrs) recognize invading viral pathogens and initiate a series of signaling cascades that lead to the expression of type i interferons (ifns) and inflammatory cytokines. during the past decade, significant progresses have been made to characterize prrs such as toll-like receptors (tlrs) and rig-i-like receptors (rlrs) and elucidate the molecular mechanisms of tlrand rlr-mediated signaling. to avoid excessive and harmful immune effects caused by over-activation of these signaling pathways, host cells adopt a number of strategies to regulate them. in addition, invading viruses also employ a variety of mechanisms to inhibit the production of type i ifns, thereby evading the supervision and clearance by the host. in this review, we briefly summarize the tlrand rlr-mediated type i ifn signaling and then focus on the mechanisms by which host cellular and viral components regulate the expression of type i ifns. organisms, from unicellular bacteria to human, are exposed to invading pathogens all the time. to protect themselves from pathogenic effects caused by the invaders, hosts have evolved immune system to detect and prevent infection by pathogens. the immune system in mammals is traditionally divided into two branches: innate immunity and adaptive immunity. the adaptive immunity, which is able to generate specific immune responses mediated by antibodies and effector t cells, is highly specific. however, it is evolved only in higher organisms and there is usually a delay of 4-7 days before the initial adaptive immunity takes effects. in contrast, the non-specific innate immunity is evolutionally conserved and begins to work minutes to hours after infection. therefore, the innate immunity constitutes the first line for defense against pathogens such as viruses. there are wide spectrums of viral pathogens that are known to infect humans, which have been a great threat to human health. the early events of innate immunity against invading viruses include the recognition of viral components, initiation of signaling pathways and transcriptional induction of type i ifns and other cytokines . the type i ifns bind to ifn receptor (ifnr) in both autocrine and paracrine manners to initiate a series of signaling events leading to the expression of hundreds of downstream genes, collectively referred to as interferon stimulated genes (isgs). proteins encoded by the isgs inhibit viral replication or cause apoptosis of infected cells, and therefore result in an antiviral effect (sadler and williams, 2008) . because type i ifns play a central role in antiviral immunity, great efforts have been made during the past decade to elucidate the mechanisms of virus-triggered type i ifn induction. however, over-produced type i ifns cause excessive and harmful immune effects to the host (theofilopoulos et al., 2005) . as a result, the production of these cytokines should be tightly regulated. on the other hand, viruses have also evolved a variety of mechanisms to inhibit the production of type i ifns, thereby evading the supervision and elimination by the innate immunity. in this review, we first briefly summarize the virus-triggered type i ifn signaling and then focus on the regulatory mechanisms exerted by cellular and viral proteins. 2 viral infection-triggered type i ifn signaling: a brief introduction as mentioned above, virus-triggered type i ifn signaling is initiated by the recognition of pathogen-associated molecular patterns (pamps) generated during viral infection and replication. so far there are at least five kinds of viral pamps that have been characterized, including double-stranded rna (dsrna), 5'triphosphorylated single-stranded rna (5'pppssrna), viral envelope glycoprotein, unmethylated cpg dna (cpg dna), and atrich double-stranded dna (the analog poly da:dt) (kumar et al., 2009; takeuchi and akira, 2009 ). these pamps are recognized by host pathogen-recognition receptors (prrs), which include toll-like receptors (tlrs), rig-i-like receptors (rlrs including rig-i and mda5), nod-like receptors (nlrs), and the recently identified cytoplasmic dna sensors dna-dependent activator of interferon-regulatory factors (dai), absent in melanoma 2 (aim2) and rna polymerase iii (pol-iii) (ablasser et al., 2009; chiu et al., 2009; mccartney and colonna, 2009 ). the prrs, each specific for a distinct ligand set, and the prrs-mediated signaling pathways have been extensively reviewed in several previous publications kawai and akira, 2009; kumar et al., 2009; takeuchi and akira, 2009; yoneyama and fujita, 2009) (table 1) . here, we choose tlr3, tlr7/8, tlr9, rlrs, dai and pol-iii-mediated signaling for discussion because they all recognize the viral nuclear acids but induce the production of type i ifns via three representative adaptor proteins. these pathways converge at the activation of several transcription factors such as interferon-regulated factor 3/7 (irf3/7) and nf-κb which collaborate to regulate transcription of type i ifns (maniatis et al., 1998; honda et al., 2006) . tlr3 is the first characterized mammalian prr and tlr3-meidated signaling has been extensively studied. upon stimulation of viral dsrna or its synthetic analog poly(i:c), the intracellular domain of tlr3 recruits the adaptor protein toll/interleukin receptor (tir) domaincontaining adaptor-inducing ifn-β (trif). trif has an n-terminal domain, a middle tir domain and a c-terminal domain called receptor-interacting protein (rip) homotypic interaction motif (rhim) yamamoto et al., 2003) . the adaptor protein trif undergoes oligomerization through its tir and rhim domains, and recruits the traf family-memberassociated nf-κb activator (tank) binding kinase 1 (tbk1) via its n-terminal domain to activate irf3/7. it is also suggested that nf-κb activating kinase (nak)associated protein 1 (nap1) and tumor necrosis factor (tnf) receptor-associated factor 3 (traf3) are involved in trif-mediated activation of irf3/7 by facilitating trif and tbk1 interaction (oganesyan et al., 2006; saha et al., 2006; ryzhakov and randow, 2007) . collectively, dsrna induces activation of irf3/7 through tlr3-trif-nap1/traf3-tbk1 pathway. trif mediates nf-κb activation through two distinct pathways. trif contains a consensus traf6-binding motif in the n-terminal region and mutation of this motif impairs trif-mediated nf-κb but not irf3 activation jiang et al., 2004) . however, tlr3 signaling in traf6-deficient macrophages is not affected (gohda et al., 2004) , which indicates the existence of functional redundancy. it has been demonstrated that trif is also capable of activating nf-κb through its c-terminal rhim, which is responsible for recruitment of rip (meylan et al., 2004) . it has been shown that poly(i:c)induced nf-κb activation is completely blocked in ripdeficient mefs (cusson-hermance et al., 2005) . in addition, overexpression of trif induces apoptosis by interacting with rip1 through a rip1/fas-associated death domain (fadd)/caspase 8-dependent and mitochondriaindependent apoptotic pathway . collectively, the tlr3-triggered trif-dependent pathways activate irf3/7 and nf-κb and induce apoptosis via tbk1, rip1 and rip1/fadd/caspase 8, respectively. in contrast to the trif-dependent signaling triggered by tlr3, tlr7/8 and tlr9-mediated signaling depends exclusively on another adaptor protein myd88 (myeloid differentiation primary response protein-88) akira et al., 2006; yoneyama and fujita, 2009 ). the myd88-dependent pathway includes a number of signaling molecules: the adaptor protein myd88, il-1rassociated kinase 4/1 (irak4/1), transforming growth factor-β-activated kinase (tak1), traf6 and tak1 binding protein-1/2 (tab1/2). upon binding to their respective ligands, tlr7/8 and tlr9 recruit myd88 and irak4 (honda et al., 2004; kawai et al., 2004) . irak4 further recruits irak1 and traf6 and thereby phosphorylates and activates irak1 . the irak1-traf6 complex then disassociates from the receptor . on one hand, the complex interacts with ikkα, traf3 and osteopontin, leading to phosphorylation and activation of irf7 (hoshino et al., 2006; shinohara et al., 2006) . on the other hand, it interacts with another complex consisting of tak1, tab1 and tab2, followed by the phosphorylation and activation of tak1. the activated tak1 subsequently phosphorylates the iκb kinases (ikks), leading to ubiquitination and degradation of iκb and activation of nf-κb . activation of tak1 also results in the activation of mapks, including c-jun nterminal kinase (jnk), leading to activation of ap-1 oganesyan et al., 2006; saha et al., 2006) . the canonical ikk complex ikkα/β/γ is essential for virus-triggered rlr-mediated nf-κb activation, and the noncanonical ikk family members tbk1 and ikkε are responsible for phosphorylation and short interfering rna (sirna) kawai and akira, 2008; kleinman et al., 2008; tlr7/8 imiquimod (r-837), resiquimod (r-838), loxoribine hemmi et al., 2002 guanosine and uridine-rich ssrna diebold et al., 2004; heil et al., 2004 human immunodeficiency virus diebold et al., 2004; heil et al., 2004 influenza a virus diebold et al., 2004; heil et kato et al., 2008 5'pppssrna hornung et al., 2006 pichlmair et al., 2006 5'triphosphate rna with a panhandle structure at 5' end schlee et al., 2009 in vitro transcribed rna kato et al., 2006; yoneyama and fujita, 2009 influenza a virus kato et al., 2006; yoneyama and fujita, 2009 vesicular stomatitis virus kato et al., 2006; yoneyama and fujita, 2009 newcastle disease virus kato et al., 2006; yoneyama and fujita, 2009 sendai virus kato et al., 2006; yoneyama and fujita, 2009 japanese encephalitis virus kato et al., 2006; yoneyama and fujita, 2009 hepatitis c virus saito et al.,2008; yoneyama and fujita, 2009 respiratory syncytial virus kato et al., 2006; yoneyama and fujita, 2009 dengue virus kato et al., 2006; yoneyama and fujita, 2009 west nile virus kato et al., 2006; yoneyama and fujita, 2009 mda5 long dsrna, poly(i:c) (about 2 kb) reovirus (long fragment of genomic dsrna) kato et al., 2008 encephalomyocarditis virus kato et al., 2006; yoneyama and fujita, 2009 theiler's encephalomyelitis virus kato et al., 2006; yoneyama and fujita, 2009 mengo virus kato et al., 2006; yoneyama and fujita, 2009 dengue virus kato et al., 2006; yoneyama and fujita, 2009 west nile virus kato et al., 2006; activation of irf3 and irf7. other studies have also demonstrated the involvement of several other signaling components in virus-induced activation of nf-κb and/or irf3, including tank, tradd, fadd and rip guo and cheng, 2007; michallet et al., 2008) . recently, we and others identified a new adapter protein called mediator of irf3 activation (mita, also known as sting), which plays a critical role in virus-induced type i ifn expression (ishikawa and barber, 2008; zhong et al., 2008) . mita has been found to localize to the outermembrane of mitochondria or endoplasmic reticulum (er). it has been demonstrated that mita acts as an adapter to recruit tbk1 and irf3 to the visa-associated complex after viral infection. in this complex, tbk1 first phosphorylates mita at ser358, which is critical for subsequent phosphorylation of irf3 . there is also evidence that a number of molecules involved in protein transportation are also required for virustriggered type i ifn production (ishikawa and barber, 2008; ishikawa et al., 2009 ). viral dna-triggered type i ifn production is mediated through at least three pathways. first, two recent publications reported that in transformed cells, at-rich doublestranded dna (poly(da:dt)) and cytoplasmic viral or bacterial dna is transcripted into rna by pol-iii and the transcribed rna, which probably contains 5'triphosphate structure and is then recognized by rig-i and signals through visa (ablasser et al., 2009; chiu et al., 2009) . second, in primary or low passage cells, transfection of dsdna regardless of its sequences or infection by dna viruses also triggers pol-iii-independent signaling, leading to the expression of type i ifns. the signaling pathway requires the signaling complex mita-tbk1-irf3 but not dai or visa (chiu et al., 2009; ishikawa et al., 2009) . however, future studies are needed to characterize the sensors and adaptors that function upstream of mita in the pol-iii-independent pathway. third, dai senses invading dna to induce type i ifns in l929 cells, which depends on tbk1 and irf3 but not visa (takaoka et al., 2007) . the adaptor protein for this process is currently unknown. it is commonly observed that prr-mediated signaling is rapidly activated after viral infection, leading to the production of type i ifns and other cytokines. however, the overproduction of type i ifns can cause unwanted or excessive immune responses that may lead to allergy, necrosis, autoimmune diseases and other harmful effects (theofilopoulos et al., 2005) . therefore, prr-mediated type i ifn signaling must be tightly regulated. for example, the c-terminus of rig-i contains a repressor domain (rd) which masks the card domain and rna helicase domain of rig-i, thereby inhibiting the activation of rig-i in uninfected cells takahasi et al., 2008; yoneyama and fujita, 2009 ). in addition to the autonomous inhibitory mechanism, the host has evolved several mechanisms to prevent unnecessary activation in steady-state cells or excessive signaling under viral infection conditions (table 2) . laboratory of genetics and physiology (lgp2) compared to rig-i and mda5, lgp2 contains an rna helicase domain and an rna binding domain but lacks the card domain. therefore, lgp2 binds to dsrna competitively with rig-i but does not initiate signaling. it has been shown that overexpression of lgp2 negatively regulates sendai virus (sev) and newcastle disease virus (ndv)-triggered induction of type i ifns, and rnaimediated knockdown of lgp2 can enhance expression of antiviral genes (yoneyama et al., 2005) . in addition, lgp2 interacts with rig-i and inhibits oligomerization of rig-i which is important for activation of the latter. lgp2 also disrupts visa-tbk1/ikkε interaction to inhibit rlrmediated type i ifn signaling (saito et al., 2007) . however, the lgp2 -/mice are resistant to ndv infection which is sensed by rig-i but sensitive to emcv infection which is sensed by mda5, suggesting that lgp2 differently regulates rig-i-and mda5-mediated signaling venkataraman et al., 2007) . recently, a study revealed the crystal structure of the repressor domain of lgp2, which suggests that lgp2 inhibits rig-i-mediated signaling by competitively binding to dsrna, while it positively regulates mda5mediated signaling by facilitating recognition of dsrna by mda5 (pippig et al., 2009) . rig-i splice variant (rig-i-sv) it has been demonstrated that the full activation of rig-i depends on its k63linked ubiquitination at k172 by trim25 or rnf135 (gack et al., 2008; oshiumi et al., 2009) . trim25 firstly interacts with rig-i and then catalyzes the ubiquitination of rig-i . the thr55 of rig-i is critical for its binding with trim25. compared to the full-length rig-i, rig-i-sv lacks the aa36-80 region. thus, rig-i-sv does not interact with trim25 to initiate signaling (gack et al., 2008) . however, rig-i-sv contains the intact rna binding and helicase domain and interacts with rig-i but not mda5. therefore, rig-i-sv inhibits rig-i-but not mda5-mediated type i ifn signaling by competitively binding to dsrna and visa with rig-i and inhibiting the oligomerization of rig-i. dihydroxyacetone kinase (dak) the protein kinase dak was found to interact with mda5 in yeast two-hybrid assays . dak is a member of the evolutionarily conserved family of dihydroxyacetone kinases from bacteria to humans (bachler et al., 2005; cabezas et al., 2005) . mammalian dak displays dual activities as flavin adenine dinucleotide (fad)-adenosine monophosphate (amp) lyase and atp-dependent pha kinase. however, the physiological functions of dak in innate antiviral response were unknown. in co-immunoprecipitation experiments, dak interacts with mda5 but not rig-i in untransfected cells or overexpressed conditions, and the card domaincontaining fragment of mda5 is sufficient for the association. expression of dak inhibits mda5-but not rig-i-or visa-mediated induction of ifn-β, while rnai knockdown of dak has an opposite effect. endogenous interaction between mda5 and dak is decreased when the cells are infected with the sendai virus, suggesting that the association is disrupted upon virus infection. it is possible that upon binding to dsrna, the conformational change of mda5 results in its higher affinity to the downstream adaptor visa . therefore, dak keeps mda5 inactive under steady-state conditions. atg5-atg12 the atg5-atg12 conjugate plays a critical role in autophagic process. the autophagy has been implicated for defense against infections with intracellular bacteria and viruses (gutierrez et al., 2004) . however, autophagosomes have also been exploited by certain viruses as a place for viral replication. a recent study has demonstrated that vsv infection induces higher level of irf3 phosphorylation and expression of ifn-β and ip10 in atg5 -/than in wild-type mefs (jounai et al., 2007) . further investigation suggests that the atg5-atg12 conjugate associates directly with the card domains of rig-i, mda5 and visa and viral infection can enhance the interaction. therefore, atg5-atg12 disrupts the card interactions between rig-i or mda5 and visa, preventing the formation of the rlr-visa complex. atg7 is a critical scaffold protein that facilitates atg12 to conjugate with atg5 (komatsu et al., 2006) . accordingly, atg7 deficient mefs produce higher amount of type i ifns in response to cytoplasmic poly(i:c) stimulation (jounai et al., 2007) . collectively, these data suggest that atg5-atg12 conjugate acts as a suppressor of rlr signaling by blocking the interaction between rlrs and visa. tbk1s compared to the full-length tbk1, the splice variant tbk1s lacks exons three to six. it has been shown that tbk1s is expressed 3-6 hours after viral infection . tbk1s interacts with rig-i and disrupts the interaction between rig-i and downstream molecules, thereby inhibiting rig-i-mediated activation of irf3. interestingly, tbk1s does not inhibit visa-or tbk1-mediated activation of ifn-β, suggesting that tbk1 functions at rig-i level as a negative feedback regulator. it should be noted that although dak and tbk1s disrupt interaction between mda5 or rig-i and visa, neither of them inhibits rlr-mediated nf-κb activation. the mechanisms for this process need further investigation. 3.1.2 sequestration of adaptor proteins nlrx1 nlrx1 belongs to a protein family containing nucleotide-binding domain (nbd) and leucine-rich repeat (lrr) (nlr), which are structurally conserved and have been demonstrated to function in defense against bacterial infection . expression of nlrx1 inhibits rlrmediated expression of cytokines such as type i ifns, il-6 and tnfα, while knockdown of nlrx1 has an opposite effect. nlrx1 was found to reside at the outermembrane of mitochondria and associate with the card domain of visa, thereby sequestering visa from rlrs and inhibiting rlrmediated signaling (moore et al., 2008) . gc1qr receptor for globular domain of complement component c1q (gc1qr) is located in mitochondria, nucleus, cytoplasm and on cell membrane . overexpression of gc1qr inhibits rig-i-mediated activation of irf3 and nf-κb and production of ifn-β and other cytokines. conversely, rnai knockdown of gc1qr enhanced the production of type i ifns. it is shown that mitochondrial gc1qr weakly interacts with visa and sev infection causes the translocation of gc1qr to mitochondria and enhances its association with visa ). thus, it has been postulated that gc1qr plays a role as a negative feedback regulator in rlrmediated signaling. myd88s myd88s is an alternatively spliced form of myd88, lacking the intermediate amino acids 110-157 of myd88. unlike myd88, myd88s does not induce irak1 phosphorylation, despite its interaction with irak1. the expression of myd88s can be detected in spleen and brain tissues and is upregulated by lps stimulation. myd88s forms dimer or oligomer with myd88 and interacts with irak1, sequestering irak1 from irak4 and inhibiting irak4-induced phosphorylation of irak1 (burns et al., 2003) . thus, myd88s is a negative feedback regulator of tlr-induced myd88-dependent signaling. traf1 traf1 belongs to the traf family. all members of this family except for traf1 contain a ring domain that bears an e3 ubiquitin ligase activity (bradley and pober, 2001) . in 293 cells that stably express tlr3, overexpressed traf1 interacts with trif and effectively inhibits poly(i:c)-induced activation of nf-κb and the ifn-β promoter. further studies suggest that cterminal part of traf1 and the tir domain of trif are responsible for their interaction. interestingly, trif induces caspase-dependent cleavage of traf1, and the cleaved n-terminal but not c-terminal fragment of traf1 shows inhibitory effect. inhibition of the cleavage of traf1 by mutating of the cleavage site or addition of caspase inhibitor impairs its ability to inhibit trifdependent signaling (su et al., 2006) . therefore, trifinduced cleavage of traf1 is required for its inhibition of trif signaling. isg56 interferon-stimulated gene 56 (isg56) is one of the first identified proteins induced by virus and type i ifns (sadler and williams, 2008) . in immunoprecipitaion and mass-spectrometry assays, isg56 was identified as a mita-interacting protein ). isg56 negatively regulates virus-triggered signaling at mita level by disrupting tbk1-mita and visa-mita interactions. consistent with these observations, inhibition of isg56 by rnai enhances cellular antiviral responses as well as the expression of type i ifns. it has been reported that isg56 acts as a suppressor of viral replication and protein translation (wang et al., 2003; terenzi et al., 2006; wacher et al., 2007) . in this context, isg56 might have multiple functions and is an important integrator of inhibition of viral replication and control of excessive antiviral responses. it is also possible that the functions of isg56 are temporally and spatially regulated during viral infection. however, more studies are required for the full understanding of these processes ). sike suppressor of ikkε (sike) contains no other recognizable domain but two coiled-coil domains. in yeast two-hybrid screens, sike was identified as an ikkεinteracting protein (huang et al., 2005) . because tbk1 functions in most types of cells and shows highly homology with ikkε which functions in limited types of cells and is viral infection inducible, studies are focused on the interaction between sike and tbk1 perry et al., 2004) . sike interacts with tbk1 in uninfected cells, sequestering tbk1 from irf3, while viral infection or poly(i:c) stimulation disrupts the interaction, thereby releasing tbk1 to interact with and phosphorylate irf3. consistently, overexpression of sike inhibits tlr3-and rlr-mediated signaling and knockdown of sike has an opposite effect (huang et al., 2005) . therefore, sike might sequester tbk1/ikkε in inactive forms under steady-state conditions to avoid unnecessary activation of these kinases. irak-m irak-m belongs to the irak family (ringwood and . however, it does not possess kinase activity like other iraks do. unlike the ubiquitous expression profile of irak1/4, the expression of irak-m is limited to monocytes or macrophages. irak-m -/macrophages show increased cytokine production when stimulated with bacteria (kobayashi et al., 2002) and further studies suggest that irak-m prevents the association of irak-1 and irak-4 with the myd88 complex, thereby negatively regulating tlr-mediated signaling in macrophages. fln29 fln29 is also an ifn-inducible protein. it contains a traf-type zinc finger domain at its n-terminus and a conserved traf6-binding motif which mediates its interaction with traf6. overexpression of fln29 inhibits trif-dependent nf-κb and mapk activations (mashima et al., 2005) . fln29 -/-mefs are highly resistant to vsv infection, and these cells produce more ifn-β than wild-type cells in response to poly(i:c). fln29-deficient mice become more susceptible to poly(i:c)-induced septic shock compared with wild-type mice. mechanistic studies show that fln29 interacts with trif, visa, traf3 and traf6 and inhibits virus-triggered signaling at traf3/6 level (sanada et al., 2008) . however, the exact mechanism still needs further investigations. shp-2 the src homology 2 (sh2) domain-containing protein tyrosine phosphatase 2 (shp-2) is an evolutionarily conserved tyrosine phosphatase (qu, 2000; neel et al., 2003) , which has been demonstrated to positively regulate the signaling triggered by some cytokines such as il-1, and negatively regulate the signaling triggered by ifn-α (you et al., 1999; you et al., 2001) . studies with shp-2 deficient cells suggest that shp-2 suppresses tlr3-but not tlr7or tlr9-mediated production of type i ifns and proinflammatory cytokines. the inhibitory function of shp-2 depends on its phosphatase activity . further investigations show that shp-2 directly binds to tbk1, and the c-terminal domain of shp-2 and the kinase domain of tbk1 are responsible for their interaction. it is possible that shp-2 prevents tbk1mediated phosphorylation of its substrates, thereby blocking trif-mediated signaling. isg15 isg15 is one of the proteins extensively induced by viral infection (theofilopoulos et al., 2005) . the ubiquitin activating enzyme (e1) ube1l and ubiquitin conjugating enzyme (e2) ubch8 catalyze isgylation of rig-i by conjugating isg15 to it, which is followed by ubiquitination and degradation of rig-i (zhao et al., 2005; kim et al., 2008) . consistent with this observation, viral infectioninduced expression of ifn-β is enhanced in ube1l -/than in wild-type mefs. also, the amount of rig-i protein is more stable in ube1l -/than in wild-type mefs (kim et al., 2008) . collectively, these data suggest that the conjugation of isg15 to rig-i can inhibit rig-i-mediated signaling by ubiquitination and degradation of rig-i. rnf125 ubch8 has been demonstrated to be associated with ubiquitination and degradation of rig-i (zhao et al., 2005) . to identify the e3 ubiquitin ligase in this process, yeast two-hybrid assays were performed with ubch8 as a bait. this effort led to the identification of rnf125 (arimoto et al., 2007) . rnf125 functions as an e3 ubiquitin ligase to catalyze ubiquitination of rig-i. thus, overexpression of rnf125 inhibits the virus-triggered and rig-i-mediated type i ifn expression, while knockdown of rnf125 has an opposite effect. furthermore, rnf125 is induced by ifn-α stimulation or viral infection, suggesting a negative feedback role played by rnf125 in innate antiviral signaling. rnf5 ring-finger protein 5 is an e3 ubiquitin ligase that has been implicated in cell motility, protein quality control in the er, cancer and degenerative myopathy (kyushiki et al., 1997; didier et al., 2003; bromberg et al., 2007; delaunay et al., 2008) . in a yeast two-hybrid assay, rnf5 was identified as a mita-interacting protein . overexpression of rnf5 inhibits virus-triggered expression of type i ifns in 293, hela as well as primary monocyte-derived macrophages and dendritic cells, while knockdown of rnf5 potentiates the expression of type i ifns upon viral infection. a further study suggests that rnf5 interacts with mita and catalyzes k48-linked ubiquitination of mita at k150 after viral infection, thereby inhibiting excessive type i ifn response. a20 a20 was initially found to negatively regulate tnftriggered nf-κb signaling (opipari et al., 1990) . a20 contains an ovarian tumor (otu) domain in its n-terminus which has deubiquitination activity, and a c-terminus with ubiquitination activity (wertz et al., 2004) . both domains are required for inhibition of tnf-induced activation of nf-κb. similarly, a20 also inhibits rlr-mediated activation of irf3 . interestingly, the c-terminal domain alone of a20 is sufficient for its inhibitory function, suggesting that a20 negatively regulates the signaling through its ubiquitination activity (saitoh et al., 2005; . moreover, macrophages derived from a20 deficient mice are incapable of terminating tlr-induced nf-κb activation (boone et al., 2005) . further investigations show that a20 functions to cleave the polyubiquitin chains of traf6, which is critical for termination of tlr-mediated activation of nf-κb . rbck1 rbcc protein interacting with pkc1 (rbck1) belongs to the e3 ubiquitin ligase family (marin and ferrus, 2002) . rbck1 contains a ubiquitin-like (ubl) domain and a ring-ibr-ring (rbr) domain in its n-or c-terminus, respectively. rbck1 interacts with the cterminal znf domain of tab2 and induces ubiquitination and degradation of tab2, thereby inhibiting tnf-, il-1and rlr-induced activation of nf-κb . further study also suggests that rbck1 interacts with irf3 and induces degradation of irf3 after viral infection, indicating a negative feedback regulation of virustriggered type i ifn signaling by rbck1 . trim30α tripartite motif (trim) family proteins are also known as "rbcc" proteins, as they contain an rbcc motif at their n-terminus consisting of a ring domain, one or two b-boxes and a coiled-coil region. trim family proteins have been demonstrated to function in the regulation of cell proliferation, differentiation, development, oncogenesis, apoptosis and antiviral responses (nisole et al., 2005) . in addition to the rbcc motif, trim30α also contains a spry domain at its c-terminus. the expression of trim30α is induced by the activation of nf-κb in various types of cells after stimulation with a variety of tlr ligands, such as poly(i:c) and cpg dna. it has been shown that trim30α targets tab2 and tab3 for k63-linked ubiquitination and lysosome-dependent degradation and inhibits autoubiquitination of traf6, contributing to the inhibition of tlr-mediated nf-κb activation. further studies suggest that trim30α transgenic mice are more resistant to endotoxin shock, whereas in vivo knockdown of trim30α by sirna reduces lpsinduced tolerance, which demonstrates that trim30α negatively regulated lps-mediated signaling in vivo and functions as a negative modulator of the tlr signaling pathway . 3.2.2 cleaving the polyubiquitin chains from signaling molecules duba like a20, duba is also a member of the deubiquitination (dub) domain-containing protein family which contains an otu domain. a screening of the rnai library targeting dub family proteins led to the identification of duba as a negative regulator of tlr3-mediated signaling (kayagaki et al., 2007) . knockdown of duba potentiates poly(i:c)-induced expression of ifn-β. conversely, overexpression of duba inhibits poly(i:c)induced or rig-i-mediated signaling. an unambiguous identification of duba-interacting proteins suggests that duba interacts with traf3, whose k63-linked polyubiquitination is required for tlr-and rlr-mediated signaling. duba cleaves the k63-linked polyubiquitin chains of traf3 and depletion of duba increases the level of ubiquinated traf3 both in steady-state cells and in ligands-stimulated cells. furthermore, overexpression of duba also impairs interaction between tbk1 and irf3, suggesting that ubiquitination of traf3 controls the activity of the tbk1-irf3 complex. cyld cyld is an otu dub family protein that has been identified to interact with ikkγ and deubiquitinate the k63-linked polyubiquitin chains of ikkγ, thereby negatively regulating tnf-induced activation of nf-κb (ea et al., 2006) . interestingly, two groups independently reported that cyld functions as a negative modulator of rig-i-mediated signaling friedman et al., 2008) . knockdown of cyld results in an enhancement in sev-triggered ifn-β secretion. experiments using cyld -/-mefs and dendritic cells (dcs) show constitutive activation of tbk1/ikkε as well as hyper-induction of type i ifns by vsv infection. immunoprecipitation experiments show that cyld coprecipitates not only with rig-i but also with tbk1 and ikkε. interestingly, tbk1 or ikkε specifically causes cyld to shift to higher molecular bands, suggesting phosphorylation of cyld by these kinases. the data indicate that cyld probably regulates the activities of ikk-family kinases and leads to inactivation of the signaling . experiments from another group show that cyld associates with the card domain of rig-i and removes k63-linked ubiquitin from rig-i. loss of cyld in dcs causes accumulation of ubiquitinated rig-i. furthermore, the accumulation of ubiquitinated rig-i in cyld-deficient cells is associated with constitutive activation of tbk1. the expression of cyld can be induced by tnf treatment or viral infection (friedman et al., 2008) . these data together suggest a working model of the mechanisms by which cyld negatively regulates the signaling. in uninfected cells, rig-i undergoes ubiquitination-deubiquitination kinetically, making rig-i inactive. upon viral infection, the e3 ubiquitin ligases trim25 and/or rnf135 mediate k63-linked ubiquitination of rig-i, which subsequently activates downstream kinase tbk1 oshiumi et al., 2009) . however, as viral infection goes on, cyld accumulates and cleaves the k63-linked ubiquitin chains of rig-i, inhibiting virus-triggered type i ifn signaling. thus, cyld is most likely a negative regulator that inhibits rig-i in both steady-state and activated state to prevent unnecessary signaling events. however, the precise mechanism that controls the balance between trim25/rnf135-mediated ubiquitination and cyldmediated deubiquitination of rig-i and the phosphorylation of cyld by tbk1 in its inhibitory function remains to be elucidated. in addition to the mechanisms mentioned above, several proteins have been described as regulators in tlr-and rlr-mediated signaling. for example, gsk3β regulates tlr-and ifnγ-mediated production of a subset of inflammatory cytokines and facilitates production of antiinflammatory cytokines (martin et al., 2005; woodgett and ohashi, 2005; hu et al., 2006) . nrdp1 mediates k63linked ubiquitination and activation of tbk1 as well as k48-linked ubiquitination and degradation of myd88, thereby enhancing production of type i ifns and suppressing production of pro-inflammatory cytokines . shp-1 interacts with the kinase domain of irak1 and inhibits irak1 activation, which may explain why shp-1 inhibits the expression of proinflammatory cytokines . however, the exact mechanisms are unclear. pin1 recognizes and binds to the ser336 phosphorylated irf3, leading to considerable conformational change of irf3. the conformational change makes irf3 accessible for binding with e3 ubiquitin ligases such as rbck1, which catalyze ubiquitination and degradation of irf3 (saitoh et al., 2006; . wd40 domain repeat protein 34 (wdr34) interacts with tak1 and negatively regulates tlr3-induced nf-κb activation, although the mechanism is unclear at this moment ). there is evidence suggesting that caspase 8 cleaves visa at d249 and trif at both d281 and d289, indicating a crosstalk between the apoptosis pathway and tlr-/rlr-mediated signaling . it is possible that these proteins function together in a temporal and spatial manner to control harmful excessive immune responses and protect host against autoimmune diseases. there are controversies about the functions of several proteins involved in virus-triggered signaling. for example, isg15 covalently binds to and stabilizes irf3, counteracting its negative regulation of rig-i kim et al., 2008) . ro52 (also called trim21) has been demonstrated to catalyze the ubiquitination of irf3 and induce its degradation, while another report suggests that ro52/trim21 interacts with pin1 and irf3 and prevents pin1-induced conformational change and degradation of irf3, thereby sustaining irf3 activation during viral infection (higgs et al., 2008; yang et al., 2009) . tank has been identified as a scaffold protein facilitating traf3-tbk1-irf3 interaction and subsequent activation of irf3 (guo and cheng, 2007; gatot et al., 2007) . however, experiments on tank-deficient mice suggest that tank is not involved in interferon responses but rather functions as a negative regulator of tlr-signaling and is critical for the prevention of autoimmune nephritis by regulating ubiquitination of traf6 (kawagoe et al., 2009) . sarm contains heat-armadillo motifs at its nterminus, two sterile α motifs (sam) in middle and a tir domain at its c-terminus and is inducible by tlr ligands and acts as a negative regulator in trif-but not myd88dependent pathway (carty et al., 2006) . however, another study showed that sarm is not involved in tlr-mediated signaling, as evidenced by the equivalent production of tnf and mcp-1 by bone marrow-derived macrophages of sarm-deficient and wild-type mice in response to various tlr ligands. it is also reported that sarm functions to restrict viral infection and neuronal injury in a brain region-specific manner (szretter et al., 2009) . further studies and more efforts are required to figure out these inconsistencies. host and virus have mutually exerted powerful selective pressure to each other throughout their evolution. for example, the type i ifn system as a fast and primary defense against viral pathogenesis serves as a strong selective pressure for viral evolution yoneyama and fujita, 2009 ). on the other hand, various viral components, which play roles in viral replication, assembly and pathogenesis, also target the molecules involved in the system to evade host immunity against viruses (bowie and unterholzner, 2008) . there is increasing evidence suggesting that viruses have evolved a number of strategies to evade the recognition by prrs, to inhibit prr-mediated signaling, and even to manipulate host signaling pathways for their own benefit (table 3) . it has been demonstrated that rlr-mediated expression of type i ifns in macrophages and cdcs represents the first line of defense against local viral infection (kumagai et al., 2007) . experiments on rig-i-or mda5-deficient mice suggest that rig-i and mda5 recognize different rna structures generated by distinct viruses . for example, rig-i recognizes 5'pppssrna, short dsrna, newcastle disease virus (ndv) and influenza a virus (iav), while mda5 recognizes long dsrna and piconaviridae family such as encephalomyocarditis virus (emcv). however, neither of them recognizes cellular mrna which is protected by a methylguanosine cap (m7gppp structure) or ribosomal rna or trna which is flanegan et al., 1977; lee et al., 1977 hornung et al., 2004 pichlmair et al., 2006 severe stack et al., 2005 keating et al., 2007 diperna et al., 2004 chen et al., 2008 schroder et al., 2008 rahman and mcfadden, 2006 roy and mocarski, 2007 hepatitis tait et al., 2000 rodriguez et al., 2002 kaposi's sarcoma-associated herpesvirus k13 ikkα/β/γ complex activation of nf-κb matta et al., 2007 monophosphated and/or modified with unusual bases (hornung et al., 2006; kato et al., 2006; bowie and unterholzner, 2008) . viruses seem to be so smart that they are aware of the tricks by which the host distinguishes self and nonself rna. a number of proteins encoded by viral genomes can process viral rna and simulate the modifications of host rna, thereby evading the supervision by prrs (furuichi and furuichi, 2000) . for example, picornaviruses such as emcv protect the 5' end of its rna with the covalently linked protein vpg (flanegan et al., 1977) . the nonstructural protein 14 (nsp14) of severe acute respiratory syndrome (sars) coronavirus functions as an n7 methyltransferase that catalyzes to form an m7gppp structure at the 5'end of its rna (von grotthuss et al., 2003; chen et al., 2009) . genomic rnas from hantaan virus (htnv), crimean-congo hemorrhagic fever virus (cchfv) and borna disease virus (bdv) are 5'monophosphorylated habjan et al., 2008) . however, the exact mechanisms for these modifications are unknown. human immunodeficiency virus (hiv) and adenovirus use host rna processing machinery to cap newly synthesized viral rna (furuichi and furuichi, 2000) . poxviruses replicate in the cytoplasm and encode their own rna capping machinery. in addition, iav 'snatches' capped 5' fragments from cellular mrna to its own genomic rna to mask its 5'ppp structure ( plotch et al., 1981) . many viruses also produce dsrna at some stage during their life cycle, which is recognized by host prrs. to avoid innate immune responses that are initiated by rlrs, some viral genomes encode dsrna-binding proteins, including vaccinia virus (vacv) e3l (chang et al., 1992) , ebola virus vp35 (cardenas et al., 2006; haasnoot et al., 2007) and hiv 1 tat (weeks et al., 1990) . these proteins shield the dsrna structures generated during infection and replication from recognition by rlrs. it is also possible for these proteins to inhibit tlr3-mediated signaling in virus-and viral dsrna-containing endosomes. taken together, these studies suggest that many viruses inhibit prr-mediated type i ifn signaling at the very beginning of prr-mediated recognition of viral nuclear acids. as mentioned above, virus-triggered type i ifn signaling depends on the interactions of various signaling molecules. to block the signaling process, many viral proteins interact with the key molecules involved in the process and thereby prevent signal transduction leading to the expression of type i ifns. for example, some viral proteins bind to rlrs directly, thereby inhibiting rlr-mediated signaling effectively. nonstructural protein 1 of iav which binds to rig-i and v proteins of picornavirus which bind to mda5 are two such examples (andrejeva et al., 2004; mibayashi et al., 2007) . the paramyxoviridae family human metapneumovirus (hmpv) g gene-encoded glycoprotein g specifically interacts with rig-i and blocks rig-imediated ifn-β induction (bao et al., 2008) . recombinant hmpv lacking the g gene replicates efficiently in vitro but its virulence is highly attenuated in vivo. a46r of vacv is a multiple tir domain-containing protein, which interacts with the adaptors trif and myd88 and inhibits tlrmediated production of type i ifns. consistent with the observation, the deletion of a46r attenuates but not abolishes viral virulence of vacv (stack et al., 2005) . n1l of vacv is associated with the kinase tbk1 (diperna et al., 2004) . other two vacv-encoded proteins, a52r which interacts with traf6 and irak2 and b14r which interacts with ikkβ inhibit production of type i ifns and inflammatory cytokines schroder et al., 2008) . hepatitis c virus (hcv) ns3, rabies virus phosphoprotein and bdv phosphoprotein interact with tbk1, leading to sequestration of tbk1 from its upstream or downstream signaling proteins (otsuka et al., 2005; unterstab et al., 2005) . the g1 protein of pathogenic hantavirus can inhibit tbk1 function by disrupting the traf3-tbk1 interaction which is required for signaling (alff p et al., 2006) . poxvirus protein n1l targets ikk complex and inhibits the activation of nf-κb and irf3 by tlrs (diperna et al., 2004) . recent studies suggest that dead-box protein 3 (ddx3) is a critical component of the tbk1/ikkε complex in both rlrs and cytoplasmic dna receptors-mediated signaling. k7r of vacv inhibits prr-mediated induction of ifn-β by targeting ddx3, thereby preventing tbk1 or ikkεmediated activation of irfs (schroder et al., 2008) . several viral proteins inhibit signaling by targeting transcription factors. for example, ns1 of west nile virus (wnv) inhibits tlr3-mediated induction of ifn-β by preventing the translocation of nf-κb and irf3 to the nucleus (wilson et al., 2008) . herpes simplex virus (hsv) infected cell protein 0 (icp0) binds to irf3 and sequesters irf3 from binding host dna (melroe et al., 2007) . there are also viral proteins homologous to host irfs that inhibit irf3 and irf7 signaling. v proteins of mumps virus and parainfluenza virus 5 act as substrates of tbk1/ikkε and compete with irf3 for phosphorylation by tbk1/ikkε (lu et al., 2008) . human herpesvirus 8 encoded irf homologue virf represses type i ifn induction by blocking the association of irf3 with the coactivators cbp and p300 (lin et al., 2001) . similarly, virf3, a kaposi's sarcoma-associated herpesvirus (kshv) viral irf homologue, dimerizes with the host irf7 and inhibits its dna binding activity . another kshv protein, k-bzip, competes with the host irf3 for binding sites in the promoters of isg genes, thereby modulating the expression of antiviral genes (lefort et al., 2007) . many viral genomes are translated into polypeptide precursors which need to be cleaved into mature and functional proteins during viral replication. the cleavage activity depends on several non-structural proteins encoded by the viruses. for example, hcv genome is translated to a polypeptide and ns3/4a is one of such proteases and responsible for the cleavage. on the other hand, such activity of these proteases provides the possibility that they may cleave the host signaling molecules as well. not surprisingly, the adaptors trif and visa are cleaved by ns3/4a into two polypeptides that impair their ability to mediate prr-induced expression of type i ifns (li et al., 2005a; li et al., 2005b; . further studies suggest that the protease precursor protein 3abc of picornavirus hepatitis a virus (hav) and ns3/4a of flavivirus gb virus b cleave visa on the mitochondrial membrane, which disrupts rlrmediated signaling yang et al., 2007) . ns5a of poliovirus induces the cleavage of mda5 by caspases, although the exact mechanism is unclear (abe et al., 2007) . in addition, a number of viral proteins destabilize irfs and target them for degradation. for example, bicp0 of bovine herpesvirus targets irf3 for degradation instead of temporarily sequestering it as icp0 from hsv does (barral et al., 2007) . flaviridae family classical swine fever virus protease n (npro) and hiv proteins vpr and vif also induce proteasomal degradation of irf3, suggesting a common mechanism used by viruses to evade antiviral responses (bauhofer et al., 2007; okumura et al., 2008) . rotavirus nsp1 antagonizes the function of irf3, irf5 and irf7 by inducing their degradation, thereby inhibiting the expression of type i ifns (barro and patton, 2007) . in contrast to evasion of the host recognition and inhibition of the signaling, it is proposed that viruses can 'hijack' and even subvert aspects of prr-mediated signaling. viruses use living cells as hosts, which means that while viral infection and replication causes dysfunction of the hosts, viruses have to prevent the hosts from apoptosis at the same time. not only is nf-κb involved in the production of ifn-β, it also inhibits apoptosis and promotes proliferation of the host cells. therefore, nf-κb is a key molecule to be regulated and used by viruses. taking the control of nf-κb activity by african swine fever virus (asfv) as an example, the asfv protein a238l is an early expressed homologue to the inhibitor of κbα (iκbα) that sequesters nf-κb in the cytoplasm, thereby inhibiting nf-κb activity (tait et al., 2000) . however, as infection and replication progress, a224l is expressed, which activates nf-κb and inhibits caspases, preventing cells from apoptosis (rodriguez et al., 2002) . similarly, the k13 protein from kahv interacts with the canonical ikkα/β/γ complex to selectively activate nf-κb (matta et al., 2007) . there are other examples of viruses that use and manipulate prr-signaling for their replication and survival. hiv employs ddx3 to export viral rna from the nucleus to the cytoplasm (yedavalli et al., 2004) . wnv may use tlr3-mediated signaling to produce cytokines to create a microenvironment favored by the viruses, as evidenced by the fact that tlr3 deficiency mice are resistant to wnv infection . collectively, viruses adopt different strategies to inhibit and manipulate prr-mediated signaling. although progresses have been made to elucidate the mechanisms in the past decade, we expect new insights into the strategies that are used by viruses to interfere with prr-mediated signaling. just as host and virus exert selective pressure on each other, studies on hosts and investigations of viruses mutually facilitate our understandings about each other. future studies will focus on systemic mechanisms by which hosts balance the expression of antiviral and inflammatory cytokines and the translation of viral evasion into benefit of human health. first, mice with deletion of some cellular negative regulators show resistance to viral infection and high viability, while mice defective in others are susceptible to viral infection-caused inflammatory responses. as a result, it is important to take a systemic view of the host regulators when using in vivo mouse models. second, much more efforts are needed to direct in vivo studies relevant to humans. for example, tlr3 deficiency does not impair the host immune response to several viruses (edelmann et al., 2004) . later, it was reported that tlr-deficient mice are more resistant to wnv infection . however, patients with mutations in tlr3 are related to hsv-associated encephalitis . finally, development of therapeutic drugs that targets cellular or viral inhibitors may provide new strategy to treat infection or immune dysfunction-caused diseases. hepatitis c virus nonstructural protein 5a modulates the tolllike receptor-myd88-dependent signaling pathway in macrophage cell lines rig-i-dependent sensing of poly(da:dt) through the induction of an rna polymerase iii-transcribed rna intermediate toll-like receptor signalling pathogen recognition and innate immunity recognition of double-stranded rna and activation of nf-kappab by toll-like receptor 3 the pathogenic ny-1 hantavirus g1 cytoplasmic tail inhibits rig-i and tbk-1 directed interferon responses phosphatase shp-1 promotes tlr-and rig-i-activated production of type i interferon by inhibiting the kinase irak1 shp-2 phosphatase negatively regulates the trif adaptor protein-dependent type i interferon and proinflammatory cytokine production the v proteins of paramyxoviruses bind the ifn-inducible rna helicase, mda-5, and inhibit its activation of the ifn-beta promoter negative regulation of the rig-i signaling by the ubiquitin ligase rnf125 from atp as substrate to adp as coenzyme: functional evolution of the nucleotide binding subunit of dihydroxyacetone kinases human metapneumovirus glycoprotein g inhibits innate immune responses mda-5 is cleaved in poliovirus-infected cells rotavirus nsp1 inhibits expression of type i interferon by antagonizing the function of interferon regulatory factors irf3, irf5, and irf7 classical swine fever virus npro interacts with interferon regulatory factor 3 and induces its proteasomal degradation the ubiquitin-modifying enzyme a20 is required for termination of toll-like receptor responses viral evasion and subversion of pattern-recognition receptor signalling tumor necrosis factor receptor-associated factors (trafs) increased expression of the e3 ubiquitin ligase rnf5 is associated with decreased survival in breast cancer identification of the rabies virus alpha/beta interferon antagonist: phosphoprotein p interferes with phosphorylation of interferon regulatory factor 3 inhibition of interleukin 1 receptor/toll-like receptor signaling through the alternatively spliced, short form of myd88 is due to its failure to recruit irak-4 identification of human and rat fad-amp lyase (cyclic fmn forming) as atp-dependent dihydroxyacetone kinases ebola virus vp35 protein binds double-stranded rna and inhibits alpha/beta interferon production induced by rig-i signaling the human adaptor sarm negatively regulates adaptor protein trif-dependent toll-like receptor signaling thsse e3l gene of vaccinia virus encodes an inhibitor of the interferon-induced, double-stranded rna-dependent protein kinase inhibition of ikappab kinase by vaccinia virus virulence factor b14 functional screen reveals sars coronavirus nonstructural protein nsp14 as a novel cap n7 methyltransferase gb virus b disrupts rig-i signaling by ns3/4a-mediated cleavage of the adaptor protein mavs rna polymerase iii detects cytosolic dna and induces type i interferons through the rig-i pathway triad3a, an e3 ubiquitin-protein ligase regulating toll-like receptors the cterminal regulatory domain is the rna 5'-triphosphate sensor of rig-i rip1 mediates the trif-dependent toll-like receptor 3-and 4-induced nf-{kappa}b activation but does not contribute to interferon regulatory factor 3 activation the er-bound ring finger protein 5 (rnf5/ rma1) causes degenerative myopathy in transgenic mice and is deregulated in inclusion body myositis negative regulation of virus-triggered ifn-beta signaling pathway by alternative splicing of tbk1 regulation of irf-3-dependent innate immunity by the papain-like protease domain of the severe acute respiratory syndrome coronavirus negative regulation of mda5-but not rig-i-mediated innate antiviral signaling by the dihydroxyacetone kinase rnf5, a ring finger protein that regulates cell motility by targeting paxillin ubiquitination and altered localization innate antiviral responses by means of tlr7-mediated recognition of single-stranded rna poxvirus protein n1l targets the i-kappab kinase complex, inhibits signaling to nf-kappab by the tumor necrosis factor superfamily of receptors, and inhibits nf-kappab and irf3 signaling by toll-like receptors activation of ikk by tnfalpha requires site-specific ubiquitination of rip1 and polyubiquitin binding by nemo does toll-like receptor 3 play a biological role in virus infections? covalent linkage of a protein to a defined nucleotide sequence at the 5'-terminus of virion and replicative intermediate rnas of poliovirus the tumour suppressor cyld is a negative regulator of rig-i-mediated antiviral response viral and cellular mrna capping: past and prospects roles of rig-i n-terminal tandem card and splice variant in trim25-mediated antiviral signal transduction trim25 ring-finger e3 ubiquitin ligase is essential for rig-i-mediated antiviral activity wdr34 is a novel tak1-associated suppressor of the il-1r/tlr3/tlr4-induced nf-kappab activation pathway reul is a novel e3 ubiquitin ligase and stimulator of retinoic-acid-inducible gene-i lipopolysaccharide-mediated interferon regulatory factor activation involves tbk1-ikkepsilon-dependent lys(63)-linked polyubiquitination and phosphorylation of tank/i-traf cutting edge: tnfr-associated factor (traf) 6 is essential for myd88-dependent pathway but not toll/il-1 receptor domain-containing adaptor-inducing ifn-beta (trif)-dependent pathway in tlr signaling modulation of the interferon antiviral response by the tbk1/ikki adaptor protein tank autophagy is a defense mechanism inhibiting bcg and mycobacterium tuberculosis survival in infected macrophages the ebola virus vp35 protein is a suppressor of rna silencing processing of genome 5' termini as a strategy of negative-strand rna viruses to avoid rig-i-dependent interferon induction mechanisms of the trif-induced interferon-stimulated response element and nf-kappab activation and apoptosis pathways species-specific recognition of single-stranded rna via toll-like receptor 7 and 8 small anti-viral compounds activate immune cells via the tlr7 myd88-dependent signaling pathway a toll-like receptor recognizes bacterial dna the roles of two ikappab kinase-related kinases in lipopolysaccharide and double stranded rna signaling and viral infection the e3 ubiquitin ligase ro52 negatively regulates ifn-beta production post-pathogen recognition by polyubiquitin-mediated degradation of irf3 type i interferon [corrected] gene induction by the interferon regulatory factor family of transcription factors role of a transductional-transcriptional processor complex involving myd88 and irf-7 in toll-like receptor signaling 5'-triphosphate rna is the ligand for rig-i sequence-specific potent induction of ifn-alpha by short interfering rna in plasmacytoid dendritic cells through tlr7 ikappab kinase-alpha is critical for interferon-alpha production induced by toll-like receptors 7 and 9 ifn-gamma suppresses il-10 production and synergizes with tlr2 by regulating gsk3 and creb/ap-1 proteins sike is an ikk epsilon/tbk1-associated suppressor of tlr3-and virustriggered irf-3 activation pathways sting is an endoplasmic reticulum adaptor that facilitates innate immune signalling sting regulates intracellular dna-mediated, type i interferon-dependent innate immunity toll-like receptor 3-mediated activation of nf-kappab and irf3 diverges at toll-il-1 receptor domain-containing adapter inducing ifn-beta inhibition of interferon regulatory factor 7 (irf7)-mediated interferon signal transduction by the kaposi's sarcoma-associated herpesvirus viral irf homolog virf3 the atg5 atg12 conjugate associates with innate antiviral immune responses length-dependent recognition of double-stranded ribonucleic acids by retinoic acidinducible gene-i and melanoma differentiation-associated gene 5 differential roles of mda5 and rig-i helicases in the recognition of rna viruses tank is a negative regulator of toll-like receptor signaling and is critical for the prevention of autoimmune nephritis toll-like receptor and rig-i-like receptor signaling the roles of tlrs, rlrs and nlrs in pathogen recognition interferon-alpha induction through toll-like receptors involves a direct interaction of irf7 with myd88 and traf6 ips-1, an adaptor triggering rig-i-and mda5-mediated type i interferon induction duba: a deubiquitinase that regulates type i interferon production irak-2 participates in multiple toll-like receptor signaling pathways to nfkappab via activation of traf6 ubiquitination negative feedback regulation of rig-i-mediated antiviral signaling by interferoninduced isg15 conjugation myd88-5 links mitochondria, microtubules, and jnk3 in neurons and regulates neuronal survival sequence-and target-independent angiogenesis suppression by sirna via tlr3 irak-m is a negative regulator of toll-like receptor signaling loss of autophagy in the central nervous system causes neurodegeneration in mice cpg motifs in bacterial dna and their immune effects tlr9-dependent recognition of mcmv by ipc and dc generates coordinated cytokine responses that activate antiviral nk cell function herpes simplex virus type 1 activates murine natural interferonproducing cells through toll-like receptor 9 alveolar macrophages are the primary interferon-alpha producer in pulmonary infection with rna viruses pathogen recognition in the innate immune response cloning, expression and mapping of a novel ring-finger gene (rnf5), a human homologue of a putative zinc-finger gene from caenorhabditis elegans detrimental contribution of the toll-like receptor (tlr)3 to influenza a virus-induced acute pneumonia a protein covalently linked to poliovirus genome rna binding of kaposi's sarcoma-associated herpesvirus k-bzip to interferonresponsive factor 3 elements modulates antiviral gene expression immune evasion by hepatitis c virus ns3/4a protease-mediated cleavage of the toll-like receptor 3 adaptor protein trif hepatitis c virus protease ns3/4a cleaves mitochondrial antiviral signaling protein off the mitochondria to evade innate immunity isg56 is a negative-feedback regulator of virustriggered signaling and cellular antiviral response hhv-8 encoded virf-1 represses the interferon antiviral response by blocking irf-3 recruitment of the cbp/p300 coactivators dissociation of a mavs/ips-1/visa/cardif-ikkepsilon molecular complex from the mitochondrial outer membrane by hepatitis c virus ns3-4a proteolytic cleavage negative regulation of the retinoic acid-inducible gene iinduced antiviral state by the ubiquitin-editing protein a20 isg15 enhances the innate antiviral response by inhibition of irf-3 degradation select paramyxoviral v proteins inhibit irf3 activation by acting as alternative substrates for inhibitor of kappab kinase epsilon (ikke)/tbk1 toll-like receptor 9-mediated recognition of herpes simplex virus-2 by plasmacytoid dendritic cells structure and function of the interferon-beta enhanceosome comparative genomics of the rbr family, including the parkinson's disease-related gene parkin and the genes of the ariadne subfamily toll-like receptormediated cytokine production is differentially regulated by glycogen synthase kinase 3 fln29, a novel interferon-and lps-inducible gene acting as a negative regulator of toll-like receptor signaling kaposi's sarcoma-associated herpesvirus (kshv) oncoprotein k13 bypasses trafs and directly interacts with the ikappab kinase complex to selectively activate nf-kappab without jnk activation viral sensors: diversity in pathogen recognition recruitment of activated irf-3 and cbp/p300 to herpes simplex virus icp0 nuclear foci: potential role in blocking ifn-beta induction rip1 is an essential mediator of toll-like receptor 3-induced nf-kappa b activation cardif is an adaptor protein in the rig-i antiviral pathway and is targeted by hepatitis c virus inhibition of retinoic acid-inducible gene imediated induction of beta interferon by the ns1 protein of influenza a virus tradd protein is an essential component of the rig-like helicase antiviral pathway nlrx1 is a regulator of mitochondrial antiviral immunity herpesviruses and the innate immune response the 'shp'ing news: sh2 domaincontaining tyrosine phosphatases in cell signaling trim family proteins: retroviral restriction and antiviral defence critical role of traf3 in the toll-like receptor-dependent and-independent antiviral response unconventional mechanism of mrna capping by the rna-dependent rna polymerase of vesicular stomatitis virus hiv-1 accessory proteins vpr and vif modulate antiviral response by targeting irf-3 for degradation structure of the reovirus outer capsid and dsrna-binding protein sigma3 at 1.8 a resolution the a20 cdna induced by tumor necrosis factor alpha encodes a novel type of zinc finger protein riplet/ rnf135, a ring finger protein, ubiquitinates rig-i to promote interferon-beta induction during the early phase of viral infection interaction between the hcv ns3 protein and the host tbk1 protein leads to inhibition of cellular antiviral responses differential requirement for tank-binding kinase-1 in type i interferon responses to toll-like receptor activation and viral infection rig-i-mediated antiviral responses to singlestranded rna bearing 5'-phosphates the regulatory domain of the rig-i family atpase lgp2 senses double-stranded rna a unique cap (m7gpppxm)-dependent influenza virion endonuclease cleaves capped rnas to generate the primers that initiate viral rna transcription the shp-2 tyrosine phosphatase: signaling mechanisms and biological functions modulation of tumor necrosis factor by microbial pathogens the antiviral adaptor proteins cardif and trif are processed and inactivated by caspases the involvement of the interleukin-1 receptorassociated kinases (iraks) in cellular signaling networks controlling inflammation african swine fever virus iap-like protein induces the activation of nuclear factor kappa b pathogen subversion of cell-intrinsic innate immunity sintbad, a novel component of innate antiviral immunity, shares a tbk1-binding domain with nap1 and tank interferon-inducible antiviral effectors regulation of antiviral responses by a direct and specific interaction between traf3 and cardif the infected cell protein 0 encoded by bovine herpesvirus 1 (bicp0) induces degradation of interferon response factor 3 and, consequently, inhibits beta interferon promoter activity regulation of innate antiviral defenses through a shared repressor domain in rig-i and lgp2 innate immunity induced by composition-dependent rig-i recognition of hepatitis c virus rna negative regulation of interferon-regulatory factor 3-dependent innate antiviral response by the prolyl isomerase pin1 a20 is a negative regulator of ifn regulatory factor 3 signaling fln29 deficiency reveals its negative regulatory role in the toll-like receptor (tlr) and retinoic acid-inducible gene i (rig-i)-like helicase signaling pathway toll/il-1 receptor domain-containing adaptor inducing ifn-beta (trif) associates with tnf receptor-associated factor 6 and tank-binding kinase 1, and activates two distinct transcription factors, nf-kappa b and ifn-regulatory factor-3, in the toll-like receptor signaling recognition of 5' triphosphate by rig-i helicase requires short blunt double-stranded rna as contained in panhandle of negative-strand virus genome trimming: a unique strategy for replication control employed by borna disease virus viral targeting of dead box protein 3 reveals its role in tbk1/ikkepsilon-mediated irf activation identification and characterization of mavs, a mitochondrial antiviral signaling protein that activates nf-kappab and irf 3 trim30 alpha negatively regulates tlr-mediated nf-kappa b activation by targeting tab2 and tab3 for degradation osteopontin expression is essential for interferon-alpha production by plasmacytoid dendritic cells vaccinia virus protein a46r targets multiple toll-like-interleukin-1 receptor adaptors and contributes to virulence tnf receptor-associated factor-1 (traf1) negatively regulates toll/ il-1 receptor domain-containing adaptor inducing ifn-beta (trif)-mediated signaling the immune adaptor molecule sarm modulates tumor necrosis factor alpha production and microglia activation in the brainstem and restricts west nile virus pathogenesis mechanism of inactivation of nf-kappa b by a viral homologue of i kappa b alpha. signal-induced release of i kappa b alpha results in binding of the viral homologue to nf-kappa b nonself rna-sensing mechanism of rig-i helicase and activation of antiviral immune responses dlm-1/zbp1) is a cytosolic dna sensor and an activator of innate immune response innate immunity to virus infection distinct induction patterns and functions of two closely related interferon-inducible human genes, isg54 and isg56 type i interferons (alpha/beta) in immunity and autoimmunity rbck1 negatively regulates tumor necrosis factor-and interleukin-1-triggered nf-kappab activation by targeting tab2/3 for degradation interleukin-1 receptor-associated kinase-1 plays an essential role for toll-like receptor (tlr)7-and tlr9-mediated interferon-{alpha} induction viral targeting of the interferon-{beta}-inducing traf family member-associated nf-{kappa}b activator (tank)-binding kinase-1 loss of dexd/h box rna helicase lgp2 manifests disparate antiviral responses mrna cap-1 methyltransferase in the sars genome mrna cap-1 methyltransferase in the sars genome the e3 ubiquitin ligase nrdp1 'preferentially' promotes tlr-mediated production of type i interferon alpha interferon induces distinct translational control programs to suppress hepatitis c virus rna replication toll-like receptor 3 mediates west nile virus entry into the brain causing lethal encephalitis a20 is a potent inhibitor of tlr3-and sendai virus-induced activation of nf-kappab and isre and ifn-beta promoter fragments of the hiv-1 tat protein specifically bind tar rna de-ubiquitination and ubiquitin ligase domains of a20 downregulate nf-kappab signalling west nile virus nonstructural protein 1 inhibits tlr3 signal transduction gsk3: an in-toll-erant protein kinase? inhibition of rig-i and mda5-dependent antiviral response by gc1qr at mitochondria visa is an adapter protein required for virus-triggered ifn-beta signaling role of adaptor trif in the myd88-independent toll-like receptor signaling pathway trim21 is essential to sustain ifn regulatory factor 3 activation during antiviral response disruption of innate immunity due to mitochondrial targeting of a picornaviral protease precursor requirement of ddx3 dead box rna helicase for hiv-1 rev-rre export function shared and unique functions of the dexd/h-box helicases rig-i, mda5, and lgp2 in antiviral innate immunity the rna helicase rig-i has an essential function in double-stranded rna-induced innate antiviral responses rna recognition and signal transduction by rig-i-like receptors modulation of the nuclear factor kappa b pathway by shp-2 tyrosine phosphatase in mediating the induction of interleukin (il)-6 by il-1 or tumor necrosis factor shp-2 tyrosine phosphatase functions as a negative regulator of the interferon-stimulated jak/stat pathway negative feedback regulation of cellular antiviral signaling by rbck1-mediated degradation of irf3 regulation of ikappab kinase-related kinases and antiviral responses by tumor suppressor cyld tlr3 deficiency in patients with herpes simplex encephalitis human isg15 conjugation targets both ifn-induced and constitutively expressed proteins functioning in diverse cellular pathways the adaptor protein mita links virussensing receptors to irf3 transcription factor activation the ubiquitin ligase rnf5 regulates antiviral responses by mediating degradation of the adaptor protein mita key: cord-327000-oyg3oyx1 authors: li, shasha; yang, jinping; zhu, zixiang; zheng, haixue title: porcine epidemic diarrhea virus and the host innate immune response date: 2020-05-11 journal: pathogens doi: 10.3390/pathogens9050367 sha: doc_id: 327000 cord_uid: oyg3oyx1 porcine epidemic diarrhea virus (pedv), a swine enteropathogenic coronavirus (cov), is the causative agent of porcine epidemic diarrhea (ped). ped causes lethal watery diarrhea in piglets, which has led to substantial economic losses in many countries and is a great threat to the global swine industry. interferons (ifns) are major cytokines involved in host innate immune defense, which induce the expression of a broad range of antiviral effectors that help host to control and antagonize viral infections. pedv infection does not elicit a robust ifn response, and some of the mechanisms used by the virus to counteract the host innate immune response have been unraveled. pedv evades the host innate immune response by two main strategies including: 1) encoding ifn antagonists to disrupt innate immune pathway, and 2) hiding its viral rna to avoid the exposure of viral rna to immune sensors. this review highlights the immune evasion mechanisms employed by pedv, which provides insights for the better understanding of pedv-host interactions and developing effective vaccines and antivirals against covs. porcine epidemic diarrhea virus (pedv) is the etiological agent of porcine epidemic diarrhea (ped) that causes an acute and highly contagious enteric disease of swine characterized by vomiting, diarrhea, dehydration, and anorexia in pigs of all ages, especially resulting in severe diarrhea and high mortality rate in piglets. in serious cases, outbreak of ped even leads to a mortality rate of 100% in pigs [1] [2] [3] . the causative agent of ped was first described in the 1970s in england [4] . in 1976, an unrecognized enteric disease was reported in several european countries [5] . the viral diarrhea was collectively designated "ped" in 1982 [6] . endemic ped had been described in both developed and developing countries, but with a low impact on the swine industry until 2010. in 2010, outbreaks of ped caused by highly pathogenic variant pedv strains occurred in china, and this was immediately reported in other asian counties, causing up to 100% mortality in suckling piglets [1, [7] [8] [9] . in april 2013, pedv entered the united states (us) for the first time and the virulent strains spread rapidly across the us [10]. apart from almost 100% mortality rate in suckling piglets, pedv infection also damaged the growth performance of finishing pigs [11] . the highly pathogenic strains of pedv spread worldwide, resulting in serious problems to the swine industry and substantial economic losses [7, 10, 12, 13] . vaccination used to be the main strategy to prevent and control the rate of pedv infection [14] , however, the current available pedv vaccines cannot provide complete protection for the pigs affected by the highly pathogenic strains. the optimal vaccines should induce efficient maternal antibodies in sows that could be transmitted to the offspring and protect neonatal suckling piglets from pedv. the vaccination 1 (s1) protein [34] . apart from apn, pedv is able to bind to sialic acids [36] . it remains unknown whether pedv uses sugar coreceptors during viral infection [34, 36] . pedv infects multiple cell lines from different species including bat and primate (human and non-human) in vitro. the ability of pedv to infect the cells of different species indicates that the virus utilizes the evolutionarily conserved cell components as receptors, thereby enhancing the potential for cross-species and potentially, zoonotic transmission [37, 38] . the highly pathogenic variant strains of pedv were identified in 2010 and caused a high morbidity of up to 100% in piglets, and since then, these strains become dominant, leading to most of the acute outbreaks of ped worldwide [1, 7, 8] . the high virulence of these strains is critically associated with the immune evasion mechanisms employed by the virus. pedv has evolved different strategies to delicately manipulate and damage the host innate immune system for their multiplication. clarification of these mechanisms is critical for understanding the host range, tropisms, pathogenesis, and for developing effective vaccines and antiviral drugs to curb the spread of pedv in pigs. in this review, we provide an overview of different mechanisms used by pedv to evade host innate immune responses. pedv is an enveloped, positive-sense, single-stranded rna virus. the pedv genome constitution represents a standard cov arrangement. the viral genome is approximately 28 kb in length, containing a 5 terminal cap, a 3 poly (a) tail, as well as seven open reading frames (orfs), including the orf1a, orf1b, s, orf3, e, m, and n genes ( figure 1 ) [39] [40] [41] . the n terminal orf1a and orf1b encode two large replicase polyprotein precursors (pp1a and pp1ab), which are subsequently processed into 16 nonstructural proteins (nsp1 to 16). orf1a encodes pp1a which is cleaved by viral proteases into 11 nonstructural proteins (nsp1-nsp11). the orf1b generates five additional nonstructural proteins (nsp12-16) that are proteolytically cleaved by the viral proteases from pp1ab [42] . nsp3 contains two papain-like protease domains (plp1 and plp2 or pl pro ) that cleave the nsp1-4 region of the replicase polyprotein at three sites. nsp5, a chymotrypsin-like enzyme also known as 3c-like protease, cleaves the polyprotein at remaining cleavage sites [43] . the c terminus of viral genome contains five orfs, encoding four structure proteins (spike protein (s), small envelope glycoprotein (e), membrane glycoprotein (m), and nucleocapsid protein (n)), as well as a hypothetical accessory protein orf3 [44] [45] [46] . the 16 nsps, together with the n protein, and several host proteins, form a large replication and transcription complex (rtc) that engages in the minus-strand rna synthesis, using viral genomic rna. these nsps play important roles in virion structure modification and the replication and transcription of pedv [47] . the c terminus of viral genome contains five orfs, encoding four structure proteins (spike protein (s), small envelope glycoprotein (e), membrane glycoprotein (m), and nucleocapsid protein (n)), as well as a hypothetical accessory protein orf3 [44] [45] [46] . the 16 nsps, together with the n protein, and several host proteins, form a large replication and transcription complex (rtc) that engages in the minus-strand rna synthesis, using viral genomic rna. these nsps play important roles in virion structure modification and the replication and transcription of pedv [47] . the viral proteins of pedv perform different biological functions during viral entry, replication cycle and propagation (table 1) . pedv s protein, a type i membrane glycoprotein protein located on the envelope of the virus, consists of an n-terminal signal peptide, a large extracellular region, a single transmembrane domain, as well as a short cytoplasmic tail [48, 49] . the ectodomain of s protein comprises s1 and s2 subunits. the n-terminal s1 region, containing n-and c-terminal domains (s1-ntd and s1-ctd), is mainly responsible for receptor binding [50] . the c-terminal membrane-anchored s2 region is mainly involved in triggering the fusion of the viral envelope with host cell membranes [48, 49] . the interaction of the cov s protein with its host cell surface receptor is a key determinant for host tropism. the s1-ctd of most known members of α-cov genus, including pedv, interacts with aminopeptidase n (apn) to entry into the target cell [32, 36, [51] [52] [53] [54] . in addition, s protein contains the epitopes that are the major targets of the neutralizing antibody. n protein is the most abundant viral protein during the early phase of infection in cov-infected cells [55] . similar to the n proteins of other covs, the pedv n protein has multiple functions, such as acting as a structural protein that forms nucleocapsid with viral genomic rna, playing important roles in viral replication, transcription, and assembly [56, 57] . the expression of the n protein in intestinal epithelial cells extends the s-phase of cell cycle, causes endoplasmic reticulum (er) stress, and upregulates interleukin-8 expression [44] . moreover, the n proteins of several α-covs and β-covs, including pedv, pdcov, sars-cov, and mouse hepatitis virus (mhv), have been identified as innate immunity antagonists [58] [59] [60] [61] . however, the involved antagonistic mechanisms are particularly different. pedv m protein participates in virion assembly and virus budding through collaboration with other viral proteins, and engages in the induction of neutralizing antibodies against pedv [62, 63] . pedv m protein is distributed throughout the cytoplasm. it induces the cell growth retardation in intestinal epithelial cells (iec) and arrests the cells in s-phase [64] . in addition, pedv m protein is identified as an interferon (ifn) antagonist with an unrecognized mechanism [65] . pedv e protein is important for the virus packaging and budding [66] . it is predominantly localized in the er, having no effect on cell growth, cell cycle and cyclin a expression in iec. however, it causes er stress and activates the nuclear factor-κb pathogens 2020, 9, 367 5 of 27 (nf-κb) pathway which is responsible for the up-regulation of il-8 and the anti-apoptotic protein bcl-2 expression [67] . orf3 has been predicted to possess multiple transmembrane domains [68] , while it is predominantly distributed in the cytoplasm [69] . orf3 also detains cells at s-phase, facilitating vesicle formation, and thus promoting pedv multiplication [69] . a recent study suggests that orf3 interacts with s protein during pedv assembly and consequently benefits viral replication [70] . cov nsps play multiple roles in the synthesis or processing of viral rna, or in virus-host interactions aiming to create an optimal environment for virus replication, such as facilitating viral entry, viral gene expression, rna synthesis, and virion release. nsp1 is a n-terminal cleavage product of orf1a polyprotein [71] , a 9-kda protein, that exists only in α-covs and β-covs [72] . the nsp1 of α-covs is not very similar to β-covs nsp1 with regard to sequence homology and size [73, 74] . based on the sequence alignment analysis of the genomes of different covs, the viral nsp1 can be regarded as a genus-specific marker [75] . moreover, β-covs nsp1 has been widely reported to inhibit host protein expression. however, the biological functions of α-covs nsp1 remain largely unknown. despite the lack of overall sequence similarity, the nsp1 of different covs shares a similar function to interfere with host protein expression [76] . these studies suggest the importance of nsp1 in the life cycle of different lineages of covs. it is shown that tgev nsp1 inhibits host gene expression and is critical for viral virulence [77] . pedv nsp1 induces the degradation of cbp and nf-κb to abate ifn response [78] , but the detailed mechanisms remain unclear. the sizes and amino acid sequence identity of nsp 2 are variable among different covs [76] . nsp2 of mhv and sars-cov are involved in viral rna synthesis [79] . pedv nsp2 has unknown functions in replication, and may implicate the virus-host interactions and virulence. nsp3 is the largest nsp protein, containing two papain-like protease (plp1 and plp2) domains, of which pedv plp2 acts as a viral deubiquitinase (dub), to negatively regulate type i ifn signaling [80] . covs plps domains exhibit multiple functions, serving as a viral protease, dub, as well as an ifn antagonist [81] . covs, like other positive-stranded rna viruses, induce membranous rearrangements of varying morphologies that are essential for rtcs anchoring [82, 83] . the cov-induced replicative structures consist of double-membrane vesicles (dmvs) and convoluted membranes (cms), which form a large reticulovesicular network that are critical for viral replication and transcription [84] [85] [86] [87] [88] [89] [90] [91] [92] [93] . among the cov nsps, nsp3, nsp4, and nsp6 include the hydrophobic transmembrane domains engaging in anchoring the viral rna synthesis components to the membranes [94] . for mers-cov and sars-cov, co-expression of nsp3 and nsp4 is required to induce dmvs [95] . sars-cov nsp6 has membrane proliferation ability as well, which also contributes to dmvs formation [96] . the structure and functions of α-cov nsp3 are largely unknown [97] . nsp4 is also a marker for cov-induced membrane structures; some results indicate that the nsp4-10 of pp1a act as a large complex through multidomain structure or scaffold during viral rna replication progress, before its cleavage into individual products [98] . covs nsp5 encodes a 3c-like proteinase (3cl pro ). the polyproteins pp1a and pp1ab are processed into individual elements of replicase by 3c-like protease and plps [99] . moreover, pedv nsp5 plays a crucial role in virus replication and also blocks host innate immune responses [100] . crystallographic or nuclear magnetic resonance structures have shown that nsp3, nsp5, nsp7, nsp8, nsp9, and nsp10 have the plprob and the adp-ribose 1 -phosphatase (adrp) activity [101] [102] [103] [104] [105] [106] [107] . the crystal structure of sars-cov nsp9 suggests that nsp9 is dimeric and it is able to bind to single-stranded rna [108] . the crystal structure of sars cov nsp10 protein suggests that nsp10 is a zinc-finger protein, which is existent exclusively in covs so far [107] . moreover, nsp7-10 have rna binding activity and nsp12 encodes a single rna-dependent rna polymerase (rdrp). the biochemical characterization and crystallization of sars cov nsp7 and nsp8 manifests that eight copies of nsp8 and eight copies of nsp7 form a supercomplex [106] . the complex is supportive for nucleic acid binding and may be associated with the processivity of viral rdrp [102, 106] . recently, structural studies have described that the sars-cov nsp12 polymerase binds to the nsp7 and nsp8 complex [109] that may increase the polymerase activity of nsp12 rdrp [110] . cov nsp13, a ntpase/helicase, is also determined to play essential roles in viral replication [111] . cov nsp14 is a multifunctional protein with 3 -5 pathogens 2020, 9, 367 6 of 27 exoribonuclease activity and n-7-methyltransferase [mtase] activity [112, 113] . nsp14 catalyzes the n7-methylation of gppp-rna to form a cap-0 structure. cov nsp15 encodes an endoribonuclease (endou), performing functions through a hexamer in many covs [114] [115] [116] . the endoribonuclease activity of nsp15 is not essential for cov replication [117, 118] . for covs, the 5 end of the viral genomic rna and subgenomic mrna (sgmrna) is supposed to have cap structures: an n-7 methylated guanosine nucleoside (m7gpppn) (cap 0) and a methyl group at the 2 -o-ribose position (cap 1) of the first nucleotide [119] . these cap structures enhance the initiation of translation of viral proteins, protect viral mrnas against cellular 5 -3 -exoribonuclease and limit the recognition of viral rna by host innate system [120, 121] . nsp13 is proposed to catalyze the first step of the 5 -capping reaction of viral rnas [122] . the methylation of the two sites in the 5 cap are catalyzed by three nsps; nsp14 (the n-7-mtase), nsp16 (the 2 -o-methyltransferase), and nsp10 [112, [123] [124] [125] [126] . in addition, the 3 -5 exoribonuclease activity of nsp14 is involved in a replicative mismatch repair system during rna synthesis, which improves the replication fidelity of cov [42] . although these nsps have been demonstrated to play essential roles in viral replication, transcription and/or post-translational polyprotein processing [127] , the nsp12-16 of pedv and other covs are poorly characterized to date, except for sars-cov. nsp7 is an ifn antagonist; nsp8 inhibits type iii ifn response; nsp9 is involved in nucleic acid binding; nsp10 enhances the inhibitory effect of nsp16 on ifn-β production. [65, [131] [132] [133] nsp12 encoding a rdrp; viral replication [134] nsp13 a ntpase/helicase that is essential for viral replication [111] nsp14 n-7-mtase; catalyzing n7-methylation of gppp-rna to form a cap-0 structure; 3 -5 exoribonuclease activity involves in a replicative mismatch repair system during rna synthesis; ifn antagonist [42, 65, 112, 113 host cells generally defend against virus infection by mounting an innate antiviral immune response to prevent the spread of the infection and aid in initiating an adaptive immune response which eventually removes the viruses from host. therefore, the first barrier to restrain viral infections is the host innate immune system, which is related to multiple proteins and mechanisms, including ifns, inflammatory cytokine, apoptosis, autophagy, and so on. the activation of type i ifn responses is composed of three stages: (1) recognition of pathogen-associated molecular pattern (pamp) by prrs; (2) secretion of type i ifns through paracrine or autocrine pathways; and (3) expression of numerous antiviral ifn-stimulated genes (isgs) which bring the host into the antiviral state [136] . at least three important prrs have been identified in recognition of viral nucleic acids, including retinoic acid-inducible gene i (rig-i)-like receptors (rlrs) (detection of viral rna in the cytoplasm) [137] , the membrane-bound toll-like receptors (tlrs) (recognition of viral rna or dna in the endosome) [138] , as well as a structurally unrelated group of viral dna sensors (e.g., cgas (cyclic gmp-amp synthase) and ifi16) localized in the host cytoplasm and/or nucleus [139] . in the cytosol, the formation of specific secondary structure of viral rna is closely related to viral rna delivery and replication. these molecular signatures are detected by rlrs including rig-i (also known as ddx58), melanoma differentiation-associated gene 5 (mda5), and laboratory of genetics and physiology-2 (lgp2) [140, 141] . prrs recognize pamps, inducing an intracellular signaling cascade, thus leading to the activation of transcription factors such as irfs and nf-κb, which in turn induce the production of ifns [142] . both rig-i and mda5 have two n-terminal card domains to interact with the card domains of the downstream adaptor proteins, and a dead/h-box rna helicase domain for rna binding. however, lgp2 does not have the n-terminalcard domains, and the involved functions remain unclear [143] [144] [145] . dsrna, as a specific secondary structure of viral rna, can be sensed by rig-i/mda5 to induce ifn-α/β production through the cascade activation of the rlr pathway [146] [147] [148] . activated rig-i/mda5 forms homo-oligomers and recruits the adaptor mitochondrial antiviral signaling (mavs) to induce the formation of signaling complex of mavs with other proteins on mitochondria. tnf receptor-associated factor 6 (traf6) associates with tnfr1-associated death domain protein (tradd), tripartite motif 14 (trim14), and pyruvate carboxylase (pc), resulting in the activation of iκb kinases (ikks). ikks (ikkα, ikkβ and ikkγ) phosphorylate nf-κb inhibitor (iκb), leading to the ubiquitination of iκb and its subsequent degradation. nf-κb is then activated and translocated into the nucleus to turn on the expression of proinflammatory and type i ifn. mavs also recruits and activates tbk1/ikkε by trafs that are pre-associated with tbk1/ikkε, via direct interaction between the sdd domain of tbk1/ikkε and the coiled-coil domain of trafs [149] . activated tbk1/ikkε phosphorylates ifn regulatory factors (irf3/irf7), that further dimerize and import into the nucleus to promote type i ifn production. on the other hand, mavs interacts with mita (also known as sting), that is located in mitochondria and the endoplasmic reticulum (er) membrane. mita interacts with the trap complex, which may be involved in recruiting tbk1 and ikkε to phosphorylate irf3. ubiquitination and deubiquitination are decisive in the regulation of rlr pathways activation [150] . upon binding to viral dsrna, rig-i and mda5 undergo conformational changes and release the n-terminal tandem card domains [151] [152] [153] . the card domains of rig-i are modified by lysine 63 (k63) polyubiquitin chains through the ubiquitin ligases trim25, rnf135, and riplet. this modification is crucial for rig-i to recruit mavs [154, 155] . in addition to the ubiquitination of rlrs, the polyubiquitylation of traf3 and traf6 also play an important role in the regulation of innate immune signaling by activation of tbk1 and ikks, respectively. the k63 polyubiquitin chains can be removed by dubs such as the tumor suppressor protein cyld, duba and a20, providing a mechanism to downregulate immune responses [156] . ubiquitination and deubiquitination are in a dynamic equilibrium to maintain immune homeostasis. type i ifns are secreted by secretory cells and peripheral cells through self-secretion or paracrine secretion manners. extracellular type i ifns bind to a heterodimeric complex composed of subunits of ifn-α receptors 1 (ifnar1) and 2 (ifnar2) located on the cell surface, which activates the tyrosine kinase 2 (tyk2)/janus kinase 1 (jak1) signal transducer. tyk2/jak1 subsequently induces the phosphorylation of transcription factors stat1 and stat2, which dimerize and in turn recruit ifn-regulatory factor 9 (irf9), to form stat1-stat2-irf9 trimerized complex (isgf3). this complex then translocates to the nucleus, where it binds to the ifn-stimulated response elements (isre motif; conserved sequence is tttcnntttc) [157] . the binding of isgf3 to isre finally triggers expression of ifn-stimulated genes (isgs) that directly or indirectly exert antiviral effects in host cells [157] . three types of ifns (types i, ii and iii) have been identified. type i and type ii ifns have been widely reported. type ii ifns only contain ifn-γ. ifn-γ is produced by natural killer (nk) cells and activated cd4 + and cd8 + t cells in response to the cytokines such as interleukin-12 (il-12) and il-18 [158] . ifn-γ binds to the type ii ifn receptor composed of two subunits, ifngr1 and ifngr2. ifngr1 and ifngr2 induce the formation of stat1-stat1 homodimers. stat1-stat1 homodimers translocate to the nucleus and bind to the promoter of the ifn-γ-activation site (gas) elements, to initiate the transcription of ifn-γ-regulated genes [159] . type iii ifns have been explored in recent years, to unravel the underlying mechanisms that manipulate host innate immune responses. type iii ifns in humans contains ifn-λ1 (interleukin 29 ), ifn-λ2 (il-28a), ifn-λ3 (il-28b), and ifn-λ4 [160] [161] [162] . their expression profiles, signaling pathways, and gene expression programs resemble those of type i ifns. the production of type i and iii ifns are both initiated through the recognition of pamps or damage associated molecular patterns (damps) by prrs [163] . despite the fact that the same transcriptional factors are required for the activation of promoters of type i and iii ifns, the nf-κb pathway is a pivotal regulator in ifn-λ production, whereas the irfs pathway dominates type i ifns expression. the promoter of ifn-λ1 includes more nf-κb binding sites compared with in the ifn-β promoter [164] . the signal transduction of type iii ifns depends on the ifn-λ-specific receptor, il-28ra chain and il-10r2 chain [161, 165] . synthetic ifn-λ binds to il-28rα and induces a conformational change within the receptor subunits, that triggers the activation of the receptor-associated tyrosine kinases (tyk2 and jak1), which then phosphorylate stat1 and stat2. stat1 and stat2 are heterodimerized and interact with irf9 to form the isgf3 transcription complex that binds to isre in the promoters of isgs, to induce the expression of hundreds of proteins with antiviral functions [166] . induction of ifn-α/β is the most rapid and effective mechanism for hosts to initiate antiviral innate immune responses. sars-cov, mhv and many other covs are sensitive to ifns. a great number of viral dsrnas intermediates are generated during covs infection that contribute to ifn production, but these covs remain highly pathogenic. as a matter of fact, covs have developed a set of elaborate mechanisms to evade or inhibit the host antiviral innate immune response during virus evolution [134, 167] . the evasive strategies utilized by pedv are classified into four major types: (1) inhibition of rlrs-mediated ifn production pathways, (2) inhibition of the activation of transcription factors responsible for ifn induction, (3) disruption of the signal cascades induced by ifn, and (4) hiding its viral rna to avoid the exposure of viral rna to immune sensors. in the past decade, accumulating evidence demonstrates that pedv n protein, nsp1, plp2, nsp5, nsp15, and nsp16 antagonize type i ifn or type iii ifns production [58, 65, 78, 80, 100, 132, 135, 168] . this explains why only weak ifns' and cytokines' expression is detected in pedv-infected cells [169, 170] . n protein, as an abundantly produced structural protein within cov-infected cells, has multiple functions, including virus replication, transcription, and assembly [56, 134] . pedv n protein has been identified as an ifn antagonist that blocks the expression of ifn-β and isgs by suppression of the pathogens 2020, 9, 367 9 of 27 irf3 and nf-κb activities [58] . pedv n protein inhibits the activation of the ifn-β promoter induced by tbk1 and its upstream rig-i, mda-5, visa, and traf3, while not affecting the activation of the ifn-β promoter driven by irf3. further experiments confirm that n directly interacts with tbk1 to obstruct the association between tbk1 and irf3, which inhibits tbk1-induced irf3 phosphorylation and ifn-β production [58] . moreover, the effect of pedv n protein on type iii ifn production has also been evaluated [168] . n protein inhibits polyinosinic-polycytidylic acid (poly(i:c))-induced ifn-λ3 production by blocking the nuclear translocation of nf-κb, but does not antagonize the type i or type ii ifn expression induced by poly(i:c) in ipec-j2 cells [168] . recent studies show that sars-cov n protein inhibits type i ifn production through suppressing trim25-mediated rig-i ubiquitination [171] . the mers-cov n protein also blocks ifn production by interacting with trim25 [171] . in addition, both mhv and sars-cov n proteins perturb the function of cellular protein activator of protein kinase r (pact), which can bind to rig-i and mda5 to activate ifn production, and thus antagonize type-i ifn signaling [61] . these results indicate the important function of the covs n protein in modulating host innate immune response. whether pedv n protein targets trim25 or pact should be investigated. although several studies have been performed to understand the pathogenicity of pedv, there remains limited information about the interaction between viral proteins and host cell factors during viral infection. cov n protein is a vital viral protein involved in virus replication. current researches have indicated that n protein interacts with many host proteins, such as hcypa [172] , proteasome subunit p42 [173] , smad3 [174] , hnrnp-a1 [175] , and the chemokine cxcl16 [176] . in the host cells, a large number of host proteins reveal various functions. however, for the virus, the genome only encodes several limited viral proteins. therefore, these viral proteins have to be multifunctional, which is pivotal to virus replication and existence. pedv nsp1 is the n-terminal cleavage product from polyproteins pp1a and pp1a/b processed by nsp3 and nsp5 [177] and is about 110 amino acids in length [74, 178] . nsp1 of many α-cov and β-cov exhibits both functional conservation and mechanistic diversity in suppressing host gene expression and ifn signaling. for sars-cov, nsp1 triggers the decay and cleavage of host mrna and inhibits host protein translation, subsequently inhibiting type i ifn production [179, 180] . sars-cov nsp1 also blocks the expression of ifn-inducible genes, by restraining the signal transduction during virus infection [181, 182] . the tgev nsp1 considerably suppresses host protein expression during viral infection [77] . structural studies show that the core structure of pedv nsp1 is highly similar to those of sars-cov nsp1 and tgev nsp1 [183] . pedv nsp1 inhibits host gene expression and three motifs (amino acids 67 to 71, 78 to 85, and 103 to 110) form a stable functional region for inhibition of host protein synthesis, differing considerably from sars-cov nsp1 [183] . pedv nsp1 has been identified as an ifn antagonist, which constrains poly (i:c)-induced ifn-β promoter activity [65] . nsp1 significantly inhibits the activation of ifn-β promoter triggered by irf3, whereas it does not inhibit irf3 phosphorylation and its nuclear translocation. nsp1 interrupts the association of irf3 with creb-binding protein (cbp), by promoting cbp degradation in the nucleus via the proteasome-dependent pathway. cbp/p300, the transcription co-activator camp responsive element binding protein (creb), forms a complex with the activated irf3 in nucleus. the irf3-cbp/p300 complex binds to the positive regulatory domain (prd) regions of the ifn-β promoter, to assemble the enhanceosome with nf-κb and other factors, which ultimately turn on the transcription of type i ifn genes [184] [185] [186] . therefore, pedv nsp1 blocks type i ifn production in the nucleus. activated nf-κb induces the production of type i ifns and proinflammatory cytokines and is important for inhibiting viral infection. pedv nsp1 has been shown to interfere with the nf-κb activity [78] and is the most potent suppressor of proinflammatory cytokines at early infection. it inhibits the phosphorylation and degradation of iκbα, and blocks p65 nuclear translocation, leading to the suppression of both ifn and the early production of pro-inflammatory cytokines [78] . moreover, pedv inhibits type iii ifn production and nsp1, nsp3, nsp5, nsp8, nsp14, nsp15, nsp16, orf3, e, m, and n are identified as type iii ifn antagonists. among these antagonists, nsp1 is the most potent suppressor [130] . pedv nsp1 blocks the nuclear translocation of irf1 and decreases the amounts of peroxisomes and then suppresses irf1-mediated type iii ifns. the conserved residues of pedv nsp1 protein are crucial for ifn suppression [130] . multiple effects of nsp1 on modulating innate immune response during pedv infection suggest the vital role of nsp1 in the pedv replication cycle. the antiviral innate immune signaling pathways are regulated by several posttranslational modifications (ptms), such as phosphorylation, ubiquitination, glycosylation, neddylation and sumoylation [187] , of which ubiquitination is a critical modification to modulate the stability and activity of prrs and other components of innate immune signaling pathways. during viral infection, a reciprocatory action (occurrence of ubiquitination and deubiquitination) helps maintain the homeostasis of host immune responses. hence, deubiquitinases (dubs) are indispensable in the regulation of virus-induced type i ifn signaling [188] . many host dubs have been reported engaging in the regulation of innate immune signaling pathways [189] [190] [191] . in recent years, a variety of viral dubs have been discovered to target key components of type i ifn pathway during various rna virus infections. for example, foot-and-mouth disease virus leader proteinase (fmdv lb pro ) [192] , and porcine reproductive and respiratory syndrome virus nsp2 (prrsv nsp2) possess ubiquitin-deconjugating activity to deubiquitinate key host components [193, 194] . to counteract host antiviral response, covs likely take advantage of dub activity to break host innate immunity. indeed, the plps of mouse hepatitis virus a59 (mhv-a59) [195] , sars [196] , and human cov nl63 have dub activity and antagonize ifn induction [197] . pedv plp2 has been reported as having a deubiquitinase activity as well, and it can be co-immunoprecipitated by rig-i and sting. as mentioned above, fmdv lb pro , mhv plp2 and sars plps all counteract host innate immune response through blocking the ubiquitination of the components of rlrs pathways. similarly, pedv plp2 removes the ubiquitinated conjugates from rig-i and sting by its dub activity, to negatively regulate type i ifn production. pedv plp2 probably interacts with rig-i and sting, which prevents the activation of rig-i and sting by hindering the recruitment of downstream signaling molecules. as expected, the interference with the ubiquitination of rig-i and sting by plp2 clearly benefits pedv replication [80] . pedv nsp3 contains two core domains of plps (plpl and plp2). it is determined that pedv plp2, but not plp1, inhibits the ifn-β promoter activation in hek293t cells. the dub activity of plp2 is highly dependent on its catalytic activity. three catalytically inactive mutants of pedv plp2 (c1729a, h1888a and d1901a) are defective in the deubiquitination of its targets and fail to impair virus-induced ifn-β production. sars-cov plp2 interacts with mdm2 (mouse double minute 2 homolog) to deubiquitinate and stabilize mdm2, approving the degradation of p53 and the suppression of ifn signaling [198] . pedv infection degrades p53 by upregulation of mdm2 expression [198] . pedv plp2 may be responsible for targeting the p53 pathway and inhibiting p53-dependent apoptosis, leading to immune evasion. a recent study determined that tgev pl1 inhibits the ifn-β expression and interferes with the rig-1and sting-mediated signaling pathway through a viral dub activity [195] . it suggests that different viral proteins are involved in the deubiquitination of host proteins for different covs. however, these studies offer a probability to design a common therapeutic against different viral dubs to reduce the replication and pathogenesis of covs. therefore, further studies are required to understand more about the substrate specificity of these viral dubs and clarify the precise functions of cov protease/dub activity. notably, 3c pro is a critical ifn antagonistic protein identified in multiple different families of viruses. the 3c pro of picornaviruses, including fmdv [199] , hepatitis a virus (hav) [200] , enteroviruses (ev71, ev-d68) and coxsackieviruses (cvb3, cv-a16, cv-a6) [201] [202] [203] [204] , antagonize innate immune signaling by targeting the critical components of the ifn pathways for proteolysis. a newly emerged picornavirus, seneca valley virus (svv), has also evolved an effective mechanism to escape host antiviral innate immune using its 3c pro . moreover, 3c pro cleaves the signaling components (mavs, trif, and tank) of type i ifn pathway and induces the degradation of the transcription factors irf3 and irf7 to constrain host antiviral response [205, 206] . cov nsp5 is called 3c-like protease (3cl pro ), that resembles the 3c pro of other rna viruses. for cov, the polyprotein precursors (pp1a and pp1b) are mainly processed to generate mature nonstructural proteins by 3cl pro . to date, the 3cl pro of covs, including pedv and pdcov, have been confirmed to antagonize type i ifn production by the cleavage of nf-κb essential modulator (nemo) and stat2 [100, 207, 208] . nemo is essential for rna virus-induced activation of nf-κb, irf3, and irf7 [209] . nemo is required for mavs-induced ikkα/β activation and is also crucial for the activation of tbk1/ikkε [149] . to establish successful infections, pedv targets nemo to subvert host innate immune responses. pedv nsp5 significantly inhibits sendai virus (sev)-induced ifn-β synthesis and the process depends on its protease activity [100] . further experiments show that pedv nsp5 inhibits rig-i/mda5 signaling and targets the upstream of tbk1. the cleavage of nemo by nsp5 is identified as responsible for this inhibitive effect. the pedv nsp5-mediated cleavage of nemo efficiently blocks nemo-mediated downstream signaling. the cleavage site within nemo that is grasped by nsp5 has been determined. of these reported immune evasion strategies employed by covs, the cleavage of innate immune adaptors is a particularly effective manner to disrupt antiviral responses. nsp5 is essential for the life cycle of pedv and other covs [210, 211] . it is a potential target for the development of anti-coronaviral therapeutics. although pedv nsp5 does not target stat2 mediated type i ifn signaling pathway, pedv nsp7 has been reported to inhibit the stat1 and stat2 induced activation of isre [212] . nsp7 competes with karyopherin α (kpna1), which is an adaptor mediating nuclear translocation of isgf3, in combination with stat1, to block isgf3 nuclear transport. however, the expression and phosphorylation of stat1 and stat2 are not affected by pedv nsp7. in fact, pedv infection degrades stat1, leading to the inhibition of ifn signaling [170] . therefore, other pedv encoded proteins likely target ifns mediated signaling. covs belong to rna viruses, which produce several rna species, such as dsrna intermediates and rna with a 5 -triphosphate during replication. these rna intermediates are potent stimulators of prrs and are associated with the organelles of viral rna replication, dmvs [213, 214] . dmvs formed from membranous rearrangements seem to sequester the replication intermediates using membrane-bound vesicles or invaginations to keep away from prrs. therefore, the form of dmvs may be a strategy for pedv to escape innate immune recognition in the cytosol (figure 2 ). however, whether dmvs alone are sufficient to shield rna from prrs remains unknown. besides, the replication organelles, the endoribonuclease activity and viral 5 end rna capping/protection mechanisms are also the critical ways of avoiding rna recognition or protecting it from degradation [132, 135] . (1) pedv nsp15 cov nsp15 has endou catalytic activity that was initially thought to play a vital role in virus replication. however, the catalytic-defective endou of mhv shows only a subtle defect in viral replication compared to wt virus in fibroblasts [215] . similar results are found for the nsp15 mutants of sars-cov and hcov-229e [216] . these findings suggest that the endou activity of nsp15 is not required for rna synthesis. recently, nsp15 has been demonstrated to act as a new ifn antagonist of covs [117, 118, 217] . recent reports indicate that covs' endou activity is essential for prevention of rna recognition by mda5, protein kinase r (pkr), and oas/rnase l system [118] . pkr and oas/rnase l recognize and destroy foreign rna in the cytosol to defend viral infections. to counteract the function of pkr and oas/rnase l, the virus hides or modifies its viral rna, to avoid the exposure of viral rna to these molecules. in all covs, the endou catalytic domain in nsp15 is highly conserved. pedv endou activity has been indicated as having an antagonistic effect on ifn signaling [135] . the endou activity of pedv nsp15 not only inhibits the type i ifn response in porcine macrophages, but also antagonizes the type iii ifn response in porcine epithelial cells. the replication of endou-mutant pedv (icpedv-enumt) is considerably impaired in porcine epithelial cells compared to the wild type pedv (icpedv-wt). the icpedv-enumt clearly induces early and robust type i and type iii ifns production, as well as isgs' expression compared with that induced by icpedv-wt. the endou-deficient pedv infected animals also show reduced viral shedding and mortality. these results indicate that the endou activity of pedv nsp15 plays a vital role in evading host antiviral innate immunity (figure 2 ) [135] . (2) pedv nsp16 cov nsp16 is a 2 -o-methyltransferase (2 -o-mtase). to evade recognition by the host immune sensors, many covs encode methyltransferases involved in the capping of viral rna. this modification makes the viral rna indistinguishable with host cell mrna, which is important to avoid the recognition of viral rna by mda5 ( figure 2 ) [121, 218] . methylation of the two sites in the 5 cap is catalyzed by three nsps, including nsp14 (the n-7-mtase), nsp16 (the 2 -o-mtase), and nsp10 [112, [123] [124] [125] [126] . for example, sars-cov nsp16 acts as a 2 -o-mtase to prevent innate immune recognition and promote viral proliferation [113, 121, 123] . pedv nsp14 and nsp16 have been identified as the viral ifn antagonists [65, 132] . the overexpression of nsp14 or nsp16 remarkably inhibits ifn-β production, but nsp16 appears to play a more important role in innate immunity regulation than nsp14 [132] . nsp16 is a highly conserved methyltransferase which contains an invariant kdke motif within the methyltransferase core [219] . this kdke motif is required to mediate its activity. notably, the mutation of any of the kdke active sit has been shown to abolish the 2 -o-mtase activity [123] . pedv nsp16 kdke motif plays a critical role in the inhibition of type i ifn production, suggesting the important role of the 2 -o-mtase in pedv-mediated immune evasion. pedv nsp16 negatively regulates rlr-mediated signal pathway activation, and inhibits the expression of the ifn-stimulated ifit family members (ifit1, ifit2, ifit3), which in turn promotes pedv replication. taken together, these results demonstrate that pedv nsp16 negatively regulates cellular antiviral response to promote viral replication [132] . screening inhibitors targeting the 2 -o-mtase of nsp16 might be a prominent strategy to inhibit cov infections and develop antivirals for treatment of the diseases caused by covs. additionally, covs nsp14 also includes the 3 -to-5 exoribonuclease (exon) activity [113] . a mutation of tgev nsp14 exon generates lower levels of dsrna than wildtype tgev and thus triggers a reduced antiviral response [220] . nsp14 exon activity is also critical for the resistance of host innate immune response in mhv-infected cells [221] . the role of nsp exon activity of pedv in counteracting host antiviral response should be investigated to uncover more functions of pedv nsps. these data suggest that pedv has evolved multiple evasive mechanisms to circumvent viral rna recognition or prevent rna degradation to establish a successful infection in the host. activity is also critical for the resistance of host innate immune response in mhv-infected cells [221] . the role of nsp exon activity of pedv in counteracting host antiviral response should be investigated to uncover more functions of pedv nsps. these data suggest that pedv has evolved multiple evasive mechanisms to circumvent viral rna recognition or prevent rna degradation to establish a successful infection in the host. heat shock protein 27 (hsp27) belongs to the small heat shock proteins family, which has been identified as a multifunctional protein involved in cytoskeletal stability, proinflammatory processes, and the inhibition of apoptosis [222, 223] . several hsps have been reported to be implicated in pedv infection in vitro and in vivo [224, 225] . indeed, the infection of many viruses up-regulates hsp27 expression by different mechanisms to delay cellular apoptosis, then supplies sufficient time for viral replication [226, 227] . however, pedv infection significantly induces the decreased expression of hsp27 in vero cells [225] and marc-145 cells [228] . hsp27 activates the phosphorylation of nf-κb, and thus promotes the mrna expression of ifn-β in marc-145 cells. as hsp27 is an upstream regulator of antiviral immune signaling, overexpression of hsp27 significantly inhibits the pedv replication. pedv has developed a strategy via mediating the suppression of hsp27 production to escape from host innate immune response [228] . hsp70, the most conserved hsp, is also important for the multiplication of several covs. the recruitment of hsp70 is thought to be a viral survival strategy for several viruses in their hosts [229] . the relationship between hsp70 and pedv should be exploited further in future. viral infection triggers host immune response to induce ifns' and inflammatory cytokines' production. released ifns elicit the expression of numerous isgs which limit viral replication in the infected cells. however, the release of excessive amounts of ifns and inflammatory cytokines will lead to autoimmune and auto-inflammatory diseases. the concomitant uncontrolled apoptosis is also one outcome that is harmful to the host. to maintain the reaction in a proper balance, hosts have evolved a series of effective mechanisms to control the antiviral innate immune response [230] . in contrast, viruses often break this balance, causing improper apoptosis reaction, which benefits viral replication. pedv infects various host cells including vero, pk-15 and marc-145 and cause obvious cytopathic effects. pedv-induced apoptosis of the infected cell has been demonstrated both in vitro and in vivo [231] . apoptosis is induced through the activation of apoptotic caspases, including caspase-2, -3, -6, -7, -8, -9, and -10 [232] . pedv infection results in obvious caspase-3 and caspase-8 activation, as well as the cleavage of apoptosis-inducing factor mitochondria-associated 1 (aifm1) and poly adp-ribose polymerase (parp), which leads to apoptotic nuclear fragmentation. pedv spike protein s1 significantly elicits host cell apoptosis, while the nsp1-16 and other structural proteins (m, n, e, s2, and orf3) have none or few effects on cell apoptosis. therefore, s1 protein is probably the critical protein mediating the apoptosis induced by pedv [128] . the multiple stages of cov replication cycle are closely associated with cellular membrane compartments, especially the endoplasmic reticulum (er). the shape and functions of er can be influenced by different physiological states and environmental conditions. when protein synthesis amounts surpass the folding capacity, the accumulation of a large amount of unfolded proteins in the er leads to er stress. consequently, cells manifest a corresponding biological reaction that is widely known as the unfolded protein response (upr) [233] . once the upr is induced, it alleviates the problems by host protein translation inhibition (by the transducer pkr-like er protein kinase (perk)-induced phosphorylation of eif2a), stimulating protein folding. if homeostasis cannot be re-established, apoptosis eventually is triggered. indeed, the activation of upr regulates a wide variety of signaling pathways, such as apoptosis, autophagy, mitogen-activated protein (map) kinase activation, and innate immune response [234] . furthermore, α-cov and β-cov may induce er stress in the infected cells [235] . pedv orf3, as the only accessory protein encoded by pedv, is thought to be related to virus production and virulence of pedv [68] . a series of studies suggest that orf3 plays multiple roles, in addition to acting as an ion channel during pedv replication. recent studies show that pedv orf3 consists of four transmembrane domains (tmds) and localizes in the cytoplasm in the aggregation manner [236] . orf3 is a transmembrane protein, and the confocal microscopy analysis indicates that the aggregated orf3 localizes in the er to induce the er stress associated with either apoptosis or autophagy. however, pedv orf3 induces the autophagy via driving conversion of lc3-i to lc3-ii, but not influencing the apoptosis. orf3-induced autophagy is dependent on er stress response. pedv orf3 triggers er stress response via the up-regulation of grp78 protein expression and the activation of the perk-eif2α signaling pathway. moreover, orf3 protein is identified as an ifn antagonist to block ifn response by an unknown mechanism in pedv-infected cells [65] . the functions of pedv orf3 should be further exploited. pedv has caused epidemic and endemic infections in pig populations in many countries and has become a major economic threat to the swine industry. previous studies have identified viral factors that target key signaling molecules in the rlrs' pathways, as well as viral factors that target the downstream signaling pathways responsible for isgs induction. of the 23 pedv-encoded proteins, at least 10 viral proteins have been identified as type i ifn antagonists [65, 78] . the mechanisms utilized by pedv nsp1, plp2, nsp5, and n protein to antagonize type i ifn production have been clarified (figure 3 , [58, 65, 78, 80, 129] ). however, the specific mechanisms of other viral proteins to inhibit type i ifn production remain largely unknown. at present, 11 pedv proteins have been identified as type iii ifns' antagonists. the suppression of type iii ifn signaling by n protein, nsp1, as well as nsp15, has been reported, while the mechanisms utilized by these viral proteins need to be further investigated. in addition, the covs replication cycle may induce the changes of er stress, cell apoptosis, autophagy pathways, which contain intricate virus-host interactions and cross-talk relationships. thus, more researches for pedv are needed to truly reflect viral evasions from innate immune defenses. the findings of pedv-host interactions will help prevent and control pedv spreading. have been clarified (figure 3 , [58, 65, 78, 80, 129] ). however, the specific mechanisms of other viral proteins to inhibit type i ifn production remain largely unknown. at present, 11 pedv proteins have been identified as type iii ifns' antagonists. the suppression of type iii ifn signaling by n protein, nsp1, as well as nsp15, has been reported, while the mechanisms utilized by these viral proteins need to be further investigated. in addition, the covs replication cycle may induce the changes of er stress, cell apoptosis, autophagy pathways, which contain intricate virus-host interactions and cross-talk relationships. thus, more researches for pedv are needed to truly reflect viral evasions from innate immune defenses. the findings of pedv-host interactions will help prevent and control pedv spreading. removes ubiquitinated conjugates from rig-i; pedv nsp5 induces cleavage of nemo; pedv n protein directly interacts with tbk1 to obstruct the association between tbk1 and irf3; pedv nsp1 causes degradation of cbp and iκbα, as well as inhibition of iκbα phosphorylation and p65 activation. pedv nsp16 inhibits type i ifn production and nsp10 enhances the inhibitory effect of nsp16 on type i ifn production. for type iii ifn, pedv n protein blocks the nuclear translocation of nf-κb; pedv nsp1 blocks the nuclear translocation of irf1 and reduces the amounts of peroxisomes. pedv nsp15 inhibits the type i ifn and type iii ifn responses by unknown mechanisms. pedv nsp7 interacts with stat1 and stat2 to block nuclear translocation of isgf3. removes ubiquitinated conjugates from rig-i; pedv nsp5 induces cleavage of nemo; pedv n protein directly interacts with tbk1 to obstruct the association between tbk1 and irf3; pedv nsp1 causes degradation of cbp and iκbα, as well as inhibition of iκbα phosphorylation and p65 activation. pedv nsp16 inhibits type i ifn production and nsp10 enhances the inhibitory effect of nsp16 on type i ifn production. for type iii ifn, pedv n protein blocks the nuclear translocation of nf-κb; pedv nsp1 blocks the nuclear translocation of irf1 and reduces the amounts of peroxisomes. pedv nsp15 inhibits the type i ifn and type iii ifn responses by unknown mechanisms. pedv nsp7 interacts with stat1 and stat2 to block nuclear translocation of isgf3. author contributions: the manuscript was prepared by s.l., j.y., z.z., and h.z., and reviewed by the co-authors. all authors have read and agreed to the published version of the manuscript. outbreak of porcine epidemic diarrhea in suckling piglets porcine epidemic diarrhea in china experimental infection of pigs with a new porcine enteric coronavirus, cv 777 an apparently new syndrome of porcine epidemic diarrhoea virus-like particles associated with porcine epidemic diarrhoea a new coronavirus-like particle associated with diarrhea in swine updated understanding of the outbreak of 2019 novel coronavirus (2019-ncov) in wuhan discovery of a novel swine enteric alphacoronavirus (seacov) in southern china discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus polymicrobial respiratory disease in pigs porcine aminopeptidase n is a functional receptor for the pedv coronavirus contribution of the porcine aminopeptidase n (cd13) receptor density to porcine epidemic diarrhea virus infection identification and comparison of receptor binding characteristics of the spike protein of two porcine epidemic diarrhea virus strains the moonlighting enzyme cd13: old and new functions to target receptor usage and cell entry of porcine epidemic diarrhea coronavirus public health: broad reception for coronavirus dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc genome organization of porcine epidemic diarrhoea virus porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines genome sequencing and analysis of a novel recombinant porcine epidemic diarrhea virus strain from henan, china sars-cov orf1b-encoded nonstructural proteins 12-16: replicative enzymes as antiviral targets the nonstructural proteins directing coronavirus rna synthesis and processing porcine epidemic diarrhea virus n protein prolongs s-phase cell cycle, induces endoplasmic reticulum stress, and up-regulates interleukin-8 expression role of proteases in the release of porcine epidemic diarrhea virus from infected cells manipulation of the porcine epidemic diarrhea virus genome using targeted rna recombination sars-coronavirus replication/transcription complexes are membrane-protected and need a host factor for activity in vitro structure, function, and evolution of coronavirus spike proteins pathogens 2020 coronavirus spike protein and tropism changes cryo-electron microscopy structure of a coronavirus spike glycoprotein trimer feline aminopeptidase n serves as a receptor for feline, canine, porcine, and human coronaviruses in serogroup i aminopeptidase n is a major receptor for the enteropathogenic coronavirus tgev transmissible gastroenteritis coronavirus, but not the related porcine respiratory coronavirus, has a sialic acid (n-glycolylneuraminic acid) binding activity human aminopeptidase n is a receptor for human coronavirus 229e sequence and phylogenetic analysis of nucleocapsid genes of porcine epidemic diarrhea virus (pedv) strains in china amino acid residues critical for rna-binding in the n-terminal domain of the nucleocapsid protein are essential determinants for the infectivity of coronavirus in cultured cells the nucleocapsid protein of coronavirus infectious bronchitis virus: crystal structure of its n-terminal domain and multimerization properties porcine epidemic diarrhea virus nucleocapsid protein antagonizes beta interferon production by sequestering the interaction between irf3 and tbk1 sars-cov nucleocapsid protein antagonizes ifn-β response by targeting initial step of ifn-β induction pathway, and its c-terminal region is critical for the antagonism mouse hepatitis coronavirus a59 nucleocapsid protein is a type i interferon antagonist the nucleocapsid proteins of mouse hepatitis virus and severe acute respiratory syndrome coronavirus share the same ifn-β antagonizing mechanism: attenuation of pact-mediated rig-i/mda5 activation expression and cellular localisation of porcine transmissible gastroenteritis virus n and m proteins by recombinant vaccinia viruses coronavirus particle assembly: primary structure requirements of the membrane protein porcine epidemic diarrhea virus m protein blocks cell cycle progression at s-phase and its subcellular localization in the porcine intestinal epithelial cells suppression of type i interferon production by porcine epidemic diarrhea virus and degradation of creb-binding protein by nsp1 coronavirus genome structure and replication porcine epidemic diarrhea virus e protein causes endoplasmic reticulum stress and up-regulates interleukin-8 expression pedv orf3 encodes an ion channel protein and regulates virus production porcine epidemic diarrhea virus orf3 gene prolongs s-phase, facilitates formation of vesicles and promotes the proliferation of attenuated pedv the accessory protein orf3 contributes to porcine epidemic diarrhea virus replication by direct binding to the spike protein the coronavirus replicase unique sars-cov protein nsp1: bioinformatics, biochemistry and potential effects on virulence structure of alphacoronavirus transmissible gastroenteritis virus nsp1 has implications for coronavirus nsp1 function and evolution unique and conserved features of genome and proteome of sars-coronavirus, an early split-off from the coronavirus group 2 lineage coronavirus nonstructural protein 1: common and distinct functions in the regulation of host and viral gene expression a conserved region of nonstructural protein 1 from alphacoronaviruses inhibits host gene expression and is critical for viral virulence inhibition of nf-κb activity by the porcine epidemic diarrhea virus nonstructural protein 1 for innate immune evasion the nsp2 replicase proteins of murine hepatitis virus and severe acute respiratory syndrome coronavirus are dispensable for viral replication the papain-like protease of porcine epidemic diarrhea virus negatively regulates type i interferon pathway by acting as a viral deubiquitinase deubiquitinating and interferon antagonism activities of coronavirus papain-like proteases modification of intracellular membrane structures for virus replication viral rna replication in association with cellular membranes sars-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum rna replication of mouse hepatitis virus takes place at double-membrane vesicles mers-coronavirus replication induces severe in vitro cytopathology and is strongly inhibited by cyclosporin a or interferon-α treatment ultrastructure and origin of membrane vesicles associated with the severe acute respiratory syndrome coronavirus replication complex qualitative and quantitative ultrastructural analysis of the membrane rearrangements induced by coronavirus morphogenesis of coronavirus hcov-nl63 in cell culture: a transmission electron microscopic study targeting membrane-bound viral rna synthesis reveals potent inhibition of diverse coronaviruses including the middle east respiratory syndrome virus ultrastructural characterization of membranous torovirus replication factories coronaviruses hijack the lc3-i-positive edemosomes, er-derived vesicles exporting short-lived erad regulators, for replication a contemporary view of coronavirus transcription expression and cleavage of middle east respiratory syndrome coronavirus nsp3-4 polyprotein induce the formation of double-membrane vesicles that mimic those associated with coronaviral rna replication severe acute respiratory syndrome coronavirus nonstructural proteins 3, 4, and 6 induce double-membrane vesicles papain-like protease 1 from transmissible gastroenteritis virus: crystal structure and enzymatic activity toward viral and cellular substrates functional and genetic analysis of coronavirus replicase-transcriptase proteins coronaviruses post-sars: update on replication and pathogenesis porcine epidemic diarrhea virus 3c-like protease regulates its interferon antagonism by cleaving nemo structure of coronavirus main proteinase reveals combination of a chymotrypsin fold with an extra alpha-helical domain structural genomics of the severe acute respiratory syndrome coronavirus: nuclear magnetic resonance structure of the protein nsp7 severe acute respiratory syndrome coronavirus papain-like protease: structure of a viral deubiquitinating enzyme structural basis of severe acute respiratory syndrome coronavirus adp-ribose-1"-phosphate dephosphorylation by a conserved domain of nsp3 the nsp9 replicase protein of sars-coronavirus, structure and functional insights insights into sars-cov transcription and replication from the structure of the nsp7-nsp8 hexadecamer crystal structure of nonstructural protein 10 from the severe acute respiratory syndrome coronavirus reveals a novel fold with two zinc-binding motifs the severe acute respiratory syndrome-coronavirus replicative protein nsp9 is a single-stranded rna-binding subunit unique in the rna virus world structure of the sars-cov nsp12 polymerase bound to nsp7 and nsp8 co-factors imbert, i. one severe acute respiratory syndrome coronavirus protein complex integrates processive rna polymerase and exonuclease activities an arginine-to-proline mutation in a domain with undefined functions within the helicase protein (nsp13) is lethal to the coronavirus infectious bronchitis virus in cultured cells functional screen reveals sars coronavirus nonstructural protein nsp14 as a novel cap n7 methyltransferase discovery of an rna virus 3 ->5 exoribonuclease that is critically involved in coronavirus rna synthesis turkey coronavirus non-structure protein nsp15-an endoribonuclease new antiviral target revealed by the hexameric structure of mouse hepatitis virus nonstructural protein nsp15 mutational analysis of the sars virus nsp15 endoribonuclease: identification of residues affecting hexamer formation early endonuclease-mediated evasion of rna sensing ensures efficient coronavirus replication coronavirus nonstructural protein 15 mediates evasion of dsrna sensors and limits apoptosis in macrophages conventional and unconventional mechanisms for capping viral mrna innate immune restriction and antagonism of viral rna lacking 2-o methylation ribose 2 -o-methylation provides a molecular signature for the distinction of self and non-self mrna dependent on the rna sensor mda5 multiple enzymatic activities associated with severe acute respiratory syndrome coronavirus helicase coronavirus nonstructural protein 16 is a cap-0 binding enzyme possessing (nucleoside-2 o)-methyltransferase activity biochemical and structural insights into the mechanisms of sars coronavirus rna ribose 2 -o-methylation by nsp16/nsp10 protein complex structural basis and functional analysis of the sars coronavirus nsp14-nsp10 complex coronavirus nsp10, a critical co-factor for activation of multiple replicative enzymes mechanisms and enzymes involved in sars coronavirus genome expression porcine epidemic diarrhea virus s1 protein is the critical inducer of apoptosis nucleocapsid interacts with npm1 and protects it from proteolytic cleavage type iii interferon restriction by porcine epidemic diarrhea virus and the role of viral protein nsp1 in irf1 signaling dimerization of coronavirus nsp9 with diverse modes enhances its nucleic acid binding affinity pedv nsp16 negatively regulates innate immunity to promote viral proliferation subcellular localization and effect on type i interferon response of porcine epidemic diarrhea virus nsp7 host factors in coronavirus replication coronavirus endoribonuclease activity in porcine epidemic diarrhea virus suppresses type i and type iii interferon responses innate immunity to virus infection viral rna detection by rig-i-like receptors toll-like receptor and rig-i-like receptor signaling intracellular detection of viral nucleic acids a nonself rna pattern: tri-p to panhandle the rna helicase rig-i has an essential function in double-stranded rna-induced innate antiviral responses interferon induction and function at the mucosal surface the laboratory of genetics and physiology 2: emerging insights into the controversial functions of this rig-i-like receptor lgp2 is a positive regulator of rig-i-and mda5-mediated antiviral responses the rna helicase lgp2 inhibits tlr-independent sensing of viral replication by retinoic acid-inducible gene-i mitochondrial control of innate immunity and inflammation snapshot: pathways of antiviral innate immunity immune evasion of porcine enteric coronaviruses and viral modulation of antiviral innate signaling mavs activates tbk1 and ikkε through trafs in nemo dependent and independent manner ubiquitylation in innate and adaptive immunity structural basis for the activation of innate immune pattern-recognition receptor rig-i by viral rna structural basis of rna recognition and activation by innate immune receptor rig-i structural insights into rna recognition by rig-i ubiquitin-induced oligomerization of the rna sensors rig-i and mda5 activates antiviral innate immune response reconstitution of the rig-i pathway reveals a pivotal role of unanchored polyubiquitin chains in innate immunity deubiquitylation and regulation of the immune response the jak-stat pathway at twenty regulation of interferon-gamma production by il-12 and il-18 an irf-3-, irf-5-, and irf-7-independent pathway of dengue viral resistance utilizes irf-1 to stimulate type i and ii interferon responses interferon-λ in the context of viral infections: production, response and therapeutic implications ifn-λs mediate antiviral protection through a distinct class ii cytokine receptor complex a variant upstream of ifnl3 (il28b) creating a new interferon gene ifnl4 is associated with impaired clearance of hepatitis c virus toll-like receptors and innate immunity the role of transposable elements in the regulation of ifn-λ1 gene expression il-28, il-29 and their class ii cytokine receptor il-28r type iii ifns: beyond antiviral protection to sense or not to sense viral rna-essentials of coronavirus innate immune evasion nucleocapsid protein from porcine epidemic diarrhea virus isolates can antagonize interferon-λ production by blocking the nuclear factor-κb nuclear translocation porcine epidemic diarrhea virus inhibits dsrna-induced interferon-β production in porcine intestinal epithelial cells by blockade of the rig-i-mediated pathway porcine epidemic diarrhea virus infection inhibits interferon signaling by targeted degradation of stat1 the severe acute respiratory syndrome coronavirus nucleocapsid inhibits type i interferon production by interfering with trim25-mediated rig-i ubiquitination nucleocapsid protein of sars coronavirus tightly binds to human cyclophilin a interactions of sars coronavirus nucleocapsid protein with the host cell proteasome subunit p42 severe acute respiratory syndrome-associated coronavirus nucleocapsid protein interacts with smad3 and modulates transforming growth factor-β signaling the nucleocapsid protein of coronavirus mouse hepatitis virus interacts with the cellular heterogeneous nuclear ribonucleoprotein a1 in vitro and in vivo cxcl16 interact with sars-cov n protein in and out cell human coronavirus 229e papain-like proteases have overlapping specificities but distinct functions in viral replication novel beta-barrel fold in the nuclear magnetic resonance structure of the replicase nonstructural protein 1 from the severe acute respiratory syndrome coronavirus severe acute respiratory syndrome coronavirus nsp1 suppresses host gene expression, including that of type i interferon, in infected cells sars coronavirus nsp1 protein induces template-dependent endonucleolytic cleavage of mrnas: viral mrnas are resistant to nsp1-induced rna cleavage severe acute respiratory syndrome coronavirus nsp1 protein suppresses host gene expression by promoting host mrna degradation severe acute respiratory syndrome coronavirus evades antiviral signaling: role of nsp1 and rational design of an attenuated strain structural basis for the inhibition of host gene expression by porcine epidemic diarrhea virus nsp1 mechanisms of activation of interferon regulator factor 3: the role of c-terminal domain phosphorylation in irf-3 dimerization and dna binding virus-dependent phosphorylation of the irf-3 transcription factor regulates nuclear translocation, transactivation potential, and proteasome-mediated degradation an atomic model of the interferon-β enhanceosome viral deubiquitinases: role in evasion of anti-viral innate immunity ubiquitin signaling in immune responses deubiquitinases in the regulation of nf-kappab signaling pathogens 2020 ubiquitin-specific protease 2b negatively regulates ifn-β production and antiviral activity by targeting tank-binding kinase 1 the leader proteinase of foot-and-mouth disease virus negatively regulates the type i interferon pathway by acting as a viral deubiquitinase arterivirus and nairovirus ovarian tumor domain-containing deubiquitinases target activated rig-i to control innate immune signaling the cysteine protease domain of porcine reproductive and respiratory syndrome virus nonstructural protein 2 possesses deubiquitinating and interferon antagonism functions plp2, a potent deubiquitinase from murine hepatitis virus, strongly inhibits cellular type i interferon production sars coronavirus papain-like protease inhibits the type i interferon signaling pathway through interaction with the sting-traf3-tbk1 complex croup is associated with the novel coronavirus nl63 p53 degradation by a coronavirus papain-like protease suppresses type i interferon signaling foot-and-mouth disease virus 3c protease cleaves nemo to impair innate immune signaling hepatitis a virus 3c protease cleaves nemo to impair induction of beta interferon enterovirus 71 3c inhibits cytokine expression through cleavage of the tak1/tab1/tab2/tab3 complex cleavage of the adaptor protein trif by enterovirus 71 3c inhibits antiviral responses mediated by toll-like receptor 3 the coxsackievirus b 3c protease cleaves mavs and trif to attenuate host type i interferon and apoptotic signaling disruption of mda5-mediated innate immune responses by the 3c proteins of coxsackievirus a16, coxsackievirus a6, and enterovirus d68 seneca valley virus suppresses host type i interferon production by targeting adaptor proteins mavs, trif, and tank for cleavage seneca valley virus 3c(pro) abrogates the irf3-and irf7-mediated innate immune response by degrading irf3 and irf7 porcine deltacoronavirus nsp5 inhibits interferon-β production through the cleavage of nemo porcine deltacoronavirus nsp5 antagonizes type i interferon signaling by cleaving stat2 pathogens 2020 the nemo adaptor bridges the nuclear factor-kappab and interferon regulatory factor signaling pathways structure and inhibition of 3c-like protease from porcine epidemic diarrhea virus structural basis for the dimerization and substrate recognition specificity of porcine epidemic diarrhea virus 3c-like protease studies on the molecular mechanism of porcine epidemic diarrhoea virus non-structural protein nsp7 inhibiting ifn-i signaling membranous replication factories induced by plus-strand rna viruses organelle-like membrane compartmentalization of positive-strand rna virus replication factories biochemical and genetic analyses of murine hepatitis virus nsp15 endoribonuclease nidovirus ribonucleases: structures and functions in viral replication old" protein with a new story: coronavirus endoribonuclease is important for evading host antiviral defenses coronavirus non-structural protein 16: evasion, attenuation, and possible treatments an rna cap (nucleoside-2 -o-)-methyltransferase in the flavivirus rna polymerase ns5: crystal structure and functional characterization mutagenesis of coronavirus nsp14 reveals its potential role in modulation of the innate immune response murine hepatitis virus nsp14 exoribonuclease activity is required for resistance to innate immunity hsp27 regulates pro-inflammatory mediator release in keratinocytes by modulating nf-kappab signaling heat shock protein 27 (hsp27): biomarker of disease and therapeutic target comparative proteome analysis of porcine jejunum tissues in response to a virulent strain of porcine epidemic diarrhea virus and its attenuated strain proteome analysis of porcine epidemic diarrhea virus (pedv)-infected vero cells identification of differentially expressed proteins in porcine alveolar macrophages infected with virulent/attenuated strains of porcine reproductive and respiratory syndrome virus epstein-barr virus upregulates phosphorylated heat shock protein 27 kda in carcinoma cells using the phosphoinositide 3-kinase/akt pathway down-regulating heat shock protein 27 is involved in porcine epidemic diarrhea virus escaping from host antiviral mechanism recruitment of hsp70 chaperones: a crucial part of viral survival strategies caspases control antiviral innate immunity porcine epidemic diarrhea virus induces caspase-independent apoptosis through activation of mitochondrial apoptosis-inducing factor caspase function in programmed cell death signal integration in the endoplasmic reticulum unfolded protein response coronavirus infection, er stress, apoptosis and innate immunity regulation of stress responses and translational control by coronavirus porcine epidemic diarrhea virus orf3 protein causes endoplasmic reticulum stress to facilitate autophagy this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we are thankful to everybody who participated in the studies. the authors declare no conflict of interest. key: cord-312001-8p7scli8 authors: majzoub, karim; wrensch, florian; baumert, thomas f. title: the innate antiviral response in animals: an evolutionary perspective from flagellates to humans date: 2019-08-16 journal: viruses doi: 10.3390/v11080758 sha: doc_id: 312001 cord_uid: 8p7scli8 animal cells have evolved dedicated molecular systems for sensing and delivering a coordinated response to viral threats. our understanding of these pathways is almost entirely defined by studies in humans or model organisms like mice, fruit flies and worms. however, new genomic and functional data from organisms such as sponges, anemones and mollusks are helping redefine our understanding of these immune systems and their evolution. in this review, we will discuss our current knowledge of the innate immune pathways involved in sensing, signaling and inducing genes to counter viral infections in vertebrate animals. we will then focus on some central conserved players of this response including toll-like receptors (tlrs), rig-i-like receptors (rlrs) and cgas-sting, attempting to put their evolution into perspective. to conclude, we will reflect on the arms race that exists between viruses and their animal hosts, illustrated by the dynamic evolution and diversification of innate immune pathways. these concepts are not only important to understand virus-host interactions in general but may also be relevant for the development of novel curative approaches against human disease. the animal kingdom, including humans, has evolved while facing constant threats from viral elements. viruses can be, in some cases, beneficial for a given animal species and drive its evolution [1] . however, their uncontrolled replication may cause disease and prove fatal to their hosts. consequently, animal cells have evolved devoted pathways which (1) sense and recognize pathogen-associated molecular patterns (pamps) and, more particularly, virus-associated molecular signatures; (2) initiate signaling cascades stemming from the site of detection, translocating the information to the nucleus; and (3) induce a transcriptional program that confers an antiviral state to the host ( figure 1 ). interestingly, a closer examination of individual factors constituting these pathways shows a different conservation status between different animal species. while genes encoding sensors and signaling platforms are generally well conserved amongst animals, virus-stimulated genes (vsgs) are clearly less so and are subject to faster evolution [2, 3] . in vertebrates, one such vsg is the secreted interferon (ifn) cytokine, that signals in an autocrine and paracrine fashion. secreted ifn molecules bind to cell-surface receptors and initiate signal transduction involving the janus kinase/signal transducer and activator of transcription (jak-stat) pathway. this pathway induces the transcription of a major antiviral program composed of hundreds of so-called ifn-stimulated genes (isgs) that comprise effectors of the cell-autonomous antiviral defense [4] . a lot of our understanding of the innate antiviral immune system in animals is a result of studies conducted in vertebrates and more particularly in mammalian species. therefore, the ifn system has been heavily studied over the last 60 years. however, during the last two decades, we came to appreciate that the ifn system, as we know it, is a vertebrate particularity. indeed, while some animal species like insects or nematodes are devoid of ifns and rely on rna interference (rnai) as the major antiviral pathway, some others, like mollusks, have conserved all the components that lead to ifn production but have no obvious homologs of type i ifn cytokines. nevertheless, these are predicted to use an ifn-like antiviral cytokine [5] . in fact, the ifn cytokine itself seems to be an evolutionary novelty, however, the pathways dictating its production existed early in metazoan evolution [3, 6] (figure 2 ). interestingly, ifn is not the only vsg induced upon viral detection in mammals. certain isgs that directly interfere with the viral life cycle like viperin are also immediately induced after viral infection in an ifn-independent fashion [7] [8] [9] . in this review, we will focus on the conserved pathways that are responsible for sensing viral pamps, signaling and inducing antiviral genes upon infection in animals. we will start by describing our current view on the immediate activation of the ifn and nf-kb pathways in vertebrate species. we will then zoom in on two important viral nucleic acid receptor families, toll-like receptors (tlrs) and rig-i-like receptors (rlrs), describe their function in viral rna detection and their conservation across animal species. next, we will focus on a central hub in the signaling pathways induced by dna viruses, the stimulator of ifn genes (sting). we will then examine how the evolutionary conflicts between viruses and host immune factors are shaping antiviral immunity in animals. viruses and transposable elements are very powerful drivers of evolution [1, 19] . however, their uncontrolled replication and spread can be catastrophic to host cells. it is therefore not surprising that every known living species on the planet has evolved measures to recognize and counteract parasitic genetic elements. it is suggested that anti-sense mediated targeting of viral nucleic acids was the most primordial strategy protocells used to fend off viral threats [18] . this is illustrated by argonaute and crispr-based defenses in bacteria, archaea and rnai systems in plants, all of which rely on anti-sense nucleic acids that program a nuclease to target and degrade the complementary invading viral genome [20] [21] [22] . because many of the core rnai machinery components can be found in all eukaryotic superkingdoms, it is thought that this antiviral defense mechanism predated the emergence of pattern-recognition receptor (prr)-based immunity ( figure 2 ) [23] . interestingly, antiviral defense in eukaryotes has diversified greatly during evolution, with some species maintaining rnai-based defenses [24] and others innovating and adopting completely novel antiviral strategies. in chordates and more particularly in vertebrate animals, the emergence of ifn-i and a recombinational adaptive immune system seems to coincide with the loss of rnai as the main antiviral mechanism in somatic cells. there is strong evidence of an intrinsic incompatibility between an antiviral rnai and the prr-ifn system. for instance, while long dsrnas can produce functional small interfering rnas (sirnas) in stem cells in the absence of ifn, the same dsrna molecule is not processed onto sirna and is sensed as a pamp in vertebrate somatic cells [25, 26] . interestingly, experimental evidence of antiviral rnai in mammals seems to be limited to specialized pluripotent cells in which the prr-ifn system is not fully deployed yet. the emergence of both the ifn-i (innate) and somatic dna recombination systems (adaptive) in vertebrates constituted a major evolutionary event that dispensed them from using rnai for antiviral purposes. interestingly, retrotransposition and selfish transposable elements were determinants in the acquisition of these two systems [3, [27] [28] [29] . the emergence of somatic dna recombination in vertebrate animals was considered an "immunological big bang" [28, 30] . indeed, somatic dna recombination in specialized b and t cell lineages provided jawed vertebrates with large repertoires of major histocompatibility complexes (mhc), t-cell receptors (tcrs) and immunoglobulins (igs). until relatively recently, adaptive immunity was believed to be exclusive to gnathostomes (jawed vertebrates). we now know that agnathans (jawless vertebrates), including lampreys and hagfish, have also evolved an equivalent adaptive immune system with specialized lymphocytes termed, vlra, vlrb and vlrc cells, in which specific variable lymphocyte receptors (vlrs) are produced through somatic leucine-rich repeat (lrr) rearrangements [30] . in both gnathostomes and agnathans, somatic recombination events in specialized cells permitted a pathogen-tailored response and endowed vertebrate species with an immune memory. until the end of the last century, most vertebrate immunologists concentrated their efforts on studying the adaptive arm of the immune system. nearly 30 years ago, charles janeway predicted the presence of an evolutionary ancient immune system that detects conserved microbial and danger signals, termed the pathogen-associated molecular patterns (pamps) and danger-associated molecular patterns (damps), respectively. janeway predicted that this innate immune system precedes and instructs the adaptive system [31, 32] and posited that pamps and damps must be sensed by germ-line encoded prrs. at that time, the innate immune components were severely understudied and tlrs, rlrs and sting's respective functions in immunity were completely unknown. this illustrates the immense leap forward the innate immunity field has experienced in the last three decades. today, we know that vertebrates share with other invertebrate animals specialized phagocytic cells that are able to discriminate between self and non-self. by recognizing general pathogen molecular patterns (pamps, e.g., viral double-stranded rna) and danger signals (damps), these cells establish an immediate and general inflammatory response translated into an antimicrobial and/or antiviral state. although mainly studied in vertebrates, the machineries responsible for these responses transcend this group and are fairly conserved in all animals. we will describe a particular arm of the innate immune pathways, the innate antiviral system that is best studied in mammals. unlike bacteria, viruses represent a unique challenge for prrs because they possess few unique signatures that could serve as pamps [33] . however, viral nucleic acids (dna or rna) could have peculiar biochemical features that differentiate them from endogenous host rna [34] . in rna molecules, for example, the lack of a 7-methylguanosine cap structure, double strandedness or the trior bi-phosphorylation at their ends are often used by prrs for self/non-self-discrimination. prrs that detect viral infection can be classified into four families: tlrs, rlrs, aim2-like receptors (alrs) and the cgas-sting sensors [35] . after viral detection, prr-mediated signaling directly or indirectly induces transcription factors, including ifn-regulatory factors (irfs) and nuclear factor k-b (nf-kb) to upregulate expression of vsgs including pro-inflammatory cytokines. another class of prrs such as double-stranded rna (dsrna) activated protein kinase r (pkr; also known as eif2ak2), adenosine deaminase acting on rna 1 (adar1) and 2 -5 -oligoadenylate synthetase 1 (oas1) also contribute to innate immunity [34] . these also recognize viral signatures; however, their main function is not necessarily to induce a transcriptional immune response, but rather to directly attack viral rna by degrading it or inhibiting its translation. for this reason, these are not usually considered receptors. viruses are obligatory intracellular parasites, therefore their detection by prrs most often occurs in the intracellular milieu. endosomal transmembrane tlrs, including tlr3, tlr7 and tlr8 recognize dsrna in the endosome lumen [36] [37] [38] . rlrs including rig-i [39] , melanoma differentiation associated gene 5 (mda5) [40] , and laboratory of genetics and physiology 2 (lgp2) [41,42] detect viral rnas in the cytosol, whereas cytosolic viral dna is mainly recognized by cyclic-gmp-amp (cgamp) synthase (cgas) [43] . therefore, the nature of the viral particle (e.g., enveloped vs. non-enveloped) and the viral genome (e.g., dna vs. rna) dictates which of these receptors recognizes the infection first ( figure 1 single-stranded rna (ssrna) is a potent tlr7 and tlr8 ligand, while tlr3 is specific for dsrna. tlr3, for example, recognizes dsrna viruses from reoviruses [36] , but can probably also recognize dsrna intermediates from (+) strand rna viruses like coxsackievirus and west nile virus (wnv) and (-) strand rna viruses like the human respiratory syncytial virus (hrsv) [46] . indeed, all rna viruses are thought to produce dsrna intermediates as part of their replication cycle, so both ssrna and dsrna viruses have the potential to be sensed by tlr3. tlr7 and 8, on the other hand, have been shown to prefer ssrna ligands from (-) strand rna viruses such as vesicular stomatitis virus (vsv) and influenza a virus (iav) [37, 47] . when it comes to rlrs, most rna viruses have been shown to be detected by rig-i or mda5, as these receptors have a high affinity to dsrnas. while rig-i prefers viral rnas bearing di-or tri-phosphate groups at their 5 while the cytosolic recognition of viral rna is almost exclusively mediated by rlrs, several proteins have been proposed to play a role in dna sensing and triggering innate immune responses, such as the dna-dependent activator of ifn-regulatory factors (dai), ddx41, rna polymerase iii, ifi16 and dna-pk [62] [63] [64] [65] [66] [67] . however, among all the proposed sensors, only cgas knock-outs can completely shut down ifn production in response to cytosolic dna [43] . the cgas protein is now thought to be the major viral dna sensor and has been shown to detect adenovirus, human papillomavirus (hpv), herpes simplex virus-1 (hsv-1) and cytomegalovirus (cmv) [43, [68] [69] [70] . aim2 has also been shown to activate the inflammasome upon dna stimulation [71] but will not be discussed in this review. rna ligands cause the endosomal transmembrane tlr3, 7 and 8 to dimerize and then to oligomerize through their cytoplasmic tir (toll/il-1 receptor) domains. this allows tlrs to recruit signaling adaptors via tir-tir interactions [35] . tlr3 recruits the adaptor protein trif (tir-domain-containing adapter-inducing ifn-β) [72, 73] . trif is able to play a dual role by inducing the ifn or the nf-kb pathways. when it comes to ifn, after activation, trif recruits the ubiquitin ligase traf3 (tumor necrosis factor receptor-associated factor 3) through an ubiquitination mechanism which in turn recruits tank-binding kinase 1 (tbk1) [74, 75] . the trif/tbk1 complex is then able to phosphorylate the transcription factor irf3, triggering its dimerization and nuclear translocation. phosphorylated irf3 dimers specifically bind to ifn-stimulated response elements (isres) present in the ifn-β gene promoter which leads to the transcription of this cytokine [76] . trif can also recruit ripk1 (receptor-interacting serine/threonine-protein kinase 1) that leads to the activation of the ikk complex, releasing the nf-kb transcription factor from its ikb inhibitory subunit and resulting in its translocation to the nucleus to induce the transcription of pro-inflammatory cytokines [17] (figure 1 ). unlike tlr3, the activation of tlr7 and tlr8 recruits the adaptor protein myd88 (myeloid differentiation primary response 88) through tir-tir domain interaction. myd88 death domains oligomerize which triggers the formation of the myddosome signaling complex consisting of myd88 and the irak family of kinases (il-1 receptor-associated kinases), irak 1, 2 and 4. through a series of phosphorylations and the help of the e3 ubiquitin ligase traf6, the myddosome is able to recruit and activate the transcription factors irf7, irf5 and nf-kb that translocate to the nucleus to induce the transcription of ifn-α genes and other proinflammatory cytokines [77] [78] [79] [80] [81] [82] [83] (figure 1 ). rlrs (rig-i, mda5 and lgp2) are characterized by a central dead-box helicase/atpase domain and a c-terminal regulatory domain (ctd) essential for rna recognition and autorepression in the absence of rna ligands. with the exception of lgp2, rlrs also possess two n-terminal caspase activation and recruitment domains (cards). upon rna binding rig-i is remodeled into an active conformation in which the ctd and helicase domains organize into a ring around the rna ligand and the card domains are exposed [84, 85] which facilitate their interactions with other card domains resulting in rig-i tetramers. although rig-i and mda5 share similar domain architectures, mda5 seems to prefer longer dsrna, assembling along these molecules to form helical, filamentous oligomers [86, 87] . a poly-ubiquitination reaction by ubiquitin ligases like riplet and trim25 (tripartite motif-containing 25), is thought to enhance rig-i and mda5 oligomerization and activation [88] [89] [90] [91] . rig-i and mda5 oligomers then serve as a scaffold for binding to the adaptor protein mavs (mitochondrial antiviral signaling protein, also known as ips-1, visa, and cardif) [92] [93] [94] . mavs has been shown to be critical for mounting an efficient immune response to infection by several rna viruses [95] . its c-terminal transmembrane domain is inserted into the outer mitochondrial membrane [96] , whereas its n-terminal card domain mediates its aggregation on the mitochondrial surface by interacting with the tandem cards of rig-i or mda5 oligomers [97, 98] . mavs aggregates then recruit several e3 ubiquitin ligases including traf2, traf5 and traf6. although traf-mediated ubiquitination is essential to activate mavs downstream signaling, the ubiquitination targets of traf remain unknown [99] . subsequently, the ubiquitin sensor nemo (nf-κb essential modulator, also known as ikkγ) [100, 101] is then recruited to the mavs/trafs complex, which in turn recruits ikk and tbk1 to the mavs complex leading to activation of nf-kb and irf3 and their translocation to the nucleus to induce the transcription of antiviral genes [99] [100] [101] [102] (figure 1 ). after trif and mavs were discovered, sting was identified as a third adaptor protein that is also able to activate irf3 and ifn production [103, 104] . sting is an endoplasmic reticulum (er) resident membrane protein with cytoplasmic c-and n-termini. sting has been shown to be essential for dna-mediated ifn production in different tissues, for example, it is crucial for host defense against the dna virus hsv-1 [105] . sting has also been shown to sense cyclic dinucleotides (cdns), which are the second messengers known to be produced by bacteria such as listeria monocytogenes [106] [107] [108] [109] . although it can bind bacterial cdns, sting is unable to bind dna and relies on an upstream sensor, cgas [43] . cgas is an enzyme that contains a nucleotidyltransferase (ntase) domain and can synthesize the second messenger 2 3 -cyclic gmp-amp (cgamp) from atp and gtp upon dna recognition ( figure 1 ). loss of cgas in various cell lines and also in vivo results in a complete loss of type i ifn induction upon dna delivery or viral infections [110, 111] . cgas preferentially binds longer dna (>45 bp) as a dimer to form stable protein-dna ladder networks responsible for strong cgamp production [112, 113] . a unique cgamp isomer termed 2 3 -cgamp with particular phosphodiester linkages is produced by cgas [114, 115] . 