key: cord-299270-fwbz3t25 authors: lemieux, m. joanne; overduin, michael title: structure and function of proteins in membranes and nanodiscs date: 2020-08-22 journal: biochim biophys acta biomembr doi: 10.1016/j.bbamem.2020.183445 sha: doc_id: 299270 cord_uid: fwbz3t25 abstract the field of membrane structural biology represents a fast-moving field with exciting developments including native nanodiscs that allow preparation of complexes of post-translationally modified proteins bound to biological lipids. this has led to conceptual advances including biological membrane:protein assemblies or “memteins” as the fundamental functional units of biological membranes. tools including cryo-electron microscopy and x-ray crystallography are maturing such that it is becoming increasingly feasible to solve structures of large, multicomponent complexes, while complementary methods including nuclear magnetic resonance spectroscopy yield unique insights into interactions and dynamics. challenges remain, including elucidating exactly how lipids and ligands are recognized at atomic resolution and transduce signals across asymmetric bilayers. in this special volume some of the latest thinking and methods are gathered through the analysis of a range of transmembrane targets. ongoing work on areas including polymer design, protein labelling and microfluidic technologies will ensure continued progress on improving resolution and throughput, providing deeper understanding of this most important group of targets. with a short sma(2:1) copolymer with a 2:1 ratio of s:ma subunits shows optimum copolymer concentrations of 0.5-1% under temperatures in the 25-37 °c range with incubation times of 1-2 hours. lower temperatures warrant longer incubations, while excessive polymer concentrations should be avoided to minimize aggregates. optimal ionic strength (300-450 mm nacl), divalent cations (<2 mm) and ph (8) (9) are evident for kcsa solubilization but can be protein and membrane dependent as they are subject to respective charge distributions. comparisons with the performance of conventional detergents suggests that the development of neutral or basic copolymers related to sma could offer advantages, providing avenues for solubilization and analysis of a broader array of biological membrane:protein assemblies (memteins). the utility of sma lipid particle (smalp) technology for analysis of memteins by mass spectrometry (ms) and x-ray diffraction methods remains limited. this problem stems from the requirements of the respective gas phase or crystalline samples and the heterogeneity of currently used sma copolymers. in order to address this, muench and colleagues have developed a method to transfer membrane proteins from smalps into amphipols or detergents for downstream analysis [3] . the homotrimeric e. coli multidrug transporter acrb was used as a test case. the vulnerability of sma to precipitation in the presence of divalent cations was exploited by using stepwise addition of mgcl 2 from 0.5 to 2 mm to cause the solubilizing copolymer to precipitate in the presence of a rescue solution composed of amphipol a8-35 or 1% n-dodecyl-β-d-maltoside (ddm) detergent. the resulting amphipol j o u r n a l p r e -p r o o f journal pre-proof and detergent complexes are more homogeneous, tolerate higher divalent cations levels, and yield observable signals by native mass spectrometry, including of phosphatidylglycerol in the amphipol-stabilized protein trimer. new methods are being applied to analyze the process of forming nanodiscs from membranes in greater detail. in particular, a microfluidic diffusional sizing (msd) system which was recently developed to detect protein hydrodynamic radii between 0.3 to 20 nm has been adapted to probe nanodisc formation [4] . this microfluidics technology requires small sample volumes (<10 μl) which are injected into a microfluidic chip. the complexes diffuse across from one side of the chamber to the other during a 15 minute time course and are then sensed via primary amines of, for example, phosphatidylethanolamine. several stages can be reliably seen from comparison of mds and dynamic light scattering (dls) data collected with increasing ratios of sma(3:1) to lipid. first the polymer inserts into bilayers until it reaches saturation. this is followed by solubilization into particles with 30 -10 nm diameters. as excess polymer is applied these become progressively smaller until reaching a limiting diameter of 6 nm. the lipid-specific association of an aggregation peptide derived from the tau protein is also demonstrated, indicating utility for detecting how fibers are nucleated on