2 3 -cgamp is a potent sting ligand and has a higher affinity to this protein than other cgamp molecules containing different phosphodiester linkages such as 2 2 -cgamp, 3 2 -cgamp or bacterial cdns [70, 115] . apart from activating sting in the cell where cgas initially detects viral dna, cgamp second messengers can also travel to neighboring cells, through gap-junctions [114] or after being packaged in newly formed virions [116, 117] . this intercellular transfer of free or packaged cgamp permits uninfected cells to mount a preventive ifn response, protecting them from infection or providing a faster response to dna viruses that encode cgas antagonists. upon cgamp binding, sting undergoes a conformational change that results in the release of its c-terminal tail (ctt) from its autoinhibitory state and in the formation of sting homodimers that translocate to perinuclear regions to colocalize with tbk1 [105, 118, 119] . tbk1 recruitment results in the phosphorylation of sting and the phosphorylated site serves as a platform for irf3 dimerization and activation which ultimately results in ifn-β induction [120] (figure 1 ). sting has also been shown to induce nf-kb, map kinase and stat6 activation, as well as the stimulation of lc3 puncta formation, a hallmark associated with autophagosome formation [119, [121] [122] [123] . however, the molecular mechanisms by which sting induces these non-ifn responses remain poorly understood. tlrs comprise an ancient family of membrane-spanning receptors that recognize ligands through their extracellular domains and initiate an intracellular response upon stimulation (see above). the toll gene was first identified as a developmentally important gene in drosophila in 1985 [124] . in the mid-1990s the discovery that this gene also plays an essential role in the ability of drosophila to resist fungal infections connected for the first time toll receptors to innate immunity [125, 126] . although in flies toll functions as a cytokine receptor, a human toll receptor (tlr4) was rapidly identified [127, 128] and shown to induce an immune response in mice after induction by lps [129] . we now know that there are ten tlrs in humans that can respond to many bacterial and viral pamps [130] . prototypical tlrs contain three structural elements, a hydrophobic ectodomain containing a variable number of lrrs, a transmembrane domain and a tir domain, which mediates downstream signaling through adaptor proteins [131] . tlrs are likely very ancient immune sentinels since two of their characteristic building blocks (lrr and tir domains) are observed in placozoans (e.g., trichoplax animals) [132] and porifera (e.g., sponges) [131] . full tlrs were detected in cnidarian species, like the starlet sea anemone (nematostella vectensis; one single tlr) [133, 134] and the acroporid corals (acropora digitifera; four tlrs) [135] (figure 2) . interestingly, both developmental and immunological roles of tlrs have been described in cnidarians. tlrs from both the sea anemone (nematostella vectensis) and the mountainous star coral (orbicella faveolata) have been shown to signal via myd88 leading to nf-kb activation [133, 136] . in the bilateria phylum, tlrs can be found in most studied species, however, their numbers vary greatly among species, ranging from a single tlr in nematodes like caenorhabditis elegans, to over two hundred in echinoderms like the pacific purple sea urchin strongylocentrotus purpuratus (figure 2 ). the expansion of the tlr repertoire in some animals like the sea urchin, reflects the adaptation of their immune arsenal to rapidly changing environmental stressors [137] . amongst a multitude of other innate immune factors in this species, such as nacht domain-lrrs and scavenger receptors, sea urchin genomes encode for 222 tlrs. among those, 211 tlrs belong to a greatly expanded set of genes with vertebrate like features, many of which seem to have duplicated recently. the high prevalence of pseudogenes (25% to 30%) among those might reflect a history of strong positive selective pressures. another phylum where tlrs have undergone a significant expansion is in mollusca [138] , like the pacific oyster crassotrea gigas [139] (figure 2 ). the pacific oyster encodes for 83 tlrs in total, potentially reflecting a highly specialized response to environmental challenges and response to pathogens. the spread of pathogens in c. gigas natural habitats occurs very quickly, which is highlighted by the mass mortality events the ostreid herpesvirus 1 (oshv1) has caused in many oyster nurseries. tlr sensing of oshv1 results in the differential regulation of more than a thousand genes, many of which are related to viral infection (e.g., cytosolic dna sensing and dna replication) [5, 139] . in contrast to the very diverse set of tlr repertoires found in other bilateria species (e.g., nematodes, sea urchins and oysters), chordates and more particularly vertebrates contain roughly equal numbers of tlrs, reflecting the reduced need for highly diversified pattern recognition due to the acquisition of adaptive immune components (figure 2 ). in general, vertebrate tlrs can be grouped into six major families [15] . the families responsible for sensing of viral pamps are the tlr3 family, which recognizes dsrna, the tlr7 family (including tlrs 7, 8 and 9) which recognizes nucleic acid motifs and the large tlr11 family (tlr11, 12, 13, 19, 20, 21, 22, 23 and 26) . the reduced number of tlrs in vertebrates does not necessarily mean that the tlr-response in those species cannot be tailored to a particular environment. a peculiar example is tlr22, one of two virus sensing tlrs present in the pufferfish takifugu rubripes. tlr22 is widely conserved among teleosts and amphibians but does not seem to be present in avian or mammalian animals, which indicates that tlr22 might be required only in vertebrates living in water [140] . in mammals, one last case of tlr adaptation and rapid evolution that is worth mentioning comes from bat species. analyses of tlr evolution in bats reveal adaptations acquired by tlrs 3, 7, 8 and 9, with unique mutations fixed in ligand-binding sites [141, 142] . these adaptations are thought to stem from the unique lifestyle of bat species, that are the only known flying mammals, and that represent important viral reservoirs [143] . evolutionary studies paint a complex and dynamic picture of the emergence and functional diversification of rlrs across the animal kingdom. initially, several studies proposed that rig-i and mda5/lgp2 evolved in animals independently through gene fusion and domain grafting events [16, 144] . for instance, it has been proposed that the two card domains have been acquired by rig-i and mda5 in two separate events: the first domain being gained by the ancestor of rig-i and mda5 before their duplication and the second acquired after their divergence [16] . these studies suggested that full-length rlrs are a vertebrate-specific evolutionary novelty, although their building blocks may have been present in closely related invertebrate animals [16, 144] . a more recent study challenges this view and finds that the rlr-based immunity is not vertebrate-specific but originated in the earliest multicellular animals [14] (figure 2 ). in this study, the authors show that rlrs functionally diversified through a series of gene duplication events, followed by protein-coding changes that modulated their rna-binding properties. using homology-based gene prediction based on confirmed human rlrs the authors were able to identify full-length rlrs in early-branching animal genomes, including porifera (e.g., sponges) and cnidaria (e.g., jellyfish). however, they were unable to identify rlrs in non-metazoan eukaryotes, including fungi and choanoflagellates [14] (figure 2 ). it is therefore proposed that the ancestral rlr (rig-i/mda5/lgp2anc) duplicated in bilateria to give rise to rig-i and mda5/lgp2 lineages, followed by a more recent duplication of the mda5/lgp2 ancestor, giving rise to mda5 and lgp2 lineages in jawed vertebrates after their split from jawless vertebrates [14] . the emergence of rlrs early in animal evolution is a very plausible scenario, since other components of the signaling pathways downstream of rlrs, like the irf genes, are also found in early metazoans [6] ( figure 2 ). another recent evidence suggesting that rlrs predated vertebrate evolution comes from studies performed in mollusks (pacific oyster; c. gigas). the invertebrate c. gigas not only encodes up to 12 rlrs, but also mavs, traf6, tbk1 and irf family proteins, which have been shown to have functional antiviral roles [5, 139, 145] (figure 2) . even though there is no consensus on the exact evolutionary history of rlrs, it is clear that these receptors (and/or their building blocks) existed very early in metazoan evolution and most importantly, they are subject to a very dynamic evolution. this is illustrated by the lineage-specific loss of rlr genes in many species. for example, although mda5 and lgp2 homologs were found in many teleost fish, rig-i homologs have only been identified in some fish species like salmon and carp [146] . rig-i is absent in the chicken genome although mda5 and lgp2 are both present [147, 148] . interestingly, chickens suffer severely from avian influenza virus (aiv) infection compared to ducks (that do possess the rig-i gene) which could be due to the loss of rig-i affecting their first line of defense in epithelial cells [148] . most studied mammals possess rig-i, however, it has been lost in at least one mammalian species; the chinese tree shrew [149] . interestingly, with the loss of rig-i, both mda5 and lgp2 have undergone strong positive selection in chinese tree shrews, and positively selected sites in mda5 endowed the substitute function for the lost rig-i [150] . another eloquent example illustrating the dynamic evolution of these receptors is the loss of all rlr genes (rig-i, mda5 and lgp2) in insects (figure 2 ). in drosophila, for example, although the nf-kb and jak/stat pathways are present and contribute to antiviral defenses [151, 152] , all components of the rlr-mavs-irf-axis have been lost. instead, drosophila like other insects and relies on the rnai mechanism as the major antiviral system protecting it from viral infections [24, 153, 154] . interestingly the rnase iii dicer-2, a central player in insect antiviral immunity, responsible for generating small interfering rnas (sirnas), also contains an n-terminal dexd/h-box helicase domain that is highly homologous to the helicase domains of vertebrate rlrs [155, 156] . moreover, dicer-2 has been shown to be responsible for the transcriptional upregulation of an antiviral gene (vago) that could function as a cytokine by activating the jak/stat pathway and triggering systemic antiviral immunity in various mosquito tissues [157, 158] . although the pathway leading to the transcriptional activation of vago is still poorly understood in insects, these studies established that dexd/h-box helicase containing proteins, like dicer and rlrs, may represent an evolutionarily conserved set of viral nucleic acid sensors that direct antiviral responses in animals [159] . one last observation exemplifying the dynamic and rapid evolution of these receptors comes from mammalian species. indeed, rlrs seem to be experiencing very recent adaptive changes in some mammals. for example, rig-i seems to have accumulated adaptive changes altering its rna-binding properties throughout mammalian evolution [160] . moreover, in humans, for example, a number of protein-coding polymorphisms have been identified in rig-i which may contribute to differences in viral susceptibility and risk of autoimmune diseases [14, 161, 162] . sting presence in animal genomes is probably more ancient than that of rlrs, since sting homologs can be found in most animal phyla including unicellular choanoflagellates ( figure 2) [12, 163] . furthermore, the ability of sting to bind cdns seems to be an ancient property. in an elegant study, kranzusch and colleagues show that a sting homolog in the starlet sea anemone n. vectensis (nvsting) is not only structurally very similar to that of human sting but is also able to bind 2 3 cgamp with very high affinity [13] . however, sting's ctt domain, which is crucial for tbk1 recruitment and downstream ifn induction, appeared only in vertebrate species [164] . consequently, nvsting lacking the ctt is unable to induce ifn-β production in response to cdns when transfected in mammalian cells [13] . the lack of a ctt domain in invertebrates does not mean that sting could not have an immune function in these animals. a first indication comes from invertebrate species like the lophotrochozoa phylum that includes the pacific oyster c. gigas and the annelid worm capitella teleta. in these animals, an unusual sting architecture can be found, where a sting domain is fused to a tir domain, known to be involved in innate immune signaling [164, 165] . the second indication that sting lacking a ctt could function in immunity comes from arthropods. recent studies in drosophila, that lack an ifn system, clearly show that sting is important for antimicrobial and antiviral nf-kb activation in this model [151, 166] (figure 2) . interestingly, the emergence of the ctt domain of sting in vertebrate species seems to coincide with the development of the ifn system. nevertheless, sting ctt domain function, which dictates downstream signaling, seems to be plastic amongst vertebrate species. in a recent study, authors show that sting ctt-dependent activation of irf3 and nf-kb varies between vertebrate species [167] . while sting ctt from mammalian species is able to induce a strong ifn-β and a weaker nf-kb response, an extension of this domain in ray-finned fish species elicits a dramatic enhancement of nf-kb activation and weaker irf3-ifn signaling [167] . another indication of sting ctt structure-function plasticity comes from bat species. a highly conserved and functionally important serine residue (s358) in sting's ctt domain is lost in bats [168] . the replacement of this critical residue in this mammalian species significantly dampens sting-dependent ifn activation. the authors of this study suggest that the lifestyle of bat species (e.g., flight induced cytosolic dna, high viral titers) imposes a strong selective pressure on sting. this results in functionally dampened sensing and signaling mechanisms to avoid ifn overactivation and to cope with high cytosolic dna content. taken together, present studies suggest an evolutionarily ancient role of sting in antiviral immunity and modulation of its structure and function to accommodate species-specific pathogen burdens. the picture is less clear for the cgas enzyme when it comes to antiviral immunity. although cgas homologs have been identified in a variety of ancient metazoan lineages [12, 163] , it is believed that the ability of cgas to bind and detect dsdna emerged in vertebrates. indeed, cgas' zinc-ribbon domain, required for dna binding and cgamp synthesis in response to dna in the cytosol, seems to be a vertebrate innovation [169] [170] [171] . interestingly, primate cgas seems to have undergone rapid evolution in this lineage, as observed by the positive selection at its nucleic acid binding interfaces [172] . these studies argue that although the cgas enzyme existed early in metazoans, its function has been repurposed for dna sensing only recently in vertebrates. clearly, cgas and sting seem to have acquired novel features throughout evolution. specifically in vertebrates cgas evolved the zinc ribbon motif to detect dna and sting evolved the ctt domain that expanded its signaling potential. as obligate intracellular parasites, viruses have evolved an array of evasion mechanisms to escape their elimination by the host's immune system. interestingly, viral antagonism is a general strategy and is not a peculiarity of animal viruses. many bacteriophages, for instance, encode crispr-cas inhibitors, termed anti-crisprs, to counter prokaryotic antiviral systems [173] . plant viruses also encode viral suppressors of rnai (vsrs) the main antiviral system in plant cells [20] . likewise, several evasion strategies and immune antagonisms by animal viruses have been described [174] [175] [176] [177] [178] . these include hiding the viral genome from immune detection, shutting off host translation or transcription machineries, inhibiting host rna processing and trafficking and interfering directly with either proteins that sense viral presence, or factors that signal the information to the nucleus. since interfering with the innate immune system is less damaging for the host than targeting vital cellular machineries (e.g., translation), many studied viruses seem to have opted for this strategy. several studies describe viral evasion mechanisms at both the recognition and sensing step (tlrs, rlrs and cgas-sting) or at the downstream signaling steps through the targeting of proteins such as mavs, tbk1, irf3, irf7 and nf-κb. evasion strategies and immune antagonisms by animal viruses are a very active area of research, that have yielded a rich literature in the past few years. we will here just give some select examples of viral strategies that curb sensing and signaling by tlrs, rlrs and cgas-sting in animals, with an obvious bias towards viruses infecting humans. for a more complete picture on the subject, readers can refer to excellent reviews, published recently, describing those strategies [174] [175] [176] [177] [178] . tlr signaling has been shown to be inhibited by the vaccinia virus (vacv) protein a46r, that targets specific tir-domain-containing adaptor proteins. a46r itself contains a tir domain which allows it to competitively interact with tir-domain-containing complexes such as myd88, trif or tram, thereby inhibiting the activation of both nfkb and irfs [179, 180] . human t-cell leukemia virus type-1 (htlv-1) is also able to interfere with tlr4-dependent signaling. the htlv-1 encoded viral protein p30 binds and disables a transcription factor, pu.1, required for tlr4 surface expression [181] . trif, an important player in the tlr signaling cascade, is a target of choice of many viruses. the ns3/4a protease of hcv and the 3c proteases of several picornaviruses such as coxsackievirus b, ev71 and hepatitis a virus (hav), can all recognize and proteolytically cleave trif, producing trif fragments that are unable to signal [182] [183] [184] [185] [186] . when it comes to rlrs, one basic strategy used by cytosolic viruses to escape surveillance is to simply prevent these receptors from accessing viral genomes. denv, for example, replicates in convoluted membranes of the er concealing its dsrna intermediates from the cytosol and thereby prevents the activation of rlrs [187] . other viruses like ebola virus (ebov) and marburg viruses encode viral protein 35 (vp35) that tightly binds and 'shields' the viral genome from detection by rig-i [188, 189] . another strategy used by viruses to 'hide' from rlrs consists of modifying the very molecular features these receptors rely on to recognize viral genomes. for example, both, hantaan viruses from the bunyaviridae family and borna disease virus (bdv) from the bornaviridae family, encode phosphatases that process the triphosphate group at their 5 genome termini, to a 5 -monophosphate to escape rig-i surveillance [190, 191] . lassa virus (lasv) from the arenaviridae family evolved a unique strategy in which its nucleoprotein (np) acquired a 3 -5 exonuclease activity, that enables it to digest free dsrna, preventing the activation of rig-i [192] . however, the most direct way of interfering with rlr function and their signaling partners is to either directly target them for cleavage and degradation or to manipulate their phosphorylation and ubiquitination statuses, which are crucial for their activation. indeed, many viruses encode proteases that directly cleave rlrs. while the 3cpro proteases of both poliovirus and ev71 cleave rig-i, the 2apro of ev71 cleaves mda5 [193, 194] . mavs, a crucial hub for both rig-i and mda5-mediated signaling is also frequently targeted and cleaved by numerous viral proteases, such as 3cpro from hav, 2apro from ev71, ns3-ns4a from hcv, 2apro and 3cpro from rhinovirus and 3cpro from coxsackievirus b3 (cvb3) [184, 185, [194] [195] [196] . mavs can also be indirectly degraded by particular viruses. for instance, measles virus (mev) can trigger a selective form of autophagy, called mitophagy, responsible for the degradation of mitochondria, which leads to a decrease of mavs abundance [197] . another example of indirect mavs degradation comes from studies with severe acute respiratory syndrome (sars)-associated coronavirus (sars-cov). this virus has evolved a strategy in which its 9b protein localizes to mitochondria and subverts the cellular e3 ubiquitin ligase atrophin-1-interacting protein 4 (aip4) to degrade mavs [198] . post-translational modifications of both mavs and rlrs have also been shown to be subverted by viruses to inhibit their downstream signaling. ns1 proteins from many influenza a virus strains (iav) interact with the host ubiquitin ligase trim25 and inhibit its oligomerization, a crucial step for its enzymatic activity of attaching lys63-linked polyubiquitin to the card domains of rig-i [24, 199] . other viruses encode deubiquitinating enzymes (dubs) to remove the lys63-linked ubiquitination off rig-i. orf64 from kaposi's sarcoma herpesvirus (kshv), papain-like protease (plp) from sars-cov, leader proteinase (lpro) from foot-and-mouth disease virus (fmdv) and the ovarian tumor (otu)-type proteins of arteriviruses and nairoviruses have all been shown to possess a deubiquitination activity and interfere with rig-i mediated signaling [177, [200] [201] [202] . rig-i and mda5 phosphorylation status can also be subverted by viruses. in normal conditions, phosphorylation of serine or threonine residues keeps rig-i and mda5 in an inactive state. upon viral infection, pp1 phosphatases are recruited to dephosphorylate specific marks on those receptors and activate them. v proteins from measles and nipah viruses (mev and niv) act as decoys and have been shown to bind pp1-α and pp1-γ, sequestering them away from mda5 and rig-i [203, 204] . similar to evasion strategies that counter the rna sensing machinery described earlier, dna viruses use numerous strategies to escape cgas-sting-dependent detection and signaling. they could either hide their viral genomes or cleave, degrade, post-translationally modify or even relocalize dna sensing and signaling factors [205] . hepatitis b virus (hbv), that causes chronic hepatitis and increases the risk of developing liver cirrhosis and hepatocellular carcinoma, has developed an array of mechanisms to inhibit the host's immune systems (reviewed in [206] ). notably, the hbv polymerase can bind to sting to block its lys63-linked ubiquitination, inhibiting the production of ifn-β [207] . moreover, even though cgas is expressed in human hepatocytes and is able to sense and signal upon transfection of naked relaxed-circular hbv dna; during a natural infection, hbv dna seems to escape cgas detection, likely due to packaging of the genome into the viral capsid [208] . kshv, another dna virus has been shown to act on both cgas and sting. several kshv proteins (e.g., orf52 and lana) can either sequestrate stimulatory dna or directly bind to cgas inhibiting its enzymatic activity [209, 210] . kshv has been also shown to encode a viral interferon regulatory factor (virf1) that interacts with sting thereby preventing tbk1 binding and sting activation by tbk1-dependent phosphorylation [211] . the ns3 protease of denv, together with its ns2b co-factor, has been shown to target the residues 93-96 (lrrg) of human sting, leading to its cleavage and degradation [212, 213] . interestingly, mouse sting lacks these lrrg residues, and ns2b/ns3 of denv is neither able to cleave the murine sting, nor to block murine ifn-β production. therefore, it has been proposed that the inability of denv to cleave mouse sting might explain its host tropism, as murine cells are not very susceptible to denv infection [212, 213] . in animals, prrs and their associated signaling pathways are early and potent cellular sensors of viral elements, that mobilize the organism's defenses by inducing an antiviral state. major advances have been made in the last two decades in the understanding of their function in mammalian immunity. new genomics data and gene editing tools can now let us interrogate prr-like pathways in poorly studied animal species and define their evolutionary trajectories. studying the evolution of immune components and their interplay with viral pathogens is extremely important since our immune responses to contemporary viruses have been shaped by our evolutionary responses to previous infections. the modern innate immune system is generally not yet optimized against modern viruses but rather was selected for by previous rounds of co-evolution with ancient viruses [214] . studying the biological arms race between host and virus, referred to as the "red queen hypothesis" [215] , in which each entity maintains a relatively constant fitness cost, will be instrumental in the fight against future infections. such studies will help us understand many aspects of viral infections including viral zoonoses, tropism, global epidemics and disease progression. furthermore, exploring these pathways and mechanisms for therapeutic purposes may offer novel strategies to cure human disease. indeed, modulating the action of the aforementioned immune sensors is proving to be an effective strategy to develop vaccines and vaccine adjuvants [216] [217] [218] [219] [220] or to treat viral infections [221] [222] [223] [224] [225] [226] . finally, the use of tlr, rlr and sting modulators, to treat inflammation, auto-immune disease [227, 228] and also in cancer immunotherapy [229] [230] [231] [232] [233] [234] [235] [236] provides an eloquent incentive to continue studying these pathways and to look ahead with great optimism. a virocentric perspective on the evolution of life evolution of innate immunity: clues from invertebrates via fish to mammals evolution of interferons and interferon receptors transcriptional regulation of antiviral interferon-stimulated genes antiviral defense and innate immune memory in the oyster dynamic evolution of immune system regulators: the history of the interferon regulatory factor family ifn regulatory factor-1 bypasses ifn-mediated antiviral effects through viperin gene induction constitutive expression of an isgf2/irf1 transgene leads to interferon-independent activation of interferon-inducible genes and resistance to virus infection transcriptional profiling of interferon regulatory factor 3 target genes: direct involvement in the regulation of interferon-stimulated genes phylogenetic analysis of the endoribonuclease dicer family early origins and evolution of micrornas and piwi-interacting rnas in animals molecular evolutionary and structural analysis of the cytosolic dna sensor cgas and sting ancient origin of cgas-sting reveals mechanism of universal 2 ,3 cgamp signaling ancient origins of vertebrate-specific innate antiviral immunity the evolution of vertebrate toll-like receptors evolution of mda-5/rig-i-dependent innate immunity: independent evolution by domain grafting nf-kappab signaling pathways in mammalian and insect innate immunity the evolution of antiviral defense systems the frustrated gene: origins of eukaryotic gene expression antiviral immunity directed by small rnas crispr-cas immunity in prokaryotes the evolutionary journey of argonaute proteins on the origin and functions of rna-mediated silencing: from protists to man rna interference to treat virus infections specific interference with gene expression induced by long, double-stranded rna in mouse embryonal teratocarcinoma cell lines antiviral rna interference in mammalian cells transposition mediated by rag1 and rag2 and its implications for the evolution of the immune system primordial emergence of the recombination activating gene 1 (rag1): sequence of the complete shark gene indicates homology to microbial integrases evolution of immune systems from viruses and transposable elements re-evaluation of the immunological big bang pattern recognition receptors and control of adaptive immunity approaching the asymptote? evolution and revolution in immunology crosstalk between cytoplasmic rig-i and sting sensing pathways discriminating self from non-self in nucleic acid sensing innate immune pattern recognition: a cell biological perspective recognition of double-stranded rna and activation of nf-kappab by toll-like receptor 3 recognition of single-stranded rna viruses by toll-like receptor 7 human tlr-7-, -8-, and -9-mediated induction of ifn-alpha/beta and -lambda is irak-4 dependent and redundant for protective immunity to viruses the rna helicase rig-i has an essential function in double-stranded rna-induced innate antiviral responses inhibition of the rna polymerase iii-mediated dsdna-sensing pathway of innate immunity by vaccinia virus protein e3 rna polymerase iii detects cytosolic dna and induces type i interferons through the rig-i pathway rig-i-dependent sensing of poly(da:dt) through the induction of an rna polymerase iii-transcribed rna intermediate dna-pk is a dna sensor for irf-3-dependent innate immunity dlm-1/zbp1) is a cytosolic dna sensor and an activator of innate immune response the interferon response to intracellular dna: why so many receptors? immunobiology ifi16 is an innate immune sensor for intracellular dna the helicase ddx41 senses intracellular dna mediated by the adaptor sting in dendritic cells adenovirus detection by the cgas/sting/tbk1 dna sensing cascade pan-viral specificity of ifn-induced genes reveals new roles for cgas in innate immunity cyclic gmp-amp is an endogenous second messenger in innate immune signaling by cytosolic dna aim2 recognizes cytosolic dsdna and forms a caspase-1-activating inflammasome with asc ticam-1, an adaptor molecule that participates in toll-like receptor 3-mediated interferon-beta induction role of adaptor trif in the myd88-independent toll-like receptor signaling pathway specificity in toll-like receptor signalling through distinct effector functions of traf3 and traf6 critical role of traf3 in the toll-like receptor-dependent and -independent antiviral response type i interferon [corrected] gene induction by the interferon regulatory factor family of transcription factors cutting edge: tnfr-associated factor (traf) 6 is essential for myd88-dependent pathway but not toll/il-1 receptor domain-containing adaptor-inducing ifn-beta (trif)-dependent pathway in tlr signaling interferon-alpha induction through toll-like receptors involves a direct interaction of irf7 with myd88 and traf6 helical assembly in the myd88-irak4-irak2 complex in tlr/il-1r signalling protein kinase ikkbeta-catalyzed phosphorylation of irf5 at ser462 induces its dimerization and nuclear translocation in myeloid cells an oligomeric signaling platform formed by the toll-like receptor signal transducers myd88 and irak-4 ikkbeta is an irf5 kinase that instigates inflammation interleukin-1 receptor-associated kinase-1 plays an essential role for toll-like receptor (tlr)7-and tlr9-mediated interferon-{alpha} induction structural basis of rna recognition and activation by innate immune receptor rig-i structural basis for the activation of innate immune pattern-recognition receptor rig-i by viral rna mda5 assembles into a polar helical filament on dsrna cooperative assembly and dynamic disassembly of mda5 filaments for viral dsrna recognition trim25 ring-finger e3 ubiquitin ligase is essential for rig-i-mediated antiviral activity ubiquitin-induced oligomerization of the rna sensors rig-i and mda5 activates antiviral innate immune response riplet/rnf135, a ring finger protein, ubiquitinates rig-i to promote interferon-beta induction during the early phase of viral infection the ubiquitin ligase riplet is essential for rig-i-dependent innate immune responses to rna virus infection ips-1, an adaptor triggering rig-i-and mda5-mediated type i interferon induction cardif is an adaptor protein in the rig-i antiviral pathway and is targeted by hepatitis c virus identification and characterization of mavs, a mitochondrial antiviral signaling protein that activates nf-kappab and irf 3 essential role of ips-1 in innate immune responses against rna viruses an autoinhibitory mechanism modulates mavs activity in antiviral innate immune response mavs forms functional prion-like aggregates to activate and propagate antiviral innate immune response structural basis for the prion-like mavs filaments in antiviral innate immunity mavs recruits multiple ubiquitin e3 ligases to activate antiviral signaling cascades activation of ikk by tnfalpha requires site-specific ubiquitination of rip1 and polyubiquitin binding by nemo sensing of lys 63-linked polyubiquitination by nemo is a key event in nf-kappab activation key role of ubc5 and lysine-63 polyubiquitination in viral activation of irf3 sting is an endoplasmic reticulum adaptor that facilitates innate immune signalling the adaptor protein mita links virus-sensing receptors to irf3 transcription factor activation sting regulates intracellular dna-mediated, type i interferon-dependent innate immunity sting is a direct innate immune sensor of cyclic di-gmp coordinated regulation of accessory genetic elements produces cyclic di-nucleotides for v. cholerae virulence mpys is required for ifn response factor 3 activation and type i ifn production in the response of cultured phagocytes to bacterial second messengers cyclic-di-amp and cyclic-di-gmp the n-ethyl-n-nitrosourea-induced goldenticket mouse mutant reveals an essential function of sting in the in vivo interferon response to listeria monocytogenes and cyclic dinucleotides cyclic gmp-amp synthase is an innate immune sensor of hiv and other retroviruses pivotal roles of cgas-cgamp signaling in antiviral defense and immune adjuvant effects cgas senses long and hmgb/tfam-bound u-turn dna by forming protein-dna ladders cyclic gmp-amp synthase is activated by double-stranded dna-induced oligomerization cgas produces a 2 -5 -linked cyclic dinucleotide second messenger that activates sting cyclic gmp-amp containing mixed phosphodiester linkages is an endogenous high-affinity ligand for sting viruses transfer the antiviral second messenger cgamp between cells transmission of innate immune signaling by packaging of cgamp in viral particles structure-function analysis of sting activation by c[g(2 ,5 )pa(3 ,5 )p] and targeting by antiviral dmxaa atg9a controls dsdna-driven dynamic translocation of sting and the innate immune response phosphorylation of innate immune adaptor proteins mavs, sting, and trif induces irf3 activation activation of stat6 by sting is critical for antiviral innate immunity cytosolic-dna-mediated, sting-dependent proinflammatory gene induction necessitates canonical nf-kappab activation through tbk1 activation of autophagy by alpha-herpesviruses in myeloid cells is mediated by cytoplasmic viral dna through a mechanism dependent on stimulator of ifn genes establishment of dorsal-ventral polarity in the drosophila embryo: the induction of polarity by the toll gene product signals from the il-1 receptor homolog, toll, can activate an immune response in a drosophila hemocyte cell line the dorsoventral regulatory gene cassette spatzle/toll/cactus controls the potent antifungal response in drosophila adults chromosomal localization of til, a gene encoding a protein related to the drosophila transmembrane receptor toll, to human chromosome 4p14 prediction of the coding sequences of unidentified human genes. i. the coding sequences of 40 new genes (kiaa0001-kiaa0040) deduced by analysis of randomly sampled cdna clones from human immature myeloid cell line kg-1 defective lps signaling in c3h/hej and c57bl/10sccr mice: mutations in tlr4 gene the role of pattern-recognition receptors in innate immunity: update on toll-like receptors evolutionary origins of toll-like receptor signaling innate immunity in the simplest animals-placozoans sea anemone model has a single toll-like receptor that can function in pathogen detection, nf-kappab signal transduction, and development the innate immune repertoire in cnidaria-ancestral complexity and stochastic gene loss differential and convergent utilization of autophagy components by positive-strand rna viruses a conserved toll-like receptor-to-nf-kappab signaling pathway in the endangered coral orbicella faveolata genomic insights into the immune system of the sea urchin massively parallel rna sequencing identifies a complex immune gene repertoire in the lophotrochozoan mytilus edulis massive expansion and functional divergence of innate immune genes in a protostome teleost tlr22 recognizes rna duplex to induce ifn and protect cells from birnaviruses adaptive evolution of virus-sensing toll-like receptor 8 in bats the evolution of bat nucleic acid-sensing toll-like receptors immune system modulation and viral persistence in bats: understanding viral spillover origin and evolution of the rig-i like rna helicase gene family characterization of the mollusc rig-i/mavs pathway reveals an archaic antiviral signalling framework in invertebrates retinoic acid-inducible gene i (rig-i)-like receptors (rlrs) in fish: current knowledge and future perspectives chicken cells sense influenza a virus infection through mda5 and cardif signaling involving lgp2 association of rig-i with innate immunity of ducks to influenza genome of the chinese tree shrew loss of rig-i leads to a functional replacement with mda5 in the chinese tree shrew the kinase ikkbeta regulates a sting-and nf-kappab-dependent antiviral response pathway in drosophila the jak-stat signaling pathway is required but not sufficient for the antiviral response of drosophila the rna silencing endonuclease argonaute 2 mediates specific antiviral immunity in drosophila melanogaster essential function in vivo for dicer-2 in host defense against rna viruses in drosophila sensing viral rnas by dicer/rig-i like atpases across species the dexd/h-box helicase dicer-2 mediates the induction of antiviral activity in drosophila secreted vago restricts west nile virus infection in culex mosquito cells by activating the jak-stat pathway dicer-2-dependent activation of culex vago occurs via the traf-rel2 signaling pathway nucleic acid sensing in invertebrate antiviral immunity the rig-i atpase core has evolved a functional requirement for allosteric stabilization by the pincer domain the selective footprints of viral pressures at the human rig-i-like receptor family evolution and functional impact of rare coding variation from deep sequencing of human exomes cyclic di-nucleotide signaling enters the eukaryote domain evolutionary origins of cgas-sting signaling toll signaling: the tireless quest for specificity analysis of drosophila sting reveals an evolutionarily conserved antimicrobial function modular architecture of the sting c-terminal tail allows interferon and nf-kappab signaling adaptation dampened sting-dependent interferon activation in bats structure of human cgas reveals a conserved family of second-messenger enzymes in innate immunity ] is the metazoan second messenger produced by dna-activated cyclic gmp-amp synthase structural mechanism of cytosolic dna sensing by cgas overlapping patterns of rapid evolution in the nucleic acid sensors cgas and oas1 suggest a common mechanism of pathogen antagonism and escape the discovery, mechanisms, and evolutionary impact of anti-crisprs decoding type i and iii interferon signalling during viral infection viral evasion of dna-stimulated innate immune responses ten strategies of interferon evasion by viruses viral evasion of intracellular dna and rna sensing viral evasion and subversion of pattern-recognition receptor signalling a46r and a52r from vaccinia virus are antagonists of host il-1 and toll-like receptor signaling vaccinia virus protein a46r targets multiple toll-like-interleukin-1 receptor adaptors and contributes to virulence the htlv-i p30 interferes with tlr4 signaling and modulates the release of pro-and anti-inflammatory cytokines from human macrophages innate immunity evasion by enteroviruses: insights into virus-host interaction toll-like receptors in antiviral innate immunity hepatitis c virus protease ns3/4a cleaves mitochondrial antiviral signaling protein off the mitochondria to evade innate immunity the coxsackievirus b 3c protease cleaves mavs and trif to attenuate host type i interferon and apoptotic signaling disruption of tlr3 signaling due to cleavage of trif by the hepatitis a virus protease-polymerase processing intermediate the dengue virus conceals double-stranded rna in the intracellular membrane to escape from an interferon response ebola virus vp35 protein binds double-stranded rna and inhibits alpha/beta interferon production induced by rig-i signaling structural basis for marburg virus vp35-mediated immune evasion mechanisms sequestration by ifit1 impairs translation of 2 o-unmethylated capped rna old world hantaviruses do not produce detectable amounts of dsrna in infected cells and the 5 termini of their genomic rnas are monophosphorylated structure of the lassa virus nucleoprotein reveals a dsrna-specific 3 to 5 exonuclease activity essential for immune suppression rig-i is cleaved during picornavirus infection enterovirus 2apro targets mda5 and mavs in infected cells cleavage of ips-1 in cells infected with human rhinovirus disruption of innate immunity due to mitochondrial targeting of a picornaviral protease precursor mitophagy enhances oncolytic measles virus replication by mitigating ddx58/rig-i-like receptor signaling sars-coronavirus open reading frame-9b suppresses innate immunity by targeting mitochondria and the mavs/traf3/traf6 signalosome influenza a virus ns1 targets the ubiquitin ligase trim25 to evade recognition by the host viral rna sensor rig-i deubiquitinating and interferon antagonism activities of coronavirus papain-like proteases inhibition of rig-i-mediated signaling by kaposi's sarcoma-associated herpesvirus-encoded deubiquitinase orf64 deubiquitinase function of arterivirus papain-like protease 2 suppresses the innate immune response in infected host cells antagonism of the phosphatase pp1 by the measles virus v protein is required for innate immune escape of mda5 measles virus suppresses rig-i-like receptor activation in dendritic cells via dc-sign-mediated inhibition of pp1 phosphatases the cgas-sting defense pathway and its counteraction by viruses immune evasion strategies during chronic hepatitis b and c virus infection. vaccines (basel) 2017, 5, 24 hepatitis b virus polymerase disrupts k63-linked ubiquitination of sting to block innate cytosolic dna-sensing pathways hepatitis b virus evasion from cyclic guanosine monophosphate-adenosine monophosphate synthase sensing in human hepatocytes inhibition of cgas dna sensing by a herpesvirus virion protein cytoplasmic isoforms of kaposi sarcoma herpesvirus lana recruit and antagonize the innate immune dna sensor cgas modulation of the cgas-sting dna sensing pathway by gammaherpesviruses denv inhibits type i ifn production in infected cells by cleaving human sting dengue virus targets the adaptor protein mita to subvert host innate immunity evolutionary conflicts between viruses and restriction factors shape immunity the evolutionary conundrum of pathogen mimicry enhanced influenza virus-like particle vaccination with a structurally optimized rig-i agonist as adjuvant pika as an adjuvant enhances specific humoral and cellular immune responses following the vaccination of mice with hbsag plus pika a tlr3 ligand that exhibits potent inhibition of influenza virus replication and has strong adjuvant activity has the potential for dual applications in an influenza pandemic as04, an aluminum salt-and tlr4 agonist-based adjuvant system, induces a transient localized innate immune response leading to enhanced adaptive immunity the tlr4 agonist, monophosphoryl lipid a, attenuates the cytokine storm associated with respiratory syncytial virus vaccine-enhanced disease sting agonists enable antiviral cross-talk between human cells and confer protection against genital herpes in mice the tlr4 antagonist eritoran protects mice from lethal influenza infection targeting innate immunity for antiviral therapy through small molecule agonists of the rlr pathway direct antiviral properties of tlr ligands against hbv replication in immune-competent hepatocytes safety, efficacy and pharmacodynamics of vesatolimod (gs-9620) in virally suppressed patients with chronic hepatitis b antibody and tlr7 agonist delay viral rebound in shiv-infected monkeys therapeutic effects of the artemisinin analog sm934 on lupus-prone mrl/lpr mice via inhibition of tlr-triggered b-cell activation and plasma cell formation tak-242 (resatorvid), a small-molecule inhibitor of toll-like receptor (tlr) 4 signaling, binds selectively to tlr4 and interferes with interactions between tlr4 and its adaptor molecules essential for the antitumor effect of immune checkpoint blockade sa-4-1bbl and monophosphoryl lipid a constitute an efficacious combination adjuvant for cancer vaccines magnitude of therapeutic sting activation determines cd8(+) t cell-mediated anti-tumor immunity immunogene therapy using immunomodulating hvj-e vector augments anti-tumor effects in murine malignant glioma promising targets for cancer immunotherapy: tlrs, rlrs, and sting-mediated innate immune pathways immunogenic cell death of human ovarian cancer cells induced by cytosolic poly(i:c) leads to myeloid cell maturation and activates nk cells sting-mediated dna sensing promotes antitumor and autoimmune responses to dying cells sting agonist formulated cancer vaccines can cure established tumors resistant to pd-1 blockade this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license funding: this work has been supported by the h2020 marie-curie actions msca-if-792661-hipshot (km). this work was also supported in part by the national institutes of health grants u19-ai123862 (tfb) by arc, paris and institut hospitalo-universitaire, strasbourg (therahcc and therahcc2.0 ihuarc ihu201301187 and ihuarc2019 to t. acknowledgments: the authors would like to thank jean-luc imler and joao t. marques for their critical reading of the manuscript. the authors apologize to colleagues whose work could not be cited due to space limitations. the authors declare no conflict of interest. key: cord-299754-tgexahwd authors: van tol, sarah; hage, adam; giraldo, maria isabel; bharaj, preeti; rajsbaum, ricardo title: the trimendous role of trims in virus–host interactions date: 2017-08-22 journal: vaccines (basel) doi: 10.3390/vaccines5030023 sha: doc_id: 299754 cord_uid: tgexahwd the innate antiviral response is integral in protecting the host against virus infection. many proteins regulate these signaling pathways including ubiquitin enzymes. the ubiquitin-activating (e1), -conjugating (e2), and -ligating (e3) enzymes work together to link ubiquitin, a small protein, onto other ubiquitin molecules or target proteins to mediate various effector functions. the tripartite motif (trim) protein family is a group of e3 ligases implicated in the regulation of a variety of cellular functions including cell cycle progression, autophagy, and innate immunity. many antiviral signaling pathways, including type-i interferon and nf-κb, are trim-regulated, thus influencing the course of infection. additionally, several trims directly restrict viral replication either through proteasome-mediated degradation of viral proteins or by interfering with different steps of the viral replication cycle. in addition, new studies suggest that trims can exert their effector functions via the synthesis of unconventional polyubiquitin chains, including unanchored (non-covalently attached) polyubiquitin chains. trim-conferred viral inhibition has selected for viruses that encode direct and indirect trim antagonists. furthermore, new evidence suggests that the same antagonists encoded by viruses may hijack trim proteins to directly promote virus replication. here, we describe numerous virus–trim interactions and novel roles of trims during virus infections. eukaryotes are constantly exposed to a variety of pathogens, including viruses. as with other environmental signals, viral invasion triggers tightly regulated intracellular signaling cascades to optimally respond to infection. mammals enact both an innate and an adaptive immune response to identify an infecting pathogen, to clear the foreign agent, and to protect against subsequent invasion. a primary mechanism for fine-tuning molecular pathways is utilization of post-translational modifications. altering the functional proteome influences protein interactions, transcriptional programs, translation, secretion, and cytoskeletal arrangement. a variety of molecules, including phosphates, sugars, lipids, or proteins, can be attached or removed enzymatically to modulate protein function. post-translational modifications thus enable rapid and reversible regulation. under positive selection [42] . this species-specific pattern of positive selection of closely related trims suggests that individual trims play specific antiviral roles. in addition to differential mrna expression upon viral infection, several trim family members are intimately involved in the antiviral response. type-i ifns and other cytokines, such as pro-inflammatory cytokines induced via the nf-κb pathway, have been noted to differentially regulate the expression of a significant population of trims [7, [45] [46] [47] [48] [49] [50] [51] . likewise, trim overexpression influences the transcription of type-i ifn, pro-inflammatory cytokines, and ifn-stimulated genes (isgs) [7, 16] . the roles of trims in viral infection include intrinsic restriction of viral pathogens, positive regulation of immune pathways that promote viral clearance, and negative regulation of antiviral pathways to limit immunopathology [6, 7, 52] . the incorporation of trim antagonists into viral genomes exemplifies the importance of trims in antiviral responses [53] [54] [55] [56] [57] [58] . here, we will focus on the role of trims in the direct and indirect inhibition of viruses and novel mechanisms of viral-mediated antagonism and hijacking of trims. excellent reviews on the roles of trims in autophagy, cancer, and other diseases have been recently published [59] [60] [61] . cells identify pathogen invasion due to the presence of pathogen-associated molecular patterns (pamps) contained in viral components, which are recognized by host pattern recognition receptors (prrs) [62] . examples of viral pamps include some envelope or capsid proteins, viral nucleic acid, or intermediates of genome replication [62] . upon pamp engagement of a prr, a signaling cascade is initiated that relies on post-translational modifications for proper coordination. these modifications include ubiquitination and phosphorylation, which facilitate the assembly of adaptor and enzymatic molecules needed to activate and inactivate transcription factors and other effector molecules [2, 62, 63] . these transitions in the transcriptional profile and functional proteome enable the cell to respond optimally to the pathogen and to communicate (e.g., cytokine secretion) with neighboring cells to limit viral replication and promote clearance. examples of pathways critical in response to viral infection include ifn induction and signaling and nf-κb activation [64] . trim e3 ligases regulate ifn production and signaling as well as nf-κb induction at multiple levels, from prr-mediated pamp recognition to regulation of transcription factors and from promotion of signaling complex assembly to degradation of inhibitors [2, 7] . in this section, we discuss trim regulation of antiviral pathways (summarized in figures 1 and 2 ). viral double-stranded rna (dsrna) or single-stranded rna (ssrna) containing 5'-triphophates produced during virus replication, in the cytoplasm of a host cell, act as a retinoic acid-inducible gene i rig-i-like receptor (rlr) agonist [65] [66] [67] [68] . rig-i and melanoma differentiation-associated protein (mda5), encoded by ddx58 and ifih1, respectively, bind distinct viral rna agonists yet they induce similar downstream antiviral pathways [69] . rlrs are atp-dependent rna helicases that have two n-terminal caspase-activated recruitment domains (cards), a central dead box, and an auto-inhibitory c-terminal domain [70] . the unique pamps recognized by these receptors enable the host to respond to a broader range of pathogens [69] . upon engagement of the pamp with the rlr, a conformational shift exposes the cards, which allows homo-oligomerization and recruitment of the rlrs to their adaptor mitochondrial antiviral signaling protein (mavs) at the mitochondrial outer membrane (mom) [66, [71] [72] [73] . a variety of factors influence the activation of rlrs downstream of pamp recognition including atp hydrolysis [66, 74] , rlr oligomerization [71, 72, 74] , and post-translational modifications [75] [76] [77] [78] [79] . interaction of the n-terminal cards of both the rlrs and mavs induces the adaptor to form prion-like aggregates and exposes domains to recruit critical ring e3 ligases including tumor necrosis factor (tnf) receptor-associated factors (trafs) 3 and 6 [80, 81] . downstream of traf6, the ubd-containing adaptor tgf-β-activated kinase 1(tak1)/mitogen activating protein 3k7 (map3k7)-binding protein (tab) 2/3 recruits the critical kinase tak1 [82] [83] [84] . tak1 auto-phosphorylates to enable the phosphorylation of nf-κb essential modulator (nemo), the regulatory domain of inhibitor of nf-κb (iκb) kinase (ikk) complex, to activate the enzymatic domains of ikkα and β [83, 85] . ikkα and β then phosphorylate iκb, resulting in the recruitment of another e3 ligase, β-trcp (β-transducin repeat containing e3 ubiquitin protein ligase). β-trcp ligates k48-linked ubiquitin to iκb, inducing the proteasome-mediated degradation of the nf-κb inhibitor [63] . once the inhibitor is destroyed, nf-κb is phosphorylated and its nuclear localization sequence is exposed allowing nuclear translocation [63, 86] . inside the nucleus, nf-κb regulates the transcription of a variety of genes including pro-inflammatory cytokines and chemokines, such as pro-il-1β, tnf-α, and il-6, and negative regulators of the pathway to limit an exacerbated inflammatory response [86] . vaccines 2017, 5, 23 5 of 36 another e3 ligase, β-trcp (β-transducin repeat containing e3 ubiquitin protein ligase). β-trcp ligates k48-linked ubiquitin to iκb, inducing the proteasome-mediated degradation of the nf-κb inhibitor [63] . once the inhibitor is destroyed, nf-κb is phosphorylated and its nuclear localization sequence is exposed allowing nuclear translocation [63, 86] . inside the nucleus, nf-κb regulates the transcription of a variety of genes including pro-inflammatory cytokines and chemokines, such as pro-il-1β, tnf-α, and il-6, and negative regulators of the pathway to limit an exacerbated inflammatory response [86] . trims play an integral role in the positive and negative regulation of antiviral pathways. trims can act as pathogen prrs, as is the case for trim21 in the recognition of non-enveloped viruses bound by immunoglobulin (ig). additionally, these trims can regulate the activation of other prrs that recognize viral pathogen-associated molecular patterns (pamps) in the cytosol (ddx41 (dead-box helicase 41), cyclic gmp-amp synthase (cgas), deah-box helicase 33 (dhx33), nucleotide-binding oligomerization domain-containing protein 2 (nod2), retinoic acid-inducible gene i (rig-i), and melanoma differentiation-associated protein (mda5)) and at membrane surfaces (toll-like receptors, tlrs). downstream of the initial pattern recognition, trims also influence the recruitment and interaction of adaptor molecules (stimulator of ifn genes (sting), mitochondrial antiviral signaling protein (mavs), tgf-β-activated kinase 1(tak1)/map3k7-binding protein (tab) 2, myeloid differentiation primary response gene 88 (myd88), tir-domain-containing adapterinducing interferon-β (trif), nf-κb essential modulator (nemo), nucleosome assembly protein (nap-1), and tumor necrosis factor (tnf) receptor-associated factors (traf) family memberassociated nf-κb activator (tank)) and enzymes (traf3, traf6, tak1, inhibitor of nf-κb (iκb) kinase (ikk) α,β,ε, tank binding kinase 1 (tbk1)) to signaling complexes in order to activate transcription factors. this includes ifn regulatory factor (irf)3 and irf7, important in type-i interferon (ifn) signaling, and nf-κb, important in expression of pro-inflammatory genes, which regulate the expression of antiviral effectors. type-i ifn production is critical for an effective antiviral response. trims play an integral role in the positive and negative regulation of antiviral pathways. trims can act as pathogen prrs, as is the case for trim21 in the recognition of non-enveloped viruses bound by immunoglobulin (ig). additionally, these trims can regulate the activation of other prrs that recognize viral pathogen-associated molecular patterns (pamps) in the cytosol (ddx41 (dead-box helicase 41), cyclic gmp-amp synthase (cgas), deah-box helicase 33 (dhx33), nucleotide-binding oligomerization domain-containing protein 2 (nod2), retinoic acid-inducible gene i (rig-i), and melanoma differentiation-associated protein (mda5)) and at membrane surfaces (toll-like receptors, tlrs). downstream of the initial pattern recognition, trims also influence the recruitment and interaction of adaptor molecules (stimulator of ifn genes (sting), mitochondrial antiviral signaling protein (mavs), tgf-β-activated kinase 1(tak1)/map3k7-binding protein (tab) 2, myeloid differentiation primary response gene 88 (myd88), tir-domain-containing adapter-inducing interferon-β (trif), nf-κb essential modulator (nemo), nucleosome assembly protein (nap-1), and tumor necrosis factor (tnf) receptor-associated factors (traf) family member-associated nf-κb activator (tank)) and enzymes (traf3, traf6, tak1, inhibitor of nf-κb (iκb) kinase (ikk) α,β,ε, tank binding kinase 1 (tbk1)) to signaling complexes in order to activate transcription factors. this includes ifn regulatory factor (irf)3 and irf7, important in type-i interferon (ifn) signaling, and nf-κb, important in expression of pro-inflammatory genes, which regulate the expression of antiviral effectors. type-i ifn production is critical for an effective antiviral response. trims in cytokine signaling. downstream of the initial pathogen recognition and induction of pro-inflammatory cytokines, trims can regulate their cytokine signaling pathways through interactions with cytokine receptor adaptors (tab2/3) and enzymatic proteins (ikkα, ikkβ and ikkε) within the signaling complexes, the activity and stability of pathway negative regulators (protein inhibitor of activated stat 3 (pias3), suppressor of cytokine signaling (socs), and influence the transcription of various cytokine-effector genes (nf-κb-induced pro-inflammatory cytokines, signal transducer and activator of transcription (stat)-induced genes, interferon stimulated genes (isgs)) or cytokine signaling regulators (tumor necrosis factor (tnf) receptors (tnfr1/2, and stat-induced genes)). in addition to activation of nf-κb, rlr signaling induces type-i ifn [73] . traf3, in cooperation with nemo, recruits and stabilizes traf family member-associated nf-κb activator (tank) or nucleosome assembly protein (nap1) which are critical in linking tank binding kinase 1 (tbk1), and in some cases inhibitor of kappa light polypeptide gene enhancer in b cells (ikkε), to the mavs signalosome [81, 82] . once activated, tbk1 and/or ikkε phosphorylate the ifn regulatory factor (irf) 3 and irf7 [87, 88] . upon phosphorylation, the irfs homodimerize and translocate to the nucleus where they bind to dna regulatory regions [87, 89] . to induce optimal ifn-β transcription, activated irf3, nf-κb, and ap-1 (activator protein 1) must translocate to the nucleus and bind to their respective regulatory regions of the ifnb1 promoter [64] . the resulting ifn-β is then secreted and signals in a paracrine and autocrine manner. binding of ifn-β to its heterodimeric receptor results in the activation of tyrosine kinases, janus kinase 1 (jak1) and tyrosine kinase 2 (tyk2), which phosphorylate signal transducer and activator of transcription (stat) 1 and stat2. following phosphorylation, stat1 and stat2 heterodimerize and associate with irf9 to form ifn-stimulated gene factor 3 (isgf3) and translocate to the nucleus [87] . within the nucleus, isgf3 binds to genes with an ifn stimulated response element (isre) in their promoter to activate transcription [87] . the resulting proteins expressed from these isgs, such as pkr (protein kinase r), mxa (myxovirus resistance gene a), isg15, and trims, are involved in creating a cellular environment prohibitive to viral entry and replication [87] . as with other immune pathways, isgf3 also promotes the transcription of type-i ifn negative regulators to mitigate deleterious effects [87] . trims play a critical role in both the positive and negative regulation of the rlr pathway to ensure optimal virus restriction while minimizing self-inflicted damage ( figure 1 ). several trims have been shown to positively regulate the receptors rig-i and mda5 [90] [91] [92] . the best characterized example of trim-mediated rig-i activation involves trim25. trim25 ligates k63-linked poly-ub chains onto the n-terminal card at k172, which induces downstream signaling [92] . additionally, trim25 catalyzes the synthesis of unanchored k63-linked poly-ub chains, which facilitate rig-i oligomerization and stabilization [77] . both oligomerization and stabilization of rig-i promotes the interaction of its cards with mavs [93] . adding complexity to this interaction, trim25 k48-linked . trims in cytokine signaling. downstream of the initial pathogen recognition and induction of pro-inflammatory cytokines, trims can regulate their cytokine signaling pathways through interactions with cytokine receptor adaptors (tab2/3) and enzymatic proteins (ikkα, ikkβ and ikkε) within the signaling complexes, the activity and stability of pathway negative regulators (protein inhibitor of activated stat 3 (pias3), suppressor of cytokine signaling (socs), and influence the transcription of various cytokine-effector genes (nf-κb-induced pro-inflammatory cytokines, signal transducer and activator of transcription (stat)-induced genes, interferon stimulated genes (isgs)) or cytokine signaling regulators (tumor necrosis factor (tnf) receptors (tnfr1/2, and stat-induced genes)). in addition to activation of nf-κb, rlr signaling induces type-i ifn [73] . traf3, in cooperation with nemo, recruits and stabilizes traf family member-associated nf-κb activator (tank) or nucleosome assembly protein (nap1) which are critical in linking tank binding kinase 1 (tbk1), and in some cases inhibitor of kappa light polypeptide gene enhancer in b cells (ikkε), to the mavs signalosome [81, 82] . once activated, tbk1 and/or ikkε phosphorylate the ifn regulatory factor (irf) 3 and irf7 [87, 88] . upon phosphorylation, the irfs homodimerize and translocate to the nucleus where they bind to dna regulatory regions [87, 89] . to induce optimal ifn-β transcription, activated irf3, nf-κb, and ap-1 (activator protein 1) must translocate to the nucleus and bind to their respective regulatory regions of the ifnb1 promoter [64] . the resulting ifn-β is then secreted and signals in a paracrine and autocrine manner. binding of ifn-β to its heterodimeric receptor results in the activation of tyrosine kinases, janus kinase 1 (jak1) and tyrosine kinase 2 (tyk2), which phosphorylate signal transducer and activator of transcription (stat) 1 and stat2. following phosphorylation, stat1 and stat2 heterodimerize and associate with irf9 to form ifn-stimulated gene factor 3 (isgf3) and translocate to the nucleus [87] . within the nucleus, isgf3 binds to genes with an ifn stimulated response element (isre) in their promoter to activate transcription [87] . the resulting proteins expressed from these isgs, such as pkr (protein kinase r), mxa (myxovirus resistance gene a), isg15, and trims, are involved in creating a cellular environment prohibitive to viral entry and replication [87] . as with other immune pathways, isgf3 also promotes the transcription of type-i ifn negative regulators to mitigate deleterious effects [87] . trims play a critical role in both the positive and negative regulation of the rlr pathway to ensure optimal virus restriction while minimizing self-inflicted damage ( figure 1 ). several trims have been shown to positively regulate the receptors rig-i and mda5 [90] [91] [92] . the best characterized example of trim-mediated rig-i activation involves trim25. trim25 ligates k63-linked poly-ub chains onto the n-terminal card at k172, which induces downstream signaling [92] . additionally, trim25 catalyzes the synthesis of unanchored k63-linked poly-ub chains, which facilitate rig-i oligomerization and stabilization [77] . both oligomerization and stabilization of rig-i promotes the interaction of its cards with mavs [93] . adding complexity to this interaction, trim25 k48-linked polyubiquitination negatively regulates rlr activation, but the ubiquitin specific protease 15 (usp15) can specifically disassemble these poly-ub chains to stabilize trim25 [94] . trims 4, 13, and 38 have also been implicated in positive regulation of the rig-i pathway. similar to trim25, trim4 also catalyzes the ligation of k63-linked poly-ub chains onto rig-i card [91] . additionally, trim38 functions as an e3 sumo (small ubiquitin-like modifier) ligase and sumoylates both rig-i and mda5 to prevent the ligation of k48-linked poly-ub chains thus stabilizing these prrs [7, 95, 96] . the capacity of multiple trims to activate rig-i suggests that ubiquitination is crucial in rig-i signaling, but the relative contribution of each trim is not well understood. perhaps multiple trims allow for redundancy in the instance that one trim is inhibited or if trims play cell-type specific roles in rlr signaling. recently trim65 was identified as an e3 ligase of mda5. unlike trim25, trim65 ubiquitinates mda5 at the rna helicase domain [90] . the covalent linkage of k63-linked poly-ub onto k743 promotes mda5 oligomerization and downstream activation of irf3 [90] . demonstrating the specificity of mda5 activation, trim65 only promotes the restriction of encephalomyocarditis virus (emcv), a picornavirus, and not vesicular stomatitis virus (vsv), a rhabdovirus [90] . in mouse cells, trim13 was shown to impair mda5-mediated activation of the ifn pathway through an unclarified mechanism [50] . another trim inhibitor of the mavs pathway is trim59, which interacts with evolutionarily conserved signaling intermediate in toll pathways (ecsit) and mavs and subsequently inhibits the transcription of irf3 and nf-κb target genes [97] . although trims have not been identified as rig-i negative regulators, their role in mda5 inhibition suggests there may be unidentified trim-mediated rig-i inhibition. the role of trim25 in the regulation of rlr pathways and/or type-i ifn induction has been shown to be conserved among different species. in fact a diverse range of vertebrates encode rig-regulating trims. in salmonids, trim25, mavs, mda5, and rig-i were induced following infection with an alphavirus although the signaling pathways were not addressed directly [98] . duck trim25 catalyzes the synthesis of unanchored poly-ub chains to activate rig-i [99] . despite lacking lysine 172 in duck rig-i, duck trim25 ubiquitinates rig-i's card domains and promotes rlr signaling [99] . in chicken cells, despite lacking a functional rig-i gene, knockdown of chicken trim25 results in reduced ifn-β upon infection with specific strains of the influenza a virus (iav) [100] , suggesting that trim25 is involved in activation of ifn signaling through a rig-i-independent mechanism, perhaps activation of mda5 or mavs. expression of chicken trim25 is induced after newcastle disease virus (ndv), poly(i:c) treatment, or poly(da:dt) treatment [101] , probably via a type-i ifn signaling-dependent pathway. similar to human trim25, trim27-l stimulates ifn-β production in response to iav infection in ducks [102] . this anti-viral benefit is absent in chickens and turkeys, as they do not carry the trim27-l gene in their trim cluster [102] . however, expression of duck trim27-l and d2card in chicken df1 cell lines was shown to facilitate ifn-β and mx1 expression [102] . this difference may account for the different pathologies in avian species as waterfowl are typically more resistant to some strains of iav as compared to chickens. downstream of rlr activation, a variety of trims promote mavs signaling. trim25 has been implicated in the k48-linked polyubiquitination of mavs, which results in its proteasome-mediated degradation and release of downstream signaling molecules (tbk1, nemo, and possibly traf3) to induce type-i ifn production [103] . recently, trim31 has been described to mediate the k63-linked ubiquitination of mavs at lysines 10, 311, and 461 [104] . this ubiquitination promotes the prion-like aggregation of mavs needed for optimal signaling, exemplified by the decrease in tbk1 and ikkε phosphorylation [104] . in a trim31-deficient murine model, trim31 knock-out mice demonstrated an increased susceptibility to vsv infection and an upregulation in ifn-β production. although rig-i was demonstrated to be required for activation of trim31 function, its role in mda5-mediated signaling was not investigated in-depth [104] . trim14 has been demonstrated to play a crucial role in linking the nf-κb and irf3 branches of rlr signaling [30] . despite lacking a ring domain, trim14 interacts with nemo via its pry-spry domain and promotes k63-linked ubiquitination of nemo, which is critical in recruiting nemo to mavs [30] . the role of trim44 stabilization of mavs has also been characterized [7, 105] . although no trim inhibitors of mavs have been described, screens of trims that inhibit mavs-mediated type-i ifn production may reveal such trims. in addition to regulating rlrs and mavs, trims modulate downstream adaptors and enzymes. several of these trims can likewise mitigate signaling downstream of other prrs that converge on shared molecules such as nemo and tak1, but trims identified to be involved at the level of signaling using rlr induction are described below. recently, the short isoform of trim9 (trim9s) was shown to bridge gsk3β ( glycogen synthase kinase 3 beta) to phosphorylated tbk1 to promote tbk1 oligomerization and activation of irf3 [106] . to facilitate the interaction between gskβ and ptbk1, trim9s must be auto-ubiquitinated [106] . this activation of tbk1-signaling occurs downstream of rlr and sting signaling [107] and biases the immune response toward the type-i ifn pathway while limiting nf-κb-induced transcription [106] . trim11's coiled-coil domain interacts with the coiled-coil domain 2 of tbk1 to prohibit the kinase's interaction with adaptors nap1 or tank [108] . impairing this interaction results in lack of ifn-β production [108] . nap1 is targeted for degradation following trim38-mediated k48-linked poly-ub, which likewise decreases activation of ifn-β [109] . interaction between the arf (adp-ribosylation factor domain) (c-terminus) domain of trim23, and both the coiled-coil 1 and lz domains of nemo, allow trim23 to facilitate k27-linked ubiquitination of nemo [110] . this ubiquitination of nemo facilitates the activation of irf3 and nf-κb signaling downstream of pathogen recognition, but not tnf-α signaling [110] . similar to the described trim9s mechanism, trim26 is able to interact with tbk1 and auto-phosphorylates k27-linked poly-ub chains to bridge tbk1 and nemo [111] . this interaction was demonstrated downstream of mavs signaling and promoted irf3 activation [111] . indirectly, trim68 antagonizes ifn-β transcription [112] . this trim is able to inhibit both toll-like receptor (tlr) and rlr-driven activation of the type-i ifn pathway [112] . several trims are likewise implicated in the inhibition of the nf-κb activation branch of prr signaling. murine specific trim30α negatively regulates the tab2/tab3 complex, which impedes the recruitment and activation of tak1, thus preventing the phosphorylation of nemo and subsequent nf-κb activation [7, 113] . through a different mechanism in the brain, the long isoform of trim9 (trim9l) inhibits β-trcp [33] . perturbation of β-trcp inhibits both canonical and non-canonical nf-κb activation [33] . the trim9l protein sequence includes a degron motif that, when phosphorylated at serine residues 76 and 80, recruits β-trcp [33] . titrating β-trcp from the nf-κb inhibitors prevents their degradation and subsequent pro-inflammatory signaling [33] . several other trims also inhibit nf-κb activation including trim11 [114] , trim29 [115] , and trim39 [116] . the mechanisms for trim11 regulation have not been clearly elucidated, but trim29 targets nemo for degradation in alveolar macrophages [115] and trim39 stabilizes cactin [116] , a nuclear, negative regulator of nf-κb. the bias of trims in the negative regulation of the nf-κb branch of rlr signaling suggests that trims may play a role in promoting the type-i ifn pathway at the cost of nf-κb activation, and further supports the hypothesis that groups of trims may have evolved as part of the antiviral type-i ifn system [48] . however, it is important to note that some studies showing negative regulatory roles of trims have only used overexpression assays with large concentrations of trim expressing vectors, which could lead to artifacts. however, it is now clear that overall trims act at several levels to regulate rlr signaling to balance viral clearance and cell survival. in addition to regulating cytosolic rna-stimulated responses, trims also regulate the pathways following cytosolic dna recognition. in the cytoplasm, the host expresses multiple dna and rna receptors aside from rlrs, including ifi16 (interferon gamma inducible protein 16), cyclic gmp-amp synthase (cgas), and ddx41. the listed double-strand (ds) dna receptors activate the adaptor molecule stimulator of ifn genes (sting) at the endoplasmic reticulum (er) [117] [118] [119] [120] [121] . upon recognition of dsdna, which can result from infection with dna viruses, cgas oligomerizes and catalyzes cyclic dinucleotide (c-gmp-amp) synthesis [95] . c-gmp-amp and other dinucleotides activate sting and induce sting dimerization [95, 119, 120] . consequently, sting recruits tbk1, which phosphorylates irf3 for type-i ifn induction [117] . the dna helicase ddx41 recognizes both cytoplasmic dsdna and cyclic dinucleotides, both of which promote ddx41 activation of sting [121, 122] . five trims are known to influence sting-mediated signaling ( figure 1 ). the pry-spry domain of trim21 interacts with ddx41's helicase domain and catalyzes ubiquitination at lysine residues 9 and 115, targeting the prr for degradation [123] . this degradation pathway occurs in myeloid dendritic cells and restricts type-i ifn induction [123] . the murine-specific trim, trim30α, ubiquitinates sting following herpes simplex virus 1 (hsv-1) and targets the adaptor protein for degradation [124] . both trim32 and trim56 facilitate k63-linked polyubiquitination of sting to promote dimerization and activation of sting-mediated antiviral responses [125, 126] . finally, trim38 sumoylates cgas and sting similar to the sumoylation of rig-i and mda5 [95] . sumoylation inhibits the ligation of k48-linked ubiquitin and results in their stabilization [95] . in addition to regulating cytosolic prr signaling, trims also modulate membrane-bound prrs including toll-like receptors (tlrs) ( figure 1 ). several tlr family members recognize viral pamps. the main tlrs involved in virus recognition include endosome-localized tlrs 3, 7, 8, and 9 [127] . tlr3 recognizes both double-stranded rna and the viral rna mimic poly(i:c), tlr7 and tlr8 recognize single-stranded rna, and tlr9 recognizes cpg [127] . tlr4 is associated mainly with the plasma membrane, although it can also be internalized in endosomes, and can recognize some viral surface antigens. tlrs 4, 7, 8, and 9 signal through iraks (interleukin-1 receptor-associated kinase) 1 and 4, which are recruited via the adaptor molecule myd88 (myeloid differentiation primary response gene 88) [127] . traf6 then re-localizes to the tlr signaling complex to activate tak1, inducing nf-κb [82, 127] similar to the pathway described in the aforementioned rlr section. tlr3 and 4 also signal through iraks 1 and 4, and interact with the adaptor trif (tir-domain-containing adapter-inducing interferon-β), resulting in the activation of traf3. ubiquitination downstream of traf3 induces tbk1-and ikkε-mediated phosphorylation of irf3 and irf7 to induce type-i ifn [127] . trim21's pry-spry domain interacts with irfs, including irf3, 5, and 7, to induce their degradation [128] [129] [130] . although trim21 may promote degradation of irfs downstream of other prrs, the interaction between the two proteins may depend on the induction of a specific tlr pathway. trim38 targets tlr signaling at multiple points. downstream of tlr2, 3, 4, or 7, trim38 targets traf6 for proteasome-mediated degradation following k48-linked polyubiquitination [131] . downstream of tlr3 and tlr4 signaling, trim38 also targets trif and nap1 for degradation [109, 132] . finally, trim56 has been shown to promote tlr3 activation via interaction with trif in a ring ligase-independent manner [133] . perhaps this interaction promotes the stability of trif to facilitate downstream signaling. disruption of adaptor protein availability thus bottlenecks antiviral signaling. nucleotide-binding domain and leucine-rich repeat-containing receptors (nlrs) are another class of cytosolic receptors that recognize both pamps and damage-associated molecular patterns (damps). damps are host-derived molecules that are expressed only after a cell experiences stress and/or damage commonly due to inflammation [134, 135] . upon activation of a nlr, the receptor assembles an inflammasome in cooperation with the adaptor asc to recruit pro-caspases [134, 135] . the nlrp3 (nlr family pyrin domain containing 3) -induced inflammasome promotes the cleavage of pro-caspase 1 to caspase 1, which then promotes a pro-apoptotic response involving the caspase-1-mediated cleavage of pro-il-1β and pro-il-18 to their active forms il-1β and il-18 [134, 135] . the secreted pro-inflammatory cytokines then promote further inflammatory responses, such as pyroptosis, which may be damaging to the host when uncontrolled [134, 135] . in some instances another nlr, nod2, may function as a cytosolic dsrna receptor and converge with the rlr pathway at the level of mavs [136, 137] . at this point only a few trims have been described to interact with nlrs in the control of viral infections. trim33 binds to and ubiquitinates dhx33, a cytosolic dsrna receptor that acts upstream of nlrp3, at lysine k218 to facilitate inflammasome activation [138] . the knockdown of trim33 diminishes the activation of caspase 1 and likewise decreases the release of il-1β and il-18 [138] . the interaction of trim33 with dhx33 relies on the b-box and coiled-coil domains [138] . in contrast, the murine-specific trim30α impairs the nlrp3 inflammasome through an unknown mechanism [139] . another nlrp3 inhibitor identified using a dextran sodium sulfate-induced colitis model is trim31 [140] . the coiled-coil domain of trim31 interacts with the leucine rich and nacht domains of nlrp3 and ligates k48-linked poly-ub chains to target nlrp3 for proteasome-mediated degradation [140] . although the authors did not evaluate virus infection directly, trim31-deficient cells stimulated with poly(i:c) express higher levels of nlrp3 compared to wild-type cells suggesting trim31 may regulate nlrp3 downstream of virus recognition [140] . trim27 is able to promote degradation of nod2 [141] , which may prohibit this receptor from recognizing dna and rna virus infection [136, 137] . the further investigation of trim interactions with nlrs is important for understanding the immune response during bacteria-virus co-infections. several trims are also involved in regulating the cytokine signaling following initial pathogen recognition ( figure 2 ). downstream of tnf-α engagement with its receptor tnfr1, the tak1 complex is recruited to activate nf-κb [63] . trim8 specifically promotes the activation of tnf-α-induced nf-κb signaling through inhibition of the nf-κb nuclear repressor protein inhibitor of activated stat (pias) 3 [142] . the trim8-mediated repression of pias likewise promotes il-6-dependent activation of stat3 [143] . specifically downstream of il-1β and tnf-α signaling in ifn-β-primed cells, trim38 targets tab2 for lysosomal degradation [49, 144] . as described above, the degradation of tab2 inhibits the recruitment and activation of tak1 which blocks pro-inflammatory signaling. zheng and colleagues showed that trim27 catalyzes k48-linked poly-ub at residues k251 and k372 of tbk1 to promote proteasome-mediated degradation downstream of ifn signaling [145] . trim27-tbk1 interactions require the coiled-coil and b-box trim domains [145] . in endothelial cells, trim28 sustains the expression of tnfr1 and tnfr2 to promote the activation of pro-inflammatory pathways [146] . the role of trim28 in the activation of the endothelium may play an important, unexplored role in immune cell trafficking in response to infection. in response to cytokines (i.e., il-12) secreted from activated dendritic cells (dcs), ifn-γ (type-ii ifns) is released from t cells and natural killer cells [147] . after this, type-ii ifn binds its receptor, and the downstream kinases jak1 and jak2 promote stat1 phosphorylation and homodimerization to form gamma activating factor, which translocates to the nucleus to bind gamma-stimulated elements in gene promoters [147] . trim24 inhibits stat1 transcription via binding to the stat1 promoter [148] . in contrast, trim8 is able to destabilize socs-1 (suppressor of cytokine signaling 1), a negative regulator of the ifn-γ signaling pathway, resulting in increased type-ii ifn signaling [149] . the regulation downstream of ifn signaling suggests that this is a negative regulatory mechanism to prevent inflammatory response overactivation. in response to type-i ifns, trim6 catalyzes the formation of k48-linked unanchored poly-ub chains to facilitate the activation of ikkε, which favors isgf3 formation due to stat1 phosphorylation at s708 [11] . this modification increases stat1-stat2 dimerization [150, 151] . this trim6-enahnced activation of ikkε may also play a role in activating ifn-β transcription and translation downstream of rlr activation [11] . trim22 induces the lysosome-mediated destruction of foxo4 and consequently impairs the transcription of ifn-β downstream of tlr3 and rlr signaling [152] . after the innate response to pathogen invasion, the host evolves an adaptive immune response. establishment of an adaptive response requires the presentation of pathogen antigens to t and b lymphocytes. although trim proteins have been mostly studied as regulators of innate immune responses and the role of trims in the regulation of antigen presentation has not been investigated intensively, several studies indicate that trims play an important role in regulating t cell activation. expression of trim22, for example, has been shown to be down-regulated upon cd28/cd2-mediated activation despite being expressed at high levels in resting t cells [153] . in contrast, trim22 expression in t cells is increased following il-2 and il-15 cytokine signaling [154] , both of which act as pro-survival signals. recently, trim24 was shown to influence the expression of th2-type cytokines but not other cd4 + t cell sub-types [155] . as th2 cells predominantly act in allergy-and parasite-induced responses while th1 cells are more important for viral clearance, deregulation of trim24 expression may impact the cd4 + population composition and the capacity of the host to efficiently clear the virus. in the past, trim27 was described as impairing the activation of cd4 + t cells via k48-linked poly-ub of pi3kc2b (phosphatidylinositol 4-phosphate 3-kinase c2 domain-containing subunit beta) [156] . this impairment was observed specifically in cd4 + t cells downstream of t cell receptor (tcr) engagement and not cd8 + t cells [156] . additionally, trim28 depletion in vivo promotes expansion of the th17 population resulting in an autoimmune phenotype [34] . following tcr-mediated activation, trim28 was phosphorylated suggesting it plays a role t cell activation [34] . throughout the lifespan of trim30 knockout mice, the ratio of cd4 + to cd8 + progressively increased and the cd4 + t cells proliferated abnormally [157] . the role of trim30 is intrinsic to cd4 + t cells, because the same defect was observed upon adoptive transfer of knockout cd4 + t cells to wild-type mice [157] . it remains to be seen whether other trims play important roles in adaptive immunity. it is likely that trims have unexplored functions in recognition of antigen presentation by the t cell receptor, and or in differentiation of cd4 + /cd8 + t cells. despite the numerous host evasion mechanisms pathogens employ, a variety of host encoded molecules, such as trims, are able to restrict viruses [2] . in addition to conferring an antiviral state indirectly by regulating cytokine production downstream of prr signaling, trims are capable of restricting the effectiveness of pathogens through direct interactions with viral proteins crucial to their entry, dissemination, or life cycle [6] . the categories of viral restriction include: inhibition of viral transcription, replication or translation, degradation or interference of viral proteins, and impairment of virus entry or exit. we next outline the various means by which hosts deploy trims to counter and clear pathogens. trim5α is an example of a trim functioning as both a direct virus restriction factor as well as a pathogen-recognition receptor ( figure 3 ). in old-world monkeys, such as african green monkeys and rhesus macaques, trim5α enables natural resistance to hiv-1 while new-world monkeys, like owl monkeys, are protected from hiv-1 by fusion of trim5 with cyclophilin a (tcypa) [158] [159] [160] [161] . progress on the topic ranges from revealing the α isoform of trim5 as the restriction factor to discerning the role in restriction of each structural motif [158, 159] . . the role of trims in retrovirus replication. trim5α oligomerization into a hexagonal lattice associates directly with hiv-1 capsids to promote premature uncoating. recognition of hiv-1 capsid by trim5α also triggers nf-κb/ap-1-mediated innate immune signaling via synthesis of unanchored k63-linked poly-ub chains that activate the tak1 kinase. one potential mechanism of trim5αmediated restriction could be involved ubiquitination of trim5α and proteasomal degradation of trim5α-capsid complexes. trim11 mobilizes cellular microtubule formation to prematurely uncoat hiv-1 and facilitate rapid release of the vrna from the viral core, resulting in inhibition of virus replication. trim19 translocates to the cytoplasm and binds daxx to prevent its degradation by the proteasome, allowing for daxx-mediated disruption of hiv-1 reverse transcription (rt). trim22 inhibits sp1, preventing ltr-mediated transcription. the connection between trim5α-mediated restriction and proteasomal-mediated degradation of hiv-1 requires further characterization. however, one potential model may involve improved proliferation of hiv-1-specific cd8 + t cells due to enhanced production of viral peptides from proteasome-mediated degradation of hiv-1 capsids. several components inherent to trim5α play a role in hiv-1 capsid restriction, including dimerization, oligomerization, and ubiquitination [162] [163] [164] . there have been different models proposed for the mechanism of restriction and all have been attributed to the interaction of the spry domain of trim5α with the retrovirus capsid. binding of trim5α to viral cores serves to prematurely uncoat the viral particle and induces early release of the viral genome. elucidation of the trim5α higher order structure has revealed the significant contributions made by each component of the trim5α protein [26, [163] [164] [165] [166] . monomeric trim5α can dimerize in an antiparallel fashion through the coiled-coil domains, thereby generating the most fundamental component needed to bind the viral capsid [163, 167] . once bound to its target, trim5α can form higher-order structures resembling a hexagonal net [164, 165, 168, 169] . trim5α and trim5 variants from several primate species, including rhesus macaques, african green monkeys, and owl monkeys, are capable of forming flexible, hexagonal frameworks encompassing hiv-1 capsid surfaces [164] . the hexagonal nets are formed from trim5α trimers and are dependent on the trim5α's b-box domain [165, 166] . these trimers have been shown to be flexible and may allow for ideal binding of the spry domains on the highly variable hiv-1 capsid [165, 166] . the ability of rhesus macaque trim5α (rhtrim5α) to form higher-order structures upon binding to the capsid also allows the formation of trim5α ring dimers, which enhances its e3-ubiquitin ligase activity and innate anti-hiv-1 activity [29] . as one potential mechanism of rhtrim5α-mediated restriction it was proposed that as the linker 2 regions of the dimer change their conformation, the spry domains that are bound to the hiv-1 capsid in their hexagonal lattice formation disturb the viral structure [163] . the induction of this conformational change disrupts the integrity of the viral core and may be responsible for pre-mature viral uncoating. capsid by trim5α also triggers nf-κb/ap-1-mediated innate immune signaling via synthesis of unanchored k63-linked poly-ub chains that activate the tak1 kinase. one potential mechanism of trim5α-mediated restriction could be involved ubiquitination of trim5α and proteasomal degradation of trim5α-capsid complexes. trim11 mobilizes cellular microtubule formation to prematurely uncoat hiv-1 and facilitate rapid release of the vrna from the viral core, resulting in inhibition of virus replication. trim19 translocates to the cytoplasm and binds daxx to prevent its degradation by the proteasome, allowing for daxx-mediated disruption of hiv-1 reverse transcription (rt). trim22 inhibits sp1, preventing ltr-mediated transcription. the connection between trim5α-mediated restriction and proteasomal-mediated degradation of hiv-1 requires further characterization. however, one potential model may involve improved proliferation of hiv-1-specific cd8 + t cells due to enhanced production of viral peptides from proteasome-mediated degradation of hiv-1 capsids. several components inherent to trim5α play a role in hiv-1 capsid restriction, including dimerization, oligomerization, and ubiquitination [162] [163] [164] . there have been different models proposed for the mechanism of restriction and all have been attributed to the interaction of the spry domain of trim5α with the retrovirus capsid. binding of trim5α to viral cores serves to prematurely uncoat the viral particle and induces early release of the viral genome. elucidation of the trim5α higher order structure has revealed the significant contributions made by each component of the trim5α protein [26, [163] [164] [165] [166] . monomeric trim5α can dimerize in an antiparallel fashion through the coiled-coil domains, thereby generating the most fundamental component needed to bind the viral capsid [163, 167] . once bound to its target, trim5α can form higher-order structures resembling a hexagonal net [164, 165, 168, 169] . trim5α and trim5 variants from several primate species, including rhesus macaques, african green monkeys, and owl monkeys, are capable of forming flexible, hexagonal frameworks encompassing hiv-1 capsid surfaces [164] . the hexagonal nets are formed from trim5α trimers and are dependent on the trim5α's b-box domain [165, 166] . these trimers have been shown to be flexible and may allow for ideal binding of the spry domains on the highly variable hiv-1 capsid [165, 166] . the ability of rhesus macaque trim5α (rhtrim5α) to form higher-order structures upon binding to the capsid also allows the formation of trim5α ring dimers, which enhances its e3-ubiquitin ligase activity and innate anti-hiv-1 activity [29] . as one potential mechanism of rhtrim5α-mediated restriction it was proposed that as the linker 2 regions of the dimer change their conformation, the spry domains that are bound to the hiv-1 capsid in their hexagonal lattice formation disturb the viral structure [163] . the induction of this conformational change disrupts the integrity of the viral core and may be responsible for pre-mature viral uncoating. proteasomal-mediated degradation of viral components is a host restriction strategy characteristic of many trim family proteins involved in host innate immunity. however, the functional role of the proteasome in trim5α-mediated hiv-1 restriction has been difficult to discern and has been subject of debate. early work with proteasomal inhibitors, like mg132, suggested a two phase restriction of hiv-1 by trim5α. in the proteasome-independent phase, trim5α inhibits nuclear entry of the reverse transcription (rt) products [170, 171] . in the second phase, trim5α can inhibit late rt products in the presence of a functional proteasome, suggesting trim5α may utilize the proteasome to disrupt the viral rt complex [170, 171] . while proteasome inhibition can block disassembly and/or degradation of viral core components, it does not appear to rescue infectivity, which has led some investigators to conclude that degradation of capsid by the proteasome is not the mechanism of trim5α-mediated restriction [172] . in addition, thus far no study has described ubiquitination sites on the hiv-1 capsids, which could potentially link degradation of the capsid with proteasomal function. however, additional studies have shown proteasomal involvement in trim5α restriction of hiv-1 by degrading trim5α itself [173] . trim5α's e3 ligase activity can facilitate auto-ubiquitination, thereby signaling for its own destruction by the host proteasomal machinery [36, 173] . these occurrences have led some to speculate on a potential model where trim5α interacts with the hiv-1 capsid leading to auto-ubiquitination, capsid uncoating, and delivery to the proteasome [173] [174] [175] [176] . despite numerous investigations centered around proteasomal involvement, controversy within the literature surrounding the exact model connecting trim5α, hiv-1 cores, and proteasomes remains, and proteasome-dependent and independent mechanism have been proposed [175, 176] . polyubiquitination through the e3 ligase activity of trim5α's ring domain may also be a factor involved in inhibition of retroviral replication [177] . in addition to its restriction activity, trim5α can induce antiviral type-i ifns. multiple trim5 orthologs induce ap-1-mediated innate immune signaling [169, 178] . in the presence of the hiv-1 capsid, the e3 ligase activity of trim5 facilitates the generation of unanchored k63-linked poly-ub chains. these k63-linked chains promote tak1 autophosphorylation and activation, resulting in the induction of ap-1-and nfκb-mediated transcription [169] . collectively, these recent findings serve to better characterize the mechanisms through which trim5α achieves inhibition of hiv-1 replication. the restriction benefits conferred by trim5α can be observed in the progression to disease upon infection with simian immunodeficiency virus (siv) in rhesus macaques. course of infection studies with either a trim5α-sensitive or -resistant strain of siv revealed that inoculation with a trim5α susceptible strain of siv provided higher survival rates for rhesus macaques. also, delayed development of the pathology associated with trim5α-sensitive siv compared to subjects treated with a trim5α-resistant strain was observed [179] . in addition to these benefits, the restriction of the trim5α sensitive siv strain correlated with prolonged maintenance of cd4 + central memory t cells [179] . the preservation of cd4 + central memory t cells has been extensively characterized as important in resisting viremia and generating ctl (cytotoxic t lymphocytes) subsets [180] [181] [182] . the prolonged presence of these cd4 + t cells in the trim5α-susceptible siv-infected treatment may be responsible for the higher survival rate [179] . in spite of this, trim5α escape mutants were present [179] . half of the subjects that received the trim5α-sensitive strain of siv still developed aids, and a third succumbed to infection at a similar time as the trim5α-resistant group [179] . sequencing of the siv capsids from these macaques revealed two mutations in the region encoding the gag protein, which resulted in nonsynonymous substitutions that mimicked the alterations made by the authors to generate their trim5α resistant strain [179] . subversion of trims is a central evolutionary strategy for viral innate immune evasion, as demonstrated by the high mutational rate of retroviruses. although first described as a viral restriction factor against hiv-1 in old-world monkeys, humans and other species are capable of utilizing trim5α to inhibit other retroviruses. human trim5α is incapable of restricting hiv-1, yet a single amino acid mutation in its pry-spry domain can confer resistance to the pathogen [183] . instead, human trim5α can bind to and restrict n tropic mouse leukemia virus (n-mlv) [183] . interestingly, the 13-amino-acid stretch in the pry-spry domain of primate trim5α is under strong positive selection [44] . trim5α paralogues have been identified in bovine [184, 185] , ovine [186] , and piscine [40] species, and have also been shown to restrict retroviruses. overall, trim5α plays a convergent role in the recognition and restriction of retroviruses. aside from its role in non-human primates, trim5α has been described functioning in a cell-type specific manner [187] [188] [189] . in contrast to conventional dc-sign + (dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin) dcs, langerhans cells (lcs) benefit from the anti-retroviral capabilities of trim5α through an increased activity of the lc autophagocytic components [187] . upon langerin-mediated hiv-1 uptake, the presence of cellular autophagosomes increases as a result of trim5α-directed assembly, leading to targeting of the viral capsid for destruction [187] . further evidence of cell-type specific hiv-1 restriction by trim5α was found in rhesus macaque dcs, which in contrast to macrophages appear to be permissive to hiv-1 infection [188] . the lack of trim5α-mediated restriction in both human and macaque conventional dcs may be due to sumoylation of trim5α, which promotes its sequestration in nuclear bodies [189] . however, this lack of trim5α-mediated restriction in dcs provides an innate immune sensing advantage by allowing recognition of hiv-1 reversed transcribed dna by cgas, resulting in type-i ifn production [189] . it remains to be seen whether hiv-1 can actively promote or enhance sumoylation of trim5α in dcs as mechanism to antagonize type-i ifn production. the ability of trim5α to promote restriction of hiv-1 may go beyond direct intervention of the viral lifecycle. trim5α has also been linked to the activity of other components of cellular innate immunity including antigen presentation to cytotoxic t lymphocytes (ctl). tcypa and rhtrim5α enhanced the ability of cd8 + t cells to identify hiv-1 infected cells, and promoted a hiv-1-specific immune response [174] . the presence of these trim5 orthologs was associated with increased associations between hiv-1 particles and the host proteasome [174] . it is speculated that this may facilitate improved hiv-1-specific ctl development, as amplified peptide concentrations could support enhanced antigen presentation to cd8 + t cells. the mechanism linking direct restriction of viral capsids with heightened cd8 + t cell activation warrants further investigation. a plethora of trim-mediated restriction mechanisms targeting hiv-1 and other retroviruses have been proposed [15] , and have been reviewed previously [6] . more recent reports acknowledged trim11 as a potent host restriction factor of hiv-1 [114, 190] . the mechanisms of trim11-mediated restriction include curbing the amount of viral reverse transcription products allowed to accumulate in the host. through an interaction with the viral capsid-nucleocapsid protein (ca-nc) complexes, trim11 promotes premature uncoating and release of the viral genetic material, reducing transduction efficiency. neither proteasomal nor lysosomal inhibitor treatments recovered viral p24 protein in the pellets of trim11 overexpressing cells, suggesting that ubiquitin-mediated degradation by the proteasome or lysosomal acidification is not required for trim11-mediated uncoating [190] . furthermore, while rhtrim5α-mediated inhibition of hiv-1 is rescued by proteasome inhibitors [170, 171] , trim11-mediated inhibition was not, indicating that trim11 and trim5α restrict hiv-1 by different mechanisms [114, 190] . in contrast, using the microtubule dynamics inhibitors nocodazole and taxol, the authors were able to demonstrate a restoration of hiv-1 capsid levels in cells supplemented with exogenous trim11, suggesting that microtubules may contribute to trim11-mediated hiv-1 restriction [190] . earlier work with the use of nocodazole and taxol in a study examining hiv-1 and trim5α also demonstrated recovered hiv-1 infectivity in the absence of functional microtubules [191] . although the exact mechanism of trim11-mediated restriction of hiv-1 in the aforementioned study has yet to be determined, the authors demonstrated that purified trim11 associated with in vitro assembled hiv-1 capsids [190] , indicating that trim11 interacts directly with the capsid and it probably does not require trim5α or other cellular proteins for promoting untimely capsid uncoating. microtubule involvement in viral uncoating has been implicated as both a host restriction and a viral propagation mechanism [192] [193] [194] [195] . functional microtubules and their associated motor proteins, like dynein, have been suggested as key components in genome release for both iav and hiv-1 [192] [193] [194] [195] . interestingly, in the case of iav, unanchored poly-ub chains contained in the virion are recognized by the host histone deacetylase 6 (hdac6), a component of the aggresome-autophagy pathway, which interacts with dynein and microtubules to promote viral uncoating [192, 195] . it will be interesting to examine whether hiv-1 utilizes a similar mechanism and whether this is mediated by trim11. additionally, several members of the trim family (trims 1, 9, 18, 36, 46, and 67) have been shown to possesses a c-terminal domain motif allowing for association with cytoskeletal elements like microtubules [31] . so far only trim1 (also called mid2) and trim18 (also called mid1) have been shown to be directly involved in microtubule stabilization [196] , and trim1 has been implicated in restriction of n-mlv [159] . whether trim1-mediated restriction is dependent on microtubules, or whether other microtubule-interacting trims may have viral restriction activity by microtubule-dependent mechanisms, remains to be seen. trim-mediated suppression of hiv-1 replication has revealed additional avenues through which trims subdue pathogens. trim22 prevents normal viral transcription events by regulating the effectiveness of the transcription factor sp1 to bind the hiv-1 long terminal repeat (ltr) promoter region [197] . this restriction was independent of trim22's e3 ligase activity and did not involve direct interaction between trim22 and sp1, implying that the observed reduction in hiv-1 ltr-mediated transcription requires additional unspecified factors [197] . trim37 exhibits anti-retroviral functions through interference of hiv-1 infection, replication, and transcription, possibly interfering with dna synthesis [198] . however further investigation into trim-mediated transcriptional inhibition will be required to reveal additional pathways in which trims play a significant role. another example of indirect inhibition of virus replication by trims is illustrated by the promyelocytic leukemia protein (pml)/trim19. pml/trim19 interferes with hiv-1 infection in human and murine fibroblasts through the reduction of reverse transcriptase products [199, 200] . this effect was dependent on two events; the translocation of pml/trim19 from the nucleus to the cytoplasm in the presence of hiv-1 infection, as well as association of the pml/trim19 cytoplasmic bodies (cb) with daxx [201] . pml/trim19 interacts with daxx in a protective fashion in order to prevent its degradation by the proteasome, thereby making pml/trim19 a necessary component for daxx-mediated inhibition of hiv-1 rt [201] . the inhibition of hiv-1 by pml/trim19 and daxx may be cell-type dependent, since studies using different cell types have shown different results [200, 201] . another well characterized trim involved in pathogen recognition is trim21, which has been shown to detect intracellular antibody-opsonized viruses (reviewed previously in [6, 7] ). some immunoglobulin-coated non-enveloped viruses are internalized into the host cell. within most cells, a high affinity antibody receptor, trim21, binds to the highly conserved fc region of virion-bound immunoglobulin (ig)g, igm, or iga [7, 202] , and targets the virus for degradation by the proteasome before the virus can transcribe its genes [203] . trim21's pry-spry domain interacts with fc residues conserved across mammalian species [204] . trim21 binding with an immunoglobulin-virion (ig-v) complex triggers tightly regulated intracellular antibody neutralization and pro-inflammatory pathways [7, 202] . upon engagement of trim21 with the ig-v complex within the cytoplasm, the e2 ube2w (ubiquitin conjugating enzyme e2 w) monoubiquitinates trim21, which promotes ube2n/ube2v2 e2 complex recruitment to trim21 for k63-linked poly-ub [12] . following poly-ub, trim21 is recruited to the proteasome to initiate degradation of the antibody-bound virus. concurrently, with proteasome-mediated degradation, poh1 de-ubiquitinates trim21 [12] . this antibody-dependent intracellular neutralization appears to be limited to non-enveloped dna and rna viruses that can enter the host cytosol with immunoglobulin attached to the virion surface [205] . the de-ubiquitinated trim21 is then able to promote both ifn induction and nf-κb activation [12, 205, 206] , hypothesized to result from the release of the viral genome into the cytoplasm for prr-mediated recognition [207] . however, some antiviral cytokines are induced independent of prrs [207] , but the pathway is not well characterized. in addition, it has also been proposed that recognition of the ig-v complex in the cytoplasm by trim21 triggers the synthesis of unanchored k63-linked poly-ub chains that activate nf-κb, ap-1 and irf3 pathways [205] . notably, when the affinity of trim21 for the fc portion of an antibody is decreased, the viral neutralization efficiency is maintained while the activation of cytokine signaling is diminished [208] . this suggests that the association of trim21 with ig-v complexes is important for triggering antiviral responses. trim21-deficient mice infected with mouse adenovirus 1 experienced lethal disease while wild-type mice were protected, exemplifying the importance of trim21 in viral neutralization in vivo [209] . most likely, these mechanisms are shared in other species due to the highly conserved fc and trim21 interacting residues, however the activity of trim21 as an intracellular antibody receptor in non-human, non-murine models has only been demonstrated in pig cells infected with foot-and-mouth disease virus [210] . trim family proteins can target several components of pathogens ranging from structural elements to factors essential for transcription and replication. a variety of influenza a virus (iav) and influenza b virus (ibv) proteins are targets of trim-mediated inhibition of virus replication (figure 4 ). despite being a target of iav-ns1 (nonstructural protein 1)-dependent ifn antagonism, trim25 interacts with the n-terminus of ibv-ns1 preventing the viral protein's c-terminal component from binding viral rna [211] . preventing ibv-ns1 from interacting with rig-i allows signaling through this rlr pathway to proceed [211] . another trim-mediated anti-iav mechanism is polyubiquitination of iav nucleoprotein (np) by trim22, which leads to np proteasome-mediated degradation and reduced virus replication [212] . the polymerase basic protein 1 (pb1) of iav is also a target of trim-mediated inhibition. pb1 is one of the three components that make up the rna-dependent rna-polymerase responsible for transcribing the eight segments of the iav genome [213] . trim32 facilitates k48-linked polyubiquitination of pb1, resulting in enhanced turnover of this viral subunit, and a reduction in viral titers [214] . aside from targeting pathogen components for proteasomal degradation via ubiquitination, some members of the trim family have been shown to enact their restriction factor capabilities through other means. the trim56 c-terminal domain, rather than its e3 ligase activity, is required to reduce iav and ibv replication. the mechanism trim56 employs to diminish vrna levels of both influenza viruses is currently unknown, although inhibition of translation through direct interaction between trim56 and the vrnas has been proposed [215] . trims have also been reported to mediate restriction against flaviviruses ( figure 5 ). the flavivirus genus comprises more than 70 viruses including a number of important human pathogens such as dengue virus (denv), zika virus (zikv), west nile virus (wnv), tick-borne encephalitis virus (tbev), japanese encephalitis virus (jev), hepatitis c virus (hcv), and yellow fever virus (yfv) [216] . flaviviruses are small enveloped viruses hosting a positive-sense single-stranded rna genome. several flaviviral proteins are associated with viral persistence, immune system evasion, or viral replication [217] . influenza viruses is currently unknown, although inhibition of translation through direct interaction between trim56 and the vrnas has been proposed [215] . trims have also been reported to mediate restriction against flaviviruses ( figure 5 ). the flavivirus genus comprises more than 70 viruses including a number of important human pathogens such as dengue virus (denv), zika virus (zikv), west nile virus (wnv), tick-borne encephalitis virus (tbev), japanese encephalitis virus (jev), hepatitis c virus (hcv), and yellow fever virus (yfv) [216] . flaviviruses are small enveloped viruses hosting a positive-sense single-stranded rna genome. several flaviviral proteins are associated with viral persistence, immune system evasion, or (2) . rna genome is released into the cytoplasm (3). the positive-sense genomic ssrna is translated into a polyprotein, which is cleaved into all structural and non-structural proteins (4). replication takes place at the surface of endoplasmic reticulum in cytoplasmic viral factories (5) . in this step, the trims restrict virus replication, degrading viral proteins such as ns2a in japanese encephalitis virus (jev) by trim52 and viral rna inhibition in jev and dengue virus (denv) by trim56. trim22 and trim79 degrade ns5 protein in hepatitis c virus (hcv) and tick-borne encephalitis virus (tbev), respectively. virus assembly occurs at the endoplasmic reticulum. the virion buds at the endoplasmic reticulum and is transported to the golgi apparatus (6) . the prm protein is cleaved in the golgi, thereby maturing the virion, which is fusion competent (7) . release of new virions by exocytosis (8) . trims and antagonism function also are used by flaviviruses. trim21 inhibits ifn-β production during jev infection. trim23 promotes yellow fever virus replication. denv short noncoding sfrnas bind trim25 to inhibit ifn expression. hcv encodes a nonstructural protein, ns5a, which inhibits the phosphorylation and nuclear translocation of stat1 in the ifn-α2-induced jak/stat pathway via their ifn sensitivitydetermining region [218, 219] . trim14's spry domain specifically interacts with ns5 of hcv and induces ns5a degradation [220] , which is an example of trim-mediated ifn-independent inhibition. trim22 specifically binds the ns5a-d1 protein (domain 1) via its spry domain and the positive-sense genomic ssrna is translated into a polyprotein, which is cleaved into all structural and non-structural proteins (4). replication takes place at the surface of endoplasmic reticulum in cytoplasmic viral factories (5) . in this step, the trims restrict virus replication, degrading viral proteins such as ns2a in japanese encephalitis virus (jev) by trim52 and viral rna inhibition in jev and dengue virus (denv) by trim56. trim22 and trim79 degrade ns5 protein in hepatitis c virus (hcv) and tick-borne encephalitis virus (tbev), respectively. virus assembly occurs at the endoplasmic reticulum. the virion buds at the endoplasmic reticulum and is transported to the golgi apparatus (6) . the prm protein is cleaved in the golgi, thereby maturing the virion, which is fusion competent (7) . release of new virions by exocytosis (8) . trims and antagonism function also are used by flaviviruses. trim21 inhibits ifn-β production during jev infection. trim23 promotes yellow fever virus replication. denv short noncoding sfrnas bind trim25 to inhibit ifn expression. hcv encodes a nonstructural protein, ns5a, which inhibits the phosphorylation and nuclear translocation of stat1 in the ifn-α2-induced jak/stat pathway via their ifn sensitivity-determining region [218, 219] . trim14's spry domain specifically interacts with ns5 of hcv and induces ns5a degradation [220] , which is an example of trim-mediated ifn-independent inhibition. trim22 specifically binds the ns5a-d1 protein (domain 1) via its spry domain and utilizes its e3 ubiquitin ligase activity to target ns5a for removal [221] . wenchun and colleagues have shown that trim52 interacts with the ns2a protein of jev and targets the protein for proteasome-mediated destruction [222] . ns2a is a small, hydrophobic transmembrane protein involved in the virus life cycle and subversion of host antiviral responses [223, 224] , including inhibition of the double-stranded rna-activated protein kinase pkr during jev infection [225] . trim52-dependent inhibition of jev ns2a protein occurs in bhk-21 and 293t cells and is therefore important for restricting jev replication [222] . recent evidence also suggests that the ring and c-terminal domains of trim56 may be important in the restriction of other flaviviruses, including yfv and denv [226] , but the mechanism of action remains elusive. trim56 might modulate post-translational modification of one or more viral proteins and/or host factors to suppress viral replication [227] . although trim56 fails to restrict hcv replication when overexpressed in human hepatoma huh7 cells [228] , trim56 overexpression in hek293 cells support some selectable hcv rna replicons at very low efficiencies [227] . perhaps trim56 facilitates degradation of viral proteins similar to mechanisms observed with trims 14 and 22 against hcv and trim52 against jev. further studies are needed to elucidate the underlying molecular mechanisms of trim56-mediated restriction of denv and yfv. interestingly, trim56 is capable of binding to the protease n pro of bovine diarrheal virus (bvdv), a pestivirus of the flaviviridae family [228] . the restriction of this viral protein is critical considering that n pro is capable of mediating irf3 degradation, thus impairing production of ifn-β [228] . trim56-mediated restriction of bvdv is specific, as closely related viruses are not likewise impaired in their replication [228] . trim79α, also known as trim30-3 or trim30d, is present only in rodents. trim79α is highly expressed in the spleen, lymph node, and bone marrow in a type-i ifn-dependent manner, and is required for effective restriction of tbev replication [229] . trim79α is an important mediator of the innate cellular response to restrict langat virus (a member of the tbev serogroup) infection by targeting the viral rna polymerase and major ifn antagonist, ns5 [229] . ns5 has a methyltransferase and rna-dependent rna polymerase activity that associates with ns3 and ns2b to form the viral replication complex. ns5 inhibits ifn-α/β-dependent responses by preventing jak-stat signaling and thus suppresses ifn-stimulated gene (isg) expression [229] [230] [231] [232] . taylor and colleagues demonstrated that trim79α interacts with ns5 from lgtv and tbev and blocks the replication of these viruses via a lysosomal-targeting mechanism. despite ns5 being the most conserved of the flaviviral proteins, trim79α did not target ns5 from wnv, nor could it inhibit wnv replication [232] . besides flaviviruses, other positive-sense rna viruses have been reported to be inhibited by trim-mediated mechanisms and can occur through both direct and indirect means. for example, trim25 acts as a cofactor for the zinc-finger antiviral protein (zap), a member of the poly(adp-ribose) polymerase family that is known to bind and promote viral rna degradation [233] . trim25 enhances zaps' ability to inhibit sindbis virus translation [233] . however, although trim25 is capable of promoting k48-and k63-linked polyubiquitination of zap isoforms, ubiquitination does not appear to affect zap antiviral activity [233] . it will be of interest to elucidate what other factors may be ubiquitinated by trim25 that could affect zap antiviral activity. trim19-iv has been shown to confer resistance to encephalomyocarditis virus (emcv) hampering viral replication and protein synthesis [234] . this ability was shown to originate from trim19-iv's c-terminus, specifically through an interaction with the viral 3d polymerase (3dpol), leading to nuclear body sequestration of the viral polymerase [234] . additionally, sumoylation of trim19-iv was required to facilitate restriction of emcv [234] . trims play an additional role in restricting dna virus replication. eight different trim proteins (trim5, 6, 11, 14, 25, 26, 31, and 41) were identified to inhibit hepatitis b virus (hbv) [235] . in particular, trim41 was the only trim from this group of eight that specifically reduced both the enhancers i and ii components of hbv. this inhibition of viral transcription required trim41's ring and pry/spry domains, implicating its e3 ligase activity [235] . trim22 is associated with hbv clearance in acutely infected chimpanzees, and possesses anti-hbv activity under physiological conditions [236] . gao and colleagues also reported that trim22 was one of the most strongly induced trim family molecules in human hepatoma hepg2 cells after treatment with ifns [237] . epstein-barr virus (ebv) replication and transcription activator (rta) protein activates ebv lytic genes for proliferation [238] . trim5α promotes the ubiquitination of rta upon interaction, thereby blocking the ebv lytic cycle [238] . the involvement of trim5α in a non-retroviral innate immune response implies trims possess diverse, situation-specific functions that have yet to be characterized. trims may also operate as recognition receptors for other components of host immunity. trim19 isoforms are able to capture varicella-zoster virus nucleoproteins to impede nuclear egress and thus block the release of new virions [239] . comprehension of the multiple roles trim-family proteins play may become critical in discerning the impact that they have on the innate immune response. as we have described, trim family proteins play an important role in innate immunity and counter pathogens [7] . in retaliation to this evolutionary pressure, pathogens have adapted to antagonize trims. mechanisms of viral-mediated trims restriction range from impairment of trim-promoted innate immune signaling complex assembly to direct hindrance of the trim proteins. interfering with trims, either directly or indirectly, undermines their intended function, and enables viruses to gain an early advantage over the host [58] . the following section highlights several recent examples that elucidate viral manipulation of trims. a broad range of rna viruses have evolved effective evasion strategies to manipulate host antiviral immunity. influenza viruses employ well-studied examples of trim antagonism ( figure 4 ). the nonstructural protein 1 (ns1) of influenza a and b virus (iav/ibv) has been characterized as a viral antagonist of host innate immunity through interactions with trim25 [57, 100, 240, 241] . as described above, trim25 plays a critical role in the activation of the rlr pathways [57, 242, 243] . the ns1 protein from iav directly interacts with the coiled-coil domain of trim25 to impede its multimerization [57, 244] . since dimerization is required for trim25 e3 ligase activity, ns1 binding to trim25 leads to impaired ubiquitination of the rig-i and downstream signaling, resulting in a reduced antiviral response [57, 244] . the trim25 interaction with iav ns1 is species-specific. human trim25 interacts with iav strains isolated from many species while chicken trim25 binds only ns1 from avian strains and murine trim25 did not bind any ns1 [100] . riplet, a close relative of trim25, lacks a b-box domain but shares homologous ring and spry domains. its predicted coiled-coil structure also binds ns1 to inhibit rig-i signaling in mice and humans [100] . aside from interacting with trim25, iav ns1 also associates with host rig-i directly [244] . as seen with iav, the n-terminal domain of ibv ns1 is also able to block the lys63-linked ubiquitination of rig-i and subsequent antiviral signaling downstream of the rlr pathway [211] . members of the coronaviridae, flaviviridae, and bunyaviridae families also encode viral proteins that inhibit trim-mediated regulation of rlr signaling. severe acute respiratory syndrome and middle east respiratory syndrome coronavirus (sars/mers-cov) are large, positive-sense single-stranded rna viruses. akin to the influenza virus ns1 protein, the nucleoprotein of sars-cov was demonstrated to interact with the spry domain of trim25, preventing the necessary interaction and subsequent ubiquitination of the rig-i card domains [245] . a similar loss of rig-i-induced ifn-β is achieved when mers-cov nucleoprotein associates with trim25 [245] . for denv, manokaran et al. compared two viral sequences (pr1 and pr-2b) and identified mutations that resulted in the increased production of subgenomic flavivirus non-coding rnas (sfrnas) by the pr-2b strain [55] ( figure 5 ). the pr-2b sfrnas were capable of binding to host trim25 and prevented usp15-mediated deubiquitination [55] , which is crucial for activation of rig-i [94] . this data provides unique molecular insight into the epidemiological fitness of denv, suggesting that denv sfrnas can bind to host proteins to promote viral evasion of innate immunity [55] . the nss protein of severe fever with thrombocytopenia syndrome virus (sftsv), a negative-sense rna virus in the family bunyaviridae, interacts directly with trim25, and indirectly with rig-i and tbk1 to isolate these signaling molecules from associating with mavs [246] . as with the other viral protein-trim25 interactions described above, downstream activation of irf3 and subsequent ifn-β production are impaired [246] . trim25 is a common target of a diverse group of rna viruses, suggesting that other pathogens may also impair trim25-mediated stimulation of the rlr pathway. similar to iav, hiv-1 encodes multiple proteins that restrict trim function ( figure 3 ). for example, hiv-1 vpr protein has been suggested to play a role in trim manipulation [114] . interestingly, protein expression levels of trim11 have shown to be under the control of vpr in a dose-dependent manner, where the presence of trim11 was decreased when the concentration of vpr was low [114] . currently, the mechanism hiv-1 vpr employs to antagonize trim11 is unknown. following infection of human neural precursor cells (hnpcs) with hiv-1, trim32 becomes upregulated, primarily due to the viral trans-activator of transcription protein (tat) [247] . this tat-mediated upregulation of trim32 induces proliferation arrest in hnpcs, eventually leading to neurodegeneration [247] . flaviviruses also encode viral proteins that antagonize trim-mediated innate immunity ( figure 5 ). yfv and denv ns5 protein have 10 amino acid residues on the n-terminus, which are essential for antagonism of type-i ifn signaling [54, 248] . yfv ns5 binds stat2 only after ifn treatment, and appears to inactivate isgf3 within the nucleus [54] . morrison et al. found in denv ns5 a glycine and a threonine residue within the n-terminus that are required for binding with ubr4 (ubiquitin protein ligase e3 component n-recognin 4) to mediate stat2 degradation [249] . ubr4, a member of the n-recognin family, is a potential e3 ligase that recognizes and degrades proteins containing destabilizing n termini. [250] . ubr4 interacts preferentially with proteolytically-processed denv ns5, but not with yfv ns5 or wnv ns5 [249] . although ubr4 does not belong to the trim family, it is possible that trim members may also be involved in stat2 degradation by ns5. for example, trim23 was identified as an essential factor in yfv replication due to its interaction and poly-ub of residue k6 on yfv-ns5, promoting binding with stat2 and inhibition of type-i ifn signaling [54] . another flavivirus, japanese encephalitis virus (jev), induces expression of trim21 in human microglial cells, which attenuates jev-induced antiviral signaling [53] . the study by manocha et al. demonstrated that trim21 overexpression suppressed phosphorylation of irf3 and activation of ifn-β, while silencing trim21 permitted efficient type-i ifn responses in jev-infected human microglial cells [53] . this study provides evidence that jev suppress the ifn-i response due to induction of trim21. finally, we recently showed that nipah virus (niv), a single-stranded negative-sense highly pathogenic rna virus (paramyxoviridae family, genus henipavirus) that causes fatal diseases in humans [251] , can inhibit trim6-mediated type-i ifn responses [56] (figure 6 ). mechanistically, the niv matrix (niv-m) structural protein, which is required for virus assembly and budding [252, 253] , targets trim6 for degradation. interestingly, niv budding requires trafficking of niv-m from the cytoplasm to the nucleus before reaching the cell membrane for virus assembly [252, 253] . the reason for niv-m trafficking to the nucleus is still unclear, however, a lysine residue (k258) in the niv-m bipartite nuclear localization signal that is conserved in divergent henipaviruses and is required for trafficking, is critical for the ifn antagonist function [56] . consistent with this, the matrix proteins of ghana, hendra and cedar viruses were also able to inhibit ifn-β induction [56] . it is currently unknown whether trim6 e3-ligase activity affects niv-m trafficking or whether it directly interferes with niv-replication. niv-m-induced trim6 degradation did not appear to require the proteasome, however. although the precise mechanism of trim6 degradation remains to be elucidated, inhibitors that recover trim6 protein levels could potentially be used as therapeutic drugs against niv infections. these findings highlight the importance of trim6 as an antiviral factor of the type-i ifn system. a variety of dna viruses also encode viral proteins that interfere with trims. alternative methods for subverting trim-induced innate immune responses can be noted through alterations made to host transcription by hepatitis b virus (hbv). the hbv-encoded protein x (hbx) methylates a single cpg in trim22's promoter region [254] . this viral-mediated epigenetic modification blocks ifn-α/γ-induced irf1, a transcriptional activator, from binding the trim22 promoter and consequently facilitates viral proliferation and escape [255] . in epstein-barr virus (ebv), kap1 (krab [kruppel-associated box domain]-associated protein 1)/trim28 regulates activation of the viral lytic cycle [256] . in cells undergoing lytic stage activation, kap1/trim28 becomes phosphorylated at serine residue 824 by ataxia telangiectasia mutated (atm). this post-translational modification impairs kap1/trim28's restriction factor function and allows ebv to transition from latency to lytic stage [256] . additionally, the anti-malarial drug chloroquine acts as an activator for atm by phosphorylating kap1/trim28, which ultimately leads to promotion of the ebv lytic cycle and escape of the virus particles [256] . mutations in gene expression serve as an additional focal point highlighting the role that trims play in a dysfunctional antiviral response. when a population of 765 hbv-infected individuals was screened, chronic hbv infection was found to correlate to a single t to c silent mutation single nucleotide polymorphism (snp) in the trim22 ring domain [257] . although the implications to patient outcomes are considerable, additional investigations into the basic biological mechanisms are warranted to discern the importance of these snps in trim-mediated innate immunity. figure 6 . trim6 is targeted by nipah and ebola viruses to enhance virus replication. ebola virus (ebov) inhibits type-i ifn production by multiple mechanisms. ebov vp35 binds and inhibits rig-i, ikkε, and tbk-1 to inhibit ifn production. trim6 ubiquitinates vp35 on k309 and promotes vp35 activity as the cofactor of the viral polymerase and enhances virus replication. additional unidentified ubiquitination sites of vp35 exist. whether trim6 enhances ebov replication by promoting viral genome replication or viral gene transcription is not known. in nipah virus infection, the viral matrix protein (niv-m) promotes trim6 degradation, resulting in reduced synthesis of k48-linked unanchored polyubiquitin chains, ikkε oligomerization, ikkε-t501 autophosphorylation, irf3 phosphorylation, and reduced ifn induction. these combined losses confer an impaired host-antiviral response. a variety of dna viruses also encode viral proteins that interfere with trims. alternative methods for subverting trim-induced innate immune responses can be noted through alterations made to host transcription by hepatitis b virus (hbv). the hbv-encoded protein x (hbx) methylates a single cpg in trim22's promoter region [254] . this viral-mediated epigenetic modification blocks ifn-α/γ-induced irf1, a transcriptional activator, from binding the trim22 promoter and consequently facilitates viral proliferation and escape [255] . in epstein-barr virus (ebv), kap1 (krab [kruppel-associated box domain]-associated protein 1)/trim28 regulates activation of the viral lytic cycle [256] . in cells undergoing lytic stage activation, kap1/trim28 becomes phosphorylated at serine residue 824 by ataxia telangiectasia mutated (atm). this post-translational modification impairs kap1/trim28's restriction factor function and allows ebv to transition from latency to lytic stage [256] . additionally, the anti-malarial drug chloroquine acts as an activator for atm by phosphorylating kap1/trim28, which ultimately leads to promotion of the ebv lytic cycle and escape of the virus particles [256] . mutations in gene expression serve as an additional focal point highlighting the role that trims play in a dysfunctional antiviral response. when a population of 765 hbv-infected individuals was screened, chronic hbv infection was found to correlate to a single t to c silent mutation single nucleotide polymorphism (snp) in the trim22 ring domain [257] . although the implications to patient outcomes are considerable, additional investigations into the basic biological mechanisms are warranted to discern the importance of these snps in trim-mediated innate immunity. in addition to impairing trim gene expression, dna viruses can mimic host proteins to prohibit trim function. the immediate early protein (icp0) of herpes simplex virus 1 (hsv-1) possesses a ring domain and facilitates the degradation of trim27 through ubiquitination [258] . this destruction of trim27 is facilitated through the host's proteasome [258] , mimicking the numerous instances of trim-mediated restriction. gammaherpesvirus mhv-68 similarly targets trim19 for proteasome-mediated degradation [239] . the ie1 proteins of human cytomegalovirus (hcmv) mimics the coiled-coil domain of trims to recruit and sequester trim19 from nuclear bodies thus impairing the activation of ifn responses [239] . so far, most studies on trims have focused on their roles as antiviral factors by directly restricting virus replication or indirectly by inducing antiviral cytokines, as described above. the fact that trims are targeted by viruses for immune evasion further highlights their important roles in protecting the host against infections. however, whether trims may directly act as host factors required for virus replication, or "pro-viral" factors, has not been addressed. some studies have shown that certain viral antagonists can hijack trims to activate their ifn antagonist activity (e.g., trim23 ubiquitinates yfv-ns5 for antagonism of stat2 function [54] ), but these are indirect effects that provide an advantage to the virus by reducing host antiviral responses. since ubiquitination of viral proteins may positively influence specific steps of the replication cycle, it would not be surprising if trims are involved in directly promoting virus replication by non-degradative ubiquitination of viral proteins. indeed, we recently reported the first example of such a role for trim6 [259] . as described above in section 2.6, trim6 is involved in antiviral type-i ifn responses by catalyzing the synthesis of unanchored k48-linked polyubiquitin chains that activate ikkε (figures 2 and 6 ). further evidence of trim6 as an antiviral factor is highlighted by our findings that the niv can inhibit ifn-i responses by targeting trim6 [56] . furthermore, knockdown of trim6 in lung a549 cell lines and primary human monocyte derived dendritic cells has shown increased replication of multiple viruses, including iav, emcv, and sendai virus (sev), most probably due to reduced type-i ifn responses [11] . in addition, trim6 a549 knockout cells showed reduced ifn responses [259] . unexpectedly, despite a defect in the ifn response, infectious ebola virus (ebov: filoviridae family) replicated less efficiently in trim6 knockout cells as compared to parental wild type (wt) cells. this observation raised the question whether trim6 may be acting as a pro-viral factor or an enhancer of virus replication. in support of this hypothesis, we found that trim6 interacts with ebov-vp35, which is a major viral ifn antagonist by targeting rig-i [260, 261] and the kinases ikkε and tbk-1 [262] . however, since vp35 also plays a critical role as the cofactor of the virus polymerase [263] , trim6 could directly affect polymerase function. mass spectrometry analysis and co-immunoprecipitation assays demonstrated that trim6 ubiquitinates vp35 on k309 [259] , a lysine residue located on its ifn antagonist domain. moreover, minigenome reporter experiments showed that trim6 can enhance vp35-mediated polymerase activity, and this effect requires the e3-ubiquitin ligase activity of trim6. although vp35 is ubiquitinated in multiple (currently unidentified) residues in addition to k309, the trim6-dependent effects on vp35 minigenome activity required an intact k309 residue. vp35 was also able to inhibit rig-i induced trim6-enhanced ifnβ in reporter assays, however the precise mechanism was not elucidated [259] . collectively, these findings suggest that trim6 is a host factor hijacked by ebov-vp35 for both immune evasion and for promoting virus replication via ubiquitination of vp35 ( figure 6 ). future studies will address whether ubiquitination of vp35 regulates virus rna replication or transcription. it remains to be seen if other trims that are targeted by viruses may also directly enhancing virus replication via ubiquitination of viral proteins. autophagy (self-eating) is a highly conserved catabolic mechanism through which eukaryotic cells deliver dispensable, or potentially dangerous, cytoplasmic material to lysosomes for degradation [264] . the process is characterized by the formation of autophagosomes, which sequester the cytoplasmic structures targeted for destruction. autophagy has been linked to a wide range of physiological processes, including cell differentiation and development, the degradation of aberrant structures and turnover of damaged organelles, as well as innate and adaptive immunity [265, 266] . a growing number of studies indicate that several trims are linked to autophagy and recent excellent reviews are available on the emerging roles of trims in autophagy [59, 60, 267, 268] . a good example of the potential role of trims in autophagy and virus infections is rhesus trim5α, which acts both as a regulator of autophagy by providing a platform for the assembly of activated ulk1 and beclin 1 (key components of the autophagy regulatory complexes) and as a receptor for selective autophagy [269, 270] . in its role as an autophagic cargo receptor, trim5α directly recognizes viral capsid sequences via its spry domain [158, [269] [270] [271] . this is an example of selective autophagy in mammalian cells, which could occur via direct substrate recognition by trims and connects autophagy with a role in defense against viral pathogens. the rhesus trim5 can execute precision autophagy of the hiv-1 capsid. in contrast, the weak affinity of human trim5 for the hiv-1 capsid precludes effective precision autophagy. as a consequence, rhesus trim5, but not human trim5, could contribute to defense against hiv-1 through precision autophagy. since this recognition depends on the c-terminal region of trims (e.g., spry domain of trim5α), other types of c-terminal domains on trims could selectively recognize diverse protein targets. thus, trim proteins, as a group, could comprise a class of broad-repertoire, high-fidelity, selective autophagic receptors. given the breadth of the role of trims in various diseases, it will be important to explore precision autophagy-in addition to bulk autophagy-as a therapeutic target against viral infections. the proposed role of trims in autophagy raise questions. for example, how many trims act as autophagic receptors and what are their specific targets? do trim proteins function as hubs connecting different signaling pathways or different systems? how is the autophagic role of trims integrated with the other functions of trims, including regulation of gene expression and pro-inflammatory signaling? what is the interplay between the e3 ligase activity of trims and precision autophagy? it is important to determine inhibitory compounds of trim proteins for their use as therapeutic tools in infectious diseases. however, because some trim proteins have simultaneous dual functions in carcinogenesis and the immune response, it should be considered that putative drugs (inhibitors of some trim proteins) for cancer therapy may affect immunological reactions as a side effect. further detailed analysis of trim proteins is needed for their use as novel therapeutics with minimal side effects. this review highlighted the roles of trims in virus-host interactions. extensive reports detail trim involvement in immune signaling and direct virus restriction. in addition, viral antagonism of trims exemplifies the importance of this protein family in antiviral responses. despite these advancements, many trims have yet to be characterized. additionally, the molecular mechanisms underlying trim-mediated virus restriction or viral protein-mediated trim inhibition are not fully elucidated. the role of trims in regulating poly-ub chain topology is also of interest. in several examples, including trims 5 [169] , 6 [11] , 21 [12] , and 25 [77] , trims facilitate the synthesis of unanchored poly-ub chains. the relative contribution of trims in synthesizing specifically unanchored poly-ub chains, versus covalently linked poly-ub chains, is unclear. classically, the e2 is considered more important in determining poly-ub chain characteristics [4, 5] , but post-translational modifications may influence the decision between covalent and non-covalent linkage [12] as may the choice of the partnering e3. perhaps trims play a unique role in the synthesis of unanchored chains. in the future, evaluating the factors that regulate e2-trim pairing may add an additional layer of complexity to trim-mediated regulation and the ubiquitin code. although one study addressed this question by testing interactions between trims and the e2-conjugases and a few trim-e2 pairs were identified [272] , the complexity of potential transient interactions, and the possibility of cell-type specific expression for the combination of trims and e2-conjugases makes this task a huge challenge. importantly, another question is how poly-ub chains of unconventional linkages may affect virus replication. for example, unanchored poly-ub chains can be incorporated into the iav virion to enable hijacking of the host's aggresomal pathway to facilitate viral replication [192] . the e2 and e3 enzymes responsible for synthesizing these poly-ub chains have not been identified, but perhaps iav hijacks a trim to make these poly-ub chains critical in efficient infection. therefore, unanchored poly-ub chains may have both pro-viral and antiviral functions [195] and elucidating how to tip the balance towards antiviral responses may help develop novel antiviral therapies. additionally, iav replication via host ubiquitin and aggresome systems relies on the host's cytoskeletal network [192] . this may further implicate the role of trims in similar pathways as several trims associate with microtubules [18, 31] . many trims assemble into cytoplasmic bodies, which