membranes. two longstanding challenges are the characterization of endogenous heteromultimeric membrane proteins and the development of conformation-specific antibodies for memteins. a study by mouro-chanteloup and coworkers of red blood cell ghosts addresses these confounding issues [5] . the individual components of erythrocyte membranes including rh proteins are well known, but their multimeric lipid complexes are dissociated in detergent-based preparations and hence no longer recognizable or extractable using conformation-specific antibodies. in contrast using sma(3:1) copolymer for solubilization preserves the native states of rh complexes, and membrane-associated cytoskeletal proteins can be gently removed by washing in very low ionic strength buffer. the biological relevant states of detergent-free rh complexes can then be isolated using gel filtration and monoclonal antibodies. the native states are preserved based on detection by conformation-dependent antibodies, paving the way for antibody screening and structure-function analysis. the extraction and purification of low abundance drug targets from membranes in stable, functional forms is a key goal for the pharmaceutical industry. the g-protein coupled receptor (gpcr) superfamily is critical, being targeted by many therapeutic agents. they respond to a diversity of signals and mediate cellular responses through interactions with heterotrimeric g proteins, gpcr kinases and arrestins. the complexity and conservation of the interactions of the phosphorylated and palmitoylated vasopressin receptor type 2, its peptide agonist vasopressin and arrestin is modeled by fanelli and coworkers, revealing coupled motions between key gpcr sites [6] . conformational changes that are induced by ligands including full agonists, partial agonist, antagonists and inverse agonists are j o u r n a l p r e -p r o o f journal pre-proof membrane-dependent. the adenosine 2a (a2a) receptor is a well-characterized gpcr from a structural perspective, although the roles of dynamics and lipids remain unclear. after expression in pichia pastoris, the wild-type a2a receptor and a pair of mutants with trp to tyr substitutions were solubilized into nanodiscs using sma(2:1) copolymer by wheatley and colleagues [7] . a fluorescent reporter (iaedans) was also attached to an introduced cys residue by the sixth helix, providing an additional probe. binding of specific ligands induces concentration-dependent changes in the fluorescence signals of trp residues, which exhibit greater steric hindrance based on fluorescence anisotropy measurements. the association with phospholipids from the plasma membrane is apparent by ms/ms analysis of the smalps. watts et al. show that the dopamine d1 receptor can be purified from human embryonic kidney (hek) cells with a yield of 255 g/l using sma(3:1) copolymer [8] . as with the a2a receptor, the purification of this gpcr was carried out at 4 °c to preserve the native state. binding studies of the protein in smalps provides estimated native-like affinities for a neurotensin peptide ligand and a specific antagonist. the transport of materials including drug molecules out of cells is carried out by the atp binding cassette (abc) family of membrane transporters. the structures and functions of these proteins are reviewed by kanelis and coworkers, with an emphasis on the intrinsically disordered regions that mediate key interactions in a phosphorylation-dependent fashion [9] . a set of abcg2 protein constructs were designed with n-terminal gfp or snap and his 6 tags and solubilized from hek cells using sma(2:1) copolymer, as were cd28 and cd86 antibiotics include lipid-specific peptides that self-associate into pores, which permeabilize bacterial membranes. however, the intact pores are difficult to resolve due to their dissociation in detergents. palmer's lab studies daptomycin, a cyclic lipopeptide consisting of 13 amino acids and a decanoic acid group that is a critical line of defense against infections caused by gram-positive bacteria. it works by binding phosphatidylglycerol-containing membranes in a calcium-dependent manner to form octameric pores. the design of alternating sma copolymers with methylamine sidechains allows octamers and tetramers to be obtained by varying on the amount of added polymer, illuminating the assembly of membrane complexes [13] . this paves the way for structure-determination of the native pore state and design of improved antibiotics to combat multi-drug resistant bacteria. aquaglyceroporin proteins allow the selective permeation of molecules across membranes. this family includes the e. coli