can be dynamic structures as described with trim32 [35] . the exact role of cytoplasmic bodies is not well characterized. possible trim-cytoplasmic body functions may include facilitation of signaling complex assembly [11] , regulation of active trim solubility, and/or generation of autophagosome-like complexes to target proteins for degradation. viruses can also disrupt or reorganize trim-cytoplasmic bodies, further supporting a role in antiviral responses. understanding how trim sub-cellular localization influences activity will also benefit the field. the applicability of the information garnered from studying trim-virus interactions may facilitate the identification of novel antiviral targets. further studies will advance our understanding of the complexities involved with trim signaling such as isoform-specific roles, cell-type specific activity, cytoplasmic body assembly and function, and post-transcriptional modification-induced trim function. finally, future studies will need to address whether other trims, in addition to trim6, may be hijacked by viruses to directly enhance replication, acting as pro-viral factors. the increasing complexity of the ubiquitin code ubiquitin enzymes in the regulation of immune responses ubiquitin-binding proteins: decoders of ubiquitin-mediated cellular functions building ubiquitin chains: e2 enzymes at work ube2d3 and ube2n are essential for rig-i-mediated mavs aggregation in antiviral innate immunity the roles of the trim e3-ubiquitin ligase family in innate antiviral immunity intrimsic immunity: positive and negative regulation of immune signaling by tripartite motif proteins ubiquitin acetylation inhibits polyubiquitin chain elongation ubiquitin modifications ubiquitin-binding domains unanchored k48-linked polyubiquitin synthesized by the e3-ubiquitin ligase trim6 stimulates the interferon-ikkepsilon kinase-mediated antiviral response sequential ubiquitination and deubiquitination enzymes synchronize the dual sensor and effector functions of trim21 trim family proteins and their emerging roles in innate immunity trim protein-mediated regulation of inflammatory and innate immune signaling and its association with antiretroviral activity trim e3 ligases interfere with early and late stages of the retroviral life cycle the e3-ligase trim family of proteins regulates signaling pathways triggered by innate immune pattern-recognition receptors structural determinants of trim protein function the tripartite motif family identifies cell compartments trim16 acts as an e3 ubiquitin ligase and can heterodimerize with other trim family members solution structure of the rbcc/trim b-box1 domain of human mid1: b-box with a ring functional role of trim e3 ligase oligomerization and regulation of catalytic activity the tripartite motif coiled-coil is an elongated antiparallel hairpin dimer structure and catalytic activation of the trim23 ring e3 ubiquitin ligase structural insights into the trim family of ubiquitin e3 ligases crystal structure of trim20 c-terminal coiled-coil/b30.2 fragment: implications for the recognition of higher order oligomers a b-box 2 surface patch important for trim5α self-association, capsid binding avidity, and retrovirus restriction the trim5alpha b-box 2 domain promotes cooperative binding to the retroviral capsid by mediating higher-order self-association tripartite motif ligases catalyze polyubiquitin chain formation through a cooperative allosteric mechanism ring dimerization links higher-order assembly of trim5alpha to synthesis of k63-linked polyubiquitin trim14 is a mitochondrial adaptor that facilitates retinoic acid-inducible gene-i-like receptor-mediated innate immune response subclassification of the rbcc/trim superfamily reveals a novel motif necessary for microtubule binding genomic analysis of the trim family reveals two groups of genes with distinct evolutionary properties negative regulation of nf-κb activity by brain-specific tripartite motif protein 9 trim28 prevents autoinflammatory t cell development in vivo 14-3-3 proteins sequester a pool of soluble trim32 ubiquitin ligase to repress autoubiquitylation and cytoplasmic body formation rapid turnover and polyubiquitylation of the retroviral restriction factor trim5 the cellular level of trim31, an rbcc protein overexpressed in gastric cancer, is regulated by multiple mechanisms including the ubiquitin-proteasome system genomics and evolution of the trim gene family identification of a genomic reservoir for new trim genes in primate genomes origin and evolution of trim proteins: new insights from the complete trim repertoire of zebrafish and pufferfish an evolutionary screen highlights canonical and noncanonical candidate antiviral genes within the primate trim gene family positive selection of the trim family regulatory region in primate genomes discordant evolution of the adjacent antiretroviral genes trim22 and trim5 in mammals positive selection of primate trim5alpha identifies a critical species-specific retroviral restriction domain human trim gene expression in response to interferons induction of trim22 by ifn-gamma involves jak and pc-plc/pkc, but not mapks and pi3k/akt/mtor pathways expression of the immune regulator tripartite-motif 21 is controlled by ifn regulatory factors type i interferon-dependent and -independent expression of tripartite motif proteins in immune cells trim38 negatively regulates tlr3/4-mediated innate immune and inflammatory responses by two sequential and distinct mechanisms trim13 is a negative regulator of mda5-mediated type i interferon production expression profiling of trim protein family in thp1-derived macrophages following tlr stimulation tripartite-motif proteins and innate immune regulation regulatory role of trim21 in the type-i interferon pathway in japanese encephalitis virus-infected human microglial cells the interferon signaling antagonist function of yellow fever virus ns5 protein is activated by type i interferon dengue subgenomic rna binds trim25 to inhibit interferon expression for epidemiological fitness the matrix protein of nipah virus targets the e3-ubiquitin ligase trim6 to inhibit the ikkepsilon kinase-mediated type-i ifn antiviral response influenza a virus ns1 targets the ubiquitin ligase trim25 to evade recognition by the host viral rna sensor rig-i viral evasion mechanisms of early antiviral responses involving regulation of ubiquitin pathways trim family proteins: roles in autophagy, immunity, and carcinogenesis precision autophagy directed by receptor regulators--emerging examples within the trim family trim proteins and diseases pattern recognition receptors and inflammation expanding role of ubiquitination in nf-κb signaling convergence of the nf-κb and irf pathways in the regulation of the innate antiviral response in vivo ligands of mda5 and rig-i in measles virus-infected cells the rna helicase rig-i has an essential function in double-stranded rna-induced innate antiviral responses 5'-triphosphate rna is the ligand for rig-i reis e sousa, c. rig-i-mediated antiviral responses to single-stranded rna bearing 5'-phosphates differential roles of mda5 and rig-i helicases in the recognition of rna viruses rig-i-like receptor regulation in virus infection and immunity ubiquitin-induced oligomerization of the rna sensors rig-i and mda5 activates antiviral innate immune response structural basis for dsrna recognition, filament formation, and antiviral signal activation by mda5 identification and characterization of mavs, a mitochondrial antiviral signaling protein that activates nf-κb and irf 3 rig-i forms signaling-competent filaments in an atp-dependent, ubiquitin-independent manner phosphorylation-mediated negative regulation of rig-i antiviral activity the ubiquitin ligase riplet is essential for rig-i-dependent innate immune responses to rna virus infection reconstitution of the rig-i pathway reveals a signaling role of unanchored polyubiquitin chains in innate immunity negative regulation of the rig-i signaling by the ubiquitin ligase rnf125 ubch8 regulates ubiquitin and isg15 conjugation to rig-i mavs forms functional prion-like aggregates to activate and propagate antiviral innate immune response mavs recruits multiple ubiquitin e3 ligases to activate antiviral signaling cascades traf molecules in cell signaling and in human diseases activation of the iκb kinase complex by traf6 requires a dimeric ubiquitin-conjugating enzyme complex and a unique polyubiquitin chain tab2 and tab3 activate the nf-κb pathway through binding to polyubiquitin chains direct activation of protein kinases by unanchored polyubiquitin chains the regulation of nf-κb subunits by phosphorylation. cells type i interferons in infectious disease are the ikks and ikk-related kinases tbk1 and ikk-epsilon similarly activated? primary activation of interferon a and interferon b gene transcription by interferon regulatory factor 3 trim65-catalized ubiquitination is essential for mda5-mediated antiviral innate immunity trim4 modulates type i interferon induction and cellular antiviral response by targeting rig-i for k63-linked ubiquitination trim25 ring-finger e3 ubiquitin ligase is essential for rig-i-mediated antiviral activity ubiquitin-mediated modulation of the cytoplasmic viral rna sensor rig-i the ubiquitin-specific protease usp15 promotes rig-i-mediated antiviral signaling by deubiquitylating trim25 multifaceted roles of trim38 in innate immune and inflammatory responses innate immunity to rna virus is regulated by temporal and reversible sumoylation of rig-i and mda5 trim59 interacts with ecsit and negatively regulates nf-κb and irf-3/7-mediated signal pathways de novo transcriptome analysis shows that sav-3 infection upregulates pattern recognition receptors of the endosomal toll-like and rig-i-like receptor signaling pathways in macrophage/dendritic like to-cells activation of duck rig-i by trim25 is independent of anchored ubiquitin species-specific inhibition of rig-i ubiquitination and ifn induction by the influenza a virus ns1 protein molecular characterization, tissue distribution and expression analysis of trim25 in gallus gallus domesticus duck trim27-l enhances mavs signaling and is absent in chickens and turkeys mavs ubiquitination by the e3 ligase trim25 and degradation by the proteasome is involved in type i interferon production after activation of the antiviral rig-i-like receptors the ubiquitin e3 ligase trim31 promotes aggregation and activation of the signaling adaptor mavs through lys63-linked polyubiquitination novel function of trim44 promotes an antiviral response by stabilizing visa trim9 short isoform preferentially promotes dna and rna virus-induced production of type i interferon by recruiting gsk3β to tbk1 crosstalk between cytoplasmic rig-i and sting sensing pathways trim11 negatively regulates ifnβ production and antiviral activity by targeting tbk1 tripartite motif-containing protein 38 negatively regulates tlr3/4-and rig-i-mediated ifn-β production and antiviral response by targeting nap1 polyubiquitin conjugation to nemo by triparite motif protein 23 (trim23) is critical in antiviral defense autoubiquitination of trim26 links tbk1 to nemo in rlr-mediated innate antiviral immune response trim68 negatively regulates ifn-β production by degrading trk fused gene, a novel driver of ifn-β downstream of anti-viral detection systems trim30 α negatively regulates tlr-mediated nf-kappa b activation by targeting tab2 and tab3 for degradation the human antiviral factor trim11 is under the regulation of hiv-1 vpr identification of a role for trim29 in the control of innate immunity in the respiratory tract trim39 negatively regulates the nfκb-mediated signaling pathway through stabilization of cactin sting is an endoplasmic reticulum adaptor that facilitates innate immune signalling sting regulates intracellular dna-mediated, type i interferon-dependent innate immunity sting is a direct innate immune sensor of cyclic di-gmp structure of sting bound to cyclic di-gmp reveals the mechanism of cyclic dinucleotide recognition by the immune system structural and functional analysis of ddx41: a bispecific immune receptor for dna and cyclic dinucleotide the helicase ddx41 recognizes the bacterial secondary messengers cyclic di-gmp and cyclic di-amp to activate a type i interferon immune response the e3 ubiquitin ligase trim21 negatively regulates the innate immune response to intracellular double-stranded dna trim30α is a negative-feedback regulator of the intracellular dna and dna virus-triggered response by targeting sting the ubiquitin ligase trim56 regulates innate immune responses to intracellular double-stranded dna trim32 protein modulates type i interferon induction and cellular antiviral response by targeting mita/sting protein for k63-linked ubiquitination the critical role of toll-like receptors-from microbial recognition to autoimmunity: a comprehensive review the e3 ubiquitin ligase ro52 negatively regulates ifn-β production post-pathogen recognition by polyubiquitin-mediated degradation of irf3 self protection from anti-viral responses-ro52 promotes degradation of the transcription factor irf7 downstream of the viral toll-like receptors tripartite motif 21 (trim21) differentially regulates the stability of interferon regulatory factor 5 (irf5) isoforms e3 ubiquitin ligase tripartite motif 38 negatively regulates tlr-mediated immune responses by proteasomal degradation of tnf receptor-associated factor 6 in macrophages hung, t. trim38 negatively regulates tlr3-mediated ifn-β signaling by targeting trif for degradation trim56 is an essential component of the tlr3 antiviral signaling pathway mechanisms and functions of inflammasomes activation and regulation of the inflammasomes activation of nucleotide oligomerization domain 2 (nod2) by human cytomegalovirus initiates innate immune responses and restricts virus replication nod1 and nod2 signaling in infection and inflammation the e3 ubiquitin ligase tripartite motif 33 is essential for cytosolic rna-induced nlrp3 inflammasome activation tripartite-motif protein 30 negatively regulates nlrp3 inflammasome activation by modulating reactive oxygen species production the e3 ubiquitin ligase trim31 attenuates nlrp3 inflammasome activation by promoting proteasomal degradation of nlrp3 trim27 negatively regulates nod2 by ubiquitination and proteasomal degradation nucleo-cytoplasmic trafficking of trim8, a novel oncogene, is involved in positive regulation of tnf induced nf-κb pathway trim8 modulates stat3 activity through negative regulation of pias3 trim38 inhibits tnfα-and il-1β-triggered nf-κb activation by mediating lysosome-dependent degradation of tab2/3 siglec1 suppresses antiviral innate immune response by inducing tbk1 degradation via the ubiquitin ligase trim27 tripartite motif-containing 28 bridges endothelial inflammation and angiogenic activity by retaining expression of tnfr-1 and -2 and vegfr2 in endothelial cells interferons and viral infections tripartite motif 24 (trim24/tif1α) tumor suppressor protein is a novel negative regulator of interferon (ifn)/signal transducers and activators of transcription (stat) signaling pathway acting through retinoic acid receptor α (rarα) inhibition trim8/gerp ring finger protein interacts with socs-1 multiple functions of the ikk-related kinase ikkepsilon in interferon-mediated antiviral immunity iκb kinase ε (ikkε) regulates the balance between type i and type ii interferon responses control of foxo4 activity and cell survival by trim22 directs tlr3-stimulated cells toward ifn type i gene induction or apoptosis the interferon-inducible staf50 gene is downregulated during t cell costimulation by cd2 and cd28 regulation of the interferon-inducible p53 target gene trim22 (staf50) in human t lymphocyte activation t-cell-intrinsic tif1α/trim24 regulates il-1r expression on th2 cells and th2 cell-mediated airway allergy tripartite motif containing protein 27 negatively regulates cd4 t cells by ubiquitinating and inhibiting the class ii pi3k-c2β tripartite motif-containing protein 30 modulates tcr-activated proliferation and effector functions in cd4+ t cells the cytoplasmic body component trim5α restricts hiv-1 infection in old world monkeys trim5α protein restricts both hiv-1 and murine leukemia virus a trim5-cyclophilin a fusion protein found in owl monkey kidney cells can restrict hiv-1 cyclophilin a retrotransposition into trim5 explains owl monkey resistance to hiv-1 trim5α requires ube2w to anchor lys63-linked ubiquitin chains and restrict reverse transcription dynamic conformational changes in the rhesus trim5α dimer dictate the potency of hiv-1 restriction primate trim5 proteins form hexagonal nets on hiv-1 capsids mechanism of b-box 2 domain-mediated higher-order assembly of the retroviral restriction factor trim5α crystal structure of the trim5α bbox2 domain from rhesus macaques describes a plastic oligomerisation interface recent insights into the mechanism and consequences of trim5α retroviral restriction hexagonal assembly of a restricting trim5αprotein trim5 is an innate immune sensor for the retrovirus capsid lattice proteasome inhibition reveals that a functional preintegration complex intermediate can be generated during restriction by diverse trim5 proteins proteasome inhibitors uncouple rhesus trim5α restriction of hiv-1 reverse transcription and infection fates of retroviral core components during unrestricted and trim5-restricted infection proteasomal degradation of trim5α during retrovirus restriction nonhuman trim5 variants enhance recognition of hiv-1-infected cells by cd8+ t cells capsid-binding retrovirus restriction factors: discovery, restriction specificity and implications for the development of novel therapeutics hiv-1 capsid recognition, and innate immune signaling trim5α-mediated ubiquitin chain conjugation is required for inhibition of hiv-1 reverse transcription and capsid destabilization trim5 retroviral restriction activity correlates with the ability to induce innate immune signaling trim5α restriction affects clinical outcome and disease progression in simian immunodeficiency virus-infected rhesus macaques preserved cd4+ central memory t cells and survival in vaccinated siv-challenged monkeys defective cd8 t cell memory following acute infection without cd4 t cell help requirement for cd4 t cell help in generating functional cd8 t cell memory a single amino acid change in the spry domain of human trim5αleads to hiv-1 restriction evolution of a cytoplasmic tripartite motif (trim) protein in cows that restricts retroviral infection isolation of an active lv1 gene from cattle indicates that tripartite motif protein-mediated innate immunity to retroviral infection is widespread among mammals ovine trim5α can restrict visna/maedi virus receptor usage dictates hiv-1 restriction by human trim5α in dendritic cell subsets lack of endogenous trim5α-mediated restriction in rhesus macaque dendritic cells endogenous trim5α function is regulated by sumoylation and nuclear sequestration for efficient innate sensing in dendritic cells an hiv-1 capsid binding protein trim11 accelerates viral uncoating functional evidence for the involvement of microtubules and dynein motor complexes in trim5α-mediated restriction of retroviruses influenza a virus uses the aggresome processing machinery for host cell entry hiv-1 uncoating is facilitated by dynein and kinesin 1 cytoplasmic dynein promotes hiv-1 uncoating unanchored ubiquitin in virus uncoating mid1 and mid2 are required for xenopus neural tube closure through the regulation of microtubule organization hiv-1 transcriptional silencing caused by trim22 inhibition of sp1 binding to the viral promoter anti-hiv-1 activity of trim 37 the interferon-induced antiviral protein pml (trim19) promotes the restriction and transcriptional silencing of lentiviruses in a context-specific, isoform-specific fashion trim19/pml restricts hiv infection in a cell type-dependent manner pml/trim19-dependent inhibition of retroviral reverse-transcription by daxx translocalized iga mediates neutralization and stimulates innate immunity inside infected cells antibodies mediate intracellular immunity through tripartite motif-containing 21 (trim21) trim21 is an igg receptor that is structurally, thermodynamically, and kinetically conserved intracellular antibody-bound pathogens stimulate immune signaling via the fc receptor trim21 simultaneous neutralization and innate immune detection of a replicating virus by trim21 trim21 promotes cgas and rig-i sensing of viral genomes during infection by antibody-opsonized virus trim21 immune signaling is more sensitive to antibody affinity than its neutralization activity intracellular antibody receptor trim21 prevents fatal viral infection swine trim21 restricts fmdv infection via an intracellular neutralization mechanism robust lys63-linked ubiquitination of rig-i promotes cytokine eruption in early influenza b virus infection trim22 inhibits influenza a virus infection by targeting the viral nucleoprotein for degradation structure of influenza a polymerase bound to the viral rna promoter trim32 senses and restricts influenza a virus by ubiquitination of pb1 polymerase the c-terminal tail of trim56 dictates antiviral restriction of influenza a and b viruses by impeding viral rna synthesis historical perspectives on flavivirus research pathogenesis of flavivirus infections: using and abusing the host cell understanding the molecular mechanism(s) of hepatitis c virus (hcv) induced interferon resistance mutations in the nonstructural protein 5a gene and response to interferon in patients with chronic hepatitis c virus 1b infection trim14 inhibits hepatitis c virus infection by spry domain-dependent targeted degradation of the viral ns5a protein interferon α(ifnα)-induced trim22 interrupts hcv replication by ubiquitinating ns5a trim52 inhibits japanese encephalitis virus replication by degrading the viral ns2a dengue virus inhibits α interferon signaling by reducing stat2 expression inhibition of interferon signaling by dengue virus blocking double-stranded rna-activated protein kinase pkr by japanese encephalitis virus nonstructural protein 2a overlapping and distinct molecular determinants dictating the antiviral activities of trim56 against flaviviruses and coronavirus identification of three interferon-inducible cellular enzymes that inhibit the replication of hepatitis c virus trim56 is a virus-and interferon-inducible e3 ubiquitin ligase that restricts pestivirus infection tick-borne flaviviruses antagonize both irf-1 and type i ifn signaling to inhibit dendritic cell function mechanisms of evasion of the type i interferon antiviral response by flaviviruses inhibition of interferon-stimulated jak-stat signaling by a tick-borne flavivirus and identification of ns5 as an interferon antagonist trim79α, an interferon-stimulated gene product, restricts tick-borne encephalitis virus replication by degrading the viral rna polymerase trim25 enhances the antiviral action of zinc-finger antiviral protein (zap) promyelocytic leukemia isoform iv confers resistance to encephalomyocarditis virus via the sequestration of 3d polymerase in nuclear bodies identification and characterization of multiple trim proteins that inhibit hepatitis b virus transcription trim22: a diverse and dynamic antiviral protein tripartite motif-containing 22 inhibits the activity of hepatitis b virus core promoter, which is dependent on nuclear-located ring domain trim5α promotes ubiquitination of rta from epstein-barr virus to attenuate lytic progression emerging role of pml nuclear bodies in innate immune signaling influenza a virus trims the type i interferon response the role of trim25 in development, disease and rna metabolism ubiquitin in influenza virus entry and innate immunity trim25 identification in the chinese goose: gene structure, tissue expression profiles, and antiviral immune responses in vivo and in vitro subcellular localizations of rig-i, trim25, and mavs complexes the severe acute respiratory syndrome coronavirus nucleocapsid inhibits type i interferon production by interfering with trim25-mediated rig-i ubiquitination hijacking of rig-i signaling proteins into virus-induced cytoplasmic structures correlates with the inhibition of type i interferon responses tripartite containing motif 32 modulates proliferation of human neural precursor cells in hiv-1 neurodegeneration ns5 of dengue virus mediates stat2 binding and degradation dengue virus co-opts ubr4 to degrade stat2 and antagonize type i interferon signaling a family of mammalian e3 hendra and nipah viruses: different and dangerous evidence for ubiquitin-regulated nuclear and subnuclear trafficking among paramyxovirinae matrix proteins ubiquitin-regulated nuclear-cytoplasmic trafficking of the nipah virus matrix protein is important for viral budding hepatitis b virus x protein: trimming antiviral defences in hepatocytes suppression of interferon-mediated anti-hbv response by single cpg methylation in the 5 -utr of trim22 chloroquine triggers epstein-barr virus replication through phosphorylation of kap1/trim28 in burkitt lymphoma cells tripartite motif-containing 22 gene-364t/c polymorphism associated with hepatitis b virus infection in chinese han population identification of trim27 as a novel degradation target of herpes simplex virus 1 icp0 the host e3-ubiquitin ligase trim6 ubiquitinates the ebola virus vp35 protein and promotes virus replication ebola virus vp35 protein binds double-stranded rna and inhibits α/β interferon production induced by rig-i signaling mutual antagonism between the ebola virus vp35 protein and the rig-i activator pact determines infection outcome ebola virus protein vp35 impairs the function of interferon regulatory factor-activating kinases ikkepsilon and tbk-1 filovirus replication and transcription. future virol eaten alive: a history of macroautophagy autophagy, immunity, and microbial adaptations autophagy fights disease through cellular self-digestion autophagy in leukocytes and other cells: mechanisms, subsystem organization, selectivity, and links to innate immunity trim-directed selective autophagy regulates immune activation trim proteins regulate autophagy and can target autophagic substrates by direct recognition trim proteins regulate autophagy: trim5 is a selective autophagy receptor mediating hiv-1 restriction specific recognition and accelerated uncoating of retroviral capsids by the trim5α restriction factor functional interactions between ubiquitin e2 enzymes and trim proteins the authors declare no conflict of interest. key: cord-307598-p54p7enk authors: schlee, martin title: master sensors of pathogenic rna – rig-i like receptors date: 2013-07-01 journal: immunobiology doi: 10.1016/j.imbio.2013.06.007 sha: doc_id: 307598 cord_uid: p54p7enk initiating the immune response to invading pathogens, the innate immune system is constituted of immune receptors (pattern recognition receptors, prr) that sense microbe-associated molecular patterns (mamps). detection of pathogens triggers intracellular defense mechanisms, such as the secretion of cytokines or chemokines to alarm neighboring cells and attract or activate immune cells. the innate immune response to viruses is mostly based on prrs that detect the unusual structure, modification or location of viral nucleic acids. most of the highly pathogenic and emerging viruses are rna genome-based viruses, which can give rise to zoonotic and epidemic diseases or cause viral hemorrhagic fever. as viral rna is located in the same compartment as host rna, prrs in the cytosol have to discriminate between viral and endogenous rna by virtue of their structure or modification. this challenging task is taken on by the homologous cytosolic dexd/h-box family helicases rig-i and mda5, which control the innate immune response to most rna viruses. this review focuses on the molecular basis for rig-i like receptor (rlr) activation by synthetic and natural ligands and will discuss controversial ligand definitions. receptors of the innate immune system sense foreign molecules and structures such as the highly conserved microbe-associated molecular patterns (mamps) like sugars, lipids, proteins, or nucleic acids of bacteria, fungi or viruses . innate immune receptor stimulation by mamps triggers intracellular defense mechanisms and the induction of innate immune responses, including secretion of cytokines and chemokines, which lead to alarming of neighboring cells and attracting immune cells. most of the known highly pathogenic and emerging viruses are rna genome-based; they give rise to epidemic and zoonotic diseases (flu, foot-and-mouth disease) or cause viral hemorrhagic fever including yellow fever, dengue, lassa fever and ebola (bray 2008) . the recognition of foreign pathogenic rna, resulting in induction of type i interferon (ifn), the most important antiviral cytokine, is therefore highly critical. innate immune cells express the endosomal toll-like receptors (tlr) 7, 8 and 9, which sense gu-rich rna and cpg-containing dna. tlr stimulation leads to secretion of type-i ifn, il-12 and assorted chemokines (diebold et al. 2003; heil et al. 2004; hemmi et al. 2000; hornung et al. 2005 hornung et al. , 2002 judge et al. 2005; krieg et al. 1995) , reviewed in (barchet et al. 2008; schlee et al. 2007 schlee et al. , 2006 . in contrast to tlr7, 8 and 9, tlr3 is expressed in more cell types (e.g. endothelial cells, fibroblasts, astrocytes) (barchet et al. 2008; schlee et al. 2007) and was found to detect long double-stranded rna (alexopoulou et al. 2001) . unlike tlrs, the rig-i-like receptors (rlr) rig-i, mda5 and lgp2 are present in the cytosol of all cell types. similar to tlrs, rig-i and mda5 induce type i ifn and chemokines (but no il12) upon activation by viral but also bacterial rna. while the endosomal rna detecting tlrs do contribute to antiviral immunity, rlrs are essential for the immune recognition of and response to most rna viruses ( fig. 1) (gitlin et al. 2006; hornung et al. 2006; kato et al. 2006; rothenfusser et al. 2005; venkataraman et al. 2007; yoneyama et al. 2004 ). this review summarizes the biological role of and ligand recognition by rlr with special focus on rig-i, which represents the most broadly studied and understood receptor to date. rig-i (retinoic acid-inducible gene i) and mda5 (melanoma differentiation-associated gene-5) are closely related dexd/h-box helicase family proteins. they consist of an n-terminal tandem caspase activation and recruitment domain (card) fused to a dexd/h-box helicase domain (composed of hel1, hel2 and hel2i) and the c-terminal domain (ctd; previously called rd = repressor domain) (luo et al. 2013; saito et al. 2007 ; yoneyama et al. but no recognition by rig-i or mda5 in bm-dc or fibroblasts (zhou and perlman 2007) , recognition by mda5, not rig-i in macrophages and microglia (roth-cross et al. 2008) . **evasion of rig-i recognition by nuclease 5 end cleavage leaving monophosphate at the 5 end of the viral genome (garcin et al. 1995; habjan et al. 2008 ). ***evasion of rig-i recognition via substitution of 5 triphosphate by vpg protein at the 5 end of the viral genome (hruby and roberts 1978; lee et al. 1977; rohayem et al. 2006 ). ****evasion of rig-i recognition by overhang at the 5 end of the viral genome (marq et al. 2010b ). weber, 2013 : papers contributing to the characterization of the real ligand structure in vivo are underlined. 2004) (fig. 2) . stimulation of rig-i or mda5 by viral rna release the associated cards, which aggregate with k63 polyubiquitin chains to card tetramers and then bind and activate the adaptor molecule mavs (jiang et al. 2012; zeng et al. 2010 zeng et al. , 2009 . mavs (also known as ips-1, cardif or visa (kawai et al. 2005; meylan et al. 2005; seth et al. 2005; xu et al. 2005) ) recruits tbk-1, which phosphorylates irf3 to induce transcription of type-i ifn genes (doyle et al. 2002; fitzgerald et al. 2003; sharma et al. 2003) . at present, the interaction of rig-i with its corresponding ligand rna is far better understood and analyzed than ligand-receptor interactions of mda5 or lgp2. both the ctd and helicase domain (which is no active rna helicase) possess rna binding sites, whereas solely the ctd harbors the critical binding pocket for the rna ligand, the features of which will be discussed card1 card2 hel2i ctd hel1 hel2 hel2i ctd hel1 hel2 rig-i/mda5 lgp2 fig. 2 . domain structure of rig-i, mda5 and lgp2. below. as of now, high-resolution structure studies have been performed of the ctd alone (cui et al. 2008; takahasi et al. 2008) , the ctd with ligand (lu et al. 2010b; wang et al. 2010) , mouse rig-i sf2 domain + non-hydrolysable atp (civril et al. 2011) , human rig-i( cards) + ligand (jiang et al. 2011; luo et al. 2011) , whole duck rig-i(ligand free) and helicase + ligand (kowalinski et al. 2011 ). the current model of rig-i ligand interaction resulting from the above-mentioned studies was extensively discussed (kolakofsky et al. 2012) . briefly, the card of non-stimulated rig-i binds to the so-called hel-2i domain within the helicase domain, mediating an auto-inhibited state. upon stimulation the ctd-bound rna interacts with hel-2i, leading to dislocation of the cards, which now become accessible for downstream interactions (card multimerization, mavs interaction, type i ifn induction) as described above. a similar activation mechanism for mda5 is thinkable. but as long as a mda5 recognition motif has not been clearly defined (discussed below) it remains unclear if the mda5 ctd mediates ligand specificity. a recently described crystal structure of mda5( cards) with a synthetic 11mer dsrna (which is not a mda5 activating ligand) revealed that the mda5 ctd does not cap the terminus of the blunt dsrna but rather binds the internal rna duplex structure (wu et al. 2013) . this would provide a prerequisite for a putative mda5 head-to-tail arrangement in a filament structure with exposed cards, which was suggested to be the mavs activating structure (wu et al. 2013) . the third rlr family member lgp2 lacks cards and does not induce type i ifn. its putative function will be discussed in the next section. the role of lgp2 in the immune response against viruses is not entirely understood. the lgp2 ctd resembles the rig-i ctd, albeit with different requirements for ligand binding, which will be discussed below (li et al. 2009b; murali et al. 2008; pippig et al. 2009 ). while lgp2 structurally shares a helicase and ctd, it lacks cards, suggesting a putative ligand sequestering role. indeed, initial reports suggested a pure immune suppressive function for lgp2 (komuro and horvath 2006; rothenfusser et al. 2005; saito et al. 2007; yoneyama et al. 2004 ). confusingly, a rig-i-suppressing activity was found to be independent of dsrna binding (li et al. 2009b ). the parainfluenzavirus type 5 v protein was reported to interact with lgp2, to stabilize a lgp2/rig-i complex and in this way to cooperatively inhibit induction by rig-i ligands (childs et al. 2012) . further studies on lgp2-deficient mice revealed that lgp2 absence impairs the immune response to viruses that are mainly detected by mda5, and can both impair or enhance rig-i mediated antiviral responses (pippig et al. 2009; satoh et al. 2010; venkataraman et al. 2007 ). suthar et al. (2012) confirmed that lgp2 contributed to sustained rlr signaling of ifn-␤ expression in myeloid cells during west nile virus (wnv) or dengue virus infection. additionally, they discovered a role for lgp2 in cd8(+) t cell survival: lgp2 modulated the sensitivity of cd8(+) t cells to cd95 ligand-mediated cell death through the control of cd95 expression during wnv or lymphocytic choriomeningitis virus infection. although the authors excluded a mda5/lgp2 interaction to be responsible for the observed cd95 modulation, it remained unclear if the effect in cd8(+) t cells occurs independently of rig-i. in conclusion, lgp2 appears to have a modulatory role in fine-tuning the innate immune response to viruses. mda5 -biological role, target pathogens and ligand structure mda5 recognizes long double stranded rna and contributes to or even dominates the immune response to double strand (dsrna) and positive strand rna [(+)ssrna] viruses ( fig. 1) (fredericksen et al. 2008; gitlin et al. 2006; kato et al. 2006; loo et al. 2008; mccartney et al. 2008; melchjorsen et al. 2010; roth-cross et al. 2008; saito et al. 2008) . it is crucial for raising innate immune responses against picornaviruses, like theiler's virus or encephalomyocarditis virus (emcv), enteroviruses, saffold virus 3, human parechovirus 1, equine rhinitis a virus or the caliciviridae family member norovirus, which escape rig-i recognition (feng et al. 2012; gitlin et al. 2006; kato et al. 2006; mccartney et al. 2008; triantafilou et al. 2012) (fig. 1) . at first view, mda5 appears to target virus types which are known to produce considerable amounts of dsrna during their replication cycle, including (+)ssrna, dsrna or dna viruses (mccartney et al. 2008; melchjorsen et al. 2010; pichlmair et al. 2009; roth-cross et al. 2008; targett-adams et al. 2008; weber et al. 2006) . correspondingly, two independent groups identified the double stranded replicative intermediates of (+)ssrna enteroviruses as mda5-stimulating rna species (feng et al. 2012; triantafilou et al. 2012) . a crystal with mda5( card) binding to dsrna could be obtained (wu et al. 2013) . however, the concept of dsrna recognition by mda5 seems incomplete. (−)ssrna paramyxoviruses express the immune suppressive v protein which binds to and inhibits mda5 directly, suggesting that also (−)ssrna viruses (which were shown not to generate long double stranded rna ) produce mda5 ligands (andrejeva et al. 2004; childs et al. 2007; luthra et al. 2011; motz et al. 2013) . in light of the above-mentioned studies, it appears unexpected that many double stranded rna species do not activate mda5. thus far only one artificial, albeit enzymatically generated, mda5-stimulating ligand (polyinosinepolycytidylic acid = poly i:c) has been described. it is composed of annealed strands of long (>7000 nt) rna polymers of inosins (polyi) and cytidines (polyc) (gitlin et al. 2006; kato et al. 2008 kato et al. , 2006 . most studies investigating the recognition of "long double stranded rna" (dsrna) in fact have used poly i:c. of note, poly i:c is a very particular "dsrna", as it was reported as the only copolymer among many other artificial dsrnas which was capable of inducing high amounts of type i ifn in mammalian cells (field et al. 1967) . the absence of well-defined mda5 ligands impairs systematic investigations of the mda5-ligand interaction. although the ctd of mda5 binds blunt-ended dsrna (li et al. 2009a; wu et al. 2013 ), mda5 is not activated by short dsrna and no contribution of the ctd in discriminating mda5 stimulating rna has been demonstrated (saito et al. 2007) . even though poly i:c also binds and can stimulate rig-i in certain cell lines and experimental settings in vitro (kato et al. 2008; yoneyama et al. 2004) , it is important to note that poly i:c indeed fails to induce ifn-alpha when injected intravenously into mda5-deficient mice or transfected in vitro into mda5-deficient peritoneal macrophages, dendritic cells or mefs (gitlin et al. 2006; kato et al. 2006) . by using poly i:c fragments of different sizes from rnase-iii digestion, kato and colleagues observed that mda5 was only stimulated by long poly i:c fragments. an alternative interpretation of these results would be that rnase-iii destroys certain secondary structures, which are required for recognition by mda5. pichlmair and colleagues concluded from testing of gelfractionated rnas of vaccina virus-infected cells that it was not the double-strandedness of rna that accounted for mda5 stimulating activity, but rather other higher order rna structures in large rna containing complexes (pichlmair et al. 2009 ). luthra et al. discovered a mrna fragment from the ss(−)rna parainfluenza virus 5 (piv5) that activated type-i ifn expression in a mda5-dependent manner (luthra et al. 2011 ). since type i ifn induction by this rna required rnase l, the authors concluded that rnase l recognizes and processes viral mrna into a mda5 activating structure. although a 432-nt-long region critical for mda5 stimulation was identified, no specific features of a minimal recognition motif were found. the observation by züst and colleagues that deficiency of the viral cap n 1 -2 o-methyltransferase in a type of (+)ssrna corona virus (murine hepatitis virus; mhv) provoked recognition of this mhv by mda5 and tlr7 (zust et al. 2011 ) suggested a 5 enddependent rna recognition by mda5. this finding would contrast the findings by luthra et al., who expressed the mda5 stimulatory mrna from a promoter, which supports normal capping (including n 1 -2 o-methylation). however, binding assays documenting the interaction/non-interaction of the 5 end of viral transcripts with mda5 were not performed. indirect effects were therefore not excluded. later studies on a n 1 -2 o-methyltransferase-lacking west nile virus did not reveal a role of mda5 in enhanced immune recognition of non-methylated cap structures (szretter et al. 2012) , suggesting that n 1 -2 o-methylation does not generally impair mda5 engagement. despite increasing amounts of high resolution crystal data on the rig-i/ligand interaction, the ligand requirements for rig-i stimulation are still controversial in the literature. by giving an overview of the history of the rig-i ligand definition, the following section aims to help the reader to understand how different read-out systems and ligand preparation methods could lead to conflicting interpretation of data. when rig-i was discovered as antiviral sensor by the fujita group (yoneyama et al. 2004) , the requirements of its rna ligand were not explored. rig-i was shown to bind to and to be stimulated by poly i:c when overexpressed in cell lines. at the same time, the group around john rossi, while developing sirnas against hiv, observed that all rnas generated by phage-polymerase in vitro transcription (kim et al. 2004 ) strongly induced type-i ifn in several human cell lines (hela, k562, hek293, jurkat, cem). by contrast, synthetic sirnas did not show any immune stimulatory effect in the same cell lines. dna template-dependent rna transcription occurs primer-independently from the 5 -to the 3 -end of rna. for this reason, rna transcripts of all known rna polymerases, including phage polymerase, possess a triphosphate at the 5 end (banerjee 1980) . by using phosphatase or rnase t1 (removes the 5 end pppg), kim et al. could show that the 5 triphosphate was the crucial type-i ifn-inducing structural element of in vitro transcribed rnas which was absent in synthetic sirnas (kim et al. 2004 ). this finding prompted us to analyze the ifn-alpha inducing capacity of in vitro transcribed 5 triphosphorylated rna (ppprna) in human blood cells . at this time, plasmacytoid dendritic cells (pdc) were presumed to be the principal type i ifn producing cells (cella et al. 1999; siegal et al. 1999) . they express tlr7 and 9, and secrete large amounts of ifn-alpha upon tlr7 stimulation with single or double stranded rna (hornung et al. 2005) . human monocytes express the rna-sensing endosomal tlr8. however, tlr8 stimulation does not induce ifn-alpha secretion (barchet et al. 2008) . unexpectedly, ppprna induced high levels of ifn-alpha not only in pdc but also in human monocytes. therefore, ppprna represented the first agent that induced ifn-alpha in human monocytes at comparable quantities to human pdc ). removal of the 5 triphosphate abrogated ifn-alpha induction by in vitro transcribed rna in monocytes but not in pdc . integration of nucleotides with modified bases (pseudouridine, 2-thio-uridine) or backbone modifications (2 o-methyl at uridines) abolished ifn-alpha induction by ppprna both in pdc and monocytes. using murine rig-i/tlr7 deficient primary cells, rig-i was identified to be crucial for ppprna mediated ifn-alpha induction in myeloid immune cells, while tlr7 was essential for ifn-alpha induction in pdc . at the same time, pichlmair and colleagues reported 5 phosphate-dependent type i ifn induction by influenza virus vrna, which contained no dsrna detectable by a dsrna specific antibody (pichlmair et al. 2006) . saito and colleagues suggested that rig-i detects (+)rna viruses (hcv) in a sequence-dependent manner (saito et al. 2008) . they screened the hcv genome for rig-i activating motifs by in vitro transcription of small domains of the hcv genome and analyzed the results for rig-i binding and -activation. a transcript from an 100 nt u-or a-rich region 8000 nt downstream of the 5 end showed an exceptional rig-i inducing activity. the presence of triphosphate at the 5 end was essential for rig-i stimulation. of note, a polyu sequence elicited a similar ifn response as the polya sequence, which could be explained by a phenomenon to be discussed below. while developing phage-polymerase transcription-generated shrna without rig-i stimulating activity, gondai and colleagues found that 5 end extension by more than one g abolished type i ifn induction (gondai et al. 2008) . the results by saito and gondai suggested a sequence-dependent rna recognition by rig-i. however, later experiments with defined synthetic rig-i ligands indicated that the work of both groups needs to be re-interpreted (schlee and hartmann 2010; schlee et al. 2009; schmidt et al. 2009 ). before 5 triphosphate was identified as the crucial rna modification to induce rig-i activation, marques and colleagues observed that synthetic blunt ended dsrna oligonucleotides can stimulate rig-i ( fig. 3 ) (marques et al. 2006) . the read-out system used consisted of the glioblastoma cell line t98g, which was transfected with blunt ended or 3 overhang-possessing sirnas. type i ifn activity in these cells was monitored 48 or 72 h after transfection by western blot analysis of the type i ifn induced protein ifit1 (p56), a very sensitive assay. in contrast to 3 overhangs possessing sirna, blunt-ended sirna induced substantial ifit1 upregulation. similar results were obtained with mrc-5 cells. sirna-mediated knockdown of rig-i in t98g indicated involvement of rig-i in the blunt-ended sirna-induced type i ifn response. by contrast, ht1080 cells and hela cells did not respond to blunt-ended dsrna, but exhibited ifit1 induction after transfection of in vitro transcribed rna. the response to blunt dsrna could be restored in ht1080 cells by priming with type i ifn. according to the described results, the rig-i stimulation motif was defined as double bluntended dsrna longer than 23 bp. single blunt-ended sirnas were less active. 5 overhangs were reported to permit detectable activity after 72 h of stimulation while 3 overhangs abolished activity ( fig. 3 ) (marques et al. 2006) . further studies analyzed the physical interaction of recombinant full-length rig-i or card-or ctd deficient mutants with synthetic blunt-ended dsrna in comparison to in vitro transcribed single stranded ppprna (ivtppp-ssrna) (cui et al. 2008) . while full length rig-i was highly activated by ivtppp-ssrna, synthetic non-phosphorylated dsrna induced rig-i to a much weaker degree. unexpectedly, for rig-i lacking the card domain, dsrna and ivtppprna showed comparable atpase activity. by contrast, interaction studies using fluorescence anisotropy with recombinant rig-i protein or the recombinant rig-i ctd domain confirmed the requirement of the 5 triphosphate for substantial interaction with full-length rig-i (cui et al. 2008 ). takahasi and colleagues reported a rig-i-dependent type i ifn response to synthetic 5 -monophosphorylated and 3 -monophosphorylated dsrna oligonucleotides in an ifn-beta-primed murine cell line and in ifnbeta-treated mouse embryonic fibroblasts (mef). the type i ifn response was monitored by ifnbeta promoter reporter assays and irf-3 dimerization ( fig. 3 ) (takahasi et al. 2008) . in this setting nonmodified synthetic dsrna did not induce type i ifn (takahasi et al. 2008 ). in accordance with earlier studies (marques et al. 2006 ), 3overhangs at the 5 -monophosphorylated end abrogated the type i ifn response, while 5 overhangs were not tested ( fig. 3) (takahasi et al. 2008) . by contrast, 2 nt 3 overhangs in 3 monophosphorylated dsrna induced a type-i ifn response (no other end structures, e.g. blunt, 5 overhang, were analyzed). unexpectedly, the authors found that monophosphorylation did not enforce rig-i binding of dsrna but increased rna stability in the cells, suggesting that increased rna stability is responsible for the particular rig-i stimulating activity (takahasi et al. 2008) . since the 5 triphosphorylated end sequence of rnas generated by phage polymerase is restricted to a conserved consensus starting nucleotide g (or a followed by g), in vitro transcription is not applicable for screening of sequence variations at the 5 end of triphosphorylated oligonucleotides. therefore, our group (+++) = activity was not compared in one figure established a method to generate synthetic triphosphorylated rnas (schlee et al. 2009 ) which is based on the standard cyclotriphosphate protocol of triphosphate synthesis (ludwig and eckstein 1989) . unexpectedly, synthetic single stranded triphosphorylated rna (ppp-ssrna) did not induce type-i ifn in human monocytes, while the "same" rna sequence generated by in vitro transcription (ivtppp-ssrna) was a strong type-i ifn inducer. sequencing of products from the ivtppp-ssrna transcription mix revealed the presence of complementary sequences and double stranded hairpin species, which were obviously generated by template-dependent rna transcription, a side activity of phage polymerase that had been reported earlier (cazenave and uhlenbeck 1994; triana-alonso et al. 1995) . transcription reaction conditions that did not allow synthesis of complementary rna abrogated rig-i activation by ivtppp-ssrna completely, suggesting that rig-i was not stimulated by the intended ssrna transcript but rather by side products (schlee et al. 2009 ). hence, hybridization of a complementary ssrna strand reconstituted rig-i stimulation by synthetic ppp-ssrna. optimal rig-i agonists appeared to be blunt ended, while 2 nt 3 overhangs at the 5 triphosphate end impaired rig-i activation by more than 70% (fig. 3) . 5 overhangs of the triphosphorylated end were not tolerated, thus demonstrating that base pairing of the nucleotide carrying the 5 triphosphate is essential for rig-i activation. the non-phosphorylated end structure had no substantial impact on rig-i stimulation, as long as the dsrna encompassed at least 19 base pairs. small (3 nt) bulge loops in the center of the sequence were tolerated. all four nucleotides constituted active triphosphorylated 5 ends of the rig-i ligand. activity of pppa, pppg and pppu differed only slightly (a = g > u), whereas pppc induced around 50% less type i ifn. of note, this sequence-dependency that was observed is based on only one dsrna sequence (nacacacacacacacacacacuuu), and remains to be verified in another sequence context. according to the public databases, no genomic viral rnas (vrna) start with pppc but most start vrna with pppa. at the same time, by testing synthetic ppp-ssrna, schmidt and colleagues (fig. 3) confirmed the importance of dsrna ). in disparity with our results, they observed that rig-i tolerates a 1 nt 5 overhang at the ppp bearing end for one tested sequences. by using phage polymerase-generated hairpin ppprna structures with intended 5 ppp overhangs, they concluded that longer (>1 nt) 5 overhangs in hairpin ppprnas are tolerated. however, this interpretation may be misleading since in vitro transcribed ppprna hairpins with overhangs (accurate transcription/identity was not analyzed by mass spectrometry) are likely to be contaminated with completely double stranded material (cazenave and uhlenbeck 1994; triana-alonso et al. 1995) . in our experience, one-time size fractionation of hairpin rna is not sufficient to exclude contamination of transcripts with small size differences completely. on the other hand, it has to be considered that hairpins are in equilibrium with their self-complementary duplex (nakano et al. 2007) , which is supposed to be a more active ligand than the monomeric hairpin (binder et al. 2011) . thus, small contaminations can cause substantial effects. schmidt et al. reported that a double stranded region of minimum 10 bp length is sufficient for rig-i activation. in their test they included three different sizes of ppp-dsrna (15, 10 and 5 bp, blunt at the pppend). curiously, a 10mer ppp-dsrna induced a stronger type-i ifn response than a 15mer. as a direct comparison of the full 19mer duplex ppprna to the 15mer and 10mer is missing, the interpretation of the result remains difficult ). altogether, it remains unclear whether just any 10mer duplex ppp-dsrna sequence can activate rig-i. it has to be considered that the possibility of non-canonical base pairings of rna (e.g. g-u wobbles) provides manifold alternatives to form double stranded structures, all of which have to be kept in mind when claiming that rna structures are single stranded. to date only a small number of synthetic ppp-dsrna sequences have been analyzed (seven in our work (schlee et al. 2009 ), one in the work of schmidt et al. (2009) ). it is possible that a stabilizing nucleotide sequence next to the 5 ppp end enables a tolerance of 1 nt 5 -ppp-overhangs. using highly purified in vitro transcribed ppprna from arenavirus sequences marq et al. (2010b) confirmed the requirement of a base paired 5 -ppp end of dsrna for rig-i activation and suggested that some arenaviruses and bunyaviruses use a prime and realign mechanism for genome synthesis, leading to 5 overhangs in order to evade rig-i recognition (marq et al. 2010b) . the need of a base-paired 5 -ppp end of dsrna was also validated by the assembly of the rig-i ligand within the rig-i ctd ppp-dsrna binding cleft (lu et al. 2010b; wang et al. 2010) . 5 pppterminal base pairing supports an essential stacking interaction with a conserved phenylalanine residue in the rna binding cleft of the ctd. in addition, in contrast to a single base pair, which allows free rotation of the following sequence, the double strand assembly stabilizes the helix in a fixed optimum position for interaction of the adjoining phosphodiester backbone with the ctd and the helicase domain (fig. 4) (kolakofsky et al. 2012) . in disparity to previous studies using type-i ifn-primed murine cells as a readout (takahasi et al. 2008) , no considerable type i ifn induction was observed after 24 h when human monocytes were transfected with 5 monophosphorylated dsrnas (schlee et al. 2009; schmidt et al. 2009 ). of note, schmidt and colleagues tested the same sequences, which were previously reported to induce type i ifn in type i ifn-primed mefs takahasi et al. 2008) . by contrast, in both studies monophosphorylated and nonphosphorylated dsrna induced a substantial atpase activity of rig-i protein at higher rna doses (schlee et al. 2009; schmidt et al. 2009 ). this indicates that rig-i activation observed by marques et al. (2006) and takahasi et al. (2008) could occur because of relatively high local rna concentrations in the cytosol. further factors, sensitizing the readout and leading to contradictory results are most likely due to the use of highly rig-i responsive cell lines (t98g) or murine cells combined with long incubation times (48-72 h (marques et al. 2006) ), pre-activation by incubation with type i ifn (marques et al. 2006; takahasi et al. 2008 ) and sensitive detection methods (ifit1 western blot, ifn-beta reporter assay, irf-3 dimerization (marques et al. 2006; takahasi et al. 2008) ). using gel shift experiments with radioactive labeled ligand, vela et al. could calculate that the triphosphate moiety of dsrna enhanced binding to the ctd 127 fold (vela et al. 2012) . the crystallization of rig-i ctd with 5 oh-dsrna by lu and colleagues revealed that binding of 5 oh-dsrna in the ctd rna binding cleft is possible (lu et al. 2010a) . although the assembly for 5 oh-dsrna resembles that of 5 ppp-dsrna, the crystal data reveal that 5 oh-dsrna binds in a different angle, and with other amino acid positions. habjan and colleagues observed that crimean-congo hemorrhagic fever virus (cchtv), hantaan virus (htnv), and borna disease virus (bdv) can prevent rig-i mediated detection of their genomes by a prime and realign mechanism and cleavage of the 5 terminal base of their genomic rna leaving monophosphorylated 5 ends (habjan et al. 2008) (fig. 1) . in contrast to 5 ppp ended genomic rna, the genomic rnas of cchtv, htnv or bdv with 5 monophosphorylated ends (prna) failed to bind or to activate rig-i when transfected into hek293 cells. this laborious procedure of (−)ssrna viruses to generate 5 monophosphorylated genomes to prevent rig-i recognition does not support the concept that prna is a preferred target structure for rig-i during viral infection. the occurrence of aberrant dsrna during phage polymerase in vitro transcription questions the data interpretation from earlier studies, which intended to identify rig-i recognition sequences based on experiments with in vitro transcribed rna. the observations of gondai and colleagues can easily be explained by the non-acceptance of 5 ppp-overhang structures (gondai et al. 2008) : shrnas consisting of a rna hairpin with base paired 5 pppends and a uu 3 -overhang induced rig-i. extra gs at the 5 -ppp end generated single stranded or mismatched 5 ppp ends (5 pppoverhang), which fail to stimulate rig-i. the finding that ppprna composed of polya or poly u stretches are equally potent rig-i inducers can be explained by the possibility that both complementary rna species are generated in the phage-polymerase transcription reaction that was intended to produce only one ssrna species and form a duplex (saito et al. 2008) . by using the same template as used by saito and colleagues for phage polymerase-mediated generation of the rig-i stimulating "poly a" rich sequence (which in fact is composed of starting gs and a), schmidt and colleagues did not receive any rig-i stimulating activity when utp or ctp were omitted in the transcription mix (saito et al. 2008; schmidt et al. 2009 ). by contrast, addition of utp and ctp yielded rig-i-activating rna as reported (saito et al. 2008; schmidt et al. 2009 ), suggesting that a double stranded polya/polyu rich sequence constitutes the rig-i activating agent. potent rig-i stimulation by this structure can be explained by the fact that poly a and poly u represent sequences that are not able to form stable secondary structures. absence of secondary structures facilitates the hybridization of complementary rnas at low temperature to uniform dsrna structures in comparison to mixed high melting g/c containing sequences. it is important to note that subgenomic (single stranded) rnas of hcv were reported to have 5 monophosphorylated ends . since rig-i activation by polyu strictly depended on the presence of 5 triphosphate (saito et al. 2008) , triphosphate-dependent rig-i stimulation in vivo can happen by recognition of replicative ppprna intermediates, which are generated during replication and are, in fact, double stranded (targett-adams et al. 2008). in summary, for rig-i recognition structural features appear to be more important than the sequences of candidate ppp-dsrna. rig-i possesses two rna binding domains (dech domain and ctd). pioneering studies involving crystal structure analysis or nmr from the hopfner and the fuijita lab identified a basic binding cleft within the ctd of rig-i (amino acids 802-925) as the crucial ppprna binding structure that determines ligand specificity (cui et al. 2008; kolakofsky et al. 2012; takahasi et al. 2008 ). as described above, ppp-dsrna bound to the ctd displaces autoinactivated cards from binding to the helicase domain, leading to liberation and activation of cards and downstream events that culminate in the induction of type i ifn [reviewed in kolakofsky et al. (2012) ]. using synthetic or highly purified in vitro transcribed triphosphorylated rna led to the resolution of the crystal structure of the rig-i ctd bound to 12mer ppp-dsrna palindromic sequences (lu et al. 2010b; wang et al. 2010 ). the crystal structures revealed an rna binding basic binding cleft with highly conserved amino acids involved in binding of the 5 terminal base pair, the triphosphate structure itself, and backbone phosphate (fig. 4) . k849 and k851 (amino acid numbers in the text always refer to human rig-i sequence) are in proximity to the gamma phosphate of the triphosphate. however, conservative k849 and k851 mutations to alanine (a) impaired rig-i activation only at very low ligand concentrations (wang et al. 2010) , suggesting a minor role of this interaction in rig-i mediated recognition of viral rna. k858, h847 and k861 were identified to interact with the beta phosphate of the triphosphate. substitution of k861 to a, which is also in contact with alpha phosphate, or double substitution of h847 and k858 to a, abrogated rig-i activation by ppp-dsrna. likewise substitution of k888, being in contact with the alpha phosphate group, inhibited recognition of ppp-dsrna by rig-i. the side chain of k907 is in contact with either the backbone phosphate between n2 and n3 (wang et al. 2010) or n3 and n4 (lu et al. 2010b ). since substitution of k907 to a abolishes rig-i activation completely (wang et al. 2010) this backbone phosphate interaction appears to be crucial for the detection of the ribose backbone of a dsrna structure. the contact of k907 to the phosphate between n2 and n3 (wang et al. 2010) or n3 and n4, as reported in another study (lu et al. 2010b ), could either depend on the incorporated oligonucleotide sequence (ppp-gacgcuagcguc (wang et al. 2010) or pppggcgcgcgcgcgcc (lu et al. 2010b )) or the crystal packaging. in summary, both studies exhibited very similar ppprna/ctd structures. of note, the amino acids mediating rna ligand binding (h847, k858, k861, k888 and k907) are 100% conserved in the rig-i sequences of vertebrates, highlighting the importance of these positions for rig-i-mediated rna-virus recognition. f853 is also essential for rig-i activation (wang et al. 2010) . it conducts the crucial stacking interaction with the 5 terminal base pair (fig. 4) , mediating the rig-i selectivity for base paired triphosphorylated rna. the stacking interaction is stabilized by base pairing of n 1 . in addition, base pairing of the interacting base pair prevents free rotation of the following rna strand and in this way can assure appropriate assembly in the ctd structure (kolakofsky et al. 2012) , another argument for the strict necessity of a base pairing at the triphosphate-bearing end of dsrna to stimulate rig-i. as suggested by the comparison of rig-i ctd of different vertebrate species, f853 can only be substituted by the functionally related tyrosine (y). h830 and c829 mediate possibly important backbone interactions with the 2 oh groups of the ribose of n 1 and n 2 , suggesting a strict discrimination of rna versus dna at those positions. the conclusion by lu and colleagues that rig-i can bind and simultaneously be stimulated by single-stranded ppprna has to be questioned, since in vitro transcribed rna (which usually contains ppp-dsrna species) was used for stimulation of cells (lu et al. 2010b ). although the rig-i ctd appears to interact only with the ppp-bearing rna strand, helix formation should be still crucial for appropriate interaction of the rna with the contacting amino acid residues of h853, k907, h830 and c829. marq and colleagues found that non-stimulatory ppp-dsrna (ppp-dsrna with 5 ppp-overhang) can bind rig-i with comparable affinity as active ligands (complete ppp-dsrna) indicating the possibility of stimulatory ("productive") and non-stimulatory ("non-productive") ligand binding modes to rig-i (marq et al. 2010a) . these data suggest that binding to rig-i is essential but not sufficient for activation of rig-i. two independent studies found that the presence of cards strongly reduce the tolerance for binding of oh-dsrna, thus represent a considerable contribution to the selective recognition 5 triphosphorylated rna (cui et al. 2008; vela et al. 2012) . additionally, based on energetic parameters of the rig-i dsrna interaction, vela et al. suggested that a relatively low affinity of full-length rig-i for dsrna and, therefore, enhanced target specificity is mediated through antagonistic domain binding between helicase and ctd (vela et al. 2012) . the ctd of the rig-i inhibiting helicase lgp2 is closely related to the rig-i ctd (li et al. 2009b; pippig et al. 2009 ). similar to rig-i, lgp2 was reported to preferentially bind to blunt ended dsrna (li et al. 2009b; murali et al. 2008; pippig et al. 2009 ), albeit in a 5 triphosphate independent manner (pippig et al. 2009 ). amino acids mediating the interaction with the 5 terminal base pair and the ribose backbone (h830 rig-i , f853 rig-i , k888 rig-i , k907 rig-i = h576 lgp2 , w604 lgp2 , k634 lgp2 , k651 lgp2 ) are conserved or at least functionally related between the rig-i and the lgp2 ctd, while triphosphate-interacting amino acids (h847, k849, k858 and k861) are missing in the lgp2 ctd. h576 lgp2 , w604 lgp2 k634 lgp2 and k651 lgp2 were found to be involved in dsrna binding of the lgp2 ctd (li et al. 2009b; pippig et al. 2009 ). conversely, the binding mode of oh-dsrna to lgp2 differed considerably from the binding mode of ppp-dsrna. confusingly, mutation of the amino acids in the lgp2 ctd corresponding to k888 rig-i (k634 lgp2 → e) and k907 rig-i (k651 lgp2 → e) led to loss of rna binding but did not impair lgp2-mediated inhibition of rig-i activation, suggesting a ligand-independent rig-i inhibiting mechanism by lgp2 (li et al. 2009b) . the rig-i ligand requirements for short dsrna described above is in contrast to the initial finding that rig-i can be activated by poly i:c (yoneyama et al. 2004 ), a dsrna polymer with monophosphates at the 5 end (grunberg-manago 1967) . in order to characterize ligand structure motifs differentiating rig-i and mda5 recognition, kato and colleagues fractionated rnase iii-digested poly i:c (resulting in 5 monophosphates and 2 nt 3 overhangs). while 7 kb poly i:c fragments (high molecular weight) were preferentially detected by mda5, fractions equal to 300 bp or smaller were exclusively recognized by rig-i (kato et al. 2008) . binder et al. (2011) found an inverse correlation: in their experiments, dsrnas of different length (40-1600 bp) were assessed for rig-i stimulating activity in rig-i transgenic huh7.5 cells. they observed that molecular weight of ppp-dsrna positively correlated with rig-i activation. of note, binder et al. compared rig-i activation at relatively low concentration with constant molarity while kato et al. analyzed rig-i activity transfecting high concentrations (1 g/ml) at constant mass concentration (binder et al. 2011; kato et al. 2008 ). binder et al. could reproduce the results of kato et al. when transfecting high amounts (0.1 pmol/well) of rna. nevertheless, both studies observed 5 triphosphate independent activation of rig-i by very long (≥100 bp) dsrna. length-dependent, 5 end-independent rig-i activation cannot be explained with the current model of ctd-mediated recognition. binder et al. proposed an alternative ctd-independent recognition mechanism (cooperative multimerization of rig-i) in which binding of one rig-i molecule facilitates the binding of a second, etc. (binder et al. 2011) . in this case, binding can only occur in a helicase-dependent manner and would enable displacement of the cards, leading to initiation of the signaling cascade. based on multi-angle light scattering and sizeexclusion chromatography-coupled small-angle x-ray scattering of rig-i/29mer dsrna complexes, beckham e al. suggested that binding of a first rig-i molecule changes the rna structure, thus constructively influencing the binding of a second rig-i molecule (beckham et al. 2013) . however, if rig-i can be activated by long dsrna in a 5 end independent manner, the question still remains why long doublestranded replicative rna intermediates purified from (+)ssrna picornaviruses, are not recognized by rig-i (feng et al. 2012) . rnase cleavage products-ligands for rig-i and mda5? malathi et al. (2007) observed that activated antiviral endoribonuclease rnase l generates small rna cleavage products from self-rna that induce type i ifn production (malathi et al. 2007) . in this study both, mda5 and rig-i were reported to contribute to recognition of small (<200 nt) rnase l cleavage products of total cellular rna. importantly, the type i ifn-induced rnase l digests single-stranded rna into rna products with 5 -oh and 3 -monophosphate groups. by reverse sequencing in a follow-up study, malathi et al. discovered hcv genome sequence-derived rnase-l cleavage products that bind to rig-i (malathi et al. 2010) . one of 15 rig-i binding rna sequences was able to significantly activate rig-i in a 3 monophosphate-dependent but 5 triphosphate-independent manner. the putative structure of rig-i activating sequence included long (>20 bp) dsrna regions but also long (>5 nt) single stranded 5 and 3 ends. as the sequence was generated by in vitro transcription and not validated by mass spectrometry, it remains unclear whether the intended structure or co-purified side products from in vitro transcription are responsible for rig-i stimulation. nevertheless, the study indicates that special rna structures exist, which can stimulate rig-i in a 3 monophosphate-dependent manner and that these kinds of structures can be generated by rnase l cleavage of rna virus genomes. of note, 3 monophosphate-dependent rig-i stimulation was also reported earlier by the fujita group (takahasi et al. 2008) . at the same time, the akira lab and the medzhitov lab reported a tlr9-independent type i ifn induction when dsdna was transfected into the cytosol of cells stetson and medzhitov 2006) . knock-down of mavs in 293t cells significantly reduced dsdna-induced type i ifn, suggesting a mavs-dependent pathway of dsdna recognition in these cells ). unexpectedly, mavs-deficient murine cells still responded to dsdna (sun et al. 2006) . importantly, the akira lab used a special type of dsdna, the heteropolymer dadt (a polymer of the alternating sequence at). cheng et al. observed that human cell lines (huh7, hek293) secrete type i ifn in a mavs and rig-idependent manner after transfection of dadt but not plasmid dna (cheng et al. 2007 ). the dadt enigma was later solved by two independent groups (ablasser et al. 2009; chiu et al. 2009 ): ablasser et al. and chiu et al. discovered that dadt functions as a template for the endogenous rna polymerase iii, which generates 5 triphosphorylated au-polymers in the cytosol (chiu et al. 2009 ). at-rich dna is in two ways a special kind of dna: first, rna polymerase iii prefers transcription of at-rich sequences; second, the resulting pppau-polymers are self-complementary and can easily anneal to ppp-dsrna. they therefore represent excellent rig-i target structures (ablasser et al. 2009 ). thus, it has to be kept in mind that "rig-i stimulating dna" needs to provide two features: first, the dna needs to be able to serve as a template for rna polymerase iii, second, the transcript has to form an appropriate rig-i ligand structure (base paired 5 ppp end + dsrna > 19 bp). most natural dsdna structures do not provide both features. some dna virus (e.g. epstein barr virus and adenovirus) encode for small rnas (eber and vai) under the control of a polymerase iii-driven promoter; these were observed to stimulate rig-i (ablasser et al. 2009; minamitani et al. 2011; samanta et al. 2008) . unlike murine cells and human monocytic cells, most human cell lines are not stimulated by dsdnas (e.g. plasmid dna or pcr products) other than dadt. this indicates the absence of a receptor in the cytosol, which can recognize dna directly in those cell lines (ablasser et al. 2009; cheng et al. 2007 ). in those cells, the rna polymerase iii/rig-i pathway constitutes the only alternative to detect cytosolic dsdna. since some dna viruses and facultative intracellular bacteria induced a mavs or rig-i-dependent type i ifn response in non-immune cells, it was suggested that in these cells the innate immune response to intracellular dsdna-containing pathogens can occur via stimulation of rig-i by pathogen-dna templated polymerase iii transcripts (ablasser et al. 2009; chiu et al. 2009 ). as will be summarized in the next section, intracellular bacteria were actually shown to release rna into the cytosol of cells, which is then recognized by rig-i (abdullah et al. 2012; hagmann et al. 2013) . in general, all rna polymerase-dependent transcribed rnas initially possess a 5 triphosphorylated start nucleotide. posttranscriptional 5 processing or modification such as capping are key features of mrna translation regulation in eucaryotes. in contrast to eucaryotes, one third of escherichia coli (bacterial) mrna remains 5 triphosphorylated (bieger and nierlich 1989) . regulation of the 5 phosphorylation status of bacterial mrna, which determines mrna decay (celesnik et al. 2007) , occurs via the pyrophosphatase rpph (deana et al. 2008 ). the 5 triphosphate moiety prevents degradation of bacterial mrna by rnase-e (celesnik et al. 2007 ). importantly, pyrophosphatase rpph preferentially targets single-stranded triphosphorylated 5 nucleotides as a substrate, consequently leaving 5 triphosphorylated dsrna (deana et al. 2008 ). this conclusion is supported by the finding that 5terminal stem-loops prolong the lifetime of bacterial rnas (emory et al. 1992; mackie 2000) . thus, base-paired 5 triphosphorylated rnas (ppp-dsrna), which are an excellent target structure for rig-i, appear to represent a characteristic molecular pattern (mamp) for bacteria. in a sirna-based approach, opitz et al. observed that legionella pneumophilae, a facultative intracellular gram-negative bacterium with type iv secretion system, raised a mavs-dependent but rig-ior mda5-independent type-i ifn response in a human endothelial cell line (a549) (opitz et al. 2006) . the involvement of mavs in the l. pneumophilae induced immune response was later affirmed in experiments with mavs-and mda5-deficient, anti-rig-i shrnaexpressing bone marrow-derived macrophage cell lines (monroe et al. 2009 ). in contrast to findings by opitz et al. (2006) , contribution of mda5 and rig-i to the type i ifn response was reported (monroe et al. 2009 ). while knock-down of rig-i substantially reduced the response to transfected purified bacterial rna, it remained an open question whether legionella-derived rna actually gains access to the cytosol of the host cell during infection. as mda5 appeared to not directly sense bacterial rna, the authors speculated about an indirect mechanism leading to activation of rig-i and mda5. importantly, dna from l. pneumophilae did not induce type i ifn in hek293 cells, thus excluding rig-i-mediated recognition of rna polymerase-iii transcripts in the host cell, as previously suggested (chiu et al. 2009; monroe et al. 2009 ). recognition of bacterial rna by rig-i was also observed for the bacterium helicobacter pylori (rad et al. 2009 ). abdullah et al. discovered that the facultative intracellular bacterium listeria monocytogenes actively secrete small rnas via its seca2 secretion system resulting in strong rig-i activating activity (abdullah et al. 2012) . recently, we visualized translocation of bacterial rna from l. monocytogenes into the cytosol of several human cell lines (hagmann et al. 2013) . previously, l. monocytogenes was reported to induce type i ifn exclusively via the sting pathway, an adaptor/receptor sensing second messenger molecules or cyclic nucleic acids either directly secreted by l. monocytogenes (cyclic di-amp) or generated downstream of cytosolic dna recognition (gmp-amp) in mammalian cells (ishikawa et al. 2009; sauer et al. 2011; sun et al. 2012; woodward et al. 2010; wu et al. 2012; zhong et al. 2008) . we found that the l. monocytogenes-triggered type i ifn response is dependent on rig-i recognition when the sting pathway is not present in cells, this being the case in tested non-immune cells (hagmann et al. 2013) . in immune cells such as the human cell line thp-1 the stingdependent recognition pathway dominates the immune response to l. monocytogenes while rig-i appears to not play any role in recognition (hagmann et al. 2013) . in this respect, data for murine cells are conflicting: while some studies excluded involvement of rig-i/mavs in l. monocytogenes recognition in macrophages (soulat et al. 2006; sun et al. 2006) another study observed a substantial contribution of the rig-i pathway (abdullah et al. 2012) . culturing conditions of bacteria and murine cells may influence the amount of transferred bacterial ppprna and the balance between rig-i and the sting pathway in murine macrophages, thus leading to controversial findings. interestingly, li et al. found that rna of commensal bacteria is recognized in a mavs-dependent manner and that mavs in cells of non-hematopoietic origin plays a dominant role in preventing dss-induced colitis (li et al. 2011) , supporting that rig-i-inducing bacterial rna indeed has access to non-immune cells in vivo and mediates important effects. this finding explained the observation by wang et al. that rig-i deficient mice easily develop colitis (wang et al. 2007) . considering the fact that mice with a defect in the type i ifn pathway exhibit a strong resistance to listeria-induced pathogenesis, it remains to be determined if the observed rig-i-dependent recognition of bacterial rna contributes more to the pathogenicity or to the clearance of listeria (auerbuch et al. 2004; o'connell et al. 2004 ). detection of viral rna -which structure stimulates rig-i? in addition to dsrna viruses positive single-strand rna [(+)ssrna] viruses also generate cytosolic dsrna species, such as replicative dsrna intermediates, during their replication (feng et al. 2012; targett-adams et al. 2008; triantafilou et al. 2012; weber et al. 2006) . together with 5 triphosphate, such rna species represent ideal rig-i target structures. since picornaviruses were shown not to activate rig-i during infection (gitlin et al. 2006; kato et al. 2006) it was presumed that picornaviruses (and caliciviruses) (fig. 1 ) are able to escape rig-i recognition because instead of a 5 triphosphate their rna genomes possess a peptide (vpg) linked via a tyrosine residue to 5 monophosphate (hruby and roberts 1978; lee et al. 1977; rohayem et al. 2006) . in line with these findings, feng et al. observed that purified picorna virus rna stimulated mda5 but not rig-i (feng et al. 2012) . additionally and rather unexpectedly, picornaviruses were observed to degrade rig-i during infection. the apparent need to degrade rig-i indicates the occurrence of some rig-i stimulating activity, the identity of which has not been clarified so far (barral et al. 2009; papon et al. 2009 ). rig-i dominates the immune response to many (−)ssrna viruses (cardenas et al. 2006; habjan et al. 2008; hornung et al. 2006; kato et al. 2005 kato et al. , 2006 loo et al. 2008; plumet et al. 2007; yoneyama et al. 2005) . however, three out of four so far analyzed (−)ssrna viruses were described not to generate double-stranded rna species during infection (no dsrna: influenza; sendai virus, sev; la crosse virus, lacv; dsrna detectable: new castle disease virus, ndv) (pichlmair et al. 2006; takeuchi et al. 2008; weber et al. 2006) . this finding may seem conflicting at first, but can be explained by the fact that the dsrna-visualizing antibody used in these studies is only able to bind dsrna longer than 40 bases (bonin et al. 2000) but rig-i can recognize ppp-dsrna ≥ 19 bp. in general, the complementary 5 and 3 terminal sequences of all (−)ssrna viruses genomes bear the potential to hybridize to so-called dsrna panhandle structures with blunt ended 5 triphosphorylated ends (fig. 1, left bottom) . a certain degree of self-complementarity cannot be avoided by (−)ssrna viruses since the same viral polymerase which recognizes its start sequence needs to start mrna transcription or replication from the (−)ssrna or (+)ssrna intermediates. for bunyaviridae, including lacv, it was validated by psoralen-crosslinking and electron microscopy that the 5 -and 3 -ends of the viral genome indeed constituted a panhandle structure in vivo (hewlett et al. 1977; raju and kolakofsky 1989) . the lacv panhandle consists of a blunt-ended 24-27 bp dsrna stretch with only a few mismatched nucleotides (raju and kolakofsky 1989) , thereby achieving almost all rig-i ligand requirements (schlee et al. 2009 ) without being detected by the 40pb rna-specific antibody. marq et al. suggested that for this reason some arenaviruses and bunyaviruses circumvent rig-i recognition by conducting a prime and realign mechanism which enables generation of 5 overhangs in their genomes (marq et al. 2010b ). as mentioned above, habjan et al. identified viruses (bornaviridae, bunyaviridae) , which escape rig-i recognition by combination of a prime and realign mechanism to 5 terminal cleavage, leaving a 5 monophosphorylated end (fig. 1 , "rlr escape"). by using replication and translation blocking agents, applying enzymatic probing and visualization by superresolution microscopy, weber et al. confirmed that rig-i indeed recognizes viral capsids of lacv upon entry into the cell by binding to the panhandle structure (weber et al. 2013) . similar to lacv, influenza virus genomic ppprna (comprising 8 segments with conserved 5 ends) forms triphosphorylated, panhandle structures, albeit with quite short double stranded stretches (about 15 bp), including mismatches/bulge loop structures (desselberger et al. 1980; hsu et al. 1987 ). this panhandle structure represents the site of rna transcription-initiation for the viral rna polymerase complex in the nucleus of the host cell (portela and digard 2002) . dauber et al. observed that the influenza panhandle is accessible for the antiviral double-stranded rnadependent protein kinase (pkr) after export from the nucleus into the cytosol of the host cell (dauber et al. 2009 ). since pkr and rig-i recognize comparable dsrna structures-short triphosphorylated dsrna (nallagatla et al. 2007; schlee et al. 2009 ), it is likely that rig-i also has access to the influenza panhandle structure in the cytosol. if the influenza panhandle is indeed sufficient to activate rig-i still remains to be verified using well-defined rig-i ligands. rehwinkel and colleagues generated mutated influenza rna polymerases which either selectively produce viral mrna or replicative genomic rna (rehwinkel et al. 2010) . using this tool, they confirmed that rig-i activation occurs exclusively by the genomic rna and not mrna of influenza (rehwinkel et al. 2010) . additionally, analysis of rig-i-bound viral rna from influenza infected cells revealed that only 5 triphosphorylated viral genomic rna coprecipitated with rig-i. in contrast to lacv and influenza, viral particles of sendai virus (sev) and measles virus (mev) which belong to the group of mononegavirales contain predominantly linear nucleocapsids because encapsidation with structural proteins prevents formation of double stranded or panhandle structures (bhella et al. 2004; gerlier and lyles 2011; loney et al. 2009 ). but sev and vsv were found to produce defective interfering (di) viral genomes during replication (kolakofsky 1976; lazzarini et al. 1981; perrault and leavitt 1978) . three kinds of di genomes were identified: di genomes with internal deletions, 5 promoter duplications with completely complementary 5 -3 ends, and hairpin di genomes "snap back", consisting of a dsrna hairpin of 100-1000 bp (fig. 1) . "panhandle" and "snap back" di rnas should result in excellent rig-i ligands. indeed, strahle et al. could correlate sendai virusinduced rig-i activation with the occurrence of snap back di genomes (di-h4) which are generated during infection without encapsidation and thus are able to form panhandle structures in infected cells (strahle et al. 2006 (strahle et al. , 2007 . in concordance with these data, by applying a deep sequencing approach after purification of rna attached to rig-i from sendai virus infected cells, baum et al. determined preferred binding of di genomes to rig-i (baum et al. 2010 ). it appears plausible that the genomic rna should be targeted by rig-i, primarily. actually, purified genomic rna from most examined (−)ssrna viruses like rabies virus , lassa virus, nipah virus, rift valley fever virus (habjan et al. 2008 ) activated rig-i. nevertheless, rna purification by denaturating agents removes rna-interacting viral nuclear proteins, allowing formation of secondary rig-i-activating rna structures which do not occur in vivo (e.g. hybridization of (−)ssrna and (+)ssrna). therefore it is uncertain if these viral genomes would activate rig-i during infection. however, the phenomenon that (−)ssrna viruses possess mechanisms to modify their genomic 5 end, preventing rig-i recognition (habjan et al. 2008; marq et al. 2010b) , implies that the dsrna structures that (can) form at the 5 end of viral genomes are critical for the viruses with rig-i escape mechanisms (arenaviridae, bunyaviridae, bornaviridae). in fact, the principle of detecting panhandle structures represents an intelligent strategy to detect (−)ssrna viruses because replication of (−)ssrna viruses necessitates highly conserved promoters at both ends of the genome, consequently yielding self-complementary 5 and 3 ends. the intolerance of vsv for artificially introduced extra nucleotides at the 5 and 3 ends of its (−)ssrna genome suggests lack of flexibility of (−)ssrna viruses concerning promoter sequences (pattnaik et al. 1992) . therefore, a blunt-ended double-stranded rna structure with 5 triphosphate, the consequence of two conserved promoters, constitutes a negative strand virus-associated molecular pattern recognized by rig-i. correctly processed viral mrna is unlikely to be targeted by rig-i, because rna viruses possess mechanisms to cap their mrna either by viral-encoded capping enzymes or cap-snatching mechanisms (fechter and brownlee 2005) leading to loss of, or masking of the 5 triphosphate. nevertheless, other studies suggest that type i ifn induction by mononegavirales (e.g. sev, vsv, mev) is associated with mrna transcription rather than replication (reviewed in gerlier and lyles (2011) ). e.g., in contrast to influenza, replicationdisabled measles virus that was still capable of transcription was observed to still activate a type i ifn response (plumet et al. 2007 ). leader and trailer rnas (lerna and trrna) represent the only non-capped 5 triphosphorylated transcripts occurring during the transcription of the genome. plumet et al. and bitko et al. observed (−) ssrna virus lerna dependent stimulation of rig-i (bitko et al. 2008; plumet et al. 2007 ). however, the viral rna which forms together with the lerna the rig-i activating dsrna species still needs to be determined. gerlier and lyles proposed that l-trrna read-through transcripts (viral l-mrna extended by the trrna template) hybridized to ppp-trrna could also reconstitute a source of perfect blunt-ended rig-i target structures during the nonreplicative transcription phase (fig. 1, left panel) (gerlier and lyles 2011) . dna genome-based viruses like adenovirus, vaccinia virus and herpesviridae (herpes simplex virus, hsv) generate copious amounts of dsrna during their life cycle ); these dsrnas are most probably generated by overlapping converging transcription (jacobs and langland 1996) . while melchjorsen et al. reported mda5 but not rig-i-dependent type i ifn induction by hsv1 (melchjorsen et al. 2010) , xing et al. reported that the hsv1 encoded us11 protein interacts with and inhibits both rig-i and mda5, indicating that hsv1-derived rna is also recognized by rig-i (xing et al. 2012 ). finally, ebv and adenovirus-encoded small rna polymerase iii transcripts (eber and vai) were described to activate rig-i (ablasser et al. 2009; minamitani et al. 2011; samanta et al. 2008) . despite the presence of crystal data, conflicting results on ligand motif definition still obscure the understanding of mda5 pathogen rna detection. as demonstrated for paramyxoviruses, conclusions from studies testing immune responses in rig-i/mda5 knock-out cells to whole viruses can be misleading as long as the function of viral proteins is unknown: viruses appear to be recognized by rig-i because they express proteins efficiently inhibiting mda5, and the opposite is also thinkable. by contrast, recent progress in the synthesis and purification of defined synthetic ligands, native preparation and visualization of natural rig-i ligands (nucleocapsids) and crystallization of rig-i/ligand complexes has greatly improved the understanding of the molecular basis of rig-idependent virus recognition. most of the studies agree on terminal base pair recognition by rig-i, which is dependent on the presence of 5 triphosphate in a physiological relevant ligand concentration range. completely end base pair-independent rig-i recognition as proposed for long (≥100 bp) dsrna needs further investigation since it can currently not be explained by the model derived from recent crystal data. naturally occurring rig-i ligands are double stranded triphosphorylated replicative intermediates of (+)ssrna viruses (formal scientific proof is missing though) and the triphosphorylated panhandle structures of genomes of (−)ssrna viruses with the exception of mononegavirales, which are able to avoid forming panhandle structures in vivo. other viruses avoid rig-i recognition by laborious procedures leading to 5 overhangs or 5 monophosphorylation. interestingly, in analogy to phage polymerases in vitro, rna polymerases of mononegavirales generate rig-i ligands due to an error-prone transcription process, therefore counteracting their -in principle -perfect camouflage against rig-i recognition. on the other hand, a successful virus has to protect its host from detrimental consequences of infection. the best examples are herpesviruses, the infection of which usually proceeds asymptomatic: >95% of adults are, for example, infected by ebv, which persists lifelong in its host without causing detrimental symptoms, making ebv a very successful virus (thorley-lawson 2005) . ebv is known for its close interaction with the immune system, limiting its own infection in the host (thorley-lawson 2005) . thus immune recognition of the virus at a certain stage of its spread in the host should be evolutionarily favored and complete immune evasion not the goal of virus adaption. the author is listed as inventor on a patent application covering structures described in a manuscript, which is cited in this review (schlee et al. 2009 ). rig-i detects infection with live listeria by sensing secreted bacterial nucleic acids rig-i-dependent sensing of poly(da:dt) through the induction of an rna polymerase iii-transcribed rna intermediate recognition of doublestranded rna and activation of nf-kappab by toll-like receptor 3 the v proteins of paramyxoviruses bind the ifn-inducible rna helicase, mda-5, and inhibit its activation of the ifn-beta promoter mice lacking the type i interferon receptor are resistant to listeria monocytogenes 5'-terminal cap structure in eucaryotic messenger ribonucleic acids accessing the therapeutic potential of immunostimulatory nucleic acids rig-i is cleaved during picornavirus infection preference of rig-i for short viral rna molecules in infected cells revealed by next-generation sequencing conformational rearrangements of rig-i receptor on formation of a multiprotein:dsrna assembly conformational flexibility in recombinant measles virus nucleocapsids visualised by cryo-negative stain electron microscopy and real-space helical reconstruction distribution of 5 -triphosphate termini on the mrna of escherichia coli molecular mechanism of signal perception and integration by the innate immune sensor retinoic acid-inducible gene-i (rig-i) cellular la protein shields nonsegmented negative-strand rna viral leader rna from rig-i and enhances virus growth by diverse mechanisms determination of preferential binding sites for anti-dsrna antibodies on double-stranded rna by scanning force microscopy highly pathogenic rna viral infections: challenges for antiviral research ebola virus vp35 protein binds double-stranded rna and inhibits alpha/beta interferon production induced by rig-i signaling rna template-directed rna synthesis by t7 rna polymerase initiation of rna decay in escherichia coli by 5 pyrophosphate removal plasmacytoid monocytes migrate to inflamed lymph nodes and produce large amounts of type i interferon double-stranded dna and doublestranded rna induce a common antiviral signaling pathway in human cells paramyxovirus v proteins interact with the rna helicase lgp2 to inhibit rig-i-dependent interferon induction mda-5, but not rig-i, is a common target for paramyxovirus v proteins rna polymerase iii detects cytosolic dna and induces type i interferons through the rig-i pathway the rig-i atpase domain structure reveals insights into atp-dependent antiviral signalling the c-terminal regulatory domain is the rna 5 -triphosphate sensor of rig-i influenza b virus ribonucleoprotein is a potent activator of the antiviral kinase pkr the bacterial enzyme rpph triggers messenger rna degradation by 5 pyrophosphate removal the 3 and 5 -terminal sequences of influenza a, b and c virus rna segments are highly conserved and show partial inverted complementarity viral infection switches non-plasmacytoid dendritic cells into high interferon producers irf3 mediates a tlr3/tlr4-specific antiviral gene program a 5 -terminal stem-loop structure can stabilize mrna in escherichia coli recognition of mrna cap structures by viral and cellular proteins mda5 detects the double-stranded rna replicative form in picornavirus-infected cells inducers of interferon and host resistance. ii. multistranded synthetic polynucleotide complexes lps-tlr4 signaling to irf-3/7 and nf-kappab involves the toll adapters tram and trif establishment and maintenance of the innate antiviral response to west nile virus involves both rig-i and mda5 signaling through ips-1 the 5 ends of hantaan virus (bunyaviridae) rnas suggest a prime-and-realign mechanism for the initiation of rna synthesis interplay between innate immunity and negative-strand rna viruses: towards a rational model essential role of mda-5 in type i ifn responses to polyriboinosinic:polyribocytidylic acid and encephalomyocarditis picornavirus shorthairpin rnas synthesized by t7 phage polymerase do not induce interferon polynucleotide phosphorylase: structure and mechanism of action processing of genome 5 termini as a strategy of negative-strand rna viruses to avoid rig-i-dependent interferon induction rig-i detects triphosphorylated rna of listeria monocytogenes during infection in non-immune cells species-specific recognition of single-stranded rna via toll-like receptor 7 and 8 a toll-like receptor recognizes bacterial dna circular forms of uukuniemi virion rna: an electron microscopic study 5 -triphosphate rna is the ligand for rig-i sequencespecific potent induction of ifn-alpha by short interfering rna in plasmacytoid dendritic cells through tlr7 quantitative expression of toll-like receptor 1-10 mrna in cellular subsets of human peripheral blood mononuclear cells and sensitivity to cpg oligodeoxynucleotides encephalomyocarditis virus rna. iii. presence of a genome-associated protein genomic rnas of influenza viruses are held in a circular conformation in virions and in infected cells by a terminal panhandle a toll-like receptor-independent antiviral response induced by double-stranded b-form dna sting regulates intracellular dna-mediated, type i interferon-dependent innate immunity when two strands are better than one: the mediators and modulators of the cellular responses to double-stranded rna structural basis of rna recognition and activation by innate immune receptor rig-i ubiquitin-induced oligomerization of the rna sensors rig-i and mda5 activates antiviral innate immune response sequence-dependent stimulation of the mammalian innate immune response by synthetic sirna cell type-specific involvement of rig-i in antiviral response length-dependent recognition of doublestranded ribonucleic acids by retinoic acid-inducible gene-i and melanoma differentiation-associated gene 5 differential roles of mda5 and rig-i helicases in the recognition of rna viruses ips-1, an adaptor triggering rig-i-and mda5-mediated type i interferon induction interferon induction by sirnas and ssrnas synthesized by phage polymerase isolation and characterization of sendai virus di-rnas a structure-based model of rig-i activation rna-and virus-independent inhibition of antiviral signaling by rna helicase lgp2 structural basis for the activation of innate immune patternrecognition receptor rig-i by viral rna cpg motifs in bacterial dna trigger direct b-cell activation the origins of defective interfering particles of the negative-strand rna viruses a protein covalently linked to poliovirus genome rna murine coronavirus induces type i interferon in oligodendrocytes through recognition by rig-i and mda5 structural basis of double-stranded rna recognition by the rig-i like receptor mda5 the rig-i-like receptor lgp2 recognizes the termini of double-stranded rna mitochondrial antiviral signaling protein (mavs) monitors commensal bacteria and induces an immune response that prevents experimental colitis paramyxovirus ultrastructure and genome packaging: cryo-electron tomography of sendai virus distinct rig-i and mda5 signaling by rna viruses in innate immunity crystal structure of rig-i c-terminal domain bound to blunt-ended double-strand rna without 5 triphosphate the structural basis of 5 triphosphate double-stranded rna recognition by rig-i c-terminal domain rapid and efficient synthesis of nucleoside 5 -0-(1-thiotriphosphates), 5 -triphosphates and 2 ,3 -cyclophosphorothioates using 2-chloro-4h-1,3,2-benzodioxaphosphorin-4-one structural insights into rna recognition by rig-i duplex rna activated atpases (dras): platforms for rna sensing, signaling and processing activation of ifn-&#946; expression by a viral mrna through rnase l and mda5 stabilization of circular rpst mrna demonstrates the 5 -end dependence of rnase e action in vivo small self-rna generated by rnase l amplifies antiviral innate immunity rnase l releases a small rna from hcv rna that refolds into a potent pamp short doublestranded rnas with an overhanging 5 ppp-nucleotide, as found in arenavirus genomes, act as rig-i decoys unpaired 5 ppp-nucleotides, as found in arenavirus double-stranded rna panhandles, are not recognized by rig-i a structural basis for discriminating between self and nonself double-stranded rnas in mammalian cells mda-5 recognition of a murine norovirus early innate recognition of herpes simplex virus in human primary macrophages is mediated via the mda5/mavsdependent and mda5/mavs/rna polymerase iii-independent pathways cardif is an adaptor protein in the rig-i antiviral pathway and is targeted by hepatitis c virus adenovirus virus-associated rnas induce type i interferon expression through a rig-i-mediated pathway identification of host cytosolic sensors and bacterial factors regulating the type i interferon response to legionella pneumophila paramyxovirus v proteins disrupt the fold of the rna sensor mda5 to inhibit antiviral signaling structure and function of lgp2, a dex(d/h) helicase that regulates the innate immunity response influence of cationic molecules on the hairpin to duplex equilibria of selfcomplementary dna and rna oligonucleotides 5 -triphosphate-dependent activation of pkr by rnas with short stem-loops type i interferon production enhances susceptibility to listeria monocytogenes infection legionella pneumophila induces ifnbeta in lung epithelial cells via ips-1 and irf3, which also control bacterial replication the viral rna recognition sensor rig-i is degraded during encephalomyocarditis virus (emcv) infection infectious defective interfering particles of vsv from transcripts of a cdna clone inverted complementary terminal sequences in single-stranded rnas and snap-back rnas from vesicular stomatitis defective interfering particles rig-i-mediated antiviral responses to single-stranded rna bearing 5 -phosphates activation of mda5 requires higher order rna structures generated during virus infection the regulatory domain of the rig-i family atpase lgp2 senses double-stranded rna cytosolic 5 -triphosphate ended viral leader transcript of measles virus as activator of the rig i-mediated interferon response the influenza virus nucleoprotein: a multifunctional rna-binding protein pivotal to virus replication extracellular and intracellular pattern recognition receptors cooperate in the recognition of helicobacter pylori the ends of la crosse virus genome and antigenome rnas within nucleocapsids are base paired rig-i detects viral genomic rna during negative-strand rna virus infection protein-primed and de novo initiation of rna synthesis by norovirus 3dpol murine coronavirus mouse hepatitis virus is recognized by mda5 and induces type i interferon in brain macrophages/microglia the rna helicase lgp2 inhibits tlr-independent sensing of viral replication by retinoic acid-inducible gene-i regulation of innate antiviral defenses through a shared repressor domain in rig-i and lgp2 innate immunity induced by composition-dependent rig-i recognition of hepatitis c virus rna epstein-barr virus-encoded small rna induces il-10 through rig-i-mediated irf-3 signaling lgp2 is a positive regulator of rig-i-and mda5-mediated antiviral responses the nethyl-n-nitrosourea-induced goldenticket mouse mutant reveals an essential function of sting in the in vivo interferon response to listeria monocytogenes and cyclic dinucleotides beyond double-stranded rna-type i ifn induction by 3prna and other viral nucleic acids the chase for the rig-i ligand-recent advances sirna and isrna: two edges of one sword recognition of 5 triphosphate by rig-i helicase requires short blunt double-stranded rna as contained in panhandle of negative-strand virus 5 -triphosphate rna requires base-paired structures to activate antiviral signaling via rig-i identification and characterization of mavs, a mitochondrial antiviral signaling protein that activates nf-kappab and irf 3 triggering the interferon antiviral response through an ikk-related pathway the nature of the principal type 1 interferon-producing cells in human blood cytoplasmic listeria monocytogenes stimulates ifn-beta synthesis without requiring the adapter protein mavs recognition of cytosolic dna activates an irf3-dependent innate immune response sendai virus defective-interfering genomes and the activation of interferon-beta activation of the beta interferon promoter by unnatural sendai virus infection requires rig-i and is inhibited by viral c proteins cyclic gmp-amp synthase is a cytosolic dna sensor that activates the type i interferon pathway the specific and essential role of mavs in antiviral innate immune responses the rig-i-like receptor lgp2 controls cd8(+) t cell survival and fitness 2 -o methylation of the viral mrna cap by west nile virus evades ifit1-dependent and -independent mechanisms of host restriction in vivo analysis of the 5 end structure of hcv subgenomic rna replicated in a huh7 cell line nonself rna-sensing mechanism of rig-i helicase and activation of antiviral immune responses sendai virus c protein plays a role in restricting pkr activation by limiting the generation of intracellular double-stranded rna pattern recognition receptors and inflammation visualization of double-stranded rna in cells supporting hepatitis c virus rna replication ebv the prototypical human tumor virus -just how bad is it? self-coded 3 -extension of run-off transcripts produces aberrant products during in vitro transcription with t7 rna polymerase visualisation of direct interaction of mda5 and the dsrna replicative intermediate form of positive strand rna viruses the thermodynamic basis for viral rna detection by the rig-i innate immune sensor loss of dexd/h box rna helicase lgp2 manifests disparate antiviral responses structural and functional insights into 5 -ppp rna pattern recognition by the innate immune receptor rig-i rig-i −/− mice develop colitis associated with downregulation of g alpha i2 doublestranded rna is produced by positive-strand rna viruses and dna viruses but not in detectable amounts by negative-strand rna viruses incoming rna virus nucleocapsids containing a 5 -triphosphorylated genome activate rig-i and antiviral signaling c-di-amp secreted by intracellular listeria monocytogenes activates a host type i interferon response structural basis for dsrna recognition, filament formation, and antiviral signal activation by mda5 cyclic gmp-amp is an endogenous second messenger in innate immune signaling by cytosolic dna herpes simplex virus 1 tegument protein us11 downmodulates the rlr signaling pathway via direct interaction with rig-i and mda-5 visa is an adapter protein required for virus-triggered ifn-beta signaling shared and unique functions of the dexd/h-box helicases rig-i, mda5, and lgp2 in antiviral innate immunity the rna helicase rig-i has an essential function in double-stranded rna-induced innate antiviral responses reconstitution of the rig-i pathway reveals a signaling role of unanchored polyubiquitin chains in innate immunity key role of ubc5 and lysine-63 polyubiquitination in viral activation of irf3 the adaptor protein mita links virus-sensing receptors to irf3 transcription factor activation mouse hepatitis virus does not induce beta interferon synthesis and does not inhibit its induction by double-stranded rna ribose 2 -omethylation provides a molecular signature for the distinction of self and nonself mrna dependent on the rna sensor mda5 i thank janos ludwig for illuminating discussions and cristina amparo hagmann for critically reading the manuscript. present work in the laboratory was supported by grants from the deutsche forschungsgemeinschaft (sfb670 and dfg research grants program schl 1930/1-1). key: cord-341324-f9g9gitn authors: rojas, josé m.; alejo, alí; martín, verónica; sevilla, noemí title: viral pathogen-induced mechanisms to antagonize mammalian interferon (ifn) signaling pathway date: 2020-10-21 journal: cell mol life sci doi: 10.1007/s00018-020-03671-z sha: doc_id: 341324 cord_uid: f9g9gitn antiviral responses of interferons (ifns) are crucial in the host immune response, playing a relevant role in controlling viralw infections. three types of ifns, type i (ifn-α, ifn-β), ii (ifn-γ) and iii (ifn-λ), are classified according to their receptor usage, mode of induction, biological activity and amino acid sequence. here, we provide a comprehensive review of type i ifn responses and different mechanisms that viruses employ to circumvent this response. in the first part, we will give an overview of the different induction and signaling cascades induced in the cell by ifn-i after virus encounter. next, highlights of some of the mechanisms used by viruses to counteract the ifn induction will be described. and finally, we will address different mechanism used by viruses to interference with the ifn signaling cascade and the blockade of ifn induced antiviral activities. innate immune responses are the first host defense against viral infections. conserved pathogen structures are recognized by pattern recognition receptors (prrs) on the host cells [1] , that recruit a variety of adaptor proteins to signal downstream and activate the ifn response. the ifn system is present in all vertebrates and is central to antiviral responses [2] . ifns are classified into three different families according to their receptor usage, mode of induction, biological activity and amino acid sequence [3] : type i, type ii and type iii ifns. type i ifns, originally identified by their antiviral activity [4] , include multiple ifn-α subtypes (13 in humans and 14 in mice), and a single ifn-β, and ifn-ε, ifn-κ, ifn-ω (humans) and ifn-ξ (mice) subtypes [5] . in mammals, type i ifn (ifn-i) response is essential for innate antiviral responses. they all bind to the same ubiquitously expressed receptor, ifnar receptor, but they differ in their biological functions, due partially to the different binding affinities to the ifnar receptor [6] . this differences in affinity results in different downstream signaling cascades [7] . for ifn-α subtypes, the quantity of the receptor on the surface of a target cell correlates also with their biological activities suggesting that the amount of ifnar expression might compensate the weak affinity of some ifn-α subtypes [6, 8] . type ii ifns include only one member, ifn-γ, secreted by activated t cells, natural killer (nk), nkt cells and dendritic cells with pro-inflammatory and immunomodulatory functions [9] . in general, type ii ifn acts as a link between the innate immune response and the activation of the adaptive immune response [10] . type iii ifns include ifn-λ1, ifn-λ2 and ifn-λ3, and, although genetically different to type i ifns and signaling through different receptors, they are induced by pprs and activates antiviral pathways similar to type i ifns [11, 12] . viruses use multiple mechanisms to by-pass the host ifn responses so that they can replicate and continue their infectious cycle. the present review will focus on how viruses interfere with ifn-i responses. viruses can act at different levels of the signaling cascades involved in ifn-i responses. they can inhibit the induction of the ifn response, block the ifn signaling, and/or interfere with the antiviral activities induced by ifn. we will review some of the emerging themes and new insights from the past years of the ifn evasion mechanism employed by viruses in these contexts. the antiviral state of an infected cells is attained by the initial induction of type i ifn expression, followed by ifn signaling transduction, which finally leads to the expression of multiple genes (fig. 1) . ifns are the main group of cytokines secreted by host cells in response to the presence of "aberrant" nucleic acids such as double-stranded rna (dsrna) molecules generated as viral intermediates during viral transcription in infected cells, to cpg dna, or uncapped ssrna with 5′ triphosphate present in some viruses. these elements are known as pathogen-associated molecular patterns (pamp) [13, 14] that can be recognized by prrs. four main types of prrs have been described to detect virus-derived genetic materials: toll-like receptors (tlrs) 3/7/8/9 [15] ; retinoic acid-inducible gene-i (rig-i) like receptors (rlrs) which include the cytosolic sensor rig-i, the melanoma differentiation-associated factor 5 (mda-5) and laboratory of genetics and physiology (lgp2) [16] ; and nucleotide oligomerization domain-like receptors (nlrs) [17] and the cytosolic dna sensors [18] . for a host to establish an antiviral state it first requires the production of type i (α/β) ifns in direct response to virus infection and recognition of virus-derived genetic material. hence, both rig-i and mda-5 sense cytosolic dsrna. rig-i also specifically senses 5′ triphosphate rna generated during infection, while mda-5 detects longer dsrna sequence generated during virus replication. rig-i and mda5 contain two caspase activation and recruitment domains (cards) at the n-terminal. activation of rig-i and mda5 liberates these card domains and drives the interaction of these tandem cards with the card of the mitochondrial activation signaling (mavs) protein [21] . mavs aggregates in filaments that provide a platform for the recruitment of the elements involved in the subsequent signaling cascade such as the tumor necrosis factor receptor-associated factor (traf) 3 and traf6, the tank binding kinase 1 (tbk1) and ikkε ultimately drives the activation of transcription factors as irf3/7, nfκb and ap-1, leading the production of type i ifn and pro-inflammatory cytokines [22] . irf3 is constitutively expressed in many cell types, and after phosphorylation, irf-3 forms a homodimer that translocates into the nucleus, activating the transcription of early transcribed type i and iii ifn genes, ifn-β, ifn all type i ifns signal through the same heterodimeric transmembrane receptor termed the ifnα receptor (ifnar), containing the subunit 1 and 2 (ifnar1 and ifnar2). in a first step, ifn-i binds with high affinity to ifnar1, and then recruit ifnar2 [34] . ifnar engagement with ifn-i promotes the induction of an antiviral state in cells. this involves the upregulation of products from a large subset of genes named ifn-stimulated genes (isg) that protect the cell from viral replication. broadly speaking, isg products modulate and mediate ifn activity in the cells. this includes for instance cooperating in prr recognition of viral pamps, stabilizing signaling complexes to improve their resistance to degradation, stopping virus entry, blocking viral capsid formation, impairing trafficking and budding of virions from the infected cells, but also modulating the ifn response to avoid the toxicity of these potent immune mediators. an important feature of the ifn signaling is the rapid speed at which the response happens, which is possible because protein synthesis is not required in an initial stage. the interaction of type i ifns with their universally expressed receptor (ifnar) elicits an intracellular signaling cascade through the janus protein kinase (jak) family members, jak and tyk2, that successively phosphorylate signal transducer and transcription activator (stat) family proteins [35] . the phosphorylated stat1/stat2 heterodimer associates with interferon regulatory factor 9 (irf9) to form the transcriptional factor complex isgf3, which translocate to the nucleus and binds the ifn-response elements (isre) in isg promoters leading to the expression of isg products [36] (fig. 2 the oligoadenylate synthetase (oas)-latent rnase (rnase l) pathway is another ifn-inducible pathway that provides the cell with an effector mechanism upon recognition of viral dsrna (reviewed in [44] ). when the oas senses dsrna activates the production of 2′,5′-oligoadenylates that act as a second messenger on the inactive monomeric rnasel [45] . the 2′,5-oligoadenylates binding to rnase l produces a catalytically active dimer that cleaves ssrna [46] . this leads to the translational arrest and prevent viral replication and spreading [47] . rna-activated protein kinase is an isg product that detects cytosolic dsrna. pkr recognition of its dsrna substrate leads to dimerization and autophosphorylation which in turn leads to the phosphorylation of the viruses has developed strategies to counteract dif-ferent steps on this signaling cascade. it is marked in red blades the main signaling targets of viruses eukaryotic initiation factor 2α (eif2α) required for translation initiation [48] . eif2α phosphorylation results in the shutdown of all translation of 5′capped mrna, thus preventing the synthesis of viral proteins. this also usually results in the formation of stress granules (sg) that consist of the accumulation of rna and proteins from the stalled translation complexes. sg formation has been linked to antiviral responses, and their formation is often inhibited in viral infections. in general, the antiviral activity of pkr is related to apoptosis induction [49], regulation of ifn-β synthesis and nf-κβ pathway [50] [51] [52] , serine kinase activity for stat1 that also regulate the ifn-i signaling pathway [53] . a family of proteins with a wide range of anti-viral functions are the interferon-induced proteins with tetratricopeptide repeats (ifits) [54] . these genes are expressed at very low basal levels, but their transcription is rapidly increased after activation by ifn signaling. ifit detect the lack of 2′-o-methylation on rnas species, a methylation absent in some viral rna but present in eukaryotic mrna [55] . ifit1 has also been shown to bind to the 5′-trip end of some viral rna [56] . ifit1 can sequester viral rna or interact with the eukaryotic translation initiation factor 3 to inhibit the translation initiation of ifit1-bound rna species. in addition to these isgs, one highly upregulated gene in the initial stage of the antiviral immune response is isg15 (interferon-stimulated gene 15), which encodes an ubiquitinlike protein involved in a post-translational process termed isgylation [57] . this process allows isg15 to bind covalently to a range of target proteins, both viral and cellular [58] , by a process that is reversible due to the action of the ubiquitin-specific protease 18 (usp18), an event regulated by type i ifn [59] . isgylation appears to modulate the activity of multiple elements involved in the ifn response. for instance, isgylation has been shown to sustain stat1 or irf3 activity [60, 61] , downregulate the turnover of ubiquitinated proteins [62] , but also to negatively regulate rig-i signaling [63] . isg15 acts during viral replication by interfering with the endogenous proteins that the virus needs to replicate. thus, isg15 conjugates to the eukaryotic factor 4e (eif4e) homologous protein (4ehp) that binds to the capped mrna, inhibiting in this way the viral rna translation of those viruses that contains a capped positivesense rna such as flaviviruses [64] . isg15 also exists as an unconjugated protein that acts as a cytokine, regulating viral replication and host responses [65, 66] . the role of the isgs members mentioned above clearly illustrates the breadth and diversity in the function of this protein group. a database has been created, called interferome (https ://www.inter ferom e.org/inter ferom e/home. jspx), in which isgs are catalogued and incorporated into a database, based on the information obtained from all published reports where cells were treated with ifn, thus, this database will allow to identify isg signatures from highthroughput data, having implications for determining the role of isgs. viruses employ mechanisms that impair isg activity to enhance their evasion from the ifn system. the mechanisms will be discussed in another section of this review. as discussed previously, prr activation leads to the production of ifn-i, -iii, and pro-inflammatory cytokines such as il-1β. the present section will be centered on how viruses affect ifn-i induction. typically, virus genetic material triggers ifn-i induction when recognized by viral nucleic acid sensors that are membrane bound or present in the cytoplasm/nucleus. this recognition leads to activation of signaling cascades that converge towards the induction of ifn-i production in infected cells. viruses are known to interfere at every point of this process. viruses can interfere with the sensing of their genetic material, impair the signaling cascade that leads to ifn-i induction, and/or antagonize the activity of the transcription factors involved in ifn-i gene expression. the present section aims at providing a nonexhaustive overview of some of the most commonly used viral mechanisms to counter ifn-i induction in the host with a focus on recent findings in the field. viruses use different mechanisms to counteract the recognition of their genetic material by the host cell so that ifns are not induced (table 1) . viruses can sequester, modify or even degrade their nucleic acids to avoid detection by prrs. for instance, during replication most flaviviruses create vesicular structures in the er membrane which physically shield the viral genetic material from cytosolic rlrs [67] [68] [69] . influenza a virus (iav) uses the nucleus for replication, atypically for an rna virus, so that its genetic material remains hidden from cytosolic rlrs [70] . vaccinia virus (vv), a large double-stranded dna virus has the peculiarity of replicating in the cytoplasm, where dna sensors like cgas are present, which potentially renders the viral genetic material susceptible to recognition by prr. vv replication, however, occurs in organelles similar to micronuclei [71] that probably render viral dna inaccessible to recognition by cytoplasmic dna sensors. rotaviruses (rv) concentrate their replication in a cytoplasmic structure called viroplasms where dsrna genome is generated for packaging so that it is not exposed to the cytoplasmic prr [72] . many viruses also encode proteins that help conceal their genetic material from prr. some viruses possess dsrna binding proteins that could potentially sequester these pamps from ppr recognition, such as vp35 from ebola virus or σ3 from reovirus [73, 74] . the encapsidation of the viral rna can also impair rlr recognition. for instance, iav nucleoprotein and polymerase prevents rig-i binding to viral rna during transit through the cytoplasm [75] . iav ns1 protein also possesses a dsrna binding site that prevents recognition by rig-i [76] . calicivirus and picornavirus ssrna ( +) is covalently linked to a capping protein that could prevent recognition of the 5′ viral rna extremity by rig-i [77] . lassa virus (lasv) nucleoprotein (np) can act as a capping enzyme with exonuclease activity specific for dsrna, which has been shown to antagonize ifn induction [78, 79] . other viruses can modify their 5′tri-p motifs recognized by rig-i to evade this cytoplasmic rna sensor. hantaan virus, crimean-congo hemorrhagic fever virus and borna disease virus can process their 5′ genome extremity to form 5′mono-p forms, evading rig-i recognition [80] . poxvirus decapping enzymes d9 and d10 can prevent the accumulation of dsrna, an intermediate necessary in viral replication, and thereby evade rlr recognition [81] . measles virus (mev) encodes for the non-structural c protein that can impair ifn response by modulating viral rna replication [82] and improving the polymerase processivity [83] , thus probably limiting the amount of viral material recognizable by cytosolic prr. another mechanism employed by viruses to limit detection by prr is to interact with these sensors to impair their activation. the kaposi's sarcoma-associated virus (kshv) uses the tegument protein orf52 to bind to cgas and inhibit cgamp production, the second messenger used for sting (stimulator of interferon response cgamp interactor 1) activation [84] . homologues of orf52 in other gammaherpesviruses have also been described to act similarly, indicating that inhibition of this prr pathway is probably shared by gammaherpesvirus. the herpes simplex virus 1 (hsv-1) tegument protein vp22 has also been shown to inhibit cgas enzymatic activity, indicating that other herpesviridae can directly target cgas [85] . other viruses can sequester prr so that they are unable to relocate to their activity site. for instance, the protein z of new world arenaviruses binds to rig-i and prevents its association with the signaling platform mavs protein [86] . severe acute respiratory syndrome coronavirus (sars-cov) m protein has been shown to associate with rig-i and can potentially sequester this prr [87] . viruses can also promote prr degradation, thus reducing the number of cellular sensors capable of detecting viral infection (fig. 3 ). this can be done directly by proteases encoded by the viral genome. foot and mouth disease virus (fmdv) l pro and 3c pro protease can reduce rig-i intracellular protein levels [88] . the 3c pro protease of other picornaviruses has also been shown to degrade rig-i [89] , indicating that this is a shared mechanism of rlr evasion by this viral family. rig-i is not the sole prr that picornavirus proteases can target; the poliovirus and enterovirus 71 (ev71) 2a pro protease can also degrade mda5 [90] . viruses also encode for proteins that indirectly promote prr degradation. the nuclear sensor of dna ifi16 is degraded during hsv-1 infection through a mechanism dependent on the viral icp0 protein that is not fully understood but probably involves targeting the dna sensor for proteasomal degradation [91] . a e3 ligase activity on the nss protein of the phlebovirus toscana virus was recently identified that allowed the ubiquitination of rig-i card domains and the subsequent proteasomal degradation of the prr [92] . the ns2b protein of the flavivirus dengue virus (denv) can target the cytosolic dna sensor cgas for lysosomal degradation. although this [98] [99] [100] mechanism could at first glance seem counterproductive for an rna virus, it actually allows denv to evade the recognition of the mitochondrial dna that becomes exposed during infection [93] . other viruses alter the post-translational modifications essential to prr signaling so that the ppr remains inactive. some viral-encoded proteins achieve this directly, as in the case of the deubiquitinase activity of coronaviruses papainlike protease (plp) that removes the k63 ubiquitin tail from rig-i that is essential for rig-i translocation to the mavs protein platform [94] . this deubiquitination activity has been characterized in several coronaviruses thus suggesting that this mechanism is central to coronavirus evasion from the ifn system [95] [96] [97] . paramyxovirus v protein binds to mda5 and impairs its dephosphorylation by blocking the atp hydrolysis necessary for mda5 folding to its active state, thus impairing the adequate activation of this prr [98] . viruses can also impair prr activity by interfering with the functionality of accessory cellular components required for prr activation. mev has been shown to act on the phosphatase pp1 required for rlr activation using two distinct mechanisms. the mev v protein can bind pp1 which prevents mda5 dephosphorylation [99] . mev infection in dendritic cells also produces recognition of the viral particle through the c-lectin receptor dc-sign [100] . this triggers a signaling cascade that results in raf-1 kinase activation and the association of the pp1 inhibitor i-1 with pp1 that prevented rlr dephosphorylation thus impairing ifn induction. several viruses have also been shown to interact with the dsrna binding protein pact that potentiates rlr activation [101, 102] . for instance, ebola virus vp35 protein, middle east respiratory syndrome viral interference with accessory cellular components involved in prr activation. mev can interfere with rlr activation by targeting the phosphatase pp1 using 2 distinct mechanisms. mev v protein can interact with pp1 to prevent the dephosphorylation of mda-5 required for activation. mev can interact on the cell surface with the c-lectin receptor dc-sign which results in the association of pp1inhibitor 1 with pp1 thus preventing rlr dephosphorylation. ebola virus vp35 protein, mers-cov 4a protein and arenavirus np can interfere with pact binding to dsrna, a mechanism that potentiates rlr activation. rlr ubiquitination is also essential for adequate activation and transport to mavs for subsequent ifn signaling events to take place. riplet and trim25 are critical to rig-i ubiquitination. iav-ns1 and denv sfrna can interfere with trim25 activity, whereas hepatitis c ns3-4a protease can cleave riplet to impair rig-i ubiquitination. the mitochondrial-targeting chaperone 14-3-3ε is responsible for rig-i translocation to the mitochondrial membrane. denv ns3 protein targets 14-3-3ε using a phosphomimetic domain that displaces activated rig-i from this chaperone. wnv ns3 possesses a similar domain coronavirus (mers-cov) 4a protein and arenavirus nucleoproteins have been shown to interfere with pact binding to rlr [103] [104] [105] . as previously stated, rig-i activation is associated to its ubiquitination with k63 ub chains that liberates its autorepressed n-terminal card domains, a mechanism dependent on the activity of the cellular proteins trim25 and riplet [106, 107] . this mechanism can be targeted by viral products such as the iav ns1 protein that impairs trim25-mediated rig-i ubiquitination [108, 109] , or the hepatitis c virus ns3-4a protease that cleaves riplet [106] . intriguingly, not only protein products appear to interfere with the rig-i activation complex. the subgenomic flavivirus rna (sfrna) generated during denv infection can bind to trim25 and prevent the deubiquitination step required for trim25 activity to take place [110] . denv uses yet another mechanism to avoid prr detection. denv ns3 protein possesses a phosphomimetic domain that binds the mitochondrial-targeting chaperone protein 14-3-3ε [111] . 14-3-3ε is responsible for rig-i translocation to the mitochondrial membrane where the subsequent steps of the ifn induction signaling cascade take place. by binding 14-3-3ε, denv ns3 displaces the activated rig-i complex and prevents ifn induction. interestingly, a similar phosphomimetic domain is also present in west nile virus (wnv) ns3 protein [111] . viruses not only interfere with the prr capable of detecting their presence during infection, they also commonly affect the activity of the signaling complexes in charge of signal transduction. indeed, this is a strategy employed by most viruses to limit ifn responses. viruses can antagonize these signaling cascades at multiple levels and through varied mechanisms (table 2 ). this can be achieved by impairing the post-translational modifications required for signaling, i.e. by altering the phosphorylation or ubiquitinylation status of signaling intermediates. for instance, sars-cov and human coronavirus nl63 (hcov-nl63) plps have been shown to prevent sting dimerization and thus subsequent activation of the tbk1 pathway possibly through the plp deubiquitinase activity [112, 113] , as sting dimerization is dependent on the attachment of k63ub chains [114] . similarly, sars-cov plp can inhibit tlr7 signaling by removing k63-ub chains from traf3 and traf6 and thus blocking tbk1 activation [115] . another strategy to prevent post-translational modification of ifn-i pathway signaling components consists of sequestering these components so that they do not reach the adequate cellular location for activation. the ns3 protein from the economically important orbivirus bluetongue virus (btv) binds to the ubiquitinbinding protein optineurin in the golgi apparatus [116] . this prevents optineurin from recruiting ubiquitinated tbk1 at the golgi apparatus, a necessary step for subsequent tbk1 phosphorylation to occur [116] . recently, it has been described that cgamp, the second messenger generated by cgas and that activates sting, can be cleaved by poxvirusencoded nucleases (named poxins) [117] . this allows poxvirus blockade of the cgas-sting signaling axis. viral proteins can also prevent the adequate formation of signaling complexes by steric hindrance. for instance, human adenovirus type 5 e1a protein has been shown to bind to sting and thus antagonize ifn signaling [118] . mers-cov and sars-cov m proteins can interact with traf3 and thus disrupt the traf3-tbk1 association [87, 119, 120] . mers-cov orf4b protein can associate with the tbk1/ikkε complex to impair signaling [121] , indicating that coronaviruses encode for multiple proteins that affect ifn induction at different stages of the signaling cascade. kshv encodes for viral interferon regulatory factor 1 (virf1), a protein that binds to sting and block the association of tbk1 with sting, thus hindering the consequent irf3 phosphorylation [122] . hsv-1 encodes for the icp27 protein that can interact with the active sting-tbk1 complex and inhibit the subsequent tbk1-mediated phosphorylation of irf3 [123] . the nss protein from some phleboviruses has also been described to antagonize ifn induction by targeting tbk1 [124] . tbk1 activity is thus commonly targeted by both rna and dna viruses to antagonize ifn induction. viruses can also promote the degradation of signaling proteins involved in ifn induction. virally-encoded proteases can directly cleave some of the components of these pathways. sting can be directly cleaved by the ns2b3 protease from several flaviviruses such as denv, zika virus, wnv, or japanese encephalitis virus (jev), but not others like yellow fever virus [125] [126] [127] . indeed, this specific cleavage could partially explain some of the host range and pathogenicity of these flaviviruses in humans, as the identified sting cleavage site for the ns2b3 protease is only partially conserved among species [125] . similarly, the 3c pro from several picornoviridae has been described to cleave mavs proteins, thus inhibiting signal transduction [128, 129] . the 3c-like protease from the arterivirus porcine reproductive respiratory syndrome virus (prrsv) and the ns3/4a protease complex from the flavivirus hepatitis c virus (hcv) have also been described to cleave mavs protein [130, 131] . other viruses encode proteins that promote the degradation of signaling complexes involved in the ifn responses. rv nsp1 and vp3 proteins have been shown to target mavs protein for proteasome-dependent degradation and thus impair ifn induction [132, 133] . sars-cov orf9b protein localizes in the mitochondrial membrane where it interacts with mavs protein and promote its degradation [134] . this mechanism probably involves the recruitment of the mavs protein cellular regulator pcbp2 [135] to the mitochondrial membrane by sars-cov orf9b protein, which favors mavs protein ubiquitination through k48-ub chains and thus subsequent proteasomal degradation. since mavs protein represents an important signaling platform in the ifn induction cascade triggered by rlr activation, some viruses use strategies to alter mitochondria structure to impair mavs protein assembly. denv ns2b3 protease can cleave mitofusins, which alters mitochondria dynamics and impair their fusion [136] . in other cases, viruses can also promote mitophagy to promote their replication and potentially impair ifn responses. mitophagy has been described in mev, hepatitis b virus (hbv) or newcastle disease virus infections [137] [138] [139] . the protein m from the human parainfluenza virus 3 has also been shown to promote mitophagy and thus trigger mavs protein degradation to antagonize ifn induction [140] . recently, iav has also been shown to employ a similar mitophagic strategy to reduce mavs protein levels through the expression of its pb1-f2 protein [141] . viruses can also act on the transcription factors that bind to the ifn-i promoter and trigger the expression of isgs. viruses use multiple strategies to impair the binding of the transcription factors to the ifn-i promoters (table 3) . they can impair the phosphorylation by the tbk1/ikkε complex of irf3/7 that activates dimerization and transport to the nucleus. for instance, the paramyxovirus c protein inhibits irf7 phosphorylation and thus its activation [142] . the denv ns2b3 protease can impair irf3 phosphorylation by masking the ikkε kinase domain necessary for irf3 activation [143] . lymphocytic choriomeningitis virus (lcmv) nucleoprotein was also shown to bind to the ikkε kinase domain, thus preventing irf3 phosphorylation [144] indicating that arenavirus np-mediated inhibition of irf3 phosphorylation [145] uses a similar mechanism. viruses not only target irf3/7 activation to antagonize ifn induction, they can also interfere with the activation of nf-κb, as this transcription factor collaborates with irf3 in the early activation of the ifn-β promoter [146] . most arenavirus nucleoproteins not only block irf3 phosphorylation but they can also impair nf-κb activity [147] . viruses can also target adaptor molecules that regulate nf-κb activity. the nf-κb essential modulator (nemo) is part of the kinase complex that controls nf-κb release from its inhibitor iκb. the 3c pro from hepatitis a virus or fmdv and the 3c-like proteases from porcine epidemic diarrhea virus or prrsv cleave nemo thus preventing iκb release from nf-κb and consequently antagonizing ifn-β production [148] [149] [150] [151] . the degradation of the transcription factors involved in ifn induction is also the target of different viral proteins, as in the case of the nsp1 from rv that promote the degradation of several irfs including irf3 and 7 [152] . rv nsp1 appears to target irfs by interacting with their dimerization domain and thus preventing their association in active form [153] . nsp1 has also been described to impair nf-κb by promoting the degradation of β-transducing repeat-containing protein (β-trcp) [154] , the protein responsible for substrate recognition of the e3 ligase complex that targets for degradation iκb, the associated inhibitor of nf-κb. similarly, to other viral products, such as hiv-1 vpu, vv a49, or epstein-barr virus lmp-1, rv nsp1 associates with β-trcp through mimicry of the phosphorylated iκb that requires degradation [155] [156] [157] [158] . rv nsp1, however, presents the particularity of not only blocking the interaction of iκb with the e3 ligase complex but also of targeting β-trcp for degradation. viruses can also impair irf3/7 activity downstream of their activation by phosphorylation. the virf1 encoded by kshv can block irf3 activity downstream of irf3 activation by impairing the recruitment of the cbp-p300 coactivators to the irf3 complex [159] . this can also be achieved by blocking the transport of activated transcription factors to the nucleus. for instance, some paramyxoviruses use their v protein to impair irf3 translocation to the nucleus [160] . jev ns5 blocks irf3 and p65 subunit from nf-κb transport to the nucleus by interacting with nuclear transport proteins [161] . the nss from the emerging bunyavirus severe fever with thrombocytopenia syndrome virus have also been described to sequester the tbk1-ikkε-irf3 complex in viral inclusion bodies to prevent the trafficking of irf3 to the nucleus [162] . some viruses also code for proteins that interfere with ifn induction in the nucleus. in many instances, the exact mechanisms of ifn antagonism by viral products in the nucleus are not fully resolved. mers-cov orf4b protein impairs ifn induction in the nucleus by a mechanism yet to be elucidated [121] . btv encodes non-structural protein 4 (ns4) that localizes in cell nucleoli and possess ifn antagonist activity [163] . mev c protein can interfere with ifn-β promoter activation in the nucleus and this property has been linked to the virus pathogenicity [164] . the nss protein from the phlebovirus sandfly fever sicilian virus can prevent irf3 activity by interacting with the dna binding domain of irf3 [165] . the protein orf36 from the murine gammaherpes virus 68 was shown to bind to irf3 and to prevent its association with the co-transcriptional activator cbp, thus impairing irf3 binding to the ifn-β promoter [166] . viruses have developed strategies to antagonize ifn induction at multiple stages of the signaling cascade. this includes limiting the recognition of their genetic material, impairing the activation of prrs or their signaling partners, promoting the degradation of key components of the signaling cascade, sequestering signaling complexes away from their site of action, or impairing dna binding of the transcription factors involved in ifn induction. in viruses, multiple mechanisms have also often evolved to target several elements in these pathways, and hence augment their capacity to evade innate immunity. viruses can suppress the ifn signaling at different levels ( table 4 ). in this section, we will discuss some of the mechanisms that viruses use to counteract the action of this signaling cascade. one of the first steps in the signaling cascade is the phosphorylation of jak1 and tyk2, relevant for initiating the jak-stat signaling. by directly promoting the dephosphorylation of the jak/stat pathway, viruses counteract the ifn response. for instance, sendai virus (respirovirus) c protein inhibits the phosphorylation of receptor-associated kinases jak1 and tyk2 by binding to the ifn receptor subunit ifn-α/β [167] . the ns5 protein of the jev blocks the tyrosine phosphorylation of tyk2 and stat1 [168] . stat1 phosphorylation is targeted by several viruses, using different mechanisms. the paramyxovirus family, that includes mev, peste des petits ruminants virus (pprv) and mumps, express v and p proteins that interact directly with stat1, inhibiting phosphorylation [169, 170] . the immediate-early protein icp27 of hsv-1 downregulate stat1 phosphorylation and prevent the accumulation of stat1 in the nucleus [171] . the denv proteins ns2a, ns4a, ns4b block the phosphorylation of stat1 [172] . the ns5 protein of hcv interacts with stat1, interfering with its phosphorylation [173] . among the reoviridae family, the nsp1 protein encoded by rotavirus block the phosphorylation of stat1 [174] , and the ns3 protein encoded by btv blocks stat1 phosphorylation [175, 176] . many viruses target stat2 to antagonize ifn signaling. thus, mev v protein binds to stat2 [177, 178] . yellow fever virus ns5 protein also binds stat2 but this interaction requires stat2 activation by ifn [179] . other example is denv ns5 protein acts as a bridge between ubr4 and stat2, driving stat2 to degradation through the proteasome [180] . the nsp11 protein of pprs degrades stat2 via proteasome [181] . the proteasome is not the only catabolic cellular machinery that viruses can highjack to degrade signaling components of the ifn signaling pathway. btv was recently shown to use ubiquitination of its ns3 protein to drive stat2 degradation by an autophagy-dependent mechanism [175] . another strategy used by viruses includes the interference with ifn signal transduction by modification of the constitutive or basal levels of molecules involved in the jak/ stat pathway that viruses members of the rubulavirus genus like simian virus 5, mumps virus or human parainfluenza virus type 2, use [182, 183] . the human papillomavirus-16 (hpv-16) expresses the viral e7 protein that binds the p48 protein blocking its translocation to the nucleus, impeding the association of irf-9 with the stat-1/stat-2 heterodimer (isgf3), and thereby inhibiting the induction of ifn-i inducible genes [184] . viruses involved in persistent infections, such as cytomegaloviruses (cmv), polyomaviruses, hcv [185] , or hsv-1 [186] use similar strategies. cmv affects the expression levels of jak1 and irf-9 [187] , and the viral large t antigen of murine polyomavirus binds to jak1 inactivating the transduction signal through ifn receptors [188] . viruses employ mechanisms that impair isg activity to enhance their evasion from the ifn system. in this section, we will discuss some of these mechanisms. as previously mentioned, some isg products enhance the recognition of viral pamps and provide the cell with effector mechanisms that block viral replication. thus, fmdv l pro cleaves g3bp1, an rna-binding protein essential to stress granules (sg) assembly, to impair the formation of these structures [189] . mev c protein has been involved in sg inhibition through blockade of pkr-induced sg by the activity of the adenosine deaminase acting on rna 1 (adar1) [190] . denv sfrna can bind to various components involved in sg assembly. this property of denv sfrna also led to impaired isg mrna translation, thus dampening ifn responses [191] . to overcome recognition by isg products, some viral mechanisms are discussed. the reovirus σ3 protein was shown to inhibit pkr activity probably through its ability to bind dsrna [192, 193] . nss protein from bunyavirus can promote pkr degradation [194] . poxvirus d9 and d10 decapping enzyme promote dsrna degradation, thus preventing pkr recognition [81] . viruses can also highjack regulatory isg pathways to evade isg product action. for instance, adar1 is an isg involved in rlr regulation. adar1 has an important physiological function as it edits adenosines to inosines in rna, a feature that destabilize the structure of complementary dsrna strands, thus preventing rlr or pkr recognition. this is an important mechanism in the prevention of autoimmunity, as it limits the recognition of cellular dsrna. viruses such as mev, vsv or hiv-1 have been shown to use adar1 function to block pkr activation and thus evade translation shutdown [195] [196] [197] . viruses can also interfere with the oas-rnase l pathway. rotavirus vp3 protein blocks rnasel activation [198] by cleaving the 2′,5′-oligoadenylates produced by oas. the ns2 protein from coronavirus murine hepatitis virus has been shown to act similarly [199] . the ifit is another isg that contribute to viral rna recognition. some viruses have developed 2′-o methyltransferase activities on their gene products to prevent translation blockade of their rna by ifit. this has been described for wnv, coronaviruses, rv and poxvirus [200] [201] [202] [203] [204] . the ifn-induced transmembrane proteins (ifitm) expression is greatly enhanced upon ifn activation, but these proteins are also expressed ubiquitously in the absence of ifn. the family of ifitm proteins has been shown to block iav, wnv and denv cell entry, a mechanism that probably involves viral hemagglutinin recognition [205] . hcov-oc43 has been shown to highjack ifitm2 and ifitm3 for cell entry. this mechanism could be important for virus entry in lower respiratory tract under inflammatory conditions induced by ifn [206] . in hiv-1, vpu and env proteins can mutate to increase infectivity and overcome ifitm1-mediated restriction of replication [207] . mutations to overcome the activity of the isg product mxgtpase have also been described for iav, indicating that multiple isg products probably exert a selective pressure on viruses. for instance, pandemic avian iav strains appear to adapt to human through evasion of the np recognition by mxa gtpase [208] . viruses have evolved strategies to dampen isg15 effects on ifn signaling. for instance, the plp from hcov-nl63, sars-cov and mers-cov have been shown to not only act as a deubiquitinase but also as a deconjugating protease for isg15 chains [94, 96, 97] . fmdv lpro has also been associated with cleavage of isg15, but instead of targeting the isopeptide bond used in isg15 conjugation, it hydrolyzes the peptide bond preceding the isgylation motif [209] . human cytomegalovirus (hcmv) tegument pul26 protein prevents isgylation [210] . pul26 activity appears to be supported by two other tegument proteins pul25 and pp65 [211] . hcmv also uses another tegument protein pul50 to affect isgylation by targeting ube1l, an important ligase responsible for isg15 linkage, for proteasomal degradation [212] . since isgylation also regulates the activation of ifnrelated pathways, some viruses have harnessed isgylation to favor their replication. hcv has also been described to use isgylation to favor its replication and develop persistent infections [213, 214] . recently, isgylation was also associated with increased replication in hbv infections [215] . multifaceted strategies have evolved in viruses to circumvent isg product activity and thus enhance their replication and spreading. these range from directly impairing the activity of isg products involved in host cellular defense to highjack the ifn modulating activity of some isg products. understanding these mechanisms of evasion will undoubtedly shed light on some of the pathogenic processes induced by viral infections. a unique strategy to inhibit the activity of ifn was described in 1995 with the identification of a poxviral secreted ifn type i binding protein (ifn-i bp) encoded by the b18r gene of vaccinia virus (vv) [216] a well characterized member of the orthopoxvirus genus that contains the strains used for the efficacious worldwide vaccination campaigns against smallpox. the protein was found to be a secreted glycoprotein of about 60 kda expressed early during infection. while its sequence is unrelated to either of the two subunits of the cellular ifn-i receptor, ifnar1 or ifnar2, the ifn-i bp was found to bind with high affinity (k d = 175 pm for hifnα2) to several subtypes of human ifn-is, inhibiting their binding to the receptor and thus abrogating their biological activity [217] . in stark contrast to the high species specificity observed for the cellular receptor, the viral protein is able to bind and inhibit the activity of ifn from different species, including mouse, rat, bovine and rabbit ligands, suggesting different interaction modes. currently, all human ifn-i molecules tested including 8 (out of 13) ifnα, ifnβ, ifnϖ as well as the more divergent ifnκ and ifnε are known to be bound and inhibited by b18 [218] [219] [220] , although with varying affinities. interestingly, murine ifnα, but not murine ifnβ are inhibited by the poxviral ifn-i bp in spite of being bound with high affinity, as assessed by surface plasmon resonance [219, 221] . competition studies using anti ifn monoclonal antibodies (mabs) showed that the binding interface of ifns with b18 is larger than the one with their cellular receptor, which could probably account for its broad species specificity and inhibitory capacity over a wide range of affinities [222] . further examinations substantiated an additional property of the poxviral ifn-i bp which is its saturable binding to the cell surface after secretion, where the protein is active and can inhibit ifn as efficiently as the secreted one [221] . this suggested that the main site of action is the cell surface, providing local tissue protection by protecting neighboring, still uninfected cells from entering into an ifn mediated antiviral state. examination of a truncated version of b18 expressed by the attenuated wyeth vaccv strain lacking its third, c-terminal immunoglobulin (ig) domain showed that cell binding capacity is mediated by the n-terminal regions of the protein [221] . additional transfection analyses with different constructs suggested that cell binding activity is mediated by ig domain 1, while ifn blocking activity requires ig domains 2 and 3 [223] site directed mutagenesis assays identified stretches of basic residues at the n terminus of b18 to mediate high affinity binding to cell surface sulfated glycosaminoglycans, preferentially heparan sulfate [224] and showed that mutants lacking gag binding activity could still bind and inhibit ifn efficiently. the ifn-i bp protein has been found to be conserved in other orthopoxviruses including cowpox virus and ectromelia virus (ectv), a natural mouse pathogen, as well as the two viruses causing significant disease in humans, monkeypox virus and variola virus, the causative agent of smallpox [219] . interestingly, the human viruses show an enhanced affinity for the human ligands, possibly reflecting the host adaptation of the virus, as occurs with other secreted cytokine binding receptors in this family. while possible orthologues can be readily found in virus species from several other poxvirus genera, these are frequently more distantly related, and their properties have not been extensively studied. the single exception to this is protein y136 of the tanapoxvirus yaba-like disease virus (yldv), a primate virus causing infection restricted to the skin. this protein, which shares only 27% aminoacid identity to the vacv b18, can bind and inhibit both human (and monkey) ifn-i as well as the more recently described family of type iii ifns [218] . the latter are a specialized group of ifns mediating antiviral response specifically at mucosal sites without compromising barrier integrity of the epithelia and promoting long-lasting humoral and cellular responses which signal through a distinct, specific heterodimeric cellular receptor (reviewed by [225] ). the authors have proposed that inhibition of these ifns might be related to the specific tissue tropism of yldv, although information on its role in vivo has not yet been provided. insights into the biological role of poxviral ifn-i bps comes from murine infection models using vaccv and ectv, the latter naturally causing fatal mousepox in susceptible mouse strains. early reports showed that deletion of ifn-i bp gene from vacv attenuated the virus in vivo both in intranasal [216] as well as intracranial [217] infection models. in ectv, absence of ifn-i bp resulted in a completely attenuated phenotype upon footpad inoculation (ld 50 reduction at least 10 7 -fold) with severely impaired dissemination of virus to its secondary replication sites, liver and spleen, as well as enhanced nk cell recruitment and both cd4 + and cd8 + t cell activation [226] crucially, these effects were shown to be dependent on ifnar signaling by the use of knockout mice. the ifn-i bp was found to bind to uninfected cells around infection foci in the liver and spleen protecting these tissues locally from ifn induced antiviral activity [227] . the biological relevance of tissue retention of this inhibitory protein was shown using recombinant ectv that express a mutated ifn-i bp unable to bind to the cell surface but still able to inhibit ifn-i efficiently. infection with these recombinants resulted in non-lethal infection as in the case of the virus lacking ifn-i bp altogether [228] . interestingly, it was found that immunization of mice with recombinant ifn-i bp could prevent the development of mousepox upon challenge [226] , probably through the development of antibodies capable of impairing its interaction with its ligands [227] and also pointing to a novel therapeutic target for the treatment of poxviral infections in humans. the structure of the complex of ectv ifn-i bp with murine ifnα-5 has been solved to high resolution (pdb entry 3oq3, deposited by fremont and lee, results to be published). comparisons with the ternary complexes of different ifn-i with their cellular receptor [229, 230] will be crucial to disentangle the structure function relationships in the interaction and inhibition of the biological activities of ifn-i ligands by the poxviral ifn-i bps. the particular properties of the poxviral ifn-i bp, especially its broad species and type specificity as well its high affinity have been instrumental to its use as a biotechnological tool. thus, b18 has been used to determine the implication of ifn-i in diverse processes, such as the monocyte-derived macrophage-mediated inhibition of human cytomegalovirus (hcmv) spread [231] . in addition, recombinant oncolytic herpes simplex viruses expressing b18 have been developed to improve their infectivity in the face of antiviral responses [232] . finally, b18 has been used to block ifnα mediated hiv associated encephalitis in a murine model [233] or to inhibit the detrimental ifn mediated effects produced by mrna exposure in induced pluripotent stem (ips) cell reprogramming [234] . secreted ifn-i bps have been exclusively found in poxviruses to date. a recent report described the murine norovirus ns1 protein, which is secreted by an unconventional caspase-3 mediated pathway, to be essential for tuft cell infection in gastrointestinal tissue through blockade of ifn type iii signaling [235] . while a direct inhibition of ifn type iii could not be demonstrated in the reporter assay used, the molecular mechanism employed by this protein remains unsolved and raises the question as to whether additional and different soluble ifn-i or ifn-iii bps might be identified in other virus species. ifn responses are a complex and important component of the innate immune system. this is reflected in the vastness and complexity of isgs roles, not only involved in antiviral responses but also in several immunomodulatory functions. viruses can disrupt ifn responses leading to the antiviral state to promote their successful replication. indeed, viruses often interfere with multiple pathways involved in the ifn response to evade innate immunity. the importance of the ifn system in host antiviral responses is highlighted by the fact that viruses dedicate some of their genetic material to encode for ifn antagonists. the viral mechanisms of ifn evasion can be mediated directly by viral gene products. viruses also often usurp components of the cellular machinery to carry out their ifn antagonistic activity. there is no doubt that understanding how viruses evade the ifn system will shed some light on the pathogenicity and allow for a better design of therapeutic approaches. the interaction of these pathogens with the ifn system can also shed some light on some of the regulatory cellular mechanisms that control the ifn response. studying the interaction of viral components with the ifn system remains essential to understand the pathogenesis of emergent viruses that threatened global health. innate immune sensing and signaling of cytosolic nucleic acids identification of the zebrafish ifn receptor: implications for the origin of the vertebrate ifn system how cells respond to interferons virus interference. i. the interferon cut, copy, move, delete: the study of human interferon genes reveal multiple mechanisms underlying their evolution in amniotes the human interferon alpha species and receptors type i interferon differential therapy for erythroleukemia: specificity of stat activation receptor density is key to the alpha2/beta interferon differential activities the dual nature of type i and type ii interferons functions of natural killer cells ifn-lambdas mediate antiviral protection through a distinct class ii cytokine receptor complex il-28, il-29 and their class ii cytokine receptor il-28r pathogen recognition and innate immunity tlr-mediated innate immune recognition toll like receptors and viruses differential roles of mda5 and rig-i helicases in the recognition of rna viruses inflammasome recognition of influenza virus is essential for adaptive immune responses irf-1 induced cell growth inhibition and interferon induction requires the activity of the protein kinase pkr deficient cytokine signaling in mouse embryo fibroblasts with a targeted deletion in the pkr gene: role of irf-1 and nf-kappab physical association between stat1 and the interferon-inducible protein kinase pkr and implications for interferon and double-stranded rna signaling pathways ifits: emerging roles as key anti-viral proteins sequestration by ifit1 impairs translation of 2‫׳‬o-unmethylated capped rna structural basis for viral 5'-ppp-rna recognition by human ifit proteins isg15, a small molecule with huge implications: regulation of mitochondrial homeostasis interferon-stimulated gene 15 and the protein isgylation system high-throughput immunoblotting ubiquitiinlike protein isg15 modifies key regulators of signal transduction positive regulation of interferon regulatory factor 3 activation by herc5 via isg15 modification acetaldehyde disrupts interferon alpha signaling in hepatitis c virus-infected liver cells by up-regulating usp18 identification and characterization of a novel isg15-ubiquitin mixed chain and its role in regulating protein homeostasis negative feedback regulation of rig-i-mediated antiviral signaling by interferon-induced isg15 conjugation isg15 modification of the eif4e cognate 4ehp enhances cap structure-binding activity of 4ehp immunoregulatory properties of isg15, an interferon-induced cytokine isg15 in antiviral immunity and beyond the dengue virus conceals double-stranded rna in the intracellular membrane to escape from an interferon response ultrastructure of kunjin virus-infected cells: colocalization of ns1 and ns3 with double-stranded rna, and of ns2b with ns3, in virus-induced membrane structures tick-borne encephalitis virus delays interferon induction and hides its double-stranded rna in intracellular membrane vesicles influenza a virus cell entry, replication, virion assembly and movement vaccinia virus dna replication occurs in endoplasmic reticulumenclosed cytoplasmic mini-nuclei regulated, stable expression and nuclear presence of reovirus double-stranded rna-binding protein sigma3 in hela cells relevance of ebola virus vp35 homo-dimerization on the type i interferon cascade inhibition influenza virus adaptation pb2-627k modulates nucleocapsid inhibition by the pathogen sensor rig-i a recombinant influenza a virus expressing an rna-binding-defective ns1 protein induces high levels of beta interferon and is attenuated in mice the genome-linked protein vpg of vertebrate viruses-a multifaceted protein cap binding and immune evasion revealed by lassa nucleoprotein structure structure of the lassa virus nucleoprotein reveals a dsrna-specific3‫׳‬ to5‫׳‬ e‫ٴٴ‬xonuclease activity essential for immune suppression processing of genome ‫׳5‬ termini as a strategy of negative-strand rna viruses to avoid rig-i-dependent interferon induction poxvirus decapping enzymes enhance virulence by preventing the accumulation of dsrna and the induction of innate antiviral responses measles virus circumvents the host interferon response by different actions of the c and v proteins the c protein is recruited to measles virus ribonucleocapsids by the phosphoprotein inhibition of cgas dna sensing by a herpesvirus virion protein herpes simplex virus 1 tegument protein vp22 abrogates cgas/ sting-mediated antiviral innate immunity z proteins of new world arenaviruses bind rig-i and interfere with type i interferon induction severe acute respiratory syndrome coronavirus m protein inhibits type i interferon production by impeding the formation of traf3.tank.tbk1/ikkepsilon complex foot-andmouth disease virus viroporin 2b antagonizes rig-i-mediated antiviral effects by inhibition of its protein expression rig-i is cleaved during picornavirus infection enterovirus 2apro targets mda5 and mavs in infected cells nuclear ifi16 induction of irf-3 signaling during herpesviral infection and degradation of ifi16 by the viral icp0 protein toscana virus non-structural protein nss acts as e3 ubiquitin ligase promoting rig-i degradation dengue virus ns2b protein targets cgas for degradation and prevents mitochondrial dna sensing during infection deubiquitinating and interferon antagonism activities of coronavirus papain-like proteases structure-guided mutagenesis alters deubiquitinating activity and attenuates pathogenesis of a murine coronavirus structural basis for the ubiquitin-linkage specificity and deisgylating activity of sars-cov papain-like protease mers-cov papain-like protease has deisgylating and deubiquitinating activities a shared interface mediates paramyxovirus interference with antiviral rna helicases mda5 and lgp2 antagonism of the phosphatase pp1 by the measles virus v protein is required for innate immune escape of mda5 measles virus suppresses rig-i-like receptor activation in dendritic cells via dc-sign-mediated inhibition of pp1 phosphatases the double-stranded rna-binding protein pact functions as a cellular activator of rig-i to facilitate innate antiviral response pact facilitates rna-induced activation of mda5 by promoting mda5 oligomerization middle east respiratory syndrome coronavirus 4a protein is a double-stranded rna-binding protein that suppresses pact-induced activation of rig-i and mda5 in the innate antiviral response mutual antagonism between the ebola virus vp35 protein and the rig-i activator pact determines infection outcome arenaviral nucleoproteins suppress pact-induced augmentation of rig-i function to inhibit type i interferon production a distinct role of riplet-mediated k63-linked polyubiquitination of the rig-i repressor domain in human antiviral innate immune responses trim25 ring-finger e3 ubiquitin ligase is essential for rig-i-mediated antiviral activity influenza a virus ns1 targets the ubiquitin ligase trim25 to evade recognition by the host viral rna sensor rig-i molecular mechanism of influenza a ns1-mediated trim25 recognition and inhibition dengue subgenomic rna binds trim25 to inhibit interferon expression for epidemiological fitness a phosphomimetic-based mechanism of dengue virus to antagonize innate immunity sars coronavirus papain-like protease inhibits the type i interferon signaling pathway through interaction with the sting-traf3-tbk1 complex coronavirus papain-like proteases negatively regulate antiviral innate immune response through disruption of sting-mediated signaling ubiquitination of sting at lysine 224 controls irf3 activation sars coronavirus papain-like protease inhibits the tlr7 signaling pathway through removing lys63-linked polyubiquitination of traf3 and traf6 the golgi apparatus acts as a platform for tbk1 activation after viral rna sensing viral and metazoan poxins are cgamp-specific nucleases that restrict cgas-sting signalling dna tumor virus oncogenes antagonize the cgas-sting dna-sensing pathway middle east respiratory syndrome coronavirus m protein suppresses type i interferon expression through the inhibition of tbk1-dependent phosphorylation of irf3 suppression of innate antiviral response by severe acute respiratory syndrome coronavirus m protein is mediated through the first transmembrane domain middle east respiratory syndrome coronavirus orf4b protein inhibits type i interferon production through both cytoplasmic and nuclear targets modulation of the cgas-sting dna sensing pathway by gammaherpesviruses hsv-1 icp27 targets the tbk1-activated sting signalsome to inhibit virusinduced type i ifn expression heartland virus nss protein disrupts host defenses by blocking the tbk1 kinase-irf3 transcription factor interaction and signaling required for interferon induction species-specific disruption of sting-dependent antiviral cellular defenses by the zika virus ns2b3 protease denv inhibits type i ifn production in infected cells by cleaving human sting dengue virus targets the adaptor protein mita to subvert host innate immunity seneca valley virus suppresses host type i interferon production by targeting adaptor proteins mavs, trif, and tank for cleavage the coxsackievirus b 3c protease cleaves mavs and trif to attenuate host type i interferon and apoptotic signaling porcine reproductive and respiratory syndrome virus 3c protease cleaves the mitochondrial antiviral signalling complex to antagonize ifn-beta expression hepatitis c virus protease ns3/4a cleaves mitochondrial antiviral signaling protein off the mitochondria to evade innate immunity mavs protein is attenuated by rotavirus nonstructural protein 1 rotavirus vp3 targets mavs for degradation to inhibit type iii interferon expression in intestinal epithelial cells sars-coronavirus open reading frame-9b suppresses innate immunity by targeting mitochondria and the mavs/traf3/traf6 signalosome pcbp2 mediates degradation of the adaptor mavs via the hect ubiquitin ligase aip4 dengue virus impairs mitochondrial fusion by cleaving mitofusins mitophagy switches cell death from apoptosis to necrosis in nsclc cells treated with oncolytic measles virus mitophagy promotes replication of oncolytic newcastle disease virus by blocking intrinsic apoptosis in lung cancer cells hepatitis b virus disrupts mitochondrial dynamics: induces fissi on and mitophagy to attenuate apoptosis the matrix protein of human parainfluenza virus type 3 induces mitophagy that suppresses interferon responses influenza a virus protein pb1-f2 impairs innate immunity by inducing mitophagy an anti-interferon activity shared by paramyxovirus c proteins: inhibition of toll-like receptor 7/9-dependent alpha interferon induction dengue virus subverts the interferon induction pathway via ns2b/3 protease-ikappab kinase epsilon interaction arenavirus nucleoprotein targets interferon regulatory factor-activating kinase ikkepsilon inhibition of the type i interferon response by the nucleoprotein of the prototypic arenavirus lymphocytic choriomeningitis virus convergence of the nf-kappab and irf pathways in the regulation of the innate antiviral response arenavirus nucleoproteins prevent activation of nuclear factor kappa b hepatitis a virus 3c protease cleaves nemo to impair induction of beta interferon footand-mouth disease virus 3c protease cleaves nemo to impair innate immune signaling porcine epidemic diarrhea virus 3c-like protease regulates its interferon antagonism by cleaving nemo arterivirus nsp4 antagonizes interferon beta production by proteolytically cleaving nemo at multiple sites diversity of interferon antagonist activities mediated by nsp1 proteins of different rotavirus strains rotavirus nsp1 mediates degradation of interferon regulatory factors through targeting of the dimerization domain rotavirus nsp1 inhibits nfkappab activation by inducing proteasome-dependent degradation of beta-trcp: a novel mechanism of ifn antagonism putative e3 ubiquitin ligase of human rotavirus inhibits nf-kappab activation by using molecular mimicry to target beta-trcp the human immunodeficiency virus type 1 vpu protein inhibits nf-kappa b activation by interfering with beta trcp-mediated degradation of ikappa b interaction of epstein-barr virus latent membrane protein 1 with scfhos/beta-trcp e3 ubiquitin ligase regulates extent of nf-kappab activation poxvirus targeting of e3 ligase beta-trcp by molecular mimicry: a mechanism to inhibit nf-kappab activation and promote immune evasion and virulence hhv-8 encoded virf-1 represses the interferon antiviral response by blocking irf-3 recruitment of the cbp/p300 coactivators inhibition of interferon regulatory factor 3 activation by paramyxovirus v protein japanese encephalitis virus ns5 inhibits type i interferon (ifn) production by blocking the nuclear translocation of ifn regulatory factor 3 and nf-kappab evasion of antiviral immunity through sequestering of tbk1/ikkepsilon/irf3 into viral inclusion bodies bluetongue virus ns4 protein is an interferon antagonist and a determinant of virus virulence measles virus c protein interferes with beta interferon transcription in the nucleus nss protein of sandfly fever sicilian phlebovirus counteracts interferon (ifn) induction by masking the dnabinding domain of ifn regulatory factor 3 conserved herpesviral kinase promotes viral persistence by inhibiting the irf-3-mediated type i interferon response respirovirus c protein inhibits activation of type i interferon receptor-associated kinases to block jak-stat signaling blocking of interferon-induced jak-stat signaling by japanese encephalitis virus ns5 through a protein tyrosine phosphatasemediated mechanism paramyxovirus v proteins interact with the rig-i/ trim25 regulatory complex and inhibit rig-i signaling the nucleoprotein and phosphoprotein of peste des petits ruminants virus inhibit interferons signaling by blocking the jak-stat pathway role for herpes simplex virus 1 icp27 in the inhibition of type i interferon signaling inhibition of alpha/beta interferon signaling by the ns4b protein of flaviviruses hepatitis c virus ns5a disrupts stat1 phosphorylation and suppresses type i interferon signaling rotavirus nsp1 protein inhibits interferon-mediated stat1 activation virus-induced autophagic degradation of stat2 as a mechanism for interferon signaling blockade induction and control of the type i interferon pathway by bluetongue virus the measles virus v protein binding site to stat2 overlaps with that of irf9 stat2 is a primary target for measles virus v protein-mediated alpha/ beta interferon signaling inhibition the interferon signaling antagonist function of yellow fever virus ns5 protein is activated by type i interferon dengue virus co-opts ubr4 to degrade stat2 and antagonize type i interferon signaling nonstructural protein 11 of porcine reproductive and respiratory syndrome virus induces stat2 degradation to inhibit interferon signaling the v protein of simian virus 5 inhibits interferon signalling by targeting stat1 for proteasome-mediated degradation association of mumps virus v protein with rack1 results in dissociation of stat-1 from the alpha interferon receptor complex the human papillomavirus type 16 e7 protein binds human interferon regulatory factor-9 via a novel pest domain required for transformation expression of hepatitis c virus proteins interferes with the antiviral action of interferon independently of pkr-mediated control of protein synthesis herpes simplex virus 1 gene products occlude the interferon signaling pathway at multiple sites human cytomegalovirus inhibits ifn-alpha-stimulated antiviral and immunoregulatory responses by blocking multiple levels of ifn-alpha signal transduction the polyoma virus t antigen interferes with interferon-inducible gene expression foot-and-mouth disease virus leader protease cleaves g3bp1 and g3bp2 and inhibits stress granule formation stress granule formation induced by measles virus is protein kinase pkr dependent and impaired by rna adenosine deaminase adar1 g3bp1, g3bp2 and caprin1 are required for translation of interferon stimulated mrnas and are targeted by a dengue virus non-coding rna inhibitory activity for the interferoninduced protein kinase is associated with the reovirus serotype 1 sigma 3 protein site-directed mutagenic analysis of reovirus sigma 3 protein binding to dsrna rift valley fever virus nss protein functions and the similarity to other bunyavirus nss proteins rna-specific adenosine deaminase adar1 suppresses measles virus-induced apoptosis and activation of protein kinase pkr double-stranded rna deaminase adar1 increases host susceptibility to virus infection adar1 interacts with pkr during human immunodeficiency virus infection of lymphocytes and contributes to viral replication silencing the alarms: innate immune antagonism by rotavirus nsp1 and vp3 homologous 2',5'-phosphodiesterases from disparate rna viruses antagonize antiviral innate immunity rotavirus open cores catalyze 5′-capping and methylation of exogenous rna: evidence that vp3 is a methyltransferase 2′-o methylation of the viral mrna cap evades host restriction by ifit family members attenuation and restoration of severe acute respiratory syndrome coronavirus mutant lacking 2'-o-methyltransferase activity biochemical and structural insights into the mechanisms of sars coronavirus rna ribose 2'-o-methylation by nsp16/nsp10 protein complex middle east respiratory syndrome coronavirus nonstructural protein 16 is necessary for interferon resistance and viral pathogenesis the ifitm proteins mediate cellular resistance to influenza a h1n1 virus, west nile virus, and dengue virus interferon induction of ifitm proteins promotes infection by human coronavirus oc43 hiv-1 mutates to evade ifitm1 restriction in vivo evasion of mxa by avian influenza viruses requires human signature in the viral nucleoprotein irreversible inactivation of isg15 by a viral leader protease enables alternative infection detection strategies consecutive inhibition of isg15 expression and isgylation by cytomegalovirus regulators the abundant tegument protein pul25 of human cytomegalovirus prevents proteasomal degradation of pul26 and supports its suppression of isgylation transmembrane protein pul50 of human cytomegalovirus inhibits isgylation by downregulating ube1l isg15, a ubiquitin-like interferon-stimulated gene, promotes hepatitis c virus production in vitro: implications for chronic infection and response to treatment the interferon stimulated gene 15 functions as a proviral factor for the hepatitis c virus and as a regulator of the ifn response interferon-stimulated gene 15 conjugation stimulates hepatitis b virus production independent of type i interferon signaling pathway in vitro vaccinia virus encodes a soluble type i interferon receptor of novel structure and broad species specificity vaccinia virus b18r gene encodes a type i interferon-binding protein that blocks interferon alpha transmembrane signaling inhibition of type i and type iii interferons by a secreted glycoprotein from yaba-like disease virus the highly virulent variola and monkeypox viruses express secreted inhibitors of type i interferon human interferon-and interferon-kappa exhibit low potency and low affinity for cell-surface ifnar and the poxvirus antagonist b18r the vaccinia virus soluble alpha/beta interferon (ifn) receptor binds to the cell surface and protects cells from the antiviral effects of ifn analysis of an interaction between the soluble vaccinia virus-coded type i interferon (ifn)-receptor and human ifn-alpha1 and ifn-alpha2 evaluating the orthopoxvirus type i interferon-binding molecule as a vaccine target in the vaccinia virus intranasal murine challenge model glycosaminoglycans mediate retention of the poxvirus type i interferon binding protein at the cell surface to locally block interferon antiviral responses type iii interferons: balancing tissue tolerance and resistance to pathogen invasion the orthopoxvirus type i ifn binding protein is essential for virulence and an effective target for vaccination antibody inhibition of a viral type 1 interferon decoy receptor cures a viral disease by restoring interferon signaling in the liver a virus-encoded type i interferon decoy receptor enables evasion of host immunity through cell-surface binding structural linkage between ligand discrimination and receptor activation by type i interferons structural basis of a unique interferon-beta signaling axis mediated via the receptor ifnar1 human monocyte-derived macrophages inhibit hcmv spread independent of classical antiviral cytokines incorporation of the b18r gene of vaccinia virus into an oncolytic herpes simplex virus improves antitumor activity the recombinant vaccinia virus gene product, b18r, neutralizes interferon alpha and alleviates histopathological complications in an hiv encephalitis mouse model highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mrna a secreted viral nonstructural protein determines intestinal norovirus pathogenesis acknowledgements this work was supported by grant rti2018-094616-b-100 from the spanish ministerio de ciencia e innovación; grant s2018/baa-4370-platesa2 from the comunidad de madrid (fondo europeo de desarrollo regional, feder) and vetbionet infraia-731014 from the euopean union h2020. key: cord-313138-y485ev30 authors: magor, katharine e.; miranzo navarro, domingo; barber, megan r.w.; petkau, kristina; fleming-canepa, ximena; blyth, graham a.d.; blaine, alysson h. title: defense genes missing from the flight division date: 2013-04-24 journal: dev comp immunol doi: 10.1016/j.dci.2013.04.010 sha: doc_id: 313138 cord_uid: y485ev30 birds have a smaller repertoire of immune genes than mammals. in our efforts to study antiviral responses to influenza in avian hosts, we have noted key genes that appear to be missing. as a result, we speculate that birds have impaired detection of viruses and intracellular pathogens. birds are missing tlr8, a detector for single-stranded rna. chickens also lack rig-i, the intracellular detector for single-stranded viral rna. riplet, an activator for rig-i, is also missing in chickens. irf3, the nuclear activator of interferon-beta in the rig-i pathway is missing in birds. downstream of interferon (ifn) signaling, some of the antiviral effectors are missing, including isg15, and isg54 and isg56 (ifits). birds have only three antibody isotypes and igd is missing. ducks, but not chickens, make an unusual truncated igy antibody that is missing the fc fragment. chickens have an expanded family of lilr leukocyte receptor genes, called chir genes, with hundreds of members, including several that encode igy fc receptors. intriguingly, lilr homologues appear to be missing in ducks, including these igy fc receptors. the truncated igy in ducks, and the duplicated igy receptor genes in chickens may both have resulted from selective pressure by a pathogen on igy fcr interactions. birds have a minimal mhc, and the tap transport and presentation of peptides on mhc class i is constrained, limiting function. perhaps removing some constraint, ducks appear to lack tapasin, a chaperone involved in loading peptides on mhc class i. finally, the absence of lymphotoxin-alpha and beta may account for the observed lack of lymph nodes in birds. as illustrated by these examples, the picture that emerges is some impairment of immune response to viruses in birds, either a cause or consequence of the host-pathogen arms race and long evolutionary relationship of birds and rna viruses. lymph node duck chicken major histocompatibility complex a b s t r a c t birds have a smaller repertoire of immune genes than mammals. in our efforts to study antiviral responses to influenza in avian hosts, we have noted key genes that appear to be missing. as a result, we speculate that birds have impaired detection of viruses and intracellular pathogens. birds are missing tlr8, a detector for single-stranded rna. chickens also lack rig-i, the intracellular detector for singlestranded viral rna. riplet, an activator for rig-i, is also missing in chickens. irf3, the nuclear activator of interferon-beta in the rig-i pathway is missing in birds. downstream of interferon (ifn) signaling, some of the antiviral effectors are missing, including isg15, and isg54 and isg56 (ifits). birds have only three antibody isotypes and igd is missing. ducks, but not chickens, make an unusual truncated igy antibody that is missing the fc fragment. chickens have an expanded family of lilr leukocyte receptor genes, called chir genes, with hundreds of members, including several that encode igy fc receptors. intriguingly, lilr homologues appear to be missing in ducks, including these igy fc receptors. the truncated igy in ducks, and the duplicated igy receptor genes in chickens may both have resulted from selective pressure by a pathogen on igy fcr interactions. birds have a minimal mhc, and the tap transport and presentation of peptides on mhc class i is constrained, limiting function. perhaps removing some constraint, ducks appear to lack tapasin, a chaperone involved in loading peptides on mhc class i. finally, the absence of lymphotoxin-alpha and beta may account for the observed lack of lymph nodes in birds. as illustrated by these examples, the picture that emerges is some impairment of immune response to viruses in birds, either a cause or consequence of the host-pathogen arms race and long evolutionary relationship of birds and rna viruses. ó 2013 elsevier ltd. all rights reserved. a survey of genomic resources demonstrates that the avian immune gene complement is reduced compared to mammals. an initial investigation of the immune genes in the chicken genome, a red jungle fowl, suggested that birds have a reduced immune gene repertoire (consortium, 2004) . as this sequence assembly and annotation has been improved, some of these missing genes have been identified, however others are clearly not present. as genomes are sequenced for other birds, including turkey (dalloul et al., 2010) , zebrafinch and duck (http:// pre.ensembl.org/anas_platyrhynchos/info/index), synteny along the chromosome allowed identification of genes. thus, immune genes could be identified even if significantly diverged. a comparison of immune genes between three species of birds, confirmed that immune genes show greater divergence between species than other genes, with higher dn/ds ratio than other parts of the genome and evidence of positive selection on specific codons within genes . the sequencing of cdna libraries as expressed sequence tags (ests) (carre et al., 2006) , and blast homology searches helped to identify the genes. nonetheless, some genes are still unaccounted for. this appears true for all birds, although species differences exist. for some of these genes missing from the avian defense arsenal, the evidence is overwhelming, while others are less certain. in all cases, the completion and quality of the genome sequence and annotation determines whether a gene can be identified or not. gaps exist in the genome sequences, and immune genes are often present in gene families, which are particularly prone to problems with assembly. est libraries are incomplete, and immune gene expression may be restricted to certain tissues or cell types, and most importantly, only following immune activation. thus, until genomes are complete and error-free it may be premature to say that a gene is not there. nonetheless, claiming that a gene is missing certainly inspires research aimed at confirming or disproving this, or demonstrating that another gene plays an analogous or compensatory role. thus, it is worth highlighting the genes that appear to be missing. the contracted immune gene repertoire of birds was discussed in recent review of the progress in avian immunology since the availability of the chicken genome (kaiser, 2010 (kaiser, , 2012 . in comparison with mammals, birds have partial repertoires of pattern recognition receptors including tlr receptors (boyd et al., 2007; brownlie and allan, 2011; cormican et al., 2009 ) and rig-like receptors (barber et al., 2010; karpala et al., 2012) . others have extensively examined the repertoire of avian cytokines (kaiser et al., 2005) and chemokines (hughes et al., 2007; kaiser et al., 2005) interferons (schultz et al., 2004) (schultz and magor, 2008) and defensins (lynn et al., 2007) noting the genes missing from these repertoires. the immunoglobulin locus been characterized in ducks (lundqvist et al., 2001) , and encodes just three antibody isotypes (magor, 2011) . finally, the chicken major histocompatibility complex (kaufman, 2013) is a minimal mhc, where only the most essential genes have been retained. these reviews of each system, although excellent, do not dwell on the genes not found. over the course of our analysis of immune systems of ducks, we have often invested significant effort to identify homologues of the chicken or mammalian immune system. despite our best efforts, some genes have eluded our search. here we will focus on components of three parts of the immune system that we are investigating in ducks (pattern recognition, antibodies and mhc) and identify the genes that are not there in the duck or the chicken or both. we will assess the strength of the data suggesting the absence of the gene, and consider the effect of the gene loss on the immune system of the animal. finally, we will speculate on the selective forces that may have led to the loss of the gene. innate immunity provides the first line of defense against pathogens. recognition of the pathogen through the molecular patterns of conserved pathogen components, or pattern recognition activates a signaling cascade to turn on genes for the effectors of the immune response. toll like receptors (tlrs) detect foreign invaders by sensing pathogen-associated molecular patterns. binding of agonists to tlrs on the cell surface, or within the endosomal compartment, activate signal transduction pathways to turn on antimicrobial peptides, cytokines, interferons and cellular killing mechanisms. birds possess genes for ten tlrs. these include two tlr1 genes, two tlr2 genes, tlr3, tlr4, tlr5, tlr7, tlr15 and tlr21. several excellent reviews have been written recently on avian tlr genes (brownlie and allan, 2011; cormican et al., 2009) . two genes are missing in comparison to fish and mammals, tlr8 and tlr9. tlr9, which detects cpg, has been functionally compensated by tlr21 (brownlie et al., 2009; keestra et al., 2010) . tlr7 and tlr8 are phylogenetically related as the product of an ancient gene duplication and both can recognize single-stranded rna, oligoribonucleotides, and nucleic acid analogues in the endosomal compartment (reviewed by cervantes et al., 2012) . tlr8, which is present in fish and mammals, is absent in birds. sequencing downstream of chicken tlr7 showed only fragments of tlr8 (philbin et al., 2005) . further, pcr evidence suggested that tlr8 was disrupted in all the galliform birds, but not anseriform birds, and there was speculation that this could account for the increased susceptibility of chickens to influenza relative to ducks (philbin et al., 2005) . to follow up on this observation, we cloned duck tlr7 cdna, and isolated a genomic clone for duck tlr7, sequenced it, and examined the region downstream. as seen for chickens, we could identify only small fragments of tlr8, and a cr1 element disrupted the gene in both ducks and chickens (macdonald et al., 2007) . tlr8 is also absent from the zebra finch (cormican et al., 2009 ) and turkey genome (ramasamy et al., 2012) . given the evolutionary distance of galliform birds and zebra finch, tlr8 is likely missing from the entire avian lineage. for several years, mouse tlr8 had been presumed non-functional, based on the lack of response to tlr7/8 agonists in the tlr7à/à mouse (hemmi et al., 2002) . however, when peripheral blood monocytes from mice are treated with selective tlr8 agonists, imidazoquinoline 3m002 and poly t oligonucleotides, mouse tlr8 activation is demonstrated while tlr7 is suppressed (gorden et al., 2006) . tlr8 is expressed in monocytes/macrophages and myeloid dcs, while tlr7 is expressed in pdcs and b cells (hornung et al., 2002) . tlr8 also plays a role in detecting bacterial rna, including rna from borrelia burgdorferi, the agent of lyme disease, inducing production of ifn-beta through irf7 (cervantes et al., 2011) . tlr8 is upregulated by the phagocytosis of mycobacterium, including the attenuated bcg vaccine strain, mycobacterium bovis, and mycobacterium tuberculosis (davila et al., 2008) . human tlr8 allelic variants are associated with increased susceptibility to pulmonary tuberculosis (davila et al., 2008) . the protective allele is associated with decreased translation of tlr8, presumably resulting in a decrease in sensing and activation, and less inflammation (davila et al., 2008) . effectively, loss of tlr8 expression is protective against tuberculosis. it is not clear why the loss of tlr8 was selected for in birds. the simplest explanation is the similarity of function between tlr7 and tlr8 rendered the second gene non-functional. in this scenario, however, there is no selection for the deletion of the gene. alternatively, tlr8 became detrimental, perhaps by recognizing self-antigens and initiating autoimmunity. negative selection would then likely lead to the loss of this receptor. tlr7 has been implicated in induction of autoimmunity (mills, 2011) . early experiments used chickens to demonstrate thyroid autoimmunity (sundick et al., 1992; wick et al., 1974) but it is not known to what extent avian species suffer autoimmunity in nature. ironically, knockout of tlr8 in mice leads to autoimmunity through overexpression and disregulation of tlr7 (demaria et al., 2010) . intriguingly, nucleic acid sensing tlrs are implicated in preventing reactivation of host retroviral elements and consequent tumor production (yu et al., 2012) . tlr7 has been directly implicated in this immunosurveillance, as lack of antibodies against endogenous retroviral elements correlates with absence of tlr7 in knockout mice strains. this crucial role of tlr7 in immunosurveillance of endogenous retroviruses would provide the selective pressure to retain tlr7 in the genome, regardless of how tlr8 was lost. since tlr8 appears to have been inactivated by a cr1 repetitive element, it is tempting to speculate that tlr8 was lost in a hypothetical reactivation of endogenous retroviral elements that disrupted the genome in a distant avian ancestor. jim kaufman has alluded to such a catastrophic 'avian big bang' in describing the loss of several avian mhc genes (kaufman and wallny, 1996) . another theoretical possibility is that tlr8 became the target of a pathogen that subverted it for its own benefit (discussed in barber, 2011) . viral subversion of tlr3 is such that its absence increases host survival from many pathogens. tlr3-induced host proinflammatory cytokines allow west nile virus to cross the blood brain barrier (wang et al., 2004) . tlr3 activity has also been implicated in influenza-induced pneumonia (le goffic et al., 2006) and morbidity from vaccinia infection (hutchens et al., 2008) . along these lines, we can envision a pathogen that subverted avian tlr8 for increased susceptibility. this could include viral targeting of tlr8 receptor for increased inflammation and pathology, or subversion of an endosomal tlr8 for entry of a mycobacterial pathogen into the cell. mycobacteria are initially engulfed by macrophages, but survive and multiply intracellularly. thus, bacterial or viral subversion of a prr may drive selection to disable the gene. whether cause or effect, the lack of tlr8 in avian monocytes/ macrophages likely does contribute to the susceptibility of birds to rna viruses (west nile virus, newcastle disease virus, influenza virus and others) and intracellular bacterial infections, including mycobacteria. mycobacterium avium is a significant pathogen of birds, particularly those raised in small flocks, while modern flock hygiene has reduced the incidence in commercial poultry. susceptibility to mycobacteriosis in birds varies, with chickens, pheasants, partridges being most susceptible, ducks and geese moderately resistant, and pigeons being very resistant (reviewed in tell et al., 2001) . rig-i is a cytoplasmic pattern recognition receptor for singlestranded 5 0 -triphosphate rna with short double-stranded conformation, such as panhandle structures of viral genomes (hornung et al., 2006; pichlmair et al., 2006; schlee et al., 2009; yoneyama et al., 2004) . both rig-i, and the related pattern recognition receptor for intracellular rna, mda5, share the same pathway signaling through mavs on the mitochondrion (fig. 1 ). after detection of viral rna by rig-i, a conformational change releases the card domains (kolakofsky et al., 2012; kowalinski et al., 2011; luo et al., 2011; takahasi et al., 2008) . trim25, an e3 ubiquitin ligase, interacts with the card domains of rig-i to activate it through attached (gack et al., 2007) or unanchored k63-polyubiquitin chains (jiang et al., 2012; zeng et al., 2010) . the relative importance of these two mechanisms in the activation of rig-i is still controversial, but activation leads to oligomerization and rig-i translocation to the mitochondria. translocation of rig-i and trim25 to the mitochondrial membrane involves the mitochondrial chaperone 14-3-3e , allowing interaction with mavs at the mitochondrion. this interaction induces prion-like aggregates of mavs (hou et al., 2011) that initiate signaling leading to irf3/7 translocation and the production of type i interferons and proinflammatory cytokines. the gene encoding rig-i, ddx58, is not annotated in the chicken genome sequence, and is missing in some fish species, but mda5 homologues are present in all vertebrate families (zou et 2009). we demonstrated that ducks have a functional rig-i (barber et al., 2010) . in contrast, the ddx58 gene appears absent in chickens by analysis of the syntenic region of the z chromosome, although we can identify the flanking gene. we also cannot find rig-i in a search of the expressed sequence tag database for chickens. thus rig-i is missing in the genome of the ancestral chicken represented by the red jungle fowl, and the sequences from modern commercial chicken breeds. our southern blots show a duck rig-i probe cross-hybridizes with pigeon dna, but not with chicken dna (barber et al., 2010) . we also cannot detect the gene in dna of turkey or partridge, suggesting that the gene is missing in galliformes (barber, 2011) . furthermore, we showed that chicken df-1 cells cannot detect rig-i ligand, but if we transfect the cells with duck rig-i we can reconstitute the pathway (barber et al., 2010) . the loss of rig-i likely contributes to the susceptibility of chickens to infection compared to ducks to a variety of singlestrand rna viruses, including influenza a virus and newcastle disease virus, both of which cause more harm in chickens than ducks. the related rna detector, mda5, can partially compensate and detect avian influenza in chicken cells to generate an interferon response (karpala et al., 2011; liniger et al., 2012) . it is difficult to speculate on the selective forces resulting in loss of rig-i in some birds. some have suggested the possible existence of a compensatory yet-to-be identified alternate receptor (karpala et al., 2011) , which could certainly facilitate the loss from the genome. rig-i was initially identified in a leukemia cell line upregulated by retinoic acid (liu et al., 2000) , and indeed it is upregulated by a variety of stress inducers. rig-i is implicated in a number of other biological events including cell proliferation, apoptosis, senescence, and acute and chronic inflammatory diseases (liu and gu, 2011) . it is possible that selection to eliminate rig-i from aberrant activation in one of these alternate roles had resulted in the loss of rig-i in galliform birds. finally, there remains the intriguing possibility that the rig-i receptor was the prey of one of the many single-strand rna viruses that infect birds, including influenza virus, newcastle disease virus, west nile virus, and coronaviruses, and was usurped for the virus' advantage. the tlr and rlr signaling pathways are targets for viral subversion (reviewed by es-saad et al., 2012; ramos and gale, 2011) . influenza virus interferes in the mammalian rig-i pathway in several places, through the action of ns1 protein (gack et al., 2009) . in a similar manner, paramyxoviruses make the v protein that interferes with signaling by the chicken mda5 receptor (childs et al., 2007) involving direct protein-protein interaction and preventing interaction with the rna ligand (motz et al., 2013) . while this interference renders the receptor non-functional during an infection, it would not necessarily lead to selection to eliminate the receptor. however, we can envision interactions with the rig-i receptor where regulation is aberrant, or excessive activation leads to death. in this scenario, loss of rig-i could provide a selective advantage to survive a lethal infection with an unknown pathogen. suggesting that rig-i is also involved in development in some capacity, rig-i knockout mice are embryonic lethal due to liver damage (kato et al., 2005) . aberrant or loss of expression of rig-i during development due to pathogen subversion, and associated embryonic lethality, could result in selective loss of this receptor. while this raises the question of how the birds without rig-i survived, we note that rig-i is not absolutely essential for development, since rig-i knockout mice have since been made on a different genetic background which are fertile and viable (wang et al., 2007) . given that rig-i expression is impaired in lethal infections (kobasa et al., 2007) , it is possible to envision a scenario by which aberrant expression of rig-i leads to death, and the gene is selectively lost in a common ancestor of chickens and turkeys. human rig-i is regulated through polyubiquitinylation, isgylation, sumolyation, and phosphorylation and alternate splicing (eisenacher and krug, 2012; loo and gale, 2011; maelfait and beyaert, 2012; oshiumi et al., 2012; wang et al., 2011) . a major on-off switch for human rig-i upon viral infection is polyubiquitination by host e3 ubiquitin ligase, tripartite motif protein 25 (trim25) (gack et al., 2007) . sequences at the t55 residue implicated in interaction with trim25 and the site of attachment of polyubiquitin chains, k172, are not conserved in duck or zebra finch rig-i (barber et al., 2010) or goose rig-i . thus activation of avian rig-i involves ubiquitination at alternate residues, or interaction with unanchored polyubiquitin chains, not attached to any protein, can activate rig-i (zeng et al., 2010) . given the lack of rig-i in chickens, the recent observation that knockdown of chicken trim25 impairs the interferon response of chicken cells is intriguing (rajsbaum et al., 2012) . perhaps trim25 is involved in the activation of chmda5. the binding of unanchored k63 polyubiquitin chains can activate human mda5 in vitro (jiang et al., 2012) . a role for trim25 in generating or attaching ubiquitin chains to mda5 could explain the importance of chtrim25 in the interferon response of chicken cells. riplet/rnf135 is a cytoplasmic e3-ligase identified by yeast two-hybrid as one of the proteins binding rig-i, and is essential for rig-i activation in human cell lines upon infection with an rna virus (oshiumi et al., 2009; oshiumi et al., 2010) . riplet shares 60.8% identity with trim25 in humans (oshiumi et al., 2009) , and also has an n-terminal ring domain and c-terminal pry/spry domain. the ring domain confers ubiquitin e3 ligase activity (nisole et al., 2005) and also contributes to other protein-protein interactions (borden, 2000) . riplet also mediates k63-polyubiquitination of rig-i (gao et al., 2009; oshiumi et al., 2010) . however, there is debate as to whether riplet interacts with the card domains of rig-i (gao et al., 2009 ) the c-terminal repressor domain of rig-i or both (oshiumi et al., 2010) . riplet is crucial for rig-i activation in cells regardless of expression of trim25 (oshiumi et al., 2010) . knockout of riplet (oshiumi et al., 2010) or trim25 (gack et al., 2007) impaired the rig-i dependent innate immune response, suggesting that both are required. knockout of riplet resulted in animals that were deficient in the production of interferon in response to rna, but not dna viruses (oshiumi et al., 2010) . riplet is present in zebra finch (taeniopygia guttata), but we were unable to find the ortholog in the chicken (gallus gallus) genome. in the duck, we have located a putative riplet coding region, but it lacks exon 1. in repeated 5 0 race experiments, all clones recovered contain sequences that correspond to an intact open reading frame, but lack the expected ring domain. in mice, deletion of the ring domain prevents rig-i activation (oshiumi et al., 2010) therefore we hypothesize that deletion of the ring domain in ducks may render it functionally inactive. nonetheless, we saw upregulation of riplet and trim25 in duck lung at 1dpi with highly pathogenic avian influenza virus (fleming-canepa x. et al., unpublished data) . because riplet may not be functional in ducks, but still highly upregulated during influenza infection, we speculate that riplet is acting as a decoy for the viral ns1. influenza a ns1 protein interacts with trim25 (gack et al., 2009) and riplet (rajsbaum et al., 2012) causing inhibition of innate immune signaling. alternatively, riplet may dimerize with other e3 ligases to function, as recently shown for trim16 (bell et al., 2012) . comparison of embryonic fibroblast cells from rig-i knockout and wild-type mice upon influenza infection, reveal genes that are downstream of rig-i signaling. these genes have been referred to as the rig-i bioset, the genes induced by influenza infection in a rig-i dependent manner. in mouse fibroblast cells, the genes include ifnb, irf3, irf7, stat1, stat2, pkr, oas, mx1, ifit2 (isg54), ifit1 (isg56) and rsad2 (viperin) (loo et al., 2008) . while the overlap between rig-i and mda5 inducible genes downstream of mavs signaling in chicken cells is unknown, we used a microarray approach to examine the genes turned on by rig-i in chicken cells. using chicken df-1 cells, transfected with duck rig-i, the expression of the rig-i gene bioset in avian species is augmented (barber et al., 2013) . we noted that some essential genes of the mouse rig-i bioset are missing in avian species, including irf3, isg15, and ifit2 (isg54) and ifit1 (isg56). interferon regulatory factor-3 is a critical player in the induction of type i ifns following virus infection (au et al., 1995) . irf3 and irf7 have different and crucial roles in the induction of infa/b . irf3 is constitutively expressed, and is activated by c-terminal phosphorylation that allows dimerization and nuclear localization (lin et al., 1998) . this led to the suggestion that irf3 was responsible for the initial upregulation of the ifnb gene, followed by interferon dependent induction of irf7. however, irf7à/à knockout mice are severely impaired in interferon production upon infection with ssrna viruses (honda et al., 2005) , suggesting the contribution of irf3 is minor. although a gene has been named irf3 in chickens (grant et al., 1995) , it is interferon inducible and more similar to irf7. others have noted the absence of irf3 in chickens (huang et al., 2010) and in avian species (cormican et al., 2009 ). it is not known which irf is translocating to the nucleus to activate interferon in the rig-i/mda5 pathway in avian species. we speculate that irf7 fulfills the nuclear translocation and activation of type i ifns in both tlr and rlr signaling, but this has not been experimentally examined. interferon stimulated gene 15 (isg15) is highly up regulated by interferon treatment and was the first ubiquitin-like modifier identified. the amino acid sequence of isg15 is similar to a linear ubiquitin dimer (reviewed in zhang and zhang, 2011) . isg15 is conjugated to proteins like ubiquitin, through a process called isgylation. among the identified isgylated substrates are interferon-induced proteins like pkr, rig-i, mxa (zhao et al., 2005) . irf3 isgylation by herc5 (the main isg15 e3 ligase in human) increases stability of irf3, exerting a positive regulation in the rig-i pathway . negative feedback on rig-i expression and signaling is mediated by isg15 conjugation to rig-i (kim et al., 2008) . isg15 is also involved in a direct antiviral mechanism where isgylation of influenza a virus ns1 protein impairs viral replication . no genes homologous to human isg15 have been annotated in any of the available avian genomes. in the chicken, genes located adjacent to human isg15 were predicted; including hes1 (homologous to human hes4) and agrn. within this syntenic region of the chicken genome no ubiquitin-like gene was present. similarly, homologs of hes1 and argn genes were found in the duck scaffolds (scaffold 1197 and 2665, respectively) but synteny analysis cannot be performed because the scaffolds do not overlap. enzymes involved in the isgylation system (including ube1l, ubch8, herc5 and usp18) are present in the chicken and duck genomes, but there is not yet any functional evidence of isgylation in these species. usp18, which is responsible for cleavage of isg15 from isgylated substrates, correlates with survival of influenza-infected chickens, indirectly suggesting some functionality of the isgylation system (uchida et al., 2012) . isg15 conjugation plays many roles in mammalian antiviral immunity, including isgylation of mx, pkr, rig-i, and irf3, and influenza ns1 protein (reviewed in skaug and chen, 2010) . however, given the absence of several isg15 targets, including rig-i in chickens, irf3 in birds, and evidence that mx is non-functional in chickens (schusser et al., 2011) and ducks (bazzigher et al., 1993) , the absence of this ubiquitin modifier in birds would be less significant. it is not known whether ns1 is modified by isg15 in avian hosts. we cannot rule out the possibility that we have failed to identify the avian isg15 homolog because of low sequence conservation with human isg15. it is also possible that another unknown ubiquitin-like modifier in birds plays the role of isg15 within the isgylation system. intriguingly, the most similar sequence to isg15 in the duck and chicken genome lies within the c-terminal end of 2 0 ,5 0 -oligoadenylate synthetase-like (oasl) gene. oasl has two tandem ubiquitin-like domains that share 37% amino acid identity to human isg15. the antiviral activity of the human p59 protein, encoded by human oasl, is dependent on the c-terminal ubiquitin-like domain (marques et al., 2008) . however, the biological function of the oasl ubiquitin-like domains is not yet clear and its role as ubiquitin-like modifier has not been described. the interferon-induced proteins with tetratricopeptide repeats (ifit) genes are highly upregulated by type i ifns or by viral infection (bluyssen et al., 1994; levy et al., 1986; wathelet et al., 1986) . the human ifit gene family consists of ifit1 (isg56), ifit2 (isg54), ifit3 (isg60), and ifit5 (isg58), while the mouse ifit family lacks ifit5 and contains ifit1, ifit2, and ifit3 (bluyssen et al., 1994) . the ifit family appears to be limited to a single gene in marsupials, birds, frogs and fish (reviewed by zhou et al., 2013) . while these proteins have served as markers of viral infection, only recently have their functions in the innate antiviral response been elucidated (daffis et al., 2010; fensterl et al., 2012; mcdermott et al., 2012; pichlmair et al., 2011; schmeisser et al., 2010) . ifit proteins reside within the cytoplasm of cells, and all contain multiple tetratricopeptide repeats (tprs) (lamb et al., 1995) . the tprs within these proteins consist of a helix-turn-helix motif and facilitate protein-protein interactions (blatch and lassle, 1999) . ifit1 and ifit2 mediate their antiviral activity by a disruption of translation via an interaction with eukaryotic initiation factor 3 (eif3) . ifit1 and ifit2 also inhibit the translation of viral mrnas lacking a 2 0 -o methylation cap structure (daffis et al., 2010) . ifit1 and ifit5 have the ability to bind to, and sequester viral 5 0 -triphosphate rna (pichlmair et al., 2011) . the crystal structure of ifit2 has revealed an rna binding domain that also may function in an antiviral context (yang et al., 2012) . the multi-functional ifit1 protein can also restrict the replication of human papilloma virus (hpv) by binding the viral helicase e1, and restricting its function in viral replication (saikia et al., 2010) . interestingly, ifit1 has also been associated with negative feedback regulation of genes upregulated during viral infection, further demonstrating the diverse function of these genes . the ifit gene family represents a significant contributor to the broad-ranged antiviral activity of interferons, and plays an important role in the cellular, innate antiviral response. in avian species, the only identifiable ifit gene encodes a protein that aligns with other ifit5 proteins in a phylogenetic tree (fig. 2) . the upregulation of ifit5 following viral infection of chicken cells expressing duck rig-i ( barber et al., 2013) or infection of ducks (vanderven et al., 2012) suggests ifit5 is an important antiviral effector in avian species. the apparent absence of an expanded ifit gene family in avian species suggests that several of the functions attributed to ifit proteins will be missing. indeed, the specific role of avian ifit5 during a viral infection is unknown. birds have only three antibody isotypes, igm, iga and igy. igy, the avian serum ig most similar to mammalian igg, is a precursor to igg and ige that has composite function of both isotypes (warr et al., 1995) . ducks make a truncated version of igy (magor et al., 1992) . in addition, birds use a single light chain gene of the k type (magor et al., 1994a; reynaud et al., 1983) . ducks have three immunoglobulin heavy chain genes arranged in the gene order ighm, igha and ighy encoding the mu, alpha and upsilon chains for igm, iga and igy, respectively (lundqvist et al., 2001; magor et al., 1999) . the igha gene, encoding alpha is inverted in the locus, and ighd (delta) is absent. despite availability of chicken, zebrafinch and turkey genomes, no other avian immunoglobulin heavy chain locus has yet been assembled. from the limited analysis that has been published for chicken igh , it shares the same organization. the transposition of igha from the 3 0 most position in the locus, to an inverted position downstream of ighm, may have also resulted in the loss of ighd. lack of ighd is evident from genomic sequencing for ducks. early studies reported a d chain in chickens (chen et al., 1982) , but it is generally accepted that there is no avian homologue of igd. since igd has been identified in teleosts (bengten et al., 2002; wilson et al., 1997) frogs (zhao et al., 2006) and reptiles (cheng et al., 2013; wei et al., 2009 ) the ighd gene was lost in birds. igd is an enigmatic antibody that exists in a wide variety of forms in different species, except birds. the function of igd is beginning to emerge from observations first made for fish, and subsequently for human igd. igd functions at the interface of innate and adaptive immune responses. in fish, igd specific b cells have been identified, and secreted igd lacks the variable region suggesting it functions more like a pattern recognition receptor (edholm et al., 2010) . igd is found on the surface of granulocytes in fish, which do not make the igd transcript, and involves a specific receptor (edholm et al., 2010) . in humans, circulating igd binds to basophils and activates antimicrobial and inflammatory factors . igd from igd+ igm-b cells binds to basophils, and can also bind to certain bacteria in the respiratory tract. the basophil binds igd through a specific receptor, and cross-link-ing of igd leads to the production of b cell activating factors (baff) and pro-inflammatory cytokines. serum igd is elevated in patients with chronic infections, and specific igd antibodies could be demonstrated in a number of these infections (reviewed by chen and cerutti, 2010) . this ancient surveillance system serves to instruct the b cells of the type of pathogens in the respiratory tract. as the specific functions of igd are elucidated, the consequences of the lack of igd antibody in birds will become evident. birds have basophils, but it is unclear whether a different ig isotype can bind to the basophil igd receptor to compensate, or whether the receptor exists in birds. indeed, it remains to be demonstrated that a homologous receptor is involved in the igd binding by basophils of humans and fish. duck igy is made in two secreted forms, a full-length form and a truncated form. the truncated form, called igydfc, lacks the fc region entirely. it arises from alternate splicing that adds an exon encoding just two amino acids after the ch1 and ch2 domains, and uses an alternate polyadenylation site (magor et al., 1992) (magor et al., 1994b) . what controls the alternate splicing is unknown, but the truncated form predominates later in the immune response. the igydfc antibodies would be expected to be defective in several processes such as antigen internalization, which is required for appropriate presentation of antigens needed to generate t cell help. the truncated igy also does not participate in complement fixation, opsonization, precipitation reactions, and reportedly also cannot participate in hemagglutination inhibition (hi) (higgins et al., 1987) . of benefit to ducks, perhaps the truncated igy helps prevent viral internalization through receptor-mediated endocytosis and subsequent infection of macrophages and other leukocytes (magor, 2011) . the chicken ig-like receptor (chir) genes (dennis et al., 2000) are counterparts of the leukocyte immunoglobulin-like receptor family (lilr). the chir genes constitute a large and diverse family of genes in the chicken, with more than 100 members located in a region syntenic to the mammalian leukocyte receptor complex fig. 2 . a phylogenetic tree showing similarity of avian and mammalian ifit sequences. sequences were aligned and phylogenetic tree generated using a maximum likelihood estimation using a program called phyml using www.phylogeny.fr. (dereeper et al., 2008) . accession numbers for the ifit sequences were: chicken ifit5 (xm_421662.3), turkey ifit5 (xm_003208028.1), zebra finch ifit5 (xm_002188552.1), human ifit1 (nm_001270927.1), mouse ifit1(nm_008331.3), human ifit2 (nm_001547.4), mouse ifit2 (nm_008332.3), human ifit3 (nm_001031683.2), mouse ifit3 (nm_010501.2), human ifit5 (nm_012420.2). note the duck ifit sequence is a partial sequence. (lrc) (laun et al., 2006; nikolaidis et al., 2005; viertlboeck and gobel, 2011; viertlboeck et al., 2005) and vast diversity within an individual (viertlboeck et al., 2010) . chir are expressed in a variety of myeloid and lymphoid cells, with individual receptors expressed in a cell-type restricted manner (viertlboeck et al., 2005) . receptor diversity includes variation within a hypervariable region, the putative binding region, alternate transcript splicing, and presence or absence of functional activation or inhibitory motif. the extensive expansion and diversification of this family in chickens, and leukocyte expression, suggests their evolution is in response to the pressure of pathogens, as suggested for human lilr and mouse pir genes (barclay and hatherley, 2008) . human lilr receptors are involved in self/non-self recognition and some engage mhc class i targets, as well as pathogen mimics of mhc proteins (anderson and allen, 2009; brown et al., 2004) . staphylococcus aureus targets the mouse inhibitory receptor pir-b for increased virulence (nakayama et al., 2012) . in turn, activating pir-a receptors may have evolved in response to the selective pressure from pathogens, as indicated by the relict itims in the pir-a gene suggesting it is derived from a pir-b ancestor (nakayama et al., 2012) . thus, counterbalance through inhibitory and activating chir proteins may have evolved in response to pathogen manipulation of immune signaling through these receptors. we have searched unsuccessfully for immunoglobulin superfamily members homologous to the chicken chir receptors in ducks. in high and low stringency southern blots, genomic dna from chickens shows an extensive pattern of hybridization, while dna from ducks shows no significant hybridization (macdonald et al., 2007) . all efforts to amplify these genes by polymerase chain reaction using several sets of degenerate primers are completely unsuccessful. our searches of the draft assembly of the duck genome, and 70,000 expressed tag sequences generated by 454 sequencing, also find no evidence of chir homologues. we cannot rule out the possibility that we have simply missed the chir genes due to weak homology, if they have evolved to be quite different in ducks. this in itself is quite intriguing. the rapid species-specific divergence of primate lilr genes, with only some genes showing clear orthologous relationships between species (canavez et al., 2001) , while others have evolved to be unique in each species is thought to reflect their species-specific interactions with pathogens. alternatively, these genes are truly absent from ducks, despite their presence in chickens. there are several examples where different vertebrates have employed different families of leukocyte receptors (parham and moffett, 2013) . for example, cattle use kir as nk cell receptors, which have undergone expansion (mcqueen et al., 2002) while horses use ly49 (takahashi et al., 2004) . although there are hundreds of chir genes, the only chir with a known ligand is chir-ab1, which functions as the chicken igy fc receptor (viertlboeck et al., 2007) . chickens have a large number of chir-ab1 genes that have varying specificities for igy . remarkably, we cannot find identifiable homologues of the chir-ab1 in ducks. duck full-length igy does not bind the chicken fc receptor (viertlboeck et al., 2007) . as noted above ducks also make a truncated igydfc that would be expected to not to bind fc receptor. göbel speculated that the loss of the igy fc fragment, and the duplication and divergence of the chicken chir-ab1 (fc receptor) family were both strategies to evade a pathogen interfering with the igy-fc receptor interaction in birds (purzel et al., 2009) . ducks evade this pathogen by production of an anti-body lacking the fc region, retaining the specificity for the antigen, as this truncated form predominates in the later immune response. in chickens, selection favored the duplication of the chir receptor family to make a large number of potential 'decoy receptors' for igy. while no known pathogen targets the fc-igy interaction in birds, this is a very interesting hypothesis. chickens may elude this pathogen through the binding of decoy receptors, while ducks may avoid the internalization of an intracellular pathogen through the production of the igydfc. the vertebrate mhc is the most dynamic part of the genome, showing repeated cycles of 'birth-and-death' evolution (kelley et al., 2005) . polygeny and polymorphism are hallmarks of the region, with varying numbers of genes between species (and sometimes between individuals). usually it is not possible to identify orthologous genes between species. the mhc class i and class ii genes are the most polymorphic genes in the vertebrate genome. the mhc of the chicken has been referred to as the 'minimal mhc' (kaufman et al., 1995) , fulfilling all the requirements of an mhc region, with a limited set of genes. the b locus, or genomic mhc region, contains just 19 genes within 92 kb (kaufman et al., 1999) . mhc class i genes flank either side of the transporters for antigen processing (tap) genes. they are referred to as the major (bf2) and minor (bf1) mhc class i loci. similarly, the mhc class ii genes (blb1 and blb2) are located on either side of tapasin (tap-bp), and in close proximity to the chaperones involved in mhc class ii loading (dma and dmb). several genes are notably absent, including the proteasome genes lmp2 and lmp7, as well as genes encoding tnf alpha, and lymphotoxin alpha and beta. kaufman argues the mhc organization critically affects function because proximity of the genes involved in antigen transport and presentation, allows their encoded proteins to evolve to work together. indeed, tap1 and tap2 genes are also polymorphic, and using a peptide translocation assay, kaufman recently showed that tap determines specificity for the linked dominant mhc class i gene (walker et al., 2011) . the limitation to one mhc class i gene in chickens, impairs defense against viral pathogens, as ability to defend against a particular pathogen is completely dependent on whether or not it can load peptides from that pathogen. the best illustration of the consequences of limited mhc class i presentation is the ability of chickens of one genotype to defend against rous sarcoma virus, while other strains cannot (wallny et al., 2006) . the duck has a functionally similar mhc class i region, with 5 mhc class i genes encoded adjacent to the tap genes (moon et al., 2005) . ducks predominantly express one gene, which is adjacent to the tap2 gene, which is also polymorphic (mesa et al., 2004) . in ducks, as in chickens, this is expected to have functional consequences for the defense against viruses. viruses can easily change the one or two epitopes that can be presented by alleles encoded by one mhc class i gene, and thus escape the cytotoxic t cells focused on these epitopes. we have been unable to identify the tapasin (tapbp) gene in the duck mhc. tapasin bridges the gap between the tap transporter and empty mhc class i molecules, bringing them into close proximity to the translocation core where peptides are loaded (sadasivan et al., 1996) . in the absence of tapasin, empty mhc class i molecules weakly associate with tap leading to binding and cell surface expression of less than optimal peptides (grandea et al., 1995). tapasin has been identified adjacent to mhc class ii in other birds, including chicken (frangoulis et al., 1999) , quail (shiina et al., 1999) , turkey (chaves et al., 2009) , pheasant and zebra finch and black grouse . also, the tapasin gene is polymorphic in chicken, turkey and pheasant (sironi et al., 2006) . through analysis of the unannotated duck genome (preensemble) we can identify the location of the duck mhc class ii genes, but a search for tapasin within proximity is unsuccessful. using primers based on the chicken sequence, or conserved regions identified in aligned avian tapasin sequences, our attempts to amplify tapasin from mallards or a domestic duck by rt-pcr or from genomic dna fail to yield a tapasin product. it is possible that failure to amplify tapasin from ducks is due to sequence divergence of tapasin between avian species. the galliform tapasin proteins are about 90% identical , but the human and chicken tapasin share only 36% amino acid identity (frangoulis et al., 1999) . in low stringency southern blot analysis, distinctive bands could be detected in chicken, however the probe does not hybridize to duck genomic dna (petkau, 2012) . although tapasin seems to play an important role in antigen presentation, certain human mhc class i alleles can function in a tapasin-independent manner (park et al., 2003; lewis et al., 1998) . a single amino acid substitution in hla-b from asp116 to tyr116 allows the latter to function in a completely tapasin independent manner (sieker et al., 2007) . it also appears that tapasin influences the peptide repertoire presented to mhc class i, favoring certain peptides over others. in the absence of tapasin antigen presentation is altered, rather than deficient, and is still sufficient to induce in an immune response (boulanger et al., 2010) . similarly, the absence of immunoproteasomes in mice, results in a change in 50% of the loaded peptide repertoire (kincaid et al., 2012) . we could speculate that loss of tapasin was advantageous in ducks. we presume that ducks, like chickens, already had constraints on presentation of antigens by mhc class i due to potential co-evolution of the tap transporters and adjacent mhc class i genes. evidence for this is that both tap1 and tap2 are polymorphic in ducks, and they express one dominant mhc class i gene (mesa et al., 2004) . perhaps the loss of tapasin permits closer interaction of the tap transporter and the specific mhc class i molecule intended for loading. proteins encoded by genes co-evolving along a haplotype reach a best fit. tapasin as the bridge between tap and mhc class i molecules could serve to bring the incorrect mhc class i into proximity of the tap transporters from the other haplotype, unless it was also evolving to keep step. the genes encoding tnf-alpha (tnf-a), and lymphotoxin-alpha and beta (lta and ltb) are missing from the avian mhc. extensive efforts by pcr, est mining, and hybridization to identify tnf-a in chickens and ducks have failed. similarly, the two genes encoding lta and ltb are missing in chickens (kaiser, 2012) , and a scan of the genome sequence shows they are also absent in ducks. lymphotoxin-alpha knockout mice lack lymph nodes (de togni et al., 1994) , and similarly chickens have no lymph nodes. primitive lymph nodes were previously described in ducks (berens von rautenfeld and burdras, 1983) and immunoglobulin transcripts were analyzed in lymphatic tissues isolated from ducks (bando and higgins, 1996; magor et al., 1994a) . however, we question whether these tissues contain recognizable lymph nodes containing secondary lymphoid tissue, as we are unable to identify anything in the lymphatic tissue that resembles a lymph node, even tracking with injected india ink. we examined isolated lymphatic tissues in ducks for mrna expression of ccl19 and ccl21, the two chemokines which are responsible for recruiting naïve t cells and dendritic cells to lymph nodes. we showed expression of these chemokines was negligible in lymphatic tissues, and abundant in spleen and influenza-infected lung tissues (fleming-canepa et al., 2011) . clearly, the lymphatic tissues of ducks are not sites of recruitment of lymphocytes and dendritic cells as expected for secondary lymphoid tissues. thus, ducks and chickens, like all other non-mammalian vertebrates (hofmann et al., 2010) , lack true lymph nodes. through comparison of the immune arsenal of ducks and chickens we highlight several immune genes that are 'mia or missing in action' from the flight divisions. we refer to these genes as mia, because we cannot say with certainty that these genes are not present, at least until the sequencing and annotation of avian genomes is more complete. if these genes are truly absent, what emerges is a picture in which chickens, missing tlr8 and rig-i, have less ability to detect rna viruses and intracellular bacteria than ducks, which lack only tlr8. in addition, birds are missing components in the rig-i pathway, and interferon-responsive antiviral effectors. chickens have a large expanded family of leukocyte receptors, which are apparently missing in ducks, which include the igy fc receptors. notably, ducks make a truncated igy that is lacking the fc region as their most abundant serum antibody. by similarity to their human homologues, the lilr receptors, other members of the chir family are presumed to be involved in self/non-self recognition, and their expansion may somehow compensate for the deficit due to the minimal avian mhc. in contrast, ducks may have lost tapasin, and with it lost some of the constraint on antigen presentation due to co-evolving linked mhc class i and tap transporter genes. whether the host-pathogen arms race is cause or consequence of these gene losses, birds have had a long evolutionary relationship with rna viruses, including those causing zoonoses. regulation of t-cell immunity by leucocyte immunoglobulin-like receptors: innate immune receptors for self on antigenpresenting cells identification of a member of the interferon regulatory factor family that binds to the interferonstimulated response element and activates expression of interferon-induced genes gene duplication and fragmentation in the zebra finch major histocompatibility complex duck lymphoid organs: their contribution to the ontogeny of igm and igy identification of avian rig-i responsive genes during influenza infection antiviral pattern recognition receptors in the natural host of influenza, ducks (anas platyrhynchos) association of rig-i with innate immunity of ducks to influenza the counterbalance theory for evolution and function of paired receptors no enhanced influenza virus resistance of murine and avian cells expressing cloned duck mx protein trim16 acts as an e3 ubiquitin ligase and can heterodimerize with other trim family members the igh locus of the channel catfish, ictalurus punctatus, contains multiple constant region gene sequences: different genes encode heavy chains of membrane and secreted igd topography, ultrastructure and phagocytic capacity of avian lymph nodes the tetratricopeptide repeat: a structural motif mediating protein-protein interactions structure, chromosome localization, and regulation of expression of the interferon-regulated mouse ifi54/ifi56 gene family ring domains: master builders of molecular scaffolds? absence of tapasin alters immunodominance against a lymphocytic choriomeningitis virus polytope viral evasion and subversion of patternrecognition receptor signalling conserved and distinct aspects of the avian toll-like receptor (tlr) system: implications for transmission and control of bird-borne zoonoses the lilr family: modulators of innate and adaptive immune pathways in health and disease avian toll-like receptors chicken tlr21 acts as a functional homologue to mammalian tlr9 in the recognition of cpg oligodeoxynucleotides comparison of chimpanzee and human leukocyte ig-like receptor genes reveals framework and rapidly evolving genes chicken genomics resource: sequencing and annotation of 35,407 ests from single and multiple tissue cdna libraries and cap3 assembly of a chicken gene index phagosomal signaling by borrelia burgdorferi in human monocytes involves toll-like receptor (tlr) 2 and tlr8 cooperativity and tlr8-mediated induction of ifn-beta tlr8: the forgotten relative revindicated defining the turkey mhc: sequence and genes of the b locus evidence for an igd homologue on chicken lymphocytes new insights into the enigma of immunoglobulin d immunoglobulin d enhances immune surveillance by activating antimicrobial, proinflammatory and b cell-stimulating programs in basophils extensive diversification of igh subclass-encoding genes and igm subclass switching in crocodilians mda-5, but not rig-i, is a common target for paramyxovirus v proteins sequence and comparative analysis of the chicken genome provide unique perspectives on vertebrate evolution the avian toll-like receptor pathway-subtle differences amidst general conformity 0 -o methylation of the viral mrna cap evades host restriction by ifit family members genetic association and expression studies indicate a role of toll-like receptor 8 in pulmonary tuberculosis abnormal development of peripheral lymphoid organs in mice deficient in lymphotoxin tlr8 deficiency leads to autoimmunity in mice paired ig-like receptor homologs in birds and mammals share a common ancestor with mammalian fc receptors phylogeny.fr: robust phylogenetic analysis for the non-specialist identification of two igd+ b cell populations in channel catfish, ictalurus punctatus regulation of rlr-mediated innate immune signaling -it is all about keeping the balance evolutionary analysis and expression profiling of zebra finch immune genes regulators of innate immunity as novel targets for panviral therapeutics interferon-induced ifit2/isg54 protects mice from lethal vsv neuropathogenesis expression of duck ccl19 and ccl21 and ccr7 receptor in lymphoid and influenza-infected tissues identification of the tapasin gene in the chicken major histocompatibility complex influenza a virus ns1 targets the ubiquitin ligase trim25 to evade recognition by the host viral rna sensor rig-i trim25 ring-finger e3 ubiquitin ligase is essential for rig-i-mediated antiviral activity reul is a novel e3 ubiquitin ligase and stimulator of retinoicacid-inducible gene-i cutting edge: activation of murine tlr8 by a combination of imidazoquinoline immune response modifiers and polyt oligodeoxynucleotides dependence of peptide binding by mhc class i molecules on their interaction with tap cirf-3, a new member of the interferon regulatory factor (irf) family that is rapidly and transiently induced by dsrna small anti-viral compounds activate immune cells via the tlr7 myd88-dependent signaling pathway bile immunoglobulin of the duck (anas platyrhynchos). ii. antibody response in influenza a virus infections b-cells need a proper house, whereas t-cells are happy in a cave: the dependence of lymphocytes on secondary lymphoid tissues during evolution type i interferon [corrected] gene induction by the interferon regulatory factor family of transcription factors irfs: master regulators of signalling by toll-like receptors and cytosolic pattern-recognition receptors irf-7 is the master regulator of type-i interferon-dependent immune responses 0 -triphosphate rna is the ligand for rig-i quantitative expression of toll-like receptor 1-10 mrna in cellular subsets of human peripheral blood mononuclear cells and sensitivity to cpg oligodeoxynucleotides mavs forms functional prion-like aggregates to activate and propagate antiviral innate immune response global characterization of interferon regulatory factor (irf) genes in vertebrates: glimpse of the diversification in evolution re-evaluation of the chicken mip family of chemokines and their receptors suggests that ccl5 is the prototypic mip family chemokine, and that different species have developed different repertoires of both the cc chemokines and their receptors tlr3 increases disease morbidity and mortality from vaccinia infection ubiquitin-induced oligomerization of the rna sensors rig-i and mda5 activates antiviral immune response advances in avian immunology-prospects for disease control: a review the long view: a bright past, a brighter future? forty years of chicken immunology pre-and post-genome a genomic analysis of chicken cytokines and chemokines identifying innate immune pathways of the chicken may lead to new antiviral therapies characterization of chicken mda5 activity: regulation of ifn-beta in the absence of rig-i functionality cell type-specific involvement of rig-i in antiviral response antigen processing and presentation: evolution from a bird's eye view the chicken b locus is a minimal essential major histocompatibility complex a ''minimal essential mhc'' and an ''unrecognized mhc'': two extremes in selection for polymorphism chicken mhc molecules, disease resistance and the evolutionary origin of birds chicken tlr21 is an innate cpg dna receptor distinct from mammalian tlr9 comparative genomics of major histocompatibility complexes negative feedback regulation of rig-i-mediated antiviral signaling by interferon-induced isg15 conjugation mice completely lacking immunoproteasomes show major changes in antigen presentation aberrant innate immune response in lethal infection of macaques with the 1918 influenza virus a structure-based model of rig-i activation structural basis for the activation of innate immune pattern-recognition receptor rig-i by viral rna tetratrico peptide repeat interactions: to tpr or not to tpr? the leukocyte receptor complex in chicken is characterized by massive expansion and diversification of immunoglobulin-like loci detrimental contribution of the toll-like receptor (tlr)3 to influenza a virus-induced acute pneumonia interferonstimulated transcription: isolation of an inducible gene and identification of its regulatory region hla-a⁄0201 presents tapdependent peptide epitopes to cytotoxic t lymphocytes in the absence of tapasin the interaction between interferon-induced protein with tetratricopeptide repeats-1 and eukaryotic elongation factor-1a isg56 is a negative-feedback regulator of virus-triggered signaling and cellular antiviral response virus-dependent phosphorylation of the irf-3 transcription factor regulates nuclear translocation, transactivation potential, and proteasome-mediated degradation chicken cells sense influenza a virus infection through mda5 and cardif signaling involving lgp2 retinoic acid inducible gene-i, more than a virus sensor the mitochondrial targeting chaperone 14-3-3epsilon regulates a rig-i translocon that mediates membrane association and innate antiviral immunity gene expression networks underlying retinoic acid-induced differentiation of acute promyelocytic leukemia cells distinct rig-i and mda5 signaling by rna viruses in innate immunity immune signaling by rig-i-like receptors the immunoglobulin heavy chain locus of the duck. genomic organization and expression of d, j, and c region genes structural insights into rna recognition by rig-i avian betadefensin nomenclature: a community proposed update genomics of antiviral defenses in the duck, a natural host of influenza and hepatitis b viruses emerging role of ubiquitination in antiviral rig-i signaling. microbiol immunoglobulin genetics and antibody responses to influenza in ducks cdna sequence and organization of the immunoglobulin light chain gene of the duck, anas platyrhynchos one gene encodes the heavy chains for three different forms of igy in the duck opposite orientation of the alpha-and upsilon-chain constant region genes in the immunoglobulin heavy chain locus of the duck structural relationship between the two igy of the duck, anas platyrhynchos: molecular genetic evidence the p59 oligoadenylate synthetase-like protein possesses antiviral activity that requires the c-terminal ubiquitin-like domain identification and validation of ifit1 as an important innate immune bottleneck evolution of nk receptors: a single ly49 and multiple kir genes in the cow the dominant mhc class i gene is adjacent to the polymorphic tap2 gene in the duck, anas platyrhynchos tlr-dependent t cell activation in autoimmunity the mhc of the duck (anas platyrhynchos) contains five differentially expressed class i genes paramyxovirus v proteins disrupt the fold of the rna sensor mda5 to inhibit antiviral signaling inhibitory receptor paired ig-like receptor b is exploited by staphylococcus aureus for virulence origin and evolution of the chicken leukocyte receptor complex trim family proteins: retroviral restriction and antiviral defence riplet/rnf135, a ring finger protein, ubiquitinates rig-i to promote interferon-beta induction during the early phase of viral infection ubiquitin-mediated modulation of the cytoplasmic viral rna sensor rig-i the ubiquitin ligase riplet is essential for rig-i-dependent innate immune responses to rna virus infection variable nk cell receptors and their mhc class i ligands in immunity, reproduction and human evolution a single polymorphic residue within the peptide-binding cleft of mhc class i molecules determines spectrum of tapasin dependence allelic diversity of tap in wild mallards identification and characterization of a functional, alternatively spliced toll-like receptor 7 (tlr7) and genomic disruption of tlr8 in chickens reis e sousa, c, 2006. rig-i-mediated antiviral responses to single-stranded rna bearing 5 0 -phosphates chicken igy binds its receptor at the ch3/ch4 interface similarly as the human iga: fc alpha ri interaction species-specific inhibition of rig-i ubiquitination and ifn induction by the influenza a virus ns1 protein expression analysis of turkey (meleagris gallopavo) toll-like receptors and molecular characterization of avian specific tlr15 rig-i like receptors and their signaling crosstalk in the regulation of antiviral immunity complete sequence of a chicken lambda light chain immunoglobulin derived from the nucleotide sequence of its mrna roles for calreticulin and a novel glycoprotein, tapasin, in the interaction of mhc class i molecules with tap the inhibitory action of p56 on select functions of e1 mediates interferon's effect on human papillomavirus dna replication recognition of 5 0 triphosphate by rig-i helicase requires short blunt double-stranded rna as contained in panhandle of negative-strand virus identification of alpha interferon-induced genes associated with antiviral activity in daudi cells and characterization of ifit3 as a novel antiviral gene the interferon system of non-mammalian vertebrates comparative immunology of agricultural birds mx is dispensable for interferon-mediated resistance of chicken cells against influenza a virus positive regulation of interferon regulatory factor 3 activation by herc5 via isg15 modification gene organization of the quail major histocompatibility complex (mhccoja) class i gene region comparative molecular dynamics analysis of tapasin-dependent and -independent mhc class i alleles single nucleotide polymorphism discovery in the avian tapasin gene emerging role of isg15 in antiviral immunity goose rig-i functions in innate immunity against newcastle disease virus infections the role of iodine in thyroid autoimmunity: from chickens to humans: a review natural killer cell receptors in the horse: evidence for the existence of multiple transcribed ly49 genes nonself rna-sensing mechanism of rig-i helicase and activation of antiviral immune responses mycobacteriosis in birds identification of host genes linked with the survivability of chickens infected with recombinant viruses possessing h5n1 surface antigens from a highly pathogenic avian influenza virus avian influenza rapidly induces antiviral genes in duck lung and intestine complexity of expressed chir genes the chicken leukocyte receptor cluster the chicken leukocyte receptor complex: a highly diverse multigene family encoding at least six structurally distinct receptor types the chicken leukocyte receptor complex encodes a primordial, activating, high-affinity igy fc receptor the chicken leukocyte receptor complex encodes a family of different affinity fcy receptors the dominantly expressed class i molecule of the chicken mhc is explained by coevolution with the polymorphic peptide transporter (tap) genes peptide motifs of the single dominantly expressed class i molecule explain the striking mhc-determined response to rous sarcoma virus in chickens sequencing of the core mhc region of black grouse (tetrao tetrix) and comparative genomics of the galliform mhc mitochondrion: an emerging platform for host antiviral signalling tolllike receptor 3 mediates west nile virus entry into the brain causing lethal encephalitis rig-i à/à mice develop colitis associated with downregulation of g alpha i2 igy: clues to the origins of modern antibodies the genome of a songbird molecular cloning, full-length sequence and preliminary characterization of a 56-kda protein induced by human interferons expression of igm, igd, and igy in a reptile, anolis carolinensis a review: the obese strain (os) of chickens: an animal model with spontaneous autoimmune thyroiditis a novel chimeric ig heavy chain from a teleost fish shares similarities to igd crystal structure of isg54 reveals a novel rna binding structure and potential functional mechanisms isolation of a 97-kb minimal essential mhc b locus from a new reverse-4d bac library of the golden pheasant the rna helicase rig-i has an essential function in double-stranded rna-induced innate antiviral responses nucleic acid-sensing toll-like receptors are essential for the control of endogenous retrovirus viremia and erv-induced tumors reconstitution of the rig-i pathway reveals a signaling role of unanchored polyubiquitin chains in innate immunity interferon-stimulated gene 15 and the protein isgylation system human isg15 conjugation targets both ifn-induced and constitutively expressed proteins functioning in diverse cellular pathways isg15 conjugation system targets the viral ns1 protein in influenza a virus-infected cells identification of igf, a hinge-region-containing ig class, and igd in xenopus tropicalis mapping of the chicken immunoglobulin heavy-chain constant region gene locus reveals an inverted alpha gene upstream of a condensed upsilon gene interferon induced ifit family genes in host antiviral defense origin and evolution of the rig-i like rna helicase gene family the idea for this review came from a conversation with martin flajnik in the beer tent at the comparative immunology workshop, held in waterloo, ontario in 2010. key: cord-346916-jj4l9ydl authors: girardi, erika; pfeffer, sebastien; baumert, thomas f.; majzoub, karim title: roadblocks and fast tracks: how rna binding proteins affect the viral rna journey in the cell date: 2020-08-23 journal: semin cell dev biol doi: 10.1016/j.semcdb.2020.08.006 sha: doc_id: 346916 cord_uid: jj4l9ydl as obligate intracellular parasites with limited coding capacity, rna viruses rely on host cells to complete their multiplication cycle. viral rnas (vrnas) are central to infection. they carry all the necessary information for a virus to synthesize its proteins, replicate and spread and could also play essential non-coding roles. regardless of its origin or tropism, vrna has by definition evolved in the presence of host rna binding proteins (rbps), which resulted in intricate and complicated interactions with these factors. while on one hand some host rbps recognize vrna as non-self and mobilize host antiviral defenses, vrna must also co-opt other host rbps to promote viral infection. focusing on pathogenic rna viruses, we will review important scenarios of rbp-vrna interactions during which host rbps recognize, modify or degrade vrnas. we will then focus on how vrna hijacks the largest ribonucleoprotein complex (rnp) in the cell, the ribosome, to selectively promote the synthesis of its proteins. we will finally reflect on how novel technologies are helping in deepening our understanding of vrna-host rbps interactions, which can be ultimately leveraged to combat everlasting viral threats. viruses are obligate intracellular parasites that strictly rely on host cells to translate and amplify their genomes [1] [2] [3] . viral rnas (vrnas) play a central role during infection as they bear all the necessary information for a virus to express its proteins, replicate and spread. unlike dna viruses where the messenger rna must be first transcribed for the synthesis of viral proteins, rna viruses use their rna molecules both for protein synthesis and as replication templates. viruses encode an array of vrna-binding proteins (vrbps), essential for different steps in the viral lifecycle, comprising translation, synthesis, packaging of the viral genome and cell-to-cell spread. importantly, vrna is never really naked in the cellular milieu. apart from its interactions with vrbps, it has evolved to co-opt specific sets of host encoded rbps. this is illustrated by the variety of described strategies that vrnas use to hijack key cellular machineries and evade the host's defense arsenal. unlike vrbps that promote infection, host encoded rbps can either have pro-or antiviral functions. indeed, a large body of work produced during the last decades describe the involvement of host encoded rbps in almost every known stage of vrna lifecycle. certain host rbps, ordinarily functioning in cellular tasks, are repurposed by vrnas to guide them through the viral lifecycle, including genome translation, synthesis, modification, localization and packaging. on the other hand, many host rbps have evolved dedicated antiviral functions, ranging from the recognition of the invading vrna to the restriction of viral replication. the ability of vrna to either subvert or get antagonized by cellular rbps determine the outcome of a viral infection. indeed, vrna-rbps interactions could dictate the permissiveness of certain cell types to infection, host range, tissue tropism, viral evolution, efficiency of viral replication, pathology of infection and immune clearance [4] . vrnas interact with numerous and very diverse rbps during infection. this is illustrated by the rich literature on the subject that has yielded many discoveries in the last decades. for example, the study of intimate vrna-rbp interactions shaped our understanding of how rna interference (rnai) functions as the main antiviral defense system in plants and invertebrates, and how bacteria defend themselves against invading phages through the crispr system [5, 6] . herein, we review the mechanisms at play when rna viruses infect their animal hosts, taking a vrna-centric view. focusing on viruses that are important human pathogens, we will describe how cellular rbps act on early steps after viral entry, driving mechanisms of vrna recognition, degradation and modification to limit or to promote viral replication and spread. we will further describe a number of crucial mechanisms during which vrna hijacks the ribosome to preferentially translate the viral program. finally, we will reflect on how new emerging techniques are allowing to get a better grasp on the diversity of host rbps-vrna interactions. elucidating the mechanisms by which rbp-vrna interactions influence viral infection, is advancing our understanding of cell biology by revealing unforeseen biological knowledge and may also provide new targets for host-directed antiviral therapies [7] . indeed, recent corona, ebola and zika virus outbreaks remind us that new innovative antiviral approaches are clearly needed, particularly for emerging rna viruses with important epidemic potential [8] [9] [10] . despite the fact that the molecular composition of rna is universal throughout life kingdoms, host immune pathways are able to differentiate and recognize non-self nucleic acids based on specific features, structures and modifications. moreover, despite the molecular mimicry set by rna viruses to resemble cellular mrnas and escape host recognition, the viral nucleic acid still needs to embark on a long journey through a hostile cell environment and must overcome the obstacles put in place by the host antiviral system in order to be translated and replicated. cellular innate immunity comprises a rather sophisticated set of host factors that discriminate molecular alterations with a high specificity and restrict rna virus infection through direct or indirect activity on the vrna. in vertebrate animals, a variety of cellular sensors have evolved to detect foreign rna sensing to activate the innate immune response, which involves transcriptional and post-transcriptional activation of genes of interferon-stimulated genes (isgs). pattern recognition receptors (prrs) are host-encoded proteins that sense molecular features of vrna molecules which are generally absent in the majority of cellular rnas. multiple molecular receptors are involved in the recognition of these so-called pathogen-associated molecular patterns (pamps) [11, 12] . a major molecular signature and weakness of all rna viruses is the long double stranded (ds) rna molecules that are generated during replication. the sensing of dsrna is believed to be sequence-independent and rather depends on the distinct molecular features of the viral dsrna structure mostly absent in uninfected host transcriptome [13] . among the first cellular proteins that detect the invading virus are the toll-like receptors (tlrs) (fig. 1) , which are transmembrane glycoproteins that are constitutively expressed or pathogen-induced, located on the plasma membrane or intracellular endosomes which are preferential entry routes for vrnas (reviewed in [14, 15] ). tlrs share strong similarities in their structures and organization. their n-terminal region recognizes ligands thanks to its extracellular or luminal domains and is connected to the c-terminal cytoplasmic domains (ctds) by a single membrane-spanning domain. among the 10 tlrs identified in humans so far, tlr7 and tlr8 recognize ssrnas and tlr3 recognizes dsrnas [12, 16, 17] . in tlr3, the n-terminal region recognizes the ligand, dsrna, in the lumen of endosomes whereas the c-terminal signalling domain resides in the cytoplasm, and upon ligand recognition, tlr3 initiates the signalling process that culminates in transcriptional induction of specific immune genes. tlr3 was believed to be a major sentinel against viral infections since it responds to a common by-product of viral replication ( table 1) . for instance, encephalomyocarditis virus (emcv) infection leads to a tlr3-dependent innate stress response, which is involved in mediating protection against virus-induced myocardial injury [18] . similarly, tlr3 recognizes dengue virus (denv), limiting its replication [19] , while zika virus (zikv)-mediated tlr3 activation was shown to deplete neural progenitor cells [20] . indeed, the protective versus pathogenic role of tlr3 in viral pathogenesis is debated [21] . another group of molecular sentinels that senses foreign rna is represented by the ifn-induced intracellular cytosolic receptors, named retinoid acid-inducible gene i (rig-i)-like receptors (rlrs), which comprise dexd/h-box helicases such as rig-i (or ddx58), melanoma differentiation-associated protein 5 (mda5 or ifih1) and laboratory of genetics and physiology 2 (lgp2 or dhx58) [22] (fig. 1 , table 1 ). all rlrs possess a central dexd/h-box rna helicase domain and a ctd which are necessary for detection of foreign rnas. rig-i and mda5 have as well two n-terminal caspase activation and recruitment domains (cards), which mediate downstream signal transduction, while lgp2 lacks them. although rig-i and mda5 share similar structural composition, their ability to detect vrna is not redundant but specific [22] ( table 1) . rig-i monitors the 5 ′ ends of rna molecules via its ctd and helicase domain in order to initiate cytokine production in response to a wide range of viruses. triphosphate (ppp) or diphosphate (pp) at the uncapped 5 ′ -end of rna molecules with partial base-pairing to a complementary strand of rna molecules are recognized and required for vrna is sensed by different families of receptors (e.g. rlrs, tlrs, pkr). vrna is edited by adars and methylated by host mettl proteins (m 6 a) or viral methyl transferases (2 ′ -o-me). vrna can also be degraded by 5 ′ -3 ′ or 3 ′ -5 ′ exonucleases (e.g. xrn1 and rna exosome respectively) or by endonucleases (e.g. dicer). rig-i activation [23] [24] [25] [26] . the typical structure of the panhandle of negative-strand viral genomes confers full rig-i ligand activity. rig-i recognizes many single stranded negative rna virus families, such as paramyxoviridae, rhabdoviridae, orthomyxoviridae, bunyaviridae and filoviridae [27] [28] [29] . rig-i also detects some positive-sense rna viruses such as dengue and zika viruses of the flaviviridae family, by recognition of nascent viral genomes containing 5 ′ -ppp groups prior to capping [30] . mda5 senses both rna length and secondary structure by recognizing uninterrupted rna duplexes longer than a few hundred base pairs [31] and forming filaments around dsrna to initiate signalling [32] . mda5 is required for immunity against several classes of viruses, including single-stranded positive rna viruses such as picornaviruses [28, 33, 34] , flaviviruses [35] and coronaviruses [36] (table 1) . the third rlr family member lgp2 lacks the amino-terminal tandem cards and thus lacks signal-transducing activity. several studies indicate a disparate regulatory role for lgp2 in either triggering or dampening the innate immune signalling pathways following rna virus infection [37] . on one hand, lgp2 can function as a feedback inhibitor of rig-i by sequestrating double-stranded but not single-stranded rnas. lgp2 negatively regulates sendai virus (sv)-induced irf-3 or nf-κb signaling acting as a natural inhibitor of antiviral responses, and as a repressor of rig-i signaling [38] . however, lgp2 has a positive effect on mda5-mediated antiviral response. lgp2-deficient mice exhibited a defect in type i ifn production in response to infection by the encephalomyocarditis virus, the replication of which activates mda5-dependent innate immune response [39] . moreover, a recent study indicated that lgp2 can inhibit antiviral rnai in mammals by competing with and preventing dicer-mediated processing of dsrnas into sirnas both in vitro and in cells [40] . interestingly, other dexd/h-box helicases, such as ddx17, ddx21, ddx6 or ddx56, are also emerging as important sentinels that influence viral sensing in multiple ways. these helicases have been shown to bind vrnas, acting either as antiviral effectors or contributing to viral replication [41] . the presence and replication of viral nucleic acids in vertebrate cells can also induce the activation of other antiviral enzymes that are both sensors and effectors ( table 1) . these also recognize viral signatures; however, their main function is not necessarily to only induce a transcriptional immune response, but rather to directly attack vrna by degrading it or inhibiting its translation. for this reason, these are not usually referred to as receptors [12, 42, 43] . among them, double-stranded rna (dsrna) activated protein kinase r (pkr; also known as eif2ak2) or adenosine deaminase and 2 ′ -5 ′ -oligoadenylate synthetase class of enzymes (oass), can bind dsrna in a sequence independent manner, recognizing the structure as a prr (fig. 1 first, pkr is a serine-threonine kinase constitutively expressed in all tissues at a basal level as an inactive monomer. viral dsrna binding to the two n-terminal rna binding motifs induces a conformational change that allows atp binding to the c-terminal kinase domain. pkr can be activated by dsrna from reoviruses [44] but can also recognize dsrna from replication intermediates of + rna viruses [45] ] (table 1) . moreover, it has been shown that highly structured rna elements from retroviruses such as human immunodeficiency virus (hiv) transactivation-response region (tar) rna hairpins [46] or dsrna formed by the antiparallel mrna transcripts of some dna viruses can also activate pkr [47] . upon dsrna binding pkr dimerizes and autophosphorylates threonine residues in its activation domains, thereby stabilizing the dimers and increasing kinase activity [48] . this in turn leads to a series of events that inhibit viral translation and replication, as will be detailed later on in this review. interestingly, pkr is an interferon stimulated gene (isg) itself and its transcription can be up-regulated upon type i interferon production by virus-infected cells, stimulating its accumulation in neighboring cells. pkr activation following infection of interferon-primed neighboring cells inhibits global protein synthesis and reduces viral spread, making this activation a key player in the innate response to viruses [49] . therefore, pkr can be considered a major molecular sentinel of antiviral innate immunity, recognizing a diverse set of stress-inducing stimuli and mounting an appropriate (fig. 3) . the second known class of dsrna sensors and effectors is represented by the 2 ′ ,5 ′ -oligoadenylate synthetases (oases) (fig. 1, table 1 ), which also act as prrs for the detection of many rna viruses [54] . the four human oas genes, oas1, oas2, oas3 and oasl (oas-like), are constitutively expressed at low levels in the cytoplasm and are activated in response to viral dsrna. as an example, oas1 protein accumulates in the cytoplasm as an inactive monomer [55] and following activation by viral dsrna, it forms a tetramer that synthesizes 2 ′ ,5 ′ -oligoadenylates (2-5 as). these 2-5as activate the ribonuclease l (rnasel) to cleave cellular and viral rnas and supress viral replication (see below). overall, the functional redundancy, provided by multiple host proteins that recognize different features of vrnas, is a successful strategy for the host to fight and contain the infection [56] . similar to pkr, the oas-rnasel axis can be antagonized by some rna viruses. for example, certain corona and rotaviruses encode proteins belonging to the phosphodiesterase family, able to cleave 2-5 a molecules, thereby preventing activation of rnasel [57] . in concert with innate immune sensors linked to ifn activation, mammalian cells can also depend on intrinsic factors that participate in the direct antiviral response to non-self nucleic acids. accumulating evidence suggests that the cellular rna surveillance pathway restricts viral infection by modulating vrna stability [58] . the presence of specific molecular features on the vrna and the binding of trans-acting antiviral host rbps can drive vrna through the host mrna quality control pathways (fig. 1 ). for instance, in mammalian cells, the non-sense mediated decay (nmd) pathway has recently been shown to function in the restriction of positive-sense (+) rna viruses such as alphaviruses in mammalian cells [59] . in fact, alphavirus rna genome resembles cellular mrnas and is translated into non-structural viral proteins (nsps) but has an untranslated 3 ′ utr, much longer than typical cellular mrnas, which is detected and degraded by the nmd pathway [59] . the 5 ′ to 3 ′ rna decay machinery can also directly target vrna for degradation in the cytoplasm. in this pathway, the monomethyl guanosine (m7g) cap structure is cleaved from mrnas by decapping enzymes exposing the 5 ′ monophosphorylated product to progressive 5 ′ →3 ′ exoribonucleolytic degradation by xrn1. the hepatitis c virus (hcv) genomic rna, which lacks a 5 ′ cap and is therefore susceptible to xrn-1 mediated decay, uses an unusual mechanism to overcome this pathway [60] . binding of the liver-abundant mir-122 to the 5 ′ utr of hcv in association with argonaute-2 (ago2) protein stabilizes the vrna and slows xrn1 decay of the viral genome in infected cells [60, 61] ( fig. 1) . other flaviviridae are able to take advantage of their susceptibility to xrn-1 mediated vrna decay. dengue, west nile, yellow fever or zika vrnas contain a highly structured 300-700 bp-long non-coding rna in their 3 ′ end. an incomplete 5 ′ -3 ′ degradation of the viral genome by the cellular xrn1 exonuclease produces the so called small flaviviral rna (sfrna). during rna degradation, xrn1 is stalled at the 3 ′ utr extremity of flaviviral vrnas, more precisely at stem-loops/pseudoknots, causing the accumulation of different species of sfrna. although, sfrna does not seem to have a direct role in viral replication per se, it contributes to viral pathogenicity in vivo partly by interfering or evading innate immune responses, by sequestering cellular rbps and inhibiting their endogenous functions [62] [63] [64] [65] [66] [67] [68] [69] [70] . interestingly, sfrna seems to function in a species-specific manner. while it facilitates replication and pathogenesis by inhibiting ifn-signalling in mammalian hosts, a recent report shows that zikv sfrna possess an anti-apoptotic function that helps the virus to disseminate and reach saliva in mosquito hosts, thus promoting productive infection and transmission [71] . it has also been proposed that sfrna could be a suppressor of rnai [72] . in the 3 ′ decay pathway, deadenylated mrna degradation is mediated by a cytoplasmic multisubunit 3 ′ -to-5 ′ exoribonuclease complex, namely the rna exosome ( fig. 1 ). this degradation pathway is emerging as a crucial effector for recognition and degradation of specific vrnas [73] . the exosome interacts with a variety of rbps, some of which are exported to the cytoplasm in response to viral infection and serve as adaptors to specifically target particular rnas. several antiviral rbps have been found to interact with the exosome, suggesting that their mechanism of action may involve exosomal degradation. for instance, the conserved rna helicase ddx17 translocates from the nucleus to the cytoplasm upon rift valley fever virus (rvfv) infection, binds a specific stem-loop structure in the vrna and recruits the exosome to degrade the vrna [74] . another example, is the zinc-finger antiviral protein (zap), which binds vrnas containing a zap response element (zre) and induces rna degradation via interaction of its n-terminal domain with host decay machinery mediated [75] (fig. 1 ). zap is constitutively expressed in many cell types and it is induced upon pathogen-sensing and in response to interferon. zap exhibits broad antiviral activity against a broad spectrum of viruses, such as alphaviruses, enteroviruses, filoviruses, influenza and porcine reproductive and respiratory virus [42] but also retroviruses [76] . for instance, zap binds regions of hiv-1 viral rna containing cpgs and induce degradation of viral rnas via interaction with the essential cellular cofactor khnyn [77] . moreover, zap antiviral activity has been linked to its subcellular localization in stress granules, which are induced upon infection by sindbis virus (sinv) for example [78] . interestingly, trim25, an e3 ubiquitin ligase with no previous known role in rna binding is emerging as a regulator of many antiviral factors, including zap [79, 80] . other ribonucleases can exhibit broad-spectrum antiviral effects through viral rna binding and degradation. the ribonuclease mcpip1 is involved in the suppression of japanese encephalitis virus (jev) and denv, but also other positive-sense rna viruses, such as sinv, hcv and emcv, and negative-sense rna virus, such as influenza virus [81, 82] . finally, two conserved endoribonucleases have been shown to play a major antiviral role in different animal species, namely rnase l and dicer ( fig. 1 , table 1 ). rnase l is composed of three major domains: an n-terminal regulatory ankyrin repeat domain (ard), a protein kinase (pk)-like domain, and a c-terminal ribonuclease domain (rnase). in the absence of 2-5 a, the ard represses the rnase domain. binding of 2-5as, generated by oases, to the ard alters the conformation of rnase l, thereby exposing protein-protein interaction domains and releasing the rnase domain from internal inhibitory sequence, triggering its dimerization and activation. rnase l cleaves single-stranded rnas at the uu/ua dinucleotides, which are relatively rare in the coding regions of cellular mrnas compared to viral rnas [55] . in the case of hcv infection, rnase l degradation of vrna produces small rna cleavage products with 3 ′ -monophosphate (3 ′ -p) and 5 ′ -hydroxyl (5 ′ -oh) at their termini, which fold back into potent pamps recognized by rig-i, amplifying the innate immune responses [83] . interestingly, some hcv genotypes escape rnasel cleavage by accumulating silent mutations at ua and uu dinucleotides, which are preferentially targeted by the nuclease [84] . the rnase iii endoribonuclease dicer is active against different rna viruses in both vertebrate and invertebrate animals (table 1 ) [6, [85] [86] [87] [88] [89] . rna interference (rnai) is a primordial form of antiviral immunity mediated by small rnas and is the major immune defence in insects, plants and nematodes species [12, 90] . in the antiviral rnai pathway, dicer is able to bind and cleave viral dsrnas into ~21 nt-long small interfering rnas (sirnas). the sirnas are subsequently loaded into the argonaute-containing rna induced silencing complex (risc) and program risc to target vrna in a sequence-specific manner, inducing vrna degradation or translational arrest. although evidence of dicer activity against certain rna viruses have been reported in undifferentiated mouse stem cells [88] , and in some other specific context [91, 92] , the physiological antiviral role of rnai and its interplay with the interferon response in vertebrate animals is still an area of active debate [93] [94] [95] . in recent years, it has been revealed that the rna code is much more complex than the primary rna sequence. in particular, covalent rna base and sugar modifications recently emerged as an additional layer in the regulation of gene expression. such modifications can change the rna structure and its interaction with other rnas or proteins, thereby regulating important steps in rna metabolism including splicing, rna stability and translation. these rna modifications are also utilized by host cells as a way to distinguish self-rnas from vrnas [96] [97] [98] . it is therefore not surprising that viruses evolved strategies to modify their own genomes and transcripts or hijack dedicated cellular enzymes responsible for these modifications. here we briefly describe three rna modifications known to be present on vrnas (adenosine-to-inosine editing, n 6 -methyladenosine (m 6 a) and 2 ′ -o-methylation) [88] [89] [90] . we expect that other rna modifications may have the ability to also influence vrna stability, translation and immune potential. deamination of adenosines to inosines (a-to-i editing) is a covalent rna modification occurring on dsrna structures, by the adenosine deaminase acting on rna (adar) enzymes (fig. 1, table 1 ). among the three mammalian adars, adar1 is expressed in its constitutive nuclear form adar1p110 and the ifn-inducible cytoplasmic one, adar1p150. both enzymes consist of a c-terminal deaminase domain, three consecutive dsrna binding motifs and one or two copies of n-terminal z-dna binding domains [99] . the consequence of the rna modification by adar1 is two-fold: first, if editing takes place in coding sequences, inosines will be misinterpreted by the translation machinery and will be read as guanosines. second, a-to-i conversion have the capacity to destabilize dsrna structures by changing the watson-crick base pairing and impairing recognition by cytoplasmic antiviral receptors, such as rlrs, pkr and oas1. for these reasons, adar proteins play a pivotal role in masking self rnas against innate immune detection by selective labelling [100] . given the opposite effects of adar1 and antiviral sensors in the control of innate immune responses to endogenous duplex rna, the ato-i editing contribution in antiviral defence remains unclear. a bias toward a-to-g mutations dependent on adar has been described for a wide range of viruses [101] . this could potentially lead to the synthesis of dysfunctional viral proteins and destabilize key rna structures such as dsrna replication intermediates. although several studies have reported high a-to-i editing levels indicative of a potential mutagenic and antiviral effect, adar activity seems to be rather proviral due to its dampening effects on the innate immune system [101] . one clear example where adar plays a proviral role is the case of hepatitis d virus (hdv). hdv contains only one orf responsible of expressing the hdv delta antigen (hdag), essential for hdv replication and spread. two forms of hdag are expressed: a short form s-hdag and one that is 19 amino acid longer (l-hdag). hdv needs both to be expressed in order to replicate and spread and this depends on adar1. in fact, adar1 recognizes a specific structure on the rod-like rna of hdv antigenomic rna and converts adenosine to inosine. replication of this mutated rna leads to the substitution of guanosine in its genome, specifically leading to the replacement of an amber codon (uag) for the small delta antigen by a tryptophan codon (ugg) which produces a protein that is 19 amino acid longer. the synthesis of this longer protein is crucial for hdv spread, therefore adar1 is a proviral host rbp essential for hdv infection [102] . in mammalian cells, n 6 -methyladenosine (m 6 a) is the most prevalent and dynamically modulated internal mrna modification. such a modification is reversible via the activity of two key enzyme classes: methyltransferases or "writers" (e.g. methyltransferase like 3 (mettl3) and 14 (mettl14)) and demethylases or "erasers" (e.g. (fto) and alkbh5) (fig. 1, table 1 ). the m 6 a writers and erasers can alter the cellular rna fate by destabilizing local rna structures or by affecting protein binding to modulate host rna stability or translation efficiency. in particular, the m 6 a readers such as yth domain-containing family proteins (ythdf1-ythdf3) play crucial roles [103] . recent reports show that m 6 a could be a regulator of the immune system, playing a role in the discrimination between endogenous and exogenous rnas [104] . indeed, several studies have identified m 6 a modifications on a variety of vrnas and this enhances global viral gene expression by increasing rna stability and translation [96, 105] . interestingly, mapping of m 6 a on the rna genomes of flaviviridae, including dengue, zika, yellow fever, and west nile virus, identified conserved regions modified by m [6] a, suggesting that this modification is a conserved regulatory mark across flaviviridae genomes [106] . another case where m 6 a appears to function in innate immune discrimination between self and non-self rnas is given by the human metapneumovirus (hmpv) [107] . m 6 a methylation of vrna promotes hmpv replication and gene expression. in the absence of m 6 a residues on the vrna, viral infection is compromised due to a rig-i-dependent increase of type i interferon [107, 108] . this notion is reinforced by the recent report of the effect of the loss of the m 6 a writer mettl3 in the mouse hematopoietic system. indeed, mettl3 knock-out results in the upregulation of mda5-rig-i, pkr and oas-rnase l pathways due to the aberrant accumulation of endogenous dsrna [109] . therefore, m6a modification appears to prevent formation of certain dsrna, so it would be interesting in future work to check whether this has implications during viral infection. the 2 ′ o-methylation (2 ′ o-me) modification is a highly abundant chemical modification found in the rna of virtually all eukaryotic species, whereby a methyl group is added to the 2 ′ -oh of the ribose ring by cellular 2 ′ -o-methyltransferases (2 ′ o-mtase) (fig. 1 ). 2 ′ o-me is enriched in the first and second transcribed nucleotides next to the n7 methylated 5 ′ cap structure of higher eukaryote mrnas. while n-7methylation is important for stability and translation of the mrna, the 2 ′ -o-methylation has been shown to antagonize the innate immune response by allowing endogenous mrnas to escape recognition by immune sensors [97, 110] . notably, 2 ′ -o-me of the first (cap 1) and often the second (cap 2) nucleotide abolishes interaction of host mrnas with rig-i and mda5 [97] . the functional importance of 5 ′ -structures and modifications in mrnas is supported by the fact that many cytoplasmic rna viruses (including picornaviruses, flaviviruses and coronaviruses) have evolved alternative 5 ′ elements. these include small viral proteins linked to the 5 ′ end of genomic rna, or encode functions associated with the formation of a 5 ′ cap that are homologous to those found in eukaryotic cells. many viruses have evolved mechanisms to generate their own cap structures with methyl ribose in 2 ′ -o position in addition to the one at the n7 position of the capped guanine to evade innate immune recognition. for instance, the coronavirus 2 ′ -o-methyltransferase activity associated with the highly conserved viral nonstructural protein 16 (nsp16) is necessary to inhibit mda5 recognition of vrna and activation of ifn pathway [111] . 2 ′ -o methylation of the 5 ′ cap of vrna also subverts mammalian antiviral responses by evading restriction by interferon-induced proteins with tetratricopeptide repeats (ifits). ifits are cytoplasmic antiviral proteins which differ in specificity against rna structures. among them, ifit1 binds 5 ′ ppp rnas [112] and non-2 ′ -o methylated viral capped transcripts [113, 114] to inhibit their translation by out-competing the cellular translation initiation apparatus [115] (table 1) . members of the flaviviruses and coronaviruses are able to evade ifit1 sensing by adding a 2 ′ o-methyl cap structure to their own mrna via viral proteins [116, 117] . interestingly, alphaviruses whose genomic rna has a 5 ′ cap lacking 2 ′ -o methylation can avoid ifit1 immune restriction and efficiently replicate thanks to a secondary structure within their 5 ′ untranslated region (utr) which alters ifit1 binding and function [118] . once it has managed to escape host cell sensing and degradation pathways, vrna must compete with endogenous mrnas for accessing the same protein synthesis machinery. rna viruses have evolved several mechanisms to bypass the highly regulated translation cycle, particularly at the initiation phase and most of these mechanisms rely on peculiar vrna-host rbps interactions. importantly, changes in abundance, modification and activity of translation factors could regulate both vrna and host mrna translation [50] . therefore, rna viruses often employ strategies that favor the selective translation of their genomes. they can accomplish that by targeting, modifying or cleaving host translation factors and/or by usurping the identity of endogenous mrnas to have a privileged access to the ribosome [119] . canonical cellular mrnas are capped at their 5 ′ end cotranscriptionally. the cap structure, consisting of a 7-methylguanosine (m 7 g) linked to the 5 ′ nucleoside of the mrna chain through a 5 ′ -5 ′ triphosphate bridge, is crucial for mrna stability and translatability. specifically, the cap protects mrna from 5 ′ -3 ′ exonucleases and ensures recognition by the cap-binding complex eukaryotic translation initiation factors (eif4f) [120] (fig. 2) . interestingly, vrna caps can be stolen ("cap-snatching") from cellular mrnas or synthesized using either a host-or virus-encoded capping apparatus, and these capping assemblies exhibit a wide diversity in organization, structure and mechanism [121] . recognition of the cap by the eif4f subunit eif4e is thought to prepare mrnas for recruitment of the 43s complex containing multiple eifs, the initiator methionine trna (met-trna i met ) as a ternary complex with eif2 and gtp (eif2-tc), and the small 40s ribosomal subunit (fig. 2) . the polyadenylated 3 ′ mrna end is recognized by a poly(a)binding protein (pabp), which is bridged to the 5 ′ end via pabp interactions with the eif4g subunit of eif4f end. the assembled 43s complex scans the 5 ′ utr of the mrna to locate the initiator start codon (aug) (fig. 2) . after aug recognition ribosomal 60s subunits joining trigger eifs release to form the 80s ribosome where elongation can proceed for synthesis of the polypeptide chain. translation is one of the most energy-expensive processes in the cell and is therefore tightly regulated. cells rapidly reprogram gene expression when subjected to stress to conserve energy and minimize damage [122] . the arrest of bulk protein synthesis is a classical reaction cells employ during viral infection and it is often characterized by a specific inhibition of translation initiation. in the never-ending arms-race, many viruses have however evolved mechanisms to overcome the cellular stress they induce. generally, translation initiation is inhibited either by the disruption of the formation of the eif4f complex or by the inhibition of eif2-tc recycling [122] (fig. 3) . one major cellular regulator of eif4f complex formation is the mechanistic target of rapamycin (mtor) pathway and will not be discussed here [122] [123] [124] . the second major mechanism that causes translational arrest is the disruption of eif2-tc recycling, caused by phosphorylation of the α subunit of eif2. mammalian cells encode four known eif2α kinases among which pkr (table 1 ) (see above). the other three eif2α kinases: haeme-regulated inhibitor (hri), general control non-derepressible protein 2 (gcn2) and pkr-like endoplasmic reticulum (er) kinase (perk), respond to various cellular stresses including heat shock, amino acid deprivation, and er stress [122] (fig. 3) . these cellular stress states can be indirect indicators of viral infections. for instance, flaviviruses like dengue and zika viruses, which replicate in close association to er-membranes, induce er stress that results in perk activation and eif2α phosphorylation early during viral infection [125, 126] (fig. 3) . in some cases, gcn2 can also be activated by vrna to arrest translation. for example, gcn2 can recognize two regions of sindbis virus (sinv) genomic rna and inhibit vrna replication by blocking early viral translation. strikingly, mice lacking gcn2 are extremely susceptible to intranasal sinv infections, demonstrating high virus titers in the brain compared to similarly infected control animals [127] . alphaviruses like sinv take advantage of eif2 inactivation and can be totally insensitive to translational arrest by eif2 phosphorylation. subgenomic mrnas of sinv and another alphavirus, semliki forest virus (sfv), initiate translation in the presence of high levels of phosphorylated eif2α, utilizing a highly stable rna hairpin loop downstream of the aug initiator codon that stalls the ribosomes on the correct site to initiate translation of their mrnas, thus bypassing the requirement for a functional eif2 [128] . as mentioned earlier, dsrna is detected by and activates pkr for the rapid inhibition of translation initiation [129] . following activation by dsrna binding, dimeriziation and auto-phosphorylation, pkr phosphorylates eif2α which locks it onto its guanine nucleotide exchange factor eif2b, thereby preventing the regeneration of the eif2-tc that is critical for canonical translation initiation [122] (fig. 3) . although initially identified because of its ability to regulate translation in response to dsrna, pkr can also be activated by other cellular stresses such as oxidative stress, cytokines or following the stimulation of tlrs [130, 131] . furthermore, upon activation, pkr not only inhibits translation initiation, but also modulates a plethora of different signal transduction pathways, including the activation of p38 mitogen-activated protein kinase (mapk), c-jun n-terminal kinase (jnk), signal transducer and activator of transcription 1 and 3 (stat1 and 3) and the tumor suppressor p53 [132] . pkr can indirectly activate the nuclear factor κb (nfκb), which has among other functions an important role in type i ifn induction [132] . selective translation is a common strategy used by rna viruses to maintain intact their protein synthesis during global translation shutoff. we will briefly describe mechanisms used by viruses to promote the selective translation of their genomes during global protein shutdown. sequence, structure and chemical modifications present in the viral genome can determine the identity of the interacting rbps serving as translation factors. selective translation can therefore be achieved when vrna shunts the lengthy and elaborate multi-step mechanisms that yield assembled and productive 80s ribosomes to gain a privileged access to the ribosome. other strategies, such as targeting and degrading translation factors, ribosomal rnas or messenger rnas might also confer a selective advantage for vrnas over endogenous ones. we will discuss mechanisms that target the translation initiation step, nevertheless it is important to note that viruses are also able to trick capped rna is recognized by eif4f complex recruiting the 43s complex to the 5 ′ utr of mrna. this is followed by scanning until a start codon in encountered, followed by the formation of 80s ribosomes before elongation. lower panel: depicted are different modes of ires-dependent translation (i, ii, iii and iv). note the minimal requirement for initiation factors depending on ires class. these can either act on endogenous mrnas, eif4f, eif2, eif3 or the ribosomal subunits. in red are listed the four known cellular eif2alpha kinases: gcn2, hri, perk and pkr. translation elongation or termination steps [133] . while some positive-sense rna viruses like flavi or coronaviruses possess a cap-structure on the 5 ′ end of their vrna, some others like caliciviruses or picornaviruses (e.g. poliovirus or rhinovirus) decorate their 5ʹend with a covalently bound viral protein, vpg. in some cases, vpg proteins linked to the 5 ′ end of positive-strand rna genomes (e.g. feline calicivirus (fcv) or human norovirus) plays the role of a cap structure, recruiting ribosomes through interaction with eif3 [134] . in fact, modifications at the 5 ′ end of vrnas largely dictate the translation initiation mode that could either be cap -dependent or -independent. coronavirus mrnas are thought to undergo a classical cap-dependent translation initiation mechanism. a small compound that prevents the formation of the eif4f complex, a hallmark of cap-dependent translation, by blocking the interaction between eif4e and eif4g is able to significantly decrease human coronavirus 229e replication and infectious virus titers [135] . to favor the translation of their vrnas over endogenous mrnas, severe acute respiratory syndrome coronaviruses (sars-cov) non-structural protein 1 (nsp1) is able to efficiently and selectively inhibit host mrna translation by binding 40s subunits [136] (fig. 3) and promote host mrna degradation. for example, sars-cov-1 nsp1 protein induces template-dependent endonucleolytic cleavage of mrnas, however viral mrnas seem to be resistant to nsp1-induced rna cleavage [137] . one mechanism permitting this selectivity have been shown for middle east respiratory syndrome coronavirus (mers-cov) where nsp1 selectively target mrnas transcribed in the nucleus and spares mrnas of cytoplasmic origin, notably vrnas [138] . flaviviruses are also thought to undergo a canonical cap-dependent initiation of translation requiring both the eif4f complex and pabp [139] [140] [141] . interestingly, in contrast to most cellular mrnas, flavivirus vrna lacks a 3 ′ poly-a tail; however, pabp is still able to associate internally to denv 3 ′ utr upstream of a conserved 3 ′ stem-loop, pabp/3 ′ utr interaction mimicking the role of mrna poly-a tail and presumably stimulating translation initiation 141]. both utr regions (5 ′ and 3 ′ ) in flaviviruses possesses secondary structures shown to stimulate viral translation [139, 142, 143] . interestingly, under cellular conditions where translation factors are limiting or eif4e is inhibited, denv has been shown to alternate between canonical cap-dependent translation initiation and a noncanonical mechanism that does not require a functional m [7] g cap [144] . although the mechanism by which flaviviruses undergo cap-independent translation initiation is not completely clear, it has been recently suggested that denv and zikv 5 ′ untranslated regions could harbor internal ribosomal entry site (ires) functions [145] . in fact, ires-dependent initiation of translation is the other major mechanism used by many viruses to initiate their translation, dispensing them from cap-dependence. ires sequences are specialized cis-acting rna elements present in viral 5 ′ utrs capable of bypassing the extensive requirement of translation initiation factors to recruit ribosomal complexes and drive protein synthesis. iress therefore confer a considerable competitive advantage to viral mrnas, freeing them from host regulatory constraints and sustaining viral protein synthesis when canonical cap-dependent translation is impaired [119] (fig. 2) . there are four known types of viral iress (i, ii, iii and iv). although iress perform a similar function, there are no known universal ires sequences. in fact, ires elements present in the genome of different families of rna viruses lack overall conserved features [146, 147] .the classification of viral iress in four types stems from their structural organization, their respective dependence on sets of translation initiation factors, and whether they use scanning or instead directly recruit ribosomes to the start codon [148] (fig. 2) . type i and ii ireses were first discovered in picornaviruses, such as poliovirus (type i) and encephalomyocarditis virus (type ii). these two ires classes interact with eif4g c-terminal region which binds to eif3 and eif4a [149] . both require eif5b, eif2 and met-trnai and are stimulated by the activity of eif1, eif1a and eif4b [150] (fig. 2) . type iii iress, exemplified those of pestiviruses and hepatitis c virus ires, require even less translation initiation factors to recruit 40s ribosomes. in fact, the hcv ires bypasses the requirement for eif4f and the scanning step of translation initiation. hcv ires is able to directly interact with eif3 and the ribosomal 40s subunit, placing the start codon present in the vrna in the ribosomal p-site [151] (fig. 2) . finally, type iv iress are amongst the most remarkable as they completely obviate the need for canonical initiation factors. these iress are common in the dicistroviridae family of viruses such as cricket paralysis virus (crpv) or drosophila c virus (dcv), which infect insects. these viruses contain a single positive-sense rna genome that encodes two non-overlapping open reading frames separated by a short intergenic region (igr). the igr region acts as an ires to initiate translation by recruiting 80s ribosomes in the absence of initiator trna i met or any canonical initiation factors, from a gcu alanine codon located in the a-site of the ribosome [152, 153] (fig. 2) . indeed, the igr rna structure mimics a trna, thus tricking the ribosome into translation initiation without the need of the eif2-gtp-met-trna complex. using shortcuts for a privileged access to the translation machinery by tricking the ribosome is not the only strategy rna viruses employ to selectively favor the synthesis of viral proteins. another effective tactic is targeting and inhibiting the translation of cellular mrnas, which frees up a large pool of ribosomes, making them readily available for vrna translation. this is generally achieved by targeting host rbps, specifically translation initiation factors or ribosomal proteins. viruses that use ires-dependent translation for example, actively provoke the shut-off of host cap-dependent translation initiation, by targeting specific initiation factors. for instance, enteroviruses including rhino and polioviruses, encode proteases that cleave eif4g, impeding the formation of the eif4f complex and therefore cap-dependent host mrna translation [154] (fig. 3) . other picornaviruses, such as emcv suppresses cap-dependent translation by activating the translational repressor 4ebp1 [155] . hypophosphorylated 4ebp1 binds eif4e preventing, eif4e-eif4g interaction, thus eif4f assembly. in fact, 4ebp hypophosphorylation is a strategy common to many viruses to inhibit host mrna translation (e.g., crpv, vsv) [156] [157] [158] (fig. 3) . targeting eif4f-associated pabp proteins is also used by some viruses to influence host translation. for example, rotavirus nsp3 interacts with eif4g and displaces pabp, therefore inhibiting host translation [159] . likewise, enterovirus and calicivirus proteases cleave pabp, and the rubella virus capsid protein binds pabp to suppress cellular mrna translation [119, 154, 159] . other translation initiation factors like eif3 can also be targeted by viral proteins. eif3-binding proteins from measles and rabies viruses inhibit host protein synthesis [160, 161] , whereas foot-and-mouth disease virus protease degrades eif3a and eif3b subunits [162, 119] . also, the coronavirus spike protein has been shown to interact with eif3f influencing translational control [163] (fig. 3) . although initiation factors are the privileged target of viral proteins to shut-off host mrna translation, some examples of viruses targeting translation elongation factors are also reported. for instance, it has been shown that ef1a and eef2 are respectively inactivated by sars-cov-1 n protein and avian reovirus p17 [164, 165] . in many of these examples, how vrna translation is able to proceed upon inactivation of these essential initiation or elongation factors remains unclear. viral rbps can directly target the ribosome to promote translation of their genomes and viral rna may also directly interact with ribosomal proteins to preferentially translate their genomes. denv ns1 protein interacts with rpl18 to promote viral protein synthesis [166] . conversely, vrna can rely on specific nonessential ribosomal proteins, dispensable for translating most host mrnas. moloney leukemia virus and sinv stop codon readthrough and frameshifting efficiency, have been shown to depend on rpl4 [167] . vsv cap-dependent mrna translation have been shown to be affected by rpl40 presence [168] (fig. 3) . also, some ires functions, like those found in polio, hcv or dicistroviridae, have been shown to be regulated by small ribosomal proteins such as rack1 and rps25 [169] [170] [171] [172] [173] (fig. 3) . interestingly, rack1 seem to be a privileged target of poxviruses to promote selective translation of viral mrnas [174] . finally, rbps that are not necessary translation factors or ribosomal can also positively influence viral translation. for example, pelo has been shown to be a host factor needed for efficient translation of viral capsids of drosophila c virus (dcv) [175] . vigilin and rrbp1, two endoplasmic reticulum localized rbps have been shown to bind denv and zikv vrnas, promoting their translation, stability and replication [176] . during the last decades, our knowledge of the intricate interactions of cellular rbps with vrnas have been a direct result of technical advances in modern molecular biology. when it comes to the study of rbps that are viral sensors or antiviral effectors, many major discoveries in how tlrs, rlrs, dicer or other rbps recognize and/or antagonize vrna, were made thanks to in vivo genetic screening technologies and innovations [177] . both, hypothesis-driven reverse genetics approaches, and forward genetic techniques have been able to yield susceptibility or resistance phenotypes, revealing genes with unanticipated and important roles in recognizing and/or targeting vrnas. the involvement of tlr 3, 9, mda5 and rig-i in the innate antiviral response wouldn't have been possible without in vivo genetic manipulation and screening of mouse models [178] [179] [180] . another classical example where genetics played a fundamental role in discovering important antiviral host rbps, is the study of the rnai pathway components. argonaute and dicer proteins' involvement in antiviral defenses across several animal and plant species was only possible thanks to genetic manipulation and screening techniques in model organisms [85, [181] [182] [183] . importantly, biochemical and structural biology approaches including x-ray crystallography and cryo-electron microscopy (cryo-em) guided these discoveries and were able to determine structurally how sensor and effector rbps interact with vrna [184] [185] [186] [187] [188] . likewise, recent technical innovations and improvements in rna isolation, manipulation and sequencing have enabled detection of new rna species and rna chemical modifications (e.g. m 6 a, 2 ′ -o-me) in vrnas, revealing an underappreciated role of these species and modifications in viral lifecycles [96, 189, 190] . thus, the use of oxidative treatment on the rna prior to its sequencing helped to unravel the existence of 2 ′ -o-methylated viral small rnas species that accumulate during sinv infection [191] . more recently, the use of single cell rna sequencing allowed to determine at an unprecedented resolution the accumulation of viral transcripts in a dynamic manner [192] . while this has so far been used to study dna viruses' transcription, it might also prove useful to study replication of rna viruses. we are still lacking a global picture of rna modifications that are present in viral rnas, and more importantly, we need to determine their exact contribution to the infectious cycle. to this end, it is crucial to develop and validate good antibodies targeting modified ribonucleotides, or to turn to emerging sequencing technologies, such as the oxford nanopore one, that allows direct rna sequencing. this strategy has recently allowed the direct detection of m 6 a by sequencing of cellular rnas [193, 194] . similarly, the last decades of research brought very detailed insights into how vrnas co-opt host ribosomes. this is partly due to the history of research in the mrna translation field. the ribosome being the most conserved and largest known rnp complex in living organisms, deciphering mrna translation has been an endeavor pursued since the birth of modern molecular biology [195] . important knowledge in our understanding of mrna translation were made in the past thanks to technical innovations and improvements. for example, in vitro translation systems, reconstituted from rabbit reticulocytes, were instrumental to understand many aspects of endogenous mrna translational control [195] . interestingly, thanks to such in vitro systems, we know since the 1970 ′ s the inhibitory role of viral dsrna or eif2 phosphorylation on translation initiation [196, 197] . in vitro translation systems coupled to reporter assays were also crucial later on to decipher the different mechanisms by which ires-dependent translation proceeds [50, 152, 198] . more recently, cryo-em approaches were able to paint a more detailed picture of how different ireses can interact and co-opt host ribosomes [150, 199, 200] . the rapid development and improvement of cryo-em techniques is expected to generate further knowledge, surpassing ires-ribosome interactions. indeed, cryo-em techniques have been lately used to unravel the structural basis of flaviviral sfrna production for example [63] . most recently, cryo-em structures of sars-cov2 nsp1 with human 40s ribosomal subunit revealed that nsp1 c-terminus binds to and obstructs the mrna entry tunnel, explaining how this protein is able to shutdown endogenous mrna translation [201] . another very recent cryo-em study was able to identify residues of nsp1 crucial for mediating translation inhibition [202] . in the future, these approaches promise to aid structure-based drug design not only against sars-cov2 but also other emerging rna viruses [201] . it is finally important to note that other novel and innovative techniques developed to study translation such as single-molecule approaches [203] or ribosome profiling by deep sequencing [204] are revealing the complexity of vrna interaction with host ribosomes. for instance, single-molecule studies of ires-mediated translation have revealed insights into the dynamics of hcv and crpv ires interaction with ribosomes and clarified decades of biochemical research, providing an outline of the conformational and compositional trajectory of the ribosome during initiation [151] . likewise, ribosome profiling revealed strategies that some rna viruses use to induce programmed ribosomal frameshifting (prf) that would have not been discovered otherwise. frameshifting events are 'programmed' because they occur at specific sequences, stimulated by cis-acting elements generally present in the vrna at rates that are orders of magnitude more frequent than nonprogrammed events [205] . cardioviruses such as emcv and its relative theiler's murine encephalomyelitis virus (tmev) induce prf but are atypical due to the absence of an appropriately positioned stimulatory rna structure in their genomes and a failure to reconstitute prf in vitro, outside of the context of virus infection. remarkably, thanks to ribosome profiling, a recent study shows that emcv frameshifting is trans-activated by viral protein 2a. as a result, the frameshifting efficiency increases from 0 to 70 % (one of the highest known in a mammalian system) over the course of infection, temporally regulating the expression levels of the viral structural and enzymatic proteins [206] . in the future, ribosome profiling techniques are expected to reveal more secrets of vrna translation, especially for viruses that annexes organelles like the er to complete their translation [207] . each time a technological leap is achieved the scope of what we know about vrnas interaction with cellular rbps has expanded. two global strategies have been used to study vrna-rbp interactions; protein-centric or rna-centric methods. protein-centric methods start with a known rbp of interest and characterize its interaction with rna. these approaches involve uv-crosslinking followed by antibody purification of the rbp of interest and identification of bound rnas, often by deep sequencing, broadly termed hits-clip (cross-linking immunoprecipitation followed by high-throughput sequencing) [208] . these methods have been used in the past in the context of viral infections to characterize the rna species that bind either known host rbps [176, [209] [210] [211] or of viral proteins [212, 213] . we will not focus further on these methods here but readers can refer to excellent reviews on the subject in the context of viral infections [214] [215] [216] . rna-centric approaches are more recent and focus on purifying an rna of interest to identify its associated proteins. a large number of the so-called "unconventional rbps" have been identified as associated to rnas thanks to these rna-centric approaches. for example, rna interactome capture (rna-ic), have largely expanded the repertoire of known rbps [217] . rna-ic entails ultraviolet crosslinking of rbps to rna in live cells, followed by collective capture of rnps with polyadenylated (poly(a)) rna on oligo(dt) beads and identification of proteins by quantitative mass spectrometry (q-ms) [217, 218] . these methods discovered new rbps that generally lack canonical rna-binding domains and are conserved across species, suggesting the existence of previously unidentified modes of rna binding and new biological functions for protein-rna interactions [217] . this method was applied to study cellular rna-interactome during sinv infection and was able to identify over 200 rbps that interacted differentially with host rna upon viral infection, amongst which, rnas encoding proviral and restriction factors [219] . although rna-ic is able to identify rbps that directly interact with rnas, one limitation of this method is its reliance on oligo(dt) to pull-down