glycerol facilitator, which mediates passive diffusion of glycerol across the inner membrane of the bacterium. although prone to aggregation, careful optimization of its expression and solubilization in various media showed that this helical bundle protein is most monodispersed in lauryl maltose neopentyl glycol [14] . this detergent j o u r n a l p r e -p r o o f journal pre-proof stabilizes the physiological tetramer, although a non-native octamer persists due to interactions between the disordered termini. the dynamics of lipids in nanodiscs formed by sma(3:1) and diisobutylene/maleic acid copolymer (dibma) copolymers, which differ in their hydrophobic groups, are compared by steinhoff and coworkers [15] . the application of electron paramagnetic resonance (epr) spectroscopy and a set of phosphatidylcholine lipids with nitroxide groups located at the 5 th , 12 th or 16 th carbon atom positions reveals that the lipids are more constrained in the more stable smalp discs. this can be explained by coarse-grain molecular dynamics simulations, which indicate that in the case of dibma a single belt of polymer contains lipids within the nanodisc with dynamics more closely resembling their mobility in a liposome. in the overduin lab, new amphipathic copolymers are being developed to broaden the utility of native nanodiscs. a series of alkylamine derivatives of sma with alternating sidechains were designed to reduce polymer sequence heterogeneity and improve resolution [16] . in the major facilitator superfamily, various solute carrier (slc) families exist. one of the challenges is to functionally characterize these transporters. in particular, a different transporter function may be present for the same transporter when it resides in different cell types. the cordat lab determined that the slc26a7 protein is a chloride/bicarbonate exchanger in kidney cells, which is in contrast to its role as a chloride channel in oocytes [17] . furthermore, they show that the abundance of this protein at the cell surface is dependent on the osmolarity of the cell culture medium, and ph of the cells. this is important considering the slc26a7 is expressed in outer medullary collecting duct cells near acid secreting cells that can render the local environment hyperosmotic compared to plasma. in human cells, both concentrative and equilibrative nucleoside transporters (cnt and ent) are found. with multiple transporters in this family having overlapping function it is important to characterize each individually. radioactive hplc was used by the young lab to determine the metabolism of nucleosides in oocytes, for which little previous study has been conducted [18] . this study revealed little metabolism occurred in oocytes and helped develop this novel means to examine nucleoside levels. next, in mice, the contribution of cnt and ents, which have overlapping specificities, were examined using radioactive j o u r n a l p r e -p r o o f hplc. nucleotide metabolites were assessed from plasma of mice with cnt or ent knockouts, illuminating the specificities of these transporters in vivo. the seca protein works to facilitate the transport of proteins destined for secretion through the secyeg translocon. despite structural information that reveals helical nucleotide binding domains, how seca interacts with diverse secretory proteins remain a question in the field. to address this, the bondar group conducted a sequence analysis of bacterial seca proteins (425), which was guided by sequence alignment, structural analysis and phylogeny [19] . they identified that seca proteins have varied length as a result of insertions and deletions, with clustering revealing three main groups. the first 220 residues housing the nucleotide binding region 1 (nbd1) is highly conserved in the family, with preferred residues at the n-terminus, which include a conserved phe at position 10, followed by hydrophobic sequences, and an arg/lys, are important for lipid anchoring. in addition they highlight the tyr residue located at the end of a helix finger structural motif, and other charged residues that may play a role in recognition of the positively charged pre-sequence. they further suggest diversity in sequence length contributes to increased conformational dynamics that may influence target interactions. the development of new tools to produce and analyze of integral membrane proteins is covered in a timely review by danmaliki and hwang [20] . these targets remain challenging to characterize at atomic resolution by any method. their analysis by nuclear magnetic j o u r n a l p r e -p r o o f journal pre-proof resonance (nmr) spectroscopy