poly(a) containing rnas. another novel method, comprehensive identification of rna binding proteins-mass spectrometry" (chirp-ms), addresses this limitation by using 20-mer oligonucleotide probes complimentary to the rna of interest. chirp-ms uses formaldehyde (fa) cross-linking prior to pull-down with biotinylated probes, able to hybridize with an rna of interest. unlike uv-crosslinking, the use of fa for crosslinking doesn't only identify direct rna binders but also their associated protein complexes. cross-linked rnp complexes are then captured with streptavidin beads prior to proteomic analysis [220] . originally developed to identify rbps associated with long non-coding rnas (lncrnas) [221] , chirp-ms was recently applied to identify rbps associated with denv and zikv vrnas [176] . chirp-ms identified viral non-structural proteins ns3 and ns5 as highly enriched with denv and zikv vrna. furthermore, this method identified 464 high confidence hits from the human proteome that were specifically and reproducibly associated with the denv or zikv vrna [222] . many of those proteins were localized to the er, in line with the known biology of flaviviral rna, being translated and replicated at specific er sites [223] . importantly, when viral infection is difficult to synchronize, chirp-ms is unable to differentiate at which step in the viral lifecycle vrna-rbps associations occur (e.g. entry, translation, replication, assembly). a recent approach addresses partly this limitation and is able to capture interactions with just the pre-replicated viral rna genome [224] . viral cross-linking and solid-phase purification (vir-clasp) relies on infection of unlabeled host cells with 4-thiouridine (4su)-labeled viral genomes [224] . irradiation of infected cells with 365 nm light generates covalent cross-links between 4su-labeled viral genomes and interacting host or viral proteins. solid-phase capture of these complexes with solid-phase reversible immobilization (spri) beads leads to purification of total rna but only proteins covalently crosslinked to the 4su-labeled viral rna. this purification precedes (lc-ms/ms) identification of the crosslinked proteins [224] . using vir-clasp, the study identifies early interactomes of different viral genomes (e.g. chikv, iav, zikv, vsv). the authors of the study find that incoming chikv vrna binds both proviral and antiviral factors. while on one hand, chikv incoming vrna hijacks the lipid-modifying enzyme fatty acid synthase (fasn) for pro-viral activity, it is on the other hand n6-methyladenosine modified, binding to ythdf1 which suppresses its replication [224] . the aforementioned rna-centric methods (rna-ic, chirp-ms, vir-clasp) all use crosslinking (uv or fa) to identify in vivo interactions by purifying the rna under denaturing conditions that remove noncovalent interactions, and subsequently extracting only the cross-linked proteins for identification [225] . other approaches, that do not require crosslinking of rnps, use proximity proteomics to identify rbp-rna interactions in live cells. these approaches rely on 'promiscuous' biotin ligases that are able to label preferentially proteins that are within 20 nm of distance with biotin [226, 227] . originally developed to identify protein-protein interactions, this approach was recently adapted to identify rna-rbps interactions. the rna-protein interaction detection (rapid) method allows to use this spatial detection constraint to detect rbps bound to rna by tagging an rna of interest with a boxb aptamer to recruit a fusion protein of λ-n and a promiscuous biotin ligase [228, 225] . the biotin sprayer binds the boxb motif through its λ-n domain and labels proteins proximal to its bound rna [225] . rapid-ms was successfully used to identify rbps that bind zikv utr sequences. interestingly, the identified rbps show enriched expression in neural tissue, which is in line with the neurotropic nature of zikv [228] . rapid pointed to a known rna-binding protein, qki, that is highly expressed in neural progenitor cells (npcs) and whose depletion reduces zikv rna levels by 90 % [228] . importantly, because these rna-centric methods rely on rna sequence specificity they can be transposed and applied to identify rbps associated with virtually any rna virus. after these proof-of-principle studies, all of these novel rna-centric approaches are expected to yield important fundamental knowledge about vrna interactions with host rbps, if applied to emerging or re-emerging rna viruses with epidemic potential such as ebola or sars-cov2 [229, 230] . rna-protein interactions are central to most cellular processes. it is thus not surprising that rna viruses and cellular genes evolved intricate vrna-rbp interactions that determine many aspects of the viral life cycle [4] . from the host cell perspective, the affinity of its rbps to the invading vrna, could be a double-edged sword. many cellular genes have evolved to specifically recognize and/or target vrnas and alert the immune system to mobilize cellular defenses. this is illustrated by the relative conservation of many vrna sensors and anti-vrna effectors across species, such as components of the rnai pathway or vrna sensors like rlrs and tlrs [12] . likewise, many viral genes and rna structures have specifically evolved to co-opt fundamental host cell machineries, without which the virus in unable to replicate. this is exemplified by the convergent evolution of many rna elements in the viral genomes, with roles in hijacking ribosomes and the cellular machinery in general [133] . in fact, structured rna elements, such as iress are shared amongst viruses from very different families, ranging from plant to animal viruses [133, 148, 231] . also, viral enzymes that can interfere with endogenous mrna lifecycles favoring vrnas translation, are also found across a wide range of viral families [119, 156] . during the last decades, since the rise of modern molecular biology we are witnessing a remarkable and accelerated development of tools and technologies that are enabling to delve deeper into fundamental cellular mechanisms. as mentioned in this review, each time a technical innovation emerged, our understanding of vrna-rbp interactions expanded. for example, proteins identified by the novel aforementioned rna-centric methods (e.g. chirp-ms, vir-clasp, rna-ic) are highly enriched with classical rbps as expected. however, hundreds of "unorthodox" rbps have also been found to bind vrnas [219, 222] . some of those unorthodox' rbps have already known cellular functions and weren't expected to have an influence on viral lifecycles by binding vrnas. for example, cellular transport motors like dyneins have been shown to have an rna binding activity that might be responsible of rnas trafficking, uncoating, reverse transcription and in some cases viral replication factory assembly [232] . another eloquent example comes from the interaction of er-resident multimolecular complexes, such as sec61 translocon complex or the oligosaccharyltransferase (ost) complex, with the lifecycle of flaviviruses [3, 233] . although these complexes have known and established roles in protein translocation and glycosylation in the er, new unexpected rna-related functions of these complexes are just emerging [234] . for example, flavivirus vrnas that are translated and replicate in close proximity to er membranes, seem to require "non-canonical" functions of the ost complex [3] . this might involve tethering viral rna genomes to the er for efficient translation and replication [176, 207, 232, 234] . future work will clarify which unconventional rbps are truly important for the viral infectious cycle. in the meantime, these systemwide approaches are uncovering new host rbps with unexpected new roles during viral lifecycles. understanding mechanistically the relevance of these newly discovered interactions is crucial in the future. alongside careful and rigorous biochemistry, this can be tackled with new complimentary cutting-edge methods (e.g. clip-seq, ribosome profiling) [204, 206, 210, 222] to determine the footprints of rbps on viral and host rnas. other new approaches to visualize, track, target or detect rnas (e.g. crispr-cas13), if applied to vrnas, will clarify the role of the newly discovered host rbp-vrna interactions [235] [236] [237] . finally, there are many reasons to look at the future of the field with optimism. indeed, continuing to dissect the intimate relationship between vrnas and host rbps with new tools, is expected to both expand our fundamental understanding of virology and cell biology, and to guide the development of much needed novel antiviral approaches. the authors report no declarations of interest. host factors in positive-strand rna virus genome replication human host factors required for influenza virus replication genetic dissection of flaviviridae host factors through genome-scale crispr screens diverse roles of host rna binding proteins in rna virus replication crispr-cas immunity in prokaryotes antiviral immunity directed by small rnas combating emerging viral threats the global distribution and burden of dengue virus: new clinical syndromes and its emergence in the western hemisphere emergence of a novel human coronavirus threatening human health sensing of rna viruses: a review of innate immune receptors involved in recognizing rna virus invasion the innate antiviral response in animals: an evolutionary perspective from flagellates to humans discriminating self from non-self in nucleic acid sensing toll-like receptor control of the adaptive immune responses toll-like receptors in antiviral innate immunity innate antiviral responses by means of tlr7-mediated recognition of single-stranded rna species-specific recognition of single-stranded rna via toll-like receptor 7 and 8 toll-like receptor 3 is an essential component of the innate stress response in virus-induced cardiac injury human tlr3 recognizes dengue virus and modulates viral replication in vitro zika virus depletes neural progenitors in human cerebral organoids through activation of the innate immune receptor tlr3 toll-like receptor 3 in viral pathogenesis: friend or foe? rig-i-like receptors: their regulation and roles in rna sensing 5'-triphosphate rna is the ligand for rig-i rig-i-mediated antiviral responses to single-stranded rna bearing 5'-phosphates recognition of 5' triphosphate by rig-i helicase requires short blunt double-stranded rna as contained in panhandle of negative-strand virus 5'-triphosphate rna requires base-paired structures to activate antiviral signaling via rig-i processing of genome 5' termini as a strategy of negative-strand rna viruses to avoid rig-i-dependent interferon induction differential roles of mda5 and rig-i helicases in the recognition of rna viruses cell type-specific involvement of rig-i in antiviral response rig-i recognizes the 5' region of dengue and zika virus genomes balancing act: mda5 in antiviral immunity and autoinflammation mda5 assembles into a polar helical filament on dsrna enterovirus 2apro targets mda5 and mavs in infected cells mda5 detects the double-stranded rna replicative form in picornavirus-infected cells how flaviviruses activate and suppress the interferon response mda5 is critical to host defense during infection with murine coronavirus lgp2 is a positive regulator of rig-i-and mda5-mediated antiviral responses the rna helicase lgp2 inhibits tlr-independent sensing of viral replication by retinoic acid-inducible gene-i loss of dexd/h box rna helicase lgp2 manifests disparate antiviral responses the rig-i-like receptor lgp2 inhibits dicer-dependent processing of long double-stranded rna and blocks rna interference in mammalian cells dead-box helicases: sensors, regulators, and effectors for antiviral defense type 1 interferons and the virus-host relationship: a lesson in détente interferon-inducible antiviral effectors involvement of the interferon-regulated antiviral proteins pkr and rnase l in reovirus-induced shutoff of cellular translation doublestranded rna is produced by positive-strand rna viruses and dna viruses but not in detectable amounts by negative-strand rna viruses rna dimerization promotes pkr dimerization and activation viral double-stranded rnas from vaccinia virus early or intermediate gene transcripts possess pkr activating function, resulting in nf-kappab activation, when the k1 protein is absent or mutated regulation of innate immunity through rna structure and the protein kinase pkr translational control in virus-infected cells a cap-to-tail guide to mrna translation strategies in virus-infected cells viral noncoding rnas: more surprises inhibition of the protein kinase pkr by the internal ribosome entry site of hepatitis c virus genomic rna protein kinase r degradation is essential for rift valley fever virus infection and is regulated by skp1-cul1-f-box (scf)fbxw11-nss e3 ligase viral encounters with 2',5'-oligoadenylate synthetase and rnase l during the interferon antiviral response new insights into the role of rnase l in innate immunity molecular mechanisms for the adaptive switching between the oas/rnase l and oasl/rig-i pathways in birds and mammals homologous 2',5'-phosphodiesterases from disparate rna viruses antagonize antiviral innate immunity attacked from all sides: rna decay in antiviral defense the host nonsense-mediated mrna decay pathway restricts mammalian rna virus replication competing and noncompeting activities of mir-122 and the 5' exonuclease xrn1 in regulation of hepatitis c virus replication stabilization of hepatitis c virus rna by an ago2-mir-122 complex zika virus produces noncoding rnas using a multipseudoknot structure that confounds a cellular exonuclease the structural basis of pathogenic subgenomic flavivirus rna (sfrna) production rna structures that resist degradation by xrn1 produce a pathogenic dengue virus rna full genome sequence and sfrna interferon antagonist activity of zika virus from recife, brazil rna structures required for production of subgenomic flavivirus rna dengue subgenomic rna binds trim25 to inhibit interferon expression for epidemiological fitness a noncoding rna produced by arthropod-borne flaviviruses inhibits the cellular exoribonuclease xrn1 and alters host mrna stability a highly structured, nuclease-resistant, noncoding rna produced by flaviviruses is required for pathogenicity an rna pseudoknot is required for production of yellow fever virus subgenomic rna by the host nuclease xrn1 zika virus noncoding rna suppresses apoptosis and is required for virus transmission by mosquitoes noncoding flavivirus rna displays rna interference suppressor activity in insect and mammalian cells viruses: overturning rna turnover stem-loop recognition by ddx17 facilitates mirna processing and antiviral defense the zinc-finger antiviral protein recruits the rna processing exosome to degrade the target mrna cg dinucleotide suppression enables antiviral defence targeting non-self rna khnyn is essential for the zinc finger antiviral protein (zap) to restrict hiv-1 containing clustered cpg dinucleotides zap's stress granule localization is correlated with its antiviral activity and induced by virus replication rna-binding activity of trim25 is mediated by its pry/ spry domain and is required for ubiquitination trim25 enhances the antiviral action of zinc-finger antiviral protein (zap) mcpip1 ribonuclease exhibits broad-spectrum antiviral effects through viral rna binding and degradation mcpip1 suppresses hepatitis c virus replication and negatively regulates virus-induced proinflammatory cytokine responses rnase l releases a small rna from hcv rna that refolds into a potent pamp activation and evasion of the antiviral 2'-5' oligoadenylate synthetase/ribonuclease l pathway by hepatitis c virus mrna essential function in vivo for dicer-2 in host defense against rna viruses in drosophila cross-species comparative analysis of dicer proteins during sindbis virus infection rna interference functions as an antiviral immunity mechanism in mammals antiviral rna interference in mammalian cells flavivirus induces and antagonizes antiviral rna interference in both mammals and mosquitoes the evolution of antiviral defense systems human virus-derived small rnas can confer antiviral immunity in mammals induction and suppression of antiviral rna interference by influenza a virus in mammalian cells viruses and rna interference: issues and controversies no evidence for viral small rna production and antiviral function of argonaute 2 in human cells rnase iii nucleases from diverse kingdoms serve as antiviral effectors making the mark: the role of adenosine modifications in the life cycle of rna viruses innate immune restriction and antagonism of viral rna lacking 2‫-׳‬o methylation rna modifications: what have we learned and where are we headed? dar proteins: double-stranded rna and z-dna binding domains editor-in-chief' of cytoplasmic innate immunity adenosine deaminases acting on rna (adars) are both antiviral and proviral hepatitis d virus replication, cold spring harb the biogenesis and precise control of rna m6a methylation the rna modification n6-methyladenosine as a novel regulator of the immune system n6-methyladenosine and viral infection n6-methyladenosine in flaviviridae viral rna genomes regulates infection n6-methyladenosine modification enables viral rna to escape recognition by rna sensor rig-i viral rna in an m6a disguise m6a modification prevents formation of endogenous doublestranded rnas and deleterious innate immune responses during hematopoietic development rna modifications modulate activation of innate toll-like receptors ribose 2'-o-methylation provides a molecular signature for the distinction of self and non-self mrna dependent on the rna sensor mda5 ifit1 is an antiviral protein that recognizes 5'-triphosphate rna sequestration by ifit1 impairs translation of 2'o-unmethylated capped rna ifit1 inhibits japanese encephalitis virus replication through binding to 5' capped 2'-o unmethylated rna ifit1: a dual sensor and effector molecule that detects non-2'-o methylated viral rna and inhibits its translation 2'-o methylation of the viral mrna cap evades host restriction by ifit family members attenuation and restoration of severe acute respiratory syndrome coronavirus mutant lacking 2'-o-methyltransferase activity a viral rna structural element alters host recognition of nonself rna viral subversion of the host protein synthesis machinery mrna capping: biological functions and applications conventional and unconventional mechanisms for capping viral mrna translation inhibition and stress granules in the antiviral immune response mtor: from growth signal integration to cancer, diabetes and ageing adapting the stress response: viral subversion of the mtor signaling pathway dengue virus modulates the unfolded protein response in a time-dependent manner flavivirus infection uncouples translation suppression from cellular stress responses antiviral effect of the mammalian translation initiation factor 2alpha kinase gcn2 against rna viruses translational resistance of late alphavirus mrna to eif2alpha phosphorylation: a strategy to overcome the antiviral effect of protein kinase pkr the dsrna protein kinase pkr: virus and cell control impact of protein kinase pkr in cell biology: from antiviral to antiproliferative action, microbiol ceramide regulates protein synthesis by a novel mechanism involving the cellular pkr activator rax the multiple faces of proteinkinase r in antiviral defense viral rna structure-based strategies to manipulate translation the genome-linked protein vpg of the norwalk virus binds eif3, suggesting its role in translation initiation complex recruitment blocking eif4e-eif4g interaction as a strategy to impair coronavirus replication▿ a twopronged strategy to suppress host protein synthesis by sars coronavirus nsp1 protein sars coronavirus nsp1 protein induces template-dependent endonucleolytic cleavage of mrnas: viral mrnas are resistant to nsp1-induced rna cleavage middle east respiratory syndrome coronavirus nsp1 inhibits host gene expression by selectively targeting mrnas transcribed in the nucleus while sparing mrnas of cytoplasmic origin rna secondary structure in the coding region of dengue virus type 2 directs translation start codon selection and is required for viral replication flavivirus rna transactions from viral entry to genome replication poly(a)-binding protein binds to the nonpolyadenylated 3' untranslated region of dengue virus and modulates translation efficiency control of translation by the 5'-and 3'-terminal regions of the dengue virus genome enhancement of dengue virus translation: role of the 3' untranslated region and the terminal 3' stem-loop domain dengue virus utilizes a novel strategy for translation initiation when cap-dependent translation is inhibited dengue and zika virus 5' untranslated regions harbor internal ribosomal entry site functions structural insights into viral ires-dependent translation mechanisms insights into structural and mechanistic features of viral ires elements viral internal ribosomal entry sites: four classes for one goal ires-induced conformational changes in the ribosome and the mechanism of translation initiation by internal ribosomal entry the structures of nonprotein-coding rnas that drive internal ribosome entry site function dynamics of ires-mediated translation factorless ribosome assembly on the internal ribosome entry site of cricket paralysis virus initiation of protein synthesis from the a site of the ribosome translational control by viral proteinases activation of the translational suppressor 4e-bp1 following infection with encephalomyocarditis virus and poliovirus tinkering with translation: protein synthesis in virus-infected cells vesicular stomatitis virus infection alters the eif4f translation initiation complex and causes dephosphorylation of the eif4e binding protein 4e-bp1 host and viral translational mechanisms during cricket paralysis virus infection poly(a)-binding protein (pabp): a common viral target measles virus n protein inhibits host translation by binding to eif3-p40 rabies virus matrix protein interplay with eif3, new insights into rabies virus pathogenesis foot-and-mouth disease virus infection induces proteolytic cleavage of ptb, eif3a,b, and pabp rna-binding proteins coronavirus spike protein inhibits host cell translation by interaction with eif3f the nucleocapsid protein of severe acute respiratory syndrome coronavirus inhibits cell cytokinesis and proliferation by interacting with translation elongation factor 1alpha avian reovirus influences phosphorylation of several factors involved in host protein translation including eukaryotic translation elongation factor 2 (eef2) in vero cells dengue virus ns1 protein interacts with the ribosomal protein rpl18: this interaction is required for viral translation and replication in huh-7 cells large ribosomal protein 4 increases efficiency of viral recoding sequences a ribosome-specialized translation initiation pathway is required for cap-dependent translation of vesicular stomatitis virus mrnas rack1 controls ires-mediated translation of viruses ribosomal protein s25 dependency reveals a common mechanism for diverse internal ribosome entry sites and ribosome shunting ribosomal protein rack1 enhances translation of poliovirus and other viral iress rack1 on and off the ribosome a complex ires at the 5'-utr of a viral mrna assembles a functional 48s complex via an uaug intermediate trans-kingdom mimicry underlies ribosome customization by a poxvirus kinase pelo is required for high efficiency viral replication an rna-centric dissection of host complexes controlling flavivirus infection genetic analysis of resistance to viral infection differential roles of mda5 and rig-i helicases in the recognition of rna viruses essential role of mda-5 in type i ifn responses to polyriboinosinic: polyribocytidylic acid and encephalomyocarditis picornavirus toll-like receptors 9 and 3 as essential components of innate immune defense against mouse cytomegalovirus infection antiviral immunity directed by small rnas an antiviral role for the rna interference machinery in caenorhabditis elegans the rna silencing endonuclease argonaute 2 mediates specific antiviral immunity in drosophila melanogaster the structure of human argonaute-2 in complex with mir-20a the structural biology of toll-like receptors structural basis for dsrna recognition, filament formation, and antiviral signal activation by mda5 a structure-based model of rig-i activation structural basis for double-stranded rna processing by dicer regulation of viral infection by the rna modification n6-methyladenosine positive-sense rna viruses reveal the complexity and dynamics of the cellular and viral epitranscriptomes during infection identification of rnase l-dependent, 3'-end-modified, viral small rnas in sindbis virus-infected mammalian cells scslam-seq reveals core features of transcription dynamics in single cells decoding the epitranscriptional landscape from native rna sequences direct rna sequencing enables m6a detection in endogenous transcript isoforms at base-specific resolution cold spring harbsynthesis and translational control: a historical perspective double-stranded poliovirus rna inhibits initiation of protein synthesis by reticulocyte lysates phosphorylation of initiation factor elf-2 and the control of reticulocyte protein synthesis translation of encephalomyocarditis virus rna: parameters influencing the selection of the internal initiation site viral ires rna structures and ribosome interactions hepatitis c virus ires rna-induced changes in the conformation of the 40s ribosomal subunit structural basis for translational shutdown and immune evasion by the nsp1 protein of sars-cov-2 sars-cov-2 nsp1 binds ribosomal mrna channel to inhibit translation quantification of mrna translation in live cells using singlemolecule imaging ribosome profiling: new views of translation, from single codons to genome scale mechanisms and implications of programmed translational frameshifting protein-directed ribosomal frameshifting temporally regulates gene expression dengue virus selectively annexes endoplasmic reticulumassociated translation machinery as a strategy for co-opting host cell protein synthesis clip identifies nova-regulated rna networks in the brain a broad rna virus survey reveals both mirna dependence and functional sequestration dissecting noncoding and pathogen rna-protein interactomes argonaute proteins regulate hiv-1 multiply spliced rna and viral production in a dicer independent manner nucleotide resolution mapping of influenza a virus nucleoprotein-rna interactions reveals rna features required for replication identification of interactions between sindbis virus capsid protein and cytoplasmic vrna as novel virulence determinants clip-related methodologies and their application to retrovirology integrated approaches to reveal mechanisms by which rna viruses reprogram the cellular environment hits-clip and par-clip advance viral mirna targetome analysis a brave new world of rnabinding proteins system-wide identification of rna-binding proteins by interactome capture system-wide profiling of rna-binding proteins uncovers key regulators of virus infection chirp-ms: rna-directed proteomic discovery systematic discovery of xist rna binding proteins an rna-centric dissection of host complexes controlling flavivirus infection rewiring cellular networks by members of the flaviviridae family discovery of widespread host protein interactions with the prereplicated genome of chikv using vir-clasp methods to study rna-protein interactions an improved smaller biotin ligase for bioid proximity labeling a promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells rna-protein interaction detection in living cells the trinity of covid-19: immunity, inflammation and intervention ebola virus disease non-canonical translation in plant rna viruses unconventional rna-binding proteins step into the virus-host battlefront a crispr screen defines a signal peptide processing pathway required by flaviviruses multifunctional roles for the protein translocation machinery in rna anchoring to the endoplasmic reticulum programmable inhibition and detection of rna viruses using cas13 development of crispr as an antiviral strategy to combat sars-cov-2 and influenza rna editing with crispr-cas13 enterovirus 2apro targets mda5 and mavs in infected cells mda5 detects the double-stranded rna replicative form in picornavirus-infected cells activation of the antiviral kinase pkr and viral countermeasures activation of rnase l is dependent on oas3 expression during infection with diverse human viruses zika virus infection induces rnai-mediated antiviral immunity in human neural progenitors and brain organoids the authors would like to thank dr. alex g. johnson for his thorough and critical reading of the manuscript. the authors apologize to colleagues whose work could not be cited due to space limitations. supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.semcdb.2020.08.006. key: cord-303189-ktl4jw8v authors: coccia, eliana m.; battistini, angela title: early ifn type i response: learning from microbial evasion strategies date: 2015-03-31 journal: seminars in immunology doi: 10.1016/j.smim.2015.03.005 sha: doc_id: 303189 cord_uid: ktl4jw8v abstract type i interferon (ifn) comprises a class of cytokines first discovered more than 50 years ago and initially characterized for their ability to interfere with viral replication and restrict locally viral propagation. as such, their induction downstream of germ-line encoded pattern recognition receptors (prrs) upon recognition of pathogen-associated molecular patterns (pamps) is a hallmark of the host antiviral response. the acknowledgment that several pamps, not just of viral origin, may induce ifn, pinpoints at these molecules as a first line of host defense against a number of invading pathogens. acting in both autocrine and paracrine manner, ifn interferes with viral replication by inducing hundreds of different ifn-stimulated genes with both direct anti-pathogenic as well as immunomodulatory activities, therefore functioning as a bridge between innate and adaptive immunity. on the other hand an inverse interference to escape the ifn system is largely exploited by pathogens through a number of tactics and tricks aimed at evading, inhibiting or manipulating the ifn pathway, that result in progression of infection or establishment of chronic disease. in this review we discuss the interplay between the ifn system and some selected clinically important and challenging viruses and bacteria, highlighting the wide array of pathogen-triggered molecular mechanisms involved in evasion strategies. the ability of the host to respond to invading pathogens relies on the activation of the innate immune system that orchestrates adaptive immune responses for pathogen clearance. in recent years, our understanding of the mechanisms involved in the activation of the innate response has evolved significantly with the identification and characterization of the mammalian system of pathogen recognition. the innate immune system detects the presence of a pathogen through a set of germline-encoded membrane-associated or cytoplasmic receptors, termed pattern recognition receptors (prrs) that are engaged by microbial-derived products named pathogenassociated molecular patterns (pamps). major classes of prr include toll-like receptors (tlrs), nucleotide-binding oligomerization domain (nod)-like receptors (nlr), c-type lectin receptors (clrs), retinoic-acid inducible gene (rig)-i-like receptors (rlrs) and a growing list of cytosolic dna-sensing receptors [1] [2] [3] [4] [5] [6] [7] . upon engagement, these receptors recruit a number of adaptor proteins to signal downstream and activate three major pathways: the nuclear factor kappa-light-chain-enhancer of activated b cells (nf-b), the mitogen-activated protein kinases (mapks) and the ifn regulatory factor (irf) pathway [8, 9] . downstream tlrs and rlrs, type i ifn and pro-inflammatory cytokines are mainly produced, while nlrs predominantly activate inflammasomes, resulting in the release of il-1 and multiple inflammatory cytokines (fig. 1) . based on the structure of their receptors, interferons are broadly classified into three groups, type i, ii and iii ifns. type i ifn comprises the largest ifn class that includes ifn-␣, constituted by several partially homologous genes and ifn-␤, ifn-, ifn-, ifnrepresented by a single gene. the ifn-␣ and ifn-␤ are the best characterized as antiviral and the most broadly expressed [10] [11] [12] and will be referred as ifn-i from now on. the ifn-␥, the only type ii ifn, is released by activated t and nk cells; type iii ifns, which include ifn-1-4, similarly to ifn-i are believed to regulate the antiviral response [13] . according to the most common view, ifn-i exerts primarily an anti-viral action while ifn-ii acts predominantly on macrophages to induce a microbicidal state against ingested intracellular, non-viral pathogens. anti-microbial ifn-i activity is not intrinsic, but mediated, in both autocrine and paracrine manner, by a unique set of induced genes named ifn-stimulated genes (isgs) [10, 11, 14] . secreted ifn-i, indeed, binds to a common heterodimeric ubiquitously expressed receptor composed of ifnar1 and ifnar2 chains, and initiates a signaling cascade that has been characterized in detail and reviewed elsewhere [10] [11] [12] . briefly, canonical ifn-i pathway results in activation of the janus kinase (jak) family (cytoplasmic tyrosine kinases) that, in turn, activate by phosphorylation the signal transducers and activators of transcription (stats) [15] . activated stats complex with irf9 to form the heterocomplex ifn-stimulated gene factor 3 (isgf3), that translocates in the nucleus, binds to upstream sequence elements named ifn-stimulated response elements (isre), and activates the transcription of isgs (fig. 2) . these genes act to promote viral clearance and establish an antiviral state in uninfected bystander cells, or to induce apoptosis and several anti-microbial mechanisms in infected cells. they also stimulate cells at the interface of innate and adaptive immunity, such as macrophages and dendritic cells (dcs) that trigger the adaptive response [16, 17] . the ability to break in innate immunity before the onset of the adaptive response is, thus, crucial for the survival of virtually all mammalian pathogens. on the other side of the coin, an essential part of the early response to pathogens is aimed at limiting their ability to hijack host cellular machinery and evade the ifnmediated antimicrobial mechanisms. nowadays, while it is largely accepted that also bacteria can induce the production of ifn-i, the role of the ifn system in the pathogenesis of bacterial infections can be either detrimental (mainly in intracellular bacterial infections) or protective (mainly in extracellular bacterial infections). the route and tropism of bacterial infection, viral co-infections and last, but not least, the balance between ifn-i and ifn-ii effects are all determinants of the different outcome [18] . the ratio between ifn-i and ii species produced in response to infection, might differ as consequence of the capacity of the pathogen to stimulate specific cell types in the infected tissues. moreover, taking into consideration that several antibacterial ifn-ii-induced genes are stimulated to a much lesser degree by ifn-i, it is quite difficult in vivo to separate the activities strictly dependent on one of the two ifn families. a more reliable view consists of a crosstalk between the two pathways: if the bacterium is able to stimulate ifn-i production, generally it occurs early after the infection as an immediate innate immune response, while ifn-ii intervenes later on when immune cells (such as t cell subsets and nk cells) are activated. also due to this complex picture, while a number of viral evasion strategies have been mechanistically defined so far, studies aimed at characterizing the bacterial components that inhibit the ifn system are only recently starting to be elucidated in molecular details [19] [20] [21] . main strategies that pathogens have evolved to disarm the ifn-i response include: (i) blocking ifn-i production by modifying, curtailing or limiting production of their pamps to make them inaccessible to prrs and/or hitting the components of prrs signaling pathways; (ii) interfering with the ifn-i signaling by inhibiting signal transducers; (iii) blocking or disturbing the action of isgs; (iv) hijacking host proteins or components of the ifn system. each of these strategies involves a number of different molecular mechanisms and the combination of more strategies may be necessary to overcome the ifn-i response by a single pathogen. depending on the nature of the pathogen, these countermeasures that tip the balance toward the pathogen, may result in increased replication or in the establishment of a persistent infection. leaving out strategies that envision ifn-i repression due to a general inhibition of cellular gene expression, reviewed elsewhere [22, 23] , here we summarize recent findings on evasion tricks utilized by pathogens to specifically subvert the ifn system. a special focus is put on few highly pathogenic bacteria and emerging or re-emerging viruses, which represent a major threat to human health due to the lack of effective vaccines and/or therapeutics. for an in-depth coverage of other pathogens and strategies to escape the host immune response, the reader is referred to more comprehensive and specific reviews [19] [20] [21] [23] [24] [25] [26] . recent years have seen major advances in our understanding of the innate response to infectious pathogens and specifically of how pathogen recognition promotes ifn-i release [8, 27, 28] . cytoplasmic and membrane-associated prrs recognize a variety of pathogen components. viral pamps mainly consist of nucleic acids originating from the uncoating of infecting virions, the transcription of viral genomes and the replication of genomic intermediates, in the form of single-stranded (ss) and double-stranded (ds) dna, and ss and ds rna, as well as viral glycoproteins. bacterial pamps include various molecules, ranging from lipoproteins, lipopolysaccharide (lps), flagellin and peptidoglycan to unique bacterial nucleic acid structures, such as cyclic dinucleotides (cdns). multiple endosome-associated tlrs (tlr3, 7, 8 and 9) are specialized in the detection of viral and bacterial nucleic acids. tlr3 recognizes dsrna, tlr7/8 are bound by ssrna and tlr9 by cpgcontaining dna. tlr2, 4 and 13, previously considered sensors only for bacterial components, are now been involved also in recognition of viral ligands and in the induction of ifn-i [29] . all tlrs contain an intracellular domain, the toll-interleukin-1 receptor (tir), which recruits one or more tir-containing adaptor proteins to transmit signals downstream. tlr3 signals through the adaptor tir domaincontaining adaptor inducing ifn-␤ (trif) to activate the two related kinases, inhibitor of kb kinase (ikk)-and tank-binding kinase 1 (tbk1) that mediates activation by phosphorylation of irf3 and irf7. tlr7/8 and tlr9 use, as adaptor, myeloid differentiation primary-response protein 88 (myd88) that then initiates signaling cascades involving il-1r-associated kinases (irak1-4) and tnfrassociated factor (traf) 3/6 proteins, which finally converge at the activation of the ib kinase (ikk) family members ikk-␣, ikk-␤, ikk-and tbk1 responsible for activation of nf-b and irf3/7 [4] . in the cytoplasm, two closely related helicases, retinoic acidinducible gene 1 (rig-i) and melanoma differentiation-associated gene-5 (mda5), recognize dsrna of many replicating viruses in a tlr-independent manner. rig-i preferentially senses short dsrna and ssrna with a 5 -triphosphate (5 -ppp rna), while mda5 recognizes long dsrna and poly i:c [30] [31] [32] . like viral rnas, bacterial rnas can possess 5 -ppp termini and secondary structures that make them rig-i agonists. consistent with this notion, rig-i can act as a sensor of bacterial rna and may help maintain homeostasis to gut microbiota [33, 34] . upon recognition of non-self rna, rig-i and mda5 are recruited to the mitochondrial antiviral signaling protein (mavs; also known as cardif, visa or ips1), which triggers a signaling cascade that leads to the activation of ikk-and tbk1 and, in turn, irf3/7 phosphorylation [35] . stimulator of ifn genes (sting), initially identified as a cytosolic dna sensor, also participates in the rig-i signalling [36] . sting interacts with the adaptor mavs at the mitochondrial associated membranes (mam) facilitating the recruitment of tbk1 and the activation of irf3 [37] . a more general role of sting in innate immune responses, not only limited to its function as adaptor of rna and dna sensors, has been now established [38] . in addition to these rna sensors, viral rnas may also be recognized by effector molecules that are themselves ifn-induced proteins with antiviral functions. these molecules include dsrnaactivated protein kinase (pkr) that binds and is activated by dsrna from viruses and bacteria [39] , ifn-induced tetratricopeptide repeat proteins 1/2/3 (ifit1, ifit2, and ifit3) that, as rig-i, bind 5 -ppp rna and may recognize viral mrnas that lack 2 -omethylation [1, 40] . a growing list of cytosolic dna sensors then recognizes dna from different sources including viruses, bacteria and apoptotic cells [3, 6, 7] . more than ten dna cytosolic receptors have been proposed so far that include dai (dna-dependent activator of irf), rna polymerase-iii, ifn-inducible interferon gamma-interferoninducible protein 16 (ifi16), sting, extrachromosomal histone h2b, leucine rich repeat (in flii) interacting protein (lrrfip1), ku70, deah box protein (dhx) 9 and dhx36, cyclic gmp-amp synthase (c-gas). as other sensors, dna sensor activation results in the production of ifn-i and proinflammatory cytokines and chemokines via the sting-tbk1-irf3 axis. in addition to the activation of irf3-and nf-b-dependent signaling cascades, cytosolic dna can also promote an apoptosis-associated speck-like protein containing a card (asc)-dependent inflammasome-mediated response resulting in the secretion of proinflammatory cytokines [3, 7, 25, 27, 28] . the intracellular nlrs scaffold large signaling complexes to mediate innate immunity and inflammatory responses. they may trigger the assembly of inflammasomes and modulate the nf-b, mapk and irf signaling pathways. in particular, nod1 and nod2 are important for immune detection of intracellular bacterial pathogens and are also involved in a variety of immune homeostatic functions [41] . upon the recognition of bacterial peptidoglycans by nod1 and nod2, receptor-interacting serine-threonine kinase 2 (rip2) is activated via cellular inhibitors of apoptosis 1 and 2 (ciap1 and 2), subsequently leading to ubiquitination of nf-b essential modulator (nemo) and the activation of the proinflammatory nf-b pathway. in parallel, the recognition of muramyl dipeptide present in all peptidoglycans, can also lead to the activation of mapk pathway via rip2, which contributes to cytokine production. for the induction of ifn-i, nod2 activated by viral rna, signals through the mitochondrial mavs independently of rip2. then, some sensors can aggregate with adaptors as apoptosisassociated speck-like protein containing a caspase recruitment domain (asc) and caspase 1 to form multimeric structures named inflammasome [42] (fig. 1) . as first line strategy to face these sensing pathways, pathogens hide detection by modifying their pamps. they may also degrade/inactivate target key signal transduction hubs by counteracting host-induced post-translational modifications required for signaling molecule activity and use concomitant different strategies as better described below and also discussed by thomas kufer and igor brodsky in this issue. we focus here on some members of coronaviruses, flaviviruses, hepaciviruses, filoviruses and retroviruses as well as bacteria whose pamps could in principle activate the ifn system, but that efficiently betray host sensing and effector pathways (fig. 3) . coronaviruses (cov) are large enveloped rna viruses, in the family coronaviridae, of both veterinary and clinical importance. two newly emerging viruses in the family, the severe acute respiratory syndrome cov (sars-cov) and the middle east respiratory syndrome cov (mers-cov), have been recently responsible for severe disease in humans (reviewed in [43] [44] [45] [46] [47] ). a common trait in their pathogenesis is the lack of induction of a robust ifn-i response in infected cells [48, 49] . to do this, cov have developed multiple strategies (recently reviewed in [50, 51] ). rig-i, mda5 and the host isgs ifit1 and 2 are critically involved in sensing of cov infection. however, as first line of hiding from recognition, cov encode several highly conserved nonstructural proteins (nsps) implicated in viral rna capping activity in order to examples of viral and bacterial antagonists that block subvert or exploit the ifn system. pathogens affect at every step the ifn system by multiple mechanisms. sites of intervention by several antagonists are indicated. ifn antagonists may prevent prr recognition by hiding or modifying pamps, may inhibit prr signaling by directly targeting adaptors and signaling effectors, may interfere with the ifn signaling by impairing signaling transducers and may block or disturb the action of isgs. some antagonists have more than one cellular target while others target common signaling molecules, effectively blocking ifn induction from a variety of pamps. see the text for more details and references. mimick n7-and 2 -o-methylated 5 cap structure of cellular mrnas [52] . these viral proteins include a rna-triphosphatase, a guanine-n7-methyltransferase, and a 2 -o-methyltransferase encoded by nsp13, 14 and 16, respectively [50, 53] . consistently, human and mouse cov mutants lacking 2 -o-methyltransferase activity induce higher expression of ifn-i and are highly sensitive to ifn-i effects [54, 55] . sars-cov nsp14 also encode a 3 ,5 exoribonuclease that is involved in rna-proofreading, but probably it also functions in degrading viral pamps, further hiding immune detection [50, 56] . similarly, nsp11 encodes a ribonuclease that may degrade viral pamps [57] . another strategy used to avoid detection is the replication in protected sites as the double membrane vesicles (dmvs) that the virus induces in the host cytoplasm. in the case of sars-cov these membranes contain the replicase complex and the viral genomic rna suggesting that replication occurs in these sites and the generated nucleic acids are soon after shielded [58, 59] . in addition, the highly basic nucleocapsid (n) protein of sars-cov has been reported to directly inhibit ifn-i production induced by both poly(i:c) or sendai virus, by a mechanism that involves steps upstream rig-i. thus, it is conceivable that by binding dsrna, n protein prevents rig-i/mda5 activation [60] . others sars-covencoded proteins, as orf-3b and orf6, may also interfere with the rlr recruitment of the adaptor mavs based on their preferential localization at the mitochondrial membrane even if their mechanism of action is not yet elucidated [61, 62] . the sars-cov membrane (m) protein blocks transcription of ifn-i when stimulated by dsrna or members of the rig-i signaling pathway including rig-i, mavs, ikk-, and tbk1, but it does not influence the transcriptional activity of the ifn promoter when irf3 or irf7 are overexpressed. the physical association of sars-cov m with rig-i, tbk1, ikk-, and traf3 suggests that m protein may prevent the formation of the functional complex with tbk1 thereby inhibiting activation of irf3/irf7 and ifn-i transcription [63] . similarly, the papain-like protease (plp) domain contained in the sars-cov nsp3 protein, an essential component of the viral replicase complex, interacts with the adaptor sting blocking the binding to mavs and the recruitment of the tbk1/irf3 complex [64] [65] [66] . finally, through its deubiquitinating activity, the plp protein removes ubiquitin from several components of the rlr pathway blocking their activation (reviewed in [50, 51, 67, 68] ). recently, two mers-cov accessory proteins, the orf-4a and orf-4b products, have also been identified as immunosuppressive factors. mers-cov 4a is a rna-binding protein that interacts in a mrna-dependent manner with pkr-associated activator (pact), a cellular dsrna-binding protein, which potently stimulates rig-iinduced ifn production by binding to the c-terminal repression domain of rig-i [69] . so, orf-4a inhibits rig-i/mda5 pathway without a direct binding to the sensor, but by perturbing the function of a stimulator of rig-i signaling as pact [70] . the orf-4bencoded accessory protein is also able to inhibit ifn-i induction in vitro, however, the mechanism involved is not yet elucidated [71] . the genus flavivirus, of the flaviridae family, includes west nile virus (wnv), dengue virus (denv), japanese encephalitis virus (jev), yellow fever virus (yfv), tick-borne encephalitis virus (tbev) and several other viruses all causing serious medical problems in humans [72] [73] [74] [75] . currently, vaccines for humans are available only for yfv, jev, and tbev and no clinically approved antiviral therapy is available for the treatment of flavivirus infections [75] . in particular, denv and wnv are re-emerging as global life-threatening human pathogens [75, 76] . both viruses are sensitive to ifn antiviral effects and the severity of the disease is mostly dependent on the ability to avoid and/or attenuate induction of ifn-i and its effector responses through several viral encoded ifn-antagonists [73, 74] . interestingly, some of these strategies are shared with cov. the most relevant prrs for the detection of wnv and denv products, described so far, are the tlr3, 7, 8 and the rlrs, rig-i/mda-5. suppression of both tlr-and rlr-mediated ifn induction has been shown to be important for viral replication [77, 78] . as cov, flaviviruses contain a cap structure that is generated by a methyltransferase mapped to the n-terminal region of the ns5 protein [74, 79] . through 2 -o-methylation of the viral mrna cap wnv, denv and jev evade ifit1-dependent and -independent mechanisms of host restriction in vitro and in vivo [80, 81] . moreover, to hide their nucleic acids during the replicative cycle, both denv and wnv induce the formation of convoluted membranes in the endoplasmic reticulum (er) and golgi apparatus that envelop the virus replication complex [82, 83] protecting viral nucleic acids from both tlr and rlr recognition, as it occurs along the infection with cov. so far, an antagonism at the level of sensing signaling has been clearly defined only for denv that fails to induce an ifn response in myeloid cells where it replicates, despite other pro-inflammatory cytokines and chemokines are produced [84, 85] . the ability to inhibit ifn-i production is due to the viral ns2b/3 protease that binds and cleaves the adaptor/sensor sting [85] [86] [87] . interestingly, first evidences indicate that the ns2b/3 proteases of other flaviviruses, as jev or yfv, are not able to cleave sting, while the ns4b of yfv can do it [68] . to the same flaviviridae family belongs the hepacivirus genus, distantly related to flaviviruses, that includes hepatitis c virus (hcv) a major cause of chronic liver disease [88, 89] . hcv uses several strategies to efficiently evade innate immunity and this escape is considered the main determinant of viral persistence that leads to a chronic infection in 70-80% of infected people [88, 90] . hcv rna can be sensed by different prrs, namely rlrs, tlrs, nlrs, and, as recently reported, by protein kinase r (pkr). rig-i is the best described sensor for the poly u/uc region located within the 3untranslated region (utr) of the viral rna, along with a 5 -ppp. this region is essential for viral replication and, thus, highly conserved among hcv genotypes. the key viral protein involved in the evasion strategies is the ns3/4a protease, which consists of ns3 and ns4a. the complex is essential for several steps in the viral cycle including viral rna replication, polyprotein processing and viral assembly [91] . taking advantage of its serine protease activity, ns3/4a cleaves the adaptor mavs, preventing its dimerization and downstream signaling [92, 93] . after cleavage, mavs dissociates from the mitochondrial associated endoplasmic reticulum membranes (mam) where upon hcv-induced rig-i activation it is recruited and colocalizes with ikk[94] , thus impairing ifn-i expression [95] . importantly, the cleavage of mavs by the viral protease has been confirmed in patients [96] . to antagonize ifn-i production, ns4b protein instead targets sting. hcv ns4b, indeed, contains a sting homology domain and interacts with sting in the er blocking sting interaction with mavs and tbk1 [97, 98] . even if the exact molecular mechanism involved in ns4b inhibition of sting signaling has not yet been defined in the context of viral infection, ns4b likely cooperates with ns3/4a in targeting the rig-i signaling pathway. interestingly, ns2b/3 and ns4b of other members of the flaviviridae family including denv and yfv as mentioned above also block sting signaling and possess the same sting homology domain, indicating a conserved mechanism of sting antagonisms between flaviviruses and hepaciviruses. intracellularly, both tlr3 and tlr7 have been shown to sense hcv rna, depending on the infected cell type considered [88] . sensing by tlr3 may occur in liver cells, as hepatocytes, and liver resident macrophages kupffer cells, while tlr7 sensing occurs predominantly in plasmacytoid dcs (pdcs) and macrophages. as reported above for the mavs adaptor, the serine protease ns3/4a also cleaves the key adaptor of tlr3, trif [99] , thus preventing an ifn response in productively-infected hepatocytes. in these cells, the hcv-induced mir-21 has been recently reported to be involved in evasion of ifn-i production and stimulation of hcv replication, upon suppression of myd88 and irak1 expression, that is required for the tlr7-mediated sensing of the virus [100] . in macrophages, myd88 signaling is instead targeted by the ns5a protein that, upon the direct binding to the adaptor, impairs the recruitment of irak-1 and cytokine production in response to tlr ligands [101] . interestingly, tlr3 and tlr7 levels are decreased in patients chronically infected with hcv [102, 103] and this has been recently correlated with increased levels of mir-758 [104] . hcv-infected cells, however, can trigger ifn-i production in non-productively-infected pdcs in a cell-cell contact-and tlr7dependent manner depending on the intracellular hcv rna level of cocultured infected cells [105] . this production of ifn-i by pdcs may thus account for the strong ifn-i response observed in the liver of infected people [106] . interestingly, a robust expression of isgs correlated with a decreased response to ifn therapy [107] supporting a pathogenetic role of a high ifn signature in chronically infected individuals where the virus has established a persistent infection. in contrast, in monocytes and macrophages upon clathrinmediated endocytosis and recognition of the virus by tlr7, hcv activates the inflammasome and not ifn-i production in an infection-independent process [108] . an association between tlr-polymorphisms and cytokine production in response to tlr7 agonist in vitro has also been reported, supporting a pathogenic role of tlr7-mediated sensing in immune cells [109] . interestingly, this cell-type dependent stimulation of ifn-i and inflammasome in response to hcv infection is also observed in human immunodeficiency virus type 1 (hiv-1) infection that as hcv can establish a persistent infection (see discussion below). pkr has been shown to sense hcv rna very early in infection even prior to rig-i sensing [110] . pkr is a dsrna binding protein that upon activation phosphorylates the ␣ subunit of the eukaryotic translation initiation factor 2 (eif2 ␣) to inhibit translation of host capped mrna but not of non-capped mrna, as that of hcv. pkr, upon binding hcv rna and independently of its kinase-activity, interacts with mavs to induce the transcription of a number of early isgs including ifn stimulated gene 15 (isg15), but not ifn-i. isg15, in turn, deubiquitinates rig-i inhibiting its functions [110] . by doing so, hcv blocks sensing by pkr and reinforces hcv evasion from rig-1 signaling. amongst rna viruses that, as hcv, can establish a persistent infection, hiv-1, a lentivirus from the retroviridae family, represents a paradigm for its ability to prevent or circumvent the innate immune response mediated by ifn-i. in spite of evidence that a sustained ifn-i response occurs in hiv-infected patients, it fails to clear the infection in the first place and to prevent the early establishment of long-lived hiv-1 reservoirs [111] [112] [113] . hiv-1 has, indeed, developed a number of strategies to block the ifn signaling and the activity of ifn-induced host restriction factors. here, we only briefly summarize these strategies some of which have been only recently discovered, leading to the identification of immune pathways, thus far, unrecognized (as recently reviewed elsewhere [114] [115] [116] [117] [118] [119] [120] ). in the past few years, the knowledge on innate immunity against hiv-1 has evolved enormously with the recent identification and the characterization of the molecular basis of retroviral recognition by prrs [116, 118, 121] . retroviral replication generates several structural and intermediate molecules as ssrna, hybrids rna/dna, ss and ds dna produced upon reverse transcriptase, that are potentially available for recognition by cellular prrs as "non-self". with the exception of pdcs, which produce high levels of ifn-i upon detection of hiv-1 ssrna by tlr7, in all other immune cells ifn-i production is prevented or barely detectable unless viral countermeasures are disabled. in conventional dcs (cdcs), monocytes and resting cd4 + t cells, indeed, hiv-1 sensing is prevented by the restriction factor sterile alpha motif (sam) and the hystidine/aspartic acid (hd) domain-containing protein1 (samhd1) that blocks reverse transcription or directly degrades genomic rna, thus preventing prr recognition [122, 123] . this samhd1mediated restriction is overcome by the viral protein vpx [124, 125] that is a hiv-2 and siv accessory protein absent in hiv-1. this apparent disadvantage is, however, effectively exploited by the virus that maintains a reward by not replicating in myeloid cells and by reducing the impact of ifn-i production in these cells. the block of productive infection in non-cycling cells where samdh1 is active, particularly in cdcs, results in lack of maturation and thus in impairment in priming of naive, hiv-1-specific t cells for optimal anti-hiv-1 immunity [126] . furthermore, in the small fraction of cdcs that become infected, the recognition of hiv-1 genomic rna by tlr8 paradoxically licenses hiv-1 transcription [127] . in this case, productive dc infection allows an increased transmission to t cells while inhibiting ifn-i production. in monocytes instead, recognition of viral rna by tlr8, does not trigger ifn-i production but, as in the case of hcv, leads to the formation of the nlrp3 inflammasome with activation of caspase-1 and il-1␤ production, favoring the establishment of an inflammatory milieu that fuels hiv-1 replication [108, 126] . in contrast, in cells that are target of a productive infection, such as macrophages and t lymphocytes, ifn-i production is prevented by an escape mechanism mediated by the hiv-1 protease that drives rig-i to the lysosomes [128] . the ssdna derived from proviral dna upon rt can, instead, be sensed by the newly identified dna sensor interferon gammainterferon-inducible protein 16 (ifi16) [129] , however, hiv exploits the host cytosolic nuclease 3 repair exonuclease1 (trex1) to digest hiv-1 dna generated during infection that, thus, does not accumulate at levels sufficient to be detected by ifi16, unless trex1 activity is blocked [130] . finally, resting cd4 + t cells are not permissive to virus replication due to the expression of an active samhd1 that, as mentioned before, degrades genomic rna and prevents efficient rt and recognition of ssdna. however, this prevention of sensing may not be complete and partial recognition of rt intermediates by the ifi16 sensor not only leads to the initiation of ifn-i production, but also to the activation of the inflammasome, triggering cell death mechanisms including pyroptosis and apoptosis [131] . finally, the viral capsid exploits two cellular proteins, cyclophilin a and cpsf6, and binds just a right amount of both to allow opening of the capsid and rt process, while preventing sensing of the viral cdna before integration with the following production of ifn-i [132, 133] . overall, viruses as hcv and hiv-1 have evolved nifty strategies to dampen the host innate response in cells where a productive infection may take place, while they induce infection-independent mechanisms in non-permissive cells to facilitate the viral life cycle and promote a chronic inflammation. the genus filoviruses from the filoviridae family are among the most virulent known human pathogens and comprise one species of marburg virus and five species of ebola viruses, including zaire ebolavirus (ebov) that is the most lethal and responsible of the recent severe outbreak of hemorrhagic fevers causing up to 90% mortality in untreated humans [134] . several immunoevasion strategies that result in total impairment of the innate immune system are responsible for most of ebov virulence [135] . a major target of these strategies that are exerted by few viral proteins, is the ifn-i response that controls in vivo filoviruses infection [136] . at the level of viral sensing, the ebov vp35 inhibits rig-i signaling [137, 138] . vp35 binds with high affinity to dsrna and 5 -ppp dsrna in a sequence-independent manner. four crystallographic structures of ebov vp35 rbd/iid from zaire and reston viruses and of marv vp35 rbd/iid have elucidated how vp35 rbd/iid dimers bind to rna strands and how the dimers mimic the rlr shape, hiding the rna recognition site [139, 140] . interestingly, the residues involved in both protein-protein interactions at the rbd/iid dimer interface and involved in dsrna binding are highly conserved among all known ebov and marv species. moreover, all residues that are important for dsrna binding are also crucial for ifn-i inhibition. as the knowledge on the importance of ifn-i in controlling the immunity against bacteria increases, studies aimed at characterizing the evasion mechanisms that these pathogens employ to evade, inhibit, or otherwise manipulate the innate immune response are arising. the mechanisms induced by bacteria for ifn-i expression in different cell types are various and reflect the heterogeneity of host-pathogen interactions established along bacterial infections. while the extracellular bacteria activate ifn signaling mainly through the interaction with molecules present on the cell surface, the intracellular bacteria are recognized as they enter a cell by cell-surface or endosome/phagosome bound receptors or by cytoplasmic pathogen sensors once they escape from these compartments [141] . here, we report only few examples of bacterial evasion mostly related to signaling pathways converging in ifn-i stimulation. a more exhaustive discussion of bacterial evasion strategies from prr-signaling pathways and inflammasome is provided by the contribution of thomas kufer and igor brodsky in this issue. to elude prr recognition many bacterial pathogens, like viruses, have modified the molecular structure of their pamps. lps, an ubiquitous component of gram-negative bacterial cell wall, is a pamp that is recognized by the tlr4/md-2 complex present on the cell surface. some bacterial species have evolved an alternative form of lps resulting in a weak antagonist of tlr4/md-2 signaling. this strategy is utilized by yersinia pestis, the causative agent of pestis infection. the acylation status of lipid a from hexa-to tetra-acylated is reduced when the temperature increases from 21 • c (flea temperature) to 37 • c (human temperature). this alteration renders lipid a less recognizable lps by tlr4 and, following transmission from fleas to humans, contributes predominantly to the virulence of the bacterium [142] . another example is shigella that, after internalization and proliferation within epithelial cells, hypoacylates lipid a to become less visible to the immune system once leave the infected epithelial cells [143] . in addition to lps, other bacterial components that may induce ifn-i, include bacterial nucleic acids and peptidoglycans. these pamps can be recognized outside or inside the host cells, leading to the activation of distinct signaling pathways [141] . intracellularly bacterial rna and dna nucleic acids are recognized by the several intracellular receptors that also sense viral pamps, while bacterial peptidoglycans are detected by nod1 and nod2 system. most of these pathways then converge in the activation of the sting/tbk1/irf3 axis [6] . as viruses, bacteria also encode proteins with enzymatic activity that interfere with the activation of adaptor molecules involved in prr signaling. this is the case of the yersinia pestis virulence factor yopj that, besides being a potent inhibitor of the nf-b and mapk signaling pathways, also inhibits tlr-mediated ifn response. as a deubiquitinating protease, yopj prevents or removes the k63polymerized ubiquitin conjugates, which are required for traf3 and traf6 activation in the signal transduction pathway leading to irf3 activation [144] . similarly, the type iii effector ospi of shigella flexneri inactivates by deamination the e2 ubiquitin ligase ubc13, a factor important for traf6 auto-polyubiquitinylation and activation [145] . another interesting example is represented by the translocated intimin receptor (tir), which is one of the first type iii effector proteins discovered in a/e pathogens including the enteropathogenic e. coli (epec), enterohemorrhagic e. coli o157:h7 (ehec), and citrobacter rodentium. in addition to the role played in the attachment to the host membrane, this factor shares sequence similarities with conserved regions present in the cytoplasmic tails of inhibitory receptors of the host immune system, such as the immunoreceptor tyrosine-based inhibition motifs (itims). tir utilizes these itimlike motifs to mimic an endogenous innate immunoregulatory mechanism. in particular, tir recruits the host src-homologyregion-2-domain-containing phosphatase 1 to the adaptor traf6 and thus prevents polyubiquitinylation and activation of traf6 in the ifn-stimulated pathway [146] . adaptor molecules downstream sensors transmit signals to classical ib kinase complex, including nemo/ikk-␥, tank and to the atypical ikk-related kinases ikk-and tbk1 that trigger activation of nf-b and irfs, respectively [147, 148] . ifn-i production downstream of these signaling pathways depends essentially on the presence and activation of irfs and their contribution changes depending on the cell type considered [9] . the irf family is presently composed of nine mammalian members namely irf1 to 9, coded by distinct but related genes that exert a number of functions in the regulation of innate and adaptive immune responses. the irfs with intrinsic antiviral function include irf3 and irf7 that are essential for the prr-mediated ifn gene transcription, but also induce some ifn effectors in an ifn-independent manner. irf9, as mentioned before, is part of the heterocomplex isgf3 that drives the expression of most isgs including irf1 (fig. 2) . although irf1 is itself an isgs, which affects different aspects of the immune response even independently from ifn-i production [149, 150] , it also represents a positive regulator of ifn-i gene expression in response to specific stimuli in a cell type specific manner [151] . moreover, irf1 plays a crucial role in regulating mavs-dependent signaling from peroxisomes [152] . in this respect, irf1 regulates the transcriptional profile of antiviral genes unique to that induced by ifn-i and cooperatively promotes an effective antiviral program against a broad spectrum of viruses [152, 153] . irf5 is instead specifically involved in inflammatory cytokines induction [9] . given the unique functions exerted by irfs, viruses have evolved strategies aimed at the specific destruction of these transcription factors. with regard to the viruses covered here, most of them inhibit irf activation either indirectly by acting on sensors and elements of the signaling pathway that activate them, as described above, or directly by impairing/hijacking irf activity (fig. 3) . as described above, the sars-cov plp affects the activation of both irf3 and nf-b not directly, as initially suggested [66, 154] , but by targeting rig-i, mavs, traf3 [63] and, as more recently reported, sting [64, 65] . interestingly, this activity is independent from plp protease activity. recently, it has been reported that the plp of mers-cov also suppresses ifn-i transcription by interfering with irf3 phosphorylation and nuclear translocation [155] . as sars-cov, mers-cov plp is a viral deubiquitinating enzyme that acts on both k48-and k63-linked ubiquitination and isg15-linked isgylation, two posttranslational modifications that play important roles in regulating the rig-i and sting/irf3 and nf-b activation [156] . whether the deubiquitination and deisgylation activity of mers-cov plp are directly responsible for inactivation of irf3/nf-b or upstream signaling pathway, it remains unclear. the wnv ns1 protein inhibits the tlr3-induced activation of ifn-i and il-6 transcription through inhibition of nuclear translocation of irf3 and nf-b [157] . recent studies indicate that this effect seems to be dependent on ns1 domains that control viral replication [158] . interestingly, in the draining lymph nodes the protein released predominantly from macrophages and dcs can inhibit the innate immune signaling pathways in uninfected cells and impairs cytokine production in response to infection [159] , thus suggesting that ns1 could also influence the development of the adaptive immune response directed to wnv. the ns2b/3 serine protease of denv, instead, blocks the serine 386 phosphorylation and nuclear translocation of irf3 by directly interacting with ikk-and masking the kinase domain [160] . two tick-borne flaviviruses, lgtv and tbev, have been recently reported to inhibit irf1 independently of their ability to antagonize ifn signaling. in particular, a weak expression of irf1 protein and nuclear localization, without reduction in irf1 mrna expression, was observed in dcs, an early cellular target of infection [161] . several hcv proteins interfere with irf activity. the hcv ns3 protease impairs irf3 activation by blocking the interaction with tbk1 and irf3 [162] . in addition, the ns2 protein inhibits, in a dose-dependent manner, ikk--and especially tbk1-induced irf3 phosphorylation [163] . the basic amino acid region 1 (br1) in the n-terminal region of the core protein is also crucial in inhibiting irf3 dimerization as well as phosphorylation induced by ndv infection and poly (i:c) [164] . interestingly, this domain has been identified as the binding region for a dead box protein the ddx3, which has been recently found to enhance the tbk1/ikk--induced ifn-␤ promoter activity upon binding to the adaptor mavs [165] . the hcv core also decreased the expression levels of ddx3 suggesting that the irf3 inhibition may be mediated by the core effect on ddx3. moreover, through binding to ddx3, the hcv core protein also promotes hcv replication. thus, the core protein appears to switch ddx3 from an ifn-inducing mode to an hcv-replication mode [166] . irf1 expression is, instead, suppressed by the hcv core at the transcriptional level. this event blocks the expression of several antiviral and immunomodulatory genes of both innate and adaptive immunity and, in doing so, facilitates the establishment of hcv persistent infection [167] . in line with this hypothesis, accumulating evidence suggests that hcv also targets dcs to control the host antiviral response and trigger persistence. as wnv ns1, hcv core is, indeed, a secreted protein found in the peripheral blood of patients with chronic infection that may thus affect directly dc functions. in this context, it has been recently reported that the core protein suppresses ifn-i production in response to tlr agonists and to rig-i stimulated by hcv pamp in a cell culture model of pdcs, through the reduced levels of irf7 and of phosphorylated stat1 protein [168] . the effect on irf7 is, however, not direct but probably mediated by the reduced levels of ifn-i production by core-stimulated pdcs. whether or not this also occurs along the natural infection and contributes to hcv persistency remains to be determined. to increase virus replication and establish viral persistence and latency, hiv-1, besides to dismantle or exploit almost all cell intrinsic innate recognition pathways, as discussed above, also directly hits irfs [113, 119, 169] . in t cells, the virion-associated accessory proteins, vif, vpr and vpu, directly target irf3 for ubiquitin-associated proteasome degradation [170] [171] [172] . recently, this vpu effect on irf3 degradation has been, however, challenged and it has been reported that vpu, instead, mediates a partial cleavage of irf3 in a caspase-dependent manner. interestingly, this cleavage produces a c-terminal fragment that can act as a negative regulator of irf3-dependent gene activation [173] . thus, hiv-1, as already reported for several viruses, can also exploit the apoptotic machinery to interfere with irf3 function [174] . in myeloid cells, instead, hiv-1 does not inhibit but, rather, stimulates both irf1 and irf7 expression. irf1 activity is, however, exploited by the virus to induce a distinct subset of isgs that despite displays intrinsic and unique antiviral actions, does not restrict viral infection [175] . irf1 is also induced in hiv-1-productively infected t cells where it may regulate viral promoter activity even in the absence of the viral transactivator tat driving initial transcription of the viral genome [176, 177] . later on, however, when viral replication is mostly accomplished by the viral transactivator, irf1 is sequestered by tat to accelerate proteasomal-mediated irf1 degradation (remoli al and battistini a, unpublished) and to quench irf1 transcriptional activity on target genes [178] . by so doing, tat disarms the unique antiviral response against viral infections that irf1 could exert [153] . a block of ifn transcription in primary cd4 + t cells may also depend on cd3/cd28-mediated activation of ikk-. we, indeed, recently reported that ikk-activation results in a peculiar pattern of irf phosphorylation in t cells, including a splicing isoform of irf3, which may function as an inhibitor of ifn-␤ expression, and phosphorylation of irf1 that blocks its activity on ifn-i promoter [179] . the ebov vp35, in addition to prevent rlr recognition, inhibits ifn-i promoter activation mediated by tbk-1/ikk-overexpression but not by a constitutively active irf3, strongly suggesting a specific inhibition of the kinase activity of tbk-1/ikk-. indeed, vp35 interacts with the tbk-1/ikk-kinase domain and functions as a substitute substrate, thus inhibiting both the kinase activity and the binding of the physiological irf3/7 substrate [180, 181] . notably, mutations of vp35 residues, involved in this ifn antagonism, do not alter the function of vp35 in viral replication and transcription [182] . in dcs, vp35 targets irf7. by interacting with the small ubiquitinlike modifier sumo e2 enzyme ubc9 and the e3 ligase pias1, vp35 promotes irf7 sumoylation, a post-translational modification that prevents irf translocation into the nucleus and, in turn, ifn-i gene transcription. a similar effect of vp35 was also reported for irf3 [183] . this vp35 activity is independent of its ability to recognize dsrna and maps to the n-terminus, which is essential for interactions with both irf7 and pias1. interestingly, sumo modification of irf3/7 is a part of the negative feedback loop of normal ifn-i signaling [184] that is exploited by ebov to weaken host innate immunity. downstream prrs, bacteria mainly target the mapk pathway and the nemo-ikk-nf-b signaling axis, which primarily induces inflammatory cytokines (reviewed in [19] and thomas kuper and igor brodsky in this issue). as an example of bacteria that target the irf pathway. listeria monocytogenes suppresses ifn-i gene induction downstream of tlr-triggered myd88 signaling pathway, acting on irf3. indeed, a mapk phosphatase renders irf3 hypophosphorylated by enhancing the formation of a mapk phosphatase-irf3-tbk-1 ternary complex in response to infection [185] . in fig. 2 is illustrated the ifn-i signaling pathway downstream the ifn-i receptor (ifnar) (a heterodimer of ifnar1 and ifnar2) that is activated upon binding of virus-infected cell-secreted ifn-i, and some isgs, including positive and negative feed-back regulators. most of the viruses here covered and some bacteria also target these pathways (fig. 3) . upstream the jak/stat pathway, the sars-cov 3a promotes serine phosphorylation within the ifnar1 degradation motif and increases ifnar1 ubiquitination [186] . the plp from sars-cov has a complex mechanism of interference with the jak/stat pathway. through its de-ubiquitinase activity upregulates the expression of the ubiquitinating enzyme e2-25k, leading to degradation of the erk kinase that, in turn, interferes with stat1 phosphorylation [187] . the orf6-encoded protein, instead, antagonizes stat1 function by interacting and sequestering in the er components of the nuclear import complex, as karyopherin alpha 2 and karyopherin beta 1. by doing so, orf-6 competes for the binding of the nuclear import complex to stat1, thus inhibiting stat1 nuclear import [188] . however, the majority of these evidences have been obtained by overexpression or stably expression of individual viral components in cell culture, which represents an experimental setting that may not accurately reflect the innate immune signaling occurring during sars-cov infection in vivo. wnv interferes with ifnar complex by promoting phosphorylation-dependent ubiquitination and degradation of ifnar1. this effect is mediated by the hydrophobic ns4a and ns4b proteins that potently induce the unfolded protein response (upr). this pathway is physiologically induced by different stimuli, including the accumulation of misfolded proteins in the er [189, 190] that, as mentioned before, represents the site of flaviviruses replication. activation of the upr pathway inhibits ifn activation and induces a general er stress response, thus facilitating viral replication. the methyltransferase domain of ns5 from langat virus and jev, instead, binds directly to ifnar through its methyltransferase domain and inhibits the activation of kinases associated to the receptor [191, 192] . several wnv non structural proteins, as ns2a, ns2b, ns3, ns4a, ns4b and ns5, have been reported to prevent the phoshorylation of jak1, tyk2 and, as a consequence, the activation of stat1/2 (recently reviewed in [73] ). likewise, expression of denv nonstructural protein ns2a, ns4a, or ns4b proteins impairs the jak/stat signaling pathway by reducing the phosphorylation and nuclear translocation of stat1 [193] . phosphorylation of stat2 is also blocked by denv ns5 through inhibition of jak1 and tyk2 activity [194] . ns5 also binds to the coiled-coil region in the first half of the human stat2 protein and acts as a bridge between ubr-4, a member of the n-recognin family, and stat2 leading to stat2 ubiquitination and proteasomal-mediated degradation [195, 196] . interestingly, only proteolytically-processed ns5 can efficiently mediate stat2 degradation, though both unprocessed and processed ns5 proteins are able to bind stat2. the jev ns5 protein greatly reduces tyk2 and, partially, stat1 phosphorylation, probably through its phosphatase activity [197] . the tbev ns5, instead, blocks stat1 phosphorylation by promoting the association with the pdz membrane protein scribble [198] . by activating the ras/raf/mek pathway, hcv replication has been shown to increase the phosphorylation of a motif contained in the cytoplasmic tail of ifnar1, which is involved in controlling the receptor ubiquitin-dependent endocytosis and attenuation of stat1/2 phosphorylation [199] . several hcv proteins have also been implicated in the regulation of the ifn response pathway interfering directly with the jak/stat signaling. however, contrasting results have been reported that probably stem from the use of different cell lines or different hcv expression/replication systems [200] [201] [202] [203] [204] . the core protein has been reported to upregulate the expression of socs3, thereby inhibiting tyrosine phosphorylation of stat1 [201] , although the decreased stat1 phoshorylation has not been detected in other studies [200, 205] . the hcv core protein, expressed alone, has been reported to directly bind to stat1 and to prevent its phosphorylation and subsequent expression of downstream isgs [206] . the ns5a, similarly, binds and prevents stat1 phosphorylation specifically in hepatocyte-derived cell lines [207] . two strategies are used by ebov vp24 to limit the jak1/tyk2mediated activation of stat1/2 and their subsequent nuclear localization. vp24 binds within the tyrosine-phosphorylated-stat1 binding region located in the c terminus of members of the npi-1 subfamily of karyopherin alpha nuclear localization signal receptors preventing their binding and shuttling to the nucleus of tyrosine-phosphorylated-stat1 [208, 209] . the crystal structure of human kpnalpha5 c terminus in complex with vp24 has been recently resolved and a unique nonclassical nuclear localization signal binding site on kpna5 has been identified. this motif is necessary for binding and efficient nuclear import of tyrosinephosphorylated-stat1 [210] . ebov vp24 can also directly bind stat1; whether the binding occurs with the unphosphorylated or phosphorylated stat1 or both isoforms it is not yet clear [211] . however, also unphosphorylated stat1 enters the nucleus to activate and sustain the expression of a number of ifn-induced immune regulatory genes, which are distinct from those activated by the phosphorylated stat1. thus, ebov vp24 binding and inhibition of either forms of stat1 may be important in the suppression of an antiviral state. notably, in spite of the high similarity of ebov and marv genome organization and high sequence homology between many of the ebov and marv encoded proteins, marv vp24 unlike ebov vp24, does not inhibit jak/stat signaling [212] . this is consistent with the observation that regions important for karyopherin alpha binding are different between these two vp24s [210] . in contrast, tyrosine phosphorylation of jak1 and stat is inhibited by the marv matrix protein vp40 that also inhibits this ifn-ii signaling. interestingly, also jak1-dependent and il-6-induced tyrosine phosphorylation of stat1 and stat3 are targeted by marv vp40 suggesting that marv may globally inhibit jak1dependent cytokine signaling with mechanisms different from that employed by ebov [212] . among the escape mechanisms used by bacteria to evade immune responses, some have been recently reported to target ifn-i signaling molecules. having found that influenza viruses replicate to a higher efficiency in cells co-infected with staphylococcus aureus, warnking et al. [213] demonstrated that an impaired stat1/stat2 dimerization is responsible for a poor induction of isg transcription in spite of an abundant secretion of ifn-i driven by the flu virus infection. similarly, the inhibition of the response to ifn-i by mycobacterium tuberculosis was observed in human macrophages and correlated with mycobacterial pathogenicity [214] . in primary cells and thp-1 cells, indeed, mycobacterium tuberculosis specifically inhibits ifn-i signal transduction pathway by impairing the activation of stat1, while the avirulent mycobacterium bovis bcg fails to do so. alteration in isgf-3 complex formation was instead observed in human macrophages infected with nonpathogenic lactobacillus rhamnosus gg where only stat1 homodimers were found. in contrast, the pathogenic streptococcus pyogenes led to formation of not only stat1 homodimers but also of isgf-3 [215] . based on these finding the authors speculated that the efficient induction of ifn-i production and related transcription factor activation by streptococci would lead to fast and effective immune responses that, however, could play a role in the pathogenesis. although the first antiviral isgs were discovered decades ago, until recently the mechanisms of action was defined for only a limited number of isg-encoded proteins. the renewed interest in the innate immune response to retroviruses with the identification of how several host restriction factors may limit retroviral infection [118] [119] [120] , as well as large-scale functional screening of isgs, have identified genes that coordinately control the infection of a range of rna and dna viruses and have begun to dissect their mechanism of action [153, 216] . interestingly, some of the most potent antiviral effectors reinforce the system by further inducing ifn or isgs. thus, by directly disarming and/or making the use of individual ifn-induced effector proteins, the antiviral effect of host cells may still be attenuated even though ifn-i is induced, and viruses can ensure protection from both autocrine and paracrine effects of secreted ifn-i. here, we will primarily focus on isgs for which viral countermeasures have been identified in the context of viruses covered in the present review. recent reviews report more extensively on isg viral antagonists and specifically on ifn-induced hiv restriction factors that are not covered here [117] [118] [119] [120] [217] [218] [219] . pkr is one of the major effectors of the ifn-i-induced antiviral state. upon activation from binding dsrna molecules via a dsrna binding domain, pkr phosphorylates the translation initiation factor, eif2␣, thus blocking cellular and viral protein synthesis in infected cells. due to its ability to bind dsrna molecules pkr is also a nucleic acid sensor, as mentioned above [220] . viruses have evolved specific mechanisms to inhibit pkr activity or escape its action downstream. these include: the production of small and highly structured rna molecules that prevent the dsrnainduced dimerization and activation of pkr; expression of proteins that bind directly to and inhibit the activity of pkr or pkr activators; proteins that behave as pseudosubstrate and competitive inhibitors of pkr [221] [222] [223] . amongst viruses covered in this review, both antiviral and proviral roles of pkr have been reported for hcv. the hcv ns5a and e2 proteins directly interfere with the antiviral action of pkr. ns5a binds directly to pkr, while the glyco-protein e2 acts as competitive substrate with eif2 ␣ for pkr binding, resulting in inhibition of pkr kinase activity and in increased hcv replication. the full length ires of hcv rna, which is recognized by pkr, may mediate either activation or inhibition of pkr [224] . moreover, ns5a, by binding to various domains of the ires, can alter the activation of pkr [225] . another indirect inhibitory strategy mediated by the hcv ires has been recently reported. upon hcv rna sensing, pkr activates the mavs/traf3/irf3 pathway that, however, does not induce ifn but a set of isgs including isg15. as mentioned, isg15 deubiquitinates rig-i to negatively control the rig-i/mavs pathway and prevent uncontrolled detrimental ifn-i expression in physiological conditions. thus, hcv hijacks a protective cellular pathway to curtail host innate response [110] . through a specific ires-mediated inhibition of eif2␣-dependent translation, the hcv ires also regulates the translational activity of pkr [224] . eif2 ␣ is, indeed, essential to the translation of capped mrna, as those of isgs, while non-capped mrnas, as those of hcv, are translated independently from this factor. a general attenuation of isgs expression by hcv ires can be achieved also through a direct activation of pkr [226] . interestingly, this attenuation of isg expression is observed in acute but not persistent infection, where, instead, a sustained isg expression occurs [206, 227] . other proteins from flaviviruses have been shown to inhibit pkr including the ns2a protein of jev that physically interacts with pkr and blocks its activation in response to several stimuli [228] . ebov vp35 also interferes with the pathway regulated by pkr by blocking and also reversing pkr activation, thereby preventing translational arrest of viral mrnas. this pkr antagonism seems to be functionally different from dsrna binding and irf3 inhibition [229] [230] [231] . ebov vp 35 also associates with pact (pkr-associated activator). this complex abolishes pact interaction with the cterminal domain of rig-i, which is required for full activation of rig-i [232] . as for retroviruses, hiv-1 infection does not activate pkr due to both viral and cellular controls (reviewed in [233] ). in vitro, pkr is activated by low amounts of tar rna, whereas high concentrations inhibit the kinase function. the viral tat protein also counteracts pkr activation by several other mechanisms: it sequesters the activating dsrna; it can act as a substrate homologue of eif2␣ preventing the pkr mediated inhibition of protein synthesis; it prevents pkr auto-phosphorylation and exploits pkr activity to get phosphorylated and increase its binding to tar rna. moreover, hiv-1 replicates only in cells that have high levels of the tar rna binding protein (trbp), a strong inhibitor of pkr activation. interestingly, during hiv-1 infection of lymphocytes, when hiv-1 replicates at high levels, increased amounts of adar1, an ifn-induced rna editing enzyme that binds to pkr to inhibit its activation, have been observed. moreover, pact contributes to pkr dephosphorylation during hiv-1 replication probably due to its binding to adar1 [234] . thus, hiv-1 has evolved to replicate in cells with high levels of trbp, to induce the expression of adar1 and to change the function of pact for pkr inhibition [235] . isg15 is an ubiquitin-like modifier that is induced rapidly by ifn-i and possesses antiviral activity against a number of viruses. isg15 antiviral functions include inhibition of virus release, isgylation of both viral and host proteins and immunomodulatory cytokine-like properties in its unconjugated and secreted form, as recently reviewed [236, 237] . more than 160 host proteins that are isgylated have been identified including irf3, pkr and rig-i. isgylation preferentially targets newly translated proteins and, as a consequence of isgylation, degradation of the target protein is reduced by competition with ubiquitin conjugation [238, 239] . to date, only few viral proteins have been shown to be isgylated and functional characterizations of isg15 conjugation has not been always verified under conditions of endogenous protein expression [237] . moreover, often, isg15-mediated protection might not be a result of direct antagonism of virus replication. as an example, isg15 has been shown to inhibit the release of hiv-1 and ebov. this effect is mediated by an ubiquitin antagonism. isg15 disrupts the ubiquitin-mediated regulation of ebov vp40, necessary to produce budding and release of vp40 vlps [240] , as well as the ubiquitination of the hiv-1 gag protein, which is required for the interaction with the cellular protein tsg101 to mediate hiv-1 budding and release [241] . several viruses have, thus, developed countermeasures against isg15 and/or its conjugation. strategies, identified so far, include viral proteins that bind isg15 or that remove isg15 from target proteins (reviewed in [237] ). sars-cov plp has both deubiquitinating and deisgylating activities [242, 243] . recently, the structural basis of recognition and processing of deubiquitin and isg15 by plp has been reported [244] . interestingly, despite mers-cov encodes a single plp similar to sars-cov, there is little to no sequence conservation among residues important for the deubiquitinating and deisgylating activity, suggesting that mers-cov plp is likely to recognize and process ubiquitin and isg15 substrates differently than sars-cov plp [155, 244] . however, by affecting this post-translational modification, both viruses may modify cellular protein localization, protein activity and stability as well as signal transduction in order to increase viral replication and severity of infection. nevertheless, although these results stem from in vitro overexpression or mutant studies, the direct evidence for isg15 antagonism by these proteins remains to be demonstrated during viral infection. despite the well-characterized role in restricting replication of several viruses, isg15 may actually also promote the replication of specific viruses, as hcv. in hcv infections both anti-viral or proviral effects exerted by isg15 could be related to the net effect of isgylation on the various viral and host proteins targeted by isg15. an antiviral effect has been reported when isg15 could conjugate to hcv ns5a, thereby enhancing the inhibitory effect of ifn-i on hcv replication [245] . in contrast, several groups have reported that isg15 and isgylation promote hcv production in a cell culture model independently of upstream ifn signaling [246, 247] . although counter-intuitive, this finding may be explained by the observed inhibition of ifn-i induction by hcv upon isg15 overexpression that negatively controls the rig-i/mavs pathway at the level of rig-i ubiquitination [110] . a negative regulation of ifn-i expression may also be mediated by usp18, an isg displaying a potent inhibitory effect on the ifn-i pathway, to prevent autoinflammatory consequences of uncontrolled ifn-i production. similarly, usp18 may dampen the detrimental role that the hyperactivation of ifn-i signaling plays in the pathogenesis of some viral and bacterial infections, including hiv-1, hcv and mycobacterium tuberculosis. usp18 is, indeed, stabilized by isg15 in an unconjugated free form [248] . in this respect, the suggested potential role of the isg15/usp18 pathway in hcv persistence is consistent with the observation that increased expression of hepatic isgs before ifn treatment is associated with an absent or poor response in patients chronically infected with hcv [249] . thus, isg15/usp18 pathway might explain the paradox that the preactivation of the endogenous ifn system, while fails to clear the infection, instead, may stimulate hcv production and blunt the effect of exogenous ifn-i. usp18 is similarly induced during some bacterial infections, including salmonella and mycobacterium tuberculosis. the decreased survival of mice that carry a point mutation in usp18 results from higher salmonella load in the spleen and liver, an increased inflammatory response and increased ifn-i signaling. similarly, these usp18 mutant mice are more susceptible to mycobacterium tuberculosis infection and have increased bacterial load in the lung and spleen, elevated inflammatory cytokine production and more severe lung pathology [250] . in line with these findings, the results of dorhoi et al. [251] have shown that ifnar1 deficient mice were protected from death upon aerogenic infection with mycobacterium tuberculosis. moreover, a rather detrimental effect of ifn-i was also found in whole blood of patients with tuberculosis, where a neutrophil-driven, ifn-inducible transcriptional signature was associated with clinical severity [252] [253] [254] . these results thus reveal that some viruses and bacteria, to push replication and persistence, utilize an opposite, but as much as effectual strategy, consisting in enhancing/perpetuating an ifn-i response by targeting negative regulators of ifn-i expression. anti-microbial molecules, such as nitric oxide (no) radicals and reactive oxygen species (ros) mediate the antibacterial properties of ifn-i. the key producers of no is inducible nitric oxide synthase (inos), an enzyme that can be induced by both ifn-i and -ii, although the latter is the conventional inducer that plays a crucial role in fighting the infection of intracellular bacteria [255] . similarly, ifn-i and -ii induce the subunits of the phagocyte nicotinamide adenine dinucleotide phosphate (nadph) oxidase, which generates ros for killing organisms [256] . thus, pathogens have evolved several ways of avoiding ros-and no-mediated killing. in spite of the fact that no data are available, so far, on the strategies exploited by bacteria to contrast the ifn-mediated transcriptional regulation of inos and nadph oxidase, a common theme for successful intracellular pathogens is the ability to avoid the colocalization with these harmful host enzymes. intracellular salmonella, which resides within a specialized membrane compartment called the salmonella-containing vacuole (scv) in macrophages, uses a t3ss called salmonella pathogenicity island 2 (spi2) to mediate protection from no and ros intermediates [257] . intracellular organisms have also developed mechanisms to detoxify and repair no-mediated damage [255] , as well as to avoid the induction of inos activity [258] . given the crucial role played by these antimicrobial enzymes in contrasting bacterial infection, it is likely that strategies pinpointed by pathogens to inhibit the ifn-driven expression of these molecules will be discovered in the nearest future. to effectively resist the continual microbial threat from the environment, vertebrates possess several defense mechanisms including innate and adaptive immunity. as an essential component of the innate immunity, the ifn system constitutes the first line of defense against a number of pathogens to clear an incoming infection and instructing an ensuing adaptive response. successful pathogens have, thus, evolved sophisticated strategies to subvert and/or exploit the host immune system where blocking the ifn response, in the first place, is required to replicate and survive. the elucidation of some of these strategies has led to the identification of several, thus far, poorly recognized features of the innate immune response. in parallel, with the enormous recent advances in the comprehension of the molecular mechanisms of innate immune responses to pathogens, specific processes by which pathogenic microorganisms subvert these innate immune pathways, including the ifn system, is becoming progressively appreciated and it is reasonable to assume that many more will be discovered in the near future. by learning from the anti-immune strategies of pathogens we can, thus, not only identify key pathogen regulators as useful target to exploit to the host advantage, but we can also unveil weaknesses of host defenses and intervene to more precisely tune the immune response. the number and diversity of pathogen strategies for counteracting at each step the ifn system is stupefying. although beyond the scope of this review to discuss all antagonisms in detail, the ones that we have here reported, represent common and recurrent strategies used by a number of pathogens. this is illustrated by the existence of both viral and bacterial examples of pamp modifications as well as of viral and bacterial proteins that share cellular-like domain or cellular-like enzymatic properties that can compete with the host counterparts to dampen their physiological activities. in this respect, common hubs in the signaling pathways downstream pathogen sensors that trigger ifn-i, as few common adaptors or cofactors and transcription factors, are attractive targets of pathogen antagonism. the recognition of the mechanisms involved in microbial countermeasures may have several translation implications. the definition of microbial ability to elude the detection, as the methylation of their rna caps, can suggest strategies to utilize methyltransferase mutants as successful vaccine candidates against a number of different viruses as already suggested for denv [259] . many of the pathogen proteins responsible for ifn-i antagonism are also determinants of virulence and pathogenesis and, as such, they are highly conserved and may, thus, constitute attractive targets for the development of promising therapeutics against various clinically relevant pathogens reducing the bias of resistance mutations. in turn, some of the cellular identified targets of pathogen proteins as well as protein interacting partners might turn out to be new drug targets for treating a range of different diseases that disarme common components of the ifn pathway. in this respect, the resolution of the crystallographic structures of viral antagonists in complex with their different viral and cellular ligands, is then crucial for the rational design of new drugs. the system biology approach and the ability to simultaneously investigate diverse pathways has led to appreciate the interconnection between these pathways also in terms of shared components and stimulation by different pathogens. thus, a single therapeutic strategy could modulate multiple pathways to the host benefit. nevertheless, each approach needs to be complemented with effective treatments that also overcome other concurrent strategies that often the same pathogen put in place. on the other side of the coin, recovery of a full innate response must be finely tuned. ifn-i is, indeed, not always protective but can instead play a pathogenic role as reported for some bacterial and viral infections, where an uncontrolled ifn production is a determinant of disease progression. even in these cases, however, insights in the mechanisms involved in turning an ifn protective response into a pathogenetic one, may be as well relevant for nonpathogen-induced diseases, as autoimmunity and inflammatory diseases. in this context, cell death and inflammasome activation have been described as crucial ifn-i-regulated events exploited by both pathogen and host to get their own advantage. despite inflammasome facilitates pathogen clearance and is beneficial to the host, in some instances, ifn-induced non-canonical nlrp3 inflammasome activation and pyroptosis appear to be detrimental due to excessive cell death, inflammation, and collateral tissue damage in vital organs [260] . so pathogen proteins themselves or modified versions of them could be used as therapeutics working in suppressing inappropriate immune activation. this would be a bright way of hijacking molecules evolved during pathogen adaptation and associated fitness, to shift the balance to the host advantage. similarly, some identified targets of viral proteins in prr signaling pathways might be turned out to be new targets for treating a range of diseases. to find the way to generally induce an ifn response that is protective against a number of different infectious diseases, may be particularly relevant during an outbreak of unknown etiology or during the arising of newly emerging and re-emerging strains. likewise, finding the key to unlock the detrimental outcome of excessive ifn-i production during an infectious disease can open the way to cure autoimmune and inflammatory diseases. the authors declare no competing financial interests. innate immune sensing and signaling of cytosolic nucleic acids signaling by myeloid c-type lectin receptors in immunity and homeostasis immune sensing of dna intracellular toll-like receptors advances in nod-like receptors (nlr) biology the interferon response to intracellular dna: why so many receptors? intracellular sensing of viral dna by the innate immune system pathogen recognition and innate immunity irfs: master regulators of signalling by toll-like receptors and cytosolic pattern-recognition receptors regulation of type i interferon responses interferon-stimulated genes: a complex web of host defenses mechanisms of type-i-and type-ii-interferon-mediated signalling charleston, type i and iii interferon production in response to rna viruses interferome: the database of interferon regulated genes the jak-stat pathway at twenty type i interferons in host defense ifn regulation and functions in myeloid dendritic cells confounding roles for type i interferons during bacterial and viral pathogenesis bacteria fighting back: how pathogens target and subvert the host innate immune system anti-immunology: evasion of the host immune system by bacterial and viral pathogens bacterial subversion of host innate immune pathways inverse interference: how viruses fight the interferon system viral tricks to grid-lock the type i interferon system viral evasion and subversion of patternrecognition receptor signalling cellular sensing of viral dna and viral evasion mechanisms microbial strategies for antagonizing toll-likereceptor signal transduction toll-like receptors and their crosstalk with other innate receptors in infection and immunity a virological view of innate immune recognition microbial sensing by toll-like receptors and intracellular nucleic acid sensors length-dependent recognition of double-stranded ribonucleic acids by retinoic acid-inducible gene-i and melanoma differentiation-associated gene 5 rig-i-mediated antiviral responses to single-stranded rna bearing 5 -phosphates 5 -triphosphate rna is the ligand for rig-i rig-i detects infection with live listeria by sensing secreted bacterial nucleic acids mitochondrial antiviral signaling protein (mavs) monitors commensal bacteria and induces an immune response that prevents experimental colitis identification and characterization of mavs, a mitochondrial antiviral signaling protein that activates nf-kappab and irf 3 sting is an endoplasmic reticulum adaptor that facilitates innate immune signalling the adaptor protein mita links virus-sensing receptors to irf3 transcription factor activation mita/sting: a central and multifaceted mediator in innate immune response dsrna-dependent protein kinase pkr and its role in stress, signaling and hcv infection ifit1: a dual sensor and effector molecule that detects non-2 -o methylated viral rna and inhibits its translation nod proteins: regulators of inflammation in health and disease mechanisms of inflammasome activation: recent advances and novel insights advancements in the battle against severe acute respiratory syndrome middle east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus study group coronaviruses: important emerging human pathogens from sars to mers: 10 years of research on highly pathogenic human coronaviruses severe acute respiratory syndrome vs. the middle east respiratory syndrome human cell tropism and innate immune system interactions of human respiratory coronavirus emc compared to those of severe acute respiratory syndrome coronavirus efficient replication of the novel human betacoronavirus emc on primary human epithelium highlights its zoonotic potential to sense or not to sense viral rna -essentials of coronavirus innate immune evasion sars coronavirus pathogenesis: host innate immune responses and viral antagonism of interferon ribose 2 -o-methylation provides a molecular signature for the distinction of self and non-self mrna dependent on the rna sensor mda5 coronavirus non-structural protein 16: evasion, attenuation, and possible treatments 2 -o methylation of the viral mrna cap evades host restriction by ifit family members attenuation and restoration of severe acute respiratory syndrome coronavirus mutant lacking 2 -o-methyltransferase activity rna 3 -end mismatch excision by the severe acute respiratory syndrome coronavirus nonstructural protein nsp10/nsp14 exoribonuclease complex endoribonuclease activities of porcine reproductive and respiratory syndrome virus nsp11 was essential for nsp11 to inhibit ifn-beta induction sars-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum mers-coronavirus replication induces severe in vitro cytopathology and is strongly inhibited by cyclosporin a or interferon-alpha treatment sars-cov nucleocapsid protein antagonizes ifnbeta response by targeting initial step of ifn-beta induction pathway, and its c-terminal region is critical for the antagonism severe acute respiratory syndrome coronavirus open reading frame (orf) 3b, orf 6, and nucleocapsid proteins function as interferon antagonists molecular determinants for subcellular localization of the severe acute respiratory syndrome coronavirus open reading frame 3b protein severe acute respiratory syndrome coronavirus m protein inhibits type i interferon production by impeding the formation of traf3.tank.tbk1/ikkepsilon complex sars coronavirus papain-like protease inhibits the type i interferon signaling pathway through interaction with the sting-traf3-tbk1 complex coronavirus papain-like proteases negatively regulate antiviral innate immune response through disruption of sting-mediated signaling severe acute respiratory syndrome coronavirus papain-like protease ubiquitin-like domain and catalytic domain regulate antagonism of irf3 and nf-kappab signaling strategies of highly pathogenic rna viruses to block dsrna detection by rig-i-like receptors: hide, mask, hit message in a bottle: lessons learned from antagonism of sting signalling during rna virus infection the double-stranded rna-binding protein pact functions as a cellular activator of rig-i to facilitate innate antiviral response middle east respiratory syndrome coronavirus 4a protein is a double-stranded rnabinding protein that suppresses pact-induced activation of rig-i and mda5 in the innate antiviral response the orf4b-encoded accessory proteins of middle east respiratory syndrome coronavirus and two related bat coronaviruses localize to the nucleus and inhibit innate immune signalling flaviviral rnas: weapons and targets in the war between virus and host immune evasion strategies of flaviviruses flavivirus rna cap methyltransferase: structure, function, and inhibition flaviviruses and flavivirus vaccines emerging viral diseases: confronting threats with new technologies innate immunity evasion by dengue virus west nile virus infection and immunity flavivirus methyltransferase: a novel antiviral target 2 -o methylation of the viral mrna cap by west nile virus evades ifit1-dependent and -independent mechanisms of host restriction in vivo ifit1 inhibits japanese encephalitis virus replication through binding to 5 capped 2 -o unmethylated rna the endoplasmic reticulum provides the membrane platform for biogenesis of the flavivirus replication complex composition and three-dimensional architecture of the dengue virus replication and assembly sites dengue virus inhibits the production of type i interferon in primary human dendritic cells inhibition of the type i interferon response in human dendritic cells by dengue virus infection requires a catalytically active ns2b3 complex denv inhibits type i ifn production in infected cells by cleaving human sting dengue virus targets the adaptor protein mita to subvert host innate immunity activation and evasion of antiviral innate immunity by hepatitis c virus the global burden of hepatitis c interferon-stimulated genes and their role in controlling hepatitis c virus nonstructural protein 3-4a: the swiss army knife of hepatitis c virus cardif is an adaptor protein in the rig-i antiviral pathway and is targeted by hepatitis c virus hepatitis c virus protease ns3/4a cleaves mitochondrial antiviral signaling protein off the mitochondria to evade innate immunity mitochondrial-associated endoplasmic reticulum membranes (mam) form innate immune synapses and are targeted by hepatitis c virus dissociation of a mavs/ips-1/visa/cardif-ikkepsilon molecular complex from the mitochondrial outer membrane by hepatitis c virus ns3-4a proteolytic cleavage cleavage of mitochondrial antiviral signaling protein in the liver of patients with chronic hepatitis c correlates with a reduced activation of the endogenous interferon system hepatitis c virus ns4b protein targets sting and abrogates rig-i-mediated type i interferon-dependent innate immunity hepatitis c virus ns4b blocks the interaction of sting and tbk1 to evade host innate immunity immune evasion by hepatitis c virus ns3/4a protease-mediated cleavage of the tolllike receptor 3 adaptor protein trif hcv-induced mir-21 contributes to evasion of host immune system by targeting myd88 and irak1 hepatitis c virus nonstructural protein 5a modulates the toll-like receptor-myd88-dependent signaling pathway in macrophage cell lines expression of toll-like receptors in chronic hepatitis c virus infection distinct toll-like receptor 3 and 7 expression in peripheral blood mononuclear cells from patients with chronic hepatitis c infection hepatitis c virus infection decreases the expression of toll-like receptors 3 and 7 via upregulation of mir-758 plasmacytoid dendritic cells sense hepatitis c virus-infected cells, produce interferon, and inhibit infection recovery, persistence, and sequelae in hepatitis c virus infection: a perspective on long-term outcome interferon-induced gene expression is a stronger predictor of treatment response than il28b genotype in patients with hepatitis c hiv and hcv activate the inflammasome in monocytes and macrophages via endosomal toll-like receptors without induction of type 1 interferon sex-specific association between x-linked toll-like receptor 7 with the outcomes of hepatitis c virus infection hepatitis c virus reveals a novel early control in acute immune response redefining the viral reservoirs that prevent hiv-1 eradication therapeutics for hiv-1 reactivation from latency hiv-1 latency: an update of molecular mechanisms and therapeutic strategies hiv-1, interferon and the interferon regulatory factor system: an interplay between induction, antiviral responses and viral evasion innate antiviral immune signaling, viral evasion and modulation by hiv-1 innate immune recognition of hiv-1 the interface between the innate interferon response and expression of host retroviral restriction factors unmasking immune sensing of retroviruses: interplay between innate sensors and host effectors type i ifn -a blunt spear in fighting hiv-1 infection interactions between hiv-1 and the cellautonomous innate immune system innate immune sensing of hiv-1 by dendritic cells the ribonuclease activity of samhd1 is required for hiv-1 restriction samhd1 host restriction factor: a link with innate immune sensing of retrovirus infection samhd1 is the dendritic-and myeloid-cell-specific hiv-1 restriction factor counteracted by vpx vpx relieves inhibition of hiv-1 infection of macrophages mediated by the samhd1 protein dendritic cells in progression and pathology of hiv infection hiv-1 exploits innate signaling by tlr8 and dc-sign for productive infection of dendritic cells rig-i-mediated antiviral signaling is inhibited in hiv-1 infection by a proteasemediated sequestration of rig-i ifi16 senses dna forms of the lentiviral replication cycle and controls hiv-1 replication lieberman, the cytosolic exonuclease trex1 inhibits the innate immune response to human immunodeficiency virus type 1 cell death by pyroptosis drives cd4 t-cell depletion in hiv-1 infection the capsids of hiv-1 and hiv-2 determine immune detection of the viral cdna by the innate sensor cgas in dendritic cells hiv-1 evades innate immune recognition through specific cofactor recruitment transmission dynamics and control of ebola virus disease (evd): a review characterization of host immune responses in ebola virus infections the role of the type i interferon response in the resistance of mice to filovirus infection evasion of interferon responses by ebola and marburg viruses filoviral immune evasion mechanisms marburg virus vp35 can both fully coat the backbone and cap the ends of dsrna for interferon antagonism structural basis for marburg virus vp35-mediated immune evasion mechanisms the yin and yang of type i interferon activity in bacterial infection structural modifications of bacterial lipopolysaccharide that facilitate gram-negative bacteria evasion of host innate immunity intracellular shigella remodels its lps to dampen the innate immune recognition and evade inflammasome activation yopj targets traf proteins to inhibit tlr-mediated nf-kappab, mapk and irf3 signal transduction the shigella flexneri effector ospi deamidates ubc13 to dampen the inflammatory response inhibition of tlr signaling by a bacterial protein containing immunoreceptor tyrosine-based inhibitory motifs triggering the interferon antiviral response through an ikk-related pathway ikkepsilon and tbk1 are essential components of the irf3 signaling pathway regulation of immunity and oncogenesis by the irf transcription factor family interferon regulatory factors in immune cell development and host response to infection evidence for licensing of ifn-gamma-induced ifn regulatory factor 1 transcription factor by myd88 in toll-like receptor-dependent gene induction program peroxisomes are signaling platforms for antiviral innate immunity a diverse range of gene products are effectors of the type i interferon antiviral response regulation of irf-3-dependent innate immunity by the papain-like protease domain of the severe acute respiratory syndrome coronavirus proteolytic processing, deubiquitinase and interferon antagonist activities of middle east respiratory syndrome coronavirus papain-like protease immunity by ubiquitylation: a reversible process of modification west nile virus nonstructural protein 1 inhibits tlr3 signal transduction abrogation of tlr3 inhibition by discrete amino acid changes in the c-terminal half of the west nile virus ns1 protein modulation of innate immune signaling by the secreted form of the west nile virus ns1 glycoprotein dengue virus subverts the interferon induction pathway via ns2b/3 protease-ikappab kinase epsilon interaction tick-borne flaviviruses antagonize both irf-1 and type i ifn signaling to inhibit dendritic cell function interaction between the hcv ns3 protein and the host tbk1 protein leads to inhibition of cellular antiviral responses hepatitis c virus ns2 protease inhibits host cell antiviral response by inhibiting ikkepsilon and tbk1 functions impairment of interferon regulatory factor-3 activation by hepatitis c virus core protein basic amino acid region 1 dead/h box 3 (ddx3) helicase binds the rig-i adaptor ips-1 to up-regulate ifn-beta-inducing potential hepatitis c virus core protein abrogates the ddx3 function that enhances ips-1-mediated ifn-beta induction repression of interferon regulatory factor 1 by hepatitis c virus core protein results in inhibition of antiviral and immunomodulatory genes hepatitis c virus core protein inhibits interferon production by a human plasmacytoid dendritic cell line and dysregulates interferon regulatory factor-7 and signal transducer and activator of transcription (stat) 1 protein expression battistini, hiv-1 targeting of ifn regulatory factors human immunodeficiency virus type 1 mediates global disruption of innate antiviral signaling and immune defenses within infected cells hiv-1 accessory proteins vpr and vif modulate antiviral response by targeting irf-3 for degradation vpu-deficient hiv strains stimulate innate immune signaling responses in target cells hiv-1 vpu induces caspase-mediated cleavage of irf3 apoptosis as an hiv strategy to escape immune attack hiv infection of dendritic cells subverts the ifn induction pathway via irf-1 and inhibits type 1 ifn production irf-1 is required for full nf-kappab transcriptional activity at the human immunodeficiency virus type 1 long terminal repeat enhancer modulation of human immunodeficiency virus 1 replication by interferon regulatory factors intracellular hiv-1 tat protein represses constitutive lmp2 transcription increasing proteasome activity by interfering with the binding of irf-1 to stat1 ikappab kinase epsilon targets interferon regulatory factor 1 in activated t lymphocytes the ebola virus vp35 protein inhibits activation of interferon regulatory factor 3 ebola virus protein vp35 impairs the function of interferon regulatory factor-activating kinases ikkepsilon and tbk-1 basic residues within the ebolavirus vp35 protein are required for its viral polymerase cofactor function ebola zaire virus blocks type i interferon production by exploiting the host sumo modification machinery virus infection triggers sumoylation of irf3 and irf7, leading to the negative regulation of type i interferon gene expression beneficial innate signaling interference for antibacterial responses by a toll-like receptor-mediated enhancement of the mkp-irf3 axis the sars coronavirus 3a protein causes endoplasmic reticulum stress and induces ligand-independent downregulation of the type 1 interferon receptor severe acute respiratory syndrome coronavirus papain-like protease suppressed alpha interferoninduced responses through downregulation of extracellular signal-regulated kinase 1-mediated signalling pathways severe acute respiratory syndrome coronavirus orf6 antagonizes stat1 function by sequestering nuclear import factors on the rough endoplasmic reticulum/golgi membrane virus-induced unfolded protein response attenuates antiviral defenses via phosphorylation-dependent degradation of the type i interferon receptor a conserved peptide in west nile virus ns4a protein contributes to proteolytic processing and is essential for replication inhibition of interferon-stimulated jak-stat signaling by a tick-borne flavivirus and identification of ns5 as an interferon antagonist identification of residues critical for the interferon antagonist function of langat virus ns5 reveals a role for the rna-dependent rna polymerase domain inhibition of alpha/beta interferon signaling by the ns4b protein of flaviviruses dengue virus ns5 inhibits interferon-alpha signaling by blocking signal transducer and activator of transcription 2 phosphorylation ns5 of dengue virus mediates stat2 binding and degradation dengue virus co-opts ubr4 to degrade stat2 and antagonize type i interferon signaling blocking of interferon-induced jak-stat signaling by japanese encephalitis virus ns5 through a protein tyrosine phosphatase-mediated mechanism tick-borne encephalitis virus ns5 associates with membrane protein scribble and impairs interferon-stimulated jak-stat signalling activation of the ras/raf/mek pathway facilitates hepatitis c virus replication via attenuation of the interferon-jak-stat pathway expression of hepatitis c virus proteins inhibits signal transduction through the jak-stat pathway ifn-alpha antagonistic activity of hcv core protein involves induction of suppressor of cytokine signaling-3 hcv structural proteins interfere with interferon-alpha jak/stat signalling pathway expression of hcv structural proteins impairs ifn-mediated antiviral response identification of the nonstructural protein 4b of hepatitis c virus as a factor that inhibits the antiviral activity of interferonalpha hepatitis c virus inhibits interferon signaling through up-regulation of protein phosphatase 2a intracellular innate immune cascades and interferon defenses that control hepatitis c virus hcv ns5a inhibits interferon-alpha signaling through suppression of stat1 phosphorylation in hepatocyte-derived cell lines ebola virus vp24 proteins inhibit the interaction of npi-1 subfamily karyopherin alpha proteins with activated stat1 ebolavirus vp24 binding to karyopherins is required for inhibition of interferon signaling ebola virus vp24 targets a unique nls binding site on karyopherin alpha 5 to selectively compete with nuclear import of phosphorylated stat1 the ebola virus interferon antagonist vp24 directly binds stat1 and has a novel, pyramidal fold marburg virus evades interferon responses by a mechanism distinct from ebola virus super-infection with staphylococcus aureus inhibits influenza virusinduced type i ifn signaling through impaired stat1-stat2 dimerization inhibition of response to alpha interferon by mycobacterium tuberculosis lactobacilli and streptococci activate nf-kappa b and stat signaling pathways in human macrophages interferon-stimulated genes: roles in viral pathogenesis host restriction factors in retroviral infection: promises in virus-host interaction intrinsic antiviral immunity evolutionary conflicts between viruses and restriction factors shape immunity protein kinase pkr and rna adenosine deaminase adar1: new roles for old players as modulators of the interferon response the dsrna protein kinase pkr: virus and cell control inhibition of pkr by rna and dna viruses activation of the antiviral kinase pkr and viral countermeasures hepatitis c virus controls interferon production through pkr activation regulation of pkr by hcv ires rna: importance of domain ii and ns5a hepatitis c virus blocks interferon effector function by inducing protein kinase r phosphorylation failure of innate and adaptive immune responses in controlling hepatitis c virus infection blocking double-stranded rna-activated protein kinase pkr by japanese encephalitis virus nonstructural protein 2a ebola virus vp35 antagonizes pkr activity through its c-terminal interferon inhibitory domain the vp35 protein of ebola virus inhibits the antiviral effect mediated by double-stranded rna-dependent protein kinase pkr ebola virus vp35 protein binds double-stranded rna and inhibits alpha/beta interferon production induced by rig-i signaling mutual antagonism between the ebola virus vp35 protein and the rig-i activator pact determines infection outcome multiple levels of pkr inhibition during hiv-1 replication the pkr activator, pact, becomes a pkr inhibitor during hiv-1 replication hiv-1 translation and its regulation by cellular factors pkr and pact the antiviral activities of isg15 interferon-induced isg15 pathway: an ongoing virus-host battle positive regulation of interferon regulatory factor 3 activation by herc5 via isg15 modification the isg15 conjugation system broadly targets newly synthesized proteins: implications for the antiviral function of isg15 isg15 inhibits ebola vp40 vlp budding in an l-domain-dependent manner by blocking nedd4 ligase activity innate antiviral response targets hiv-1 release by the induction of ubiquitin-like protein isg15 the papain-like protease from the severe acute respiratory syndrome coronavirus is a deubiquitinating enzyme deubiquitinating and interferon antagonism activities of coronavirus papain-like proteases structural basis for the ubiquitin-linkage specificity and deisgylating activity of sars-cov papain-like protease inhibition of hepatitis c virus replication by ifn-mediated isgylation of hcv-ns5a the interferon stimulated gene 15 functions as a proviral factor for the hepatitis c virus and as a regulator of the ifn response isg15, a ubiquitin-like interferon-stimulated gene, promotes hepatitis c virus production in vitro: implications for chronic infection and response to treatment human intracellular isg15 prevents interferon-alpha/beta overamplification and auto-inflammation activation of endogenous type i ifn signaling contributes to persistent hcv infection contribution of increased isg15, isgylation and deregulated type i ifn signaling in usp18 mutant mice during the course of bacterial infections type i ifn signaling triggers immunopathology in tuberculosis-susceptible mice by modulating lung phagocyte dynamics genome-wide expression profiling identifies type 1 interferon response pathways in active tuberculosis an interferon-inducible neutrophil-driven blood transcriptional signature in human tuberculosis common patterns and disease-related signatures in tuberculosis and sarcoidosis inducible nitric oxide synthase and control of intracellular bacterial pathogens nadph oxidases: an overview from structure to innate immunity-associated pathologies salmonella pathogenicity island 2-dependent evasion of the phagocyte nadph oxidase modulation of inducible nitric oxide synthase expression by the attaching and effacing bacterial pathogen citrobacter rodentium in infected mice rational design of a live attenuated dengue vaccine: 2 -o-methyltransferase mutants are highly attenuated and immunogenic in mice and macaques role of type i interferons in inflammasome activation, cell death, and disease during microbial infection we apologize to the many colleagues whose data and influence have been overlooked due to space or our knowledge limitations. a special thank to members of the e.m. coccia's and a. battistini's laboratory for helpful discussion and critical reading of the manuscript and eugenio morassi for preparing drawings. our work is supported in part by grant rf-2010235199 from italian ministry of health (to emc) and from istituto superiore di sanità (to ab). key: cord-307914-lgprrwee authors: bartok, eva; hartmann, gunther title: immune sensing mechanisms that discriminate self from altered self and foreign nucleic acids date: 2020-07-14 journal: immunity doi: 10.1016/j.immuni.2020.06.014 sha: doc_id: 307914 cord_uid: lgprrwee all lifeforms have developed highly sophisticated systems equipped to detect altered self and non-self nucleic acids (na). in vertebrates, na-sensing receptors safeguard the integrity of the organism by detecting pathogens, dyshomeostasis and damage, and inducing appropriate responses to eliminate pathogens and reconstitute homeostasis. effector mechanisms include i) immune signaling, ii) restriction of na functions such as inhibition of mrna translation, and iii) cell death pathways. an appropriate effector response is necessary for host defense, but dysregulated na-sensing can lead to devastating autoimmune and autoinflammatory disease. their inherent biochemical similarity renders the reliable distinction between self na under homeostatic conditions and altered or exogenous na particularly challenging. in this review, we provide an overview of recent progress in our understanding of the closely coordinated and regulated network of innate immune receptors, restriction factors, and nucleases to effectively respond to pathogens and maintain host integrity. all lifeforms have developed highly sophisticated systems equipped to detect altered self and non-self nucleic acids (na). in vertebrates, na-sensing receptors safeguard the integrity of the organism by detecting pathogens, dyshomeostasis and damage, and inducing appropriate responses to eliminate pathogens and reconstitute homeostasis. effector mechanisms include i) immune signaling, ii) restriction of na functions such as inhibition of mrna translation, and iii) cell death pathways. an appropriate effector response is necessary for host defense, but dysregulated na-sensing can lead to devastating autoimmune and autoinflammatory disease. their inherent biochemical similarity renders the reliable distinction between self na under homeostatic conditions and altered or exogenous na particularly challenging. in this review, we provide an overview of recent progress in our understanding of the closely coordinated and regulated network of innate immune receptors, restriction factors, and nucleases to effectively respond to pathogens and maintain host integrity. nucleic acids (na) are a common building block of life, yet detection of exogenous genetic material is essential to host defense. the immune sensors employed by our cells to distinguish between self and non-self na are both effective and ancient, with recent publications revealing that even bacteria utilize sensors surprisingly similar to our own, such as cgamp synthase (cgas) and toll-interleukin receptor (tir) domain-containing proteins for anti-phage defense (cohen et al., 2019; doron et al., 2018) . although there are also sequence-based, adaptive forms of na sensing such as crispr/cas and rna interference (rnai), which are integral to host defense in other kingdoms and phyla (berkhout, 2018; hampton et al., 2020) , chordates principally rely on a discreet but powerful system of germline-encoded na sensors that are activated by molecular hallmarks of non-self or altered self na (schlee and hartmann, 2016) . sensor activation triggers a transcriptional form of host defense, including type-i interferon (ifn-i) release and the autocrine and paracrine induction of interferon-stimulated genes (isg), known as the antiviral state. in turn, to avoid immunodetection, pathogens and viruses in particular have engaged in a type of ''nucleic acid arms race'' to avoid sensing by the host. pathogens can sequester their na (e.g., in replication organelles), mask them with characteristics of self (e.g., viral cap-snatching), or even directly disable host signaling. indeed, recent publications indicate that, while rnai is present in vertebrate cells and active in embryonic cells (li et al., 2013a; maillard et al., 2013) , it can be rendered ineffective by the anti-rnai mechanisms of many viruses (li et al., 2016; qiu et al., 2017) . thus, it is tempting to speculate that this disabling of rnai provided the evolutionary pressure leading to the dominance of the type-i interferon (ifn-i) signaling in vertebrate antiviral defense, a system which is both exceptionally potent and reciprocally antagonistic with rnai (maillard et al., 2016; seo et al., 2013) . it is interesting to note that, although many of the receptors activating type-i ifn signaling are highly conserved evolutionarily, their downstream signaling is unique to chordates. analogously, caspases and metacaspases are ancient participants in programmed cell death (bell and megeney, 2017 ), yet inflammatory caspase activation by inflammasome proteins, some of which can also be activated directly or indirectly by na, is unique to vertebrates (maltez and miao, 2016) . in particular, dna sensing by inflammasomes is subject to strong evolutionary pressure and divergence even among mammalian species (brunette et al., 2012; gaidt et al., 2017) . indeed, the functionalization of crispr/cas systems as a tool for genomic editing have revealed important differences in human and murine na sensing, including distinct cell subset expression patterns of na-sensing toll-like receptors (tlrs) or species differences in the structural requirements for the detection of cyclic dinucleotide cgamp by sting. in this review, we will focus on the specific molecular mechanisms employed by the nucleic acid sensors of the vertebrate innate immune system to distinguish between physiologically present and pathogen-derived or pathogenically altered na. we primarily focus on the distinction of self versus non self, and the molecular structure, availability, and localization of na ligands. our aim is to provide sufficient background to understand these findings within the broader context of the evolving field of nucleic acid immunity. special emphasis is placed on what we think is essential information for someone who is new to the field, such as the spectrum of antiviral responses elicited (see box 1, effector functions), the multiple issues around the first na ligand ''poly(i:c)'' (see box 2, polyi:c), or the advantages and disadvantages of using the most common enzymatic method for rna synthesis, in vitro transcription (ivt), and its implications for rig-i activation (see box 3, in-vitro transcription). principles of nucleic acid sensing: localization, structure, and availability unlike fundamentally exogenous, microbial substances, such as flagellin or lps, nucleic acids are common to all forms of life, whether pathogen or host. effective na sensing thus critically depends on specific and sensitive detection of pathogenic or altered na among abundant, physiological endogenous molecules. the central principles underlying this distinction are na (1) structure, (2) localization, and (3) availability ( figure 1 ). structural characteristics of na include their length, base pairing, secondary structure, 5 0 -and 3 0 -termini, and modifications. the localization of na refers to their cellular and subcellular compartmentalization, monitored by a system of strategically placed na sensors. endosomal na sensors, i.e., the na-sensing tlrs, are primarily expressed in phagocytic, professional im-mune cells and are ideally located to sense ligands released by the hydrolytic degradation of pathogens in the endophagosomal compartment (blasius and beutler, 2010; brubaker et al., 2015) . this compartmentalization is controlled by trafficking and proteolytic receptor activation in the endosomal compartment, and alterations of receptor localization can lead to fatal autoinflammation (mouchess et al., 2011) . cytosolic na sensors, including rig-i, mda5, and cgas, are broadly expressed in nucleated cells where they sense cell-intrinsic infection. correspondingly, the type-i ifn receptor (ifnar) is also ubiquitously expressed (gonzá lez-navajas et al., 2012) , allowing, in theory, any nucleated cell to sense viral infection and enter an antiviral state via autocrine or paracrine ifn. moreover, recent research has also revealed the importance of nuclear na sensing (gentili et al., 2019; volkman et al., 2019) , revising the long-standing box 1. effector functions of nucleic acid sensing: signaling, restriction, and cell death upon na binding, immune sensing receptors activate an overwhelmingly transcriptional response. these signaling transduction cascades include nuclear factor-kb (nf-kb), interferon regulatory factors(irf) 3 and 7 and activator protein-1 (ap-1) (maniatis et al., 1998; marié et al., 1998) , which cooperatively transcribe chemokines and cytokines as well as type-i and, at mucosal barriers, type-iii interferons (onoguchi et al., 2007; ye et al., 2019) . this results in a complexly orchestrated response, including the induction of interferon-stimulated genes (isg) and the anti-viral state in infected and bystander cells, as well as the activation and the recruitment of innate and adaptive immune cells to the site of infection (gonzá lez-navajas et al., 2012) . in contrast, na restriction factors do not induce transcriptional signaling but rather serve to directly restrict the function of pathogen nucleic acids via their sequestration, destruction, and processing and/or presentation for na immune sensing receptors. these factors are generally isgs, and their presence is indicative of the anti-viral state. restriction mechanisms can be highly specific, such as the sequestration of 2 0 o-unmethylated capped rna by ifit1 (daffis et al., 2010) or global, such as protein kinase r activation, which upon binding of long double-stranded(ds)rna induces the inhibition of cellular transcription and translation, halting the proliferation of cell and virus alike (levin and london, 1978) . the ultima ratio of global pathogen restriction in host defense is cell death, which simultaneously restricts pathogen replication, releases inflammatory mediators, and terminates transcriptional signaling (maelfait et al., 2020) . na-sensing can trigger a variety of forms of programmed cell death (pcd) from immunologically silent to proinflammatory. apoptosis can result from overwhelming activation of na sensors, such as sting tang et al., 2016) or mavs kaneda, 2013; matsushima-miyagi et al., 2012) , particularly in cancer cells. necroptosis, a lytic proinflammatory form of pcd, can be directly induced by the activation of the zbp-1 by viral or endogenous z-form rna (maelfait et al., 2017; thapa et al., 2016) but also indirectly by combined tnf and ifn exposure downstream of sting or mavs hyperactivation (brault et al., 2018) . pyroptosis, a rapid, lytic form of pcd accompanied by the release of pyrogenic cytokines, results from inflammasome activation and can be triggered directly by na-sensing inflammasome proteins or indirectly downstream of na sensing. in particular, the nlrp3 inflammasome is activated by cellular dyshomeostasis and thus induces pyroptosis secondary to other cell death pathways(de vasconcelos and lamkanfi, 2020), as has been described downstream of sting, mavs, and zbp-1 activation, as well as viral lytic cell death (da costa et al., 2019; franchi et al., 2014; gaidt et al., 2017; kuriakose et al., 2016) . since canonical inflammasome assembly induces caspase-1-mediated proteolytic activation of both the pyroptotic effector gasdermin d as well as cytokines from the il-1 family, it sits at the crossroads of signaling and effector functions (de vasconcelos and lamkanfi, 2020) . however, it should be noted that this type of signaling is fundamentally different from the ifn and isg response: inflammasome activation is post-translational, not transcriptional and, theoretically, does not even requiring a living cell (franklin et al., 2018) . nonetheless, inflammasome activation is of critical importance to the immune response to many viral infections, since il-1 family cytokines support immune cell recruitment, nk-cell activation and the formation of anti-viral cd8+ t cell responses (reviewed in kanneganti, 2010) . since the type-i ifn response is inherently transcriptional, na-induced cell death also acts as an important limiter of antiviral responses. several studies have demonstrated that apoptotic caspases directly inhibit cgas/sting signaling (rongvaux et al., 2014; white et al., 2014) , with a recent report showing that activated caspase 3 can cleave cgas, mavs, and irf3 (ning et al., 2019) . similar mechanisms have been demonstrated for pyroptosis. activated caspase 1 has been reported to cleave cgas , and activation of gasdermin d-mediated pyroptosis can also resolve cgas/sting signaling through k+ efflux (banerjee et al., 2018) . although many recent studies to date have focused on the effect of pcd on cgas/sting, the reciprocal interplay between na sensing and cell death is undoubtedly important for all na-sensing pathways and continues to be the focus of intense research. paradigm that the nuclear compartment is ''immune privileged.'' indeed, given that the nucleus is the site the replication of most dna viruses, as well as retroviral genomic integration, these nuclear sensors are perhaps the last line of defense for our genomic integrity. na availability is the net result of the entry or generation of na structures in a specific compartment versus their sequestration (e.g., by pathogen vacuoles), shielding from receptor binding (e.g., by viral proteins), or degradation by nucleases. pathogen sequestration can be countered by host proteins, such as interferon-inducible gtpases (meunier and broz, 2016) , which release ligands into the cytosol. moreover, host and viral nucleases can act to limit or enhance the availability of potential na ligands. indeed, as we and others have shown, the same host nuclease (e.g., dnase 2, rnase t2) can potentially enhance the activity of some receptors while limiting the activity of others, thus acting as a rheostat for na sensor activation. although these principles are generally effective, the inherent risks of this strategy are clear. inappropriate or uncontrolled sensing of self na drives autoinflammatory disease, ranging from rare and devastating monogenetic forms of type-i interferonopathy (rodero and crow, 2016) to the inevitable senescence of our cells (gl€ uck et al., 2017; yang et al., 2017) . nonetheless, na sensing provides a clear evolutionary advantage in that it is essential for host survival, as demonstrated by numerous genetic models of pathogen infection. na sensors can be categorized into (1) bona fide immune sensing receptors that act to detect pathogenic na and signal their presence to the host and (2) anti-viral restriction factors that act directly upon viral na to limit viral replication but are not linked to the transcription of classical immune-related genes such as cytokines (schlee and hartmann, 2016) . the search for immune receptors detecting exogenous rna has led to the discovery of both endosomal and cytosolic rnasensing mechanisms (figure 2 ). the transmembrane tlrs tlr3, tlr7, tlr8, and, in lower vertebrates and rodents, tlr13, are all genetically proven endosomal rna immune sensors (alexopoulou et al., 2001; diebold et al., 2004; heil et al., 2004; oldenburg et al., 2012) . tlr10 has been reported to act as an anti-inflammatory receptor of double-stranded (ds-)rna, but further studies will be needed to corroborate this finding . tlr3 was the first immune sensing receptor of polyi:c discovered (alexopoulou et al., 2001) and is a sequence-independent sensor of the ribose-phosphate backbone of dsrna > 35bp liu et al., 2008) and incomplete dsrna stem structures of sufficient length within ssrna molecules (tatematsu et al., 2013) . tlr3 signaling is distinct from other na-sensing tlrs in several respects: instead of myd88, tlr3 signals via trif to induce ifn-b and nf-kb signaling (oshiumi et al., 2003; yamamoto et al., 2003) , with recent research indicating that the nutrient sensor mtorc2 is also critically required ; tlr3 is expressed in non-immune cells, including fibroblasts, endothelial cells, oligodendrocytes, astrocytes, and neurons (bsibsi et al., 2002; lafon et al., 2006; matsumoto et al., 2002; zimmer et al., 2011) , and it has been reported that, in several of these cell types, tlr3 can also be localized and signal from the cell surface, (jack et al., 2005; matsumoto et al., 2002; pohar et al., 2013) although this would presumably require an acidic environment for dsrna binding (liu et al., 2008) . despite the seeming redundancy of dsrna sensing (see box 2, polyi:c), humans expressing hypomorphic variants of tlr3 are susceptible to hsv-1 encephalitis (zhang et al., 2007) and severe influenza pneumonitis (lim et al., 2019) in box 2. poly i:c generated by the annealing of enzymatically generated inosine and cytosine homopolymers (michelson et al., 1967) , polyi:c is by far the most commonly used rna ligand in research. clinical applications for polyi:c were explored soon after the discovery that it could produce a robust ifn response (field et al., 1967; hilleman, 1970) , and clinical trials using polyi:c in tumor immunotherapy continue today (e.g., nct02166905, nct03358719). moreover, over the last 50 years, intense research has focused on characterizing the immune sensor(s) polyi:c activates. this popularity is remarkable given that polyi:c is neither a physiological ligand nor is its mode of action well defined. rather, its widespread use results from its amenability to enzymatic synthesis (michelson et al., 1967) and its unique ability to induce a robust type-i ifn response compared to other annealed homopolymers (field et al., 1967) , a feature which remains poorly understood. as a long dsrna with a 5 0 diphosphate terminus, polyi:c is known to activate the sensing receptors tlr3, mda5, and rig-i (alexopoulou et al., 2001; gitlin et al., 2006; kato et al., 2008; yoneyama et al., 2005) , accessory proteins such including lgp2 and members of the ddx and dhx families (reviewed in oshiumi et al., 2016) as well as the restriction factors pkr and the oas family (farrell et al., 1978; hovanessian et al., 1977; zilberstein et al., 1978) . in theory, any receptor that binds dsrna could be activated by polyi:c, including further restriction factors, such as zbp-1 (z-rna), although characterizing the specific activity would clearly require multiple gene deletions. this plethora of potential receptors likely contributes to its high toxicity profile, which is reduced in the more selective derivative polyi:c12u (junt and barchet, 2015) . despite over 50 years of research, many open questions about the molecule's bioactivity remain: why does low-molecular weight polyi:c (< 300bp) preferentially activate rig-i given that rig-i senses the 5 0 diphosphate terminus ? why is polyi:c a robust activator of mda5 while many other defined, long dsrnas such as poly(a:u) are not (colby and chamberlin, 1969; pichlmair et al., 2009) ? does the mesh-like structure of poly(i:c) contribute to its immune stimulatory activity, as opposed to just its length (pichlmair et al., 2009) ? why does the polyi:c derivative poly i:c12u (ampligen) only activate tlr3 but not rig-i or mda-5 (gowen et al., 2007) ? childhood, and tlr3 à/à mice demonstrate a susceptibility to poliovirus , hsv-1 (davey et al., 2010) , and mcmv (tabeta et al., 2004) . moreover, perhaps due to its broader expression, tlr3 has a preeminent role in the cns , where it contributes to both protective and deleterious neuroinflammation during host defense (mé nager et al., 2009; perales-linares and navas-martin, 2013; sato et al., 2018; wang et al., 2004) . in contrast, tlr7, tlr8, and tlr13 signal via the adaptor myd88 and are restrictively expressed in immune cells (diebold et al., 2004; heil et al., 2004; hemmi et al., 2002; hornung et al., 2002; shi et al., 2011) . tlr7 and tlr8 result from a gene duplication (roach et al., 2005) and demonstrate numerous structural and functional similarities. both receptors can be activated by imidazoquinoline compounds and polyu and gu-rich ssrna (judge et al., 2005; jurk et al., 2002) . however, selective ligands have also been reported (forsbach et al., 2008; lu et al., 2012; ostendorf et al., 2020) . while tlr7 is functional in both mice and humans, the role of tlr8 in mice remains unclear (alexopoulou et al., 2012; heil et al., 2004) . in humans, tlr7 and tlr8 demonstrate a differential expression pattern (hornung et al., 2002) . tlr8 is highly expressed on monocytes, conventional dcs and neutrophils, whereas tlr7 is predominantly expressed in plasmacytoid dendritic cells(pdc) and b cells. in human monocytes, tlr8 activation leads to the release of ifn-b and proinflammatory cytokines, including il12p70, il6, and tnf (bergstrøm et al., 2015; cushing et al., 2017) . very recently, the type-i-interferon-inducible tlr adaptor interacting with slc15a4 on the lysosome (tasl) has been reported as an additional signaling component linking endolysosomal tlr7 and tlr8 to interferon regulatory factor-5 (irf5) and ifn-b induction (heinz et al., 2020) . tasl recruits and activates irf5, and loss of tasl specifically impairs the activation of the irf pathway without affecting nf-kb and mapk signaling, revealing a mechanistic analogy with sting, mavs, and trif. in pdcs, tlr7 activation strongly induces ifn-a via the myd8-traf6-irf7 pathway (honda:2004jg. kawai et al., 2004) . tlr7 is also weakly expressed in primary monocytes, where it can also activate downstream signaling (de marcken et al., 2019; gantier et al., 2008) . the crystal structures of the ligand-bound ectodomains of tlr7 and tlr8 revealed that both bound rna degradation products (shibata et al., 2016; tanji et al., 2015; zhang et al., 2016) . in their first binding pocket, tlr7 binds guanosine and tlr8, uridine, respectively, while the second binds short di-or trinucleotides. subsequent studies demonstrate that tlr8 activation by rna ligands critically requires upstream endosomal rnase activity (greulich et al., 2019; ostendorf et al., 2020) . since endosomal rnase expression varies substantially between cell types, their activity adds another layer to our understanding of tlr7 and tlr8 specific ligands. tlr13 is expressed in non-mammalian vertebrates, marsupials, and rodents but not in primates (hidmark et al., 2012; oldenburg et al., 2012) . two studies identify the sequence cggaaagacc and acggaaagacccc within 23s rrna of several species of bacteria as a tlr13-activating motif oldenburg et al., 2012) . the crystal structure of the ectodomain of tlr13 reveals that it senses both rna sequence and stem-loop conformation (song et al., 2015) . to date, tlr13 is the only completely sequence-specific vertebrate rna immune sensor identified. the observation that the tlr3 à/à mouse still responds to polyi:c provided the first indication of intracellular rna immune sensing receptors (diebold et al., 2003) . the presence of cytosolic rna with hallmarks of non self can indicate viral replication , intracellular bacterial infection (hagmann et al., 2013; monroe et al., 2009) or the escape of rna from the endolysosome via transport or rupture (nguyen et al., 2017; watanabe et al., 2011) . two related dexd/h box rna helicases rig-i and mda-5 (yoneyama et al., 2004) , also known as rig-i like receptors (rlrs), sense cytosolic polyi:c and mount a phage-encoded dna-dependent rna polymerases are commonly used for in-vitro transcription (ivt) of dna into rna from linear dsdna or a plasmid dna template containing the appropriate promoter sequence (green and sambrook, 2020) . this method allows the rapid, cheap, and efficient generation of long rna molecules, many of which would be prohibitively expensive to produce synthetically. as such, ivt is a popular, often kit-based, method for producing mrna and crispr guide rna for use with recombinant cas proteins. however, since ivt utilizes promoter-based unprimed rna polymerization, it creates transcripts with a 5 0 triphosphate terminus, much like other nascent viral rna transcripts. we and others used ivt-generated rna to demonstrate that 5 0 triphosphate rna activated rig-i (hornung et al., 2006; pichlmair et al., 2006) . however, the rig-i activating species within the ivt is not its main product, 5 0 triphosphate ssrna. 