is complicated by the requirement for many milligrams of pure protein containing 2 h, 13 c and 15 n isotope labels to ensure high sensitivity and resolution, as well as the complex dynamics within the bilayer that broaden signals from transmembrane regions. advanced isotope labelling protocols allow chemical groups such as methyl and methylene groups and 19 f nuclei on specific residue types to be resolved, yielding structural information from such moieties. successes include an array of  barrel structures, which have been solved in various detergent micelles, as well as -helical structures that are inherently more challenging as they exhibit greater chemical shift degeneracy and fewer observable distances between secondary structure elements. hence nmr is well-positioned to provide unique insights into dynamics and interactions, as well as structures of proteins that are small or difficult to crystallize. single pass transmembrane proteins pose challenges for structural biology, being particularly dynamic, prone to aggregation and dependent on lipid microenvironments. consequently, very few have been characterized at high resolution. ramamoorthy and colleagues solubilized the full length form of the microsomal protein cytb5, which includes a heme-containing soluble domain, transmembrane helix and flexible linker, into nanodiscs using an 18-residue amphipathic helical peptide and various synthetic lipids [21] . through expression in e. coli the four tryptophan residues could be labelled with fluorine-19 for detection by 19 f-nmr spectroscopy. due to the rapid tumbling of the ~8 nm diameter nanodiscs, the 19 multi-domain proteins that reversibly bind membrane surfaces provide technical challenges for unravelling biological pathways. the phafin2 protein plays a critical role in inducing autophagy, but its membrane recognition mechanism is unclear. this modular protein contains a pleckstrin homology (ph) domain and a (fab1, yotb, vac1, and eea1) fyve domain, both of which bind phosphoinositol 3-phosphate (pi3p), a key signaling lipid that is found in endocytic membranes. the capelluto group showed that its fyve domain is indispensable for constitutive and specific pi3p recognition [16] . in contrast, its ph domain binds acidic lipids such as phosphatidylserine or pi3p when they are present in bilayers (but not as soluble lipids). the ph domain is autoinhibited by interactions with a conserved acidic c-terminal motif. thus the multivalent and tightly regulated binding of proteins to membrane surfaces can deciphered via the individual interactions. in the future soluble and homogeneous nanodiscs may offer opportunities to reveal the native binding mechanisms via concerted recognition of multiple lipids and protein elements that are heavily phosphorylated. together, these studies illustrate the challenges and potential of the growing field of membrane structural biology. membranes have traditionally been the least well understood components of the cellular ecosystem, and new tools have been sorely needed. the coming years promise further gains as the interfaces between proteins and lipids become better understood, and are certain to yield many more secrets into memtein formation and function. our ability to probe such mechanisms in native states and recombinant forms will in turn lead to improved understanding and exploitation of membrane targets for applications including synthetic biology and drug discovery. isolation of intramembrane proteases in membrane-like environments factors influencing the solubilization of membrane proteins from escherichia coli membranes by styrene-maleic acid copolymers styrene maleic-acid lipid particles (smalps) into detergent or amphipols: an exchange protocol for membrane protein characterisation microfluidic diffusional sizing probes lipid nanodiscs formation detergent-free isolation of native red blood cell membrane complexes dynamics and structural communication in the ternary complex of fully phosphorylated v2 vasopressin receptor, vasopressin, and beta-arrestin 1 ligand-induced conformational changes in a smalp-encapsulated gpcr detergent-free extraction of a functional low-expressing gpcr from a human cell line intrinsically disordered regions regulate the activities of atp binding cassette transporters application of fluorescence correlation spectroscopy to study substrate binding in styrene maleic acid lipid copolymer encapsulated abcg2 expression and detergent free purification and reconstitution of the plant plasma membrane na(+)/h(+) antiporter sos1 solution structure of the cytoplasmic domain of nhap2 a k(+)/h(+) antiporter from vibrio cholera