3 0 end complementarity can prime a fold back reaction resulting in the production of complementary strands and rna hairpin structures (cazenave and uhlenbeck, 1994; triana-alonso et al., 1995) . as we and others subsequently showed, 5 0 triphosphate base-paired, blunt-ended rna, a side product within an ivt, is in fact the true rna species activating rig-i schmidt et al., 2009 ) reviewed in (schlee, 2013) . unless modified nucleotides or specific sequences precluding the formation of the complementary strand are used, ivt can potentially generate rig-i ligands from a variety of templates (hornung et al., 2006; kim et al., 2004; schlee et al., 2009; wienert et al., 2018) . on the one hand, this can lead to unwanted immunostimulatory effects during the generation of mrna, grna, or sirna. on the other, it means that extreme caution should be used when inferring that a particular sequence or endogenous rna species activates rig-i if ivt rna was used to model its activity. most endogenous rna species are processed post-transcriptionally and thus lack accessible 5 0 triphosphate moieties (gebhardt et al., 2017; schlee and hartmann, 2016) , thus using 5 0 triphosphate ivt rna is prone to creating immunostimulatory artifacts. immunity 53, july 14, 2020 57 type-i ifn response via the mitochondrial adaptor protein mavs (kawai et al., 2005; seth et al., 2005) . while rig-i is activated by shorter (< 300bp, low-molecular weight, lmw) poly i:c molecules, mda5 requires longer dsrna (> 300bp, high-molecular weight, hmw) polyi:c (gitlin et al., 2006; kato et al., 2005; yoneyama et al., 2005) . this overlapping yet differential activity also applies to the sensing of viral replication (loo et al., 2008; schlee, 2013) . both sensors contribute to the immune response to dsrna viruses, such as reoviridae (loo et al., 2008) , but rig-i-mediated sensing dominates the response to many (-) ssrna viruses such as influenza, which form shorter dsrna panhandles in their genomes (rehwinkel et al., 2010; schlee et al., 2009) , whereas mda5 has a greater role in the host defense against (+) ssrna viruses, many of which are known to generate large amounts of dsrna during replication . whereas subsequent studies have made substantial advances in characterizing the precise molecular patterns activating rig-i (see rna motifs distinguishing self from non self), the motifs necessary to activate mda5 still remain ill defined (hartmann, 2017) . both rna sensors are expressed in almost all primary nucleated cells (hartmann, 2017; ida-hosonuma et al., 2005) . upon activation, rig-i and mda-5 oligomerize, thereby inducing the polymerization of mavs into fibrillar structures (hou et al., 2011; xu et al., 2014) and recruiting traf2, traf6, ikk, and tbk1, which in turn activate nf-kb and irf3 and/or irf7 signaling (kawai et al., 2005; seth et al., 2005) and the transcription of proinflammatory cytokines and type-i and type-iii ifn. in addition to transcriptional signaling, mavs complexes can associate with fas-associated protein with death domain (fadd), receptor-interacting serine/threonine-protein kinase 1 (rip1), and caspase 8 (kawai et al., 2005; yang et al., 2019) . in this context, caspase 8 has been reported to induce apoptosis downstream of mavs (el maadidi et al., 2014) but also to terminate signaling without cell death (rajput et al., 2011; sears et al., 2011; yang et al., 2019) . several studies have noted that malignantly transformed cells are more sensitive to rlr-induced cell death, although the precise molecular determinants for this difference remain unknown (hartmann, 2017; kumar et al., 2015) . while it is well established that inflammasome activation is important for antiviral defense (kanneganti, 2010) , the rna sensors involved are still the subject of intense research. to date, three inflammasome-building nlrs have been reported to participate in cytosolic rna sensing, with all three requiring accessory rna-binding proteins. via the adaptor protein dhx33, nlrp3 is reported to sense viral dsrna, rnasel degradation products, and polyi:c, leading to inflammasome formation, caspase-1 cleavage and the release of il-1b and il-18 from in human thp-1 cells and monocyte-derived macrophages (chakrabarti et al., 2015; mitoma et al., 2013) . via the rna figure 1 . principles of self versus non-self or altered-self nucleic acid recognition unlike pathogen-specific molecules such as lps, nucleic acids in pathogens and the host are biochemically similar. for a reliable distinction of self versus nonself and altered-self nucleic acid recognition, information about the molecular structure, the availability, and the localization is integrated. the localization of nucleic acid receptors on different cell types, immune cells as well as non-immune cells, contribute as well. lists and cell types depicted are not comprehensive but just represent examples to better illustrate the principles. non-comprehensive overview of the most relevant rna sensing receptors at the relevant localizations, with their downstream signaling molecules and some of the functional outcomes and secondary consequences as applicable (e.g., rig-i sensing of rnasel degradation products). rna sensing receptors are in gray, signaling molecules in yellow, nucleases in green, inflammasome pathways is purple, and cell death pathways in orange. unlike other tlrs, tlr3 signals from the cell surface and the endolysosome. tlr3 is a sequence-independent sensor of the ribose-phosphate backbone of dsrna > 35bp and of incomplete dsrna stem structures of sufficient length within ssrna molecules. tlr3 signals via trif to induce ifn-b and nf-kb signaling. tlr7 and tlr8 are activated by rna degradation products, with the first pocket binding guanosine and uridine (tlr7 and tlr8, respectively), and the second pocket binding short di-or trinucleotides. tlr8 activation requires upstream endosomal rnase activity (rnaset2, rnase2) and, due to homology, rnase activity is likely required for tlr7 as well. tlr7 activation induces ifn-a via the myd88-traf6-irf7 pathway. tlr8 activation releases ifn-b and proinflammatory cytokines via a tak1-ikkb-irf5 pathway. tlr adaptor interacting with slc15a4 on the lysosome (tasl) was reported as a signaling component linking endolysosomal tlr7 and 8to irf5. tlr13 recognizes bacterial 23s rrna in a sequence-specific manner. upon activation, the cytosolic immune sensors rig-i and mda-5 oligomerize thereby inducing polymerization of mavs into fibrillar structures leading to the recruitment of traf2, traf6, ikk, and tbk1, which then activate nf-kb and irf3 and/or irf7 signaling. in addition, mavs complexes can associate with fadd, rip1, and caspase 8 and induce apoptosis downstream of mavs. ddx3, dhx15, dhx36, and ddx60 enhance rig-i signaling. dhx29 acts as co-receptor for both rig-i and mda5. lgp2 supports filament formation by mda5 but may compete with rig-i for ligand. cytosolic rna sensors with direct anti-viral activity include protein kinase r (pkr) and the 2'-5 0 -oligoadenylate synthetase system (oas) which both bind dsrna > 30bp including polyi:c. pkr phosphorylates elf2a and inhibits cap-dependent translation of viral and host mrna. oas induces the formation of 2 0 5 0 oligoadenylate which acts as a second messenger to activate ribonuclease l (rnase l), which in turn degrades cellular rna and viral rna to smaller rna molecules that can be sensed by rig-i and dhx33. dhx33 activates the nlrp3 inflammasome, inducing pyroptotic cell death. ifn-induced proteins with tetratricopeptide repeats (ifit) sequester viral mrna and block their translation by sensing 5 0 termini. ifit1 binds mrna with a cap0 structure (7mgpppnn), and ifit1b binds cap0, to a lesser extent cap1 (7mgpppnmn) structures but not cap2 (7mgpppnmnm) structures. b binds uncapped 5 0 triphosphate rna, and ifit2 which binds au-rich rna. ddx17 can bind and sequester stem loop structures from some rna viruses. adenosine deaminase acting on rna 1 (adar1) catalyzes the c6 deamination of adenosine to inosine in base-paired regions of rna, and the resulting non-synonymous coding causes amino acid substitutions and potentially renders viral proteins non-functional. the host rna decay machinery includes nonsense-mediated mrna decay (nmd), 5 0 -3 0 rna degradation and the 3 0 -5 0 rna exonuclease machinery (rna exosome). nmd targets mrna transcripts with a long 3 0 utr but also senses viral rna. the 5 0 -3 0 degradation machinery with the decapping enzymes dcp1 and dcp2 and the 5 0 -3 0 exonuclease xrn1 (xrn-dcps) is involved in physiological cellular mrna turnover and exhibits antiviral activity. the superkiller viralicidic activity 2-like (skiv2l) and zinc-finger antiviral protein (zap) support the binding and transport vrna to the rna exosome for degradation. the isg and viral restriction factor z-dna binding protein 1 (zbp-1) is a death receptor downstream of dsrna sensing. zbp-1 (legend continued on next page) ll immunity 53, july 14, 2020 59 helicase dhx9, the previously uncharacterized nlrp9b (human homolog nlrp9) forms an inflammasome in intestinal epithelial cells in response to rotavirus, leading to the maturation of il-18 and the activation of gasdermin d-mediated pyroptosis (zhu et al., 2017) . via the rna-binding accessory protein dhx15, nlrp6 has been reported to sense dsrna in intestinal epithelial cells . however, this does not result in caspase-1 activation but rather mavs-dependent type-i and iii ifn induction. this function of nlrp6 is reportedly restricted to the gastrointestinal tract, as nlrp6 à/à mice demonstrate increased mortality and viremia after oral but not systemic viral infection. since nlrp6 can form an inflammasome (elinav et al., 2011; shen et al., 2019) , how dhx15 induces nlrp6-mavs interaction rather than oligomerization with the inflammasome adaptor asc remains unclear. other dexd/h-box proteins have been reported to act as accessory proteins to cytosolic rna signaling (oshiumi et al., 2016) . ddx3, dhx15, dhx36, and ddx60 have been reported to enhance rig-i signaling (miyashita et al., 2011; oshiumi et al., 2010; pattabhi et al., 2019; yoo et al., 2014) , whereas dhx29 acts as a co-receptor for both rig-i (sugimoto et al., 2014) and mda5 . the individual contributions of these proteins to rna sensing remain unclear and occasionally contradictory. in one study, ddx3 has been found to induce mavs activation independently from rig-i and mda-5 (gringhuis et al., 2017) ; in another, it has been found to support rig-i signaling (oshiumi et al., 2010) , and, in another, it was even found to inhibit ifn-i release (loureiro et al., 2018) . likewise, the role of ddx60 is also controversial, with one group finding that it promotes rlr signaling and another reporting that it has no role (goubau et al., 2015; miyashita et al., 2011) . future studies will be needed to resolve these differences. lgp2, a dexd/h-box protein from the rlr family, contains the rlr c-terminal domain (ctd) and helicase domains but lacks the card necessary for downstream signaling (schlee, 2013) . accumulating evidence indicates that lgp2 acts (1) as a structural accessory protein that supports filament building and activation of mda5 (bruns et al., 2014; deddouche et al., 2014; satoh et al., 2010) and (2) as an interferon-inducible inhibitor of rnai (takahashi et al., 2018b; van der veen et al., 2018) . lgp2 has been reported to competitively bind dsrna and inhibit the rna silencing enhancer, tar-rna binding protein (trbp) (takahashi et al., 2018b; , an accessory protein of dicer. this mechanism also highlights the inherent antagonism between dicer-mediated cleavage of dsrna during rnai and dsrna sensing by the innate immune system. in addition to the dexd/h-box protein, zink-finger protein zcchc3 has been reported to bind viral dsrna and act as a cofactor for rig-i and mda5, and zcchc3 à/à mice are more susceptible to rna virus infection (lian et al., 2018b) . the same group has also reported in parallel that zcchc3 acts as a cofactor for dsdna sensing and that zcchc3 à/à mice are also more susceptible to lethal herpes simplex virus type 1 or vaccinia virus infection (lian et al., 2018a) . here, further studies will be necessary to determine how zcchc3 fulfils such diverse roles in host sensing. protein kinase r (pkr) and the 2'-5 0 -oligoadenylate synthetase system (oas) are the first rna sensors discovered in the cytosol. both sense dsrna > 30bp and are activated by polyi:c (see box 2, polyi:c). pkr phosphorylates elf2a and thus inhibits cap-dependent translation of viral and host mrna (levin and london, 1978) . oas binding of dsrna leads to the production of 2'5 0 oligoadenylate (hovanessian et al., 1977; zilberstein et al., 1978) , a second messenger activating latent ribonuclease l (rnase l) (zhou et al., 1993) . rnasel, in turn, degrades cellular and viral rna. global rnase-l degradation does not only prevent viral replication but also generates smaller rna molecules sensed by rlrs (malathi et al., 2007 (malathi et al., , 2010 and dhx33-mediated activation of nlrp3, inducing pyroptotic cell death (mitoma et al., 2013) . in cells without nlrp3, rnase l can also trigger apoptosis to prevent viral propagation (castelli et al., 1997) . the host rna decay machinery also contributes to antiviral defense, and the role of nonsense-mediated mrna decay (nmd), 5 0 -3 0 rna degradation and the 3 0 -5 0 rna exonuclease machinery (rna exosome) are all the subject of current, intense research. to guard the cell against aberrant self mrna, nmd targets mrna transcripts with a long 3 0 utr, indicative of a mutation leading to a premature stop codon (molleston and cherry, 2017) . however, nmd also forms an important intrinsic barrier to viral infection (balistreri et al., 2014; fontaine et al., 2018; . conceivably, nmd senses viral rna via several complementary mechanisms, including the unusual translation dynamics of polycistronic viral transcripts, instability due to non-standard codon usage (hia et al., 2019) and targeting by nmd-associated proteins . the 5 0 -3 0 degradation machinery, comprising the decapping enzymes dcp1 and dcp2 and the 5 0 -3 0 exonuclease xrn1 (xrn-dcps), is primarily involved in physiological cellular mrna turnover. however, xrn1 is also specifically targeted by and inhibited by flaviviruses (molleston and cherry, 2017) , demonstrative of antiviral activity. one recent study demonstrates that xrn1-dcps specifically colocalize with the rna of newcastle disease virus and encephalomyocarditis virus (emcv) and repress viral replication (ng et al., 2020) , although it is still unknown how xrn1-dcps are recruited to viral rna. recruitment to the rna exosome is better understood. superkiller viralicidic activity 2-like (skiv2l) (aly et al., 2016) and zincfinger antiviral protein (zap) (gao et al., 2002; guo et al., 2007) support the binding and transport viral rna to the rna exosome for degradation. how skiv2l specifically targets viral rna is still unknown. however, recent studies have demonstrated that zap directly senses hiv-1 rna via its relative abundance in cg dinucleotides (ficarelli et al., 2020; takata et al., 2017) , thus revealing activates multiple programmed cell death pathways, including pyroptosis, apoptosis, and necroptosis, termed pan-optosis. upon dsrna binding, zbp-1 interacts with ripk3 supported by caspase-6, resulting in mlkl activation and necroptosis, in caspase-8-induced apoptosis and in nlrp3 dependent inflammasome activation and pyroptosis. three inflammasome-building nlrs participate in cytosolic rna sensing: dhx33 via nlrp3, the rna helicase dhx9 via nlrp9b (human homolog nlrp9), and the rna-binding accessory protein dhx15, besides its supporting function for rig-i-like helicases, via nlrp6. a sequence-dependent mechanism for self versus non-self discrimination. of note, the rna exosome is also involved in the degradation of immunostimulatory endogenous rna transcripts, and hypopmorphic variants of skiv2l induce type-i interferonopathy that is dependent on rig-i (eckard et al., 2014) . we and others recently demonstrated that rnaset2 contributes to the activation of tlr8 (greulich et al., 2019; ostendorf et al., 2020) . however, hypomorphic variants of rnaset2 are associated with a rare form of proinflammatory cystic leukoencephalopathy (henneke et al., 2009) , likely resulting from cytosolic sensor activation by accumulating rna (haud et al., 2011) . our work also demonstrates that rnase2, a member of the rnasea family only expressed in apes and old-world monkeys, synergistically participates in uridine release from ssrna to activate tlr8 in monocytes (ostendorf et al., 2020) . antimicrobial functions have also been attributed to other members of the rnasea family (lu et al., 2018) . members of the ifn-induced protein with tetratricopeptide repeats (ifit) family specifically sequester viral mrna and thus block their translation by sensing 5 0 termini with markers on non self. ifit1 binds mrna with a cap0 structure (7mgpppnn), and ifit1b binds cap0, to a lesser extent cap1 (7mgpppnmn) structures but not cap2 (7mgpppnmnm) structures. ifit5 binds uncapped 5 0 triphosphate rna (abbas et al., 2013; habjan et al., 2013; kumar et al., 2014) , and ifit2 binds au-rich rna . in addition to the ifits, ddx17 can bind and sequester stem loop structures from the rift valley fever virus in drosophila and humans (moy et al., 2014) , and it seems highly likely that other dexd/h box rna helicases can perform similar functions. adenosine deaminase acting on rna 1 (adar1) catalyzes the c6 adenosine-to-inosine deamination of base-paired regions of rna (george et al., 2014) . due to their c6 carbonyl group, these new inosines are decoded non-synonymously as guanosines, causing amino acid substitutions and potentially rendering viral proteins non-functional (samuel, 2011) . in addition, the introduction of less stable i-u wobble pairs changes base-pairing dynamics ( spa cková and ré blová , 2018). these alterations of the secondary structure affect both viral regulation and host innate immune sensing, acting in a manner that can be pro-or antiviral (samuel, 2019; 2011) . in addition to the aforementioned cell death pathways, the isg and viral restriction factor z-dna binding protein 1 (zbp-1) is a dedicated death receptor downstream of dsrna sensing. zbp-1 was originally described as a dsdna sensor capable of inducing type-ifn. however, subsequent studies have demonstrated that zbp-1 is a dsrna sensor that activates multiple pcd pathways, including pyroptosis, apoptosis and necroptosis, termed pan-optosis (malireddi et al., 2019) . upon dsrna binding, zbp-1 enters a homotypic rhim-rhim interaction with the kinase ripk3 supported by caspase-6 (zheng et al., 2020). this can result in mlkl activation and necroptosis (maelfait et al., 2017; thapa et al., 2016) , caspase-8-induced apoptosis thapa et al., 2016) and, in nlrp3 expressing cells, inflammasome activation and pyroptosis (kuriakose et al., 2016) . how these normally hierarchical forms of pcd are coordinated after zbp-1 activation is not yet understood (maelfait et al., 2020) . nonetheless, zbp-1-mediated pcd has been shown to be important to controlling murine influenza a virus (iav) infection. moreover, human h1n1 pandemic iav but not seasonal iav has been demonstrated to suppress ripk3-mediated necroptosis, indicating that inhibiting this cell death pathway also contributes to viral virulence in humans . rna motifs distinguishing self from non self cytosolic long dsrna is considered a hallmark molecular pattern of viral infection: it is generally absent from the cytosol of uninfected cells, yet it forms the genome of dsrna viruses and is generated during the replication of ssrna and dna viruses (son et al., 2015; weber et al., 2006) . however, recent research indicates that the absence of endogenous cytosolic dsrna is not coincidental. our genome contains a large number of complementarily inverted mobile elements, particularly alu elements, which could potentially form long dsrna (reich and bass, 2019) , and their overwhelming absence in the cytosol results from both strong purifying transcriptomic selection (barak et al., 2020) and a-to-i editing of the remaining transcripts by adar1 (ahmad et al., 2018; chung et al., 2018) . in addition, mitochondria utilize an rna helicase, suv3, and polynucleotide phosphorylase (pnpase) to actively eliminate dsrna species resulting from bidirectional transcription thus preventing innate immune activation (dhir et al., 2018; pajak et al., 2019) . the importance of this sophisticated protective machinery becomes evident when it breaks down: hypomorphic variants of adar1 and hypermorphic variants of the dsrna sensor mda5 cause devastating forms of type-i interferonopathy (rodero and crow, 2016) resulting from the sensing of alu elements (ahmad et al., 2018; chung et al., 2018) . however, the contribution of adar1 to antiviral defense is less clear. a-to-i conversion destroys the coding potential of viral rna, thus inhibiting viral replication, yet it also destabilizes long dsrna structures indicative of replicating pathogens (samuel, 2011) . the same reciprocal antagonism in dsrna recognition occurs between the rnai system and rna sensing receptors. dicer-mediated processing of dsrna interferes with viral replication yet destroys the very structure which immune sensors recognize. the critical importance of dsrna as a molecular pattern is underscored by the convergent evolution of immune sensors and restriction factors it activates, making dsrna a ''promiscuous'' ligand with diverse downstream effects (hartmann, 2017 ) (see box 2, poly i:c). unfortunately, the precise requirements for the dsrna length of many of these sensors remains unknown. to date, we are unaware of any publications detailing a minimal ligand for the 8 ddx and dhx proteins detailed in this review, adar1, lgp2, or zbp-1. we hope future studies will characterize the precise nature of the dsrna activating these sensors. based on our knowledge to date, cytosolic dsrna sensing via its ribose-phosphate backbone requires dsrna > 30bp, including pkr, oas, and mda5 (> 300bp for polyi:c). detection of dsrna < 30bp requires amenable 5 0 termini (see below), which contain further information on the origin of dsrna (5 0 phosphorylation, capping, cap structure, etc.). this cutoff strongly suggests the presence of physiological endogenous dsrna < 30bp in our cytosol. nonetheless, mda5 ligands, while intensely researched, remain ill defined (hartmann, 2017). the length requirement of > 300bp has been determined for polyi:c (kato et al., 2008) but remains unknown for viral and endogenous ligands (schlee and hartmann, 2016) . indeed, while most inverted alu elements form dsrna of approximately 300bp, they are only weakly stimulatory for non-ags mda5 variants when compared to polyi:c (ahmad et al., 2018) . endosomal dsrna also represents an optimal recognition motif for viral infection. as it is more resistant to endosomal rnases than ssrna (ostendorf et al., 2020) , dsrna provides an important indication of viral infection in efferocytosed cells. here, it is interesting to note that the dsrna length required for tlr3 dimerization falls from > 90bp in the endosome to 39-48bp in late endolysosome , at lower ph and after longer rnase exposure. thus, endosomal sensing seemingly results from a combined calculus of length and rnase resistance. the precise length requirement at the cell surface is unclear, but presumably > 90bp. of note, tlr3 can also sense dsrna structures within long > 600 nt ssrna of poliovirus (tatematsu et al., 2013) , which would be indicative of a likely bulge tolerance between the two parts of the tlr3 dimer. however, reports of tlr3 activation with shorter rna, e.g., sirna < 30bp, are limited to mouse tlr3 (weber et al., 2012) , and one study on the activation of tlr3 by mrna is based on in vitro transcription (karikó et al., 2004) , which may have led to the generation of longer dsrna as well as rig-i ligands (see box 3, in-vitro transcription). nonetheless, certain endogenous rna species, such as unedited alu elements capable of activating mda5, may also activate tlr3. the 5 0 terminus contains a wealth of information about rna origin. in higher eukaryotes, endogenous mrna receives a m7gpppn cap, further modified by 2'o-methylations to m7gpppnm (cap 1) and m7gpppnmnm (cap 2). the capping process controls gene expression by modulating nuclear export, splicing, protein translation and mrna turnover (dimitrova et al., 2019) . of note, 2'o-methylation coevolved with the ifn system and is even used to specifically regulate isg expression (williams et al., 2020) . in addition to the extensive modification of mrna, small nuclear rnas (snrnas) receive methylguanosine caps which are hypermethylated into trimethylguanosine in the cytoplasm, and ribosomal rna (rrna) and transfer rna(trna) are both generated via processing of precursors which creates 5 0 monophosphate (5 0 p) termini and are extensively 2'o-methylated. thus, capping, n1 and n2 2'o-methylation of capped rna, and the presence of 5 0 p termini can all be viewed as hallmarks of self. the cytosolic dsrna sensor rig-i can be activated by dsrna > 18-19 bp but critically requires that the 5 0 terminus is compatible with its c-terminal domain (ctd). strikingly, the ctd binding cleft accommodates rna bearing signs of ''non self'': it binds 5 0 triphosphate (5 0 ppp) (hornung et al., 2006; schlee et al., 2009 ) and 5 0 diphosphate (5 0 pp) ) and, to a lesser extent 5 0 oh termini (binder et al., 2011; marques et al., 2006) , but not 5 0 p (ren et al., 2019) . moreover, while a cap0 structure reduces rig-i activity, 2'o-methylation (cap1, cap2) abrogates it completely (schuberth-wagner et al., 2015) . detection of cap0 rna allows rig-i to sense viruses that code their own capping enzymes but do not have methyltransferase activity, such as sindbis virus (hefti et al., 1975) . moreover, the discrimination between 5 0 p and 5 0 oh for rig-i is of particular importance for the dsrna products of rnase ac-tivity. the rnases processing trna and rrna create 5 0 p rna, as does dicer. in contrast, rnasel products have 5 0 oh ends and can thus activate rig-i (malathi et al., 2007) . the rig-i response to 5 0 ppp dsrna is strongly reduced by 3 0 overhangs and abrogated by 5 0 overhangs at the 5 0 ppp end of the molecule . blunt dsrna occurs naturally in the panhandle genome of (-) ssrna viruses. however, rrna, trna, and dicer products all contain 5 0 or 3 0 overhangs, a further preclusion to rig-i activation. sensing of the 5 0 terminus is also a strategy followed by members of the ifit family in their sensing of ssrna. ifit1 sequesters ssrna or dsrna with a 5 0 overhang of > 5nt that have a 5 0 ppp or a cap0 but not a cap1 structure. ifit1b sequesters 5 0 ppp, cap0 >> cap1 but not cap2, and ifit5 senses 5 0 ppp ssrna or dsrna with an overhang of at least 3nt (abbas et al., 2013) . in order to escape immunodetection, a number of viruses conceal the 5 0 termini of their rna or disguise themselves with molecular markers of self. some (-) ssrna viruses, including hantavirus and bunyavirus, process their 5 0 termini to 5 0 p, avoiding detecting by rig-i and the ifits. arenaviruses use primeand-realign mechanisms to generate rna with 5 0 overhangs, which allows for ifit binding but may even act as a competitive inhibitor of rig-i activity (marq et al., 2011) . others code for their own 2'o-methyltransferases (daffis et al., 2010) , and some even perform ''cap snatching,'' removing the host 5 0 g7m cap along with 10-14nt and integrating it into their own mrna. given the exquisite sensitivity of 5 0 terminus sensing, it is hard to imagine the presence of many endogenous ligands capable of activating rig-i. however, hypomorphic variants of the skiv2l subunit of the rna exosome drive type-i ifn mediated autoinflammation via rig-i (eckard et al., 2014) , which can recognize rna generated by inositol-requiring enzyme 1(ire-1) during the unfolded protein response. (see figure 2) . however, to date the identity and structure of these transcripts remain unknown. another strategy to discriminate self from host is the sensing sequence-specific motifs. in the cytosol, ifit2 senses au-rich rna , although the precise function of this sensing is unclear, as au-rich elements also occur in our transcriptomes (elliott and ladomery, 2017) . zap, which ferries rna to the rna exosome, binds cg-rich motifs in rna (takata et al., 2017) , a strategy enabled by the cg-suppression of the vertebrate genome (karlin and mrá zek, 1997 ) that may explain why some viruses actively avoid cg motifs (schlee and hartmann, 2016) . in the endosome, tlr13 senses a genuinely sequencespecific motif: a(cggaaagacc)cc within 23s rrna. it is not clear why such as sequence-specific receptor evolved, but there are different possible explanations for its disappearance in higher mammals. oldenburg et al. (2012) has demonstrated that a known methylation coded by an erythromycin-resistance gene leading to cggmaaagacc could abrogate tlr13 activity. this gene is of ancient origin in bacteria and could explain a loss of utility for tlr13. while li and chen (2012) demonstrate that this methylation has no effect, they find that this base within 23s rrna is exceptionally sequence-specific, i.e., that acggbaagacccc (b = not a) could no longer activate tlr13. while it is remarkable that no further study has resolved this controversy, it seems clear ll that tlr13 is an exceptionally mutation-and/or modification-sensitive receptor, which may account for its evolutionary loss. human tlr8 has been suggested as an evolutionary replacement of murine tlr13 (kr€ uger et al., 2015) . while tlr8 is not active in mice, in humans, it is essential for recognition of bacterial rna (eigenbrod and dalpke, 2015) . however, this tlr8-tlr13 equivalency only applies to pathogens containing the tlr13 consensus sequence. tlr8 also senses plasmodia rna in humans, (coch et al., 2019) , which activates tlr7, not tlr13 in mice. indeed, how tlr8 senses non-self rna is an unresolved question. two studies have made clear that tlr8 senses rnase degradation products rather than ssrna (greulich et al., 2019; ostendorf et al., 2020) . in line with previous reports, it is clear that the degraded ssrna must contain uridine since free uridine is required for the first binding pocket (tanji et al., 2015) . one study has postulated that uuru is a minimal motif for tlr8 activation (greulich et al., 2019) , in line with its known stimulatory activity (forsbach et al., 2008) . however, under uridine supplementation, the di-or trinucleotide rna for the second binding pocket of tlr8 does not require uridine for activity (ostendorf et al., 2020; shibata et al., 2016) . thus, the precise requirements for the second binding pocket and the true nature of any minimal tlr8 ligand remain unclear. moreover, given the broad number of conceivable di-or trinucleotide ligands for the second binding pocket, it seems unlikely that such motifs are not contained in self rna, or that they could all be rendered inactive by modifications such as pseudouridine or 2'o-methylation (freund et al., 2019) . in addition, it is unclear which inhibitory modifications affect tlr8 activation and which inhibit upstream rnases. 2'o-methylation is a known inhibitor of tlr8 (and tlr7) activity (freund et al., 2019) but also of upstream rnaset2 and rnase2 (ostendorf et al., 2020 ). conceivably, self rna does not avoid immunorecognition in the classical sense but rather contains inhibitory motifs perhaps similar to those synthetically designed for tlr7 and tlr8 (schmitt et al., 2017) using 2'o-methylation, but further studies will be necessary to determine if these sequences have natural counterparts in our self rna. endosomal sensing of dna tlr9 was the first immune na sensor identified (hemmi et al., 2000) and is the only endolysosomal dna receptor. despite extensive transient trafficking, the signaling competent form of tlr9 is exclusively localized to the endolysosome, and mislocalization of tlr9 to the cell surface induces strong autoinflammation (mouchess et al., 2011) . the availability of tlr9 ligands in the endolysosome is tightly regulated by dnases (see nucleases and the sensing of self versus non-self dna). tlr9 preferentially detects ssdna containing unmethylated cytosine-phosphate-guanine (cpg) motifs which are less frequent in eukaryotic self dna (methylation of the c5 carbon of cytosine) compared to bacterial dna hartmann and krieg, 2000; krieg et al., 1995) , with a recent study identifying highly conserved stimulatory cpg-dna fragments in multiple 16s and 23s rdna sequences in bacterial genomes (liu et al., 2020) . moreover, a recent structural study reports that tlr9 has two binding sites, one binding ssdna with an unmethylated cpg motif and the second binding short ssdna carrying a 5 0 hydroxyl . the second site prefers 5 0 hydroxyl ssdna with a cytosine at the second position from the 5 0 end (5 0 -xcx dna). combined cpg and 5 0 -xcx dna binding cooperatively promotes tlr9 dimerization and activation. in line with this, previously established potent tlr9 ligands such as odn2006 contain both structural elements, unmethylated cpg motifs and a cytosine at the second position from the 5 0 end . previous work on stimulatory tlr9 ligands reveal important species-specific differences. in recent years, the sequence specificity of dna ligands for both humans and mice has been further investigated (pohar et al., 2017a (pohar et al., , 2017b . in these studies, the minimal dna sequence for human tlr9 has been defined as , and the 5 0 -tcg was found to be essential for activation, consistent with the two binding sites identified by ohto et al. (2018) . interestingly, mouse tlr9 has a much lower requirement for the 5 0 -xcx sequence than human tlr9 pohar et al., 2017b) . in fact, mouse tlr9 can be dimerized by cpg dna alone due to tlr9-cpg dna interactions unique to mice . not only ligand specificity but also the cellular expression pattern differs between murine and human tlr9. whereas, in the mouse, tlr9 is widely expressed in immune cells, including myeloid cells, in humans, it is restricted to b cells and pdc (barchet et al., 2008) , and their downstream immune effects differ accordingly. notably, tasl was recently identified as an additional signaling protein linking tlr9 to irf5 (heinz et al., 2020) . the discovery that dsdna induces type-i ifn came 40 years after dsrna stetson and medzhitov, 2006) . moreover, the first pathway identified involved rna but not dna sensing (ablasser et al., 2009; chiu et al., 2009 ). like polyi:c, poly(da:dt) was used as a standard dna stimulus due to its strong ifn-i activation and its amenability to enzymatic synthesis. however, poly(da:dt) provides a template for transcription of au-rich rna by the pol iii pathway. these polyua transcripts bear 5 0 triphosphate and fold into dsrna, thus acting as rig-i ligands and inducing type-i ifn independently of a true dna sensor. at the end of 2012, the cgamp synthase (cgas)-stimulator of interferon genes (sting) pathway was identified as the principle cytosolic dsdna sensor inducing type-i ifn wu et al., 2013) . a number of other candidate receptors have previously been proposed and may have accessory and/or cell-type-specific functions (see figure 3 and box 4, cytosolic dna and type-i ifn). the carboxyl terminus of cgas binds to the ribose-phosphate backbone of dsdna in a sequence-independent manner, inducing dimerization and formation of the cyclic dinucleotide (cdn) c[g(2 0 -5 0 )pa(3 0 -5ʹ)p]) (2'3 0 cgamp). 2'3 0 cgamp then acts as a second messenger (ablasser et al., 2013a; gao et al., 2013a) , activating sting, followed by tbk1 and irf3 phosphorylation. the downstream sensor sting itself is also a cytosolic (burdette et al., 2011; gao et al., 2013b) . notably, murine sting was far more reactive to bacterial cdn than human sting (ablasser et al., 2013a; gao et al., 2013b) , with hsting h232 showing near-perfect selectivity for 2'3 0 -cgamp (gao et al., 2013b) . nevertheless, several natural polymorphisms in human sting influence the sensitivity to 2'3 0 -cgamp and 3 0 3'-cdn (yi et al., 2013) , and the most common sting variant, hsting r232 shows a moderate selectivity for 2'3 0 -cgamp (gao et al., 2013b) . further studies will be needed to elucidate the relevance of human sting as a direct cdn sensor in host-pathogen interaction. the role of the pyhin proteins ifi16 and p204 in sting activation has been controversial (see box 4, cytosolic dna and type-i ifn). human ifi16 and its reported ortholog, murine p204, are coded by the aim2-like receptor (alr) locus, an evolutionary hotbed (see dna-induced inflammasome activation). an initial publication has demonstrated that rnai targeting human ifi16 and murine p204 reduces ifn-i induction from human thp-1 cells, murine embryonic fibroblasts, and raw264.7 macrophages, respectively (unterholzner et al., 2010) . however, the importance of p204 has been challenged by subsequent studies (brunette et al., 2012; gray et al., 2016) , causing some confusion in the field. further studies using genome editing in human cells could provide clear evidence for the role of ifi16 in ifn-induction in keratinocytes and macrophages, both in supporting cgas signaling and cgas-independent dna sensing (almine et al., 2017; jønsson et al., 2017) . however, due to the genetic diversity of the alr locus, it is possible that this role in dna sensing is unique to primates. non-comprehensive overview of the most relevant dna-sensing receptors at the relevant localization, their downstream signaling molecules, and the functional outcome and secondary consequences as applicable (e.g., sting sensing of cgas product 2 0 3 0 cgamp). dna-sensing receptors are in gray, signaling molecules in yellow, nucleases in green, and inflammasome pathways is purple. the signaling competent form of tlr9 is exclusively localized to the endolysosome and preferentially detects ssdna containing unmethylated cytosine-phosphate-guanine (cpg) motifs which are less frequent in eukaryotic self dna compared to bacterial dna. tlr9 has two binding sites, one site binding ssdna with an unmethylated cpg motif and the second site binding short ssdna carrying a 5 0 hydroxyl, both cooperatively promoting tlr9 dimerization and signaling, including the recently identified signaling component tasl linking signaling to downstream irf5. tlr9 ligands in the endolysosome are tightly regulated by endolysosomal dnase ii, pld3, and pld4 which coordinately degrade single-and double-stranded dna. cytosolic dna sensing includes the rna sensor rig-i which detects rna transcribed from poly(da:dt) by pol iii. the cgamp synthase (cgas)/stimulator of interferon genes (sting) pathway is the principle cytosolic dsdna sensor pathway, activating a type-i ifn response, and via sting a nlrp3 dependent inflammasome response. availability of cytosolic dna for this pathway is regulated by dnase iii (trex1). accessory proteins for cgas are hmgb1, the gtpase-activating protein sh3 domain-binding protein 1 (g3bp1), tfam, and cchc-type zinc-finger protein 3 (zcchc3) which bind, bend, and stabilize dsdna in a manner amenable to the nucleation of cgas dimers along the dsdna strand. cgas is also localized in the nucleus where it senses foreign but not self dna. here, non-pou domain-containing octamer binding protein (nono), but also hmgb1 and tfam, assist in binding of dna to cgas. ifi16 is a nuclear restriction factor, binding, and silencing viral or transfected dna. the dna damage protein rad50 upon stimulation with dsdna induces proil1b via the card9-bcl10 pathway. in the human system, a sting-independent dna sensing pathway via the dna damage response protein dna-dependent protein kinase (dna-pk) senses linear dna, and signals via irf3/irf7 and hspa8/hsc70. absent in melanoma (aim2) is the principle cytosolic dsdna sensor responsible for inflammasome activation. several family members of the apolipoprotein b editing complex (apobec) act as restriction factors by performing c-to-u editing on first(minus) strand cdna, resulting in g-to-a mutations in the plus strand. the sterile alpha-motif (sam) and histidine-aspartate (hd) domaincontaining protein 1 (samhd1) is a deoxynucleoside triphosphate (dntp) triphosphohydrolase removing tripolyphosphate moieties from dntps thereby reducing the intracellular dntp concentration and inhibiting reverse transcription. in addition, a recent publication has identified a sting-independent dna sensing pathway (sidsp) in human but not murine cells via dna-dependent protein kinase (dna-pk) (burleigh et al., 2020) , a protein previously associated with sting-dependent responses (see box 4, cytosolic dna and type-i ifn). although dna-pk is important for sensing dna damage, sidsp does not occur downstream dna damage but requires the introduction of foreign dna. in contrast to cgas, dna-pk requires linear ligands, suggesting that it senses dsdna termini. its signaling pathway requires irf3 and/or irf7 but not tbk1 and possibly the dna-pk dependent phosphorylation of hspa8 and hsc70. of note, a much earlier study has observed that dna-pk could directly phosphorylate irf3 (karpova et al., 2002) . to date, it remains unclear whether the sting-dependent dna-pk pathway in mice is related to sidsp in humans or what would be the basis for a species-specific requirement for sting activity. another dna damage protein that has been implicated in dna sensing is rad50. upon stimulation with dsrna, rad50 has been reported to induce proil1b mrna via the card9-bcl10 pathway, thus providing proil1b for concurrent dna-induced inflammasome activation (see dna-induced inflammasome activation) (roth et al., 2014) . cytosolic mislocalization of dsdna was long thought to be required for its sensing (schlee and hartmann, 2016) . however, many dna viruses replicate in the nucleus , and it is also the site of retroviral integration into genomic dna (volkman and stetson, 2014) . thus, despite the wealth of nuclear self dna, host defense in this compartment would be advanta-geous. several recent publications have demonstrated that cgas also localizes to the nucleus where it senses foreign but not self dna (gentili et al., 2019; volkman et al., 2019) as well as inhibiting homologous recombination and dna repair liu et al., 2018) , processes requiring direct interaction with nuclear self dna. one study has observed nuclear ''tethering'' of cgas via its ntase domain preventing activation by self dna, but how this tethering is regulated or relaxed to allow cgas to perform its various nuclear functions, such as the sensing of hiv-2 (lahaye et al., 2018) , remains unclear. ifi16 is also reported to participate in nuclear retroviral sensing as an inflammasome-forming protein (see next section). furthermore, several reports provide evidence that ifi16 works as a nuclear restriction factor, efficiently binding and silencing viral or transfected dna orzalli et al., 2013) . likewise, another alr pyhin1(ifix) is reported to restrict herpesvirus in the nucleus (diner et al., 2015a) . future studies will be necessary to characterize how these and other dna sensors act in the nuclear compartment. three dna-sensing inflammasomes have been described to date (see figure 3 ): the absent in melanoma 2 (aim2), ifi16 inflammasomes, and nlrp3 activation secondary to stingmediated cell lysis. aim2 and ifi16 are both alrs (brunette et al., 2012; unterholzner et al., 2010) , which can bind dna via their hin200 domain(s) and engage in homotypic pyrin-pyrin domain interaction with the inflammasome adaptor protein asc. the alr locus is genetically diverse: unique to mammals, it contains 1 gene in marsupials and cows, 5 in humans, 4 in rats, and 13 in mice, and is absent in monotremes and bats (ahn et al., a large number of receptors have been implicated in the immune sensing of cytosolic dna, including dhx9, dhx36, ddx41, dai (zbp-1), mre11, dna-pk/ku70/ku80, the alrs pyhin1, ifi16(p204), and the nucleotidyltransferase cgas (diner et al., 2015b; unterholzner, 2013) . the type-i ifn response to cytosolic dna was described relatively late in the history of the field stetson and medzhitov, 2006) . here, the two principle ligands used were an annealed, synthetic, mixedsequence 45-mer designed to avoid cpg motifs, known as interferon-stimulatory dna (isd) (stetson and medzhitov, 2006) and the dsdna sequence poly(da-dt):poly(dt-da), known as poly(da:dt) synthesized in an enzymatic reaction . soon after, it was established that stimulator of interferon genes (sting) was involved dna-mediated activation of irf3 (ishikawa and barber, 2008; zhong et al., 2008) , and all of the putative receptors, except for dhx9 and dhx36, were reported to act upstream of sting in some manner. while it is entirely possible that redundancies in function, species or cell-specific effects or other differences can account for the plethora of dna sensors postulated, these somewhat contradictory studies still pose a conundrum for the field. moreover, two groups also found that poly(da:dt) also activated rig-i after its transcription into rna by rnase polymerase iii (ablasser et al., 2009; chiu et al., 2009) , opening the question whether other dna ligands may be indirectly sensed by other pathways. with the discovery of cgas wu et al., 2013) , it became clear that cgas-sting was the principle pathway of cytosolic dna sensing in mammalian cells. however, the advent of crispr-cas9 genome editing in 2013 has also provided important genetic proof of the function of putative human dna receptors: human ifi16 has been shown to act as an important dna sensor upstream of sting (almine et al., 2017; jønsson et al., 2017) . however, one study removed the entire alr locus in mice (aim2 + 12 paralogs), without affecting the ifn response to cytosolic dna (gray et al., 2016) , effectively dispelling any notion that an alr is required for dna-induced ifn in mice. of note, a very recent publication has proposed a further cytosolic dna sensing pathway which is active in human but not murine cells, dna-pk (burleigh et al., 2020) . however, in contrast to previous reports in murine cells, this study demonstrates that human dna-pk acts independently of sting. altogether, there seem to be important differences in mammalian dna signaling, and we hope that further studies will genetically investigate putative dna sensing pathways in humans (and other non-murine species) and clear up the controversies remaining in the field. immunity 53, july 14, 2020 65 2016; brunette et al., 2012; cridland et al., 2012; khare et al., 2014) . aim2 is the best conserved gene in this locus and the principle cytosolic dsdna sensor for inflammasome formation in mice (b€ urckst€ ummer et al., 2009; fernandes-alnemri et al., 2009; . however, aim2 is a pseudogene in several mammalian species, including cows and dogs (cridland et al., 2012) . moreover, in human monocytes and the monocytic model cell line, transdifferentiated blaer1, dna-induced inflammasome activation is dependent on nlrp3, not aim2 (-gaidt et al., 2017 (-gaidt et al., , 2018 , and direct sting activation can also activate nlrp3 in monocytes. here, sting activation leads to a lytic form of cell death and k + efflux in turn activating nlrp3. however, whether this form of sting-mediated cell death occurs in other cells is unknown. in cells connected by gap junctions, this seems implausible since bystander sting activation would theoretically induce a wave of cellular lysis and pyroptosis. moreover, aim2 acts as a dna-sensing inflammasome protein in other human myeloid cell types, including thp-1 cells (gaidt et al., 2017) and human macrophages (su et al., 2018) . human ifi16 forms a nuclear inflammasome in t cells, sensing hiv-1 cdna and inducing pyroptosis . ifi16 also senses kaposi sarcoma-associated herpesvirus (ksvh) in primary human endothelial cells (hmvec) and the monocytic cell line thp-1 (kerur et al., 2011; singh et al., 2013) . since pyroptosis is the predominant form of pcd (95%) of quiescent cd4 + t cell during hiv-1 infection, ifi16-mediated pyroptosis may be of particular pathophysiological relevance for this disease . although p204 has been reported to the murine ortholog ifi16, we are unaware of a report of p204 directly building an inflammasome, and the recent publication of the ifi204 à/à mouse indicates a distinct role in lps sensing (yi et al., 2018) . although it is conceivable that other alrs in the human and murine locus containing hin200 and pyrin domains could build inflammasomes, none have been reported to date. indeed, we are only beginning to understand the function of other alrs. the human alr pyrin-only protein 3 does not contain a hin200 domain and is reported to act as an inflammasome inhibitor (khare et al., 2014) . human pyhin1 was reported to act as an ifn-i-inducing dna sensor (see box 4, cytosolic dna and type-i ifn) but has also recently been shown to control tnf and il-6 release downstream of sting activation (massa et al., 2020) . better understanding of the mammalian alrs is not only important to our own antiviral defense but also that of livestock and important mammalian viral reservoirs, such as bats for paramyxovirues and coronaviruses (drexler et al., 2012; li et al., 2005) . accessory proteins: cgas activation requires oligomerization (li et al., 2013b) , even forming large protein foci through liquidphase separation (du and chen, 2018) , as previously described for p-bodies and stress granules (shin and brangwynne, 2017) . several accessory proteins have been reported that could contribute to lowering the activation energy for phase transition (see figure 3 ). the dna structural proteins, hmgb1 and tfam, have been reported to bind, bend, and stabilize dsdna facilitating the nucleation of cgas dimers along the dsdna strand (andreeva et al., 2017) . other identified cofactors include cchc-type zinc-finger protein 3 (zcchc3) (lian et al., 2018a) , reported by the same group to act as an accessory protein to the rlrs (lian et al., 2018b) , and gtpase-activating protein sh3 domain-binding protein 1 (g3bp1) (liu et al., 2019) . for nuclear cgas activation, the host protein non-pou domain-containing octamer binding protein (nono) supports the binding of hiv-2 dna to cgas (lahaye et al., 2018) . theoretically, these different cofactors could influence the activation state and multimeric structure of cgas. there are multiple reports of ifi16 acting as a viral restriction factor by suppressing viral promoters and interfering with replication (lo cigno et al., 2015; gariano et al., 2012; johnson et al., 2014) . nuclear ifi16 has been reported to bind and sequester unchromatinized dna and promote its integration into heterochromatin, thus restricting hsv-1 infection orzalli et al., 2013) , with one recent publication showing that it forms filamentous structures with viral dna, thus blocking the activity of hsv-1 rna pol ii (merkl and knipe, 2019) . the apolipoprotein b editing complex (apobec) family was discovered via a c-to-u base modification in apolipoprotein b (apob) mrna leading to a premature stop codon (arias et al., 2012) . the seven proteins(a-g) of the rapidly evolving primate apobec3 (or a3) subfamily are isgs countering retroviruses and endogenous retroelements (ito et al., 2020) . several apobec3s act as restriction factors for hiv-1, performing c-to-u editing on first(minus) strand cdna that result in plusstrand g-to-a mutations. apobecg3 mutates preferentially tgg motifs, converting them to a stop codon (tag) and is targeted by the hiv accessory-protein vif (along with apobec3d, f, and h). apobec3 proteins are also active against other viruses, including parvovirus, hbv, htlv, and rsv (arias et al., 2012) . however, the drawbacks of this editing strategy include evolution of more virulent viral strains and genomic mutagenesis, which for apobec3b has been linked to several forms of cancer (burns et al., 2013) . the sterile alpha-motif (sam) and histidine-aspartate (hd) domain-containing protein 1 (samhd1) is a deoxynucleoside triphosphate (dntp) triphosphohydrolase that removes tripolyphosphate moieties from dntps (goldstone et al., 2011) . samhd1 was initially identified as a myeloid-cell specific hiv-1 restriction factor and target of the lentivirus auxiliary protein vpx (laguette et al., 2011) . samhd1 decreases the concentration of intracellular dntps in myeloid cells to inhibit reverse transcription (goldstone et al., 2011) , and also inhibits the replication of dna viruses, such as hsv-1 (hollenbaugh et al., 2013) . hypomorphic variants of samhd1 induce type-i interferonopathy in patients and mice, which is dependent on the cgas-sting pathway (maelfait et al., 2016) . similarly, hypomorphic variants of the principal cytosolic nuclease in mammalian cells, dnase three-prime repair exonuclease 1 (trex1 or dnase iii), are linked to type-i interferonopathy as well as familial chilblain lupus (fcl) and systemic lupus erythematosus (sle) (rodero and crow, 2016) . trex1 degrades ss-and dsdna (grieves et al., 2015) , and trex1-deficiency leads to cytosolic accumulation of dna ligands (stetson et al., 2008) and activation of the cgas-sting pathway (ablasser et al., 2014; gehrke et al., 2013) . trex1 à/à mice develop systemic inflammation (morita et al., 2004 ) ameliorated by simultaneous deficiency in sting or cgas (gray et al., 2015) . moreover, modifications such as uv-light exposure render dna trex1-resistant and potentiate cgas activation (gehrke et al., 2013) . dnase ii is the predominant endolysosomal endonuclease and also degrades ss-and dsdna (drew, 1984) . dnase ii deficiency leads to dsdna accumulation which enters the cytoplasm, causing overwhelming inflammation (kawane et al., 2006; yoshida et al., 2005) . patients with expressing hypomorphic dnase ii present also present with type-i interferonopathy (rodero et al., 2017) , and dnase ii deficiency is embryonically lethal but can be rescued by ifnar deficiency (kawane et al., 2006) . of note, cgas-sting and aim2 both contribute to the phenotype of dnase ii deficiency (ahn et al., 2012; baum et al., 2017; jakobs et al., 2015) . recently, two further endosomal nucleases have been discovered, phospholipase d3 (pld3) and d4 pld4. unlike dnase ii, pld3 and pld4 are exonucleases which degrade ssdna from the 5 0 terminus (gavin et al., 2018) . this discovery has its origins in genome-wide association studies linking pld3 to alzheimer disease and pld4 to rheumatoid arthritis and systemic sclerosis. pld4-deficient mice demonstrate elevations in ifn-g and splenomegaly. pld3 and pld4 activity requires a 5 0 oh terminus and the absence of secondary structure, both features of dnase ii cleavage products, enabling it to act cooperatively with dnase ii (see next section). nucleases and the sensing of self versus non-self dna initially, sensing of dsdna was thought to rely entirely on subcellular localization (see figures 1 and 3) . while dna does not undergo the same exquisite processing as rna, nuclear dna sensing clearly depends on hallmarks of self and non self, and dna modifications can strongly affect the availability of cytosolic and endosomal dna. dna sensing is tightly controlled by its availability. cytosolic dsdna must contend with the high-affinity 3 0 -exonuclease trex1. similarly, activation of tlr9 is antagonized by the 5 0 -exonuclease activity of pld3/4 in the endosome. exonuclease activity is an elegant strategy for controlled sensor activity: (1) the na ligand is degraded base by base, allowing for a gradual removal of ligands, (2) whereas inhibiting endonucleases requires extensive modification to a substrate, exonucleases can be blocked by a simple modification on the 5 0 or 3 0 terminus, which can, in turn, be removed by an endonuclease. cytosolic dsdna becomes potently stimulatory with modifications that inhibit trex degradation, e.g., 8'oh guanosine after uv irradiation (gehrke et al., 2013) . endosomal pld3 and 4 only degrades ssdna and is inhibited by a 5 0 p-terminus (gavin et al., 2018) . dnase i, trex1, and caspase-activated dnase perform 5 0 p-3 0 oh cleavage of phosphodiester bonds. thus, incoming endosomal dna will be predominantly (1) double-stranded and (2) have a 5 0 p terminus, rendering it inaccessible to pld3 and 4. in contrast, dnase ii is a 5 0 oh-3 0 p endonuclease with activity on ss-and dsdna and is thus critically required to render dna accessible to pld3 and pld4. furthermore, phosphorothioate (pto) modification also inhibits pld3 and 4 activity 20-to-50-fold. pto-linkage has been employed for decades to potentiate stimulatory oligonucleotides and also occurs naturally in bacterial dna, rendering it a better stimulus for tlr9. however, single pto modifications do not inhibit the endonuclease dnase ii. in parallel to the role of rnase t2 and rnase 2 for tlr8, dnase ii does not inhibit tlr9 activity. rather, it is critically required for the generation of tlr9 ligands from complex natural ligands (chan et al., 2015; pawaria et al., 2015) by releasing ssdna from genomic dsdna and generating 5 0 oh termini for the second binding pocket . as with dsrna, length is a key determinant of whether dsdna is stimulatory. trex1 is non-processive, so longer dsdna will have a longer half-life in the cytosol (hö ss et al., 1999) . longer dsdna is more easily bent into structures amenable to dimer formation and cgas-dependent phase transformation (andreeva et al., 2017; du and chen, 2018; hooy and sohn, 2018) . for ifi16 and aim2, longer naked dsdna stretches are more amenable to the oligomerization of adjacent pyrin domains, a process which supports hin200-dsdna binding and stability (morrone et al., 2015 (morrone et al., , 2014 , provides a platform for the pyrinpyrin interaction with asc during inflammasome activity (matyszewski et al., 2018) , and effectively sequesters and neutralizes long dsdna molecules. the influence of dsdna length on receptor oligomerization, signal strength, and duration may account for the distinct signaling outcomes, e.g., restriction, cytokine induction, cell death, observed with different stimuli and cell types. for humans, cgas require dsdna of at least 45bp and aim2 requires 80bp, whereas murine cgas sensing can occur with shorter sequences (r25 bp) (fernandes-alnemri et al., 2009; jin et al., 2012; luecke et al., 2017) . at the structural level, 39bp was shown to be the minimal length for the assembly of two human cgas dimers (andreeva et al., 2017) . whereas ifi16 is reported to require at least 60-70bp for ifn activation (unterholzner et al., 2010) , we are not aware of a study determining the minimal length necessary for inflammasome activation and other ifi16 effector functions. another important aspect of dsdna sensing is packaging. an essential distinction between self and non self in the nucleus is the tight winding of chromosomal dna (gentili et al., 2019; volkman et al., 2019) , which allows cgas binding but not activation. this seems difficult to reconcile a priori with the activation of cytosolic cgas by micronuclei (harding et al., 2017; mackenzie et al., 2017) . possibly, micronuclei contain unwound regions or other features amenable to cgas sensing, or cgas activation has different requirements in the cytosol. understanding this process is of great importance to studies of cgas-mediated cellular senescence. a notable exception to the length requirement is cgas activation by g-rich y-form dna, base-paired dna r 12 bp containing an unpaired g in adjacent ssdna . this mechanism could explain various forms of ssdna sensing attributed to cgas, including secondary-structured ssdna originating from retroviral genomes (coquel et al., 2018; herzner et al., 2015) . although direct sensing of y-dna was demonstrated in a cellfree system , an accessory protein may contribute in cellulo as has been observed for many other ligands. regardless, y-dna-mediated cgas activation provides evidence of 5 0 -and 3 0 -terminal sensing (here, of guanosines) of cytosolic dsdna. another recent example is the sting-independent dna-pk pathway, which fitting its function as a sensor of dna damage and due to its requirement for linear dsdna, seems interacts with dsdna termini, although the precise structural features required remain unknown (burleigh et al., 2020) . immunity 53, july 14, 2020 67 sting is can be activated cell intrinsically and in bystander cells by endogenous 2'3 0 cgamp (ablasser et al., 2013b; gao et al., 2013a) . 2'3 0 cgamp is targeted by hydrolysing poxins expressed by members of the poxviridae, such as vaccinia virus (eaglesham et al., 2019) . clearly, inhibiting cgas-sting signaling is of advantage to dna viruses. in contrast, certain human sting alleles have evolved to tolerate bacterial cdn while retaining sensitivity for endogenous cgamp. this intentional desensitization to non self is highly unusual and thus likely indicative of a strong evolutionary advantage for not avoiding ifn induction in response to certain bacteria. here, it should be noted that the role of ifn in bacterial defense remains rather unclear and differs starkly between bacterial species (boxx and cheng, 2016) . there is abundant self ss-and dsdna in the endolysosome of phagocytes, requiring a strategy beyond sensing of the ribosephosphate backbone. to avoid unnecessary activation: (1) tlr9 signaling is strictly contained within the endolysosomal compartment via an intricate system of trafficking and proteolytic activation, (2) tlr9 activation is regulated by the activity of endosomal dnases (see previous section), and (3) tlr9 senses unmethylated cpg dinucleotides within ssrna. cpg motifs are suppressed in eukaryotic dna (krieg, 2002) , since their methylation can drive their hydrolytic deamination (shen et al., 1994) . however, unmethylated cpg-rich dna can be found in bacteria, viruses, and fungal pathogens. the tremendous progress outlined above suggests a tight functional integration of innate immune responses and cell-autonomous defense mechanisms in response to exogenous na. in this context, na-related research to date has followed three, almost completely separate, principle research directions: immune sensing of na, antiviral restriction factors, and na metabolism. we now understand that those three principles are all integral parts of a tightly controlled na defense system. impairment or failure of this system caused by dysregulation, genetic alterations, or pathogen challenge causes erroneous detection of self na resulting in autoinflammation. therefore, an improved understanding of the intricate mechanisms that govern the distinction of endogenous and exogenous na as summarized in this review will guide better diagnostic procedures and treatments for inflammatory and infectious diseases. in particular, this insight allows for targeted activation of functionally distinct na-sensing pathways in order to elicit immune response pathways that have not been previously accessible for treatment. gunther hartmann is a founder of rigontec gmbh which was acquired by msd in 2017. gunther hartmann is an inventor of the following patents: au2009203061b2 , us10238682b2, us20180127454a1, us20160210400a1, us7776344b2, us8003619b2, us8076068b2, us8815503b2, us20070065467a1, hrp20151198t1, us20050134838a1, au2001270134a1, za200201959b, ep2338499a1, ep1764108a1, us20110184045a1, ep2408918b8, hk1225755a1, au2011244863a1, ma38598b1 , au2011203218a1, jp2017006144a, ep1688147a1. structural basis for viral 5 0 -ppp-rna recognition by human ifit proteins rig-i-dependent sensing of poly(da:dt) through the induction of an rna polymerase iii-transcribed rna intermediate cgas produces a 2 0 -5 0 -linked cyclic dinucleotide second messenger that activates sting cell intrinsic immunity spreads to bystander cells via the intercellular transfer of cgamp trex1 deficiency triggers cell-autonomous immunity in a cgas-dependent manner breaching self-tolerance to alu duplex rna underlies mda5-mediated inflammation sting manifests self dna-dependent inflammatory disease unique loss of the pyhin gene family in bats amongst mammals: implications for inflammasome sensing recognition of double-stranded rna and activation of nf-kappab by toll-like receptor 3 ifi16 and cgas cooperate in the activation of sting during dna sensing in human keratinocytes rna exosome complex regulates stability of the hepatitis b virus x-mrna transcript in a non-stop-mediated (nsd) rna quality control mechanism accessing the therapeutic potential of immunostimulatory nucleic acids sting contributes to abnormal bone formation induced by deficiency of dnase ii in mice evolution of caspase-mediated cell death and differentiation: twins separated at birth tlr8 senses staphylococcus aureus rna in human primary monocytes and macrophages and induces ifn-b production via a tak1-ikkb-irf5 signaling pathway rnai-mediated antiviral immunity in mammals proapoptotic signaling induced by rig-i and mda-5 results in type i interferon-independent apoptosis in human melanoma cells molecular mechanism of signal perception and integration by the innate immune sensor retinoic acidinducible gene-i (rig-i) intracellular toll-like receptors the roles of type i interferon in bacterial infection intracellular nucleic acid sensing triggers necroptosis through synergistic type i ifn and tnf signaling innate immune pattern recognition: a cell biological perspective extensive evolutionary and functional diversity among mammalian aim2-like receptors the innate immune sensor lgp2 activates antiviral signaling by regulating mda5-rna interaction and filament assembly broad expression of toll-like receptors in the human central nervous system an orthogonal proteomic-genomic screen identifies aim2 as a cytoplasmic dna sensor for the inflammasome sting is a direct innate immune sensor of cyclic di-gmp human dna-pk activates a sting-independent dna sensing pathway evidence for apobec3b mutagenesis in multiple human cancers a study of the interferon antiviral mechanism: apoptosis activation by the 2-5a system rna template-directed rna synthesis by t7 rna polymerase rnase l activates the nlrp3 inflammasome during viral infections dnase ii-dependent dna digestion is required for dna sensing by tlr9 rna polymerase iii detects cytosolic dna and induces type i interferons through the rig-i pathway human adar1 prevents endogenous rna from triggering translational shutdown the nuclear dna sensor ifi16 acts as a restriction factor for human papillomavirus replication through epigenetic modifications of the viral promoters higher activation of tlr9 in plasmacytoid dendritic cells by microbial dna compared with self-dna based on cpg-specific recognition of phosphodiester dna human tlr8 senses rna from plasmodium falciparum-infected red blood cells which is uniquely required for the ifn-g response in nk cells cyclic gmp-amp signalling protects bacteria against viral infection the specificity of interferon induction in chick embryo cells by helical rna samhd1 acts at stalled replication forks to prevent interferon induction the mammalian pyhin gene family: phylogeny, evolution and expression irak4 kinase activity controls tolllike receptor-induced inflammation through the transcription factor irf5 in primary human monocytes rna viruses promote activation of the nlrp3 inflammasome through cytopathogenic effect-induced potassium efflux 2 0 -o methylation of the viral mrna cap evades host restriction by ifit family members cutting edge: priming of cd8 t cell immunity to herpes simplex virus type 1 requires cognate tlr3 expression in vivo tlr7 and tlr8 activate distinct pathways in monocytes during rna virus infection recent insights on inflammasomes, gasdermin pores, and pyroptosis identification of an lgp2-associated mda5 agonist in picornavirus-infected cells mitochondrial double-stranded rna triggers antiviral signalling in humans viral infection switches non-plasmacytoid dendritic cells into high interferon producers innate antiviral responses by means of tlr7-mediated recognition of singlestranded rna rna 2 0 -o-methylation (nm) modification in human diseases the functional interactome of pyhin immune regulators reveals ifix is a sensor of viral dna the emerging role of nuclear viral dna sensors cell death by pyroptosis drives cd4 t-cell depletion in hiv-1 infection systematic discovery of antiphage defense systems in the microbial pangenome structural specificities of five commonly used dna nucleases bats host major mammalian paramyxoviruses dna-induced liquid phase condensation of cgas activates innate immune signaling viral and metazoan poxins are cgamp-specific nucleases that restrict cgas-sting signalling the skiv2l rna exosome limits activation of the rig-i-like receptors bacterial rna: an underestimated stimulus for innate immune responses nlrp6 inflammasome regulates colonic microbial ecology and risk for colitis molecular biology of rna interferon action: two distinct pathways for inhibition of protein synthesis by double-stranded rna aim2 activates the inflammasome and cell death in response to cytoplasmic dna cpg dinucleotides inhibit hiv-1 replication through zinc finger antiviral protein (zap)-dependent and -independent mechanisms inducers of interferon and host resistance. ii. multistranded synthetic polynucleotide complexes the cellular nmd pathway restricts zika virus infection and is targeted by the viral capsid protein identification of rna sequence motifs stimulating sequence-specific tlr8-dependent immune responses cytosolic double-stranded rna activates the nlrp3 inflammasome via mavs-induced membrane permeabilization and k+ efflux the intra-and extracellular functions of asc specks rna modifications modulate activation of innate toll-like receptors the dna inflammasome in human myeloid cells is initiated by a sting-cell death program upstream of nlrp3 modeling primary human monocytes with the trans-differentiation cell line blaer1 tlr7 is involved in sequence-specific sensing of singlestranded rnas in human macrophages inhibition of retroviral rna production by zap, a ccch-type zinc finger protein ] is the metazoan second messenger produced by dna-activated cyclic gmp-amp synthase structure-function analysis of sting activation by c the intracellular dna sensor ifi16 gene acts as restriction factor for human cytomegalovirus replication pld3 and pld4 are single-stranded acid exonucleases that regulate endosomal nucleic-acid sensing discrimination of self and non-self ribonucleic acids oxidative damage of dna confers resistance to cytosolic nuclease trex1 degradation and potentiates stingdependent immune sensing the n-terminal domain of cgas determines preferential association with centromeric dna and innate immune activation in the nucleus an rna editor, adenosine deaminase acting on double-stranded rna (adar1) essential role of mda-5 in type i ifn responses to polyriboinosinic:polyribocytidylic acid and encephalomyocarditis picornavirus innate immune sensing of cytosolic chromatin fragments through cgas promotes senescence hiv-1 restriction factor samhd1 is a deoxynucleoside triphosphate triphosphohydrolase immunomodulatory functions of type i interferons antiviral immunity via rig-i-mediated recognition of rna bearing 5 0 -diphosphates mouse superkiller-2-like helicase ddx60 is dispensable for type i ifn induction and immunity to multiple viruses tlr3 is essential for the induction of protective immunity against punta toro virus infection by the double-stranded rna (dsrna), poly(i:c12u), but not poly(i:c): differential recognition of synthetic dsrna molecules cutting edge: cgas is required for lethal autoimmune disease in the trex1-deficient mouse model of aicardi-goutiè res syndrome the aim2-like receptors are dispensable for the interferon response to intracellular dna vitro transcription systems. cold spring harb. protoc. 2020. published online tlr8 is a sensor of rnase t2 degradation products exonuclease trex1 degrades double-stranded dna to prevent spontaneous lupus-like inflammatory disease hiv-1 blocks the signaling adaptor mavs to evade antiviral host defense after sensing of abortive hiv-1 rna by the host helicase ddx3 signalling strength determines proapoptotic functions of sting the zinc-finger antiviral protein recruits the rna processing exosome to degrade the target mrna sequestration by ifit1 impairs translation of 2'o-unmethylated capped rna rig-i detects triphosphorylated rna of listeria monocytogenes during infection in non-immune cells the arms race between bacteria and their phage foes mitotic progression following dna damage enables pattern recognition within micronuclei nucleic acid immunity mechanism and function of a newly identified cpg dna motif in human primary b cells delineation of a cpg phosphorothioate oligodeoxynucleotide for activating primate immune responses in vitro and in vivo pandemic h1n1 influenza a viruses suppress immunogenic ripk3-driven dendritic cell death rnaset2 mutant zebrafish model familial cystic leukoencephalopathy and reveal a role for rnase t2 in degrading ribosomal rna 5 0 nucleotide sequence of sindbis viral rna species-specific recognition of single-stranded rna via toll-like receptor 7 and 8 tasl is the slc15a4-associated adaptor for irf5 activation by tlr7-9 a toll-like receptor recognizes bacterial dna small anti-viral compounds activate immune cells via the tlr7 myd88-dependent signaling pathway rnaset2-deficient cystic leukoencephalopathy resembles congenital cytomegalovirus brain infection sequence-specific activation of the dna sensor cgas by y-form dna structures as found in primary hiv-1 cdna codon bias confers stability to human mrnas a human dna editing enzyme homologous to the escherichia coli dnaq/mutd protein mavs forms functional prion-like aggregates to activate and propagate antiviral innate immune response synthesis of low molecular weight inhibitor of protein synthesis with enzyme from interferon-treated cells the alpha/beta interferon response controls tissue tropism and pathogenicity of poliovirus structural basis for species-specific activation of mouse toll-like receptor 9 a toll-like receptor-independent antiviral response induced by double-stranded b-form dna sting is an endoplasmic reticulum adaptor that facilitates innate immune signalling retroviruses drive the rapid evolution of mammalian apobec3 genes tlr signaling tailors innate immune responses in human microglia and astrocytes aim2 drives joint inflammation in a self-dna triggered model of chronic polyarthritis chromatin-bound cgas is an inhibitor of dna repair and hence accelerates genome destabilization and cell death structures of the hin domain:dna complexes reveal ligand binding and activation mechanisms of the aim2 inflammasome and ifi16 receptor ifi16 restricts hsv-1 replication by accumulating on the hsv-1 genome, repressing hsv-1 gene expression, and directly or indirectly modulating histone modifications ifi16 is required for dna sensing in human macrophages by promoting production and function of cgamp sequence-dependent stimulation of the mammalian innate immune response by synthetic sirna translating nucleic acid-sensing pathways into therapies human tlr7 or tlr8 independently confer responsiveness to the antiviral compound r-848 the rig-i/mavs signaling pathway in cancer cell-selective apoptosis central roles of nlrs and inflammasomes in viral infection mrna is an endogenous ligand for toll-like receptor 3 compositional differences within and between eukaryotic genomes interferon regulatory factor-3 is an in vivo target of dna-pk cell type-specific involvement of rig-i in antiviral response differential roles of mda5 and rig-i helicases in the recognition of rna viruses length-dependent recognition of double-stranded ribonucleic acids by retinoic acid-inducible gene-i and melanoma differentiation-associated gene 5 interferon-alpha induction through toll-like receptors involves a direct interaction of irf7 with myd88 and traf6 ips-1, an adaptor triggering rig-i-and mda5-mediated type i interferon induction chronic polyarthritis caused by mammalian dna that escapes from degradation in macrophages samhd1 is the dendritic-and myeloid-cell-specific hiv-1 restriction factor counteracted by vpx nono detects the nuclear hiv capsid to promote cgas-mediated innate immune activation recognition of double-stranded rna and regulation of interferon pathway by toll-like receptor 10 the tlr3 signaling complex forms by cooperative receptor dimerization regulation of protein synthesis: activation by double-stranded rna of a protein kinase that phosphorylates eukaryotic initiation factor 2 sequence specific detection of bacterial 23s ribosomal rna by tlr13 bats are natural reservoirs of sars-like coronaviruses rna interference functions as an antiviral immunity mechanism in mammals cyclic gmp-amp synthase is activated by double-stranded dna-induced oligomerization induction and suppression of antiviral rna interference by influenza a virus in mammalian cells identification of antiviral roles for the exon-junction complex and nonsense-mediated decay in flaviviral infection zcchc3 is a co-sensor of cgas for dsdna recognition in innate immune response the zinc-finger protein zcchc3 binds rna and facilitates viral rna sensing and activation of the rig-i-like receptors severe influenza pneumonitis in children with inherited tlr3 deficiency structural basis of toll-like receptor 3 signaling with double-stranded rna nuclear cgas suppresses dna repair and promotes tumorigenesis g3bp1 promotes dna binding and activation of cgas the discovery of potent immunostimulatory cpg-odns widely distributed in bacterial genomes distinct rig-i and mda5 signaling by rna viruses in innate immunity ddx3 suppresses type i interferons and favors viral replication during arenavirus infection vtx-2337 is a novel tlr8 agonist that activates nk cells and augments adcc immune modulation by human secreted rnases at the extracellular space cgas is activated by dna in a length-dependent manner a novel mitochondrial mavs/caspase-8 platform links rna virus-induced innate antiviral signaling to bax/bak-independent apoptosis cgas surveillance of micronuclei links genome instability to innate immunity restriction by samhd1 limits cgas/sting-dependent innate and adaptive immune responses to hiv-1 sensing of viral and endogenous rna by zbp1/dai induces necroptosis nucleic acid sensors and programmed cell death antiviral rna interference in mammalian cells inactivation of the type i interferon pathway reveals long double-stranded rna-mediated rna interference in mammalian cells small self-rna generated by rnase l amplifies antiviral innate immunity rnase l releases a small rna from hcv rna that refolds into a potent pamp zbp1 and tak1: master regulators of nlrp3 inflammasome/pyroptosis, apoptosis, and necroptosis (pan-optosis) reassessing the evolutionary importance of inflammasomes structure and function of the interferon-beta enhanceosome differential viral induction of distinct interferon-alpha genes by positive feedback through interferon regulatory factor-7 short double-stranded rnas with an overhanging 5 0 ppp-nucleotide, as found in arenavirus genomes, act as rig-i decoys a structural basis for discriminating between self and nonself double-stranded rnas in mammalian cells pyhin1 regulates pro-inflammatory cytokine induction rather than innate immune dna sensing in airway epithelial cells establishment of a monoclonal antibody against human toll-like receptor 3 that blocks double-stranded rna-mediated signaling trail and noxa are selectively upregulated in prostate cancer cells downstream of the rig-i/mavs signaling pathway by nonreplicating sendai virus particles digital signaling network drives the assembly of the aim2-asc inflammasome toll-like receptor 3 (tlr3) plays a major role in the formation of rabies virus negri bodies role for a filamentous nuclear assembly of ifi16, dna, and host factors in restriction of herpesviral infection interferon-inducible gtpases in cell autonomous and innate immunity synthetic polynucleotides the dhx33 rna helicase senses cytosolic rna and activates the nlrp3 inflammasome ddx60, a dexd/h box helicase, is a novel antiviral factor promoting rig-i-like receptor-mediated signaling attacked from all sides: rna decay in antiviral defense identification of host cytosolic sensors and bacterial factors regulating the type i interferon response to legionella pneumophila ifi16 dna sensor is required for death of lymphoid cd4 t cells abortively infected with hiv gene-targeted mice lacking the trex1 (dnase iii) 3 0 ->5 0 dna exonuclease develop inflammatory myocarditis cooperative assembly of ifi16 filaments on dsdna provides insights into host defense strategy assembly-driven activation of the aim2 foreign-dsdna sensor provides a polymerization template for downstream asc transmembrane mutations in toll-like receptor 9 bypass the requirement for ectodomain proteolysis and induce fatal inflammation stem-loop recognition by ddx17 facilitates mirna processing and antiviral defense spatio-temporal characterization of the antiviral activity of the xrn1-dcp1/2 aggregation against cytoplasmic rna viruses to prevent cell death sidt2 transports extracellular dsrna into the cytoplasm for innate immune recognition apoptotic caspases suppress type i interferon production via the cleavage of cgas, mavs, and irf3 ripk3 activates parallel pathways of mlkl-driven necroptosis and fadd-mediated apoptosis to protect against influenza a virus toll-like receptor 9 contains two dna binding sites that function cooperatively to promote receptor dimerization and activation tlr13 recognizes bacterial 23s rrna devoid of erythromycin resistance-forming modification viral infections activate types i and iii interferon genes through a common mechanism nuclear interferon-inducible protein 16 promotes silencing of herpesviral and transfected dna ti-cam-1, an adaptor molecule that participates in toll-like receptor 3-mediated interferon-beta induction dead/h box 3 (ddx3) helicase binds the rig-i adaptor ips-1 to up-regulate ifn-betainducing potential the tlr3/ticam-1 pathway is mandatory for innate immune responses to poliovirus infection accessory factors of cytoplasmic viral rna sensors required for antiviral innate immune response immune sensing of synthetic, bacterial, and protozoan rna by toll-like receptor 8 requires coordinated processing by rnase t2 and rnase 2 defects of mitochondrial rna turnover lead to the accumulation of double-stranded rna in vivo dhx15 is a coreceptor for rlr signaling that promotes antiviral defense against rna virus infection cutting edge: dnase ii deficiency prevents activation of autoreactive b cells by double-stranded dna endogenous ligands toll-like receptor 3 in viral pathogenesis: friend or foe? rig-i-mediated antiviral responses to single-stranded rna bearing 5 0 -phosphates activation of mda5 requires higher-order rna structures generated during virus infection the role of unc93b1 protein in surface localization of tlr3 receptor and in cell priming to nucleic acid agonists short single-stranded dna degradation products augment the activation of toll-like receptor 9 selectivity of human tlr9 for double cpg motifs and implications for the recognition of genomic dna human virus-derived small rnas can confer antiviral immunity in mammals rig-i rna helicase activation of irf3 transcription factor is negatively regulated by caspase-8-mediated cleavage of the rip1 protein rig-i detects viral genomic rna during negative-strand rna virus infection mapping the dsrna world rig-i selectively discriminates against 5 0 -monophosphate rna the evolution of vertebrate toll-like receptors type i interferon-mediated monogenic autoinflammation: the type i interferonopathies, a conceptual overview type i interferon-mediated autoinflammation due to dnase ii deficiency apoptotic caspases prevent the induction of type i interferons by mitochondrial dna rad50-card9 interactions link cytosolic dna sensing to il-1b production adenosine deaminases acting on rna (adars) are both antiviral and proviral adenosine deaminase acting on rna (adar1), a suppressor of double-stranded rna-triggered innate immune responses combating herpesvirus encephalitis by potentiating a tlr3-mtorc2 axis lgp2 is a positive regulator of rig-i-and mda5-mediated antiviral responses master sensors of pathogenic rna -rig-i like receptors discriminating self from non-self in nucleic acid sensing recognition of 5 0 triphosphate by rig-i helicase requires short blunt double-stranded rna as contained in panhandle of negative-strand virus dna virus replication compartments 0 -triphosphate rna requires base-paired structures to activate antiviral signaling via rig-i identification of an optimized 2 0 -o-methylated trinucleotide rna motif inhibiting toll-like receptors 7 and 8 a conserved histidine in the rna sensor rig-i controls immune tolerance to n1-2'o-methylated self rna caspase-8-mediated cleavage inhibits irf-3 protein by facilitating its proteasome-mediated degradation reciprocal inhibition between intracellular antiviral signaling and the rnai machinery in mammalian cells identification and characterization of mavs, a mitochondrial antiviral signaling protein that activates nf-kappab and irf 3 the rate of hydrolytic deamination of 5-methylcytosine in double-stranded dna molecular mechanism for nlrp6 inflammasome assembly and activation a novel toll-like receptor that recognizes vesicular stomatitis virus guanosine and its modified derivatives are endogenous ligands for tlr7 liquid phase condensation in cell physiology and disease kaposi's sarcoma-associated herpesvirus latency in endothelial and b cells activates gamma interferoninducible protein 16-mediated inflammasomes double-stranded rna is detected by immunofluorescence analysis in rna and dna virus infections, including those by negative-stranded rna viruses structural basis for specific recognition of single-stranded rna by toll-like receptor 13 role of inosine à uracil base pairs in the canonical rna duplexes recognition of cytosolic dna activates an irf3-dependent innate immune response trex1 prevents cell-intrinsic initiation of autoimmunity immune checkpoint inhibition overcomes adcp-induced immunosuppression by macrophages helicase proteins dhx29 and rig-i cosense cytosolic nucleic acids in the human airway system cyclic gmp-amp synthase is a cytosolic dna sensor that activates the type i interferon pathway toll-like receptors 9 and 3 as essential components of innate immune defense against mouse cytomegalovirus infection lgp2 virus sensor regulates gene expression network mediated by trbp-bound micrornas virus sensor rig-i represses rna interference by interacting with trbp through lgp2 in mammalian cells cg dinucleotide suppression enables antiviral defence targeting non-self rna agonist-mediated activation of sting induces apoptosis in malignant b cells toll-like receptor 8 senses degradation products of single-stranded rna toll-like receptor 3 recognizes incomplete stem structures in single-stranded viral rna dai senses influenza a virus genomic rna and activates ripk3-dependent cell death self-coded 3 0 -extension of run-off transcripts produces aberrant products during in vitro transcription with t7 rna polymerase the interferon response to intracellular dna: why so many receptors? ifi16 is an innate immune sensor for intracellular dna the rig-i-like receptor lgp2 inhibits dicer-dependent processing of long double-stranded rna and blocks rna interference in mammalian cells the enemy within: endogenous retroelements and autoimmune disease tight nuclear tethering of cgas is essential for preventing autoreactivity toll-like receptor 3 mediates west nile virus entry into the brain causing lethal encephalitis nlrp6 regulates intestinal antiviral innate immunity inflammasome activation triggers caspase-1-mediated cleavage of cgas to regulate responses to dna virus infection raftlin is involved in the nucleocapture complex to induce poly(i:c)-mediated tlr3 activation double-stranded rna is produced by positive-strand rna viruses and dna viruses but not in detectable amounts by negative-strand rna viruses toll-like receptor (tlr) 3 immune modulation by unformulated small interfering rna or dna and the role of cd14 (in tlr-mediated effects) apoptotic caspases suppress mtdna-induced sting-mediated type i ifn production in vitro-transcribed guide rnas trigger an innate immune response via the rig-i pathway the mrna cap 2 0 -o-methyltransferase cmtr1 regulates the expression of cyclic gmp-amp is an endogenous second messenger in innate immune signaling by cytosolic dna structural basis for the prion-like mavs filaments in antiviral innate immunity role of adaptor trif in the myd88-independent toll-like receptor signaling pathway crystal structure of isg54 reveals a novel rna binding structure and potential functional mechanisms cgas is essential for cellular senescence control of antiviral innate immune response by protein geranylgeranylation interferon-l orchestrates innate and adaptive mucosal immune responses single nucleotide polymorphisms of human sting can affect innate immune response to cyclic dinucleotides p204 is required for canonical lipopolysaccharide-induced tlr4 signaling in mice the rna helicase rig-i has an essential function in double-stranded rna-induced innate antiviral responses shared and unique functions of the dexd/h-box helicases rig-i, mda5, and lgp2 in antiviral innate immunity dhx36 enhances rig-i signaling by facilitating pkr-mediated antiviral stress granule formation lethal anemia caused by interferon-beta produced in mouse embryos carrying undigested dna tlr3 deficiency in patients with herpes simplex encephalitis tlr3 immunity to infection in mice and humans the adaptor protein mita links virussensing receptors to irf3 transcription factor activation expression cloning of 2-5a-dependent rnaase: a uniquely regulated mediator of interferon action nlrp9b inflammasome restricts rotavirus infection in intestinal epithelial cells dhx29 functions as an rna co-sensor for mda5-mediated emcv-specific antiviral immunity isolation of two interferon-induced translational inhibitors: a protein kinase and an oligo-isoadenylate synthetase activation of endothelial toll-like receptor 3 impairs endothelial function key: cord-350836-1enteev7 authors: brisse, morgan; ly, hinh title: comparative structure and function analysis of the rig-i-like receptors: rig-i and mda5 date: 2019-07-17 journal: front immunol doi: 10.3389/fimmu.2019.01586 sha: doc_id: 350836 cord_uid: 1enteev7 rig-i (retinoic acid-inducible gene i) and mda5 (melanoma differentiation-associated protein 5), collectively known as the rig-i-like receptors (rlrs), are key protein sensors of the pathogen-associated molecular patterns (pamps) in the form of viral double-stranded rna (dsrna) motifs to induce expression of type 1 interferons (ifn1) (ifnα and ifnβ) and other pro-inflammatory cytokines during the early stage of viral infection. while rig-i and mda5 share many genetic, structural and functional similarities, there is increasing evidence that they can have significantly different strategies to recognize different pathogens, pamps, and in different host species. this review article discusses the similarities and differences between rig-i and mda5 from multiple perspectives, including their structures, evolution and functional relationships with other cellular proteins, their differential mechanisms of distinguishing between host and viral dsrnas and interactions with host and viral protein factors, and their immunogenic signaling. a comprehensive comparative analysis can help inform future studies of rig-i and mda5 in order to fully understand their functions in order to optimize potential therapeutic approaches targeting them. rig-i (retinoic acid-inducible gene i) encoded by the ddx58 gene in the human genome (1, 2) and mda5 (melanoma differentiation-associated protein 5) encoded by the ifih1 gene (3, 4) are known as important protein initiators of earliest immune responses to viral infection. a relatively large body of work has focused on understanding their roles in triggering the same innate immune pathway as they indeed share many similarities at a structural and functional level. however, it is becoming increasingly clear that there are unique differences between rig-i and mda5, such as their activation mechanisms and contextual functionalities, that need to be considered in order to fully appreciate their individual function. a comprehensive analysis of multiple aspects of rig-i and mda5 from their evolutionary origins and behavior among different species to their structures and molecular signaling will allow for a more nuanced understanding of their functional purposes. the innate immune response is a combination of non-specific defense mechanisms by the host that are critical for early detection and inhibition of pathogen growth before the adaptive immune response has time to produce proper cell-mediated immunity, such as the development of antibodies and cytotoxic t-lymphocyte responses (ctl) against the invading pathogen and/or the pathogen-infected cells (5) . cells of the innate immune arm, such as leukocytes and epithelial cells, are able recognize general components of the microbes (e.g., viruses) that are shared among related microbes. these microbial structures are called pathogen-associated molecular patterns (pamps) (e.g., viral dsrna) that are specifically recognized by the cellular pattern recognition receptors (prrs) (e.g., rig-i, mda5, or tolllike receptors tlrs) which are then activated (figure 1) . the specific signaling mechanisms of rig-i and mda5 activation will be discussed in detail below. here, the cascade of event leading to ifn1 production is briefly summarized. upon binding to pamp (e.g., dsrna), the activated rig-i and mda5 interact with the mitochondrial antiviral signaling proteins (mavs), which forms a multilayered protein complex contain several different proteins (6) (7) (8) (9) . the mavs complex then catalyzes the interaction of inhibitor of nuclear factor kappa-b kinase subunit epsilon (ikkε) and the serine/threonine-protein kinase 1 (tbk1) (10) (11) (12) , which phosphorylate the transcription factors interferon regulatory factors 3 and 7 (irf3 and irf7) (13). phosphorylated p-irf7 (14) and -pirf3 (15) factors then dimerize and translocate into the nucleus, where they activate the expression of the type 1 interferon genes (ifn1: ifnα and ifnβ). ifn1 proteins are then exported out of the cell to activate ifn1 signaling cascade by binding to their receptor (the ifnα/β receptor or ifnar) either on the same cells or neighboring cells in an autocrine or paracrine fashion. this results in the production of more ifn1 (in a positive feedback loop) and a variety of interferon-stimulated genes (isgs), which mediate vasodilation near the site of the pathogen infection and uptake of fluid, recruitment of innate immune cells, such as macrophages, neutrophils, and dendritic cells to the site of the infection that is aided by chemokine gradients to mediate innate immune cell-mediated killing of the infected cells (16). rig-i and mda5 appear to differentially induce ifn1 in response to different viral pathogens (17), with rig-i generally responding most potently to negative-strand rna viruses, such as influenza viruses (18, 19) , bunyaviruses (20, 21), filoviruses (22), and rhabdoviruses (18, 23) as well as the positive-stranded japanese encephalitis virus (18), while mda5 is activated during infection by positive-strand picornaviruses (18, 24, 25) and arteriviruses (26, 27) as well as by hepatitis d virus (28), kaposi's sarcoma-associated herpesvirus (kshv) (29). rig-i and mda5 may also play a role in recognizing non-viral pathogens, as mda5 has been found to respond to malaria (30) (figure 2) . neither are individually critical in reovirus (24) and in dengue virus infection (24, 31) but the presence of either in combination with toll-like receptor 3 (tlr3) is critical to have effective anti-viral repsonses (32). each serves an additive role during west nile virus infection (33), which is likely mediated by the production of multiple pamp species in the infected cells (34). indeed, rig-i and mda5 have also been shown to recognize different sections of the same viral genome due to their differing preferences for rna binding (35), illustrating how rig-i and mda5 can act both independently and synergistically. this has also been shown functionally in viruses where both rig-i and mda5 have been found to be essential to induce the necessary levels of ifnβ signaling for antiviral control against paramyxovirus (18, 36-38) and rotavirus infections (39). while rig-i and mda5 participate in the ifn1 signaling pathway (40), it is clear from animal modeling that they might be functionally distinct. while c57bl/6 mda5 ko mice exhibit no obvious phenotypes (18), c57bl/6 rig-i ko have high embryonic lethality as they don't live past 3 weeks of birth and experience growth retardation and liver degeneration (18, 41) . furthermore, when rig-i ko mice are back crossed onto the more genetically flexible 129s1 strain (18), these mice can spontaneously develop colitis symptoms (42). clinical cases with mutations in rig-i and mda5 have distinct autoimmune presentations, with rig-i mutations being associated with atypical singleton-merten syndrome, while mda5 mutations have been linked to classical singleton-merten syndrome, aicardi-goutières syndrome, systemic lupus erythematosus, type 1 diabetes and graves disease (43, 44) (figure 2 ). there is growing evidence that overt innate-immune interferon signaling plays a critical role in the development of other forms of autoimmune conditions (45). taken together, this suggests that rig-i and mda5 may differ significantly in their roles during development as well as in responding to different types of viral infection that is partially dependent on the pamps that are available in any given context. there is also increasing evidence that rig-i and mda5 have additional distinct molecular functionalities in immune signaling (43). it is well-established that the interferon regulatory factor (irf) and innate immune nfκb cytokine signaling pathways have many areas of cross-regulation and expression (46). accordingly, both rig-i and mda5 have been shown to activate nfκb signaling during rsv infection, but only rig-i appears to act upstream of the canonical iκbα-nfκb pathway (47, 48) (figure 1) . while both are known to activate nfκb mediated expression of il-6 and pro-il-1β through the interaction of card9 with bcl10 (49, 50), the independence of mda5 from the iκbα pathway suggests that it influences nfκb signaling in other as yet uncharacterized ways (43). a possible explanation for mda5's independence from the iκbα pathway may be that mda5-mediated nfκb (but not irf) signaling requires trim25, which activates rig-i by ubiquitination (to be discussed in detail below). this potentially implicates trim25 in other mechanisms besides activating rig-i (51, 52). rig-i (but not mda5) also induces inflammasome assembly-mediated cleavage and maturation of pro-il-1β by caspase 1 (24, 34, 53) . finally, rig-i has been shown to inhibit rnai complexes mediated by the endoribonuclease dicer, which is encoded by the dicer1 gene and cleaves dsrna and pre-micro rna into short single-stranded rna fragments known as small interfering rna (sirna) and microrna figure 1 | rig-i/mda5 signaling pathway rig-i and mda5 are first activated by recognition of pamp dsrna, which causes them to interact with mavs. following the activation of mavs by rig-i/mda5, a molecular cascade involves the interaction of ikkε and tbk1, which is followed by phosphorylation of the transcription factors irf3 and irf7, ensure to translocate the phosphorylated p-irf3 and p-irf7 into the nucleus, where they dimerize and bind to transcription factor binding sites of the ifnα and ifnβ genes to activate their transcriptions. expression and exportation of these genes into the cellular milieu trigger the ifn1 signaling cascade in an autocrine or paracrine fashion to induce expression of hundreds of interferon stimulated genes (isgs) and inflammatory genes to confer antiviral resistance. rig-i and mda5 also activate the nf-κb pathway. rig-i appears to act upstream of the canonical pathway, which results in the translocation of the two functional nf-κb units (p50 and p65) into the nucleus, while mda5 appears to affect nf-κb expression independently from this pathway. figure created using biorender software. (54), by interacting with the probable atp-dependent rna helicase dhx58 (also known as the laboratory of genetics and physiology 2 lgp2 protein), which inhibits dicer (55) as well as the dicer-complex protein trbp (56) . lgp2 has been shown to exhibit conflicting effects on rig-i and mda5 signaling (57) (58) (59) , and future studies are needed in order to clarify these regulatory mechanisms. rig-i and mda5 are expressed in all cell types (60) , but are most well-known for their functions inside innate immune cells, such as macrophages, neutrophils, and dendritic cells, as well as in other cells like mucosal epithelial cells. they are classified as atp-dependent dexd/h box rna helicases. their structure figure 2 | venn diagram comparing the signaling and functional similarities and differences between rig-i and mda5. is highly helical and consists of two caspase activating and recruiting domains (card) at the n terminus of ∼85 amino acids each, followed by a flexible hinge region and the helicase domain that consists of the reca-like hel1 and hel2 domains with an atp binding and hydrolyzing domain at their interface ( figures 3a,b) . in particular, the structure of the atp binding site distinguishes rig-i and mda5 from other helicase proteins, such as dicer. unlike other dexd/h box helicases where rna binding catalyzes the atp binding site to become structurally organized, the atp binding site in rig-i and mda5 remains comparatively open and structurally dynamic following rna binding. this is aided by the atp binding site being formed by an interface between the two hel domains, which are relatively far apart (64) . these structural features are connected by another flexible hinge region to the unique and predominantly β-sheet c terminal domain (ctd), which recognizes and binds to rna (65) . the ctd in rig-i and mda5 contains a zinc binding domain that is related to those of the gdp/gtp exchange factors (66) . each protein also contains a positively charged groove within this domain that recognizes dsrna and this groove is structurally unique in each protein, potentially explaining their different rna binding preferences (66) . rig-i primarily recognizes short double-stranded rna with 5 ′ triphosphate groups (67) (68) (69) (70) (71) (72) (73) (74) (75) , while mda5 primarily recognizes long double-stranded rna (76) (77) (78) (79) (to be discussed in detail below.) it is notable in this regard that the hel-ctd motifs adopt different orientations relative to dsrna in rig-i and mda5. specifically, the rig-i hel-ctd domain is tilted relative to dsrna with the ctd interacting with the 5 ′ and 3 ′ ends of the dsrna (61), whereas the mda5 hel-ctd domain runs parallel to the rna strand (figures 3c,d) . the series of steps required for rig-i and mda5 activation have been described in depth elsewhere (80) (81) (82) (83) (84) . briefly summarized, these proteins endogenously exist in the cytoplasm of the cell in a phosphorylated and inactivated conformation when they are not activated by pamp (dsrna) (85) (86) (87) (figures 4a,f) . phosphorylation is mediated at the n terminal card domains (s8 and t170) of rig-i by pkc-α/β (88, 89) and at the c terminal rna interaction domain (s854, s855, and t770) by ckβ (90) . on the other hand, mda5 is phosphorylated at s828 by riok3 (91) as well as by other yet unknown kinases (92, 93) . rig-i is also acetylated at k909 in its c terminal domain that requires deacetylation by hdac6 to be able to recognize rna in its activated form (94) . upon recognition of pamp (dsrna), rig-i unfolds into an open and activated state that is mediated by the flexible hinge regions between the card domains and the helicase domain, and between the helicase and the c terminal domain (64, 87, (95) (96) (97) (98) (figure 4b) . on the contrary, there is evidence to suggest that mda5 has a more dynamic structure (99) . unlike a model of rig-i activation described above, mda5 exists in a conformational equilibrium between close and open forms, with close forms favored in the dsrna unliganded state. while not yet formally demonstrated, it is possible that mda5 may be inhibited in the absence of the dsrna ligand by its structural dynamics, which may prevent strong protein-protein interactions ( figure 4f) . however, upon binding to dsrna ligand, mda5 adopts an open and activated form, which is perhaps more conducive for protein-protein interactions (figures 4g,h) . once the c terminal domains have been de-phosphorylated, the e3 ubiquitin ligase riplet attaches ubiquitin peptides onto the c terminal domain of rig-i at residues k849 and k851 (100, 101) . it was previously shown that ubiquitination by riplet was necessary for opening rig-i and for ubiquitination of the card domain (102) . however, in-situ studies found that dsrna was sufficient to weaken the interaction between purified rig-i c terminal domain and rig-i card domains (86) and that dsrna was necessary for riplet ubiquitination (103) , calling into question the sequential order for rig-i activation ( figure 4c ). following de-phosphorylation of the card domain by the phosphatase pp1-α/γ (92) , this domain is polyubiquinated at k172 by the e3 trim25 ubiquitin ligase (104) , which itself is activated by caspase 12 (105) (figure 4d ). trim25 interacting with rig-i may also be mediated by their mutual interactions with certain host long non-coding rna (lncrna), which occurs outside of the dsrna recognizing domain in the ctd of rig-i (106) . a recent study showed that riplet rather than trim25 was primarily responsible for ubiquitinating and activating rig-i (103). however, there are several factors to take into consideration with this study. these recent results were obtained using ko 293t and mouse embryonic fibroblast (mef) cells and that it was not clear whether k63 ubiquitination occurred at other known lysine sites in rig-i. the question remains whether riplet can ubiquitinate other lysine residues in the absence of trim25. additionally, in-situ experiments comparing rig-i ubiquitination by riplet and trim25 utilized an e2 enzyme (103) that had been found to be specific for riplet (107) . while the e2 that utilizes trim25 has not yet been identified, trim25 has been shown to ubiquitinate rig-i in-situ when a general mixture of e2 proteins was used (108) . the protein levels of trim25 may also have to be at a certain level in order for it to productively ubiquitinate rig-i, as the ubiquitin protease usp15 deubiquitinates trim25 at later time points in viral infection (109) . finally, trim25 has been found to be essential for rig-i activation and ifn signaling in-vitro and in-vivo. for the former, sirna-mediated knock-down (110, 111) , cellular knockout (112) and inhibition by viral protein (109, (113) (114) (115) (116) conditions for trim25 in multiple cell types have been shown to change rig-i cellular localization (110) and to negatively affect rig-i k63 ubiquitination, association with mavs and ifn signaling [when the constitutively active rig-i card domain was overexpressed (109, (112) (113) (114) (115) (116) or during viral infection (109, 111, 114) ]. viral inhibition of trim25 may even be a source of a positive selection during the evolution of certain viruses, as ns1 iav proteins have been found to interact with species specific trim25 (114) . for the latter, mefs from trim25 ko mice have significantly downregulated ifn1 production upon viral infection (113) and ko mice for nlrp12, which is a competitive interactor with trim25 to rig-i, show increased interferon production and more resistance to viral infection (117) . the known contributions of trim25 to innate immunity have recently been summarized elsewhere (52) . it is clear that both riplet and trim25 can mediate k63linked polyubiquitination. however, it has also been found that in-situ incubation of purified rig-i card domains with ubiquitin can be activated by free and unlinked k63 polyubiquitin chains (118) , calling into question whether trim25 only attaches k63-linked ubiquitin motifs to rig-i-card or if it also catalyzes the formation of unlinked k63 polyubiquitination chains (119) . a possible explanation for these differing results is that rig-i has been shown to be covalently k63 ubiquitinated by trim25 when analyzed by mass spectrometry from cells (104) , while experiments that demonstrate non-covalent k63 ubiquitination are those involve primarily interactions with purified proteins. it has also been recently found that rig-i is k63 ubiquitinated at k164 and that it may be functionally redundant to k172 (120, 121) , with their ubiquitination possibly upregulating the k63 ubiquitination of the other 6 lysine residues in rig-i (121). however, it is unknown whether trim25 ubiquitinates k164 or any of the other rig-i lysine residues. notably, these additional lysine residues in the card and c terminal domains of rig-i and mda5 are known to be k27 and k48 ubiquitinated [which are associated with degradation of rig-i (122, 123) and mda5 (123) ], but the four listed above appear to be the essential residues for activation of rig-i (122, 124) . the presence of k63 ubiquitin modifications on mda5 is more controversial. independent studies have found that mda5 is (125, 126) or is not (126) k63 polyubiquitinated. it has also been independently found that trim25 does not affect ubiquitination of mda5 (without distinguishing between k63 and k48 polyubiquitination) (104) and for trim25 to increase k63 ubiquitination (125) , the only apparent difference in the experimental models being the usage of hek293t (104, 126) vs. hek293 (125) cells. trim65 has also been recently found to be essential for mda5 activation by k63 polyubiquitination at k743 (127) . it is clear that additional studies are needed in order to clarify the ubiquitination mechanisms of mda5. upon binding to pamp (dsrna), rig-i oligomerizes with other rig-i/dsrna complexes to form helical oligomers (128) in a 2:2 complex using the purified rig-i protein (87) , where the activating ubiquitin motifs serve as a scaffold to link the oligomers together (118) . these oligomers have been found to be necessary under normal conditions to activate rig-i. this may be due to the helical structure of the rig-i oligomers closely matching those formed by mavs (63) , which is known to form filaments in-vitro (129, 130) mediated by its own card domains (131, 132) . a structural model of mavs activation by rig-i has been proposed of stacking mavs card domains on top of rig-i card domains to extend the rig-i helix (133) . the minimum length of dsrna found to activate rig-i is 13 base pairs, which is equivalent to the minimum length to facilitate the formation of a 2-rig-i/dsrna dimer (75) . that being said, shorter (∼10 bp) 5 ′ ppp stem loop dsrna complexes that have previously been used to obtain x-ray crystallographic structures of rig-i interacting with dsrna (61, 134, 135) (figure 3c ) can also activate ifnβ signaling in cells (135, 136) and in mice (136) . furthermore, a549 cells that were transfected with rig-i plasmid 6 h prior to rna transfection had a minimum dsrna length of only 8-10 bp required for activation (75) . this indicates that rig-i oligomerization may not be necessary for activation of the ifnβ pathway under some experimental conditions, which need to be further investigated. mda5 has also been shown to oligomerize to form long rnaassociated filaments in vitro (62, 137, 138) ( figures 3d, 4h) , which may be aided by chaperone proteins (139). given that the k743 residue found to have been ubiquitinated by trim65 (127) is located on the surface of hel2, it is possible that k63 ubiquitin residues may also help stabilize mda5-dsrna filaments (140). however, mda5 also spontaneously forms filaments and induce mavs to form filaments independently of ubiquitin in-situ. it is also thought that the formation of longer filaments by mda5 may be mediated by a longer linkage region between card2 and hel1 than in rig-i by 50 amino acids (the length of which is wellconserved across species), allowing for the association of more card domains in an oligomer (133) . the formation of longer filaments by rig-i has been more controversial, giving rise to two alternate models of rig-i activation: formation of individual single unit of rig-i with short dsrna monomers (leaving a free dsrna end, such as a hairpin loop), which then oligomerizes via card tetramerization that is linked by their ubiquitin chains, or filamentation on longer dsrna. like mda5, rig-i can form filaments in-situ independent of ubiquitin (141, 142) and induces mavs to also form filaments (142) , and mavs is known to form filaments in-vitro (129, 130) mediated by its own card domains (131, 132) . however, rig-i filamentation on an rna template (forming "beads on a string") as opposed to smallerscale oligomerization hasn't yet been shown to occur in-vitro. part of the reasons for the suggestion that rig-i was strongly activated by shorter dsrna was based the comparison on mass equivalents of rna species as there were less 5 ′ triphosphorylated ends for longer dsrnas with greater mass than shorter dsrnas with more 5 ′ triphosphorylated ends (76) . however, when rna species were normalized by molar equivalence, dsrna length appeared to be positively correlated with rig-i signaling (141) (142) (143) , which became insignificant at around 500 bp (141, 143) . it is significantly shorter than the length of dsrna that activates mda5, which forms filaments on 2,000 bp dsrna (137). the kinetics of rig-i and mda5 interacting with dsrna (which will be discussed in detail below) might possibly explain the decrease in dsrna length efficiency to activate rig-i as compared to mda5, as rig-i seems to first recognize the 5 ′ ppp end before sliding down the length of the dsrna (144), whereas mda5 dynamically associates and disassociates along the length of long dsrna (137). meanwhile, it is still unclear whether rig-i can preferentially be activated by longer dsrna independently of its unknown ability to form filaments in-vitro (145) . once fully activated and oligomerized, the rig-i card domain can then interact with mavs (146) (147) (148) (149) (figures 4e,i) , which is part of a protein complex containing a variety of other cellular proteins (6) (7) (8) (9) . while the mda5 card domain has much weaker direct association with mavs than the rig-i card domain, it is sufficient to lead to its activation and potentiates activation of mavs by rig-i (146), the mechanisms of which have yet to be determined. the activated mavs complex then initiates a molecular cascade which eventually results in expression of ifn1 (150) (figure 2) . interestingly, full length rig-i, when overexpressed, has been found to associate with mavs in the absence of activating dsrna and the interaction can be ablated by phosphorylation at s8 and t170 (87) , suggesting that the card phosphorylation sites function at least in part to prevent association of the inactive form of rig-i with mavs. furthermore, the crystal structure of the interaction between the rig-i card and mavs card domains shows the rig-i card2 domain interacting with mavs card domains on the outside of the tetramer and the rig-i card1 (1-87) domain facing toward the center of the tetramer (63) (figure 3e ). nmr solution structures of rig-i card2 also shows that t170 (which is required for dephosphorylation by pp1-α/γ) is largely buried within the card2 domain in a section that would be in closer contact with the helicase domains, suggesting that dephosphorylation of t170 affects an interaction domain between card2 and the c terminus (151) . furthermore, nmr of a c terminal construct of rig-i with the card2 domain shows stable interactions of card2 and the c terminal domain (151) . what all this may mean is that, while the card1 domain of rig-i is somewhat exposed in its inactivated form and therefore can be shown to interact with mavs, full exposure and engagement of both rig-i card domains (card1 and card2) with the card domain of mavs is necessary in order to induce ifn1 signaling. the card domains of rig-i also appear to be generally structurally stable, as electron microscopic structures have been obtained of the full length rig-i bound to blunt-ended dsrna showing both card domains exposed (87) . on the contrary, the card domains of mda5 may be comparatively more flexible than those of rig-i in order to mediate long mda5-dsrna filament formation (99) . the activated mavs complex induces association of the inhibitor of nuclear factor kappa-b kinase subunit epsilon (ikkε) and the serine/threonine-protein kinase 1 (tbk1) (10-12), which collectively phosphorylate the interferon regulatory factors 3 and 7 (irf3 and irf7) (13) (figure 1) . ikkε and tbk1 also interact with a number of other co-factors (152, 153) , such as the deadbox helicase 3 (ddx3) (154) . the activated p-irf3 (15) and p-irf7 (14) then translocate into the nucleus and dimerize, where they then act as the primary transcription factors for ifnα and ifnβ, respectively. existing evidence suggests that ifnα is more primarily produced in the earliest time points following rig-i/mda5 activation, while ifnβ is produced later and is responsible for more robust anti-viral control throughout the innate immune response period (155) . there is also a distinction between innate immune cell types for ifn1 production, as cells like fibroblasts and conventional dendritic cells produce ifnα and ifnβ (41, 156), while neutrophils only produce ifnβ (157) and plasmacytoid dendritic cells only produce ifnα primarily through the tlr signaling pathways (41, 158). signaling through rig-i is also known to be essential for the process of tlrmediated phagocytosis by macrophages (159) . interferons are then secreted out of the cell, where they bind to their own receptor (ifnar) and activate the janus kinase/signal transducer and activator of transcription proteins (jak/stat) signaling pathways, which result in a positive feedback signaling loop to further increase rig-i/mda5 expression and activation (160) and ifn1 production (161, 162) . expression levels of rig-i and mda5 have consistently been found to be upregulated downstream of type i (163, 164) and type ii (165, 166) ifn signals. mda5 upregulation has additionally been found to occur independently of cytokine expression at least during picornavirus infection (167). one of the most obvious distinctions between rig-i and mda5 is in the rna species to which they bind for activation (figures 1, 5) . rig-i has the highest affinity for short dsrna that is tri-phosphorylated at the 5 ′ end (67-75), with rig-i having been found to directly interact with the 5 ′ tri-phosphate group of the dsrna (71, 73) . while rig-i can bind to ss-5 ′ tri-phosphorylated rna (69), rig-i cannot be activated by it (69, 168, 169) , likely due to a conformational need to recognize double-stranded rna. as a result, rig-i is greatly attenuated by a 5 ′ overhang as well as those with a 3 ′ overhanging the 5 ′ tri-phosphate end (170) . in fact, a single unpaired 5 ′ triphosphorylated nucleotide is sufficient to competitively inhibit rig-i, which has been exploited by rna viruses to evade rig-i recognition and ifn1 signaling (171) . the unique preference of rig-i for 5 ′ tri-phosphorylated rna can be explained by the specific orientation that the rig-i c terminus adopts when directly interacting with the 5 ′ tri-phosphate group of the 5 ′ tri-phosphorylated dsrna (71, 73) as compared to unphosphorylated blunt-ended dsrna (172) . the minimally required and exclusionary features of the 5 ′ and 3 ′ dsrna ends for rig-i activation have proven to be complex. certain studies suggest that a 5 ′ diphosphate group is the minimum feature required for rig-i binding and activation, with 5 ′ monophosphate dsrna failing to productively activate rig-i as compared to 5 ′ di and tri-phosphate dsrna (173) . frontiers in immunology | www.frontiersin.org additionally, rig-i poorly distinguishes between dsrnas with either 5 ′ tri-phosphate and 5 ′ diphosphate group. when the free energies of each interaction are calculated, the affinity for 5 ′ triphosphate being lowered by disassociation of magnesium from the rig-i/dsrna complex. both are significantly more favorable for binding rig-i monophosphate dsrna (174) . this similarity in affinity appears to be important in the context of infection with viruses that produce 5 ′ diphosphate rnas, such as reoviruses (173) . likewise, the difference of energic binding between monophosphate dsrna and bi-and triphosphate dsrnas is likely important for distinction between self (host) and non-self (foreign) rna, the mechanisms of which will be discussed in detail below. the atp hydrolysis functions of rig-i have been shown to drive rapid disassociation from certain rna features, such as 5 ′ monophosphate dsrna (174, 175) and 5 ′ oh rna (144, 176) , which is particularly important for 5 ′ monophosphate dsrna because it is found in mrna after decapping during the mrna degradation process (177) . on the other hand, other studies have shown that rig-i can interact with monophosphate dsrna to a certain degree, as has been found to be the case for short synthetic dsrna with a 5 ′ and 3 ′ monophosphate group (69), poly(i:c) digested with rnase iii (76) [which generates 5 ′ mono-phosphate/3 ′ -oh dsrna (178) ] and hcv rna (179) and mitochondrial rna [in the p53 deficient mice (180) ] digested with rnase l [which produces 5 ′ oh and 3 ′ mono-phosphate dsrna at subnanomolar levels (181) , as has been found to be the case for hcv rna (179) .] it appears that the 5 ′ monophosphate is the determinate feature for rig-i activation independently of the 5 ′ or 3 ′ oh group in all these cases. a possible explanation for the discrepancy between the studies was that higher order rna structures might compensate for the less optimal 5 ′ and 3 ′ ends, as monophosphate dsrna that did not contain stem-loop structures did not activate rig-i and rna regions repetitive in certain nucleotides had been found to be critical for rig-i activation (179) . future studies are required to further characterize the behavior of rig-i with these rna species. as previously mentioned, mda5 preferentially associates with long dsrna (76) (77) (78) (79) . the crystal structure and molecular modeling of mda5/dsrna complex suggest that it can recognize the entire first turn of the blunt-ended dsrna (182) in a similar way as lgp2 can (183) . like rig-i and mda5, lgp2 belongs to the atp-dependent dexd/h box rna helicases (184) , which is structurally similar to rig-i and mda5 but lacks the card domains at the n terminus (185) . mda5 has also been found to be activated by the digested products of rnase l specifically from parainfluenza virus (186) . the presence of certain repetitive rna elements appears to be another contributing factor in determining interaction of rna with rig-i and mda5, which has recently been described in detail elsewhere (187) . while rig-i and mda5 are mostly implicated in the immune response to rna viruses, it has also been found to be activated by 5 ′ tri-phosphorylated dsrna intermediates generated by cellular rna polymerase iii from at-rich dna sequences (188) and during infection with epstein-barr virus (a dna virus) (189) . rig-i has additional binding preferences for certain nucleotide motifs, such as uridine-rich 5 ′ tri-phosphorylated hairpin rna (190) , synthetic au-rich hairpins (191) and those naturally found in the genomes of sendai virus defective-interfering (di) particles (192) , measles (193) , influenza a virus (iav) (194) and in kshv rna transcripts (195) , and poly (u/uc) regions (196) and poly (a/ag) regions (197) in the antisense hepatitis c virus (hcv) genome. it is of particular interest that the poly (a/ag) hcv regions are located significantly downstream of the 5 ′ triphosphate group (197) , thus potentially implicating other parts of rig-i (e.g., helicase domain) as potential rna interacting domains. repetitive rna elements may also be important in allowing for interaction of inhibitory rnas that do not have 5 ′ or 3 ′ features needed for full activation of rig-i, as has been shown to be the case with ga-rich regions in circular longnon-coding rna lnc-lsm3b (198) . these specific interactions explain their primary role as anti-viral receptors, as these viral motifs are mostly not found in cellular rnas (199) . rig-i and mda5 have been particularly implicated in their response to rna genomes of viral defective interfering (di) particles, as these defective viral genomes (dvgs) have originally been found to induce interferon signaling (150) . di particles are produced by many viruses during infection, and while they are similar in many regards to standard viral particles, such as in appearance and composition, they cannot productively infect cells (200) . this is largely thought to be due to the presence of large and deleterious deletions in the dvg of di particles (201) . some dvg rnas have also been noted to have "copy-back" motifs in which one end of the genome can base pair with an inverted copy at the opposite end of the genome, which may be due to stalled and aberrant replication (202, 203) . copy-back rna motifs specifically seem to be important for rlr activation in that they tend to contain hairpin motifs and 5 ′ tri-phosphate groups, as has been found for sendai (204) (205) (206) , measles (35, 207), and chikungunya (35) dvg rnas in activating rig-i. in the case of iav, dvg rnas might even be more potent activators of rig-i than the full-length viral genome. cells that were blocked from viral protein synthesis experienced rig-i mediated ifn1 expression when infected with iav stocks grown in chicken embryonic eggs (which produced higher relative quantities of di particles with dvg rnas) but not with iav grown in cell culture, indicating that rig-i activation by the genomes from primarily non-di iav particles may require active viral rna synthesis (208) . a potential explanation to this observation is that rig-i appears to be activated by the full viral genome via its panhandle structure, the affinity of which is lowered by the presence of mismatched and unpaired nucleotides in this region of the viral genome that is conserved across influenza virus strains (209) . however, the overall panhandle structure is conserved between dvgs (205) and the full length viral genome (209) , and deletions within dvgs are monogenic and internal (210) . the specific molecular mechanisms of enhanced rig-i signaling by iav dvgs have yet to be elucidated, although the level of exposure of the panhandle may play a role. while the full extent of mda5 interacting with di rna is currently unknown, mda5 appears to be more predominantly activated by dvg rna than rig-i specifically in dendritic cells early in the viral infection cycle (211) , which may be a contributor toward the phenomenon of di particles enhancing dendritic cell maturation (212) . the comparative abilities for di particles vs. infectious virions to activate rig-i and mda5 have important implications for understanding viral pathogenesis and for vaccine development. there is a burgeoning interest in this regard, especially in populations which are typically more challenging to achieve successful preventative vaccination, such as elderly populations with iav vaccination (213) . elderly populations in general do not develop as strong of memory immune responses to vaccines as their younger counterparts (214) (215) (216) (217) and have been found to have decreased rig-i mediated ifn1 signaling (218) . correspondingly, the influenza vaccine has been shown to decrease in effectiveness in older populations as the influenza season progresses (219) . a di-vaccine that strongly activates innate immune cells and increases the adaptive immune response could therefore potentially boost the immune responses to vaccines in more vulnerable populations. additionally, di particles have shown to be an important contributor of viral persistence (200, 220, 221) . this raises the question of whether a viral infection may alternate between producing primarily infectious virions which eventually activates the innate immune response and producing primarily di particles which requires less cellular activity but may initiate an even stronger innate immune response (222) (223) (224) . taken altogether, di particles provide yet another layer of distinction between rig-i and mda5 in terms of how each recognizes different species of dsrna. the preference for specific rna species by rig-i and mda5 allow for them to distinguish between viral rna and host rna in most circumstances (225) , although the specific mechanisms of distinction are not as clear for mda5 as for rig-i. studies from clinical cases of mda5 mutations provide contradictory models, with certain mutations found in aicardi-goutières syndrome (ags) increasing mda5 avidity for self rna (226) with alu retroelements found to be significantly enriched for interaction with ags mda5 mutations (227) . the modification of dsrna by host cells may be a primary inhibitor of mda5 activation by host rna as knockout of adenosine deaminase (adar1), which weakens dsrna structures, allows wild-type mda5 to be activated by alu retroelements (227) . however, other mda5 mutations decrease affinity for known mda5 ligands and atpase activity, yet still demonstrate increased ifnβ expression (228, 229) . for rig-i, a highly conserved residue in the c-terminal rna binding pocket (h830) has been found to sterically exclude canonical self-rna by the means of the n 1 -2 ′ o-methyl self-rna motif, also known as cap1 rna (61, 230) . this results in a low binding affinity of rig-i to cellular cap1 rna and decreased atpase activity as compared to pamp (dsrna) (61, 231) . flaviviruses take advantage of this precise discrimination by encoding a viral 2 ′ -o-methyltransferase capable of n 1 -2 ′ o-methylating its positive-strand rna genome in order to evade rig-i recognition and ifn1 activation (230) . conversely, the mutations e373a and c268f found in the rig-i protein in patients with auto-immune disorder singleton-merten syndrome confer the ability of the protein to recognize cap1 rna and become activated by atp dependent and independent mechanisms, respectively (232) . furthermore, the e373q mutation of rig-i, which was designed to constitutively bind atp, was found to increase the affinity of rig-i with ribosomal rna (233) . it is noteworthy that host rna contains additional internal rna modifications and non-watson-crick base pairing which can inhibit activation of the other known dsrna-sensing protein, the interferon-induced double-stranded rna-activated protein kinase (pkr) (234) , and it is known that synthetic 5 ′ triphosphorylated rna containing pseudouridine, 2-thiouridine or 2 ′ -o-methylated uridine has significantly decreased ability to activate rig-i (67), which has been demonstrated to occur by preventing rig-i filament formation in-situ (142) . n-6-methyladenosine (m6a) nucleotides, which are well-known nucleotide modifications among viruses (235) , have also been found to ablate dsrna binding to rig-i (236) . it has been demonstrated that certain rna-dna hybrid constructs with ribonucleotides at positions 2 and 5 of the dna strand can bind to rig-i and activate its atpase activity (75) . atpase activity is necessary for full activation of rig-i and expression of ifnβ (75, 237) , so the minimum requirement of a motif not found in host rna for atpase activity has significant implications for the distinction between self and non-self rnas. expanding on this observation, exogenous atpase activity may also be sufficient to potentiate rig-i and mda5, as lgp2 atpase mutant mice are significantly more susceptible to viral infection even in the presence of functional rig-i and mda5 (238) . however, this model is further complicated by certain rna-dna hybrids that are able to bind rig-i and activate atpase activity, but don't induce ifnβ expression (75) . it is currently undetermined whether such hybrids can sterically inhibit rig-i due to the presence of mostly dntps or whether they inhibit rig-i in a yet undescribed way. recent kinetic studies of rig-i and mda5 activation by pamp (dsrna) help illustrate how atpase activity is critical for their function and distinction between host (self) and foreign (non-self) rna. rig-i binding to atp is sufficient for interaction with dsrna (144, 176) . rig-i atpase activity is inhibited in the absence of pamp (dsrna) by a helical arm that blocks the atpase site (239) . upon interaction with pamp (dsrna), the helical arm shifts and the two helicase domains are brought together to form an active atpase site (239) . rig-i then catalyzes atp to break the 5 ′ ppp dsrna interactions within seconds. atp is then rapidly hydrolyzed to facilitate translocation of rig-i to the opposite dsrna end, after which the rig-i oligomers can form (144) . on the other hand, atp hydrolysis drives rapid disassociation of rig-i from host rna features. these features include dsrna with a 5 ′ monophosphate group (174, 175) that is found in mrna after decapping during the mrna degradation process (177) and 3 ′ overhang rna (144, 170) found in mirna (240) as well as other rna motifs, such as 5 ′ oh rna (144, 176) found in bacteria (241). furthermore, an impaired atpase functionality increases the promiscuity of rig-i binding these host rna motifs (144, 176, 242) . similar atpase functions have been found during mda5 filamentous formation. the c terminus of mda5 is critical to form organized helical filaments (138) and atp binding drives association and hydrolysis and disassociation from foreign dsrna [with little coordination being observed between neighboring mda5 proteins (137)] in a manner that involves mda5 twisting along its flexible and hydrophobic interface domains (243) . taken together, atpase activity may be directed toward rapid disassociation from host dsrna and degradation of rna-dna hybrids, but primarily act on the translocation pathway upon interaction with pamp (dsrna). it is also possible that host and hybrid dsrnas could inactivate rig-i independently of their ability to bind the c-terminus and activate atpase activity. this has been shown, for example, for a hybrid rna that has one strand consists mostly of dna except at positions 2 and 5, which appears to bind rig-i and activate its atpase activity but doesn't activate ifn1 signaling (75) . future studies are needed in order to determine these differential interaction mechanisms. contrary to the traditional paradigm, there is increasing evidence to suggest that rig-i and mda5 interact with certain host rna motifs, resulting in auto-activation or auto-inhibition of the irf pathway ( figure 5) . one of the most strongly supported models is activation by host and viral circular rnas (circrna). originally found in a variety of pathogen genomes, circrnas in eukaryotic cells were first thought to be byproducts of the pre-mrna splicing process. however, they have later been found to be produced by a non-canonical "backsplicing" process and there is increasing evidence to suggest that they play some important regulatory roles (244) , suggesting that they may have specifically evolved for this purpose. rig-i was first found to interact with circrna produced in situ (245) . interestingly, the minimum component required for rig-i activation is an intron of pathogenic origin to be spliced out during the circularization process. as human introns have been found to be associated with many rna binding proteins, it is speculated that these proteins may have prevented circularization of this particular synthetic circrna used in this study (245) and that host rna binding proteins normally prevent endogenous circrnas from being detected by the innate immune system. nevertheless, some viral infections can potentially expose these endogenous circrnas for immune detection, as has recently been found to be the case for a novel host-derived circrna (lnc-lsm3b) that is ifn-inducible and shows a down-regulation of its binding to host proteins during viral infection and therefore appears to compete with viral dsrna as an inhibitor of the rig-i signaling feedback loop (198) . similar inhibitory mechanisms have also been noted for rna products of the exonuclease skiv2l (246) . finally, recent studies have found that hepatitis c virus (hcv) infection increases the expression of certain cellular rnas that can inhibit rig-i function. hcv infection increased the mrna levels of hepatic selenoprotein, which was able to bind to rig-i through a hairpin structure and inactivated it during viral infection (247) . infection by hcv, vesicular stomatitis virus (vsv), or sendai virus, or direct exposure of cells to type 1 and 3 interferons increases expression of the cellular long non-coding rna (lncrna), namely lncatv, which similarly inhibits rig-i function by directly interacting with it in order to promote virus replication (248) . in addition to the greatly increased implications of rig-i and mda5 modulation, these findings also have significant implications in characterizing new biomarkers of disease, as increased serum selenoprotein level has been found to significantly associate with treatment failure of anti-viral drugs in hcv patients, and can possibly explain the increased prevalence of type 2 diabetes in hcv patients (247) . cellular rna has also been found to activate rlr signaling during viral infection. vault rnas, which are transcribed from four genes and are normally found in large ribonucleoprotein complexes in cytoplasmic "vaults, " are significantly enriched for binding to rig-i during infection with kshv (29). this may be due partly to viral infection-induced reduction in the level of cellular triphosphatase dusp11, which dephosphorylates the 5 ′ ppp group on the vault rnas, as they could only be immunogenic (in the absence of viral infection) by the addition of the 5 ′ ppp group. rig-i and mda5 have also been found to be activated by rna microparticles produced in situ by rolling circle transcription, generating tandem repeat rna strands (249) . retrotransposons may also be able to activate both rig-i and mda5, as both can be activated by line1 rna independently of dna sensing mechanisms and retrotransposition (250) . viral infections can also induce recognition of host rnas. herpes simplex virus 1 (hsv1) infection, for example, has been shown to induce translocation of the host pseudogene rna5sp141 ribosomal rna into the cytosol to bind to rig-i. knockdown of rna5sp141 decreased cytokine signaling during infection with hsv and ebv as well as influenza a virus (iav) (251) . rig-i has also been found to be activated by hairpin rna structures generated by cleavage of rna by rnase l, which has been demonstrated to occur during hcv infection (179) as well as from mitochondrial dsrna produced in p53 deficient mice (180) . the mitochondria, in particular, may be an important source of immunostimulatory host dsrna. viral infections are well-known to cause mitochondrial damage (252) . knockdown and hepatocyte-specific conditional ko of mitochondrial rna degrading enzymes resulted in the increase of cytoplasmic mitochondrial dsrna which was able to activate mda5 (253) . additionally, extracellular vesicles (ev) secreted by apoptotic endothelial cells were found to contain long interspersed nuclear element (line) and short interspersed nuclear element (sine) rnas that are products of rna polymerase iii and were able to activate rig-i signaling (254) . collectively, these findings demonstrate the many unique ways by which cellular rnas can modulate rig-i and mda5 functions as well as the potential implications of rig-i activation by pharmaceuticals as an anti-viral or generalized immunotherapy, though much caution and studies would still be needed to determine the appropriate levels of rig-i and mda5 activation. given that rig-i and mda5 are critical for activating expression of ifn1 during viral infection, there is much interest in studying the interactions of these cellular proteins with viral factors (rnas or proteins), as the ability to modulate interferon expression is a major evolutionary driving force in viral evolution (255, 256) . there are many mechanisms viruses have evolved to evade rig-i and mda5 signaling, which have been discussed at length elsewhere (257, 258) . such mechanisms are of particular importance to segmented rna viruses, providing potentially more dsrnas for rig-i and mda5 activation (259) . iav and the other orthomyxoviruses are unique in that they replicate in the nucleus of the cells (260) , preventing the viral rna from being detected by the prrs. however, recent preliminary evidence seems to suggest that rig-i may also endogenously be present in the nucleus and performs similar viral rna binding and activation of the ifn1 pathway (261) , yet this finding has yet to be replicated by other laboratories. there is also increasing evidence to suggest that rna processing is another mechanism of immune modulation. certain bunyaviruses can cleave the 5 ′ tri-phosphate group from their genomic rna (262) in order to avoid immune detection. rig-i has also been found to be subjected to negative modulation by rnai during iav infection (263) . on the contrary, nucleoproteins from the sendai virus (264) regulate the number of di particles being produced, and iav nucleoproteins also regulate the production of abortive replication rna (208) , mini viral rnas (265) and dvg rna (208) , all of which are immunostimulatory. the semliki forest virus (sfv) polymerase has even been found to convert host rna into 5 ′ -ppp dsrna to induce ifn1 expression (266) . this raises an intriguing possibility that induction of ifn1 may actually benefit some viruses under certain circumstances despite ifn1 signaling negatively regulating viral replication. the viral rna levels and localization throughout the viral life cycle might also play an important role in immune evasion (267) . control of viral rna levels by viral exoribonucleases in particular illustrates the complicated balance between viral production and immune evasion for optimal viral propagation, as has found to be the case for arenaviral nucleoproteins (nps) (268, 269) and nonstructural proteins found in coronaviruses (270, 271) . finally, viral infection has the capability to disrupt processes of the cell's basic functions, such as transcription and translation, thereby affecting viral replication and immune signaling in complicated ways (258) . one of the most significant ways viruses modulate rig-i and mda5 signaling is through their viral proteins (272) (figure 6) . the respiratory syncytial virus (rsv) non-structural protein (ns2) protein and the z matrix proteins of pathogenic arenaviruses interact with the rig-i card domains to block its interaction with mavs (273, 274) . the hsv1 deamidase ul37 specifically targets rig-i through its helicase domain, abrogating its ability to bind to rna (275) . the iav polymerase components also interact directly with rig-i (276), though their biological significance has yet to be determined as they don't significantly affect ifn1 production. on the other hand, rna binding appears to be an important bridge between the interaction of rig-i with other viral proteins, as the nucleoproteins (nps) of iav (276) and arenaviruses (277, 278) both interact with rig-i through viral rna. the ns1 protein of rotaviruses targets rig-i for degradation that is independent of proteasomes (279) . the v protein of paramyxoviruses inhibits mda5 (40) by targeting a unique feature of the atp binding pocket in mda5 (280) and by inhibiting mda5 card dephosphorylation (93) , but can also inhibit rig-i by interacting with the card domain to prevent its ubiquitination by trim25 (281) . finally, the us11 protein of hsv1 (282) and the arenaviral z matrix proteins (274) directly interact with and inhibit rig-i and mda5 in a similar fashion. there are also many other viral proteins that can regulate proteins in the rig-i and mda5 pathways, which have been discussed in detail elsewhere (44, 53, 59, 96, 257, 283) . it is important to consider the different regulatory mechanisms of rig-i and mda5 when considering their different functionalities (figures 4, 6) . one of the key differences between these proteins is in their post-translation modifications (96) . ubiquitination of rig-i is necessary for its activation (118) and is a point of negative regulation by host proteins (117, 284, 285) , viral proteins (281, 286, 287) and ubiquitin mimics (288) as well as positively regulated by influenza b ns1 protein (289) and another ubiquitin mimic (290) . on the contrary, mda5 is more well-known to be negatively regulated by ubiquitination (291) , with positive regulation by k63 ubiquitination being more controversial. while the deubiquitinase usp3 inhibits mda5 as well as rig-i, it is thought that this may be due to usp3 directly binding the mda5 card domain to prevent rna filamentation (284) . this raises the question of how rig-i can maintain its stability outside of the proteasome, as ubiquitination at other lysine residues in rig-i besides k172 induces proteasomal degradation (291) (292) (293) . this proteasomal degradation may be mediated by a p62 autophagic complex that associates with lrrc25/isg15 (294) and sqstm1 (295) and also mediates mitophagy and downregulation of mavs signaling during measles virus infection (296) . one key observation is that, while both rig-i and mda5 are cleaved during picornavirus infection, this cleavage is mediated by the viral proteinase 3c pro (297) and is independent of the proteasome (298) for rig-i, whereas it is mediated by cellular caspases and the proteasome for mda5 (299) . mda5 is also cleaved by caspases during apoptosis (4), though it hasn't been shown whether this is mediated by mda5's ubiquitination sites. the ubiquitin linkage site may be a determinate of function, as the ubiquitin ligases rnf122 (300) and stub1 (293, 301) have been shown to negatively regulate rig-i catalyzed k48linked ubiquitination as opposed to the known k63-linked ubiquitination at the k172, k849 and k851 activating sites, and rnf125 has also been proposed to k48 ubiquitinate rig-i (291) (though it hasn't been shown directly) (59) . trim40 has also been shown to negatively regulate rig-i and mda5 by k27 and k48 ubiquitination (123) . substantiating the possibility that k63 ubiquitination on rig-i may be functionally distinct from its other ubiquitination sites by protecting it from degradation is the finding that the ns1 protein of west nile virus (wnv) targets both rig-i and mda5 for degradation by proteasomes. additionally, ns1 inhibited k63 ubiquitination of rig-i, but mda5 was not found to be k63 ubiquitinated (126) . heat shock protein 90-alpha (hsp90) has been found to protect rig-i from proteasomal degradation, but it is unknown which type of ubiquitination that is inhibited by hsp90 (302) . taken together, the experimental evidence suggests that rig-i may be protected from proteasome degradation despite its activating ubiquitin moieties (52) . this warrants further studies for mechanistic elucidation. rig-i and mda5 additionally interact with different cellular co-factors, contributing to their differential regulations of function. rig-i is well-known for being potentiated by proteins that also bind dsrna, such as (pact) (303, 304) , which was first discovered as a protein activator of pkr, the serine/threonineprotein kinase 1 (tbk1) (305) (306) (307) (308) (309) and the oligoadenylate synthetase l (oasl) (310) . pact in particular has some functional similarities to rig-i, as they each contain three distinct rna binding domains (311) and interact with many of the same cellular co-factors, such as pkr (312) and dicer (312, 313) . because of the important role of pact in augmenting rig-i function, it is a prime target for inhibition of rig-i signaling by several viral proteins from diverse families of viruses (314) (315) (316) , the molecular mechanisms of pact inhibition by these viral proteins can vary and still need to be characterized in detail in future studies. similarly, the host ribonucleoprotein raver1 can increase affinity of mda5 for dsrna (317) , and the zinc-finger protein zcchc3 has recently been found do so for both rig-i and mda5 (125) in similar mechanisms to the other known rna-binding proteins. on the contrary, the human hemoglobin subunit beta (hb) has recently been suggested to decrease mda5 signaling by competing for long dsrna, while hb can enhance rig-i signaling by increasing k63 ubiquitination on rig-i (318) . several host factors interacting with rig-i and mda5 do so by yet undescribed mechanisms. pkr [which is also activated by pact (319, 320) and is sequestered by the cellular helicase dhx36 protein to form stress granules (321, 322) along with rig-i (323, 324) and trim25 (324)] appears to have a novel and yet uncharacterized function in enhancing mda5-dependent mavs signaling that is dependent on the kinase activity of pkr (325) . additionally, the porcine interferon-inducible oligoadenylate synthetase-like protein (poasl) has also been found to interact with and inhibit mda5 by an unknown mechanism (326) . the rig-i card domain interacts with mavs to induce interferon signaling, so proteins that disrupt this interaction [as it has been proposed for the atg5 and atg12 autophagy proteins (59) ] can specifically inhibit rig-i signaling. however, other cellular proteins, such as the complement protein gc1qr (327) and tarbp2 (328) that interact directly with mavs, inhibit both rig-i and mda5. lactate and hexokinase have also recently been found to inhibit rig-i and mda5 by interacting with mavs, which may be significant in explaining the interplay between metabolism and immune signaling as glycolysis was found to be greatly decreased upon rlr signaling (329) . likewise, cellular proteins, such as nlrc5 (330) that interacts with the rig-i and mda5 card domains have been shown to block interaction of both rig-i and mda5 with mavs. contrarily, dhx15 has been identified as a rig-i cofactor that interacts with the rig-i card domains and with pamp (dsrna), thereby increasing rig-i atpase activity (331) . additionally, adp-ribosylation factor proteins can block rig-i and mda5 from interacting with pamps and thereby inhibit their activation (332, 333) . lastly, the green tea molecule egcg has also been shown to inhibit the atpase function of rig-i (334) . the similarities and differences between rig-i and mda5 modulations and signaling are complex and will need to be elucidated further in future studies. despite their structural and mechanistic differences, it is important to emphasize that existing phylogenetic analysis indicates that rig-i and mda5 come from a common origin that is also shared among several other protein families (figure 7) . the linkage of the helicase and dexd/h box protein appear to be ancient, as orthologs of these proteins are found in the archaea kingdom (335, 336) . mda5 orthologs are found in most vertebrates (184) , while rig-i orthologs are only found in mammals, ducks, geese and some selected fish and reptiles (184, (337) (338) (339) (340) (341) (342) (343) (figures 2, 7) . it is therefore likely that mda5 evolved first, perhaps from a common ancestor with the closely related lgp2 helicase family (184) , which is structural similar to rig-i and mda5 but lacks the card domains at its n terminus (185) . lgp2 orthologs are also only found in vertebrates while the next closest related family of proteins (dicer) are more ancient proteins. it has therefore been proposed that the rig-i helicase-dexd/h complex may have been duplicated from mda5 in the common ancestor of vertebrates (184) . the association of the two card domains appears to have followed, as individual card domains are found in a variety of vertebrates that also encode caspases (344, 345) , but only rig-i, mda5, and certain members of the nacht family of ntpases (346) have two card domains. phylogenetic analysis has shown that the helicase-dexd/h and card2 have strong co-evolution history (347, 348) , while card1 has evolved more independently (184) . card2 appears to have been grafted onto the rig-i helicase-dexd/h complex first, with the card2-mda5 being duplicated from this event. finally, card1 was grafted onto the card2-helicase-dexd/h complex in separate events for rig-i and mda5 (184) . in mammals, positive selection can be seen in the flexible hinge region connecting the card domains to the helicase in rig-i and mda5. rig-i contains an additional site of positive selection within the hel1 structural motif (n421), while most of the unique positive selection sites for mda5 are in regions specific to it, including a 29 amino acid insertion in hel2 (349) . while rig-i and mda5 may both originate from common ancestors of vertebrates, there is increasing evidence to suggest , and related dexd/h-box helicases are shown as a phylogenetic tree, along with their lowest level of biological taxonomy that these proteins are found in present day. in short, the precursor of the mda5 helicase-ctd likely originated from a common ancestor with the precursor for lgp2, which was then duplicated to create the helicase-ctd precursor of rig-i in the common ancestor of vertebrates. card2 was then grafted onto the helicase-ctd protein, and this protein was duplicated to create the card2-helicase-ctd precursor of mda5. finally, card1 was grafted onto these proteins in separate events, forming the modern-day rig-i and mda5. that proteins with similar functions may have evolved separately in other species from ancient helicase-dexd/h proteins, implicating rna-mediated defense responses as a potentially universal biological function. a rig-i homolog has recently been found in a planarian that is able to activate downstream inflammatory genes in the absence of the traditional card domains (350) , and a similar homolog in caenorhabditis elegans has been proposed to mediate anti-viral rnai by complexing with dicer and catalyzing their translocation on the viral genome (351) . additionally, insects have been found to primarily respond to rna viruses by rnai mediated by dicer proteins (352) . dicer may potentially mediate dsrna-activated anti-viral signaling pathways that is independent of rnai pathways, as has been found to be the case for the expanded cag-repeat dsrna (353) . pattern recognition receptors (prrs) that respond to viral rna have not yet been found outside of the animal kingdom, as rlrlike proteins in prokaryotes do not have card domains and the prrs in plants found so far are surface-receptor kinases that respond to external molecular elements of bacteria (354) [similar to the mammalian toll-like receptors (tlrs)]. however, rna silencing has been demonstrated to be an important anti-viral strategy in plants (354, 355) and certain arabidopsis mutants appear to be more susceptible to infection by rna viruses (356) . rig-i (357) and mda5 (357, 358) are known to influence antiviral signaling in zebrafish (danio rerio) and other fish species (357, (359) (360) (361) through the canonical mavs signaling pathway. fish rig-i like receptors (rlrs) have been shown to be regulated by the expression of alternate splicing isoforms (358, 362) , which have also been found to occur with a dominant-negative splice variant of the human rig-i (363). rig-i and mda5 have also been found to participate in anti-viral signaling in ducks (364) (365) (366) (367) and geese (340, 368, 369) , and mda5 alone in chickens (370) (371) (372) and other birds (373) . the observation across species of rlr's performing compensatory mechanisms when a function or a pathway protein is absent is reiterated in birds, as mda5 has been found to sense short and long dsrna in chickens (372) and in the chinese shrew (374) , both of which lack rig-i. additionally, trim25 activates rig-i in ducks (364) and in the chinese goose (375) in the absence of the k172 activating ubiquitin binding site that is conserved in primates and some rodents (364) . finally, the rainbow trout (oncorhynchus mykiss) has been found to express a lgp2 variant in addition to the canonical lgp2 that contains an incomplete c-terminal domain of rig-i (376). the differential presence of prrs may also influence viral evolution. a mutation in the iav polymerase subunit pb2 found in avian-adapted h1n1 strains decreases the inhibition of human rig-i function by iav nucleoproteins, which may indicate a differential selective pressure for viruses that propagate in species that don't contain rig-i (377) . the evolutionary pattern and compensatory mechanisms of rlrs across species implicate them as critical for anti-viral function, and that evolutionary forces drive the available pathway proteins to meet these functional needs. future studies need to be done to further differentiate rlr function among the different species, as this will provide critical information concerning the various methods of disease control by targeting the pathogen by these important host proteins. there is also increasing evidence for other rna-sensing dexd/h helicases serving important roles in anti-pathogen immune sensing, which have recently been reviewed elsewhere (187) . some rna helicase (ddx) proteins appear to serve as complex proteins upon interacting with viral rna. ddx3 is a well-known example, being suspected of being a transcription factor for ifn-β (378), associating with spliceosomes and the stress-induced p-bodies to influence mrna splicing and decay, respectively (322, 378) , and interacting with the mavs complex during viral infection conditions (378, 379) . in particular, ddx3 associating with mavs has been found to be important for anti-viral control against several viruses (378) (379) (380) , and since the two ddx3 homologs are found on the x and y chromosomes, they may contribute to immunological differences between genders (381). this is a repeated theme, as dhx9 (382), dhx15 (383) , and a complex consisting of ddx1/ddx21/dhx36 (384) have also been found to associate with the mavs complex to enhance ifn1 signaling, while dhx33 interacts with mavs independently of viral infection (385) . ddx proteins can also activate other proteins in the irf pathway. multiple ddx proteins can interact with ikkε, with ddx3 being phosphorylated by ikkε to induce irf3 interaction with the tbk1-ikkε complex (378) , and ddx19 blocking this interaction to inhibit ifn1 signaling (386) . similar control mechanisms have been demonstrated for ddx3 interacting with viral proteins. for example, ddx3 has recently been found to associate with arenaviral nps to increase viral rna synthesis and ifn1 expression (387) . additionally, the np of the 1918 h1n1 iav pandemic strain has been shown to target ddx3 for degradation as a potential mechanism of virulence (388) . dhx15 (389) and dhx33 have also been found to activate nfκb and mapk signaling pathways. finally, ddx60 has been shown to act as a cofactor for rig-i (390, 391) and dhx29 for mda5 (392) . taken altogether, these cellular proteins have likely evolved to regulate rig-i and mda5 signaling from their common dexd/h helicase predecessors. as our capacity to study the molecular mechanisms and to purposefully modulate immune responses increases in specificity, so will our needs to characterize the differences between related immune signaling proteins. the concept of personalized medicine derives from the idea that we can therapeutically intervene in a situation that is designed around the individual's unique characteristics. while this is an achievable realm of medicine in the future, an immediate step is to determine the functions of some critical proteins, such as the rig-i and mda5 of the innate immune arm. examining their structural and functional similarities and differences at multiple levels will allow for a deeper level of appreciation of these proteins, which may be exploited therapeutically to differentially modulate rig-i and mda5 signalings by different rna ligands (43, 191, 393, 394) or other pharmaceutical compounds (395) rig-i, a human homolog gene of rna helicase, is induced by retinoic acid during the differentiation of acute promyelocytic leukemia cell retinoic acid-inducible gene-i is induced in endothelial cells by lps and regulates expression of cox-2 mda-5: an interferon-inducible putative rna helicase with double-stranded rnadependent atpase activity and melanoma growth-suppressive properties overexpression of helicard, a card-containing helicase cleaved during apoptosis, accelerates dna degradation interferons and viruses: an interplay between induction, signalling, antiviral responses and virus countermeasures mechanisms of mavs regulation at the mitochondrial membrane mavs coordination of antiviral innate immunity assembly of the whip-trim14-ppp6c mitochondrial complex promotes rig-i-mediated antiviral signaling zyxin stabilizes rig-i and mavs interactions and promotes type i interferon response. sci rep ikkε and tbk1 are essential components of the irf3 signaling pathway multiple functions of the ikk-related kinase ikke in interferon-mediated antiviral immunity trim25 in the regulation of the antiviral innate immunity. front immunol molecular mechanisms of dicer: endonuclease and enzymatic activity the rig-i-like receptor lgp2 inhibits dicer-dependent processing of long double-stranded rna and blocks rna interference in mammalian cells virus sensor rig-i represses rna interference by interacting with trbp through lgp2 in mammalian cells lgp2: positive about viral sensing mda5 and lgp2: accomplices and antagonists of antiviral signal transduction negative regulators of the rig-i-like receptor signaling pathway tissue-based map of the human proteome structural basis for m7g recognition and 2'-o-methyl discrimination in capped rnas by the innate immune receptor rig-i structural basis for dsrna recognition, filament formation, and antiviral signal activation by mda5 molecular imprinting as a signal-activation mechanism of the viral rna sensor rig-i parts, assembly and operation of the rig-i family of motors the rna helicase rig-i has an essential function in doublestranded rna-induced innate antiviral responses the c-terminal regulatory domain is the rna 5'-triphosphate sensor of rig-i 5 ′ -triphosphate rna is the ligand for rig-i rig-i-mediated antiviral responses to single-stranded rna bearing 5'-phosphates nonself rna-sensing mechanism of rig-i helicase and activation of antiviral immune responses cytosolic viral sensor rig-i is a 5'-triphosphate-dependent translocase on doublestranded rna the structural basis of 5' triphosphate double-stranded rna recognition by rig-i c-terminal domain rig-i detects viral genomic rna during negative-strand rna virus infection structural and functional insights into 5'-ppp rna pattern recognition by the innate immune receptor rig-i the thermodynamic basis for viral rna detection by the rig-i innate immune sensor rig-i atpase activity and discrimination of self-rna versus non-self-rna length-dependent recognition of doublestranded ribonucleic acids by retinoic acid -inducible gene-i and melanoma differentiation -associated gene 5 activation of mda5 requires higher-order rna structures generated during virus infection mda5 detects the double-stranded rna replicative form in picornavirusinfected cells visualisation of direct interaction of mda5 and the dsrna replicative intermediate form of positive strand rna viruses a structure-based model of rig-i activation toward a crystal-clear view of the viral rna sensing and response by rig-i-like receptors pattern recognition and signaling mechanisms of rig-i and mda5 antiviral rna recognition and assembly by rlr family innate immune sensors post-translational control of intracellular pathogen sensing pathways essential role of the n-terminal domain in the regulation of rig-i atpase activity structural basis for the activation of innate immune pattern-recognition receptor rig-i by viral rna structural and biochemical studies of rig-i antiviral signaling negative role of rig-i serine 8 phosphorylation in the regulation of interferon-beta production conventional protein kinase c-α (pkc-α) and pkc-β negatively regulate rig-i antiviral signal transduction phosphorylation of rig-i by casein kinase ii inhibits its antiviral response riok3-mediated phosphorylation of mda5 interferes with its assembly and attenuates the innate immune response dephosphorylation of the rna sensors rig-i and mda5 by the phosphatase pp1 is essential for innate immune signaling antagonism of the phosphatase pp1 by the measles virus v protein is required for innate immune escape of mda5 hdac6 regulates cellular viral rna sensing by deacetylation of rig-i the mechanism of atp-dependent rna unwinding by dead box proteins mechanisms of rig-i-like receptor activation and manipulation by viral pathogens the catcher in the rig-i viral rna detection by rig-i-like receptors mda5 cooperatively forms dimers and atp-sensitive filaments upon binding double-stranded rna riplet/rnf135, a ring finger protein, ubiquitinates rig-i to promote interferon-beta induction during the early phase of viral infection reul is a novel e3 ubiquitin ligase and stimulator of retinoic-acid-inducible gene-i a distinct role of riplet-mediated k63-linked polyubiquitination of the rig-i repressor domain in human antiviral innate immune responses ubiquitindependent and -independent roles of e3 ligase riplet in innate immunity trim25 ringfinger e3 ubiquitin ligase is essential for rig-i-mediated antiviral activity caspase-12 controls west nile virus infection via the viral rna receptor rig-i the long noncoding rna lnczc3h7a promotes a trim25-mediated rig-i antiviral innate immune response ube2d3 and ube2n are essential for rig-i-mediated mavs aggregation in antiviral innate immunity ndr2 promotes the antiviral immune response via facilitating trim25-mediated rig-i activation in macrophages the ubiquitin-specific protease usp15 promotes rig-imediated antiviral signaling by deubiquitylating trim25 the mitochondrial targeting chaperone 14-3-3ε regulates a rig-i translocon that mediates membrane association and innate antiviral immunity trim4 modulates type i interferon induction and cellular antiviral response by targeting rig-i for k63-linked ubiquitination mechanism of trim25 catalytic activation in the antiviral rig-i pathway influenza a virus ns1 targets the ubiquitin ligase trim25 to evade recognition by the host viral rna sensor rig-i species-specific inhibition of rig-i ubiquitination and ifn induction by the influenza a virus ns1 protein the human papillomavirus e6 oncoprotein targets usp15 and trim25 to suppress rig-i-mediated innate immune signaling molecular mechanism of influenza a ns1-mediated trim25 recognition and inhibition nlrp12 regulates anti-viral rig-i activation via interaction with trim25 reconstitution of the rig-i pathway reveals a signaling role of unanchored polyubiquitin chains in innate immunity emerging role of ubiquitination in antiviral rig-i signaling a hierarchical mechanism of rig-i ubiquitination provides sensitivity, robustness and synergy in antiviral immune responses stratified ubiquitination of rig-i creates robust immune response and induces selective gene expression ubiquitin-mediated modulation of the cytoplasmic viral rna sensor rig-i the e3 ubiquitin ligase trim40 attenuates antiviral immune responses by targeting mda5 and rig-i. cell rep regulation of rig-i activation by k63-linked polyubiquitination. front immunol the zincfinger protein zcchc3 binds rna and facilitates viral rna sensing and activation of the rig-i-like receptors west nile virus ns1 antagonizes interferon beta production by targeting rig-i and mda5 trim65-catalized ubiquitination is essential for mda5-mediated antiviral innate immunity structural basis for ubiquitinmediated antiviral signal activation by rig-i mavs self-association mediates antiviral innate immune signaling mavs forms functional prion-like aggregates to activate and propagate antiviral innate immune response structural basis for the prion-like mavs filaments in antiviral innate immunity solid-state nmr resonance assignments of the filament-forming card domain of the innate immunity signaling protein mavs how rig-i like receptors activate mavs visualizing the determinants of viral rna recognition by innate immune sensor rig-i defining the functional determinants for rna surveillance by rig-i a minimal rna ligand for potent rig-i activation in living mice atpasedriven oligomerization of rig-i on rna allows optimal activation of type-i interferon rig-i forms signaling-competent filaments in an atp-dependent, ubiquitin-independent manner molecular mechanism of signal perception and integration by the innate immune sensor retinoic acid-inducible gene-i (rig-i) rig-i uses an atpase-powered translocation-throttling mechanism for kinetic proofreading of rnas and oligomerization filament assemblies in foreign nucleic acid sensors ips-1, an adaptor triggering rig-i-and mda5-mediated type i interferon induction visa is an adapter protein required for virus-triggered ifn-β signaling identification and characterization of mavs, a mitochondrial antiviral signaling protein that activates nf-κb and irf3 a common polymorphism in the caspase recruitment domain of rig-i modifies the innate immune response of human dendritic cells defective interfering particles with covalently linked [+/-]rna induce interferon structure and dynamics of the second card of human rig-i provide mechanistic insights into regulation of rig-i activation pattern recognition receptors and inflammation mitochondria in innate immunity viral targeting of dead box protein 3 reveals its role in tbk1/ikkepsilon-mediated irf activation blockade of interferon beta, but not interferon alpha, signaling controls persistent viral infection the effects of dendritic cell hypersensitivity on persistent viral infection a role for neutrophils in viral respiratory disease. front immunol plasmacytoid dendritic cells: sensing nucleic acids in viral infection and autoimmune diseases an essential role for rig-i in toll-like receptor-stimulated phagocytosis the long noncoding rna neat1 exerts antihantaviral effects by acting as positive feedback for rig-i signaling differential viral induction of distinct interferonalpha genes by positive feedback through interferon regulatory factor-7 the interferon response circuit: induction and suppression by pathogenic viruses h5n1 influenza virus-induced mediators upregulate rig-i in uninfected cells by paracrine effects contributing to amplified cytokine cascades fundamental properties of the mammalian innate immune system revealed by multispecies comparison of type i interferon responses retinoic acidinducible gene-i is induced by interferon-γ and regulates the expression of interferon-γ stimulated gene 15 in mcf-7 cells expression of retinoic acid-inducible gene-i in vascular smooth muscle cells stimulated with interferon-γ ′ -triphosphate rna requires base-paired structures to activate antiviral signaling via rig-i unpaired 5' ppp-nucleotides, as found in arenavirus double-stranded rna panhandles, are not recognized by rig-i recognition of 5' triphosphate by rig-i helicase requires short blunt double-stranded rna as contained in panhandle of negative-strand virus short double-stranded rnas with an overhanging 5' ppp-nucleotide, as found in arenavirus genomes, act as rig-i decoys crystal structure of rig-i c-terminal domain bound to blunt-ended double-strand rna without 5' triphosphate antiviral immunity via rig-i-mediated recognition of rna bearing 5 ′ -diphosphates energetics of preferential binding of retinoic acid-inducible gene-i to double-stranded viral rnas with 5 ′ tri-/diphosphate over 5 ′ monophosphate rig-i selectively discriminates against 5'-monophosphate rna establishing the role of atp for the function of the rig-i innate immune sensor a mammalian pre-mrna 5' end capping quality control mechanism and an unexpected link of capping to pre-mrna processing rnase iii: genetics and function; structure and mechanism rnase l releases a small rna from hcv rna that refolds into a potent pamp activation of innate immunity by mitochondrial dsrna in mouse cells lacking p53 protein new insights into the role of rnase l in innate immunity structural basis of double-stranded rna recognition by the rig-i like receptor mda5 the rig-i-like receptor lgp2 recognizes the termini of doublestranded rna evolution of mda-5/rig-i-dependent innate immunity: independent evolution by domain grafting regulation of innate antiviral defenses through a shared repressor domain in rig-i and lgp2 activation of ifn-β expression by a viral mrna through rnase l and mda5 rig-i and other rna sensors in antiviral immunity rna polymerase iii detects cytosolic dna and induces type i interferons through the rig-i pathway rig-i-dependent sensing of poly(da:dt) through the induction of an rna polymerase iii-transcribed rna intermediate sequence-specific modifications enhance the broad-spectrum antiviral response activated by rig-i agonists rig-i activation by a designer short rna ligand protects human immune cells against dengue virus infection without causing cytotoxicity identification of a natural viral rna motif that optimizes sensing of viral rna by rig-i in vivo ligands of mda5 and rig-i in measles virus-infected cells the 3' untranslated regions of influenza genomic sequences are 5'ppp-independent ligands for rig-i rig-i detects kaposi's sarcoma-associated herpesvirus transcripts in a rna polymerase iii-independent manner innate immunity induced by composition-dependent rig-i recognition of hepatitis c virus rna nucleotide sequences and modifications that determine rig-i/rna binding and signaling activities self-recognition of an inducible host lncrna by rig-i feedback restricts innate immune response discrimination of self and non-self ribonucleic acids defective viral particles and viral disease processes defective interfering viruses further characterization of sendai virus di-rnas: a model for their generation structure and origin of a novel class of defective interfering particle of vesicular stomatitis virus activation of the beta interferon promoter by unnatural sendai virus infection requires rig-i and is inhibited by viral c proteins preference of rig-i for short viral rna molecules in infected cells revealed by nextgeneration sequencing differential recognition of viral rna by rig-i. virulence pact-and rig-i-dependent activation of type i interferon production by a defective interfering rna derived from measles virus vaccine inhibition of ongoing influenza a virus replication reveals different mechanisms of rig-i activation influenza a virus panhandle structure is directly involved in rig-i activation and interferon induction the structure of influenza virus defective interfering (di) rnas and their progenitor genes mda5 participates in the detection of paramyxovirus infection and is essential for the early activation of dendritic cells in response to sendai virus defective interfering particles a novel role for viral-defective interfering particles in enhancing dendritic cell maturation defective interfering influenza virus rnas: time to reevaluate their clinical potential as broad-spectrum antivirals? biology of immune responses to vaccines in elderly persons vaccination in the elderly: an immunological perspective vaccination in the elderly vaccination in the elderly: the challenge of immune changes with aging aging impairs both primary and secondary rig-i signaling for interferon induction in human monocytes within-season influenza vaccine waning suggests potential net benefits to delayed vaccination in older adults in the united states viral persistence: mechanisms and consequences defective (interfering) viral genomes re-explored: impact on antiviral immunity and virus persistence multiple-hit inhibition of infection by defective interfering particles a novel phosphoserine motif in the lcmv matrix protein z regulates the release of infectious virus and defective interfering particles hostdriven phosphorylation appears to regulate the budding activity of the lassa virus matrix protein rigorous detection: exposing virus through rna sensing gain-of-function mutations in ifih1 cause a spectrum of human disease phenotypes associated with upregulated type i interferon signaling breaching self-tolerance to alu duplex rna underlies mda5-mediated inflammation aicardi-goutières syndrome is caused by ifih1 mutations autoimmune disorders associated with gain of function of the intracellular sensor mda5 a conserved histidine in the rna sensor rig-i controls immune tolerance to n1-2 ′ o-methylated self rna highresolution hdx-ms reveals distinct mechanisms of rna recognition and activation by rig-i and mda5 hdx-ms reveals dysregulated checkpoints that compromise discrimination against self rna during rig-i mediated autoimmunity atp hydrolysis by the viral rna sensor rig-i prevents unintentional recognition of self-rna a brilliant disguise for self rna: 5'-end and internal modifications of primary transcripts suppress elements of innate immunity rna modifications go viral rnas containing modified nucleotides fail to trigger rig-i conformational changes for innate immune signaling links between recognition and degradation of cytoplasmic viral rna in innate immune response lgp2 is a positive regulator of rig-i-and mda5-mediated antiviral responses the rig-i atpase domain structure reveals insights into atp-dependent antiviral signalling kinetic discrimination of self/non-self rna by the atpase activity of rig-i and mda5 cryo-em structures of mda5-dsrna filaments at different stages of atp hydrolysis circular rnas: a novel type of non-coding rna and their potential implications in antiviral immunity sensing self and foreign circular rnas by intron identity the skiv2l rna exosome limits activation of the rig-i-like receptors induction of selenoprotein p mrna during hepatitis c virus infection inhibits rig-i-mediated antiviral immunity human long non-coding rna lncatv promotes viral replication by restricting rig-i-mediated innate immunity immunostimulatory effects triggered by self-assembled microspheres with tandem repeats of polymerized rna strands line1 contributes to autoimmunity through both rig-i-and mda5-mediated rna sensing pathways viral unmasking of cellular 5s rrna pseudogene transcripts induces rig-i-mediated immunity mitochondrial dynamics and viral infections: a close nexus mitochondrial double-stranded rna triggers antiviral signalling in humans apoptotic endothelial cells release small extracellular vesicles loaded with immunostimulatory viral-like interferons and viruses: an evolutionary arms race of molecular interactions viral evasion strategies in type i ifn signaling -a summary of recent developments viral evasion of intracellular dna and rna sensing ten strategies of interferon evasion by viruses segmented negative-strand rna viruses and rig-i: divide (your genome) and rule influenza a: understanding the viral life cycle nuclear-resident rig-i senses viral replication inducing antiviral immunity processing of genome 5 ′ termini as a strategy of negative-strand rna viruses to avoid rig-i-dependent interferon induction the microrna mir-485 targets host and influenza virus transcripts to regulate antiviral immunity and restrict viral replication a single amino acid substitution within the paramyxovirus sendai virus nucleoprotein is a critical determinant for production of interferon-betainducing copyback-type defective interfering genomes mini viral rnas act as innate immune agonists during influenza virus infection rig-i and mda-5 detection of viral rna-dependent rna polymerase activity restricts positive-strand rna virus replication visualization of arenavirus rna species in individual cells by singlemolecule fluorescence in situ hybridization (smfish) suggests a model of cyclical infection and clearance during persistence structure of the lassa virus nucleoprotein reveals a dsrna-specific 3' to 5' exonuclease activity essential for immune suppression in vitro and in vivo characterizations of pichinde viral nucleoprotein exoribonuclease functions structural basis and functional analysis of the sars coronavirus nsp14-nsp10 complex characterization of a bafinivirus exoribonuclease activity regulation of rig-i-like receptor signaling by host and viral proteins human respiratory syncytial virus non-structural protein ns2 antagonizes the activation of beta interferon transcription by interacting with rig-i the z proteins of pathogenic but not non-pathogenic arenaviruses inhibit rig-i-like receptor-dependent interferon production a viral deamidase targets the helicase domain of rig-i to block rna-induced activation interactions between the influenza a virus rna polymerase components and retinoic acid-inducible gene i exonuclease domain of the lassa virus nucleoprotein is critical to avoid rig-i signaling and to inhibit the innate immune response arenaviral nucleoproteins suppress pact-induced augmentation of rig-i function to inhibit type i interferon production rotavirus non-structural protein 1 antagonizes innate immune response by interacting with retinoic acid inducible gene i paramyxovirus v proteins disrupt the fold of the rna sensor mda5 to inhibit antiviral signaling paramyxovirus v proteins interact with the rig-i/trim25 regulatory complex and inhibit rig-i signaling herpes simplex virus 1 tegument protein us11 downmodulates the rlr signaling pathway via direct interaction with rig-i and mda-5 host and viral modulation of rig-i-mediated antiviral immunity. front immunol usp3 inhibits type i interferon signaling by deubiquitinating rig-i-like receptors usp14 promotes k63-linked rig-i deubiquitination and suppresses antiviral immune responses mechanism of inhibiting type i interferon induction by hepatitis b virus x protein porcine deltacoronavirus nucleocapsid protein suppressed ifn-β production by interfering porcine rig-i dsrna-binding and k63-linked polyubiquitination ubiquitin-like modifier fat10 attenuates rig-i mediated antiviral signaling by segregating activated rig-i from its signaling platform robust lys63-linked ubiquitination of rig-i promotes cytokine eruption in early influenza b virus infection antiviral activity of human oasl protein is mediated by enhancing signaling of the rig-i rna sensor negative regulation of the rig-i signaling by the ubiquitin ligase rnf125 induction of siglec-g by rna viruses inhibits the innate immune response by promoting rig-i degradation cytoplasmic stat4 promotes antiviral type i ifn production by blocking chipmediated degradation of rig-i kai lrrc25 inhibits type i ifn signaling by targeting isg15-associated rig-i for autophagic degradation lrrc59 modulates type i interferon signaling by restraining the sqstm1/p62-mediated autophagic degradation of pattern recognition receptor ddx58/rig-i mitophagy enhances oncolytic measles virus replication by mitigating ddx58/rig-ilike receptor signaling rig-i is cleaved during picornavirus infection foot-andmouth disease virus viroporin 2b antagonizes rig-i-mediated antiviral effects by inhibition of its protein expression mda-5 is cleaved in poliovirus-infected cells rnf122 suppresses antiviral type i interferon production by targeting rig-i cards to mediate rig-i degradation mll5 suppresses antiviral innate immune response by facilitating stub1-mediated rig-i degradation the levels of retinoic acid-inducible gene i are regulated by heat shock protein 90-alpha pact, a double-stranded rna binding protein acts as a positive regulator for type i interferon gene induced by newcastle disease virus the double-stranded rna-binding protein pact functions as a cellular activator of rig-i to facilitate innate antiviral response tbk1-associated protein in endolysosomes (tape)/cc2d1a is a key regulator linking rig-i-like receptors to antiviral immunity cell type-specific subcellular localization of phospho-tbk1 in response to cytoplasmic viral dna zika virus disrupts phospho-tbk1 localization and mitosis in human neuroepithelial stem cells and radial glia zebrafish mvp recruits and degrades tbk1 to suppress ifn production the role of optineurin in antiviral type i interferon production structural and functional analysis reveals that human oasl binds dsrna to enhance rig-i signaling reversal of the interferon-sensitive phenotype of a vaccinia virus lacking e3l by expression of the reovirus s4 gene human trbp and pact directly interact with each other and associate with dicer to facilitate the production of small interfering rna approaching the rna ligand for rig-i? viral inhibitions of pact-induced rig-i activation hemorrhagic fever-causing arenaviruses: lethal pathogens and potent immune suppressors porcine deltacoronavirus nucleocapsid protein antagonizes ifn-β production by impairing dsrna and pact binding to rig-i raver1 is a coactivator of mda5-mediated cellular antiviral response human hemoglobin subunit beta functions as a pleiotropic regulator of the rig-i/mda5-mediated antiviral innate immune responses pact, a protein activator of the interferoninduced protein kinase, pkr the role of pact in mediating gene induction, pkr activation, and apoptosis in response to diverse stimuli dhx36 enhances rig-i signaling by facilitating pkrmediated antiviral stress granule formation regulation of antiviral innate immune signaling by stress-induced rna granules critical role of an antiviral stress granule containing rig-i and pkr in viral detection and innate immunity subcellular localizations of rig-i, trim25, and mavs complexes pkr transduces mda5-dependent signals for type i ifn induction interferon-inducible oligoadenylate synthetase-like protein acts as an antiviral effector against classical swine fever virus via the mda5-mediated type i interferon-signaling pathway inhibition of rig-i and mda5-dependent antiviral response by gc1qr at mitochondria tarbp2 negatively regulates ifn-β production and innate antiviral response by targeting mavs lactate is a natural suppressor of rlr signaling by targeting mavs nlrc5 negatively regulates the nf-kappab and type i interferon signaling pathways dhx15 is a coreceptor for rlr signaling that promotes antiviral defense against rna virus infection arflike protein 16 (arl16) inhibits rig-i by binding with its c-terminal domain in a gtp-dependent manner negative regulation of melanoma differentiation-associated gene 5 (mda5)-dependent antiviral innate immune responses by arf-like protein 5b green tea catechin, epigallocatechin gallate, suppresses signaling by the dsrna innate immune receptor rig-i crystal structure of a dead box protein from the hyperthermophile methanococcus jannaschii unlocking the dead-box: a key to cryptococcal virulence? sensors of infection: viral nucleic acid prrs in fish association of rig-i with innate immunity of ducks to influenza pathogen recognition receptors in channel catfish: ii. identification, phylogeny and expression of retinoic acid-inducible gene i (rig-i)-like receptors (rlrs) goose rig-i functions in innate immunity against newcastle disease virus infections innate immunity of finfish: primordial conservation and function of viral rna sensors in teleosts comparative study on pattern recognition receptors in non-teleost ray-finned fishes and their evolutionary significance in primitive vertebrates genomic analysis and adaptive evolution of the rig-i-like and nod-like receptors in reptiles nod-lrr proteins: role in host-microbial interactions and inflammatory disease a phylogenetic and functional overview of inflammatory caspases and caspase-1-related card-only proteins nacht-lrr proteins (nlrs) in bacterial infection and immunity molecular evolution of glutamate receptors: a primitive signaling mechanism that existed before plants and animals diverged the origin of polynucleotide phosphorylase domains rig-i-like receptors evolved adaptively in mammals, with parallel evolution at lgp2 and rig-i identification and characterization of an atypical rig-i encoded by planarian dugesia japonica and its essential role in the immune response caenorhabditis elegans rig-i homolog mediates antiviral rna interference downstream of dicer-dependent biogenesis of viral small interfering rnas nucleic acid-induced antiviral immunity in invertebrates: an evolutionary perspective non-self ' mutation: double-stranded rna elicits antiviral pathogenic response in a drosophila model of expanded cag repeat neurodegenerative diseases plant pattern-recognition receptors rna silencing and its suppression: novel insights from in planta analyses the immunity regulator bak1 contributes to resistance against diverse rna viruses svcv infection triggers fish ifn response through rlr signaling pathway alternative splicing transcripts of zebrafish lgp2 gene differentially contribute to ifn antiviral response inducible microrna-3570 feedback inhibits the rig-i-dependent innate immune response to rhabdovirus in teleost fish by targeting mavs/ips-1 microrna-210 participates in regulating rig-i signaling pathway via targeting duba in miiuy croaker after poly(i:c) stimulation mda5 and lgp2 acts as a key regulator though activating nf-κb and irf3 in rlrs signaling of mandarinfish higher antiviral response of rig-i through enhancing rig-i/mavs-mediated signaling by its long insertion variant in zebrafish roles of rig-i n-terminal tandem card and splice variant in trim25-mediated antiviral signal transduction activation of duck rig-i by trim25 is independent of anchored ubiquitin expression of immune genes rig-i and mx in mallard ducks infected with low pathogenic avian influenza (lpai): a dataset duck rig-i restricts duck enteritis virus infection in vivo cellular and molecular study on duck spleen infected by duck tembusu virus identification and expression profiling analysis of goose melanoma differentiation associated gene 5 (mda5) gene goose mavs functions in rig-i-mediated ifn-β signaling activation characterization of chicken mda5 activity: regulation of ifnβ in the absence of rig-i functionality chicken cells sense influenza a virus infection through mda5 and cardif signaling involving lgp2 chicken mda5 senses short double-stranded rna with implications for antiviral response against avian influenza viruses in chicken cloning, expression and bioinformatics analysis of a putative pigeon melanoma differentiation-associated gene 5 loss of rig-i leads to a functional replacement with mda5 in the chinese tree shrew trim25 identification in the chinese goose: gene structure, tissue expression profiles, and antiviral immune responses in vivo and in vitro expression and functional characterization of the rig-i-like receptors mda5 and lgp2 in rainbow trout (oncorhynchus mykiss) influenza virus adaptation pb2-627k modulates nucleocapsid inhibition by the pathogen sensor rig-i multiple functions of ddx3 rna helicase in gene regulation, tumorigenesis, and viral infection rna helicase ddx3: at the crossroad of viral replication and antiviral immunity hiv-1 blocks the signaling adaptor mavs to evade antiviral host defense after sensing of abortive hiv-1 rna by the host helicase ddx3 the rna helicase ddx3x is an essential mediator of innate antimicrobial immunity dhx9 pairs with ips-1 to sense double-stranded rna in myeloid dendritic cells dhx15 senses doublestranded rna in myeloid dendritic cells ddx1, ddx21, and dhx36 helicases form a complex with the adaptor molecule trif to sense dsrna in dendritic cells the interaction between the helicase dhx33 and ips-1 as a novel pathway to sense double-stranded rna and rna viruses in myeloid dendritic cells ddx19 inhibits type i interferon production by disrupting tbk1-ikkε-irf3 interactions and promoting tbk1 and ikkε degradation ddx3 suppresses type i interferons and favors viral replication during arenavirus infection codegradation of interferon signaling factor ddx3 by pb1-f2 as a basis for high virulence of 1918 pandemic influenza the deah-box rna helicase dhx15 activates nf-κb and mapk signaling downstream of mavs during antiviral responses ddx60, a dexd/h box helicase, is a novel antiviral factor promoting rig-i-like receptor-mediated signaling ddx60 is involved in rig-i-dependent and independent antiviral responses, and its function is attenuated by virus-induced egfr activation dhx29 functions as an rna cosensor for mda5-mediated emcv-specific antiviral immunity activation of rig-i signaling to increase the pro-inflammatory phenotype of a tumor immunotherapeutic effects of intratumoral nanoplexed poly i:c rig-i-like receptors as novel targets for panantivirals and vaccine adjuvants against emerging and re-emerging viral infections the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2019 brisse and ly. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-131093-osukknqr authors: suzen, neslihan; mirkes, evgeny m.; gorban, alexander n. title: informational space of meaning for scientific texts date: 2020-04-28 journal: nan doi: nan sha: doc_id: 131093 cord_uid: osukknqr in natural language processing, automatic extracting the meaning of texts constitutes an important problem. our focus is the computational analysis of meaning of short scientific texts (abstracts or brief reports). in this paper, a vector space model is developed for quantifying the meaning of words and texts. we introduce the meaning space, in which the meaning of a word is represented by a vector of relative information gain (rig) about the subject categories that the text belongs to, which can be obtained from observing the word in the text. this new approach is applied to construct the meaning space based on leicester scientific corpus (lsc) and leicester scientific dictionary-core (lscdc). the lsc is a scientific corpus of 1,673,350 abstracts and the lscdc is a scientific dictionary which words are extracted from the lsc. each text in the lsc belongs to at least one of 252 subject categories of web of science (wos). these categories are used in construction of vectors of information gains. the meaning space is described and statistically analysed for the lsc with the lscdc. the usefulness of the proposed representation model is evaluated through top-ranked words in each category. the most informative n words are ordered. we demonstrated that rig-based word ranking is much more useful than ranking based on raw word frequency in determining the science-specific meaning and importance of a word. the proposed model based on rig is shown to have ability to stand out topic-specific words in categories. the most informative words are presented for 252 categories. the new scientific dictionary and the 103,998 x 252 word-category rig matrix are available online. analysis of the meaning space provides us with a tool to further explore quantifying the meaning of a text using more complex and context-dependent meaning models that use co-occurrence of words and their combinations. automatic analysis of text meaning is one of the main problems in natural language processing (nlp). this work is focused on the computational analysis of the meaning of short scientific texts (abstracts or brief reports). the starting point is a combination of a simple bag of word (bow) model with the holistic approach to the text meaning: the text is considered as a collection of words, the meaning of the text is hidden in a situation of use, which is evaluated as a whole. a space of meaning for words is created from the analysis of situations of their use and then, after detailed analysis of this space (including dimensionality reduction and clustering) we will return to the texts and introduce more complex models including words co-occurrence analysis, combination of word's meaning etc. first of all, we have to consider the "meaning of meaning". this is an extremely deeply discussed topic, since antiquity till modern time (see, for example, [1, 2, 3, 4] ), but the consensus is still on the way. we start from the wittgenstein formulation: "meaning is use" or, in more detail, "for a large class of cases though not for all in which we employ the word 'meaning' it can be defined thus: the meaning of a word is its use in the language"[5, §43]. this idea was widely discussed. this paper aims to propose an approach to computational analysis of meaning for a large family of texts. the texts we work with (abstracts or brief reports) have well defined dominant communicative function: 2 there is a representation of a situation on the sender's 'blackboard of the consciousness' (a representation 1 of a situation 1). a text related to this situation is generated by the sender (translation 1). this text is transmitted to the receiver and transformed by him into a representation of a situation (representation 2 of a situation 2). the situation can be the situation in a real world, the imaginary situation in a possible world, an impossible situation in an impossible world, a chimeric situation combined from several possible or imaginary situation, and so on. we do not study the relations of representation to reality, but only consider the chain: representation 1 → text → representation 2. this is the representative function. an elementary basic scheme of the correspondent act of communication is presented in figure 1 .1. in this scheme, we see two representations of the situation on the blackboard of consciousness: the sender's representation of the situation and the receiver's representation of the situation. moreover, the represented situations can be different. in fact, they are always different, and special tools are invented and used to make them as close as possible when necessary. the situations are not compulsory real. they can be real, possibly real or imaginary, and even impossible. it is necessary to stress that the sender's and the receiver's representations never coincide and: • do not represent any situation 'in detail' and, therefore, can represent parts (or projections, let us recall the plato's cave allegory) of many different situations at the same time; • can include internal contradictions and, therefore, can represent nothing possible in reality; • can partially represent different situations, that is, can be 'chimeric' combinations of different possible real or imaginary situations. there are two 'translation' operations in the scheme figure 1 .1: (i) from the sender's representation of the situation to the text of the message and (ii) from the text of 3 informational space of meaning for scientific texts the message to the the receiver's representation. both these operations depend on much wider context of the communication including experience of the sender and receiver. of course, the standard scientific communication assumes that there may be many receivers and the sender can be not a single person. this one-to-many or even many-to-many communication adds more situations and representations and may also add some less trivial multi-agent structures with additional communication channels. we consider using the language to transmit information about the represented situations (figure 1 .1) and neglect many other uses of the language, from military orders to psychological manipulations. the scheme of the act of communication (figure 1 .1) includes just very basic elements and can be elaborated in much more detail. here we should refer to the classical works of g.p. shchedrovitsky [6, 7] and j. habermas [8, 9] . for our purposes in this work, the basic scheme (figure 1.1) is sufficient. according to shchedrovitsky [6] , at the level of 'simple communication' there is no 'meaning' different from the processes of understanding themselves, which correlate and connect the elements of the text message with each other and with the elements of the situation being restored. meaning, for our analysis, is hidden in the relationship between the representation of situations on the 'blackboard of the consciousness' and the texts of the messages. that is, a formal analysis of meaning requires the formalisation of translation operations presented in the scheme of a communication act (figure 1.1) . moreover, we can state that we understand the meaning of meaning if and only if we can produce such a translation. this translation is context-dependent, the unique experience of the sender and the receiver is involved in this context, so the task of "reproducing the translation" is not fully feasible. moreover, understanding can be represented as a reflexive game [10] with different levels (the sender prepares a message taking into account the experience of the receiver, his goals and tools, and guesses that the receiver takes into account the experience of the sender, his goals and tools, and... analogously, the receiver tries to understand the message taking into account..., etc.) the relation between the text and the representation of the situation cannot be considered as a bijection (both for sender's and receiver's representation). it is many-to-many correspondence: each text corresponds to many situations and each situation can have many representing texts. moreover, the further consistent formalisation requires the notion of fuzzy many-to-many correspondence elaborated for relational databases [11] . according to mel'uk [12, 13, 14, 15] , the natural language is "the meaning to text and text to meaning transformer". he accepted a very strong hypothesis that we are able to describe meaning in a special semantic language. we prefer to be more flexible at this point and characterise a situation "behind the text" by a set of attributes, the method of this characterisation can be changed and does not give a unique and exhaustive presentation of it. despite the multiplicity of possible translations, creating of a plausible translation (one of many possible versions) and description of the cloud of such versions of translation can be challenging. this problem resembles the translation problem for natural languages. now, after impressive progress of machine translation, it seems to be a very attractive idea to apply the modern machine learning tools and encoder-decoder approach [16] to analysis and simulation the translation operations representation 1 → text → representation 2 (figure 1.1) . huge digitized collections of texts exist and are available online. on the contrary, unfortunately, there is no generally accepted common tools for working directly with representations of situations. various philosophical and logical aspects of this problem were discussed previously by many authors (see, for example, the book 'representation and reality ' [17] ). we do not have a universal toolbox for work with all representations of situations and cannot propose a general solution to this problem. such a solution, perhaps, is impossible in a finite closed form despite many efforts over decades. our goal is more modest. we will provide computational analysis of relations between texts of messages and representations of situations for a large collection of brief scientific texts. to do this, these representations must be standardised, at least in part, and expressed in the form of diagrams, specially organized texts or other means. the simplest approach is to replace the situation representations with the values of some attributes. this approach is not only the simplest, but also quite universal. many forms of more specific descriptions of situations can be transformed into vectors of attributes. the choice of attributes can be very broad. a classical collection of examples is provided by various version of sentiment analysis. we aim to provide another basic example specific to scientific texts: a list of scientific subject categories that the text belongs to. the list of 252 possible categories is generally accepted and standardised by wos. of course, the variety of possible extensions and modifications of the set of attributes characterising the situation is virtually infinite. initially, in the act of communication, the situation is not represented by a universally conventional set of attributes. the introduction of attributes is an additional operation external to the communication and is not included in the scheme of simple communication (figure 1.1) . moreover, an additional operation must be performed for the selected set of attributes: evaluating their values. this operation can be done either on the sender's side ( figure 1 .2), the receiver's side ( figure 1.3) , or by combinations of these approaches. for example, categorisation of a brief scientific texts is a result of combined efforts: the authors select the categories by their choice of the journal, of the keywords, or by the pointing the categories directly, then the editors can have their own choice, then wos can finalise the list of subject categories for this text. for most information services, the choice of subject categories is the result of an understanding of the text by many agents and conflicts of understanding are possible. even on a famous and very 'liberal' preprint server, arxiv, moderators can sometimes change the category selected by the authors. for example, an author may decide that his paper belong to the category 'condensed matter', whereas the moderator may look through the paper and understand that the main category is not 'condensed matter' research but rather 'nonlinear science' (this was a real life example). this simple example is important because it demonstrates that the content of the text may differ from its meaning: the text contained an explicit reference to 'condensed matter', but this content was questioned by the moderator, since in his understanding the research refers mainly to nonlinear science, and not to condensed matter. there are important differences between the concepts of 'meaning' and 'content' [6] , which are often confused (just as understanding the situation behind the text is often confused with recognising the content of the text). in the general case, agents who are looking for the meaning of the text can be both humans or computer systems. the latter understand the text in the sense that they define the attributes of the situation behind the text. in our analysis below, the starting point is the combination of the text with the list of the subject categories the text belongs to (1, 673 ,350 abstracts and 252 categories). the core idea of this approach goes back to the lexical approach of sir francis galton. he selected the personality-descriptive terms and stated the problem of their interrelations for real persons. this work was continued by thurstone [18] . he selected sixty adjectives (attributes of a person that are in common use). the respondents (1300 persons) were asked to imagine a person they knew well and to select the adjectives that can best describe this person. that is, a person was described by a 60-dimensional boolean vector. the coordinates correspond to the attributes, the value is 1, if the attribute was selected to characterise the person, and 0 otherwise. factor analysis gave five factors. after many years of development and discussions, the modern five-factor personality model became one of the common tools in psychodiagnosis [19, 20] . in psycholinguistics, osgood with co-workers [21] used a similar approach for creation of the 3d space of meaning by extraction of three 'coordinates of meaning' from the evaluation of the 'affective meaning' of words (objects) by people. these three coordinates are three extracted factors: evaluation, potency, and activity. of course, the researches started from many different scales and these three were extracted by factor analysis. galton, thurstone, osgood and their followers asked respondents to evaluate a single object or person. nevertheless, we can guess that these evaluations were related to some situations with this single object or person, not just to an isolated abstract object. the people evaluated not the abstract 'terms' but the psychologically meaningful situations behind these terms. these situations were the sources of the 'affective meaning' or the personality evaluations. for example, if we evaluate a person as accurate, reliable, and friendly then we have in mind some situations where these properties were demonstrated. the same, if we evaluate a 'dog' as strong, good, and active (or, say, weak, bad and active), we have in mind a dominant situation which we associate with a dog. the 'affective meaning' or psychological properties do not seem reasonable tools for description of the situations behind scientific texts. in our world of abstracts and brief scientific texts, there is another, scientifically specific description of the situation of use -the categories of the text. there are 252 web of science (wos) categories for the leicester scientific corpus (lsc), to which the text could belong (see table b .1). these categories can intersect: a text can belong to several categories. we will use these 252 binary attributes (the text belongs to a given category, or does not belong to it) as a basic description of the situation. the categories evaluate the situation (the research area) related to the text as a whole, not as a results of the combination of words' meaning. in this holistic approach, we define the general meaning of a word in short scientific texts as the information that the use of this word in texts carries about the categories to which 7 informational space of meaning for scientific texts these texts belong. more specifically, that is the relative information gain (rig) about the subject categories that the text belongs to, which can be obtained from observing the word in the text. this rig is defined for each word and each category. thus, a meaning of a word is represented by a 252-dimensional vector of rigs. we create and study this space of meanings. (1) we intend to analyse the meaning of scientific texts. (2) we considered the specific world of the texts -the abstracts of research papers. (3) we narrowed the whole word of abstracts to a sample: 1,673,350 texts from the leicester scientific corpus (lsc) [22] . (4) we characterize the research situations behind the text by 252 binary attributes -the scientific wos categories. thus, to follow this way, we need a triad: dictionary, texts, and multidimensional evaluation of the situation of use presented by the categories. we prepared the first two elements in the previous work and the results are available online [23, 24, 25] . now, we start to create the space of meaning. in our case study, we employed very simple attributes for description of the text usage situation, the research subject categories of the text. this list of attributes can be modified and extended. the level of detail of the meaning space can vary greatly within the framework of the proposed approach. let us take a quick look at some relevant ideas about quantifying the meaning of words. quantifying the meanings of words in a metric space might be used to measure the meanings of texts in the same metric as a bow. a key issue in understanding the meaning of texts is to use a precise metric based on words' meanings. in classical psycholinguistic studies it is common to allocate words in a metric space based on their semantic connotations [26, 27] . semantic space model is a representation technique where each word is assigned to a point in high dimensional vector space. vector space model (vsm) is one of the most attractive models for researchers since it makes semantics computable [28] . osgood hypothesised 3-dimensional semantic space to quantify connotative meanings in his theory of semantic differential concerning psychological and behavioural aspects [21, 29, 30 ]. the semantic space in his work was built by, in his words, 'three orthogonal bipolar dimensions': evaluation (e), potency (p) and activity (a) where each word is uniquely located on. following this method of semantic differential, many studies have been attempted by both psychologists and linguists to identify new dimensions of semantic space and to measure the meaning [21, 27, 31, 32] . the structures of semantic spaces are constructed differently by various researchers. from the perspective of distributional linguistics, a semantic space model is a representation technique for contextual similarity of words by their co-occurrence counts. distributional hypothesis was introduced by harris [33] and distributional semantic models (dsm) were then proposed to represent word semantic by distributional vectors [28, 34, 35, 36] . this idea claims that words' similarity can be characterised by their distribution of contexts [37, 38] . the model offers that each word is represented by distribution of its contexts and the distribution of contexts 8 informational space of meaning for scientific texts can be learnt from the co-occurance. the axes in the space are determined by local word co-occurrences and the similarity of a word is measured by its position found by counting co-occurrences to other words in this semantic space [39] . this means that a word's distributional context is represented by a vector of co-occurrences with other context words in a window, where a window can be a certain number of words or lemmas (e.g. words, phrases, sentence, paragraph or document). researchers in cognitive studies and information retrieval noted that usage of raw co-occurrence counts is problematic as semantic similarity will have frequency bias [32] . it is proposed that degrees of similarity between word occurances can be assigned. different approaches are used to avoid this problem by weighining of elements of the vector. latent semantic analysis (lsa) is one of vector space models in nlp, in particular dsm, for estimating and representing the meaning of word based on statistical computations [40, 41] . in lsa, word senses (or meanings) are approximated in high dimensional space by its effect on the meaning of contexts in which it occurs [42] . relationship between texts based on their words and relationship between words based on their appearances in texts are analysed simultaneously in order to extract relations of words in terms of their contexts. lsa has been used for an adequate theory of word meaning by researchers from a wide range of research areas including psychology, philosophy, linguistics, information retrieval and cognitive science [43, 44, 45] . in cognitive science, the focus is to model human memory by activating the meaning potentials by other words in the context under the assumption that cognitive components of meaning of word are linked in a semantic-based network and changes dynamically [46] . it is assumed that human knowledge acquisition actually follows the same process that lsa does: checking events in their internal and external environments and deriving the knowledge from a high dimensional semantic space by a procedure like dimension reduction [47, 48] . here, the semantic space is used as a basis for all cognitive processing. although lsa supplies a usefull simulation of human cognitive processes, it is argued that lsa knowledge base does not provide a complete modelling of cognition [47] . there are limitations in modification of context and updating the model of semantic dimensions -in this knowledge base -which are characteristic of analytic thinking and dynamic structure of the human cognitive processes [49] . even if this problem is solved, there are other fundamental semantic problems for lsa such as polysemous words. in lsa, when each word is represented as a single context-free vector in the semantic space, different meanings or senses of a word is not taken into account [40] . this problem matches the task of characterisation of word meaning by its dictionary senses in word sense disambiguation (wsd). wsd is defined as a task to determine the word sense (meaning) by the use of the word in a context in nlp and machine learning. in traditional word sense studies, meaning of a word is characterised by mutually disjoint senses covered in dictionaries as the best fit to the its dictionary senses [50] . by both linguistics and psychologists, it has been argued that clear distinctions of senses can be difficult in certain contexts due to fluctuations of meaning in context [44, 46, 51] , especially for polysemous words. hanks (lexigropher) pointed this problem in his paper where he questioned 'do word meaning exist?' [46] as: informational space of meaning for scientific texts "...words have meaning potentials, rather than just meaning. the meaning potential of each word is made up of a number of components, which may be activated cognitively by other words in the context in which it is used. these cognitive components are linked in a network which provides the whole semantic base of the language, with enormous dynamic potential for saying new things and relating the unknown to the known." the problem of 'fluctuation of meaning in context' is also important in theories of mental representation of word senses in psychology. this was very well discussed by kintsch [40, 47, 52, 53] who stressed the complexity of representation of polygamous words into a single vector in the semantic space in lsa. he questioned 'how is the meaning of words represented in the mind?' and discussed the problem in the aspects of 'mental lexicon' and 'generative lexicon' approaches to the representation of meaning [51] . he came up with the result that both mental lexicon and generative lexicon approaches have limitations in representation of the meanings when word meanings are constructed by their explicit definitions due to multiple senses of words and the flexibility of word meanings. he then discussed the implicit way to define word meaning: relations of the word to other words in the context. according to his research, lsa allows us to modify word meaning by situating the meaning as a vector in high-dimensional semantic space. in this case, the full meaning of the word is not defined, but it is explained in a relational system by only its semantic relationships with other words. he argued that standard composition rule for vectors in lsa does not distinguish the different meanings of a word; therefore, word meanings should be modified according to the different context -where it appears in -by context-sensitive composition algorithms. polysemy is one of the characteristics of words in all natural languages. psycholinguistic studies approach this phenomenon to answer questions of how to represent multiple senses in mental lexicon and how to activate senses during language comprehension [45] . the mental lexicon here can be considered as a mental repertoire containing the list of meanings or senses in the mind. linguistics proposed several approaches for sense representation in mental lexicon, basically classified as seperate sense representation and single core representation. even though some polysemy studies argue the discreteness of sense storage in mental lexicon [44, 54, 55] , the majority of studies suggests that polysemous senses can overlap in their mental representations [50, 56, 57, 58, 59] . moreover, polysemy of words is one of major focuses in distributional semantics and it is yet to be studied [60, 61, 62] . some researches in distributional semantics have made modelling the differences of meanings in two occurrences of a word in different contexts possible by developing specialized models for word meaning [63, 64, 65, 66] . such methods do not approach to word meaning by considering disjoint senses. alternative models were purposed in which word meaning is not just extracted by pre-defined senses, but from the links between words and their window-based context words. to extract 'contexeualised meaning' of a word or a set of words, co-occurance vectors are constructed and vector operations are used [64, 65, 67, 68] . a probabilistic method that word meaning is modelled as a probability distribution over latent dimensions (senses) was applied by [65, 67] . contexualized meaning was build as a change in original sense distribution. cruys, 10 informational space of meaning for scientific texts poibeau and korhonen then purposed a model in which latent space is used to identify important dimensions for a context and adapt to vector of words constructed by the dependency relations with window-based context words [69]. in academic disciplines, the notion of meaning of a word was analysed in many works ranging from psychology to linguistics, philosophy to pedagogy and computer science [70, 71] . technical innovations in computerised methods and extensive psycholinguistic and neurolinguistic experiments have made investigating word meanings in different perspectives and linking between the language and cognition, and the language in people's mind possible. there is no unique way to represent meanings that can be used in all theories of lexical semantics from different perspectives. semantics studies require different semantic representations on the formalism for meaning of word [72] . according to kintsch, philosophers work with meaning of concepts instead of words, psychologists mostly study concept formation than vocabulary acquisition and linguistics work on meaning of word [47] . but, at this point precise representing and approximating the meaning of concept or specially a text such as sentences, passages or documents are still active problems in nlp and all other disciplines concerning with 'meaning'. in this research, we specifically focus on meanings in scientific texts. we concern with how meaning can be extracted by analysing the large scientific corpus. our fundamental assumption is that the meaning of a text can be extracted from the occurrence of its words in texts across the scientific categories. we hypothesize that there is a great connection between the meaning in a text and the vocabulary used in the text; however, we cannot say that each word has the same importance in all research disciplines. in fact, words have scientifically specific meaning in texts based on differences of use in subject categories and these meanings can be estimated from their occurrences in texts within categories. difference in word meanings for categories correlates with the difference in distribution of words across categories. as they are scientific texts, we consider that occurrence of these words in texts of categories can be used in characterisation of word meaning for science. our approach to quantifying the meaning of a word differs from measuring its meaning on the basis of human sensations and feelings, as in psycholinguistic studies. although measuring the meaning of a word in context by characterization through its dictionary meanings has many important implications in computational linguistics and psycholinguistic research, we do not focus here on dictionary meanings. rather, we create a model for word representation that allows us to extract the meaning of a word through its importance in various scientific fields without distinguishing its dictionary meanings. we approach the meaning of a word through the predictive power of a corpus analytical procedure under the assumption that the meaning of a word is determined by its use in scientific disciplines. this actually matches the statistical semantics hypothesis that 'statistical patterns of human word usage can be utilised to figure out what people mean' [28] . we can also reword this as 'statistical patterns of word usage in scientific fields can be used to figure out what a text means '. 11 informational space of meaning for scientific texts in these relations, the meaning of a word is defined as a vector of rigs from the word to a category. given such information, meaning can be defined for each word and then for research text [23] . a natural way to formalise this is to represent words as vectors and texts as sets of vectors in a specially constructed space. differences in the distributions of vectors reflect differences in meaning of texts. this technique allowed us to represent each word by a distribution of numerical values over categories and meaning in text through a vector space model, that is, quantifying of meaning. in many semantic studies, the vector space is obtained by co-occurrence of words as discussed before. there are currently two broad vsms based on co-occurrence: word-word and word-document where vectors are (normalised) frequency counts and dimensions are contexts (words or documents) [73] . vectors are called context vectors in this case, and words are represented by the context vectors. in distributional hypothesis, these vectors are used to compute vector similarity. however, cooccurrence models are plagued with efficiency in real-word applications [73] . there are two main problems in the usage of such approaches: first is the dimensionality in contexts vectors and the second is sparse data problem. in the first problem, the dimension of co-occurance matrix will tend to be extremely big for large data. in the second problem, as the vast majority of words occurs in a very small fraction of set of contexts [74] , the majority of the entities of vectors will be zero. therefore, the co-occurance matrix will not give reliable results for large data and brief texts. additional to these two problems, specifically, usage of co-occurrence is not appropriate for the representation of scientific texts due to multidisciplinary researches in the collection [23] . therefore, we introduce a new vector space to represent word meaning based on words' informational importance in the subject categories. we begin by creating a space to represent words meaning. the meaning space is defined as a vector space, in which coordinates correspond to the subject categories. a word is represented by a vector of rig about the subject categories that the text belongs to, which can be obtained from observing the word in the text. this approach allows us to identify the importance of the word for the corresponding category in terms of information gained when separating the corresponding category from its complement (like, for example, separating texts in category 'algebra' from the text that do not belong to this category). to define rigs, we consider the following two attributes of text d for a given word w j and a given category c k : the text d is in the category c k : attribute values are yes (c k (d) = 1) or no (c k (d) = 0); w j (d): the word is in the text: attribute values are yes (w j (d) = 1) or no (w j (d) = 0). the corpus is considered as a probabilistic sample space (the space of equally probable elementary results, each of which is a random selection of text from the corpus). rig measures the (normalized) information about the value of c k (d), which can be extracted from the value w j (d) (i.e. from observing the word w j in the text d) for a text d from the corpus. as we have a number of word vectors, it is convenient to organise the vectors into a matrix. these vectors are used to construct word-category rig matrix, in which rows correspond to words and columns correspond to categories. each entry in the matrix corresponds to a pair (category,word). its value for the pair 12 (c k , w j ) shows the rig on the belonging of a text from the corpus to the category c k from observing the word w j in this text. word-category rig vectors estimate the meaning of words as their importance in the research fields. thus, row vectors in the word-category rig matrix indicate words' scientific meanings. this approach computes a distributional representation (rigs) for a word across all research subjects (rigs in categories). following to the distributional semantic hypothesis, if words have similar row vectors in the word-category rig matrix, they tend to have similar meanings. the hypothesis is that if texts have a similar distributions of word meanings -similar clouds of word meanings vectors -then they tend to have similar meanings. we note that proposed hypothesis does not require an explicit distinguishing between homonymy and polysemy for words; it only requires linking the meaning of words to their importance in categories. with this approach, vocabulary meanings do not directly affect the representation of the meaning of the word. rather, the meaning of a word is characterized through its measured information content in various scientific subject categories. in this research, we present the first stage of 'quantifying of meaning': construction of the meaning space and representing word meaning as a vector of rigs for categories in this space. such an understanding of meaning of words can help analyse the meaning of the texts. having quantified meaning of words, one can represent all words in a corpus and then texts in the meaning space. specifically, each text in the corpus is a cloud of rig vectors and the text meaning can be later estimated and constructed by these distributions. analysis of texts will be focused in the next stage of the research. text analysis will be the next stage of the research. the earliest (preparatory) stage of the project was presented in [24, 23] . the empirical analysis of this research is based on the leicester scientific corpus which includes 1,673,350 texts [22] and leicester scientific dictionary-core (lscdc) of 103,998 words [75] . the main hypothesis for construction of the meaning space is: meaning is the vector of information gains from the word to the categories assigned to the text. we used 252 categories of wos. we evaluated the meaning space and representation of word meaning in this space through top-ranked words in each category. we constructed the word-category rig matrix for the lsc [76] . the most informative words in each category are presented. it is shown that the proposed representation technique stands out topic-specific words in categories. we compared this approach with the representation technique where words are represented by vectors of their raw frequencies in categories. words are ranked by both frequencies and rigs in categories. we demonstrated that frequencies are not much useful for identifying the most informative words in categories. we concluded that frequency is not much important in this sense. for each word in the lscdc, the sum and maximum of rigs in categories are calculated and added at the end of the word-category rig matrix. words can be ordered by their informativeness in scientific texts by these two criteria. the most informative n words for scientific texts can be extracted by ordering/sorting words in column of the sum or maximum of rigs. we compared these two ordering criteria by counting the number of matches in the top n words, where n ranges from 100 to 50,000. we concluded that the majority of the first 100 words do not match, 13 informational space of meaning for scientific texts with 28% matched words. the intersection of words reaches to approximately 50% for the top 1,000 words, and then 99% for the top 50,000 words. finally, we created a scientific thesaurus in which the most informative words were selected from the lscdc by their average rigs in categories. the thesaurus was called leicester scientific thesaurus (lsct). lsct contains the most informative 5,000 words in the corpus lsc. these words are considered as the most meaningful words in science. the full list of words in lsct is available online [76]. this paper is organised as follows. in section 2, the meaning space is constructed and the representation of words by vectors in the meaning space is discussed. given the representation of words by vectors of rigs, we look at words ordered by their rigs in each category. in section 3, we present the first findings of the new representation technique and the anomalies detected in the data by this model. to avoid a possible abnormal appearances of the words in the categories, we apply a further cleaning procedure of the lsc. the latest versions of the lsc, dictionaries leicester scientific dictionary (lscd) and the lscdc are described [22, 75, 77] . finally, we construct the word-category rig matrix for the lsc [76] and discuss the experimental results in this section. in section 4, we introduce the leicester scientific thesaurus (lsct), in which there are 5,000 of the lscdc words selected by their average rigs in categories. in section 5, the conclusion and outlook are summarised. in this section, we discuss the architecture of our approach to estimating the word meaning in a collection of documents. we assume that the dataset is a large corpus of natural language scientific texts and each text in the corpus belongs to at least one subject category. we hypothesize that words have scientifically specific meaning in categories and the meaning can be estimated by information gains from the word to the category. before inquiring into the measurement of the meaning, we will mention how to represent each word as a vector of frequencies in categories. we then introduce a new approach to word meaning, in which each word is represented by a vector of rigs in the meaning space. in this section, we review how to represent a word in a vector space model by using appearances this word in texts belonging to subject categories. a word representation method is defined in order to indicate term absence/presence in texts of categories. each word is represented by a vector of frequencies in categories. that is, the number of presence of a word is calculated by how frequently this word is observed in texts belonging to the category. each entry of the vector consists of the number of texts containing the word in the corresponding category. it is noteworthy that texts in a corpus do not necessarily belong to a single category as they are likely to correspond to multidisciplinary studies, specifically 14 informational space of meaning for scientific texts in a corpus of scientific researches. in other words, categories may not be mutually exclusive. for every word w j from the dictionary (j = 1, ..., n ) and every text d i from the corpus (i = 1, ..., m ) the indicator w j (d i ) is defined. if the word w j occurs in the text d i (once or more), then w j (d i ) = 1. otherwise, w j (d i ) = 1. let d k be a set of texts in the category c k . the frequency of the word w j in the category c k is this w jk is the number of texts containing the word w j in the category c k . the vector of frequencies is defined for each word w j from the dictionary. let us use the notation − → w j for it. coordinates of this vectors are w jk , where index k = 1, ..., k corresponds to the subject categories. thus, each word w j in the corpus is represented by a vector of frequencies w jk denoted by − → w j = (w j1 , w j2 , ..., w jk ), where k is the number of categories in the corpus. the collection of vectors, with all words and categories in the entire corpus, can be shown in a table. each entry w jk of the table 2 .1 corresponds to a word and a category. category c 1 c 2 · · · c k w 1 w 11 w 12 · · · w 1k w 2 w 21 w 22 · · · w 2k . . . . . . . . . . . . the number of documents in the category c k is |d k |. importantly, as each text usually has more than one word, and several different words can belong to the same text. to simplify the notation for further calculations, we now define the set of texts containing the word w j as d j . we note that |d j | ≤ k w jk and equality holds in the case when categories are mutually exclusive. the number of texts in the categories varies widely, so w jk is expected to increase as the number of texts in a category increases. this does not necessarily mean that a word rarely appearing in a category is less important for this category than for other categories in which the word appears more frequently (see the definition of 15 information gain in the next section). therefore, direct usage of frequencies may result inappropriate findings in quantification of words' meanings. given the collection of vectors, various schemes for normalisation can be performed to adjust the vectors − → w j to a common scale. the simplest and the most popular approach for normalisation is transformation to a vector where the sum of the elements is 1, that is normalisation to unite l 1 norm. for the mutually exclusive categories, this normalisation is related to the law of total probability. the objective of this normalisation scheme is to make vectors comparable by rescaling them to the same length in the l 1 norm. for a given vector − → w j , the normalisation can be performed as p jk = w jk i w ji where k p jk = 1. it should be stressed that when categories are not exclusive, k w jk is not the total number of texts containing the word w j . in other words, texts containing the word could be counted more than once in the sum. in similar way, the column vectors can be normalised as however, this representation does not indicate the proportion of exact number of texts in the category. a reasonable normalisation can also be obtained in two-steps: (1) normalize each frequency: (2) normalize the matrix to the unite sum in rows. as a result, w jk will be transformed into w jk in calculation of rigs below, the estimation of probabilities are used based on the table of frequencies. for ranking of words in categories, the raw frequencies were also used and compared to rig-based ranking. having a collection of frequency vectors, it is easy to calculate the vectors of information gains (from observing the word in the text to categories which the text belongs to). these vectors will quantify the meaning the words. the hypothesis here is that the informational content of a word about each category can be measured by comparing the appearance of a word in texts of a given category and its appearance in texts not related to this category (i.e, how the presence/absence of the word in texts can help to separate the category from its set-theoretical complement). a general concept for computing information is the shannon entropy introduced by shannon [78]. information gain (ig) is a common feature selection criterion in machine learning used, in particular, for evaluation of word goodness [79, 80] . the information gain is the measure of the information extracted about one random 16 variable if the value of another random variable is known. it is closely related to the mutual information, that measures the statistical dependence between two random variables. the larger value of the gain means the stronger relationship between the variables. the information gain of random variable a with values (or states) a 1 , . . . , a n from the random variable b with values (or states) b 1 , . . . , b m is defined as: where p (a = a i ) is probability of observing the value a i of the random variable a, p (b = b j ) is probability of observing the value b j of the random variable b, p (a = a i |b = b j ) is conditional probability of observing the value a i of the random variable a given the value b j of the random variable b. ig(a|b) measures the number of bits of information obtained for prediction of a value of the variable a by knowing the value of the variable b. in the concept of text categorisation, the information gain measures how important a given word is for category prediction. a larger gain indicates that the probability to find the word in the texts inside the category differs considerably from the probability to find it in the text outside this category. if the categories are mutually exclusive then we can consider them as values of a categoric feature c of the text with values c i and define the information gain ig(c, w) from observing a word w in the text about the value of c [80] by the textbook formula: where {c i } is the set of classes in the target space, p (c i ) is the probability of observing the i th class, p (w) is the probability that the term w appears, p (w) is the probability that w does not appear, p (c i |w) is the conditional probability of observing the i th class given that the term w appears, and p (c i |w) is the conditional probability of observing the i th class given that the term w does not appear. ig(c, w) measures the number of bits of information obtained for prediction of classes c i by knowing the presence and absence of a term w in documents of classes. the quantity ig(c, w) measures the amount of information provided by a word when splitting the documents into classes but only in the case of mutually exclusive classes, that is, each text is assigned to a single class only. on the contrary, the scientific texts belong very often to several categories. the research subject categories are not mutually exclusive and this approach cannot be used directly. unlike this approach, we start from measuring how a word is informative for a category in terms of its ability to separate the corresponding category from its settheoretical complement. we hypothesize that the topic-specific words in categories have larger information gain than other words and such words are expected to have less gain in most other categories. therefore, we approach to this problem by 17 since words are obviously not mutually exclusive (one text usually contains several different words) we cannot consider the occurrence of different words as values of a random variable to use (1) directly. to evaluate the information gain of the category c k from the word w j it is necessary to introduce for each word w j a random boolean variable with two states: w j denotes the presence of the word in texts of the category c k and w j denotes the absence of the word w j in texts of the category c k . contingency 2 × 2 table to calculate information gain of the category c k from the word w j is presented in table 2 .3. it used the raw frequencies w jk introduced in previous subsection. table 2 .3 can be used to calculate two information gains: the word w j from the category c k and the category c k from the word w j . both information gains have a meaning for different problems. the goal of this research is to evaluate informativeness of words for category identification and use this informativeness for word ranking and text representations. therefore, we will consider information gain of the category c k from the word w j : ig(c k , w j ). this information gain evaluates the number of bits extracted from presence/absence of the word w j in the text for prediction of belonging of this text to the category c k . one may expect that if a word is a very topic-specific for a category, it appears in texts belonging to this category more frequently than in texts which do not belong to this category; and the major part of texts belonging to this category contains the word. for each category, c k , a function is defined on texts that takes the value 1, if the text belongs to the category c k , and 0 otherwise. for each word, w j , a function is informational space of meaning for scientific texts defined on texts that takes the value 1 if the word w j belongs to the text, and 0 otherwise. we use for these functions the same notations c k and w j . consider the corpus as a probabilistic sample space (the space of equally probable elementary outcomes). for the boolean random variables, c k and w j , the joint probability distribution is defined according to table 2 .3, the entropy and information gains can be defined as follows. the information gain about the category c k from the word w j , ig(c k , w j ), is the amount of information on belonging of a text from the corpus to the category c k from observing the word w j in the text. it can be calculated as [78] : where h(c k ) is the shannon entropy of c k and h(c k |w j ) is the conditional entropy of c k given the observing the word w j . entropies h(c k ) and h(c k |w j ) are where p (c k ) is the probability that the text belongs to the category c k , p (c k ) is the probability that the text does not belong to the category c k and where • p (w j ) is the probability that the word w j appears in a text from the corpus; • p (w j ) is the probability that the word w j does not appear in a text from the corpus; • p (c k |w j ) is the probability that a text belongs to the category c k under the condition that it contains the word w j ; • p (c k |w j ) is the probability that a text does not belong to the category c k under the condition that it contains the word w j ; • p (c k |w j ) is the probability that a text belongs to the category c k under the condition that it does not contain the word w j ; • p (c k |w j ) is the probability that a text does not belong to the category c k under the condition that it does not contain the word w j . information about whether an element belongs to a set. high value of the informational gain ig(c k , w j ) (3) does not mean, in general, that the large proportion of information about whether a text belongs to the category c k can be extracted from observing the word w j in this text. this proportion depends on the value of the entropy h(c k ) (6). the relative information gain (rig) measures this proportion directly. it provides a normalised measure of the information gain with regard to the entropy of c k . rig is defined as the value of rig(c k |w j ) will be 0 when h(c k ) = h(c k |w j ) and 1 when h(c k |w j ) = 0. in the first case, the presence/absence of the given word w j does not contain information for the category c k . so, this word is uninformative. in the second case, using the word in the category provides exactly h(c k ) bits of information. that is, presence or absence of a word resolves exactly the question of belonging the text to the category. rig(c k |w j ) can be equal to 1 in two cases: • all texts with the word w j belong to the category c k and all texts without the word w j do not belong to the category c k ; • all texts with the word w j do not belong to the category c k and all texts without the word w j belong to the category c k ; we expect higher rig(c k |w j ) for the topic-specific words of the category c k . for simplicity, we denote rig(c k |w j ) by rig jk . given the word w j , rig jk is used to form vector − −− → rig j , where each component of the vector corresponds to a category. therefore, each word is represented by a vector of rigs. it is obvious that the dimension of vector for each word is the number of categories k (for the wos subject categories k = 252). for the word w j , this vector is the set of vectors − −− → rig j can be used to form the word-category rig matrix, in which each column corresponds to a category c k and each row corresponds to a word w j . each component rig jk corresponds to a pair (c k , w j ) and its value is the rig from the word w j to the category c k . the structure of the word-category rig matrix is demonstrated in table 2 .4. in the word-category rig matrix, a row vector represents the corresponding word as a vector of rigs for categories. we defined the meaning space as the vector space of such vectors − −− → rig j . the dimension of this space is the number of categories and each coordinate is the rig from a word to this category. note that in the word-category rig matrix, a column vector represents rigs of all words in an individual category. if we choose an arbitrary category, the 20 words can be ordered by their rigs from the most informative word to the least informative one. we expect that the topic-specific words will appear at the top of the list. the words can be ordered by their informativeness in the whole corpus of scientific texts as well as they are ordered in each category. a norm or a more general proximity measure in the meaning space is needed to compare the meaningfulness of words across all categories. two criteria were tested for measuring informativeness of words in the corpus of scientific texts: the sum (l 1 norm) and the maximum (l ∞ norm) of rigs in categories. for a given word w j , the sum s j and the maximum m j of rigs are calculated from the word-category rig matrix as: and the sum s j is a measure of the average informativeness of a word (this word has the informativeness s j /k on average), whereas the maximum m j is a measure of the maximal informativeness of the word across the categories (this word is not more informative than m j in any category). now, the words in the dictionary can be ordered by their s j or m j . for each of these ordered lists of words, the most informative (meaningful) n words for scientific texts can be selected based on one of these two criteria. the higher the value of the criterion (s j or m j ), the more informative the word is. this section describes the experimental details and the analysis done to show the performance of the vector representation method described in section 2. the dataset used in this study is the leicester scientific corpus (lsc) [22] . the lsc contains a collection of abstracts of research articles and proceeding papers with metadata such as authors, title, categories, research areas and times cited. each record (text) in the dataset is assigned to at least one of the wos categories. the leicester scientific dictionary-core (lscdc) is the collection of unique words appearing in 10 or more documents in the lsc [75] . for each word w j and category c k , rig jk is calculated and the word-category rig matrix for the lsc was formed as described in section 2. in each category, 21 informational space of meaning for scientific texts a list of words where words are sorted in descending order by their rigs can be created. the higher the relative information a word gained in a category, the more important the word is in terms of being topic-specific for the category. therefore, one could look at the top n words in categories in order to get a good grasp of the representation method. the visualisation of the top words in each category is carried out with the word clouds. having calculated the frequencies of words in the categories (table 2. 1), we compare the purposed method with the commonly-used approach based on raw frequency. at first, the procedure of word representation was applied to the lsc version 1 [24] with the dictionary [25]. to visualise top words in each categories in a convenient way, we looked at word clouds. the font size of each word in a word cloud is proportional to its rig in the category. intuitively, the more informative the word is, the bigger size the word appears in word clouds. for example, from figure 3 .1, it can be seen that the most informative 6 words for the category 'acoustics' are 'acoust', 'ultrasound', 'speech', 'nois', 'sound' and 'frequenc'. the majority of papers in acoustics is expected to include these words which are absent or at least less frequent in many other categories. these words are inferred to be informative for the category 'acoustics'. however, this method detected anomalies in some categories. anomalies here refers to words that do not conform to the expected set of words to be appearing in a subject category. such words can appear in any category frequently regardless of being a topic-specific word. these words are likely to be potential anomalies 22 informational space of meaning for scientific texts generated by inappropriate joints of words, phrases or sentences to the texts of abstracts. as shown in figure 3 .2, for the category 'chemistry, applied', words 'elsevi', 'ltd', 'acid', 'reserv' and 'right' stand out in the word cloud. we see that trends in majority of words in the word cloud agree with each other as being related to the subject. however, 'elsevi', 'ltd', 'reserv' and 'right' seem like more prominent and unusual (non-specific) for chemistry. in fact, the experiments were preliminary, but we discovered alarms indicating anomalies by our representation technique. to understand why these words arose and how they can be avoided, we checked the abstracts containing such words. our review showed that these words appeared in copyright notices such as published by elsevier ltd. or 'all rights reserved', and they were added at the footer of abstracts. in order to have a comprehensive understanding of their appearance as being informative for only some categories, for instance in chemistry, we compared distributions of 'elsevier', 'right' and 'reserve' in categories. for each word, categories are ordered by the number of documents containing the word, and the first 20 categories are presented in figure 3 .3. when we consider the list of categories ordered by the number of documents in the entire corpus, we conclude that not all categories in the list of top categories appear in the charts. this is because usage of copyright notices is much more noticeable in some categories such as chemistry. for instance, the rank of the category 'engineering, electrical & electronic' is 1 in the corpus; however, one can see that this category has rank 15 for the word 'elsevier'. to show that not all categories have the same/similar distribution of use of copyright notices, we presented this subsection provides the description of procedure of additional cleaning and correction for the lsc and lscdc. many conferences and journals put copyright notices, permission policies or conference names below abstract of papers. such footers were added to abstracts in many records in web of science database and so in the lsc during processing and storage of the original data (see table 3 .1). 26 it is really a huge and practically impossible task to find out with the help of human inspection which notifications were added in the texts of 1,673,824 abstracts in [24] . once a sample of abstracts containing publishing houses names was browsed, we found that there are much more scenarios to consider. some examples of these scenarios are presented in table 3 .2. as such expressions are more frequent in some categories than in others, a cleaning procedure is needed to avoid possible abnormal appearances of words in categories. a quick look at the scenarios is sufficient to conclude that clearing such sentences or phrases cannot be fully automated. human intervention is needed to identify and list them to avoid deleting useful information from the data. individual notices with different appearances were identified by sampling of abstracts based on keyword search. a keyword search refers to browsing words, phrases or sentences to list different appearances of them in order to delete all identified appearances from abstracts. the position of notices was also taken into account since they appeared either at the beginning (by mistake) or at the end of the text. we used several specially developed procedures successively to clean them. for instance, when removing notices in the form of '(c) published by elsevier', we first checked the appearance of 'crown copyright (c) published by elsevier'. it can also appear in the form of 'published by elsevier', thus we consider all cases based on empirical study. during cleaning, we removed copyright notices, names of conferences, names of journals, authors' rights, licenses and permission policies identified. to give an insight, table 3 .3 presents the number of document containing some notices before cleaning. these notices were completely removed after cleaning. we note that names of publishing houses could appear inside the text, in this case we did not remove them. more examples of notices that were removed from abstracts can be found in appendix a.1. to display the initial result of the cleaning, we present the word cloud and histogram of rigs for the category 'chemistry, applied' in figure 3.6. one can see that words 'elsevi', 'ltd', 'acid', 'reserv' and 'right' do not appear in the list of top words as was in the word cloud before cleaning (see figure 3 .2). instead, the cloud gives greater prominence to words that are related to specific topics and likely to be more informative for the category. the word 'acid' has been preserved in the list of the most informative words. it is noteworthy that, in both versions of the lsc, the number of subject categories is 252. all categories and the number of documents assigned to the corresponding category are presented in table b .1. same information for research areas was provided in table c. 1. the distribution of length of abstracts is displayed in figure 3 .7. there is no noticeable difference between distributions for two versions and the average length of texts is 176 words. the latest version of the leicester scientific dictionary (lscd) was developed by extracting words from the new version of the lsc [77]. the procedure applied to process the lsc in creation the lscd was the same as described in [23] . the new version of the lscd contains 972,060 unique words with the number of texts that a word appears in. a new version of the core list, lscdc, was created from the lscd by removing words appearing in no more than 10 texts of the lsc [75]. all steps applied were the same as for the previous version of the lscdc and can be found in [23] . based on the decision to clean copyright notices, we expect that words such as 'elsevier', 'reserved', 'ltd', 'right' and 'springer' will not appear frequently in the lsc as they did before. in fact, the number of appearance of these words decreased after cleaning (see table 3 .4). the results indicated that some words, for instance 'right' and 'reserve', are still relatively frequent in the corpus. this is because these words are specific for some categories. to give an insight, we compared top categories for three words 'elsevi', 'reserv' and 'right' (see figure 3 .8). the results for the word 'right' indicate that it is frequently used in medicine related categories such as 'neuroscience' and 'surgery', and in social science categories such as 'law' and 'political science'. this is an expected result as it can appear to determine the side of organs such as 'right hippocampus' or 'right hemisphere' in medicine; and the normative rules in such disciplines as law and ethics. 'elsevier' and 'reserv' are much more uniformly distributed to the categories when the rank of categories is taken into account. for 'reserv', one can identify categories related to biosciences such as 'ecology', 'zoology' and 'environmental studies'. specifically, this word occurs to indicate 'nature reserves'. recall that a representation technique for words was introduced in section 2. the vectors of frequencies in subject categories were obtained for each word. the frequency associated to a category was computed by counting texts containing the word in this category. subject categories are used to categorise papers in the wos collection; however, documents do not necessarily belong to a unique category due to interdisciplinary studies. in other words, categories are not exclusive in wos and so in the lsc. in the lsc, texts belong to at least 1 and a maximum of 6 categories out of a total of 252 subject categories (see figure 3 .9). it is noteworthy that our consideration is to count the number of times a word appears in texts of a category rather than analysing exclusivity of categories. therefore, in this stage, we just looked at the frequency of texts with these words in categories. the vectors of frequencies in categories are built for 103,998 lscdc words and 252 subject categories. each row represents a word of the lscdc in 252-dimensional space, that is, each word is represented by a vector of frequencies in 252 categories. for each category, a frequency distribution can be obtained for the set of words. the distribution indicates words used in texts of each category and the most frequently used words can be sorted in categories. to illustrate this, the most frequent 10 words for categories 'astronomy & astrophysics', 'mathematics' and 'asian studies' with frequencies are displayed in table 3 .5. a table containing all words and categories are included in [76] . one can expect that not all words in the table indicate a topic in the related subject. as an example, words 'use', 'also', 'studi' and 'paper' are frequent words in the lscdc and so in categories. these non-topic specific words occur many times in abstracts without indicating subject specificity. therefore, using the frequencies of words in categories may not reflect how specific a word is to a category. 32 informational space of meaning for scientific texts on the basis of exploratory work by the frequency table, we concluded that the use of word frequencies in categories does not provide much information about the category. to be specific, we expected that 'use' is not a topic-specific word as it appears in all 252 categories and it is likely to be used in almost all texts. this means that the meaning of a word in the text cannot be directly extracted from the frequency. aiming at this result, we must now apply a different perspective to measure the importance of words for categories, with a special attention given to the hypothesis that each word in the lscdc has scientifically specific meaning in categories and the meaning can be extracted from the information of words for 252 subject categories in the lsc. thus, as described in section 2, words were represented in a 252dimensional meaning space. rigs for each word in 252 categories were calculated and vectors of words were formed. we then represented these vectors in the word-category rig matrix. for each word in the word-category rig matrix, the sum s j and maximum m j of rigs in categories were calculated and added at the end of the matrix. the word-category rig matrix can be found in [76] . one can extract the most informative n words for scientific texts by ordering/sorting the column of words based on their s j or m j . the experimental results presented in this section were obtained using abstracts of academic research papers in the lsc [22] . we used words from the core dictionary lscdc [75] . having calculated rigs for each word and created the word-category rig matrix, we evaluate the representation model by checking words in each category. that is, we consider the list of words with their rigs in the corresponding category. those words that have larger rig are more informative in the category. being 'more informative' here allows for the interpretation of being 'more specific' to the category's topic. for each category, words are sorted by their rigs and the top 100 words are shown in the word clouds. the bigger font size the word in word clouds, the more informative it is. word clouds for the top 100 most informative words and histograms of rigs for the top 10 most informative words for each of 252 categories can be found in [81] . the most informative 100 words with their rigs for each of categories are presented in appendix e and [81] . in general, the rig based method proves to be more sensitive than the frequency based method in identifying topic-specific words of a category. this means that representing words in meaning space has the advantage of transforming words to efficient vectors with a benefit of considerably lower dimension than the standard word representation schemes. to illustrate this result, we choose categories 'biochemistry & molecular biology', 'economics' and 'mathematics' and compare two word clouds that are formed by using raw frequencies and rigs in categories (see figures 3.10, 3.11 and 3.12). it can be seen from the figures that the majority of the most frequent words in all three categories are frequent words in the entire corpus. these words are not topic-specific for categories as they appear in almost all abstracts. the frequent but non-informative words can be considered as generalised service words of science and deserve special analysis. this proves that raw frequency is not much important to identify scientifically specific meanings of words. therefore, by representing words as vector of rigs, we can avoid such frequency bias. the most informative words in categories for rig representation are topic-related in the corresponding category. we interpret these results as evidence for the usefulness of the rig based representation. words that are expected to be used together have very close values of rigs. in 'health care sciences & services', 'health' and 'care' are top words and rigs for these words are so close (see figure d .1). another example is 'xrd' and 'difract' in 'material science, ceramics'. 'xrd' is actually abbreviation of 'x-ray diffraction'; therefore, they appear together as 'x-ray diffraction (xrd)' for most of cases in the category (see figure d .2). we can extract some stylistic properties in texts of categories. for instance, in computer science related categories the word 'paper' has the highest rigs (see a casual observation indicates that while the most informative words in some categories have similar rigs, differences in values of rigs are much more noticeable for the most informative words in some other categories. to give an insight, we present the categories 'chemistry, medicinal' and 'engineering, chemical' in figure 3 .13. in 'chemistry, medicinal', the word 'compound' can be easily separated from the other words, while in 'engineering, chemical', there is a slight decrease in rigs informational space of meaning for scientific texts for the top 10 most informative words. however, in general we did not observe any explicit rule for this property. finally, we formed two lists of words that arranged in descending order based on the sum and maximum of their rigs in 252 categories. the top 100 words in two lists are displayed by word clouds in figure 3 .14 and figure 3 .15. histograms in the figures show the most informative 10 words in the lists. we found that the most informative 10 words in two lists are completely different, as shown in the figures. from words clouds, one can see that the majority of the first 100 words do not match. we then compared two lists by counting the number of matches in the top n words, where n ranges from 100 to 50,000. the numbers of matched words for different n are presented in table 3 .6. as can be seen, 18% of words match for the top 50 most informative words. this proportion increases to approximately 50% for the top 1,000 words and to 58% for the top 2,000 words. the intersection of lists reaches to approximately 99% for the top 50,000 words. from these results, one can conclude that two lists are different in the top words. when higher number of words is taken into account, lists become more similar in terms of words included. however, the rank of words are not similar. any of these criteria for selecting the most informative words can be used depending on the task and the information required. 35 informational space of meaning for scientific texts the numbers s j and m j are differently distributed for words. we observed from the lists that many words have low s j and m j . figure 3.16 and figure 3 .17 show the distribution of s j and m j for words in the logarithmic scale. supper-exponential picks near zero rigs are noticeable for both criteria. we can see that the trend is going down almost linearly beyond the picks. the bottom 10 least informative words in two lists are presented in table 3 .7. one may consider words having almost zero s j or m j as less meaningful words for scientific texts. in this section, we introduce a scientific thesaurus of english: leicester scientific thesaurus (lsct). lsct is a list of 5,000 words which are created by arranging words of lscdc in their informativeness in the scientific corpus. the procedure for creation of the thesaurus is described in detail. under the assumption that not all words having very low rigs are informative in categories, we search a cut-off point for rig to create a list of words that can be considered as relatively meaningful in scientific texts. in other words, we extract meaningful words for science from the lscdc to build a scientific thesaurus. before moving on the decision taken to determine the number of words for the thesaurus, we recall the notion 'informativeness' and investigate further the criteria of s j and m j to arrange words of lscdc in their informativeness. having the top 100 words in two lists where words are descending ordered by their s j and m j , we see that the criteria of maximum is more likely to stand out some words that are frequently used in specific categories such as categories 'dance', 'music', 'soil science' and 'theatre' (see table 4 .1) and are relatively rarely used outside them. indeed, we expect drastic differences in rigs of such words for these categories. for instance, one of the most informative word 'dance' is used in 154 categories, but the rig from this word to the category 'dance' is very distinguishable from all others (see table 4 .2). this is actually an expected result, since the word 'danc' is likely to be informative for categories related to the performing arts. to compare the meaningfulness of words across all categories, we tested two norms in the meaning space, l 1 (s j or the sum of rigs) and l ∞ (m j or the maximal rig). after a series of trials, we decided to use l 1 . this choice cannot be proven formally but the ordering words by m j lead to some words that are very specific in only one category but stand out in the list of the most informative words on average. the sum can be considered as more appropriate measure for general scientific thesaurus. when creating an lsct, we consider ordering the lscdc words by the sum of their rigs in categories. the meaningfulness of words was evaluated by the average informativeness of words in the categories. given the dictionary lscdc, the procedure to create the lsct is: • sort the words of the lscdc by their s j in descending order. • take the top 5,000 words. to find the number of words to be contained in the thesaurus, we initially follow an empirical procedure: (1) having arranged list of words in descending order by s j , take a sub-list of the top m words, denoted by t m (2) create the histogram of s j for the words in this sub-list (3) check the trend in the histogram (4) take words when the exponential pick is avoided and the histogram follows roughly linear trend. we begin with investigating the top 50,000 words in arranged list as it is almost the half of the 103,998 words of the lscdc. as the trend in the histogram for 50,000 words was showing the same behaviour with the histogram of 103,998 words (see figure 3 .16 and figure 4 .1 (a)), there was no point to check a number between 50,000 and 103,998. we then decreased the number m to 10,000, 5,000, 2,000, 1,500 and finally 1,000. all histograms are presented in figure 4 .1. we see a substantial change in the trend of the histogram when we take the subset of 5,000 words. the trend at that point is almost linear. after that, the first bin in the histogram is slightly becoming smaller and finally it disappears for 1,000 words. in this step, we also checked the minimum of the sum of rigs in the lists t m to make sure that the minimal average informativeness in the list (to be selected) is not so close to zero. these values are displayed in the table 4 .3. we can see from the table that the minimal rig is decreased less than half from 1,000 to 2,000, while it decreased faster (more than halved) from 5,000 to 10,000. finally, to support our selection of the number of words for the lsct and for evaluation of the result, we consider the following heuristic suggestion: the majority of words in the lsct appears in the list of top informative words in the categories. this does not mean that all informative words in categories should appear in the lsct, but we expect that most of top n words in categories will be included in 42 informational space of meaning for scientific texts we consider the matches of the list t m with the most informative words in categories defined by the sum of rigs. for collection c k,n of n most informative words in the category k, we define x n = k k=1 c k,n . then we test the coverage of the list t m by x n . for each category in the word-category rig matrix (a column in table 2 .4), order in descending by their rigs. this gives a list of words sorted from the most informative to the least informative for this specific category. then, individual collections c k,n are formed for each category. the set c k,n was formed with different numbers of words (n). we built the collections containing the most informative 100, 200, 300, 400 and 500 words in each category, and x n is created by uniting them for each n. the numbers of words and the minimal rigs of words in x n are presented in table 4 .4. the minimal rigs are checked to avoid zero/near-zero rig in lists. one can see from the table that words in categories are not completely different. for instance, if all c k,100 do not intersect then there should be 25,200 words in the list x 100 , but there are just 6,254 words in this union, which is almost four times less. for other n, the result is similar, and the values of x n follow almost a linear trend (see figure 4 .2). that is, the intersection k k=1 c k,n is not empty. the intersection may be pairwise or q-wise for different q. the coverage is calculated by counting the number of matches words of the list t m and words of x n . table 4 .5 illustrates the numbers of matches when n is 100, 200 and 500. up to top 2,000 words, the words are concordant in the lists t m and x n , suggesting that the most informative words are highly consistent. in fact, words in the lists are in agreement for the case where 500 words in each category are considered as informative for categories. given the list x 100 , the majority of the words (3,992 words) in the t 5,000 can be covered by words of x 100 . this trend changes and goes down when we consider the percentage of words found in 10,000 and 50,000 words. however, in this stage we have to consider the total number of words in x n . for instance, the number of words in x 100 is 6,254. in this case, the matches cannot be more than this number for the list t 10,000 . similar conclusions 44 were obtained by comparing the number of matches for 5,000, 10,000 and 50,000 words for n=200. we examined various heuristic criteria to evaluate how many words are suitable for inclusion in a thesaurus. since we want to keep the size of thesaurus reasonable, and pay attention not to loose many words in case there might be informative words having not very high rigs, we decided to include these 5,000 words (t 5,000 ) in the scientific thesaurus. this thesaurus is called leicester scientific thesaurus (lsct). it is published online [76]. in this work, we have studied the first stage of 'quantifying of meaning' for scientific texts: constructing the space of meaning. we have introduced the meaning space for scientific texts based on computational analysis of situations of words' use. the situation of use of the word is described by the absence/presence of the word in the text in scientific subject categories. the meaning of the text is hidden in the situations of usage and should be extracted by evaluating the situation related to the text as a whole. this research is done based on 1,673,350 texts from the lsc and its 103,998 words listed in the lscdc [22, 75] . a text in the lsc belongs to at least one and at most six of 252 web of science categories presented in table b .1. that is, categories can intersect. the situation of use is described by these 252 binary attributes of the text. these attributes have the form: a text is present (or not present) in a category. the meaning of a word is determined by categorising texts that contain the word and texts that do not. it is represented by the 252-dimensional vector of rig about the categories that the text belongs to, which can be obtained from observing the word in the text. this representation is demonstrated in table 2 .4. each text in the lsc can be considered as a cloud of these rig vectors. we begin with representing each word as a vector of frequencies in categories (table 2.1). components of a vector are the number of texts containing the word and belonging to the corresponding category. then we moved on to representing the meaning of a word as a rigs vector about categories. we consider the corpus (lsc) as a probabilistic sample space (the space of equal probable elementary outcomes). the function is defined on texts that takes the value 1, if the text belongs to the category c k , and 0 otherwise. similarly, for each word w j , a function is defined on texts that takes the value 1 if the word w j belongs to the text, and 0 otherwise. both functions can be considered as the random boolean variables. the information gain ig(c k , w j ) about the category c k from the word w j is calculated by (3), (4) and (5). ig(c k , w j ) measures the amount of information extracted from observing the word w j in the text on prediction of belonging of this text to the category c k . the rig rig(c k , w j ) is calculated by 46 informational space of meaning for scientific texts (8) that provides us a normalised measure of information gain giving the ability of comparing information gains for different categories. vectors of rigs are denoted by − −− → rig j for a word w j . − −− → rig j vectors for all words are presented in a word-category rig matrix (see the structure in table 2 .4) (available online [76]). a column vector of the matrix contains rigs for all words in an individual category and a row vector represents the corresponding word's meaning as a vector of rigs for categories. the meaning space has been described as a 252-dimensional vector space, where vectors are − −− → rig j . beyond the representation of words, the word-category rig matrix can be also used for the ordering words in a category from the most informative to the least informative as well as identifying the most informative words in the science for different subjects and their combinations. ranking of words in a scientific corpus are performed based on two criteria: sum of rigs (s j ) and maximum of rigs (m j ) in a row vector. calculations are done by (9) and (10). given an ordered list of words, the top n words are considered as the most informative n words in the scientific corpus. the lsc and lscdc were created and available online [23, 24, 25]. the proposed word representation technique was applied to this version of the corpus. the evaluation of the model is done based on checking the most informative words in each category. word clouds are generated using words in lists for each category (for example, see figure 3 .1 and figure 3 .2). the higher rig a word has, the bigger font size of the word is in the cloud. the clouds demonstrated that our methodology is able to identify topic specific words for categories, and most of the top words are related to the category subjects. we note, however, that some words that were not expected to be appearing as the most informative words were prominent for some categories (figure 3 .2). we concluded that words occurring in copyright notices, permission policies and the names of journals and organisations are added at the footer of abstracts in wos database (see table 3 .1, table 3.2 and table a .1). such joints result in anomalies in the word clouds and our representation technique was able to detect them. a further cleaning on identified phrases, sentences and paragraphs was performed to avoid possible abnormal appearances of words in the lists. this is done by sampling of texts based on keywords search and then deleting them from the texts. after cleaning procedure, new versions of the lsc and the lscdc are created by the same pre-processing steps as for the previous versions and can be found in [22, 75] . words of lscdc were represented by vectors of rigs in 252-dimensional meaning space as described before. the word-category matrix for the lsc was formed with the collection of all words of the lscdc [76]. the sum s j and the maximum m j of rigs in categories are calculated and added at the end of the matrix. word clouds with the top 100 words and histograms of the most informative 10 words for each category are presented in [81] . the most informative 100 words for each category with their rigs can be found in appendix e and [81]. the proposed model of rig-based word representation is analysed through these top ranked words in each category. we have evaluated the meaning space by comparing our approach to traditional frequency-based model. words in each category were also ranked and ordered by their raw frequencies in categories. it is proven that frequencies is not much important and efficient to represent scientifically specific meanings of words as the most frequent words are not topic related words such as 'use', 'studi' and 'result'. figure 3 .12 compare two approaches using word clouds for three categories. the word clouds demonstrated that the information gainbased method is capable of standing topic-specific words out. this proves that the frequency is not much important in identifying such words. by representing words in the meaning space, we have shown by the human inspection that the top words in categories are topic-related in the corresponding category. it can therefore be viewed as an evidence of the usefulness of the meaning space and the representing words in this space. s j and m j have been calculated for the lscdc words and two lists of words are created with words that are in descending orders by their s j and m j . the lists enable sorting the most important n words in science. we have compared these lists. the number of matches in the top n words in two lists are counted, where n is ranges from 100 to 50,000 (table 3 .6). the top 10 words in two lists are completely different and only 28% of words match in the first 100 words. this follows approximately 50% for the first 1,000 and 58% for the first 2,000 words. this concludes that two lists are not the same for the top words ( figure 3 .14 and figure 3 .15), however, both criteria can be used for selection of top n words regarding to task and the information required. many words in the lists have low s j and m j values. the plot of the number of words for s j and m j indicate a super exponential picks near zero s j and m j (see figure 3 .16 and figure 3 .17). the trend beyond the pick is going down almost linearly. those words with near zero values can be considered as less meaningful words for scientific texts. finally, a scientific thesaurus of english, named leicester scientific thesaurus (lsct), has been introduced. the thesaurus contains of the most informative 5,000 words from the lscdc. words in the lsct are selected by their average rigs in categories. that is, the top 5,000 most informative words in the lscdc, where words are arranged by their s j are considered as the most meaningful 5,000 words in scientific texts. the full list of words in the lsct with their s j can be found in [76] . the next focus of the research in 'quantifying of meaning' will be extraction of the meaning of text in scientific corpus from the clouds of words in the meaning space and study of more complex models in which co-occurrence of words and combination of word's meaning will be used. this, we follow the road: corpus of texts + categories → meaning space for words → geometric representation of the meaning of texts. the first two technical steps were done: the corpus of texts was collected and cleaned, and the meaning of words was represented and analysed in the meaning space. the next step will be analysis of the meaning of texts. the analysis of dictionaries is not finalised yet. this work was focused on the most informative words. they are the main scientific content words. but, for example, the frequent but non-informative words (like 'use') can be considered as generalised service words of science and deserve special analysis. it is also very desirable to extend the set of attributes for representation of the situation behind the text (figures 1.2, 1.3 ). the first choice, the research subject categories, is simple and natural, but it may be useful to enrich this list of attributes. [62] asher, n., van dance 74 appendix d. word clouds and histograms for categories. word clouds presenting the top 100 words ordered by their rigs and histograms of rigs for the first 10 words in the word clouds for 252 categories in lsc. .9 × 10 −3 2 farmer 8.7 × 10 −2 52 payment 9.9 × 10 −3 3 market 8.6 × 10 −2 53 agribusi 9.8 × 10 −3 4 farm 8.3 × 10 −2 54 economi 9.6 × 10 −3 5 price 8.1 × 10 −2 55 wtp 9.6 × 10 −3 6 food 7.2 × 10 −2 56 method 9.4 × 10 −3 7 polici 6 × 10 −2 57 choic 9.3 × 10 −3 8 product 4.8 × 10 −2 58 competit 9.3 × 10 −3 9 econom 3.6 × 10 −2 59 domest 9.1 × 10 −3 10 household 3.5 × 10 −2 60 conclus 8.8 × 10 −3 11 consum 3.3 × 10 −2 61 clinic 8.8 × 10 −3 12 incom 3.3 × 10 −2 62 nutrit 8.6 × 10 −3 13 countri 3.2 × 10 −2 63 veget 8.6 × 10 −3 14 sector 3. 1.3 × 10 −2 76 polyphenol 6.9 × 10 −3 27 feed 1.2 × 10 −2 77 genotyp 6.9 × 10 −3 28 rice 1.2 × 10 −2 78 grown 6.9 × 10 −3 29 season 1.2 × 10 −2 79 greenhous 6.8 × 10 −3 30 irrig 1.2 × 10 −2 80 anim 6.8 × 10 −3 31 veget 1.2 × 10 −2 81 intak 6.7 × 10 −3 32 nitrogen 1.2 × 10 −2 82 studi 6.7 × 10 −3 33 livestock 1.1 × 10 −2 83 ferment 6.6 × 10 −3 34 phenol 1.1 × 10 −2 84 corn 6.4 × 10 −3 35 growth 1.1 × 10 −2 85 meat 6.4 × 10 −3 36 harvest 1 × 10 −2 86 grow 6.4 × 10 −3 37 concentr 1 × 10 −2 87 fresh 6.4 × 10 −3 38 milk 1 × 10 −2 88 pakistan 6.3 × 10 −3 39 grass 9.9 × 10 −3 89 plot 6.3 × 10 −3 40 weight 9.9 × 10 −3 90 dietari 6.2 × 10 −3 41 grain 9.9 × 10 −3 91 per 6.2 × 10 −3 42 antioxid 9.8 × 10 −3 92 anthocyanin 6.1 × 10 −3 43 nutrit 9.7 × 10 −3 93 bean 6.1 × 10 −3 44 dairi 9.6 × 10 −3 94 tillag 6 × 10 −3 45 soybean 9.5 × 10 −3 95 rumin 6 × 10 −3 46 graze 9.5 × 10 −3 96 winter 5.9 × 10 −3 47 biomass 9.3 × 10 −3 97 potato 5.8 × 10 −3 48 cattl 9.2 × 10 −3 98 rumen 5.8 × 10 −3 49 manur 9.1 × 10 −3 99 compound 5.8 × 10 −3 50 trait 8.5 × 10 −3 100 grassland 5.8 × 10 −3 2.2 × 10 −2 83 tillag 1 × 10 −2 34 growth 2 × 10 −2 84 differ 9.9 × 10 −3 35 genet 1.9 × 10 −2 85 manag 9.9 × 10 −3 36 shoot 1.9 × 10 −2 86 three 9.7 × 10 −3 37 patient 1.9 × 10 −2 87 four 9.7 × 10 −3 38 field 1.8 × 10 −2 88 manur 9.6 × 10 −3 39 qtl 1.8 × 10 −2 89 grower 9.6 × 10 −3 40 veget 1.8 × 10 −2 90 qualiti 9.5 × 10 −3 41 grow 1.8 × 10 −2 91 tomato 9.4 × 10 −3 42 nutrient 1.8 × 10 −2 92 barley 9.4 × 10 −3 43 speci 1.8 × 10 −2 93 chromosom 9.2 × 10 −3 44 triticum 1.7 × 10 −2 94 method 9.1 × 10 −3 45 leav 1.7 × 10 −2 95 popul 9 × 10 −3 46 water 1.7 × 10 −2 96 treatment 8.9 × 10 −3 47 inocul 1.5 × 10 −2 97 spring 8.9 × 10 −3 48 paper 1.5 × 10 −2 98 suscept 8.8 × 10 −3 49 nitrogen 1.5 × 10 −2 99 fresh 8.7 × 10 −3 50 orchard 1.5 × 10 −2 100 progeni 8.7 × 10 −3 2.2 × 10 −2 87 cavernosum 1 × 10 −2 38 spermatogen 2 × 10 −2 88 glutathion 1 × 10 −2 39 intracytoplasm 1.9 × 10 −2 89 superoxid 9.8 × 10 −3 40 normal 1.9 × 10 −2 90 assay 9.8 × 10 −3 41 divid 1.9 × 10 −2 91 express 9.7 × 10 −3 42 level 1.9 × 10 −2 92 oestradiol 9.7 × 10 −3 43 iief 1.8 × 10 −2 93 fragment 9.6 × 10 −3 44 asthenozoosperm 1.7 × 10 −2 94 albuginea 9.6 × 10 −3 45 decreas 1.7 × 10 −2 95 simul 9.5 × 10 −3 46 normozoosperm 1.7 × 10 −2 96 stimul 9.5 × 10 −3 47 cauda 1.6 × 10 −2 97 ielt 9.5 × 10 −3 48 leydig 1.6 × 10 −2 98 lutein 9.5 × 10 −3 49 serum 1.6 × 10 −2 99 method 9.5 × 10 −3 50 icsi 1.6 × 10 −2 100 peroxid 9.5 × 10 −3 anthropolog 4.7 × 10 −2 52 radiocarbon 9.8 × 10 −3 3 ethnograph 4.5 × 10 −2 53 stone 9.8 × 10 −3 4 social 4.3 × 10 −2 54 live 9.7 × 10 −3 5 polit 3.1 × 10 −2 55 chronolog 9.6 × 10 −3 6 cultur 3.1 × 10 −2 56 individu 9.5 × 10 −3 7 argu 2.7 × 10 −2 57 world 9.1 × 10 −3 8 articl 2.4 × 10 −2 58 earli 9.1 × 10 −3 9 peopl 2.3 × 10 −2 59 debat 9 × 10 −3 10 centuri 2.1 × 10 −2 60 effect 9 × 10 −3 11 histor 2.1 × 10 −2 61 interpret 9 × 10 −3 12 neolith 2 × 10 −2 62 pleistocen 9 × 10 −3 13 site 2 × 10 −2 63 understand 8.7 × 10 −3 14 excav 1.9 × 10 −2 64 middl 8.7 × 10 −3 15 societi 1.9 × 10 −2 65 moral 8.6 × 10 −3 16 fieldwork 1. 1.9 × 10 −2 81 mesolith 1 × 10 −2 32 southern 1.9 × 10 −2 82 place 1 × 10 −2 33 interpret 1.9 × 10 −2 83 polit 1 × 10 −2 34 evid 1.8 × 10 −2 84 iron 1 × 10 −2 35 cal 1.8 × 10 −2 85 town 1 × 10 −2 36 tomb 1.8 × 10 −2 86 decor 1 × 10 −2 37 effect 1.8 × 10 −2 87 record 1 × 10 −2 38 context 1.7 × 10 −2 88 conclus 9.8 × 10 −3 39 remain 1.6 × 10 −2 89 taphonom 9.8 × 10 −3 40 mediev 1.6 × 10 −2 90 prehistori 9.7 × 10 −3 41 artifact 1.6 × 10 −2 91 east 9.7 × 10 −3 42 middl 1.6 × 10 −2 92 europ 9.7 × 10 −3 43 occup 1.6 × 10 −2 93 reduc 9.7 × 10 −3 44 patient 1.5 × 10 −2 94 isotop 9.6 × 10 −3 45 hunter 1.5 × 10 −2 95 histori 9.5 × 10 −3 46 northern 1.5 × 10 −2 96 templ 9.5 × 10 −3 47 zooarchaeolog 1.5 × 10 −2 97 citi 9.5 × 10 −3 48 social 1.5 × 10 −2 98 earliest 9.4 × 10 −3 49 cemeteri 1.4 × 10 −2 99 bone 9.4 × 10 −3 50 north 1.4 × 10 −2 100 central 9.4 × 10 −3 1.3 × 10 −2 82 sampl 6.9 × 10 −3 33 cell 1.2 × 10 −2 83 histori 6.9 × 10 −3 34 tradit 1.2 × 10 −2 84 detect 6.8 × 10 −3 35 typolog 1.2 × 10 −2 85 decreas 6.7 × 10 −3 36 sustain 1.1 × 10 −2 86 idea 6.7 × 10 −3 37 twentieth 1 × 10 −2 87 new 6.7 × 10 −3 38 conserv 9.7 × 10 −3 88 nineteenth 6.6 × 10 −3 39 earthen 9.6 × 10 −3 89 technolog 6.6 × 10 −3 40 digit 9.6 × 10 −3 90 induc 6.6 × 10 −3 41 preserv 9.5 × 10 −3 91 creation 6.6 × 10 −3 42 conclus 9.4 × 10 −3 92 acid 6.6 × 10 −3 43 show 9.3 × 10 −3 93 higher 6.5 × 10 −3 44 residenti 9.2 × 10 −3 94 survey 6.5 × 10 −3 45 villag 9.1 × 10 −3 95 dwell 6.4 × 10 −3 46 environ 9 × 10 −3 96 way 6.3 × 10 −3 47 today 8.9 × 10 −3 97 social 6.3 × 10 −3 48 clinic 8.8 × 10 −3 98 obtain 6.3 × 10 −3 49 artist 8.7 × 10 −3 99 high 6.1 × 10 −3 50 materi 8.7 × 10 −3 100 gene 6.1 × 10 −3 1.2 × 10 −2 87 beauti 6.9 × 10 −3 38 decor 1.2 × 10 −2 88 protein 6.9 × 10 −3 39 increas 1.2 × 10 −2 89 higher 6.9 × 10 −3 40 project 1.2 × 10 −2 90 low 6.9 × 10 −3 41 depict 1.2 × 10 −2 91 michelangelo 6.8 × 10 −3 42 explor 1.2 × 10 −2 92 mediev 6.7 × 10 −3 43 high 1.1 × 10 −2 93 christ 6.7 × 10 −3 44 method 1.1 × 10 −2 94 notion 6.6 × 10 −3 45 twentieth 1.1 × 10 −2 95 improv 6.6 × 10 −3 46 narrat 1.1 × 10 −2 96 induc 6.6 × 10 −3 47 social 1.1 × 10 −2 97 ratio 6.5 × 10 −3 48 effect 1 × 10 −2 98 style 6.5 × 10 −3 49 conclus 1 × 10 −2 99 roman 6.5 × 10 −3 50 music 1 × 10 −2 100 diseas 6.3 × 10 −3 telescop 9.9 × 10 −2 53 gev 1.8 × 10 −2 4 stellar 9.8 × 10 −2 54 baryon 1.8 × 10 −2 5 galact 6.6 × 10 −2 55 spectra 1.8 × 10 −2 6 mass 5.9 × 10 −2 56 near 1.7 × 10 −2 7 cosmolog 5.4 × 10 −2 57 relativist 1.6 × 10 −2 8 luminos 5.1 × 10 −2 58 space 1.6 × 10 −2 9 redshift 5 × 10 −2 59 astronom 1.6 × 10 −2 10 observ 4.9 × 10 −2 60 model 1.6 × 10 −2 11 cosmic 4.5 × 10 −2 61 giant 1.6 × 10 −2 12 gravit 4.5 × 10 −2 62 larg 1.6 × 10 −2 13 accret 3.9 × 10 −2 63 angular 1.6 × 10 −2 14 observatori 3.7 × 10 −2 64 dot 1.6 × 10 −2 15 circl 3.6 × 10 −2 65 agn 1. propos 7 × 10 −2 52 examin 1 × 10 −2 3 system 5.7 × 10 −2 53 trajectori 9.9 × 10 −3 4 control 4.4 × 10 −2 54 year 9.9 × 10 −3 5 robot 3.9 × 10 −2 55 constraint 9.5 × 10 −3 6 problem 3.8 × 10 −2 56 background 9.4 × 10 −3 7 algorithm 3.7 × 10 −2 57 converg 9.3 × 10 −3 8 simul 3.6 × 10 −2 58 motion 9.2 × 10 −3 9 lyapunov 2.6 × 10 −2 59 acid 9.2 × 10 −3 10 nonlinear 2.6 × 10 −2 60 inequ 9.1 × 10 −3 11 design 2.4 × 10 −2 61 assess 9.1 × 10 −3 12 conclus 2.3 × 10 −2 62 network 9 × 10 −3 13 studi 2. 1.9 × 10 −2 81 nest 1 × 10 −2 32 season 1.8 × 10 −2 82 prey 1 × 10 −2 33 patient 1.8 × 10 −2 83 woodland 9.9 × 10 −3 34 endem 1.8 × 10 −2 84 mammal 9.9 × 10 −3 35 north 1.8 × 10 −2 85 clinic 9.9 × 10 −3 36 extinct 1.8 × 10 −2 86 suggest 9.9 × 10 −3 37 assemblag 1.8 × 10 −2 87 america 9.8 × 10 −3 38 plant 1.7 × 10 −2 88 invertebr 9.8 × 10 −3 39 spatial 1.7 × 10 −2 89 within 9.8 × 10 −3 40 anthropogen 1.7 × 10 −2 90 disturb 9.7 × 10 −3 41 predat 1.7 × 10 −2 91 cell 9.7 × 10 −3 42 loci 1.7 × 10 −2 92 taxonom 9.7 × 10 −3 43 tree 1.6 × 10 −2 93 mountain 9.6 × 10 −3 44 northern 1.6 × 10 −2 94 nonnat 9.6 × 10 −3 45 river 1.6 × 10 −2 95 locat 9.6 × 10 −3 46 invas 1.5 × 10 −2 96 indic 9.5 × 10 −3 47 southern 1.5 × 10 −2 97 locus 9.4 × 10 −3 48 threat 1.5 × 10 −2 98 forag 9.3 × 10 −3 49 survey 1.5 × 10 −2 99 monitor 9.3 × 10 −3 50 rang 1.5 × 10 −2 100 freshwat 9.3 × 10 −3 bind 4.2 × 10 −2 52 signal 6.5 × 10 −3 3 cell 3.8 × 10 −2 53 phosphoryl 6.4 × 10 −3 4 membran 2 × 10 −2 54 target 6.3 × 10 −3 5 paper 1.9 × 10 −2 55 fold 6.3 × 10 −3 6 conform 1.6 × 10 −2 56 bilay 6.2 × 10 −3 7 angstrom 1.6 × 10 −2 57 suggest 5.8 × 10 −3 8 molecular 1.6 × 10 −2 58 kinas 5.8 × 10 −3 9 molecul 1.5 × 10 −2 59 play 5.7 × 10 −3 10 induc 1.5 × 10 −2 60 strand 5.6 × 10 −3 11 activ 1.5 × 10 −2 61 coli 5.6 × 10 −3 12 interact product 2.9 × 10 −2 54 concentr 8 × 10 −3 5 ferment 2.9 × 10 −2 55 virus 7.6 × 10 −3 6 cell 2.9 × 10 −2 56 fold 7.6 × 10 −3 7 protein 2.8 × 10 −2 57 sugar 7.5 × 10 −3 8 sequenc 2.7 × 10 −2 58 16s 7.3 × 10 −3 9 enzym 2.6 × 10 −2 59 medium 7.3 × 10 −3 10 express 2.3 × 10 −2 60 transcriptom 7.2 × 10 −3 11 paper 2.2 × 10 −2 61 hydrolysi 7 × 10 −3 12 cultur 2 × 10 −2 62 bacterium 6.9 × 10 −3 13 acid 2 × 10 −2 63 specif 6.9 × 10 −3 14 biomass 2 × 10 −2 64 mutant 6.8 × 10 −3 15 coli 2 × 10 −2 65 propos 6.7 × 10 −3 16 bacteria 1.8 × 10 −2 66 biotechnolog 6.6 × 10 −3 17 escherichia 1.8 × 10 −2 67 human 6.6 × 10 −3 18 microbi 1.6 × 10 −2 68 rrna 6.6 × 10 −3 19 bacteri 1.6 × 10 −2 69 biosynthesi 6.6 × 10 −3 20 produc 1.6 × 10 −2 70 amino 6.5 × 10 −3 21 recombin 1. 2.1 × 10 −2 77 anteced 9.8 × 10 −3 28 countri 2.1 × 10 −2 78 intent 9.7 × 10 −3 29 invest 2 × 10 −2 79 survey 9.7 × 10 −3 30 entrepreneuri 2 × 10 −2 80 theoret 9.5 × 10 −3 31 paper 1.9 × 10 −2 81 diseas 9.5 × 10 −3 32 cell 1.9 × 10 −2 82 financ 9.2 × 10 −3 33 relationship 1.8 × 10 −2 83 strategi 9.2 × 10 −3 34 practic 1.8 × 10 −2 84 stakehold 9.1 × 10 −3 35 sector 1.7 × 10 −2 85 multin 9 × 10 −3 36 entrepreneurship 1.7 × 10 −2 86 product 9 × 10 −3 37 perspect 1.7 × 10 −2 87 paramet 9 × 10 −3 38 patient 1.7 × 10 −2 88 technolog 8.9 × 10 −3 39 entrepreneur 1.5 × 10 −2 89 observ 8.9 × 10 −3 40 theori 1. crisi 4.4 × 10 −2 59 institut 1.1 × 10 −2 10 return 3.8 × 10 −2 60 incent 1.1 × 10 −2 11 capit 3.7 × 10 −2 61 protein 1 × 10 −2 12 compani 3.5 × 10 −2 62 auditor 1 × 10 −2 13 econom 3.5 × 10 −2 63 conclus 9.9 × 10 −3 14 invest 3.5 × 10 −2 64 experiment 9.9 × 10 −3 15 trade 3.3 × 10 −2 65 czech 9.9 × 10 −3 16 equiti 3.3 × 10 −2 66 industri 9.9 × 10 −3 17 economi 3.2 × 10 −2 67 impact 9.8 × 10 −3 18 corpor 3.2 × 10 −2 68 evid 9.5 × 10 −3 19 credit 3 × 10 −2 69 audit 9.5 × 10 −3 20 financ 3 × 10 −2 70 public 9.5 × 10 −3 21 countri 3 × 10 −2 71 valuat 9.1 × 10 −3 22 portfolio 3 × 10 −2 72 disclosur 9.1 × 10 −3 23 find 2.9 × 10 −2 73 romania 9 × 10 −3 24 empir 2.8 × 10 −2 74 transact 9 × 10 −3 25 debt 2.7 × 10 −2 75 diseas 8.9 × 10 −3 26 paper 2. oil 1.8 × 10 −2 57 fatti 6.9 × 10 −3 8 chromatographi 1.8 × 10 −2 58 gas 6.7 × 10 −3 9 antioxid 1.8 × 10 −2 59 synthesi 6.7 × 10 −3 10 reaction 1.7 × 10 −2 60 radic 6.6 × 10 −3 11 phenol 1.6 × 10 −2 61 outcom 6.5 × 10 −3 12 nmr 1.5 × 10 −2 62 polyphenol 6.5 × 10 −3 13 food 1. propos 8.9 × 10 −2 52 decreas 1 × 10 −2 3 algorithm 7.9 × 10 −2 53 robust 1 × 10 −2 4 problem 4 × 10 −2 54 concentr 1 × 10 −2 5 learn 3.4 × 10 −2 55 associ 1 × 10 −2 6 base 3 × 10 −2 56 age 9.9 × 10 −3 7 approach 2.8 × 10 −2 57 model 9.9 × 10 −3 8 fuzzi 2.7 × 10 −2 58 signific 9.5 × 10 −3 9 dataset 2.7 × 10 −2 59 can 9.5 × 10 −3 10 comput 2.5 × 10 −2 60 vector 9.4 × 10 −3 11 studi 2.5 × 10 −2 61 induc 9.4 × 10 −3 12 conclus 2.4 × 10 −2 62 investig 9.3 × 10 −3 13 real 2.2 × 10 −2 63 scene 9 × 10 −3 14 classif 2.2 × 10 −2 64 experi 9 × 10 −3 15 task 2.2 × 10 −2 65 materi 8.9 × 10 −3 16 art 2 × 10 −2 66 speci 8.7 × 10 −3 17 robot 2 × 10 −2 67 clinic 8.6 × 10 −3 18 featur 1.9 × 10 −2 68 report 8. 1 × 10 −2 94 provid 6.9 × 10 −3 45 observ 1 × 10 −2 95 video 6.9 × 10 −3 46 deploy 1 × 10 −2 96 report 6.9 × 10 −3 47 task 1 × 10 −2 97 onlin 6.9 × 10 −3 48 data 1 × 10 −2 98 environ 6.9 × 10 −3 49 age 1 × 10 −2 99 issu 6.9 × 10 −3 50 dataset 1 × 10 −2 100 scenario 6.9 × 10 −3 7 × 10 −3 79 discret 3.9 × 10 −3 30 induc 6.9 × 10 −3 80 virtual 3.9 × 10 −3 31 decreas 6.8 × 10 −3 81 servic 3.9 × 10 −3 32 suggest 6.7 × 10 −3 82 order 3.9 × 10 −3 33 associ 6.5 × 10 −3 83 examin 3.9 × 10 −3 34 machin 6.4 × 10 −3 84 appli 3. 1.1 × 10 −2 84 run 6.9 × 10 −3 35 clinic 1 × 10 −2 85 represent 6.8 × 10 −3 36 abstract 1 × 10 −2 86 enabl 6.8 × 10 −3 37 protein 1 × 10 −2 87 examin 6.7 × 10 −3 38 tool 1 × 10 −2 88 check 6.7 × 10 −3 39 runtim 1 × 10 −2 89 reaction 6.6 × 10 −3 40 servic 9.9 × 10 −3 90 indic 6.6 × 10 −3 41 concentr 9.6 × 10 −3 91 background 6.5 × 10 −3 42 investig 9.6 × 10 −3 92 make 6.5 × 10 −3 43 video 9.5 × 10 −3 93 support 6.4 × 10 −3 44 acid 9.5 × 10 −3 94 server 6.4 × 10 −3 45 set 9.4 × 10 −3 95 group 6.2 × 10 −3 46 graph 9.3 × 10 −3 96 platform 6.2 × 10 −3 47 age 9.3 × 10 −3 97 higher 6.1 × 10 −3 48 inform 9.3 × 10 −3 98 domain 6 × 10 −3 49 diseas 9.2 × 10 −3 99 provid 6 × 10 −3 50 queri 9 × 10 −3 100 featur 5.9 × 10 −3 propos 6.7 × 10 −2 52 surfac 9.7 × 10 −3 3 algorithm 5.7 × 10 −2 53 diseas 9.6 × 10 −3 4 user 3.4 × 10 −2 54 introduc 9.6 × 10 −3 5 comput 3.3 × 10 −2 55 queri 9.5 × 10 −3 6 problem 3 × 10 −2 56 indic 9.5 × 10 −3 7 conclus 2.7 × 10 −2 57 web 9.3 × 10 −3 8 network 2.5 × 10 −2 58 materi 9.3 × 10 −3 9 studi 2.5 × 10 −2 59 solv 9 × 10 −3 10 base 2.2 × 10 −2 60 effici 8.9 × 10 −3 11 patient 1.9 × 10 −2 61 gene 8.9 × 10 −3 12 secur 1. 1 × 10 −2 95 exist 6.9 × 10 −3 46 node 1 × 10 −2 96 mass 6.7 × 10 −3 47 automat 1 × 10 −2 97 platform 6.7 × 10 −3 48 framework 9.9 × 10 −3 98 messag 6.7 × 10 −3 49 speci 9.9 × 10 −3 99 prepar 6.7 × 10 −3 50 decreas 9.8 × 10 −3 100 resourc 6.7 × 10 −3 icu 9.9 × 10 −2 53 injur 2 × 10 −2 4 injuri 9.4 × 10 −2 54 sever 2 × 10 −2 5 hospit 8.4 × 10 −2 55 shock 2 × 10 −2 6 care 8 × 10 −2 56 death 2 × 10 −2 7 mortal 6.7 × 10 −2 57 odd 2 × 10 −2 8 trauma 6.7 × 10 −2 58 inhospit 2 × 10 −2 9 ventil 6.5 × 10 −2 59 adult 2 × 10 −2 10 outcom 6.4 × 10 −2 60 surviv 1.9 × 10 −2 11 admiss 5.6 × 10 −2 61 iss 1.8 × 10 −2 12 resuscit 5.5 × 10 −2 62 ohca 1.8 × 10 −2 13 prospect 4.9 × 10 −2 63 critic 1. 1.9 × 10 −2 79 perform 9.9 × 10 −3 30 union 1.9 × 10 −2 80 subsaharan 9.9 × 10 −3 31 argu 1.9 × 10 −2 81 mortal 9.9 × 10 −3 32 born 1.9 × 10 −2 82 europ 9.6 × 10 −3 33 transnat 1.9 × 10 −2 83 abroad 9.3 × 10 −3 34 refuge 1. 1.7 × 10 −2 94 find 9.9 × 10 −3 45 question 1.7 × 10 −2 95 peer 9.9 × 10 −3 46 lectur 1.6 × 10 −2 96 nation 9.9 × 10 −3 47 elearn 1.6 × 10 −2 97 peopl 9.7 × 10 −3 48 instructor 1.6 × 10 −2 98 acid 9.7 × 10 −3 49 collabor 1.6 × 10 −2 99 work 9.6 × 10 −3 50 questionnair 1.6 × 10 −2 100 issu 9.6 × 10 −3 aerodynam 3.6 × 10 −2 56 moon 1 × 10 −2 7 satellit 3.2 × 10 −2 57 diseas 9.9 × 10 −3 8 paper 3 × 10 −2 58 motion 9.6 × 10 −3 9 simul 2.5 × 10 −2 59 flowfield 9.5 × 10 −3 10 maneuv 2.5 × 10 −2 60 track 9.5 × 10 −3 11 space 2.5 × 10 −2 61 hyperson 9.3 × 10 −3 12 earth 2. 6.8 × 10 −3 79 propos 4 × 10 −3 30 regener 6.8 × 10 −3 80 bci 3.9 × 10 −3 31 signal 6.7 × 10 −3 81 postur 3.9 × 10 −3 32 knee 6.7 × 10 −3 82 algorithm 3.9 × 10 −3 33 brain 6.5 × 10 −3 83 compar 3.8 × 10 −3 34 stiff 6.4 × 10 −3 84 hemodynam 3.8 × 10 −3 35 joint 6.3 × 10 −3 85 dental 3.8 × 10 −3 36 ecg 6.2 × 10 −3 86 plant 3.8 × 10 −3 37 cell 6.1 × 10 −3 87 temperatur 3.8 × 10 −3 38 noninvas 5.9 × 10 −3 88 method 3.7 × 10 −3 39 rehabilit 5.9 × 10 −3 89 hip 3.7 × 10 −3 40 biomed 5.8 × 10 −3 90 regen 3.7 × 10 −3 41 walk 5.8 × 10 −3 91 ventricular 3.6 × 10 −3 42 tomographi 5.7 × 10 −3 92 perform 3.6 × 10 −3 43 load 5.7 × 10 −3 93 attach 3.6 × 10 −3 44 limb 5.7 × 10 −3 94 ultrasound 3.6 × 10 −3 45 forc 5.7 × 10 −3 95 neural 3.6 × 10 −3 46 physiolog 5.6 × 10 −3 96 registr 3.6 × 10 −3 47 emg 5.5 × 10 −3 97 base 3.5 × 10 −3 48 osteogen 5.3 × 10 −3 98 promis 3.5 × 10 −3 49 reconstruct 5.3 × 10 −3 99 biomimet 3.5 × 10 −3 50 deliveri 5.2 × 10 −3 100 modulus 3.5 × 10 −3 machin 3.9 × 10 −2 52 surfac 6.1 × 10 −3 3 paper 3 × 10 −2 53 strength 6.1 × 10 −3 4 process 2.7 × 10 −2 54 treatment 6.1 × 10 −3 5 workpiec 1.9 × 10 −2 55 popul 6.1 × 10 −3 6 cut 1.7 × 10 −2 56 custom 5.9 × 10 −3 7 patient 1.7 × 10 −2 57 cancer 5.9 × 10 −3 8 weld 1.6 × 10 −2 58 compani 5.7 × 10 −3 9 conclus 1.6 × 10 −2 59 aluminum 5.6 × 10 −3 10 industri 1.5 × 10 −2 60 deform 5.6 × 10 −3 11 steel 1.5 × 10 −2 61 taguchi 5.5 × 10 −3 12 tool 1.4 × 10 −2 62 grind 5.5 × 10 −3 13 alloy 1. 