characterization of multimeric daptomycin bound to lipid nanodiscs formed by calcium-tolerant styrene-maleic acid co-polymer solution structure and oligomeric state of the e. coliglycerol facilitator lipid dynamics in nanoparticles formed by maleic acid-containing copolymers: epr spectroscopy and molecular dynamics simulations the effect of hydrophobic alkyl sidechains on size and solution behaviors of nanodiscs formed by alternating styrene maleamic copolymer slc26a7 protein is a chloride/bicarbonate exchanger and its abundance is osmolarity-and ph-dependent in renal epithelial cells hplc reveals novel features of nucleoside and nucleobase homeostasis, nucleoside metabolism and nucleoside transport diversity and sequence motifs of the bacterial seca protein motor solution nmr spectroscopy of membrane proteins expression, purification, and functional reconstitution of (19)f-labeled cytochrome b5 in peptide nanodiscs for nmr studies where he serves as director of nanuc, canada's national nmr centre. his research focuses on membrane structural biology and the discovery of ligands of proteins involved in cell adhesion, signaling and endocytosis. he studies desmosomal protein interactions, phosphoinositide recognition by signaling proteins, and ligand binding by novel drug targets including calcium/calmodulin dependent ser/thr kinases and gtpase regulators. he co-developed the moda program to identify membrane binding domains and the smalp system for detergent-free purification of native membrane proteins into stable as director of the membrane protein disease research group she leads a multidisciplinary research program focused on membrane protease structure and function. she is internationally recognized as a leader in membrane protein crystallography having solved two distinct membrane protein crystal structures key: cord-287093-9mertwj7 authors: netherton, christopher l; wileman, tom title: virus factories, double membrane vesicles and viroplasm generated in animal cells date: 2011-10-12 journal: curr opin virol doi: 10.1016/j.coviro.2011.09.008 sha: doc_id: 287093 cord_uid: 9mertwj7 many viruses reorganise cellular membrane compartments and the cytoskeleton to generate subcellular microenvironments called virus factories or ‘viroplasm’. these create a platform to concentrate replicase proteins, virus genomes and host proteins required for replication and also protect against antiviral defences. there is growing interest in understanding how viruses induce such large changes in cellular organisation, and recent studies are beginning to reveal the relationship between virus factories and viroplasm and the cellular structures that house them. in this review, we discuss how three supergroups of (+)rna viruses generate replication sites from membrane-bound organelles and highlight research on perinuclear factories induced by the nucleocytoplasmic large dna viruses. many viruses replicate within subcellular microenvironments or 'mini-organelles' called virus factories or 'viroplasm'. formation of these structures involves rearrangement of host cell membranes and cytoskeleton and induces a 'cytopathic effect' indicative of virus infection. it is generally believed that factories and viroplasm create a platform to concentrate replicase proteins, virus genomes and host proteins required for replication, and at the same time physically separate replication sites from a myriad of cellular antiviral defences. the subversion of membrane trafficking pathways during the formation of replication sites may add further benefit by slowing the transport of immunomodulatory proteins to the surface of infected cells to protect against immune responses, while disruption of the cytoskeleton may enhance release of viruses from cells. there is growing interest in understanding how viruses induce such large changes in cellular organisation, and recent advances in electron microscopy coupled with tomography and live cell imaging are beginning to reveal the relationship between virus factories and viroplasm and the cellular structures that house them. in this review, we discuss how three supergroups of (+)strand rna viruses generate replication sites from membrane-bound organelles and highlight research on perinuclear factories induced by the nucleocytoplasmic large dna viruses (ncldv). the rna-dependent rna polymerases (rdrp) of the (+)strand rna viruses are targeted to the cytoplasmic face of membrane-bound organelles and subsequent assembly of the replicase complex induces membrane curvature and the formation of densely packed membrane vesicles (reviewed in [1, 2] ) ( figure 1 ). the alphaviruses generate