6.9 × 10 −3 90 inhibit 4.1 × 10 −3 41 express 6.9 × 10 −3 91 fabric 4 × 10 −3 42 report 6.8 × 10 −3 92 assay 4 × 10 −3 43 may 6.7 × 10 −3 93 day 4 × 10 −3 44 hard 6.6 × 10 −3 94 demand 4 × 10 −3 45 cost 6.6 × 10 −3 95 problem 3.9 × 10 −3 46 propos 6.6 × 10 −3 96 heat 3.9 × 10 −3 47 microstructur 6.5 × 10 −3 97 bind 3.9 × 10 −3 48 finit 6.4 × 10 −3 98 male 3.9 × 10 −3 49 engin 6.3 × 10 −3 99 sheet 3.9 × 10 −3 50 year 6.2 × 10 −3 100 metal 3.9 × 10 −3 8.6 × 10 −3 72 order 3.9 × 10 −3 23 base 8.3 × 10 −3 73 evid 3.9 × 10 −3 24 system 8.2 × 10 −3 74 popul 3.9 × 10 −3 25 exampl 7.9 × 10 −3 75 bind 3.9 × 10 −3 26 signific 7.2 × 10 −3 76 vitro 3.9 × 10 −3 27 group 7.1 × 10 −3 77 solut 3.8 × 10 −3 28 background 6.6 × 10 −3 78 mice 3.8 × 10 −3 29 comput 5.9 × 10 −3 79 therapeut 3.7 × 10 −3 30 activ 5.9 × 10 −3 80 pathway 3.6 × 10 −3 31 cancer 5.9 × 10 −3 81 cours 3.6 × 10 −3 32 machin 5.9 × 10 −3 82 beta 3.5 × 10 −3 33 report 5.8 × 10 −3 83 observ 3.5 × 10 −3 34 nonlinear 5.7 × 10 −3 84 day 3.5 × 10 −3 35 may 5.7 × 10 −3 85 learn 3.4 × 10 −3 36 studi 5.6 × 10 −3 86 male 3.4 × 10 −3 37 acid 5.5 × 10 −3 87 process 3.4 × 10 −3 38 model 5. 2.2 × 10 −2 77 perform 1.1 × 10 −2 28 context 2 × 10 −2 78 contest 1.1 × 10 −2 29 latino 2 × 10 −2 79 use 1.1 × 10 −2 30 communiti 2 × 10 −2 80 struggl 1 × 10 −2 31 contemporari 2 × 10 −2 81 school 1 × 10 −2 32 citizenship 1.9 × 10 −2 82 econom 1 × 10 −2 33 african 1.9 × 10 −2 83 diaspora 1 × 10 −2 34 countri 1.9 × 10 −2 84 right 1 × 10 −2 35 peopl 1.8 × 10 −2 85 temperatur 1 × 10 −2 36 muslim 1.8 × 10 −2 86 among 1 × 10 −2 37 racist 1.8 × 10 −2 87 obtain 9.9 × 10 −3 38 interview 1.8 × 10 −2 88 gender 9.9 × 10 −3 39 postraci 1.7 × 10 −2 89 perceiv 9.9 × 10 −3 40 result 1.7 × 10 −2 90 british 9.9 × 10 −3 41 accultur 1.7 × 10 −2 91 group 9.8 × 10 −3 42 ethnograph 1.6 × 10 −2 92 discurs 9.6 × 10 −3 43 attitud 1.6 × 10 −2 93 understand 9.6 × 10 −3 44 debat 1.6 × 10 −2 94 state 9.6 × 10 −3 45 migrat 1.6 × 10 −2 95 surfac 9.4 × 10 −3 46 narrat 1.6 × 10 −2 96 antiracist 9.4 × 10 −3 47 religi 1.5 × 10 −2 97 themselv 9.4 × 10 −3 48 explor 1.5 × 10 −2 98 histor 9.3 × 10 −3 49 usa 1.5 × 10 −2 99 nationalist 9.3 × 10 −3 50 educ 1.4 × 10 −2 100 war 9.2 × 10 −3 phylogenet 8 × 10 −2 53 patient 1.8 × 10 −2 4 genet 6.9 × 10 −2 54 dna 1.8 × 10 −2 5 popul 6.1 × 10 −2 55 sexual 1.7 × 10 −2 6 evolut 6 × 10 −2 56 nuclear 1.7 × 10 −2 7 diverg 5.9 × 10 −2 57 monophyli 1.6 × 10 −2 8 lineag 5.6 × 10 −2 58 evid 1.6 × 10 −2 9 clade 5 × 10 −2 59 plant 1.6 × 10 −2 10 phylogeni 4.9 × 10 −2 60 taxon 1.5 × 10 −2 11 taxa 4.9 × 10 −2 61 polymorph 1.5 × 10 −2 12 divers 4.8 × 10 −2 62 conserv 1. 1.9 × 10 −2 85 literari 1 × 10 −2 36 director 1.9 × 10 −2 86 compar 1 × 10 −2 37 bbc 1.9 × 10 −2 87 discuss 1 × 10 −2 38 portray 1.8 × 10 −2 88 theatric 1 × 10 −2 39 news 1.8 × 10 −2 89 theme 1 × 10 −2 40 popular 1.8 × 10 −2 90 applic 1 × 10 −2 41 screenplay 1.8 × 10 −2 91 industri 1 × 10 −2 42 american 1.7 × 10 −2 92 higher 1 × 10 −2 43 writer 1.7 × 10 −2 93 focus 9.8 × 10 −3 44 imagin 1.7 × 10 −2 94 histor 9.7 × 10 −3 45 way 1.7 × 10 −2 95 evalu 9.6 × 10 −3 46 artist 1.7 × 10 −2 96 effect 9.6 × 10 −3 47 style 1.6 × 10 −2 97 temperatur 9.5 × 10 −3 48 filmic 1.6 × 10 −2 98 debat 9.5 × 10 −3 49 comedi 1.6 × 10 −2 99 observ 9.4 × 10 −3 50 theatr 1.6 × 10 −2 100 discurs 9.3 × 10 −3 1.6 × 10 −2 95 bass 9.9 × 10 −3 46 popul 1.6 × 10 −2 96 pelag 9.8 × 10 −3 47 hatch 1.6 × 10 −2 97 conserv 9.8 × 10 −3 48 weight 1.5 × 10 −2 98 total 9.5 × 10 −3 49 salmonid 1.5 × 10 −2 99 anguillarum 9.5 × 10 −3 50 tilapia 1.4 × 10 −2 100 flounder 9.4 × 10 −3 folk 9 × 10 −2 53 vernacular 1.5 × 10 −2 4 folklorist 8.2 × 10 −2 54 today 1.5 × 10 −2 5 centuri 6.5 × 10 −2 55 histori 1.5 × 10 −2 6 cultur 6.1 × 10 −2 56 measur 1.5 × 10 −2 7 ritual 5.3 × 10 −2 57 anthropolog 1.5 × 10 −2 8 tradit 5.3 × 10 −2 58 alan 1.5 × 10 −2 9 legend 4.6 × 10 −2 59 supernatur 1.5 × 10 −2 10 narrat 4.3 × 10 −2 60 written 1.5 × 10 −2 11 result 4.2 × 10 −2 61 witch 1.4 × 10 −2 12 indigen 3.5 × 10 −2 62 postmediev 1.4 × 10 −2 13 song 3. antioxid 4 × 10 −2 54 solubl 1.1 × 10 −2 5 milk 3.8 × 10 −2 55 anthocyanin 1.1 × 10 −2 6 product 3.6 × 10 −2 56 raw 1.1 × 10 −2 7 extract 3.2 × 10 −2 57 respect 1.1 × 10 −2 8 chromatographi 2.9 × 10 −2 58 grape 1.1 × 10 −2 9 phenol 2.9 × 10 −2 59 moistur 1 × 10 −2 10 concentr 2.9 × 10 −2 60 whey 1 × 10 −2 11 compound 2.6 × 10 −2 61 lipid 1 × 10 −2 12 sensori 2.4 × 10 −2 62 min 1 × 10 −2 13 sampl 2.4 × 10 −2 63 contamin 9.9 × 10 −3 14 fruit 2.3 × 10 −2 64 monocytogen 9.8 × 10 −3 15 ferment 2.2 × 10 −2 65 flavor 9.7 × 10 −3 16 oil 2.2 × 10 −2 66 activ 9.7 × 10 −3 17 meat 2.2 × 10 −2 67 liquid 9.6 × 10 −3 18 hplc 2 × 10 −2 68 commerci 9.6 × 10 −3 19 flour 1.9 × 10 −2 69 water 9.6 × 10 −3 20 storag 1.9 × 10 −2 70 qualiti 9.6 × 10 −3 21 fatti 1.8 × 10 −2 71 edibl 9.6 × 10 −3 22 dri 1.8 × 10 −2 72 shelf 9.5 × 10 −3 23 dairi 1.8 × 10 −2 73 isol 9.4 × 10 −3 24 polyphenol 1.7 × 10 −2 74 bacteria 9.4 × 10 −3 25 fat 1.7 × 10 −2 75 highest 9.4 × 10 −3 26 starch 1.7 × 10 −2 76 textur 9.2 × 10 −3 27 patient 1.7 × 10 −2 77 enzym 9.2 × 10 −3 28 paper 1.7 × 10 −2 78 digest 9.1 × 10 −3 29 cook 1.6 × 10 −2 79 bread 9 × 10 −3 30 dpph 1.5 × 10 −2 80 salmonella 9 × 10 −3 31 juic 1.5 × 10 −2 81 lactic 8.9 × 10 −3 32 wine 1.4 × 10 −2 82 contain 8.8 × 10 −3 33 spectrometri 1. background 7 × 10 −2 56 recurr 2 × 10 −2 7 diseas 6.7 × 10 −2 57 bile 2 × 10 −2 8 endoscop 6.7 × 10 −2 58 infect 2 × 10 −2 9 bowel 6.6 × 10 −2 59 diagnos 1.9 × 10 −2 10 resect 4.7 × 10 −2 60 postop 1.9 × 10 −2 11 cirrhosi 4.6 × 10 −2 61 prospect 1.9 × 10 −2 12 clinic 4.2 × 10 −2 62 abdomin 1.9 × 10 −2 13 crohn 4.2 × 10 −2 63 multivari 1.9 × 10 −2 14 gastric 4 × 10 −2 64 pylori 1.8 × 10 −2 15 hepatocellular 3.9 × 10 −2 65 hospit 1.8 × 10 −2 16 underw 3.9 × 10 −2 66 age 1.8 × 10 −2 17 pancreat 3.8 × 10 −2 67 fibrosi 1.8 × 10 −2 18 coliti 3.7 × 10 −2 68 hbv 1.7 × 10 −2 19 endoscopi 3.7 × 10 −2 69 rectal 1.7 × 10 −2 20 cancer 3.7 × 10 −2 70 transplant 1.7 × 10 −2 21 ulcer 3.5 × 10 −2 71 helicobact 1.7 × 10 −2 22 treatment 3.4 × 10 −2 72 virus 1.7 × 10 −2 23 therapi 3.4 × 10 −2 73 serum 1.6 × 10 −2 24 gastrointestin 3.3 × 10 −2 74 result 1.6 × 10 −2 25 colorect 3.2 × 10 −2 75 treat 1.6 × 10 −2 26 carcinoma 3.2 × 10 −2 76 includ 1.6 × 10 −2 27 retrospect 3.1 × 10 −2 77 factor 1.6 × 10 −2 28 paper 3.1 × 10 −2 78 lesion 1.6 × 10 −2 29 chronic 3.1 × 10 −2 79 nonalcohol 1.6 × 10 −2 30 associ 2.9 × 10 −2 80 bleed 1.5 × 10 −2 31 hcc 2.9 × 10 −2 81 cohort 1.5 × 10 −2 32 esophag 2.9 × 10 −2 82 mortal 1.5 × 10 −2 33 ibd 2.9 × 10 −2 83 structur 1.5 × 10 −2 34 surgeri 2.8 × 10 −2 84 group 1.5 × 10 −2 35 outcom 2.7 × 10 −2 85 score 1.5 × 10 −2 36 complic 2.7 × 10 −2 86 propos 1. 2 × 10 −2 82 epigenet 1 × 10 −2 33 delet 2 × 10 −2 83 chromatin 9.9 × 10 −3 34 microsatellit 1.9 × 10 −2 84 missens 9.9 × 10 −3 35 transcriptom 1.9 × 10 −2 85 specif 9.7 × 10 −3 36 autosom 1.9 × 10 −2 86 solut 9.5 × 10 −3 37 cell 1.8 × 10 −2 87 regulatori 9.4 × 10 −3 38 phylogenet 1.8 × 10 −2 88 analysi 9.1 × 10 −3 39 suggest 1.8 × 10 −2 89 previous 9 × 10 −3 40 molecular 1.7 × 10 −2 90 evolut 8.9 × 10 −3 41 divers 1. .4 × 10 −2 51 radar 1.7 × 10 −2 2 rock 8.5 × 10 −2 52 northern 1.6 × 10 −2 3 mantl 6.9 × 10 −2 53 constrain 1.6 × 10 −2 4 crust 5.2 × 10 −2 54 model 1.6 × 10 −2 5 crustal 4.9 × 10 −2 55 orogen 1.6 × 10 −2 6 earthquak 4.7 × 10 −2 56 composit 1.6 × 10 −2 7 zone 4.6 × 10 −2 57 conclus 1.5 × 10 −2 8 isotop 4.5 × 10 −2 58 belt 1.5 × 10 −2 9 subduct 4.2 × 10 −2 59 upper 1.5 × 10 −2 10 tecton 4.1 × 10 −2 60 near 1.5 × 10 −2 11 geolog 4 × 10 −2 61 emplac 1.5 × 10 −2 12 lithospher 4 × 10 −2 62 estim 1.5 × 10 −2 13 earth 3.9 × 10 −2 63 fluid 1. 1.9 × 10 −2 70 livelihood 1 × 10 −2 21 explor 1.9 × 10 −2 71 clinic 1 × 10 −2 22 actor 1.9 × 10 −2 72 world 1 × 10 −2 23 territori 1.8 × 10 −2 73 cultur 1 × 10 −2 24 region 1.8 × 10 −2 74 result 1 × 10 −2 25 space 1.8 × 10 −2 75 develop 9.9 × 10 −3 26 patient 1.7 × 10 −2 76 particular 9.9 × 10 −3 27 communiti 1.7 × 10 −2 77 emerg 9.7 × 10 −3 28 context 1.7 × 10 −2 78 ecolog 9.7 × 10 −3 29 focus 1.7 × 10 −2 79 question 9.6 × 10 −3 30 public 1.7 × 10 −2 80 conceptu 9.6 × 10 −3 31 discours 1.6 × 10 −2 81 concept 9.3 × 10 −3 32 paper 1.6 × 10 −2 82 residenti 9.3 × 10 −3 33 socio 1.6 × 10 −2 83 protein 9.1 × 10 −3 34 nation 1.6 × 10 −2 84 map 9 × 10 −3 35 plan 1.6 × 10 −2 85 within 8.9 × 10 −3 36 understand 1.6 × 10 −2 86 resid 8.9 × 10 −3 37 engag 1.5 × 10 −2 87 natur 8. glacial 4.2 × 10 −2 56 topograph 1.6 × 10 −2 7 spatial 4 × 10 −2 57 eastern 1.6 × 10 −2 8 land 3.9 × 10 −2 58 south 1.6 × 10 −2 9 glacier 3.6 × 10 −2 59 geograph 1.6 × 10 −2 10 pleistocen 3.4 × 10 −2 60 period 1.6 × 10 −2 11 veget 3.3 × 10 −2 61 stratigraph 1.5 × 10 −2 12 sea 3.3 × 10 −2 62 season 1.5 × 10 −2 13 river 3.1 × 10 −2 63 east 1.5 × 10 −2 14 region 3. 3.6 × 10 −2 72 method 2 × 10 −2 23 sandston 3.6 × 10 −2 73 geochemistri 2 × 10 −2 24 metamorph 3.5 × 10 −2 74 clastic 2 × 10 −2 25 jurass 3.4 × 10 −2 75 grain 2 × 10 −2 26 middl 3.3 × 10 −2 76 ocean 2 × 10 −2 27 continent 3.3 × 10 −2 77 paleozo 1.9 × 10 −2 28 shallow 3.3 × 10 −2 78 uplift 1.9 × 10 −2 29 crust 3.2 × 10 −2 79 form 1.9 × 10 −2 30 earli 3.2 × 10 −2 80 date 1.9 × 10 −2 31 north 3.2 × 10 −2 81 siliciclast 1.9 × 10 −2 32 fossil 3.2 × 10 −2 82 fauna 1.9 × 10 −2 33 southern 3.2 × 10 −2 83 eocen 1.9 × 10 −2 34 limeston 3.1 × 10 −2 84 biostratigraph 1.9 × 10 −2 35 northern 3 × 10 −2 85 cambrian 1.9 × 10 −2 36 interpret 3 × 10 −2 86 occur 1.8 × 10 −2 37 belt 3 × 10 −2 87 coeval 1.8 × 10 −2 38 sea 3 × 10 −2 88 western 1.8 × 10 −2 39 orogen 2.9 × 10 −2 89 terran 1.8 × 10 −2 40 eastern 2.9 × 10 −2 90 central 1. dementia 7.5 × 10 −2 54 caregiv 1.6 × 10 −2 5 cognit 6.9 × 10 −2 55 frail 1.6 × 10 −2 6 adult 6.5 × 10 −2 56 adjust 1.5 × 10 −2 7 conclus 6 × 10 −2 57 daili 1.5 × 10 −2 8 particip 5.7 × 10 −2 58 comorbid 1.5 × 10 −2 9 geriatr 5.2 × 10 −2 59 result 1.5 × 10 −2 10 alzheim 5 × 10 −2 60 memori 1. 1.9 × 10 −2 93 includ 1 × 10 −2 44 symptom 1.9 × 10 −2 94 sex 1 × 10 −2 45 men 1.9 × 10 −2 95 walk 1 × 10 −2 46 signific 1.9 × 10 −2 96 brain 1 × 10 −2 47 cohort 1.8 × 10 −2 97 odd 1 × 10 −2 48 popul 1.8 × 10 −2 98 logist 1 × 10 −2 49 background 1.8 × 10 −2 99 interview 9.9 × 10 −3 50 group 1.8 × 10 −2 100 amyloid 9.9 × 10 −3 sustain 3.7 × 10 −2 52 system 7.2 × 10 −3 3 environment 3.3 × 10 −2 53 oper 7.2 × 10 −3 4 renew 2.7 × 10 −2 54 ghg 6.9 × 10 −3 5 co2 2.3 × 10 −2 55 yield 6.8 × 10 −3 6 product 2.3 × 10 −2 56 develop 6.7 × 10 −3 7 econom 2.2 × 10 −2 57 gene 6.7 × 10 −3 8 fuel 2.2 × 10 −2 58 plant 6.5 × 10 −3 9 patient 1.9 × 10 −2 59 biodiesel 6.5 × 10 −3 10 wast 1.8 × 10 −2 60 climat 6.5 × 10 −3 11 turbin 1.7 × 10 −2 61 solvent 6.4 × 10 −3 12 carbon 1.6 × 10 −2 62 age 6.3 × 10 −3 13 catalyst 1.6 × 10 −2 63 express 6.3 × 10 −3 14 solar 1.6 × 10 −2 64 eco 6.2 × 10 −3 15 wind 1.5 × 10 −2 65 catalyt 6.1 × 10 −3 16 biomass 1.5 × 10 −2 66 save 6 × 10 −3 17 electr 1.5 × 10 −2 67 temperatur 5.8 × 10 −3 18 effici 1. 1.6 × 10 −2 92 expenditur 9.9 × 10 −3 43 year 1.6 × 10 −2 93 payment 9.9 × 10 −3 44 regress 1.6 × 10 −2 94 address 9.9 × 10 −3 45 data 1.6 × 10 −2 95 live 9.8 × 10 −3 46 incom 1.5 × 10 −2 96 satisfact 9.8 × 10 −3 47 background 1.5 × 10 −2 97 identifi 9.8 × 10 −3 48 access 1.5 × 10 −2 98 report 9.8 × 10 −3 49 implement 1.5 × 10 −2 99 receiv 9.7 × 10 −3 50 ill 1.5 × 10 −2 100 energi 9.5 × 10 −3 philosoph 3.6 × 10 −2 55 polit 1 × 10 −2 6 ethic 3.1 × 10 −2 56 context 9.6 × 10 −3 7 result 3 × 10 −2 57 discuss 9.6 × 10 −3 8 argument 2.9 × 10 −2 58 decreas 9.6 × 10 −3 9 scientist 2.8 × 10 −2 59 british 9.6 × 10 −3 10 articl 2.5 × 10 −2 60 higher 9.4 × 10 −3 11 claim 2.3 × 10 −2 61 draw 9.4 × 10 −3 12 philosophi 2.3 × 10 −2 62 obtain 9.3 × 10 −3 13 epistem 2.2 × 10 −2 63 measur 9.2 × 10 −3 14 view 2 × 10 −2 64 darwin 9.2 × 10 −3 15 histori 2 × 10 −2 65 contemporari 9.1 × 10 −3 16 theori 1.9 × 10 −2 66 temperatur 9.1 × 10 −3 17 essay 1.9 × 10 −2 67 increas 9 × 10 −3 18 nineteenth 1. research 3.9 × 10 −2 56 motiv 1 × 10 −2 7 athlet 3.5 × 10 −2 57 author 1 × 10 −2 8 market 3.2 × 10 −2 58 tour 1 × 10 −2 9 social 3.2 × 10 −2 59 explor 1 × 10 −2 10 visitor 2.7 × 10 −2 60 leagu 1 × 10 −2 11 footbal 2.2 × 10 −2 61 simul 9.9 × 10 −3 12 travel librarian 3.7 × 10 −2 54 provid 9.2 × 10 −3 5 citat 3.7 × 10 −2 55 collect 9.1 × 10 −3 6 user 3.2 × 10 −2 56 protein 9.1 × 10 −3 7 journal 2.6 × 10 −2 57 explor 9 × 10 −3 8 public 2.5 × 10 −2 58 bibliograph 9 × 10 −3 9 social 2.5 × 10 −2 59 induc 9 × 10 −3 10 scienc 2.4 × 10 −2 60 purpos 9 × 10 −3 11 web 2.3 × 10 −2 61 data 8.9 × 10 −3 12 academ conclus 9.6 × 10 −2 53 therapeut 1.7 × 10 −2 4 treatment 6.6 × 10 −2 54 drug 1.7 × 10 −2 5 extract 6.6 × 10 −2 55 serum 1.6 × 10 −2 6 herbal 6.2 × 10 −2 56 cytotox 1.6 × 10 −2 7 tradit 6 × 10 −2 57 western 1.5 × 10 −2 8 chines 5.3 × 10 −2 58 aim 1.5 × 10 −2 9 rat 5.2 × 10 −2 59 express 1.5 × 10 −2 10 activ 5 × 10 −2 60 day 1.5 × 10 −2 11 effect 5 × 10 −2 61 materi 1.5 × 10 −2 12 inhibit 4.9 × 10 −2 62 radix 1. argu 6.6 × 10 −2 56 studi 1.7 × 10 −2 7 judici 6.2 × 10 −2 57 normat 1.7 × 10 −2 8 suprem 6.2 × 10 −2 58 case 1.6 × 10 −2 9 right 5.5 × 10 −2 59 oblig 1.6 × 10 −2 10 legisl 5.2 × 10 −2 60 wto 1.6 × 10 −2 11 justic 5.1 × 10 −2 61 union 1.5 × 10 −2 12 doctrin 5 × 10 −2 62 privat 1.5 × 10 −2 13 rule 4.2 × 10 −2 63 cell 1.5 × 10 −2 14 feder 3.8 × 10 −2 64 concern 1.5 × 10 −2 15 statut 3.7 × 10 −2 65 prosecut 1. write 7.5 × 10 −2 58 uber 3 × 10 −2 9 die 6.9 × 10 −2 59 book 2.9 × 10 −2 10 das 6.9 × 10 −2 60 anderen 2.9 × 10 −2 11 text 6.8 × 10 −2 61 deutschen 2.9 × 10 −2 12 narrat 6.8 × 10 −2 62 werk 2.9 × 10 −2 13 articl 6.7 × 10 −2 63 zwei 2. medic 3.6 × 10 −2 52 access 6.8 × 10 −3 3 inform 3.1 × 10 −2 53 water 6.7 × 10 −3 4 data 2.5 × 10 −2 54 cell 6.5 × 10 −3 5 care 2.3 × 10 −2 55 servic 6.3 × 10 −3 6 clinic 2.3 × 10 −2 56 person 6.3 × 10 −3 7 patient 2.1 × 10 −2 57 communic 6.2 × 10 −3 8 healthcar 2.1 × 10 −2 58 concentr 6.1 × 10 −3 9 method 2 × 10 −2 59 nurs 6 × 10 −3 10 object 1.9 × 10 −2 60 background 6 × 10 −3 11 ehr 1.9 × 10 −2 61 train 5.9 × 10 −3 12 user 1.8 × 10 −2 62 evalu 5.8 × 10 −3 13 base 1. 1.6 × 10 −2 71 renal 7 × 10 −3 22 object 1.6 × 10 −2 72 abbott 7 × 10 −3 23 healthi 1.6 × 10 −2 73 spectrometri 6.9 × 10 −3 24 paper 1.6 × 10 −2 74 determin 6.9 × 10 −3 25 carcinoma 1.6 × 10 −2 75 level 6.9 × 10 −3 26 marker 1.5 × 10 −2 76 protein 6.8 × 10 −3 27 detect 1.5 × 10 −2 77 benign 6.8 × 10 −3 28 context 1.5 × 10 −2 78 quantif 6.7 × 10 −3 29 specimen 1.4 × 10 −2 79 negat 6.7 × 10 −3 30 needl 1.4 × 10 −2 80 specif 6.6 × 10 −3 31 cell 1.3 × 10 −2 81 compar 6.6 × 10 −3 32 evalu 1.3 × 10 −2 82 prognost 6.5 × 10 −3 33 routin 1.3 × 10 −2 83 cholesterol 6.5 × 10 −3 34 signific 1.2 × 10 −2 84 kit 6.5 × 10 −3 35 pathologist 1.2 × 10 −2 85 hemoglobin 6.5 × 10 −3 36 stain 1.2 × 10 −2 86 simul 6.4 × 10 −3 37 neoplasm 1.2 × 10 −2 87 subject 6.4 × 10 −3 38 test 1.2 × 10 −2 88 tandem 6.4 × 10 −3 39 diagnos 1.2 × 10 −2 89 medicin 6.4 × 10 −3 40 preanalyt 1.2 × 10 −2 90 surfac 6.4 × 10 −3 41 chromatographi 1.2 × 10 −2 91 tissu 6.3 × 10 −3 42 malign 1.2 × 10 −2 92 antibodi 6.3 × 10 −3 43 pcr 1.1 × 10 −2 93 biobank 6.3 × 10 −3 44 imprecis 1.1 × 10 −2 94 rare 6.2 × 10 −3 45 immunohistochem 1.1 × 10 −2 95 assess 6.2 × 10 −3 46 correl 1.1 × 10 −2 96 valu 6.1 × 10 −3 47 elisa 1.1 × 10 −2 97 autom 6.1 × 10 −3 48 cytomorpholog 9.9 × 10 −3 98 diabet 5.9 × 10 −3 49 sensit 9.9 × 10 −3 99 studi 5.9 × 10 −3 50 biopsi 9.7 × 10 −3 100 interassay 5.9 × 10 −3 2 × 10 −2 77 day 1.1 × 10 −2 28 includ 2 × 10 −2 78 nation 1 × 10 −2 29 assess 1.9 × 10 −2 79 admit 1 × 10 −2 30 diagnos 1.9 × 10 −2 80 prevent 1 × 10 −2 31 studi 1.9 × 10 −2 81 odd 1 × 10 −2 32 symptom 1.8 × 10 −2 82 incid 1 × 10 −2 33 receiv 1.8 × 10 −2 83 death 1 × 10 −2 34 retrospect 1.7 × 10 −2 84 treat 1 × 10 −2 35 women 1.7 × 10 −2 85 advers 9.7 × 10 −3 36 propos 1.7 × 10 −2 86 case 9.6 × 10 −3 37 mortal 1.7 × 10 −2 87 medicin 9.5 × 10 −3 38 among 1.7 × 10 −2 88 section 9.5 × 10 −3 39 signific 1.7 × 10 −2 89 surgeri 9.3 × 10 −3 40 therapi 1.7 × 10 −2 90 prospect 9.3 × 10 −3 41 chronic 1.6 × 10 −2 91 common 9.3 × 10 −3 42 score 1.6 × 10 −2 92 complic 9.3 × 10 −3 43 diabet 1.6 × 10 −2 93 men 9.2 × 10 −3 44 preval 1.6 × 10 −2 94 hypertens 9.1 × 10 −3 45 pain 1.5 × 10 −2 95 interv 9.1 × 10 −3 46 trial 1.5 × 10 −2 96 baselin 9.1 × 10 −3 47 total 1.5 × 10 −2 97 older 9 × 10 −3 48 januari 1.4 × 10 −2 98 mean 9 × 10 −3 49 cohort 1.4 × 10 −2 99 demograph 9 × 10 −3 50 follow 1.4 × 10 −2 100 heart 8.8 × 10 −3 str 3.5 × 10 −2 56 report 7.9 × 10 −3 7 casework 3.4 × 10 −2 57 accident 7.9 × 10 −3 8 toxicolog 2.6 × 10 −2 58 sex 7.8 × 10 −3 9 victim 2.6 × 10 −2 59 profil 7.6 × 10 −3 10 legal 2.5 × 10 −2 60 cadav 7.5 × 10 −3 11 crime 2.3 × 10 −2 61 pmi 7.5 × 10 −3 12 sampl 2.2 × 10 −2 62 detect 7.4 × 10 −3 13 crimin 2 × 10 −2 63 anthropologist 7.4 × 10 −3 14 suicid 1.8 × 10 −2 64 medic 7.3 × 10 −3 15 homicid 1.8 × 10 −2 65 examin 7.3 × 10 −3 16 dna 1.8 × 10 −2 66 evid 7.2 × 10 −3 17 fatal 1.7 × 10 −2 67 toxic 7.1 × 10 −3 18 medico 1.6 × 10 −2 68 caus 6.8 × 10 −3 19 male 1.5 × 10 −2 69 noael 6.8 × 10 −3 20 suspect 1.5 × 10 −2 70 human 6.8 × 10 −3 21 identif 1.4 × 10 −2 71 function 6.8 × 10 −3 22 strs 1.3 × 10 −2 72 mortem 6.6 × 10 −3 23 injuri 1.3 × 10 −2 73 year 6.3 × 10 −3 24 substanc 1.3 × 10 −2 74 murder 6.2 × 10 −3 25 blood 1.2 × 10 −2 75 assault 6.2 × 10 −3 26 scene 1.2 × 10 −2 76 amelogenin 6.2 × 10 −3 27 abus 1.2 × 10 −2 77 hair 6.2 × 10 −3 28 polic 1.2 × 10 −2 78 medicin 6.1 × 10 −3 29 firearm 1.1 × 10 −2 79 pmct 5.9 × 10 −3 30 tandem 1.1 × 10 −2 80 exposur 5.9 × 10 −3 31 femal 1.1 × 10 −2 81 popul 5.9 × 10 −3 32 antemortem 1.1 × 10 −2 82 d12s391 5.9 × 10 −3 33 anthropolog 1.1 × 10 −2 83 weapon 5.8 × 10 −3 34 pathologist 1.1 × 10 −2 84 autosom 5.8 × 10 −3 35 kit 1.1 × 10 −2 85 repeat 5.8 × 10 −3 36 corps 9.9 × 10 −3 86 seiz 5.8 × 10 −3 37 medicoleg 9.8 × 10 −3 87 structur 5.8 × 10 −3 38 skelet 9.8 × 10 −3 88 skull 5.7 × 10 −3 39 court 9.5 × 10 −3 89 trauma 5.7 × 10 −3 40 intox 9.4 × 10 −3 90 ancestri 5.6 × 10 −3 41 drug 9.3 × 10 −3 91 properti 5.6 × 10 −3 42 allel 9.3 × 10 −3 92 age 5.6 × 10 −3 43 powerplex 9.2 × 10 −3 93 multiplex 5.5 × 10 −3 44 discrimin 9 × 10 −3 94 spectrometri 5.5 × 10 −3 45 sudden 8.8 × 10 −3 95 lethal 5.5 × 10 −3 46 deceas 8.6 × 10 −3 96 poison 5.5 × 10 −3 47 fingermark 8. climat 9.5 × 10 −2 52 land 2 × 10 −2 3 aerosol 6.7 × 10 −2 53 vertic 2 × 10 −2 4 season 5.8 × 10 −2 54 atlant 2 × 10 −2 5 precipit 5.4 × 10 −2 55 patient 2 × 10 −2 6 meteorolog 5.2 × 10 −2 56 scale 2 × 10 −2 7 wind 5.1 × 10 −2 57 resolut 2 × 10 −2 8 region 5 × 10 −2 58 ensembl 2 × 10 −2 9 ocean 5 × 10 −2 59 microphys 1.9 × 10 −2 10 weather 5 × 10 −2 60 wrf 1.8 × 10 −2 11 summer 4.9 × 10 −2 61 radiat 1.8 × 10 −2 12 air 4.6 × 10 −2 62 intercomparison 1.8 × 10 −2 13 tropospher 4.6 × 10 −2 63 sst 1.8 × 10 −2 14 warm 4.6 × 10 −2 64 pollut 1.7 × 10 −2 15 tropic 4.4 × 10 −2 65 event 1.7 × 10 −2 16 sea 4.4 × 10 −2 66 synopt 1.7 × 10 −2 17 forecast 4.2 × 10 −2 67 zonal 1.7 × 10 −2 18 winter 3.9 × 10 −2 68 altitud 1.7 × 10 −2 19 satellit 3.7 × 10 −2 69 anthropogen 1.7 × 10 −2 20 rainfal 3.7 × 10 −2 70 averag 1.7 × 10 −2 21 cloud 3.6 × 10 −2 71 estim 1.7 × 10 −2 22 observ 3.5 × 10 −2 72 rain 1.6 × 10 −2 23 convect 3.4 × 10 −2 73 midlatitud 1.6 × 10 −2 24 climatolog 3.3 × 10 −2 74 eastern 1.6 × 10 −2 25 global 3.1 × 10 −2 75 impact 1.6 × 10 −2 26 north 3.1 × 10 −2 76 diurnal 1.6 × 10 −2 27 reanalysi 3 × 10 −2 77 east 1.6 × 10 −2 28 circul 2.9 × 10 −2 78 simul 1.6 × 10 −2 29 model 2.9 × 10 −2 79 water 1.6 × 10 −2 30 cyclon 2.9 × 10 −2 80 data 1.6 × 10 −2 31 station 2.9 × 10 −2 81 conclus 1.6 × 10 −2 32 pacif 2.7 × 10 −2 82 variat 1.6 × 10 −2 33 period 2.6 × 10 −2 83 humid 1.6 × 10 −2 34 temperatur 2.6 × 10 −2 84 south 1.5 × 10 −2 35 southern 2.5 × 10 −2 85 nino 1.5 × 10 −2 36 variabl 2.5 × 10 −2 86 enso 1.5 × 10 −2 37 monsoon 2.5 × 10 −2 87 mesoscal 1. 8.8 × 10 −3 77 paper 3.9 × 10 −3 28 confoc 8.7 × 10 −3 78 flim 3.9 × 10 −3 29 tomographi 8.7 × 10 −3 79 nanoscal 3.9 × 10 −3 30 laser 8.6 × 10 −3 80 organell 3.9 × 10 −3 31 tissu 8.5 × 10 −3 81 model 3.9 × 10 −3 32 scatter 8.4 × 10 −3 82 correct 3.9 × 10 −3 33 surfac 8.4 × 10 −3 83 focal 3.8 × 10 −3 34 holographi 7.6 × 10 −3 84 wavefront 3.8 × 10 −3 35 detector 7.5 × 10 −3 85 hologram 3.8 × 10 −3 36 structur 7.5 × 10 −3 86 allow 3.8 × 10 −3 37 section 7.3 × 10 −3 87 vacuol 3.8 × 10 −3 38 eel 7.2 × 10 −3 88 immunoreact 3.7 × 10 −3 39 stem 6.8 × 10 −3 89 tomograph 3.7 × 10 −3 40 thin 6.5 × 10 −3 90 year 3.7 × 10 −3 41 cytoplasm 6.3 × 10 −3 91 dentin 3.7 × 10 −3 42 fib 6.2 × 10 −3 92 subcellular 3.6 × 10 −3 43 contrast 6.2 × 10 −3 93 risk 3.6 × 10 −3 44 dark 6.2 × 10 −3 94 manag 3.6 × 10 −3 45 conclus 6.1 × 10 −3 95 defocus 3.6 × 10 −3 46 stain 6 × 10 −3 96 holder 3.6 × 10 −3 47 patient 5.9 × 10 −3 97 particip 3.6 × 10 −3 48 multiphoton 5.8 × 10 −3 98 axi 3.5 × 10 −3 49 illumin 5.6 × 10 −3 99 dimension 3.5 × 10 −3 50 monochrom 5.6 × 10 −3 100 microstructur 3.4 × 10 −3 .4 × 10 −2 51 recoveri 6 × 10 −3 2 coal 7.2 × 10 −2 52 background 6 × 10 −3 3 ore 6.2 × 10 −2 53 porphyri 5.9 × 10 −3 4 rock 5.5 × 10 −2 54 orebodi 5.9 × 10 −3 5 miner 4.4 × 10 −2 55 depth 5.9 × 10 −3 6 flotat 3.8 × 10 −2 56 sandston 5.8 × 10 −3 7 geolog 2.8 × 10 −2 57 pit 5.7 × 10 −3 8 underground 2.4 × 10 −2 week 2 × 10 −2 85 malnutrit 1.1 × 10 −2 36 studi 2 × 10 −2 86 cardiovascular 1.1 × 10 −2 37 insulin 2 × 10 −2 87 plasma 1 × 10 −2 38 assess 2 × 10 −2 88 content 1 × 10 −2 39 fruit 1.9 × 10 −2 89 lipoprotein 1 × 10 −2 40 antioxid 1.9 × 10 −2 90 trial 1 × 10 −2 41 adipos 1.8 × 10 −2 91 polyunsatur 1 × 10 −2 42 risk 1.8 × 10 −2 92 calori 9.9 × 10 −3 43 subject 1.7 × 10 −2 93 concentr 9.9 × 10 −3 44 serum 1.7 × 10 −2 94 breakfast 9.9 × 10 −3 45 group 1.7 × 10 −2 95 hdl 9.6 × 10 −3 46 intervent 1.7 × 10 −2 96 ffq 9.6 × 10 −3 47 background 1.7 × 10 −2 97 infant 9.4 × 10 −3 48 fed 1.6 × 10 −2 98 effect 9.2 × 10 −3 49 anthropometr 1.6 × 10 −2 99 decreas 8.9 × 10 −3 50 mass 1.6 × 10 −2 100 daili 8.9 × 10 −3 1.5 × 10 −1 51 activ 9.7 × 10 −3 2 laser 7.6 × 10 −2 52 risk 9.7 × 10 −3 3 wavelength 5.4 × 10 −2 53 band 9.5 × 10 −3 4 photon 4.2 × 10 −2 54 telescop 9.4 × 10 −3 5 fiber 3.8 × 10 −2 55 assess 9.3 × 10 −3 6 light 2.9 × 10 −2 56 silicon 9.2 × 10 −3 7 beam 2.8 × 10 −2 57 aim 9.1 × 10 −3 8 conclus 2.5 × 10 −2 nestl 7 × 10 −2 56 owl 1.9 × 10 −2 7 season 5.8 × 10 −2 57 songbird 1.9 × 10 −2 8 popul 5.6 × 10 −2 58 bill 1.9 × 10 −2 9 brood 5.4 × 10 −2 59 tit 1.8 × 10 −2 10 avian 5.2 × 10 −2 60 adult 1.8 × 10 −2 11 forag 5.2 × 10 −2 61 record 1.8 × 10 −2 12 chick 4.3 × 10 −2 62 juvenil 1.8 × 10 −2 13 migratori 4.2 × 10 −2 63 eastern 1.7 × 10 −2 14 winter 3.9 × 10 −2 64 paper 1.7 × 10 −2 15 conserv 3.9 × 10 −2 65 tern 1.7 × 10 −2 16 clutch 3.8 × 10 −2 66 landscap 1.7 × 10 −2 17 prey 3.6 × 10 −2 67 southern 1.7 × 10 −2 18 reproduct 3.6 × 10 −2 68 raptor 1.6 × 10 −2 19 plumag 3.6 × 10 −2 69 recaptur 1.6 × 10 −2 20 fledg 3.6 × 10 −2 70 endem 1.6 × 10 −2 21 egg 3.5 × 10 −2 71 eurasian 1.5 × 10 −2 22 site 3.4 × 10 −2 72 variat 1.5 × 10 −2 23 predat 3.2 × 10 −2 73 eagl 1.5 × 10 −2 24 passerin 3.2 × 10 −2 74 patient 1.5 × 10 −2 25 area 3.2 × 10 −2 75 mate 1.5 × 10 −2 26 territori 3.2 × 10 −2 76 sex 1.5 × 10 −2 27 warbler 3.2 × 10 −2 77 gull 1.5 × 10 −2 28 ecolog 3.2 × 10 −2 78 distanc 1.5 × 10 −2 29 abund 3. postop 7.9 × 10 −2 56 studi 2.7 × 10 −2 7 pain 7.8 × 10 −2 57 method 2.7 × 10 −2 8 hip 7.4 × 10 −2 58 later 2.6 × 10 −2 9 conclus 6.6 × 10 −2 59 distal 2.6 × 10 −2 10 surgic 6.6 × 10 −2 60 foot 2.5 × 10 −2 11 fractur 6.2 × 10 −2 61 group 2.5 × 10 −2 12 femor 6.2 × 10 −2 62 signific 2.5 × 10 −2 13 background 6.1 × 10 −2 63 treat 2.5 × 10 −2 14 score 5. 4.4 × 10 −2 77 acl 2 × 10 −2 28 underw 4.3 × 10 −2 78 motion 1.9 × 10 −2 29 tibial 4.2 × 10 −2 79 limb 1.9 × 10 −2 30 purpos 4.2 × 10 −2 80 loosen 1.9 × 10 −2 31 follow 4 × 10 −2 81 valgus 1.9 × 10 −2 32 posterior 4 × 10 −2 82 procedur 1.9 × 10 −2 33 month 4 × 10 −2 83 cadaver 1.8 × 10 −2 34 retrospect 4 × 10 −2 84 elbow 1.8 × 10 −2 35 medial 3.9 × 10 −2 85 reconstruct 1.8 × 10 −2 36 shoulder 3.8 × 10 −2 86 proxim 1.8 × 10 −2 37 complic 3.7 × 10 −2 87 tha 1.8 × 10 −2 38 mean 3.6 × 10 −2 88 heal 1.8 × 10 −2 39 spine 3.6 × 10 −2 89 patellar 1.6 × 10 −2 40 tka 3.6 × 10 −2 90 tibia 1.6 × 10 −2 41 tendon 3.5 × 10 −2 91 summari 1.6 × 10 −2 42 age 3.5 × 10 −2 92 rotat 1.6 × 10 −2 43 orthopaed 3.4 × 10 −2 93 disloc 1.6 × 10 −2 44 ankl 3.4 × 10 −2 94 scoliosi 1.6 × 10 −2 45 arthroscop 3.1 × 10 −2 95 consecut 1.6 × 10 −2 46 lumbar 3.1 × 10 −2 96 anatom 1.6 × 10 −2 47 revis 3.1 × 10 −2 97 tear 1.6 × 10 −2 48 cruciat 3 × 10 −2 98 disabl 1.6 × 10 −2 49 screw 2.9 × 10 −2 99 degen 1.6 × 10 −2 50 paper 2.9 × 10 −2 100 result 1.5 × 10 −2 cue 3.6 × 10 −2 54 cell 9.8 × 10 −3 5 behavior 3.5 × 10 −2 55 evid 9.7 × 10 −3 6 suggest 3 × 10 −2 56 train 9.7 × 10 −3 7 respons 2.9 × 10 −2 57 reinforc 9.6 × 10 −3 8 particip 2.8 × 10 −2 58 elicit 9.6 × 10 −3 9 cognit 2.6 × 10 −2 59 cortisol 9.5 × 10 −3 10 whether 2.3 × 10 −2 60 auditori 9.3 × 10 −3 11 erp 2.1 × 10 −2 61 avers 9.3 × 10 −3 particip 7.4 × 10 −2 52 cbt 1.8 × 10 −2 3 depress 6.8 × 10 −2 53 among 1.7 × 10 −2 4 symptom 6.7 × 10 −2 54 paper 1.7 × 10 −2 5 anxieti 5.7 × 10 −2 55 youth 1.7 × 10 −2 6 cognit 5.2 × 10 −2 56 trauma 1.6 × 10 −2 7 examin 4.9 × 10 −2 57 cell 1.6 × 10 −2 8 intervent 4.4 × 10 −2 58 relat parent 9.7 × 10 −2 53 relat 1.7 × 10 −2 4 age 8.7 × 10 −2 54 hyperact 1.6 × 10 −2 5 child 8.7 × 10 −2 55 anxieti 1.6 × 10 −2 6 autism 8.3 × 10 −2 56 relationship 1.6 × 10 −2 7 examin 5.6 × 10 −2 57 cell 1.6 × 10 −2 8 youth 5.6 × 10 −2 58 languag 1.5 × 10 −2 9 asd 5.4 × 10 −2 59 month 1.5 × 10 −2 10 social 5 × 10 −2 60 suggest 1.4 × 10 −2 11 year 4.9 × 10 −2 61 toddler unconsci 6 × 10 −2 55 idea 1.5 × 10 −2 6 countertransfer 5.9 × 10 −2 56 ferenczi 1.5 × 10 −2 7 psychoanalyst 5.8 × 10 −2 57 enact 1.5 × 10 −2 8 psychic 5.6 × 10 −2 polici 9.7 × 10 −2 53 focus 9.9 × 10 −3 4 articl 5.3 × 10 −2 54 make 9.8 × 10 −3 5 polit 4.7 × 10 −2 55 author 9.7 × 10 −3 6 reform 3.7 × 10 −2 56 issu 9.6 × 10 −3 7 sector 3.7 × 10 −2 57 government 9.6 × 10 −3 8 social 3.6 × 10 −2 propos 4.2 × 10 −2 54 treatment 9.6 × 10 −3 5 actuat 3.4 × 10 −2 55 protein 9.6 × 10 −3 6 humanoid 3.2 × 10 −2 56 wheel articl 5.5 × 10 −2 51 justic 9.8 × 10 −3 2 argu 5.3 × 10 −2 52 religion 9.6 × 10 −3 3 ethic 4.8 × 10 −2 53 issu 9.6 × 10 −3 4 social 4.2 × 10 −2 54 paramet 9.5 × 10 −3 5 polici 3.9 × 10 −2 55 oblig 9.4 × 10 −3 6 polit 2.9 × 10 −2 56 surfac 9.4 × 10 −3 7 moral 2.8 × 10 −2 57 individu 9.3 × 10 −3 8 welfar 2.6 × 10 −2 the meaning of meaning: a study of the influence of language upon thought and of the sea 6.5 × 10 −2 53 popul 1.7 × 10 −2 4 marin 6 × 10 −2 54 gulf 1.7 × 10 −2 5 habitat 5.1 × 10 −2 55 diatom 1.6 × 10 −2 6 water 4.7 × 10 −2 56 pacif 1.6 × 10 −2 7 abund 4.6 × 10 −2 57 larval 1.6 × 10 −2 8 coastal 3.8 × 10 −2 58 reproduct 1.5 × 10 −2 9 benthic 3.6 × 10 −2 59 mediterranean 1.5 × 10 −2 10 ecosystem 3.6 × 10 −2 60 chlorophyl 1.5 × 10 −2 11 river 3.2 × 10 −2 61 mussel 1.5 × 10 −2 12 atlant 3.2 × 10 −2 62 plankton 1.5 × 10 −2 13 fisheri 3. 2 × 10 −2 76 attach 1 × 10 −2 27 coat 2 × 10 −2 77 exhibit 1 × 10 −2 28 regener 2 × 10 −2 78 biolog 1 × 10 −2 29 bioactiv 2 × 10 −2 79 gelatin 1 × 10 −2 30 engin 2 × 10 −2 80 synthes 1 × 10 −2 31 load 1.9 × 10 −2 81 sem 1 × 10 −2 32 glycol 1.9 × 10 −2 82 dox 9.9 × 10 −3 33 chitosan 1.9 × 10 −2 83 fluoresc 9.7 × 10 −3 34 encapsul 1.8 × 10 −2 84 extracellular 9.6 × 10 −3 35 osteoblast 1.8 × 10 −2 85 titanium 9.5 × 10 −3 36 biodegrad 1.8 × 10 −2 86 gel 9.4 × 10 −3 37 polymer 1.7 × 10 −2 87 methacryl 9.3 × 10 −3 38 materi 1.7 × 10 −2 88 caprolacton 9 × 10 −3 39 osteogen 1.6 × 10 −2 89 doxorubicin 9 × 10 −3 40 phosphat 1.6 × 10 −2 90 spectroscopi 8.8 × 10 −3 41 viabil 1.6 × 10 −2 91 immobil 8.6 × 10 −3 42 promis 1.6 × 10 −2 92 assay 8.6 × 10 −3 43 fabric 1 2.1 × 10 −2 81 densiti 1 × 10 −2 32 densif 2 × 10 −2 82 hydroxyapatit 1 × 10 −2 33 spectroscopi 2 × 10 −2 83 diseas 1 × 10 −2 34 particl 2 × 10 −2 84 associ 1 × 10 −2 35 size 1.9 × 10 −2 85 character 1 × 10 −2 36 oxid 1.9 × 10 −2 86 paper 1 × 10 −2 37 conclus 1.9 × 10 −2 87 zrb2 9.9 × 10 −3 38 poros 1.8 × 10 −2 88 chemic 9.8 × 10 −3 39 crystallin 1.8 × 10 −2 89 exhibit 9.7 × 10 −3 40 calcin 1.8 × 10 −2 90 b2o3 9.7 × 10 −3 41 structur 1.8 × 10 −2 91 investig 9.7 × 10 −3 42 fabric 1.8 × 10 −2 92 level 9.6 × 10 −3 43 mpa 1.8 × 10 −2 93 mullit 9.6 × 10 −3 44 patient 1.7 × 10 −2 94 pure 9.6 × 10 −3 45 coat 1.6 × 10 −2 95 mol 9.6 × 10 −3 46 morpholog 1.6 × 10 −2 96 mechan 9.5 × 10 −3 47 sio2 1.6 × 10 −2 97 dispers 9.5 × 10 −3 48 zirconia 1.5 × 10 −2 98 vol 9.5 × 10 −3 49 precursor 1.5 × 10 −2 99 silic 9.5 × 10 −3 50 zro2 1.4 × 10 −2 100 dens 9.4 × 10 −3 207 3.5 × 10 −2 51 surfac 6.8 × 10 −3 2 crack 3 × 10 −2 52 activ 6.6 × 10 −3 3 materi 2.8 × 10 −2 53 uniaxi 6.6 × 10 −3 4 stress 2.3 × 10 −2 54 asphalt 6.4 × 10 −3 5 load 2.2 × 10 −2 55 diffract 6.1 × 10 −3 6 specimen 2.1 × 10 −2 56 damag 6.1 × 10 −3 7 tensil 2.1 × 10 −2 57 group 6 × 10 −3 8 strain 1.8 × 10 −2 58 ductil 6 × 10 −3 9 test 1.7 × 10 −2 59 displac 5.9 × 10 −3 10 weld 1.7 × 10 −2 60 popul 5.9 × 10 −3 11 strength 1.7 × 10 −2 61 plate 5.9 × 10 −3 12 deform 1.6 × 10 −2 62 harden 5.5 × 10 −3 13 patient 1.6 × 10 −2 63 cell 5.4 × 10 −3 14 microstructur 1 51 trial 9.7 × 10 −3 2 express 4 × 10 −2 52 stem 9.7 × 10 −3 3 patient 3.9 × 10 −2 53 background 9.5 × 10 −3 4 diseas 3.1 × 10 −2 54 mrna 9.4 × 10 −3 5 treatment 2.8 × 10 −2 55 temperatur 9.3 × 10 −3 6 clinic 2.7 × 10 −2 56 target 9.2 × 10 −3 7 paper 2.7 × 10 −2 57 mous 9.1 × 10 −3 8 protein 2.5 × 10 −2 58 upregul 9 × 10 −3 9 conclus 2.5 × 10 −2 59 week 8.9 × 10 −3 10 induc 2.3 × 10 −2 60 chronic 8.8 × 10 −3 11 mice 2.3 × 10 −2 61 mesenchym 8.8 × 10 −3 12 therapeut 2.2 × 10 −2 62 endotheli 8.7 × 10 −3 13 therapi 2.1 × 10 −2 63 drug 8.4 × 10 −3 14 studi 2.1 × 10 −2 64 downregul 8.3 × 10 −3 15 tissu 2 × 10 −2 65 marker 8.1 × 10 −3 16 tumor 2 × 10 −2 66 liver 7.9 × 10 −3 17 gene 1.9 × 10 −2 67 injuri 7.9 × 10 −3 18 signific 1.9 × 10 −2 68 inflamm 7.9 × 10 −3 19 immun 1.8 × 10 −2 69 bone 7.8 × 10 −3 20 cancer 1.8 × 10 −2 70 administr 7.8 × 10 −3 21 inhibit 1.8 × 10 −2 71 diabet 7.7 × 10 −3 22 human 1.7 × 10 −2 72 control 7.7 × 10 −3 23 vaccin 1.7 × 10 −2 73 inhibitor 7.7 × 10 −3 24 blood 1.7 × 10 −2 74 factor 7.7 × 10 −3 25 rat 1.5 × 10 −2 75 increas 7.7 × 10 −3 26 prolifer 1.5 × 10 −2 76 anim 7.6 × 10 −3 27 vitro 1.5 × 10 −2 77 simul 7.4 × 10 −3 28 receptor 1.5 × 10 −2 78 energi 7.3 × 10 −3 29 associ 1.4 × 10 −2 79 progress 7.1 × 10 −3 30 treat 1.4 × 10 −2 80 vascular 7 × 10 −3 31 vivo 1.3 × 10 −2 81 polymeras 7 × 10 −3 32 level 1.3 × 10 −2 82 surviv 6.9 × 10 −3 33 aim 1.3 × 10 −2 83 interleukin 6.8 × 10 −3 34 may 1.3 × 10 −2 84 inject 6.8 × 10 −3 35 group 1.2 × 10 −2 85 stimul 6.7 × 10 −3 36 apoptosi 1.2 × 10 −2 86 infect 6.7 × 10 −3 37 blot 1.2 × 10 −2 87 normal 6.7 × 10 −3 38 serum 1.2 × 10 −2 88 kinas 6.7 × 10 −3 39 antibodi 1.2 × 10 −2 89 transplant 6.7 × 10 −3 40 dose 1.2 × 10 −2 90 beta 6.7 × 10 −3 41 efficaci 1.2 × 10 −2 91 carcinoma 6.6 × 10 −3 42 assay 1.2 × 10 −2 92 pcr 6.6 × 10 −3 43 activ 1.1 × 10 −2 93 day 6.5 × 10 −3 44 regul 1.1 × 10 −2 94 prevent 6.5 × 10 −3 45 propos 1 × 10 −2 95 cellular 6.4 × 10 −3 46 inflammatori 1 × 10 −2 96 marrow 6.4 × 10 −3 47 antigen 1 × 10 −2 97 follow 6.3 × 10 −3 48 pathway 1 × 10 −2 98 pathogenesi 6.3 × 10 −3 49 mediat 9.9 × 10 −3 99 effect 6.3 × 10 −3 50 cytokin 9.8 × 10 −3 100 stain 6.3 × 10 −3 224 2.1 × 10 −2 72 teach 1 × 10 −2 23 orchestra 2 × 10 −2 73 discours 1 × 10 −2 24 aesthet 2 × 10 −2 74 scholar 1 × 10 −2 25 improvis 1.9 × 10 −2 75 manuscript 1 × 10 −2 26 explor 1.9 × 10 −2 76 way 1 × 10 −2 27 vocal 1.8 × 10 −2 77 theme 9.9 × 10 −3 28 notat 1.8 × 10 −2 78 histor 9.8 × 10 −3 29 singer 1.7 × 10 −2 79 quartet 9.8 × 10 −3 30 audienc 1.7 × 10 −2 80 temperatur 9.7 × 10 −3 31 work 1.7 × 10 −2 81 musicologist 9.6 × 10 −3 32 student 1.6 × 10 −2 82 eighteenth 9.5 × 10 −3 33 musicolog 1.6 × 10 −2 83 narrat 9.5 × 10 −3 34 teacher 1.5 × 10 −2 84 context 9.4 × 10 −3 35 voic 1.5 × 10 −2 85 creat 9.4 × 10 −3 36 school 1.5 × 10 −2 86 increas 9.4 × 10 −3 37 tonal 1.5 × 10 −2 87 musicianship 9.4 × 10 −3 38 contemporari 1 surfac 3.9 × 10 −2 54 high 1 × 10 −2 5 film 2.9 × 10 −2 55 carbon 1 × 10 −2 6 devic 2.6 × 10 −2 56 background 1 × 10 −2 7 nanostructur 2.5 × 10 −2 57 tio2 9.8 × 10 −3 8 layer 2.2 × 10 −2 58 assess 9.8 × 10 −3 9 microscopi 2.2 × 10 −2 59 risk 9.8 × 10 −3 10 metal 2.2 × 10 −2 60 particl 9.6 × 10 −3 11 graphen 2.2 × 10 −2 61 selfassembl 9.6 × 10 −3 12 properti 8.2 × 10 −3 9 accid 3.1 × 10 −2 59 dosimet 8.2 × 10 −3 10 code 3 × 10 −2 60 radon 8.2 × 10 −3 11 mev 2.9 × 10 −2 61 co60 8. 1.8 × 10 −2 89 depress 9.9 × 10 −3 40 team 1.7 × 10 −2 90 compet 9.9 × 10 −3 41 manag 1.7 × 10 −2 91 understand 9.6 × 10 −3 42 perceiv 1.7 × 10 −2 92 distress 9.5 × 10 −3 43 mental 1.7 × 10 −2 93 paramet 9.5 × 10 −3 44 life 1.6 × 10 −2 94 focus 9.3 × 10 −3 45 unit 1.6 × 10 −2 95 servic 9.2 × 10 −3 46 studi 1.6 × 10 −2 96 infant 9.2 × 10 −3 47 experienc 1.5 × 10 −2 97 live 9.2 × 10 −3 48 famili 1.5 × 10 −2 98 psycholog 9.1 × 10 −3 49 midwiv 1.5 × 10 −2 99 assess 9.1 × 10 −3 50 find 1.5 × 10 −2 100 conduct 9 × 10 −3 women 2.1 × 10 −1 51 mother 2.1 × 10 −2 2 pregnanc 1.6 × 10 −1 52 reproduct 2.1 × 10 −2 3 conclus 1.3 × 10 −1 53 prenat 2 × 10 −2 4 gestat 1 × 10 −1 54 placenta 1.9 × 10 −2 5 object 8.9 × 10 −2 55 intervent 1.9 × 10 −2 6 matern 7.9 × 10 −2 56 woman 1.9 × 10 −2 7 birth 7.3 × 10 −2 57 method 1.9 × 10 −2 8 outcom 5.9 × 10 −2 58 ultrasound 1.9 × 10 −2 9 fetal 5.9 × 10 −2 59 care 1.9 × 10 −2 10 vagin 4.8 × 10 −2 60 contracept 1.9 × 10 −2 11 obstetr 4.8 × 10 −2 61 underw 1.9 × 10 −2 12 ovarian 4.7 × 10 −2 62 hormon 1. numer 3.7 × 10 −2 54 age 1 × 10 −2 5 turbul 3.7 × 10 −2 55 magnetohydrodynam 1 × 10 −2 6 veloc 3.6 × 10 −2 56 steadi 9.7 × 10 −3 7 reynold 2.8 × 10 −2 57 cyclotron 9.6 × 10 −3 8 equat 2.8 × 10 −2 58 motion 9.6 × 10 −3 9 particl 2.7 × 10 −2 59 energi 9.4 × 10 −3 10 wave 2.6 × 10 −2 60 analyt 9.2 × 10 −3 11 tokamak 2.6 × 10 −2 61 navier 9.2 × 10 −3 12 instabl 2.5 × 10 −2 62 forc 9.2 × 10 −3 13 simul 2.3 × 10 −2 63 transit 9 × 10 −3 14 field 2.1 × 10 −2 64 divertor 9 × 10 −3 15 regim 2 × 10 −2 65 convect 8.9 × 10 −3 16 vortic 2 × 10 −2 66 propag 8.9 × 10 −3 17 discharg 2 × 10 −2 67 activ 8.7 × 10 −3 18 patient 1. collis 4 × 10 −2 59 spin 1 × 10 −2 10 gev 3.9 × 10 −2 scalar 7.3 × 10 −2 51 collis 1.9 × 10 −2 2 quark 6.4 × 10 −2 52 conclus 1.9 × 10 −2 3 higg 5.9 × 10 −2 53 increas 1.9 × 10 −2 4 gaug 5.9 × 10 −2 54 string 1.9 × 10 −2 5 lhc 5.9 × 10 −2 55 black 1.9 × 10 −2 6 cosmolog 5.3 × 10 −2 56 proton 1.8 × 10 −2 7 gev 5.2 × 10 −2 57 nucleon 1.8 × 10 −2 8 boson 5.2 × 10 −2 induc 3.7 × 10 −2 53 pathway 1.1 × 10 −2 4 rat 3.6 × 10 −2 54 studi 1 × 10 −2 5 express 3 × 10 −2 55 base 1 × 10 −2 6 increas 3 × 10 −2 56 inhibitor 1 × 10 −2 7 paper 2.7 × 10 −2 57 signal 1 × 10 −2 8 receptor 2.5 × 10 −2 58 kinas 1 × 10 −2 9 regul 2.4 × 10 −2 59 glucos 9.9 × 10 −3 10 respons 2.4 × 10 −2 60 propos 9.7 × 10 −3 11 physiolog 2.4 × 10 −2 61 mechan 9.5 × 10 −3 12 protein 2.4 × 10 −2 articl 3.4 × 10 −2 56 temperatur 1 × 10 −2 7 social 3.4 × 10 −2 57 trade 1 × 10 −2 8 polit 3.3 × 10 −2 58 labour 9.9 × 10 −3 9 urban 3.1 × 10 −2 59 hous 9.8 × 10 −3 10 economi 2.1 × 10 −2 72 pollin 1 × 10 −2 23 patient 2 × 10 −2 73 auxin 1 × 10 −2 24 breed 1.9 × 10 −2 74 model 1 × 10 −2 25 isol 1.9 × 10 −2 75 biomass 9.9 × 10 −3 26 genet 1.9 × 10 −2 76 oryza 9.9 × 10 −3 27 accumul 1.9 × 10 −2 77 dri 9.7 × 10 −3 28 genus 1.9 × 10 −2 78 morpholog 9.7 × 10 −3 29 rice 1.8 × 10 −2 79 aba 9.6 × 10 −3 30 veget 1.7 × 10 −2 80 taxonom 9.5 × 10 −3 31 grown 1.7 × 10 −2 81 marker 9.5 × 10 −3 32 tree 1.7 × 10 −2 82 weed 9.5 × 10 −3 33 chloroplast 1.6 × 10 −2 83 infloresc 9.4 × 10 −3 34 photosynthesi 1.6 × 10 −2 84 antioxid 9.2 × 10 −3 35 transgen 1.6 × 10 −2 85 forest 9.1 × 10 −3 36 drought 1.6 × 10 −2 86 product 9.1 × 10 −3 37 wheat 1.6 × 10 −2 87 comput 9.1 × 10 −3 38 mutant 1.5 × 10 −2 88 maiz 9 × 10 −3 39 transcript 1.5 × 10 −2 89 plastid 9 × 10 −3 40 wild 1.5 × 10 −2 90 qtl 9 × 10 −3 41 toler 1.5 × 10 −2 91 greenhous 8.9 × 10 −3 42 pollen 1. argu 9.3 × 10 −2 57 blur 2 × 10 −2 8 emili 7.9 × 10 −2 58 letter 2 × 10 −2 9 literari 7.5 × 10 −2 59 artist 2 × 10 −2 10 text 7.4 × 10 −2 60 pen 2 × 10 −2 11 lyric 6.3 × 10 −2 61 wife 2 × 10 −2 implic 3.9 × 10 −2 55 perspect 9.6 × 10 −3 6 psycholog 3.4 × 10 −2 56 negat 9.3 × 10 −3 7 examin 3.4 × 10 −2 57 offend 9.1 × 10 −3 8 perceiv 3.2 × 10 −2 58 temperatur 9 × 10 −3 9 relationship conclus 7.5 × 10 −2 54 relat 2 × 10 −2 5 addict 6.7 × 10 −2 55 month 2 × 10 −2 6 drug 6.7 × 10 −2 56 gambler 1.9 × 10 −2 7 smoke 6.5 × 10 −2 57 male 1.9 × 10 −2 8 particip 6.2 × 10 −2 58 youth 1.9 × 10 −2 9 abus 6.1 × 10 −2 59 misus 1.9 × 10 −2 10 abstin 5.8 × 10 −2 60 report 1.9 × 10 −2 11 smoker 5.3 × 10 −2 61 social 1.9 × 10 −2 12 background 1.6 × 10 −1 51 nrtl 9.9 × 10 −3 2 temperatur 8.8 × 10 −2 52 pipe 9.9 × 10 −3 3 thermal 7.3 × 10 −2 53 calcul 9.8 × 10 −3 4 flow 6 × 10 −2 54 mass 9.6 × 10 −3 5 transfer 5.1 × 10 −2 55 age 9.6 × 10 −3 6 cool 4.3 × 10 −2 56 obtain 9.5 × 10 −3 7 fluid 4.1 × 10 −2 57 protein 9.5 × 10 −3 8 pressur 4 × 10 −2 58 isotherm 9.5 × 10 −3 9 experiment 3.7 × 10 −2 59 drop 9.5 × 10 −3 10 convect traffic 8.4 × 10 −2 52 batteri 9.7 × 10 −3 3 paper 6.4 × 10 −2 53 demand 9.6 × 10 −3 4 road 5.8 × 10 −2 54 plan 9.1 × 10 −3 5 travel 4 × 10 −2 55 scheme 9 × 10 −3 6 transport 3.3 × 10 −2 56 asphalt 9 × 10 −3 7 propos 2.9 × 10 −2 57 intersect 8.9 × 10 −3 8 car 2.9 × 10 −2 58 crash 1.6 × 10 −2 97 coinfect 9.9 × 10 −3 48 pathogen 1.5 × 10 −2 98 stool 9.9 × 10 −3 49 countri 1.5 × 10 −2 99 larval 9.9 × 10 −3 50 area 1.5 × 10 −2 100 diagnosi 9.7 × 10 −3 313 social 3.5 × 10 −2 59 draw 1 × 10 −2 10 metropolitan 3.4 × 10 −2 60 suburban 1 × 10 −2 11 polit 3.1 × 10 −2 61 rural 1 × 10 −2 12 articl prostat 7.4 × 10 −2 56 proteinuria 2 × 10 −2 7 bladder 6.6 × 10 −2 57 treat 2 × 10 −2 8 dialysi 5.9 × 10 −2 58 nephropathi 2 × 10 −2 9 outcom 5.3 × 10 −2 59 dysfunct 2 × 10 −2 10 glomerular replic 8.7 × 10 −2 54 entri 1.6 × 10 −2 5 hiv 8.7 × 10 −2 55 nucleotid 1.6 × 10 −2 6 host 6.6 × 10 −2 56 immunodefici 1.6 × 10 −2 7 protein 6.5 × 10 −2 57 titer 1.6 × 10 −2 8 rna 6.3 × 10 −2 58 inhibit 1.6 × 10 −2 9 cell 6.1 × 10 −2 59 mediat 1.6 × 10 −2 10 antivir 5.8 × 10 −2 60 epitop 1.6 × 10 −2 11 genom 5.5 × 10 −2 61 hbv 1.6 × 10 −2 12 immun hydrolog 6.9 × 10 −2 52 drink 1.1 × 10 −2 3 river 6.8 × 10 −2 53 eros 1.1 × 10 −2 4 groundwat 6 × 10 −2 54 coastal 1.1 × 10 −2 5 soil 3.8 × 10 −2 55 storm 1 × 10 −2 6 rainfal 3.8 × 10 −2 56 ecosystem 1 × 10 −2 7 runoff 3.8 × 10 −2 57 estim 1 × 10 −2 8 flow 3.7 × 10 −2 58 impact 1 × 10 −2 9 flood 3.5 × 10 −2 59 resourc 1 × 10 −2 10 catchment habitat 3.9 × 10 −2 54 abund 1 × 10 −2 5 femal 3.9 × 10 −2 55 conserv 1 × 10 −2 6 male 3.8 × 10 −2 56 applic 1 × 10 −2 7 describ 2.6 × 10 −2 57 insect 1 × 10 −2 8 anim 2.5 × 10 −2 58 simul key: cord-343963-99rd3o79 authors: wong, mun-teng; chen, steve s-l title: emerging roles of interferon-stimulated genes in the innate immune response to hepatitis c virus infection date: 2014-12-29 journal: cell mol immunol doi: 10.1038/cmi.2014.127 sha: doc_id: 343963 cord_uid: 99rd3o79 infection with hepatitis c virus (hcv), a major viral cause of chronic liver disease, frequently progresses to steatosis and cirrhosis, which can lead to hepatocellular carcinoma. hcv infection strongly induces host responses, such as the activation of the unfolded protein response, autophagy and the innate immune response. upon hcv infection, the host induces the interferon (ifn)-mediated frontline defense to limit virus replication. conversely, hcv employs diverse strategies to escape host innate immune surveillance. type i ifn elicits its antiviral actions by inducing a wide array of ifn-stimulated genes (isgs). nevertheless, the mechanisms by which these isgs participate in ifn-mediated anti-hcv actions remain largely unknown. in this review, we first outline the signaling pathways known to be involved in the production of type i ifn and isgs and the tactics that hcv uses to subvert innate immunity. then, we summarize the effector mechanisms of scaffold isgs known to modulate ifn function in hcv replication. we also highlight the potential functions of emerging isgs, which were identified from genome-wide sirna screens, in hcv replication. finally, we discuss the functions of several cellular determinants critical for regulating host immunity in hcv replication. this review will provide a basis for understanding the complexity and functionality of the pleiotropic ifn system in hcv infection. elucidation of the specificity and the mode of action of these emerging isgs will also help to identify novel cellular targets against which effective hcv therapeutics can be developed. hepatitis c virus (hcv) infects more than 170 million people worldwide and represents a heavy burden to global health, with the highest prevalence rates found in africa and the eastern mediterranean. 1 acute hcv infection is asymptomatic, and in 70% of infected individuals, the virus persists and progresses to chronic liver diseases, including fibrosis, steatosis, cirrhosis and hepatocellular carcinoma. 2, 3 furthermore, hcv is a major cause of type i mixed cryoglobulinemia, which occurs in 10% of patients. 4 using the hcv genotype 2a isolate japanese fulminant hepatitis-1 genome-based cell culture-derived infectious hcv (hcvcc), 5 zhong et al. 6 demonstrated that hcv and cells coevolve in vitro during chronically persistent infection, which involves the selection of viral mutants with increased infectivity and cells with resistance to viral entry and/or rna replication. in this coevolution process, hcv exhibits multifaceted interactions with the host cells, and these cellular stress responses subsequently affect virus replication. for instance, infection with hcvcc or expression of the japanese fulminant hepatitis-1 genome has been shown to trigger cytopathic effects, endoplasmic reticulum (er) stress, the unfolded protein response (upr), autophagy and the innate immune response. [7] [8] [9] [10] [11] [12] [13] [14] in the competition between this virus and host cells, viral infection often triggers a first-line host defense through the production of type i interferon (ifn), which is a broadly acting antiviral cytokine, and inflammatory cytokines. these cytokines confer an antiviral state on the host cells, thereby interfering with viral replication. 15, 16 with the ability to enhance the immune response for virus clearance or to inhibit viral replication, ifn-based therapies have been used to treat hcvinfected patients for over two decades. 17 to guard against viral infection, the host cell has developed multiple restriction strategies to limit viral infection. the expression of many of these restriction factors is subject to transcriptional regulation by ifn. 13, 14 upon infection by viruses such as hcv, viral rna is first sensed by cellular pattern recognition receptors (prrs), and the prr-mediated recruitment of adaptor proteins and the activation of downstream signaling lead to ifn production. 16, 18, 19 after binding to its receptor (ifnar) complex present on the cell surface, ifn triggers the janus kinase (jak)/signal transducer and activator of transcription (stat) pathway to drive the synthesis of over 300 ifn-stimulated genes (isgs), which block virus replication at different phases of the virus replication cycle. [19] [20] [21] [22] these isgs are usually not synthesized at the basal state, but are induced to express and mediate the antiviral effector functions of ifn upon viral infection or ifn treatment. in response, viruses have developed elaborate strategies to escape the ifn antiviral system by blocking the expression or antiviral functions of ifn. 19, 23, 24 therefore, the host and hcv maintain a homeostatic state, allowing tightly restricted viral replication without killing the host. 13, [25] [26] [27] recent studies on genome-wide sirna screens have also added new candidates to this growing list of anti-hcv isgs. these findings highlight the complexity and pleiotropic roles of ifn and its induced isgs in modulating innate immunity and virus replication. nevertheless, the complete spectrum of isgs and their functionality in suppressing hcv replication have yet to be defined. 28, 29 in this review, we focus on the molecular aspects of the type i ifn system and its effector mechanisms in modulating hcv replication. first, we briefly discuss the signaling triggered by the retinoic acid-inducible gene 1-like receptor (rlr) and the toll-like receptor (tlr), which leads to type i ifn synthesis and ifn-mediated signaling pathway activation, resulting in the expression of a variety of effector isgs. we also summarize the strategies that hcv uses to escape ifn antiviral surveillance. additionally, we highlight what is currently known regarding the pivotal isgs in viral infections, with an emphasis on their anti-hcv activities, and the emerging isgs identified from recent genome-wide sirna screens in relation to anti-hcv activities. finally, we discuss the potential functions of several critical cellular factors, such as high-mobility group box 1 (hmgb1) and immunity-related gtpase family m (irgm), and cellular pathways, such as upr and autophagy, during hcv infection. although these cellular determinants are not stimulated by ifn, these factors critically control the host immune response. therefore, these determinants may also play crucial roles in modifying hcv replication. this review provides a perspective for a better understanding of the anti-hcv mechanisms of ifn, isgs and several critical cellular determinants known to contribute to the regulation of innate immunity. the gathered information not only provides a clearer picture for the specificity, functionality and complexity of the ifn system and its effector mechanisms in the control of hcv infection, but also helps to identify novel cellular targets against which efficacious therapeutic strategies can be developed. clinically, the identification of new isgs will also help to optimize the current ifn-based therapy and to provide a basis for more accurate predictions of ifn treatment outcomes. hcv is an enveloped, positive-sense, single-stranded rna virus classified within the genus hepacivirus in the flaviviridae family. 3 currently, hcv isolates are classified into seven major genotypes, i.e., genotypes 1 through 7, and an array of subtypes. 30 hcv genotypes differ by 20%-35% in genome sequence, 31 whereas subtypes within each genotype can differ by least 15%. 30 genotype 1 is the most prevalent (46%), followed by genotype 3 (30%); genotypes 2, 4 and 6 (cumulatively approximately 22%); and genotype 5 (less than 1%). 32 different genotypes exhibit distinct geographic distributions. genotype 1 predominates in america and europe, genotype 2 in japan, genotype 3 in asia, genotype 4 in africa and middle east and genotype 6 in southeast asia. 32 hcv is transmitted via blood transfusion, intravenous drug abuse, unsafe therapeutic injection, liver transplantation and other risk factors. 33 the combination of pegylated ifn-a and ribavirin is the standard therapy for hcv infection. however, this treatment is associated with side effects, and the efficacy of this regimen varies among genotypes, limiting the success rate of this treatment. 34 compared with genotype 2, infection with genotypes 1a and 1b results in more severe liver disease and low responsiveness to ifn therapy. seventy-one percent of patients with genotype 2 infection respond to ifn therapy, whereas only 28% of genotype 1a and 26% of genotype 1b show a response. 35 patients infected with genotype 6 generally show higher sustained virological responses to ifn therapy than genotype 1infected patients, 36 whereas genotype 3-infected patients show a lower sustained virological response compared with genotype 1-infected patients. 37 the heterogeneity of hcv genotypes also translates to differences in the manifestation of liver disease. 38 for example, hepatic steatosis is most common in patients infected with genotype 3 and is attributed to its core protein. 37 recently, the use of active direct-acting antiviral molecules to block hcv infection has led to substantial improvements in sustained virological response rates in genotype 1-infected patients. however, the use of these drugs may allow selection of resistant variants if direct-acting antiviral monotherapy is adopted, and a high relapse rate occurs after direct-acting antiviral treatment is discontinued. 39 the approximately 9.6-kb hcv genome contains a single open reading frame flanked by untranslated regions (utrs) at its 59 and 39 ends (figure 1 ). 3 the internal ribosome entry site (ires) located in the 59-utr directs cap-independent translation, whereas the 39-utr contains sequences critical for viral replication and translation. 40 the 39-utr (positioned at nucleotides 9389-9679 of the hcv genome) contains a poly(u/uc) (pu/uc) tract located at nucleotide positions 9436-9600, which was identified as an hcv pathogen-associated molecular pattern (pamp) that triggers rlr-mediated type i ifn production ( figure 1 ). 22 translation of the hcv genomic rna produces a single polyprotein of approximately 3000 amino acids, which is further processed by cellular and viral proteases to yield the structural proteins core, e1 and e2 and the non-structural (ns) proteins p7, ns2, ns3, ns4a, ns4b, ns5a and ns5b (figure 1 ). 3 ns proteins participate in different phases of the viral replication cycle (figure 1 ). for example, ns3 to ns5b proteins are important for rna replication. 3 jones et al. 41 showed that p7 and ns2 are required for entry and assembly of the virus. other researchers have also reported that ns2, 42-45 ns3/4a 46 and ns5a 47, 48 are involved in virion assembly and production. hepatocytes are the primary target cells for hcv replication. upon infection, the virus particle circulating in the blood biochemically resembles the very low-density lipoprotein, which is rich in apolipoprotein (apo) e and apob. 3, 49 first, the apolipoprotein-associated lipoviral particle (lvp) attaches to glycosaminoglycan and low-density lipoprotein receptor and then interacts with cluster of differentiation 81 (cd81) and scavenger receptor class b number 1 (figure 2 ). 50 the lvp is subsequently translocated to the tight junction of hepatocytes where the lvp binds to the tight junction proteins claudin-1 and occludin followed by internalization of the hcv particle via ph-dependent endocytosis, which occurs on the plasma membrane ( figure 2 ). 50 in addition to these receptors, cell surface molecules, such as epidermal growth factor receptor, ephrin receptor a2 and niemann-pick c1-like l1 cholesterol uptake receptor, are also essential for virus internalization. 50 subsequent to internalization, the acidic ph in the endosome triggers fusion of the viral envelope with the endosomal membrane, allowing the release of the viral genome into the cytoplasm ( figure 2 ). 50 hcv genomic translation occurs at the rough er, and hcv rna replicates in an er-derived or er-associated lipid-rich environment termed the membranous web ( figure 2 ). 51 all hcv ns proteins except for ns2 are involved in viral rna replication. the ns proteins are colocalized with the replicating viral rna on a light density, detergent-resistant cytoplasmic membrane structure termed a 'lipid raft'. 52 lipid droplets (lds), which comprise a neutral lipid core with a single phospholipid layer, serve as energy storage sites and reservoirs of neutral lipids in adipose tissue and hepatocytes. 53 lds are indispensable for viral rna replication and infectious virus formation. 54 during the initial stage of virus assembly, hcv core protein interacts with lds, 55 and the viral replication complex is also directed to lds in an ns5a-and core-dependent manner, allowing encapsidation of the viral rna by the core protein and assembly of the nucleocapsid ( figure 2 ). 47, 54 additionally, the interaction of ns3 and ns5a with actin and tubulin in the microtubule network mediates translocation of the hcv replication complex to lds. 56 the late stage of virus assembly, which occurs in the lumen of the er, involves the acquisition of a lipid envelope, the embed assembly and budding liver cell figure 2 hcv replication cycle. as shown, the hcv lvp is coated with apob and apoe, which are marked by light green and light blue stripes, respectively, on its surface. the lvp attaches to srb1 and to cd81 and further interacts with the tight junction protein claudin-1 and with occludin. virus entry into cell proceeds through receptor-mediated endocytosis at the cell surface. subsequent to internalization, the viral envelope fuses with the endosomal membrane under acidic ph, and the viral genome is uncoated and released into the cytoplasm. to dissect these two events, internalization and fusion are conventionally depicted as two seemingly separate steps in the cytoplasm. viral rna is translated at the er to produce the polyprotein, which is subsequently processed into mature structural and non-structural proteins. viral non-structural proteins, in conjunction with host factors, form a membranous web where viral rna replication occurs. hcv particle assembly most likely initiates near the er and ld where core protein and viral rna accumulate. finally, hcv particles are secreted into the extracellular milieu via the secretory pathway. viral replication and assembly occur in the proximity of lds and in lipid raft microdomains, which are shown in the inserted dashed rectangle. apo, apolipoprotein; cd81, cluster of differentiation 81; er, endoplasmic reticulum; hcv, hepatitis c virus; ld, lipid droplet; lvp, lipoviral particle. ding of e1 and e2 into the envelope and the formation of the nascent virion ( figure 2 ). then, the nascent virus particles associate with apob, apoe and other very low-density lipoprotein lipids to form lvps. 50 finally, lvps are released from cells through the very low-density lipoprotein secretion pathway or the endosome secretory pathway ( figure 2 ). 50 similar to the ns proteins, p7 plays numerous crucial functions in virion assembly and egress. 57 p7, an integral, oligomeric membrane protein consisting of 63 amino acids, is grouped into the family of viroporins that form membrane pores or channels. 58 in functioning as an ion channel, p7 modulates membrane permeability to facilitate virus entry by promoting virus uncoating and to enhance assembly or release. 58 p7 conducts ions across the membrane, and this channel activity can be abrogated by the drug amantadine and iminosugar derivatives. 59, 60 during maturation and egress, the ion channel activity of p7 maintains the ph gradients within the secretory pathway and thereby stabilizes the hcv particle. 50 in addition, p7 has been shown to be necessary for capsid assembly and envelopment because mutations in p7 result in the accumulation of incompletely assembled capsids that are unable to encapsidate viral rna. 61 the ifn systems constitute the first-line defense mechanism against viral infection in humans. 62 based on their antiviral properties, ifns are grouped into three classes: type i, ii and iii ifns. 63 in humans, type i ifns comprise a large group of molecules encoded by multiple genes, mainly ifn-a and ifn-b, and other genes such as ifn-e, ifn-k and ifn-v. 64 ifn-a and ifn-b combat viruses directly by inhibiting virus replication or indirectly by inducing the innate immune response. 63 most cell types can elicit a type i ifn response by activating the tlr, rlr and jak-stat pathways. 64, 65 type ii ifn contains only one member, ifn-c. unlike type i ifns, which are elicited as a direct response to viral infection, ifn-c is secreted by natural killer cells and mitogenically activated t cells. 63 ifn-c exerts potent anti-hcv activity in vitro and mediates antiviral t-cell responses. it has also been reported that ifn-c inhibits hcv infection by downregulating claudin-1 and cd81. 66 type iii ifns consist of three members, termed ifn-l1, ifn-l2 and ifn-l3 or il-29 (l1) and il-28a/b (l2/3). as with type i ifns, viral infection also directly activates type iii ifns. however, the antiviral properties and the mechanisms of action of type iii ifns remain unknown. type iii ifns can be secreted by many cell types, but their receptors show a limited tissue distribution. 63 hcv infection results in type iii ifn induction predominantly in the human liver. despite modulation by the irf3 and nf-kb pathways for induction of type iii and type i ifns, these two systems upregulate distinct subsets of isgs with different kinetics of induction. 67 during hcv infection, cells produce type i ifn to counteract viral infection, to modulate viral replication and to activate natural killer cells, dendritic cells and kupffer cells. 68 recognition of pamps by prrs, including tlrs and rlrs, triggers ifn synthesis and ifn-mediated cascade signaling pathways, leading to the production of type i ifn and a wide range of isgs to mediate ifn antiviral activity ( figure 3 ). 13, [20] [21] [22] upon virus infection, tlrs and rlrs operate through different signaling pathways, depending on the nature of the viral signals. tlrs are expressed and localized in the intracellular compartment, similar to endosomes, or on the cell surface. 69, 70 unlike rlrs, tlrs potentially detect viral double-stranded rna (dsrna) released by cells into the extracellular milieu ( figure 3 ). 69 three types of tlrs, i.e., tlr3, tlr7 and tlr9, are involved in the recognition of virus infections. tlr3 detects the dsrna formed during the replication of positive-stranded rna viruses, whereas tlr7 recognizes the urine-rich ribonucleotide region of rna, and tlr9 senses dna pamp motifs encoding cpg dinucleotides. 69 upon binding to a pamp, tlr3 dimerizes and initiates the binding of its cytosolic toll-il-1 receptor to the adaptor protein toll-il-1 receptor domain-containing adaptor inducing ifn-b (trif), resulting in the association of tlr3 with trif ( figure 3 ). 13, 14, 19 this interaction leads to the recruitment of tumor necrosis factor (tnf) receptor-associated factor (traf) 6, traf3 and the traf family member nuclear factor kappa-light-chain-enhancer of activated b cells (nf-kb) activator-binding kinase 1 (tbk1), resulting in the phosphorylation and activation of ifn regulatory factor (irf) 3 by tbk1 and by inhibitor of kappa b (ikb) kinase-related kinase (ikk)e. 19, 70 after phosphorylation, the irf3 protein dimerizes and is translocated into the nucleus to form an enhanceosome complex with nf-kb and other transcription factors, thereby inducing the expression of target genes, such as ifns. 70 moreover, the binding of viral dsrna to tlrs also activates nf-kb activity and pro-inflammatory cytokine synthesis through the interaction of trif with receptor-interacting protein-1. tlr7 and tlr9 bind to myeloid differentiation pro-inflammatory response 88 (myd88) and activate il-1 receptor-associated kinase (irak) and traf6, followed by the activation of ikka/b, which in turn activates nf-kb through phosphorylation, polyubiquitination and proteasomal degradation of its associated inhibitor ikba. migration of nf-kb into the nucleus results in ifn production ( figure 3 ). 70 the rlr receptors consist of rig-i, melanoma differentiation-associated protein 5 (mda5) and laboratory of genetics and physiology-2. 13,16,18 rig-i recognizes the hcv replication intermediate dsrna within hours of infection, which triggers the downstream signaling before the viral protein is extensively synthesized (figure 3 ). 71 rig-i senses the short, non-self dsrnas with 59-triphosphates, whereas rnas lacking 59-ppp, such as picornaviruses, are recognized by mda5. 72 both rig-i and mda5 contain two n-terminal caspase activator and recruitment domains (cards). 24 the recognition of dsrna by rig-i is dependent upon the atp-driven translocase activity of cards and helicases, and binding to dsrna induces conformational changes in rig-i that facilitate its oligomerization and translocation from the cytosol to the mitochondrial surface. 73, 74 on the mitochondria, rig-i engages via its tandem cards with the card of its downstream effector, mitochondria antiviral signaling protein (mavs), which is also termed ifn-b promoter stimulator-1, virus-induced signaling adaptor or card adaptor inducing ifn-b ( figure 3 ). 16, 18 the chaperone protein 14-3-3e and the ring finger domaincontaining e3 ubiquitin (ub) ligase triple motif-containing protein (trim) 25 also participate in this process. trim25 mediates the ubiquitination of rig-i at position lys-63, which is important for mavs binding and for ifn production. 74 the interaction between rig-i and mavs promotes the formation of a signaling complex on the mitochondrial surface that recruits and activates the downstream classical ikk complex, ikka/ikkb, and two non-classical ikk-related kinases, tbk1 and ikke. 75, 76 activation of tbk1 and ikke leads to the phosphorylation, dimerization and nuclear translocation of the transcription factor irf3 ( figure 3 ). 14 traf3, traf6 and mitogen-activator protein kinase/extracellular signal-regulated kinase (erk) kinase 1 (mekk1) are also recruited to mavs to activate nf-kb. 24 the canonical ikka and ikkb induce nf-kb-dependent gene transcription via phosphorylation, polyubiquitination and proteasomal degradation of ikba, thereby resulting in the release and nuclear migration of nf-kb. nf-kb activation involves the interaction of card9 with b-cell lymphoma/leukemia 10 protein. activated nf-kb and irf3 are translocated into the nucleus to form an enhanceosome, thereby stimulating the expression of ifn and inflammatory cytokines with the help of other cellular factors, such as activating transcription protein 2 and c-jun. 63 then, secreted ifn binds to ifnar on the cell surface and triggers the jak-stat signaling pathway ( figure 3 ). 75, 77 following ifnar receptor binding, tyrosine kinase-2 and jak1 are activated and phosphorylate stat1 and stat2 to form a heterodimer, which subsequently recruits irf9 to form the transcription factor ifn-stimulated gene factor 3 (isgf3). 75 then, isgf3 is translocated into the nucleus, binds to ifnsensitive responsive element (isre) and transactivates the expression of various isgs, such as 29-59 oligoadenylatesynthetase (2-5oas)/29,59-linked oligoadenylate (2-5a)-dependent, latent endoribonuclease (rnase l), dsrna-dependent protein kinase r (pkr), and irf7 ( figure 3 ). 78 acute hcv infections can be spontaneously cleared in some infected individuals, suggesting that the innate immunity induced by hcv pamp sensing can control acute viral infection. 19, 23 however, 80% of acutely infected people are not effectively cleared of hcv infection, and these patients may further develop chronic infection, suggesting that hcv has developed strategies to escape or to counteract the host immune response, leading to the emergence of resistance to ifn therapy. in this regard, several hcv proteins have been shown to block host antiviral responses, resulting in progression to chronic hcv infection ( figure 4 ). 13, 23, 24 obtaining further data regarding hcv evasion of host innate immunity will certainly improve ifn-based therapy outcomes. core protein hcv core protein is involved in the formation of the viral nucleocapsid and modulates many cellular functions, including transcription and signal transduction. expression of the full-length hcv genome or core protein downregulates ifn signaling by depressing stat1 tyrosine phosphorylation, which then blocks stat1 heterodimerization with stat2 and inhibits ifn signal transduction and isg expression ( figure 4) . 79 in addition, expression of core protein induces synthesis of suppressor of cytokine signaling 3 (socs3) in hepg2 cells. 80 socs3 is an important repressor of the jak-stat pathway due to its ability to inhibit stat1 phosphorylation ( figure 4 ). 77, 80 thus, hcv core protein induces socs3 and suppresses ifn-mediated isg expression. 79-81 socs3 expression is upregulated in chronically hcv-infected patients who are ifn non-responders compared with responders. 80 core protein expression has also been demonstrated to inhibit irf1 synthesis, transcriptionally repressing several isgs, such as il-15, il-12 and pkr. 82 many viruses use molecular mimicry as an important immune evasion strategy to promote virus survival and persistence. 83 viruses express proteins that are structurally similar to host defense proteins, and these viral proteins can act as immune modulators. 83 hcv employs this molecular mimicry strategy to resist type i ifn through its e2 envelope protein. 84 ,85 e2 comprises a 12-amino acid sequence identical to eukaryotic initiation factor 2a (eif2a) and pkr. 84 this domain operates to prevent pkr-dependent phosphorylation of eif2a and repression of protein synthesis, thus possessing an ability to resist type i ifn treatment ( figure 4 ). 84, 85 ns3/4a the hcv ns3/4a protease is not only responsible for the maturation of ns proteins, viral rna replication and virion morphogenesis but is also important for suppressing the host antiviral system. 13, 19, 23, 24, 86 the ns3/4a complex is anchored to the intracellular membrane through the ns4a transmembrane domain and the amphipathic a-helix at the ns3 n-terminus. 87 all these domains facilitate cleavage of their two cellular targets, mavs and trif, which act as key players in type i ifn production ( figure 4 ). 88, 89 mavs is an essential antiviral signaling protein in the rlr system and, therefore, is an ideal target for viral immune evasion. ns4a serves as the primary membrane subcellular targeting subunit to escort ns3/4a to mavs. 86 ns3/4a binds to mavs on mitochondria 88 and cleaves mavs at cys-508, resulting in the dislocation of the n-terminal portion of mavs from mitochondria and the suppression of ifn production ( figure 4 ). 71 the hydrophobic amino acid stretch in the ns3 amphipathic a-helix is also required for controlling rig-i signaling. 86 cleavage of mavs and reduction of ifn levels have been observed in chronically hcv-infected patients. 90 thus, ns3/4a-mediated cleavage of mavs rig-i signaling impairs ifn synthesis. 90 additionally, the ns3/4a protease also cleaves trif, an adaptor protein linking tlr3 to kinases responsible for activating irf3 and nf-kb ( figure 4 ). 89, 91 cleavage of trif interferes with poly(i:c)-activated tlr signaling and irf3 and nf-kb activation, thereby limiting the expression of multiple host defense genes and enhancing hcv persistence. 89 stimulator of interferon gene (sting), which is also known as mediator of irf3 activation (mita), is a 42-kda protein mainly localized to the er. 92 in response to dsdna transfection or dna virus infection, sting plays a crucial role in the activation of transcription pathways, essential for effective innate immune signaling. 92 upon dsdna stimulation, sting polymerizes and translocates from the er to a cytoplasmic punctate structure where the sting polymer provides a platform to connect tbk1 with irf3, which phosphorylates irf3, thereby triggering downstream signaling. 93 in viral infection, ns4b from yellow fever virus (yfv) blocks the induction of the ifn production pathway through an interaction with sting. 94 ns2b3 from dengue virus (denv) acts as a protease to cleave sting, thereby shutting down ifn signaling. 95 in hcv infection, ns4b interacts with and sequesters sting on the er to inhibit the association of sting with tbk, suppressing ifn signaling ( figure 4 ). 96, 97 therefore, targeting sting to inhibit innate immunity might be beneficial for virus survival. the mature hcv ns5a is present as two phosphoproteins, the hypophosphorylated p56 and hyperphosphorylated p58. 98, 99 ns5a phosphorylation occurs at multiple serine residues, such as serine 225, 229 and 232 upstream of the ifn sensitivity determining region (isdr) of ns5a, which spans residues 237-276 (based on genotype 1b hcv-j strain), and these serine residues are important for hyperphosphorylation. 98, 100 the functional and locational significance of ns5a p56 and p58 remains unclear; however, maintenance of these two forms at a specific ratio is critical for hcv replication. 100 functions as a pleiotropic protein that modulates the host environment to favor virus replication and persistence. 102 additionally, ns5a binds to myd88, which is a major adaptor molecule in the tlr pathway, and inhibits the recruitment of irak1 to myd88, attenuating tlr signaling and impairing cytokine production. 103 a sequence within the ns5a isdr, which spans residues 237-302, was shown to be responsible for interaction with the death domain of myd88 in macrophage cells. 103 pkr is an ifn-induced gene product that is activated by binding to dsrnas commonly produced during viral replication. ns5a rescues hcv replication in ifn-treated cells and inhibits ifn antiviral activity by binding to pkr and blocking pkr autophosphorylation and eif2a phosphorylation. 23, 104 ns5a expression is sufficient to rescue the replication of an ifn-sensitive virus. 102 the interaction of pkr with ns5a requires the isdr that overlaps a broader pkr-binding region, residues 234-366, and results in the inhibition of pkr activation and resistance to ifn in hcv-expressing cells. 104, 105 consistent with this mechanism, mutations in or deletion of isdr correlate with sensitivity to ifn-a-mediated antiviral activity. 102, 105, 106 moreover, meta-analysis and long-term follow-up support the association of this isdr region with the outcome of ifn therapy. this region, which encompasses a genetically flexible domain that allows mutations to occur, is the key site of adaptation to ifn therapy and influences the fitness of hcv rna replication. 23 in contrast, other studies suggest that the inhibitory effect of ns5a on ifn may be independent of pkr. ns5a increases expression of il-8, also known as chemokine cxcl8, by upregulating the il-8 promoter, which in turn, inhibits ifn antiviral activity and facilitates virus infection. 107, 108 in a cell culture model, il-8-positive cells are associated with chronic hcv infection, and il-8 removal mitigates hcv replication. 108 importantly, the serum level of il-8 is elevated in chronic hepatitis c patients compared with control individuals or is higher in ifn non-responders relative to responders. 23 these observations suggest that ns5a expression increases il-8 production, which somehow perturbs the ifn antiviral pathway. moreover, ns5a impedes the 2-5oas/rnase l system to inhibit ifn signaling. 109 the 2-5oas/rnase l antiviral pathway is present in virtually every cell. 110 this pathway involves the activation of a latent endoribonuclease and degrades hcv mrna with a dsrna structure during replication. 110 ns5a physically binds to 2-5oas through amino acid residues 1-40 of ns5a. 109 a single point mutation at amino acid 37 of ns5a affects the ns5a and 2-5oas binding and the antiviral activity of the 2-5oas/rnase l system. 109 thus, ns5a inhibits ifn antiviral activity in an isdr-independent manner. moreover, ifn-resistant strains, such as genotypes 1a and 1b, have fewer rnase l recognition sites in their genomes than the ifn-sensitive strains, such as genotypes 2 and 3, providing a means for ifn-resistant strains to escape from nucleotylic cleavage. 110 apoptosis also plays a key role in the host defense system by restricting viral spread and persistence. blocking apoptosis could be critical for the establishment of life-long persistence in the host organism. ns5a was shown to block the activation of caspase 3 and to inhibit proteolytic cleavage of the death substrate poly(adp-ribose) polymerase in tnf-a-induced cells. 111 adenovirus infection in ns5a-transgenic mice downregulates and upregulates the expression of t-box transcription factor 21 and trans-acting t cell-specific transcription factor 3, respectively, resulting in lower ifn-c expression and a delay in virus clearance. 112 furthermore, stable expression of ns5a in the human hepatoma cell line huh7 decreases sensitivity to tnf-a-mediated apoptosis, and activation of caspase-3, 8 and 9 by tnf-a is inhibited in ns5a-expressing cells. 113 thus, ns5a protects cells from tnf-a-mediated apoptotic death. hcv-induced er stress hcv protein expression can induce an er stress response and lead to calcium release from the er, which in turn activates the cyclic amp responsive element-binding protein that binds to the cyclic amp responsive element in the promoter of protein phosphatase 2a (pp2a), resulting in upregulation of pp2a. 114 expressed in essentially all cell types, pp2a is a serine/threonine phosphatase that is involved in multiple cellular processes, such as the cell cycle, signal transduction and stress response. 115, 116 increased expression of pp2a has been observed in a cell line inducibly overexpressing hcv protein, in liver extracts from hcv transgenic mice and in liver biopsies from patients with chronic hepatitis c. 117 duong et al. 117 showed that upregulation of pp2a by hcv can inhibit the enzymatic activity of protein arginine methyltransferase 1 (prmt1), which leads to decreased methylation of stat1. hypomethylated stat1 is more prone to bind to protein inhibitor of activated stat1 and inhibits stat1 dimerization, resulting in impaired nuclear translocation into the nucleus, binding of stat1 to the isre, and isg production. 117 thus, hcv-induced pp2a activation disrupts the ifna-induced antiviral state, leading to hcv evasion of innate immunity. these authors also showed that prmt1 can methylate hcv ns3 at arginine 467, resulting in the inhibition of ns3 helicase activity. 118 therefore, hcv-mediated pp2a upregulation enhances ns3 helicase activity by inhibiting prmt1 enzymatic activity, which in turn facilitates virus replication. 118 human genomes encode hundreds of isgs, 23 and the first isg, 54k, was discovered more than 25 years ago. 119 synthesis of some isgs can be triggered by viral infection without ifn production. some isg products can directly regulate cellular processes, such as protein synthesis and cell growth, survival and apoptosis, whereas others may modify the ifn antiviral activity against invading viruses. 120 the gene products of isgs can target many steps in the hcv replication cycle to limit viral replication. 23 many pamp receptors and their subsequent sig-naling partners are also isgs. 23 isgs expressed at the basal level provide antiviral surveillance before ifn activation or therapy; however, their levels markedly increase after ifn production. 23 in the innate immune response to virus infection, viral rna acts not only as a inducer of the production of ifn and its effector functions but also as a substrate and product for cellular enzymes, such as pkr and 2-5oas/rnase l. 121 the inverse correlation between the upregulated expression of isgs, such as oas-like (oasl), isg15 and viperin, in liver biopsies from hcv-infected patients and infected hepatocytes and decreased viral rna levels suggest the anti-hcv activities of these isgs. 122 in this section, we will highlight the involvement of isgs that are critical for modulating innate immunity in hcv replication, and the potential functions of these isgs, as outlined in table 1 . rig-i rig-i, which is encoded by the ddx58 gene and which belongs to the dexd/h box rna helicase (ddx) family, is a key player in the defense against invasion by many rna viruses. 72 rig-i senses the intracellular viral components and initiates antiviral responses to stimulate ifn production (table 1 ). in turn, ifn activates the transcription of rig-i, hence forming a positive feedback loop for amplifying antiviral signals. 123 studies have shown that rig-i is essential for eliciting an immune response against vesicular stomatitis virus (vsv), sendai virus, newcastle disease virus (ndv) and hepatitis c virus. 72, 124, 125 knockout of the rig-i gene in mouse embryonic fibroblasts severely limits type i ifn production and isg activation, thereby potentiating viral replication; conversely, overexpression of rig-i restricts viral replication. 72 in addition, upregulation of rig-i gene expression has been observed in ifn-treated human dendritic cells, suggesting that rig-i serves as an isg. 126 rig-i contains two tandem cards at its n-terminal region, with a repressor domain in its c-terminal region. the card is responsible for downstream signaling and activation of type i ifn after recognition of non-self rna, whereas the repressor domain is essential for the autoregulation and recognition of viral rnas. 127 without viral stimulation, the card interacts with the helicase domain, placing rig-i in an auto-inhibitory state and disabling signal transduction. 127 upon binding to viral rna, rig-i undergoes conformational changes that expose the card, allowing rig-i to be ubiquitinated. 127 rig-i is ubiquitinated by two different ligases, trim25 and ring finger protein 125. trim25 ubiquitinates rig-i at lysine 172 to mediate the antiviral response, whereas ubiquitination by ring finger protein 125 regulates the degradation of rig-i by the proteosome, thereby downregulating rig-i-mediated signaling. 123 ubiquitination by trim25 induces rig-i to form a tetramer, promoting the cards of rig-i to engage with the cards of mavs. this results in the accumulation of mavs on the mitochondrial membrane and the activation of ikk and tbk1, which, in turn, activates the transcription of nf-kb, irf3 and irf7 to promote ifn production. 123 moreover, ubiquitination by trim25 also prevents cards from interacting with the helicase domain to reinstate the auto-inhibitory state. 127 ubiquitination at lysine 172 is crucial for rig-i function because a mutation at this residue renders rig-i unable to bind to mavs, thus abrogating downstream signaling. 128 ddx60 is also a dexd/h box helicase whose function remains unclear. 129 ddx60 slightly resembles the yeast ski protein, which is a cofactor of the rna exosome required for controlling host rna quality. 129 ddx60, which is the human homolog of yeast ski, and the rna exosome exhibit antiviral activity against monkey leukemia virus and sindbis virus (sinv). 129 ddx60 expression is upregulated during infections of measles virus (mev) and hcv. 28, 129 the ddx60 mrna level is robustly upregulated in human fetal liver cells within 24-48 h after hcv infection, providing a means to initiate the antiviral mechanism. 130 unlike rig-i, ddx60 does not contain the cards to interact with mavs. 129 after viral infection, ddx60 is induced and binds to rig-i as well as mda5 and laboratory of genetics and physiology-2 and promotes the binding of rig-i to dsrna. 28,129 ddx60 is essential for type i ifn expression during dna virus infection and is induced to suppress viral replication in a rlr-dependent manner (table 1 ). 129 ddx60 knockdown reduces the expression of type i ifn after hcv, hiv and yfv infections. 28 irf1 irf1 was first identified as a transcriptional activator of the ifn-a/b gene. in unstimulated cells, irf1 is expressed at a low level; however, its expression is increased by the induction of ifn-a/b, tnf-a, il-1 and viral infection. 131 nevertheless, the precise pathway leading to irf1 activation by virus infection remains elusive. irf1 activation may proceed through a pkrdependent pathway after virus infection. pkr indirectly phosphorylates irf1 and activates its dna-binding properties. thus, activated irf1 regulates the promoter function of ifna/b promoter and acts as a modulator of many isgs by binding to the isre in the promoter region, thereby regulating viral replication (table 1) . 82, 132 irf1 controls the ifn antiviral response by affecting a set of isgs, such as irf7 and irf3. 131 irf1 cooperates with irf3 and irf7 to regulate cellular antiviral genes, such as ifn-a/b. hcv infection increases the level of irf1, which may affect other irf pathways and isg expression, thereby leading to a reduction in viral replication. 132 irf1 overexpression induces an antiviral state that affects various viruses, including ndv, vsv and hcv. 133, 134 the expression level of irf1 is reduced in cells harboring hcv subgenomic replicons (sgrs), whereas irf1 overexpression in these cells increases the isre activity and attenuates hcv replication. 134 additionally, hcv infection mediates irf1 expression, thus affecting the intracellular level of hcv rna. 134 however, hcv may evade the irf1 anti-hcv effect through core-mediated suppression of irf1 synthesis. 82 irf7 is an essential transcription factor for the induction of ifn-a/b and isgs. all of the elements of ifn responses, either innate or adaptive immunity, are regulated by irf7. 135 irf7 is constitutively expressed in certain cells, such as macrophage and plasmacytoid dendritic cells, priming these cells for rapid ifn production. 23 during infection, ifn-a/b binds to its receptor and activates the jak-stat pathway, resulting in irf7 expression. 23 then, irf7 is phosphorylated, forms a heterodimer with irf3 and is translocated into the nucleus. 136 in the nucleus, the irf7-irf3 heterodimer binds to the irf elements in the promoter region of ifn-a genes, leading to enhanced expression of the ifn-a subtype and a diverse range of isgs (table 1) . 23, 135 in turn, these events increase the abundance of rig-i and viral pamp signaling components, whereas sustained signaling serves to amplify ifn production. 23, 135 moreover, irf7 induces expression of other isgs without activating ifn signaling. 137 thus, irf7-mediated transcriptional cascades serve as an intrinsic antiviral mechanism allowing rapid isg expression before ifn production. irf7 plays an important role in eliminating hcv infection. sirna knockdown of irf7 decreases ifn-a production and increases the hcv titer. 138, 139 mice lacking irf7 show rapidly lethal infection by west nile virus (wnv) and high virus burdens. 140 irf7 deficiency represses the induction and accumulation of ifn-a, thus favoring wnv replication. 140 although hcv seems to suppress the basal expression of irf7, tlr7 stimulation activates irf7 and suppresses hcv replication. 138 this observation suggests that hcv may only partially inhibit irf7 activity in hcv-expressing cells. 138 pkr pkr, which is also known as eif2ak2, is a serine/threonine kinase that phosphorylates eif2a in response to virus infection. this ifn-inducible kinase has two distinct activities: autophosphorylation, resulting in its activation, and phosphorylation and inactivation of eif2a. through phosphorylation events, pkr mediates the inhibition of translation initiation of both cellular and viral mrna. [141] [142] [143] it is well documented that the anti-hcv activity of pkr occurs through its translational control (table 1) . [144] [145] [146] however, viruses have evolved elaborate strategies to counteract the detrimental effects of pkr. hcv ires activity has been shown to be resistant to pkr activation in cells harboring hcv sgr and in the hcv infection model (table 1) . [147] [148] [149] mechanistically, viruses may use their proteins to impede the dsrna-dependent pathway in various ways, such as sequestering dsrna, inhibiting pkr activation, producing pkr pseudosubstrates, activating antagonist phosphatases and degrading pkr. 142 as indicated above, hcv employs ns5a and e2 to antagonize pkr function, resulting in resistance to ifn and a blockade of the pkr-mediated inhibition of viral protein synthesis (table 1) . analogous to alphaviruses sinv and semliki forest virus, 150 hcv can activate pkr and eif2a phosphorylation to enhance its own viral protein translation (table 1) . 151, 152 compared with other previously studied dsrnas, domains iii-iv of the hcv ires were shown to bind to the n-terminal dsrnabinding domain of pkr, leading to increased pkr autophosphorylation and activation. 152 additionally, cap-dependent but not hcv ires-mediated translation is inhibited by pkr and eif2a phosphorylation. 152 these results indicate that while escaping the deleterious effects of pkr activation, hcv can employ its structured ires to direct its own protein translation. karamichali et al. 153 demonstrated that activated pkr or silencing pkr upregulates or downregulates hcv ires activity (table 1) . these authors further showed that the inhibitory effect of ns5a on ires-dependent translation occurs through pkr inactivation. 153 in contrast, hcv can translate its viral protein via a bacterial-like pathway that uses eif5b, which is an analog of bacterial if2, and eif3, instead of eif2a and its gtpase-activating protein eif5, as the initiation factor (table 1) . 154 the use of eif2a-independent translation initiation provides an alternative tactic for hcv translation when eif2a is inactivated by phosphorylation under stress conditions. many lines of evidence have revealed that hcv-mediated phosphorylation and activation of pkr, in turn, inhibit its downstream target, eif2a, and attenuate the expression of host cellular proteins, including isgs, without any inhibitory effects on viral ires-mediated viral protein translation (table 1) . 143, 155, 156 pkr knockdown in hcv-infected cells restores isg expression and enhances the antiviral effect of ifn. 155 these results demonstrate that hcv escapes ifn antiviral activity by promoting the phosphorylation of pkr and inhibiting the production of antiviral isg proteins, thus providing an interesting pathway for the virus to evade the ifn antiviral response. furthermore, accumulating evidence has revealed that pkr and eif2a participate in the formation of stress granules (sgs). sgs are large, dynamic structures between 50 to 200 nm in size, that form in the cytoplasm when cells undergo extracellular stresses, including viral infections. 157 sg formation is important for the posttranscriptional regulation of gene expression. 157 sgs contain stalled translation pre-initiation complexes, including cellular mrnas, translational initiation factors, the small subunit of the ribosome and many cellular rna binding proteins, such as t-cell-restricted intracellular antigen 1 (tia-1), the homologous tia-1-related protein tiar and rasgap-sh3 domain binding protein 1 (g3bp1), involved in regulating mrna functions. [158] [159] [160] [161] many viruses, including hcv, can modulate sg assembly and co-opt sgs to promote their own protein synthesis. 162, 163 hcv induces sg formation via eif2a phosphorylation (table 1) . 156, 164 consistent with this notion, upregulation of the regulatory subunit of protein phosphatase 1 that dephosphorylates eif2a and growth arrest dna damage-inducible protein 34, inhibits sg formation. 164 these results indicate the importance of eif2a phosphorylation in hcv-induced sg formation. moreover, garaigorta et al. 156 demonstrated that hcvinduced sg formation is ifn-and pkr-dependent and is inversely correlated with the induction of isg proteins, such as myxovirus resistance gene a (mxa) and ub-like (ubl)specific protease 18 (usp18), in hcv-infected cells without affecting the mrna levels of these isgs. furthermore, the sg proteins tia-1, tiar and g3bp1 have been shown to play a critical role in hcv replication and infectious virus production. 156 in support of this finding, g3bp1 was also reported to be essential for hcv rna replication, presumably through its relocalization to lds or its interaction with ns5b. 165, 166 the results of garaigorta et al. 156 demonstrated that hcv hijacks pkr phosphorylation-triggered sg formation to downregulate the translation of antiviral isgs, thereby promoting viral rna replication, virus assembly and egression. oas and rnase l upon sensing and activation by the pamp of viral dsrna, certain ifn-stimulated 2-5oas proteins can synthesize 2-5a from atp. after binding to 2-5a short oligoadenylates, a ubiquitous, latent endonuclease, rnase l, is activated through dimerization and degrades either cellular or viral rnas, resulting in the inhibition of protein synthesis, cellular apoptosis and impaired virus propagation. 121, 167, 168 therefore, the oas/ rnase l pathway represents a critical arm of ifn's antiviral effector mechanism against many viruses, including hcv. 121 depending on the specific rna substrates and the extent of enzymatic activity, rnase l can block different types of viruses through different mechanisms, such as apoptosis, or through the 'suppressor of virus rna' derived from cellular or viral rna. 168 nevertheless, some members of the oas family can exert antiviral activity independent of rnase l. 169 the oas system has been reported to exert anti-hcv effects through the rnase l pathway. the ua and uu dinucleotides within loops of predicted stem-loop structures in the viral rna is prone to cleavage by rnase l (table 1) . 110 additionally, the sensitivity of hcv infection to ifn therapy correlates with the efficiency of rnase l-mediated viral rna cleavage. 110 the anti-hcv activity of oas1 p46 and oas3 p100 in the oas family occurs in an rnase l-dependent fashion (table 1) . 170 hcv replication is suppressed in hcvcc-infected huh7 cells co-cultured with hepatic stellate cells (lx-2) treated with 59ppp-dsrna or incubated with conditioned medium from lx2 cells stimulated with 59ppp-dsrna. 125 in these hcvccinfected cells, the expression of oas-1 and mxa is upregulated. 125 the two different domains in oas-like a (oasla), a major isoform in human liver that is induced by hcv, contribute to the antiviral activity. the n-terminal oas homology domain, which lacks the cleavage activity, impairs cell proliferation as well as viral replication, whereas the c-terminal ublike domain impedes hcv replication without affecting cell growth (table 1 ). 171 the ifn-stimulated gene 20 kda protein (isg20) has emerged as a second ifn-regulated rnase that inhibits rna virus replication. 172,173 isg20, along with the closely related isg20l1 and isg20l2, belongs to the yeast rna exonuclease 4 homolog subfamily within the deddh exonuclease family and members of this superfamily possess both rnase and dnase activities. 173 the 39-59 exonuclease activity of isg20 demonstrates a greater preference for single-stranded rna than for single-stranded dna. 173 isg20 overexpression restricts infection by encephalomyocarditis virus, vsv, influenza virus (infv), human immunodeficiency virus (hiv), yfv, picornavirus and hcv. 172,173 isg20 has been reported to impair hcv genotype 1b sgr replication in hek293 cells (table 1) . 146 in addition, isg20 can hinder genotype 2a viral rna replication either in sgr or hcvcc infection, and its anti-hcv effect is not shared with isg20l1 and isg20l2 (table 1) . 172 apart from degrading viral rna through its 39-59 exonuclease activity, 146,172 the anti-hcv mechanism of isg20 in hcv replication remains poorly understood despite its possible action on cellular factors. 173 adar rna-specific adenosine deaminase (adar) is constitutively expressed in normal cells as an inactive form. 174 however, viral infection triggers the two mammalian adar genes, adar1 and adar2, to express two active proteins, adar1 and adar2. 174 adar catalyzes adenosine to inosine editing in rnas that possess double-stranded structures. 174, 175 because i is recognized as guanosine by rna polymerase, a to i editing causes nucleotide substitution as well as dsrna destabilization because of the reduced stability of i:u mismatch base pair compared with the normal base pair. 174, 175 the rna editing ability of adar affects many biological processes, including viral replication and persistence, apoptosis, ion channel function and the posttranscriptional modification of genes. 174, 176 only the adar1 transcription level is induced by ifn treatment and by pathogen infections. 177 in addition, adar1, but not adar2, affects the stability of hcv replicon rna (table 1) . 148 in hcv sgr replication, ifn-a treatment decreases viral rna replication and concomitantly increases adar1 expression, suggesting that adar1 possesses an antiviral activity in the hcv rna replicon. 148 adar1 knockdown conversely increases the hcv replicon rna. 148 loss of hcv rna by adar1 may be due to several reasons. 148 first, an i base-specific rnase might target mutated viral rna. second, the mutated rna might lead to insufficient replication and genome instability. 148 third, the cellular mrna involved in viral replication may also be targeted by adar1. thus, the rna editing ability of adar1 negatively affects hcv rna replication, representing a potent strategy in anti-hcv therapy. in sharp contrast, the replication of hepatitis delta virus (hdv) benefits from adar1 editing. 178 the editing of hdv rna by adar1 converts the uag stop codon to a uig tryptophan codon, allowing the synthesis of a larger hdv antigen. 179 without viral rna editing, the hdv genome cannot be packaged into a virion. 179 nonetheless, adar overexpression increases rna editing but decreases hdv replication. 180 ifn-inducible transmembrane protein (ifitm) family ifitm family members, including ifitm1, ifitm2 and ifitm3, inhibit, in an ifitm-specific manner, the replication of diverse pathogenic membrane-enveloped viruses, including marburg virus and ebola (ebov) filoviruses; severe acute respiratory coronavirus; hiv; rift valley fever virus (rvfv); respiratory syncytial virus; reovirus; flaviviruses, including denv and wnv; and hcv. [181] [182] [183] [184] [185] [186] in contrast, ifitms show no inhibitory effects on entry of amphotropic mouse leukemia virus, machupo virus, lassa virus and lymphocytic choriomeningitis virus. 183 ifitms are topologically located at different intracellular membrane compartments. ifitm2 and ifitm3, which are type ii transmembrane proteins, are primarily localized to endosomes and lysosomes, 187, 188 whereas ifitm1 also localizes to the cell periphery. 189, 190 ifit3 interacts with tbk1, irf3 and other ifitm members and enhances ifn signaling. 191, 192 lipid raft membranes, which are enriched in cholesterol and sphingolipids, play vital roles in cellular pathways and in virus entry, assembly and budding. 193, 194 vesicle-associated membrane protein-associated protein a (vapa) and oxysterolbinding protein (osbp) modulate the intracellular trafficking and de novo synthesis of cholesterol. 195 vapa interacts with osbp to regulate the transfer of cholesterol from the er to other organelles. 196, 197 the regulation of intracellular cholesterol homeostasis, particularly in the endosomal compartment, is critical for the entry of viruses such as ebov and marburg viruses. 198 ifitms have been demonstrated to interfere with virus infection by blocking virus-endosome fusion (table 1) , 183, 184 presumably through the modification of cellular membrane properties, such as fluidity and spontaneous curvature. 184, 188, 199 amini-bavil-olyaee et al. 188 demonstrated that the interaction of ifitm3 with vapa antagonizes the association of vapa with osbp, thereby inducing the accumulation of cholesterol in multivesicular bodies and in late endosomes. the disruption of intracellular cholesterol homeostasis subsequently impairs the membrane fusion of intraluminal virion-containing vesicles and endosomes, resulting in a block of vsv release into the cytosol. 188 using immortalized human hepatocytes and huh7 infection models, raychoudhuri demonstrated that ifitm1 expression inhibits hcv replication but not at virus entry. 200 later, wilkins et al. 186 identified that ifitm1 is a hepatocyte tight junction protein whose antiviral action occurs through modification of the interactions of the hcv coreceptors cd81 and occludin, thereby inhibiting hcv entry (table 1) . 186 this study represents an interesting mode of antiviral innate immunity; an isg can exert its anti-hcv action by disrupting viral coreceptor associations. the ifn-induced protein with tetratricopeptide repeats (ifits) family represents a class of isgs featured by their unique helix-turn-helix motifs, known as tetratricopeptide repeats. ifits mediate a broad range of protein-protein interactions; in particular, the tetratricopeptide repeat motif is critical for modulating protein translational initiation and transport, cell proliferation and migration, virus replication, and antiviral signaling. [201] [202] [203] [204] proteins in the ifit family have been linked to ifn antiviral functions, including those against wnv and lymphocytic choriomeningitis virus. 205 ifit3 plays an important role in modulating innate immunity by bridging tbk1 to mavs on mitochondria as ifit3 expression facilitates the association of its tetratricopeptide repeat motif with the n terminus of tbk1, thereby enhancing irf3-mediated gene expression. 192 ifit1, which is also known as isg56, belongs to a family that also contains other stress-induced, structurally related proteins, p60, p58 and p54, in humans. ifit1 acts as a negativefeedback regulator for sendai virus-triggered induction of type 1 ifn antiviral signaling transduction, presumably through its interaction with the adapter protein sting and through disruption of the normal association between sting/mita and mavs or tbk1. 203 moreover, ifit1/2 preferentially targets mutants of poxvirus, coronavirus, and wnv that lack 29-o methylation in their viral rna cap, thereby rendering these mutant viruses unable to replicate. 204 this study addresses the mechanism by which 29-o methylation of the 59 cap of viral rna renders viruses insensitive to ifit-mediated host innate antiviral activity. wang et al. 147 demonstrated that ifit1 mediates its ifn antiviral activity and blocks hcv rna replication, presumably by targeting an eif3-dependent step in viral ires-mediated translation (table 1) . 147 in immortalized human hepatocyte and huh7 infection models, raychoudhuri documented that ifit1 expression inhibits hcv replication by suppressing hcv ires-mediated transcription. 200 conversely, ifit1 knockdown facilitates hcv replication. 200 these results suggest that ifit1 restricts hcv infection primarily at the viral translation/replication site. protein posttranslational modifications by ub and ubl modifiers not only play important roles in numerous cellular processes, such as protein localization, interaction, activity and degradation, signal transduction, vesicular trafficking and dna damage repair, [206] [207] [208] but also modulate pathogen-host interactions, such as the viral replication cycle and the host antiviral response. 208-210 isg15, which was the first ubl protein modifier identified, 211 is post-translationally attached via its c terminus to the lysine residues of isgs and to hundreds of target proteins involved in different pathways. 212, 213 similar to its ub homolog, isg15 is linked to proteins via a tightly regulated process known as 'isgylation', and the activating e1 (ube1l), conjugating e2 (ubch8), and ligating e3 (ceb1) enzymes catalyze these sequential events. 211, 214 isg15, together with its conjugation e3 ligase (ceb1) and its deconjugation enzyme usp18, are in the same isg15/usp18 ubl pathway. isgylation modulates signal transduction pathways and host antiviral responses. isg15 exerts its modulatory roles by inhibiting virus release, isoylating viral proteins, or modifying host proteins. 214 isg15 targets many cellular proteins, including jak1, stat1 and many isgs. three antiviral effector molecules, irf3, rig-i and pkr, are also modified by isgylation. 214 activated irf3 is stabilized by isgylation and therefore, positively regulates type i ifn signaling. 215, 216 the isg15 conjugation-mediated reduction of the non-isgylated rig-i correlates with the reduced ndv-triggered ifn response. 123 additionally, viral rna-independent pkr activation requires the isgylation of pkr. 217 isg15 expression enhances ifn-mediated antiviral activity against many viruses, including hiv and sinv. 214 overexpressing isg15 in ifn-a/b receptor knockout mice decreases sinv replication and protects the mice from sinv-induced lethality. 218 isg15 2/2 mice are more susceptible to infection by many rna and dna viruses, such as infv and herpes simplex virus (hsv) type 1, and the protection effect of isg15 from sinv infection is dependent on isgylation. 219 lu et al. 215 demonstrated that induction of isg15 expression in ndv-infected cells counteracts the ub-mediated degradation of irf3 and enhances the ndv-mediated host innate antiviral response. their findings revealed a feedback mechanism of isg15 in enhanced antiviral immunity. despite functioning as an antiviral molecule, isgylation of the antiviral rig-i enzyme inhibits ifn signaling in mouse embryonic fibroblast cells. 123 using the genotype 2a j6/japanese fulminant hepatitis-1 chimeric hcv infectious model, chen et al. 220 unexpectedly found that isg15 acts as a pro-hcv regulator because increased isg15/isgylation facilitates hcv production, whereas blocking isgylation decreases virus production (table 1) . moreover, knockdown of ube1l, the e1 activating enzyme, inhibits hcv replication, particularly hcv egress, without affecting ifn-mediated isg expression in hcv-infected cells. 220 using the hcv-huh7. 25 .cd81 infection system, arnaud et al. dissected the acute ifn response to hcv infection into early, pkr, and late, rig-i, phases. 221 hcv infection rapidly induces the expression of many irf3-dependent genes, including isg15, through a pkr-dependent mechanism before the rig-i phase, which recruits mavs. 221 then, isg15 induction blocks hcv rna-mediated rig-i activation by inhibiting rig-i ubiquitination, thereby negatively controlling the rig-i/mavs pathway. 221 these studies illustrate that hcv may exploit isg15 to antagonize host innate immunity and to promote viral replication. the deconjugation of usp15 from its target proteins is catalyzed by usp18 (mouse ortholog ubp43). 222 usp18 can function in both isg15-dependent and isg15-independent modes. usp18 was shown to bind to ifnar2 and attenuate the jak-stat pathway, thereby negatively regulating ifn signaling (table 1) . 223 reduced usp18 expression results in increased antiviral activity against many viruses, such as sinv, hepatitis b virus and vsv, in usp18 knockout mice. 218, [223] [224] [225] usp18 knockdown is concomitant with increased cellular protein isgylation, prolonged stat1 tyrosine phosphorylation and enhanced isg expression, thus greatly enhancing the anti-hcv potency of ifn. 226 all these studies suggest that usp18 disruption can impede its negative regulatory effect on ifn signaling, resulting in sustained jak-stat activity and antiviral activity. consistent with these observations, murray et al. 227 demonstrated that ifn-a signaling and isg induction were greatly increased when ups18 was knocked down in both hcv sgr-and hcvcc-infected huh7 cells. however, usp18 knockdown did not have a significant effect on anti-hcv activity. 227 these observations suggest a slight dependency of ifn-mediated antiviral activity on usp18 activity. 227 additionally, usp18 upregulation is predictive of a nonsustainable viral response to ifn treatment. 34, 228, 229 the expression levels of ups18 and isg15 increase in liver biopsy specimens from chronically hcv-infected patients who do not respond to ifn-based therapy, inferring that hcv hijacks the isg15/usp15 pathway to evade the antiviral immune response and to facilitate its replication (table 1) . 34, 230 this observation also explains, at least partially, the failure of ifn-based treatments in non-responders, although non-responders express higher levels of isgs, particularly isg15, compared with ifn responders. 34, 230 taken together, these findings demonstrate that usp18 is an attractive target for the development of anti-hcv therapeutics. viperin, which stands for virus inhibitory protein, endoplasmic reticulum-associated, ifn-inducible, plays crucial roles in virus replication, signaling and the immune response. 231, 232 the viperin protein sequence is highly conserved, and all viperin homologs contain three functional domains: the amphipathic, n-terminal domain, which mediates er and ld association; the central cxxxcxxc motif, which is functionally important for fe-s cluster formation; and the highly conserved c-terminal domain, which is essential for antiviral activity. [231] [232] [233] in addition to type i, type ii and type iii ifns, dsdna and dsrna analogs, bacteria, lipopolysaccharide, poly(i:c) and a broad spectrum of dna and rna viruses can induce viperin expression. 231, 232, 234 viperin expression regulates many cellular functions, such as forming lds and reducing membrane fluidity. 231 viperin possesses antiviral activity against diverse families of dna and rna viruses, including infv, hiv, sinv, the flaviviruses japanese encephalitis virus, denv and wnv, and the hepacivirus hcv (table 1) . 231, 232, 234 viperin functions in different ways to defend against virus infections. for instance, viperin alters membrane fluidity by interacting with farnesyl diphosphate synthase, which is an enzyme essential for isoprenoid biosynthesis, thus disrupting the formation of lipid rafts, the sites of infv budding, leading to interference with virus release from the cell surface. 235 the induction of viperin into hiv-1-infected cells disrupts lipid rafts, causing viperin redistribution to cd81 compartments, where hiv-1 buds in human macrophages. 236 the radical s-adenosyl-methionine enzymatic activity of viperin is required for the inhibition of hiv production. 236 in cells infected with japanese encephalitis virus, the antiviral function of viperin is attenuated due to its degradation by the proteasome-mediated protein degradation system. 237 in contrast, viperin enhances human cytomegalovirus infection through its interaction with the viral mitochondrial inhibitor of apoptosis vmia protein, resulting in viperin relocalization from the er to mitochondria. 238 in mitochondria, viperin interacts with the mitochondrial trifunctional protein and reduces cellular atp generation, resulting in the disruption of the actin cytoskeleton and enhancement of the virus infection. 238 viperin is upregulated in huh7 cells transfected with either poly(i:c) or hcv rna, 239 and transient expression of viperin in hcv sgr replicating cells significantly decreases hcv replication. 239 the putative radical s-adenosyl-methionine enzymatic activity of viperin is required for this anti-hcv activity. 146 helbig et al. 240 further demonstrated that the restriction of hcvcc replication by viperin depends on both the n-terminal amphipathic a-helix and the c-terminal domain. the anti-hcv function of viperin coincides with its binding to ns5a at the ld interface, whereas ns5a normally associates with the human homolog of the 33-kda vesicleassociated membrane protein-associated protein (hvap-33), which is a pro-viral cellular factor, at the viral replication complex. 239 the interaction of hcv ns5a with hvap-33 was previously shown to be critical for the formation of the viral replication complex. 52 therefore, the association between viperin and hvap-33 requires both of their c-terminal domains, which then disturbs the interaction of hvap-33 with ns5a and inhibits hcv replication (table 1) . 241 together, these findings imply that viperin hinders viral rna replication by perturbing the interaction between hvap-33 and ns5a. by conducting bioinformatic analyses of murine bone marrowderived macrophages, liu et al. showed that cholesterol-25-hydroxylase (ch25h), which is an ifn-a-and ifn-c-stimulated isg, can mitigate the replication of many membraneenveloped viruses, including hiv, vsv, hsv and murine c-herpesvirus, and many pathogenic viruses, such as rvfv, ebov, russian spring-summer encephalitis virus and nipah virus in vitro and in vivo. 242 these viruses contain different structural characteristics in their fusion proteins. for instance, hiv and ebov contain class i fusion peptides, rvfv and russian spring-summer encephalitis virus harbor class ii peptides, and vsv and hsv belong to class iii fusion proteins. 243, 244 the broadly antiviral action of the ch25h gene product is mediated by the ability of its enzymatic product, 25hydroxycholesterol, to inhibit ph-dependent and ph-independent membrane fusion between cells and viruses, as typified by vsv and hiv, respectively (table 1) . 242 this study not only demonstrates that ifn can confer an antiviral state to host and/ or target cells by inducing a natural oxysterol inhibitor but also suggests that modification of membrane oxysterols can be used as a potential antiviral approach. 242 determining whether this broad antiviral isg can block hcv-mediated membrane fusion would be interesting. several genome-wide sirna screens were recently performed to identify isgs or ifn-mediated effector genes (iegs) that mediate ifn antiviral functions. these studies have identified many new isgs or iegs and have revealed interesting features of the actions of isgs. using an overexpression screen approach, schoggins et al. 28 demonstrated that each virus exhibits a unique but partially overlapping profile of antiviral isg expression. the expression levels of isgs may vary depending on viral infection or on the time, dose, or cell type used for ifn treatment. 28 in hcv infection, higher expression levels of 36 unique isgs were found to correlate with a reduction in the hcv viral load. 245 schoggins et al. 28 further observed that multiple isg genes could target each viral species with a range of inhibitory activities. a set of effectors, including irf1, c6orf150 (also known as mb21d1), heparanase, rig-i, mda5 and ifitm3, exert broad antiviral activities against different viruses, including hcv, yfv, wnv, chikungunya virus, venezuelan equine encephalitis virus and hiv-1. 28 however, other effectors, such as ddx60, ifn-inducible proteins 44l and 6, ifitm2, mapk kinasekinase 14, moloney leukemia virus 10, nicotinamidephosphoribosyltransferase, oasl, receptor transporter protein 4, three prime repair exonuclease 1 and protein unc-84 homolog b, display species-specific antiviral effector functions. 28 these results also demonstrated that different isgs can exert additive antiviral effects on virus replication. 28 remarkably, several isgs, such as adar, family with sequence similarity 46, member c, lymphocyte antigen 6e and mucolipin-2, can enhance the replication of certain viruses. 28 certainly, further characterizing how these isgs antagonize ifn-mediated antiviral functions and determining which steps of virus replication are targeted by these isgs are important. these findings indicate the complexity of the type i ifn-mediated innate immune response in virus replication. performing a sirna-based 'gain of function' screen, metz et al. 246 identified several new anti-hcv isgs in addition to those previously reported anti-hcv isgs. this study demonstrated that both ifn-a and ifn-c can upregulate the expression of several isgs, including ifit3, trim14, phospholipid scramblase 1 and inducible nitric oxide synthase 2. these isgs possess anti-hcv activity, although the precise roles of these isgs in hcv replication are not understood. 246 this study also reported a substantial overlap in antiviral innate immune responses triggered by either cytokine. 246 however, some isgs are more specifically induced by ifn-a or by ifn-c. for instance, phospholipid scramblase 1 and nitric oxide synthase 2 primarily function as ifn-c-mediated anti-hcv effectors. 246 moreover, different isgs function additively or synergistically to interfere with hcv infection, 246 indicating that the combinatorial and concerted actions of multiple effectors mediate repression of hcv replication. in addition to the signaling molecules involved in the ifn/ jak-stat/isgpathway, the majority of genes identified by fusco et al. are not transcriptionally activated by ifn. 247 in contrast to the notion that isgs target specific virus replication steps, some of these genes can exert ifn-mediated antiviral effects at multiple steps of the hcv replication cycle. 247 for instance, dipeptidyl-peptidase 4/cd26/adenosine deaminase complexing protein 2 blocks virus entry, initial rna replication, and amplified translation. myst1 histone acetyltransferase inhibits hcv entry, translation, rna replication and virion release, and protein phosphatase 3, catalytic subunit, b isoform (ppp3cb) impairs virus entry, initial rna replication and subsequent translation. taken together, these findings reveal that these ifn-insensitive iegs, together with isgs, constitute the host cellular genes mediating the antiviral activity of ifn against viral replication. a functional genomic screen has shown that several new genes comprising the u4/u6.u5 tri-small nuclear ribonucleoprotein (snrnp) possess the ability to mediate ifn antiviral activity. 248 u4/u6.u5 tri-snrnp is the major component of human spliceosome complexes involved in mrna processing. 249 this genomic screen demonstrated that squamous cell carcinoma antigen recognized by t cells (sart1) is a u4/ u6.u5 tri-snrnp-specific factor required for ifn-a-mediated anti-hcv activity, although sart1 is not induced by ifna. 248 the anti-hcv activity of sart1 acts by regulating the expression of isgs, such as mxa, oas and pkr, either in the presence or absence of exogenous ifn-a. 248 this genetic screen links an unappreciated role of rna processing to the control of antiviral immunity. in this section, we discuss recent findings regarding the roles of several cellular factors and/or machinery involved in the immune response in modulating hcv replication. although these determinants are not directly induced or activated by ifn, knowledge of their interplay with the host immune response will help to elucidate their effects on hcv infection. the functions of these cellular determinants in hcv infection are summarized in table 2 . ikka hcv can co-opt an intrinsic innate pathway and hijack cellular lipid metabolism to facilitate its assembly. ikka was initially identified as a critical factor for hcv replication in a genomewide rna interference screen. 250 subsequently, hcv infection was shown to activate ikka through the interaction of the viral genome 39-utr with dead box polypeptide 3, x-linked (ddx3x). 251 ikka translocates into the nucleus and induces the cbp/p300-mediated expression of lipogenic genes, including sterol regulatory element-binding proteins, followed by the promotion of core-mediated ld formation and the enhancement of hcv assembly ( table 2 ). 251 dansako demonstrated that upon hcv expression, class a scavenger receptor type 1 (msr1) expressed on the plasma membrane of infected and adjacent uninfected cells can bind to dsrna released from infected cells and mediate its endocytosis and transport to endosomes where the dsrna is sensed by tlr3 and initiates a local antiviral ifn response to restrict hcv replication. 252 the msr1-mediated binding, transport, and release of dsrna at the acidified endosome requires a stretch of conserved basic residues within the c terminus of the collagen superfamily domain of msr1. 252 therefore, msr1 acts as a key element for the tlr3-mediated prr, thereby rendering both infected and uninfected hepatocytes refractory to hcv replication (table 2) . hmgb1, which is an abundant nuclear protein that mediates activation of host immune responses and inflammation, represents a prototype damage-associated molecular pattern that participates in the pathogenesis of diverse pathogens. 253, 254 hmgb1 is passively released by cell injury or ischemia without pathogen invasion, but is actively secreted from stimulated immune cells, such as natural killer cells, macrophages and mature dendritic cells. 253 many types of tlrs, such as tlr2, tlr4 and tlr9, can act as receptors for hmgb1. 253 the production of reactive oxygen species can mediate translocation from the nucleus to the cytoplasm and the subsequent release of hmgb1. 253, 255 interestingly, it has been shown that hcv core and ns5a can trigger oxidative stress in infected cells. [256] [257] [258] jung et al. 259 demonstrated that hcv infection causes the nuclear-to-cytoplasmic translocation of hmgb1 and its release into the extracellular milieu. tlr4 acts as a major component of the receptor complex that recognizes lipopolysaccharide lps and plays a role in the production of pro-inflammatory cytokines and antiviral ifns via signaling myd88 and the tlr adapter protein trif. 260 jung et al. 259 also demonstrated that hmgb1 interacts with tlr4 to activate ifn signaling (table 2 ). because hmgb1 is present at higher levels in the sera of patients with chronic hepatitis and cirrhosis compared with those detected in control individuals, 261 the results of jung et al. may help to elucidate the potential inhibitory action of hmgb1 in hcv propagation in chronically hcv-infected patients. 259 autophagy autophagy is a conserved 'self-eating' process that engulfs and delivers cytoplasmic cargos and invading pathogens within double-or multiple-membrane autophagosomal structures to lysosomes for degradation. [262] [263] [264] [265] the purpose of autophagic induction is to maintain cellular homeostasis in the host when the host undergoes extracellular or intracellular stresses. autophagy plays pivotal roles in the stress response, nutrient deprivation, damaged organelles, unfolded protein aggregation, intracellular quality control and cell death. [262] [263] [264] [265] the autophagic process requires two ubl conjugation complexes: autophagy-related gene (atg) 12-atg5-atg16l and microtubule-associated protein 1 light chain 3-phosphatidylethanolamine. 262, 266 autophagy has emerged as an immune regulator that commands the innate and adaptive immune responses against intracellular viruses. [267] [268] [269] [270] [271] autophagy also participates in the modulation of virus-host interactions. in contrast, viruses can subvert the host autophagic pathway to potentiate their own growth. 272, 273 analogously, hcv is able to subvert the host autophagic machinery and enhance viral growth, including rna replication, 274 translation of the incoming viral rna genome 275 and the release of infectious viruses ( table 2) . 276 two laboratories have independently demonstrated that hcv can activate autophagy via er stress-mediated induction of the upr and that upr-autophagy is required for hcv replication. 7,277 hcv ns3, ns4b, ns5a and ns5b have also been implicated in the induction of autophagy. 12, 27 huang et al. 278 showed that hcv induces er stress and inhibits the akttuberous sclerosis-mtor complex 1 signaling pathway, resulting in autophagy activation. in contrast, shrivastava et al. 279 demonstrated that hcv induces autophagy by stimulating beclin mrna expression and by activating mtor signaling, which may enhance hepatocyte growth. ke and chen 277 demonstrated that in the context of hcv infection or without hcv infection, activation of the upr and autophagy downregulates innate immunity; in contrast, disruption of the upr and autophagy upregulates innate immunity. these results demonstrate that hcv hijacks upr and autophagy to stimulate viral rna replication by suppressing immune antiviral immunity. the upr-autophagy pathway represents a unique mode of reversible control in the innate immunity capacity in target cells. 10, 27 subsequently, shrivastava et al. 280 found that beclin or atg7 gene silencing in genotype 1a h77 strain hcv-infected immortalized human hepatocyte upregulates ifn signaling and isg expression, which are concurrent with apoptotic cell death. together, the results from these two groups suggest that autophagy may protect hcv-infected cells from the damage caused by excessive ifn antiviral stimulation, thereby promoting hcv rna replication. furthermore, a specific mode of autophagy, termed 'mitophagy', was recently reported to play a critical role in hcv replication and in the elimination of damaged mitochondria in infected cells in a parkin-dependent manner. 281 knockdown of parkin and pink gene expression suppresses viral rna replication (table 2 ). 281 these results suggest a critical role for mitophagy in hcv replication. nevertheless, the molecular basis for the roles of autophagy and mitophagy in suppressing innate antiviral immunity in hcv infection has yet to be investigated. a recent study has demonstrated that many different families of rna viruses can target the autophagy network to promote viral growth. 282 among these targets is irgm, which modulates autophagy by interacting with many autophagy-associated proteins, such as atg5, atg10 and light chain 3c. strikingly, irgm knockdown impairs autophagy induced by many viruses, such as hcv, mev and hiv-1, resulting in mitigated viral replication (table 2 ). 282 moreover, the c protein of mev, ns3 of hcv, and nef of hiv-1 were shown to induce autophagy by interacting with irgm. these results suggest that rna viruses have evolved to use a common strategy to target a critical molecule in autophagy to benefit their growth. microrna is a class of endogenous small non-coding rnas that bind to the 39-utr of target mrnas to control gene expression. 283 micrornas also participate in innate and adaptive immunity response by binding to their complementarily mrnas and regulating the expression and translation of their target genes. 284 for example, mir-155 regulates the host antiviral immune response by promoting type i ifn, whereas mir-16 enhances mrna degradation. 285 mir-21 was shown to be upregulated in liver samples from hepatocellular carcinoma patients and in hcv-infected cells. 286 during hcv infection, mir-21 expression is activated by the pkce/jnk/cjun and pkca/erk/cfos pathways. 287 cjun and cfos form the ap-1 protein, which binds to the mir-21 promoter and activates mir-21 expression. 287 mir-21 upregulation was shown to suppress the expression of myd88 and irak1, which are two genes involved in the tlr signaling cascade, thereby repressing the production of type i ifn and isg and promoting hcv replication ( table 2) . 287 these results indicate that hcv usurps mir-21 to enhance its replication. likewise, mir-21 also increases the production of hiv, vsv and enterovirus 71 by suppressing type i ifn production. 287 the mechanisms by which viruses and cells coevolve and the tactics each party employs to establish the dynamic equilibrium are emerging as a fascinating area in hcv-host interaction research. previous studies that aimed to understand the hcv cell coevolution process have revealed several interesting aspects of virus-host cell interactions, such as er stress, upr, autophagy and innate antiviral immunity responses in hcv replication. 11, 12, 27 further determining how the virus-cell interplay subsequently reshapes the host defense mechanisms and how virus replication is modulated in response to these cellular stresses will be important for gaining a complete understanding of the molecular basis of the hcv-host interaction in the pathogenesis of hcv infection. viral infection can trigger the ifn-mediated frontline host defense mechanism, including the production of a wide range of isgs to limit virus replication. many studies have also hitherto demonstrated that some of the identified isgs can exert broad antiviral activities against a diverse spectrum of viruses, whereas other isgs may have virus type-specific functions. the majority of studied isgs mediate ifn antiviral activities, acting as negative regulators in virus replication. strikingly, some isgs function as negative modifiers in the innate immune response, thereby promoting virus replication. nevertheless, the modes of action of most of the isgs remain unclear. although most identified isgs target individual steps of virus replication, some isgs seem to act at multiple stages of the virus replication cycle. determining the mechanisms by which these isgs function at different steps of the virus replication cycle would be interesting. current studies have indicated that different types of ifns may substantially overlap in mediating their innate immune response by activating the same set of isgs. however, the induction of some isgs may be unique to only one type of ifn, indicating the specificity in the induction of these isgs by ifns. clearly, different isgs can additively or synergistically suppress hcv replication, suggesting that inhibiting hcv replication depends on the combinatorial effects of individual isgs induced by ifn under the specific context of hcv infection. therefore, ifn-mediated suppression of hcv replication is not caused by a single isg but rather by the concerted actions of multiple isgs. moreover, gene expression profiling of hepatocytes from chronically hcv-infected patients treated with ifn has consistently shown differences between ifn-responders and ifnnon-responders. for instance, the levels of specific isgs, such as isg15 and usp18, and viral sensors, such as rig-i, mda5 and laboratory of genetics and physiology-2, are upregulated in patients with non-sustained virological responses compared with patients with sustained virological responses. 34, 228 therefore, profiling gene expression for cytoplasmic viral sensors and related regulators involved in the innate antiviral immune response can identify new isgs that can be used as markers for predicting the clinical outcome of ifn therapy. in conclusion, the emergence of complexity in the highly pleiotropic type i ifn system in hcv infection reveals that the host has evolved to erect multiple checkpoints for anti-hcv innate immune surveillance to ensure that hcv is under tight control at all times, even when a single effector fails to confer antiviral activity, without drastically downgrading the overall efficacy of the ifn treatment. therefore, further deciphering which isgs and/or iegs are induced by ifns upon hcv infection and the specificity and action of these isgs and other cellular immune regulators on hcv replication will not only provide insights into how ifn functions to obstruct hcv replication but will also reveal novel cellular targets against which effective and efficacious anti-hcv therapeutics can be developed. unscrambling hepatitis c virus-host interactions replication of hepatitis c virus hepatitis c virus infection and mixed cryoglobulinemia production of infectious hepatitis c virus in tissue culture from a cloned viral genome persistent hepatitis c virus infection in vitro: coevolution of virus and host perturbation of autophagic pathway by hepatitis c virus viruses and the autophagy machinery impact of the autophagy machinery on hepatitis c virus infection autophagy: a novel guardian of hcv against innate immune response hepatitis c virus and cellular stress response: implications to molecular pathogenesis of liver diseases autophagy in hepatitis c virus-host interactions: potential roles and therapeutic targets for liver-associated diseases regulation of hepatic innate immunity by hepatitis c virus immune responses to hcv and other hepatitis viruses toll-like receptor and rig-i-like receptor signaling innate immune modulation by rna viruses: emerging insights from functional genomics treatment of chronic non-a, non-b hepatitis with recombinant human alpha interferon. a preliminary report innate immune recognition of viral infection activation and evasion of antiviral innate immunity by hepatitis c virus regulation of innate immunity against hepatitis c virus infection induction and evasion of innate antiviral responses by hepatitis c virus innate immunity induced by composition-dependent rig-i recognition of hepatitis c virus rna evasion of intracellular host defence by hepatitis c virus hepatitis c virus evasion from rig-i-dependent hepatic innate immunity viral determinants of resistance to treatment in patients with hepatitis c innate immune responses in hepatitis c virus infection suppression of innate antiviral immunity after hepatitis c virus infection: role of the unfolded protein response and autophagy a diverse range of gene products are effectors of the type i interferon antiviral response intrinsic antiviral immunity expanded classification of hepatitis c virus into 7 genotypes and 67 subtypes: updated criteria and genotype assignment web resource consensus proposals for a unified system of nomenclature of hepatitis c virus genotypes global distribution and prevalence of hepatitis c virus genotypes global burden of hepatitis c: considerations for healthcare providers in the united states hepatic gene expression discriminates responders and nonresponders in treatment of chronic hepatitis c viral infection hepatitis c virus genotypes in the united states: epidemiology, pathogenicity, and response to interferon therapy. collaborative study group systematic review: epidemiology of hepatitis c genotype 6 and its management hepatitis c virus infection and liver steatosis clinical significance of hepatitis c virus genotypes treatment failure and resistance with direct-acting antiviral drugs against hepatitis c virus characterization of the terminal regions of hepatitis c viral rna: identification of conserved sequences in the 59 untranslated region and poly(a) tails at the 39 end hepatitis c virus p7 and ns2 proteins are essential for production of infectious virus structural and functional characterization of nonstructural protein 2 for its role in hepatitis c virus assembly hepatitis c virus ns2 protein contributes to virus particle assembly via opposing epistatic interactions with the e1-e2 glycoprotein and ns3-ns4a enzyme complexes trans-complementation of an ns2 defect in a late step in hepatitis c virus (hcv) particle assembly and maturation determinants of the hepatitis c virus nonstructural protein 2 protease domain required for production of infectious virus ns3 helicase domains involved in infectious intracellular hepatitis c virus particle assembly essential role of domain iii of nonstructural protein 5a for hepatitis c virus infectious particle assembly interaction of hepatitis c virus nonstructural protein 5a with core protein is critical for the production of infectious virus particles hepatitis c virus and other flaviviridae viruses enter cells via low density lipoprotein receptor the ins and outs of hepatitis c virus entry and assembly replication of hepatitis c virus rna occurs in a membrane-bound replication complex containing nonstructural viral proteins and rna interactions between viral nonstructural proteins and host protein hvap-33 mediate the formation of hepatitis c virus rna replication complex on lipid raft lipid droplets: a unified view of a dynamic organelle the lipid droplet is an important organelle for hepatitis c virus production structural determinants that target the hepatitis c virus core protein to lipid droplets association of hepatitis c virus replication complexes with microtubules and actin filaments is dependent on the interaction of ns3 and ns5a hepatitis c virus p7 protein is crucial for assembly and release of infectious virions the elusive function of the hepatitis c virus p7 protein the p7 protein of hepatitis c virus forms an ion channel that is blocked by the antiviral drug antiviral effects of amantadine and iminosugar derivatives against hepatitis c virus hepatitis c virus p7 is critical for capsid assembly and envelopment viruses and interferons interferons and viruses: an interplay between induction, signalling, antiviral responses and virus countermeasures induction and function of type i and iii interferon in response to viral infection viruses and interferon: a fight for supremacy inhibition of hepatitis c virus infection by interferon-gamma through downregulating claudin-1 hcv infection induces a unique hepatic innate immune response associated with robust production of type iii interferons pathogenesis of chronic viral hepatitis: differential roles of t cells and nk cells innate immunity to virus infection toll-like receptors and viruses: induction of innate antiviral immune responses viral and therapeutic control of ifn-beta promoter stimulator 1 during hepatitis c virus infection differential roles of mda5 and rig-i helicases in the recognition of rna viruses cytosolic viral sensor rig-i is a 59-triphosphate-dependent translocase on double-stranded rna the mitochondrial targeting chaperone 14-3-3 regulates a rig-i translocon that mediates membrane association and innate antiviral immunity hcv innate immune responses cardif is an adaptor protein in the rig-i antiviral pathway and is targeted by hepatitis c virus regulation of jak-stat signalling in the immune system down-regulation of the internal ribosome entry site (ires)-mediated translation of the hepatitis c virus: critical role of binding of the stem-loop iiid domain of ires and the viral core protein hepatitis c virus core protein blocks interferon signaling by interaction with the stat1 sh2 domain ifn-alpha antagonistic activity of hcv core protein involves induction of suppressor of cytokine signaling-3 analysis of interferon signaling by infectious hepatitis c virus clones with substitutions of core amino acids 70 and 91 repression of interferon regulatory factor 1 by hepatitis c virus core protein results in inhibition of antiviral and immunomodulatory genes viral immune evasion: a masterpiece of evolution inhibition of the interferon-inducible protein kinase pkr by hcv e2 protein hepatitis c virus and interferon resistance control of innate immune signaling and membrane targeting by the hepatitis c virus ns3/4a protease are governed by the ns3 helix a 0 structural determinants for membrane association and dynamic organization of the hepatitis c virus ns3-4a complex hepatitis c virus protease ns3/4a cleaves mitochondrial antiviral signaling protein off the mitochondria to evade innate immunity immune evasion by hepatitis c virus ns3/4a protease-mediated cleavage of the toll-like receptor 3 adaptor protein trif cleavage of mitochondrial antiviral signaling protein in the liver of patients with chronic hepatitis c correlates with a reduced activation of the endogenous interferon system toll-like receptor 3-mediated activation of nf-kb and irf3 diverges at toll-il-1 receptor domain-containing adapter inducing ifn-b sting is an endoplasmic reticulum adaptor that facilitates innate immune signalling sting specifies irf3 phosphorylation by tbk1 in the cytosolic dna signaling pathway sting regulates intracellular dnamediated, type i interferon-dependent innate immunity denv inhibits type i ifn production in infected cells by cleaving human sting hepatitis c virus ns4b blocks the interaction of sting and tbk1 to evade host innate immunity hepatitis c virus ns4b protein targets sting and abrogates rig-i-mediated type i interferon-dependent innate immunity phosphorylation of hepatitis c virus-encoded nonstructural protein ns5a phosphorylation of the hepatitis c virus ns5a protein in vitro and in vivo: properties of the ns5a-associated kinase phosphorylation of hepatitis c virus ns5a nonstructural protein: a new paradigm for phosphorylation-dependent viral rna replication? reduction of hepatitis c virus ns5a hyperphosphorylation by selective inhibition of cellular kinases activates viral rna replication in cell culture hepatitis c virus ns5a: tales of a promiscuous protein hepatitis c virus nonstructural protein 5a modulates the toll-like receptor-myd88-dependent signaling pathway in macrophage cell lines evidence that hepatitis c virus resistance to interferon is mediated through repression of the pkr protein kinase by the nonstructural 5a protein how hepatitis c virus counteracts the interferon response: the jury is still out on ns5a control of pkr protein kinase by hepatitis c virus nonstructural 5a protein: molecular mechanisms of kinase regulation hepatitis c virus nonstructural 5a protein induces interleukin-8, leading to partial inhibition of the interferoninduced antiviral response relationships between hepatitis c virus replication and cxcl-8 production in vitro hepatitis c virus ns5a protein interacts with 29,59-oligoadenylate synthetase and inhibits antiviral activity of ifn in an ifn sensitivity-determining region-independent manner activation and evasion of the antiviral 29-59 oligoadenylate synthetase/ribonuclease l pathway by hepatitis c virus mrna hepatitis c virus ns5a protein protects against tnf-alpha mediated apoptotic cell death inhibition of intrahepatic gamma interferon production by hepatitis c virus nonstructural protein 5a in transgenic mice hepatitis c virus nonstructural protein 5a inhibits tumor necrosis factor-a-mediated apoptosis in huh7 cells activation of endoplasmic reticulum stress response by hepatitis viruses upregulates protein phosphatase 2a protein phosphatase 2a: a highly regulated family of serine/threonine phosphatases implicated in cell growth and signalling pp2a targeting by viral proteins: a widespread biological strategy from dna/rna tumor viruses to hiv-1 hepatitis c virus inhibits interferon signaling through upregulation of protein phosphatase 2a upregulation of protein phosphatase 2ac by hepatitis c virus modulates ns3 helicase activity through inhibition of protein arginine methyltransferase 1 transcriptional induction of two genes in human cells by b interferon functional classification of interferon-stimulated genes identified using microarrays a central role for rna in the induction and biological activities of type 1 interferons targeted impairment of innate antiviral responses in the liver of chronic hepatitis c patients negative feedback regulation of rig-i-mediated antiviral signaling by interferoninduced isg15 conjugation immune signaling by rig-i-like receptors retinoic acid inducible gene-i (rig-i) signaling of hepatic stellate cells inhibits hepatitis c virus replication in hepatocytes interferon-b pretreatment of conventional and plasmacytoid human dendritic cells enhances their activation by influenza virus a structure-based model of rig-i activation emerging role of ubiquitination in antiviral rig-i signaling ddx60, a dexd/h box helicase, is a novel antiviral factor promoting rig-i-like receptor-mediated signaling hepatitis c virus induces interferon-and interferon-stimulated genes in primary liver cultures irf family of transcription factors as regulators of host defense regulation of pkr and irf-1 during hepatitis c virus rna replication constitutive expression of an isgf2/irf1 transgene leads to interferon-independent activation of interferon-inducible genes and resistance to virus infection regulation of hepatitis c virus replication by interferon regulatory factor 1 irf-7 is the master regulator of type-i interferon-dependent immune responses hepatitis c virus infection impairs irf-7 translocation and alpha interferon synthesis in immortalized human hepatocytes irf-3, irf-5, and irf-7 coordinately regulate the type i ifn response in myeloid dendritic cells downstream of mavs signaling hepatitis c virus inhibits intracellular interferon alpha expression in human hepatic cell lines serum-derived hepatitis c virus infectivity in interferon regulatory factor-7-suppressed human primary hepatocytes interferon regulatory factor irf-7 induces the antiviral alpha interferon response and protects against lethal west nile virus infection antiviral actions of interferons impact of protein kinase pkr in cell biology: from antiviral to antiproliferative action dsrna-dependent protein kinase pkr and its role in stress, signaling and hcv infection pkr protein kinase is activated by hepatitis c virus and inhibits viral replication through translational control pkr-dependent mechanisms of gene expression from a subgenomic hepatitis c virus clone identification of three interferon-inducible cellular enzymes that inhibit the replication of hepatitis c virus alpha interferon induces distinct translational control programs to suppress hepatitis c virus rna replication new antiviral pathway that mediates hepatitis c virus replicon interferon sensitivity through adar1 hepatitis c virus controls interferon production through pkr activation translational resistance of late alphavirus mrna to eif2a phosphorylation: a strategy to overcome the antiviral effect of protein kinase pkr initiation of protein synthesis by hepatitis c virus is refractory to reduced eif2.gtp.met-trna(i)(met) ternary complex availability translational insensitivity to potent activation of pkr by hcv ires rna hcv ns5a co-operates with pkr in modulating hcv ires-dependent translation eukaryotic translation initiation machinery can operate in a bacterial-like mode without eif2 hepatitis c virus blocks interferon effector function by inducing protein kinase r phosphorylation hepatitis c virus (hcv) induces formation of stress granules whose proteins regulate hcv rna replication and virus assembly and egress p bodies, stress granules, and viral life cycles stress granules: sites of mrna triage that regulate mrna stability and translatability stress granules eukaryotic stress granules: the ins and outs of translation regulation of stress granules and p-bodies during rna virus infection diversion of stress granules and p-bodies during viral infection dynamic oscillation of translation and stress granule formation mark the cellular response to virus infection hepatitis c virus hijacks p-body and stress granule components around lipid droplets hepatitis c virus coopts ras-gtpase-activating protein-binding protein 1 for its genome replication viral encounters with 29,59-oligoadenylate synthetase and rnase l during the interferon antiviral response new insights into the role of rnase l in innate immunity the oligoadenylate synthetase family: an ancient protein family with multiple antiviral activities the ribonuclease ldependent antiviral roles of human 29,59-oligoadenylate synthetase family members against hepatitis c virus 59-oligoadenylate synthetaselike gene highly induced by hepatitis c virus infection in human liver is inhibitory to viral replication in vitro antiviral activities of isg20 in positive-strand rna virus infections isg20, an actor of the innate immune response adenosine deaminases acting on rna (adars) are both antiviral and proviral adenosine deaminases acting on rna, rna editing, and interferon action rna-specific adenosine deaminase adar1 suppresses measles virus-induced apoptosis and activation of protein kinase pkr expression of interferoninducible rna adenosine deaminase adar1 during pathogen infection and mouse embryo development involves tissueselective promoter utilization and alternative splicing rna editing of hepatitis delta virus antigenome by dsrna-adenosine deaminase a specific base transition occurs on replicating hepatitis delta virus rna rna editing in hepatitis delta virus the ifitm proteins mediate cellular resistance to influenza a h1n1 virus, west nile virus, and dengue virus ifitm3 limits the severity of acute influenza in mice the broad-spectrum antiviral functions of ifit and ifitm proteins ifitms restrict the replication of multiple pathogenic viruses distinct patterns of ifitm-mediated restriction of filoviruses, sars coronavirus, and influenza a virus ifitm1 is a tight junction protein that inhibits hepatitis c virus entry ifitm3 inhibits influenza a virus infection by preventing cytosolic entry the antiviral effector ifitm3 disrupts intracellular cholesterol homeostasis to block viral entry ifitm-2 and ifitm-3 but not ifitm-1 restrict rift valley fever virus the cd225 domain of ifitm3 is required for both ifitm protein association and inhibition of influenza a virus and dengue virus replication ifit1 is an antiviral protein that recognizes 59-triphosphate rna ifn-induced tpr protein ifit3 potentiates antiviral signaling by bridging mavs and tbk1 pathogens: raft hijackers cholesterol-binding viral proteins in virus entry and morphogenesis the diverse functions of oxysterolbinding proteins lipid traffic: floppy drives and a superhighway short-range intracellular trafficking of small molecules across endoplasmic reticulum junctions ebola virus entry requires the cholesterol transporter niemann-pick c1 ifitm proteins restrict viral membrane hemifusion isg56 and ifitm1 proteins inhibit hepatitis c virus replication tpr proteins: the versatile helix the isg56/ifit1 gene family isg56 is a negativefeedback regulator of virus-triggered signaling and cellular antiviral response 29-o methylation of the viral mrna cap evades host restriction by ifit family members coordinated regulation and widespread cellular expression of interferon-stimulated genes (isg) isg-49, isg-54, and isg-56 in the central nervous system after infection with distinct viruses tracing the history of the ubiquitin proteolytic system: the pioneering article the ubiquitin code interplay between viruses and host sumoylation pathways viral evasion mechanisms of early antiviral responses involving regulation of ubiquitin pathways ubiquitin-like protein modifiers and their potential for antiviral and anti-hcv therapy isg15 and immune diseases human isg15 conjugation targets both ifn-induced and constitutively expressed proteins functioning in diverse cellular pathways proteomic identification of proteins conjugated to isg15 in mouse and human cells the antiviral activities of isg15 isg15 enhances the innate antiviral response by inhibition of irf-3 degradation positive regulation of interferon regulatory factor 3 activation by herc5 via isg15 modification activation of double-stranded rna-activated protein kinase (pkr) by interferon-stimulated gene 15 (isg15) modification down-regulates protein translation identification of interferon-stimulated gene 15 as an antiviral molecule during sindbis virus infection in vivo ifn-stimulated gene 15 functions as a critical antiviral molecule against influenza, herpes, and sindbis viruses isg15, a ubiquitin-like interferon-stimulated gene, promotes hepatitis c virus production in vitro: implications for chronic infection and response to treatment hepatitis c virus reveals a novel early control in acute immune response ubp43 (usp18) specifically removes isg15 from conjugated proteins ubp43 is a novel regulator of interferon signaling independent of its isg15 isopeptidase activity the level of hepatitis b virus replication is not affected by protein isg15 modification but is reduced by inhibition of ubp43 (usp18) expression isg15 inhibits nedd4 ubiquitin e3 activity and enhances the innate antiviral response silencing of usp18 potentiates the antiviral activity of interferon against hepatitis c virus infection knockdown of usp18 increases a 2a interferon signaling and induction of interferon-stimulating genes but does not increase antiviral activity in huh7 cells potential relevance of cytoplasmic viral sensors and related regulators involving innate immunity in antiviral response interferon signaling and treatment outcome in chronic hepatitis c the isg15/usp18 ubiquitin-like pathway (isgylation system) in hepatitis c virus infection and resistance to interferon therapy viperin: a multifunctional, interferon-inducible protein that regulates virus replication the role of viperin in the innate antiviral response the interferon inducible gene: viperin viperin, a key player in the antiviral response the interferon-inducible protein viperin inhibits influenza virus release by perturbing lipid rafts hiv-1 infection of human macrophages directly induces viperin which inhibits viral production the cellular antiviral protein viperin is attenuated by proteasome-mediated protein degradation in japanese encephalitis virus-infected cells human cytomegalovirus directly induces the antiviral protein viperin to enhance infectivity analysis of isg expression in chronic hepatitis c identifies viperin as a potential antiviral effector the antiviral protein viperin inhibits hepatitis c virus replication via interaction with nonstructural protein 5a viperin inhibits hepatitis c virus replication by interfering with binding of ns5a to host protein hvap-33 interferon-inducible cholesterol-25-hydroxylase broadly inhibits viral entry by production of 25-hydroxycholesterol virus membrane-fusion proteins: more than one way to make a hairpin class ii enveloped viruses a novel unsupervised method to identify genes important in the antiviral response: application to interferon/ribavirin in hepatitis c patients identification of type i and type ii interferon-induced effectors controlling hepatitis c virus replication a genetic screen identifies interferon-a effector genes required to suppress hepatitis c virus replication a functional genomic screen reveals novel host genes that mediate interferon-alpha's effects against hepatitis c virus pre-mrna splicing: awash in a sea of proteins a genome-wide genetic screen for host factors required for hepatitis c virus propagation hepatitis c virus infection activates an innate pathway involving ikk-a in lipogenesis and viral assembly class a scavenger receptor 1 (msr1) restricts hepatitis c virus replication by mediating toll-like receptor 3 recognition of viral rnas produced in neighboring cells hmgb1 is a therapeutic target for sterile inflammation and infection release of chromatin protein hmgb1 by necrotic cells triggers inflammation high-mobility group box 1, oxidative stress, and disease human hepatitis c virus ns5a protein alters intracellular calcium levels, induces oxidative stress, and activates stat-3 and nf-kb hepatitis c virus core protein inhibits mitochondrial electron transport and increases reactive oxygen species (ros) production hepatitis c virus protein expression causes calcium-mediated mitochondrial bioenergetic dysfunction and nitro-oxidative stress hepatitis c virus infection is blocked by hmgb1 released from virus-infected cells pattern recognition receptors and inflammation serum high mobility group box chromosomal protein 1 is associated with clinicopathologic features in patients with hepatocellular carcinoma autophagy fights disease through cellular self-digestion self-eating and selfkilling: crosstalk between autophagy and apoptosis autophagy: process and function autophagy: from phenomenology to molecular understanding in less than a decade autophagy and the integrated stress response autophagy protects against sindbis virus infection of the central nervous system autophagy is an essential component of drosophila immunity against vesicular stomatitis virus enhancing immunity through autophagy eating the enemy within: autophagy in infectious diseases autophagy genes in immunity autophagy, immunity, and microbial adaptations autophagy in immunity and inflammation induction of incomplete autophagic response by hepatitis c virus via the unfolded protein response the autophagy machinery is required to initiate hepatitis c virus replication knockdown of autophagy-related gene decreases the production of infectious hepatitis c virus particles activation of the unfolded protein response and autophagy after hepatitis c virus infection suppresses innate antiviral immunity in vitro hepatitis c virus inhibits akt-tuberous sclerosis complex (tsc), the mechanistic target of rapamycin (mtor) pathway, through endoplasmic reticulum stress to induce autophagy hepatitis c virus upregulates beclin1 for induction of autophagy and activates mtor signaling knockdown of autophagy enhances the innate immune response in hepatitis c virus-infected hepatocytes hepatitis c virus induces the mitochondrial translocation of parkin and subsequent mitophagy autophagy and rna virus interactomes reveal irgm as a common target micrornas and immunity: novel players in the regulation of normal immune function and inflammation how do micrornas regulate gene expression? microrna regulation of innate immune responses in epithelial cells regulation of gene expression by microrna in hcv infection and hcv-mediated hepatocellular carcinoma hcv-induced mir-21 contributes to evasion of host immune system by targeting myd88 and irak1 this study was supported by research grants from the ministry of science and technology (101-2320-b-001-022-my3) and academia sinica, taipei. this manuscript was edited for the english language by american journal experts (aje). attribution-noncommercial-noderivs 3.0 unported license. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in the credit line; if the material is not included under the creative commons license, users will need to obtain permission from the license holder to reproduce the material. to view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/3.0/