membrane invaginations called spherules [3] , while the flaviviruses and coronaviruses generate networks of double membrane vesicles (dmvs) connected to a complex of convoluted membranes (cm) derived from the endoplasmic reticulum (er). the picornaviruses also generate dmvs and a heterogeneous series of membrane vesicles and membrane rosettes [4, 5] . the formation of spherules, and possibly dmvs, parallels mechanisms of capsid assembly where ordered assembly of replicase proteins induces membrane curvature and invaginations of uniform diameter [2] . the more complex membrane rearrangements involving er networks and cm may involve additional recruitment of host proteins that modulate snap (soluble nsf [n-ethylmaleimide sensitive factor] attachment protein) receptor (snare) proteins and the rab and arf gtpases that control the secretory pathway (reviewed in [6] ). rdrp supergroup 3 viruses such as the animal alphaviruses, semliki forest virus (sfv) and sindbis virus produce 50 nm diameter invaginations called spherules that are aligned along the inside face of the limiting membrane of endosomes and lysosomes [7] . flock house virus (fhv) is a member of the nodaviridae family (rdrp supergroup 1), which generates spherules in the outer membrane of mitochondria [8] . each spherule contains approximately 100 copies of the replicase protein packed along the inner membrane surface [9 ] . tomographic models for formation of sperules and double membrane vesicles during replication of (+)strand rna viruses. panel 1: spherule produced by alphaviruses. replicase proteins (red spheres) are recruited to the cytoplasmic face of membrane-bound organelles. assembly of replicase proteins induces membrane curvature and invagination forming a spherule. the spherule remains connected to the limiting membrane of the organelle and a pore allows new genomes to enter the cytosol (adapted from [2] ). panel 2: virus-induced vesicles and double membrane vesicles generated by denv flavivirus. replicase proteins (red spheres) are recruited to the cytoplasmic face of membrane-bound organelles. assembly of replicase proteins induces membrane curvature and invagination into the er forming a large spherule. the invagination remains connected to the limiting membrane of the organelle and a pore allows new genomes to enter the cytosol. close apposition of er membranes leads to the formation of dmvs connected to the er by convoluted membranes (cm) (adapted from [10 ] ). this may close the pore leading to the cytosol. panel 3: virus-induced vesicles and double membrane vesicles generated by sars-cov coronavirus. replicase proteins (red spheres) are recruited to the cytoplasmic face of membrane-bound organelles. assembly of replicase proteins induces membrane curvature and invagination into the er forming a large spherule. it has been difficult to find evidence for a pore connecting invaginations to cytosol. close apposition of er membranes leads to the formation of dmvs connected to the er by cm. these may exclude replicase proteins and become sites for storage of viral rna. in some cases the close apposition of er membranes is lost and single membraned vesicles arranged in vesicle packets (vp) appear within membrane networks connected to the er (adapted from [11 ] ). reconstructions show that spherule membranes are continuous with the outer mitochondrial membrane and that a membrane neck connecting the lumen of the spherule to the cytosol surrounds a channel wide enough to allow passage of (+)rna into the cytosol [9 ] . rubella virus is the only member of the rubivirus genus of the togaviridae, which are distantly related to the alphaviruses. assembly of the rubella rdrp on the cytoplasmic face of endosomes and lysosomes generates spherules containing replicase proteins and double-stranded rna. in some cases, the spherules grow into large vacuoles and rigid membrane rods and sheets possibly coated with rdrp. freeze fracture studies and tomographic reconstitution have identified inter-connections between the vacuoles [7] and protein bridges connecting vacuoles to er. the flavivirus genus are part of rdrp supergroup 2. yellow fever virus, west nile virus and dengue virus (denv) generate 80-100 nm diameter invaginations into the er. tomographic reconstructions of membranes induced by denv show a continuous network of er membrane connected to spherical vesicles and cm [10 ] . virus-induced vesicles contain replicase proteins and dsrna and are found within the lumen of the er. most have double membranes suggesting that they are formed from invaginations into er cisternae [10 ] . each spherical vesicle is connected to the er membrane by a neck with a pore opening to the cytosol that could allow transit of viral rna. in some images, the pores lie adjacent to sites of virus assembly making it possible that viral rna passes directly from the spherical vesicles to budding viruses [10 ] . arterivirus and coronaviruses also generate densely packed membrane vesicles. three-dimensional reconstructions of vesicles induced by severe acute respiratory syndrome coronavirus (sars-cov) show dmvs between 150 and 300 nm in size bounded by two tightly apposed membranes connected to er [11 ] . sars-cov also induces cm containing small tubular and reticular membranes connected to the er. later during infection cells contain 'vesicular packets' where single membraned vesicles are surrounded by a common outer er membrane [11 ] . the interior of the dmvs contains dsrna but surprisingly, unlike dmvs generated by flaviviruses, the replicase proteins are absent from the dmvs but locate to the cm that lie between dmvs. this suggests that virus replication occurs in the cm rather than dmvs. neck-like structures extending from dmvs to the outer er membrane are visible, but evidence for a pore connected to the cytosol is lacking. enveloped sars-cov can be detected in the vesicular packets. this suggests that replication may take place on cm and that genomes are transferred to vesicular packets for envelopment and budding, while excess viral rna may be stored in dmvs. picornaviruses generate densely packed dmvs between 200 and 400 nm in diameter, a series of single membraned vesicles resulting from fragmentation of the golgi, and autophagosomes possibly generated as a bystander response to infection [11 ,12-16] . the nature and relative numbers of vesicles vary greatly depending on the picornavirus family and it is not clear which population of vesicles house the replication complex. the dmvs generated during picornavirus replication lack an obvious opening to the cytosol making it possible that, as suggested for coronaviruses, they are a by-product of replicase assembly and are used as a storage site for viral rna. lipid biosynthesis plays an important role in both alphavirus replication and nodavirus replication as cerulenin treatment inhibits alphavirus and nodavirus replication [17, 18] and both fhv and sfv appear to upregulate phosphatidylcholine synthesis [18] . similarly, hepatitis c virus (hcv) replication requires fatty acid synthesis and geranylgeranylation [19] and cerulenin also inhibits poliovirus (pv) viral rna synthesis [20] and coxsackievirus b3 replication [21] . this suggests that lipid synthesis is required for efficient replication of many different (+)strand rna viruses. viral proteins that generate dmvs are beginning to be identified. when expressed separately the picornavirus 2bc, 2c and 3a proteins generate er vesicles and tubules but these differ from the vesicles produced during infection. for pv, coexpression of 2bc and 3a can generate dmvs similar to those seen in infected cells [16] . the arterivirus nsp3 and the coronavirus equivalent nsp4 proteins are multispanning er membrane proteins. coexpression of equine arterivirus nsp2 and 3 [22] can generate dmvs from the er, and protein interactions involving cysteine residues in the first loop domain are required for generating correct curvature [23] . dmv formation may also require host proteins that regulate the formation of vesicles within the er and golgi. inhibition of sar1 and arf1 gtpases inhibits rna replication, and inhibition of the arf gtp exchange factor, gbf1, by brefeldin a reduces the number of dmvs in infected cells [24 ] . the downstream effector of arf1 required by coronaviruses remains unknown but may be the phosphatidylinositol-4-kinase-iiib (pi4kiiib) shown to play a role in picornavirus and hcv replication (see below). for enteroviruses such as pv and coxsackieviruses, the nonstructural protein 3a may play a crucial role in the recruitment of the rdrp, 3d pol , to the cytoplasmic face of membrane-bound organelles by increasing the recruitment of pi4kiiib [25 ] . the mechanism hinges on the ability of the enterovirus 3a protein to modulate the activity of the arf1 gtpase and its guanine nucleotide exchange factor gbf1. arf1 and gbf1 play a central role in regulating membrane traffic between the er and golgi. activation of arf1 by gbf1 catalyses recruitment of cop1 proteins to the golgi to facilitate formation of the cop1-coated vesicles that carry proteins from the golgi to the er. binding of enterovirus 3a protein to gbf1 activates arf1 and increases recruitment of an alternative arf1 effector protein, pi4kiiib, to membranes [24 ] . since 3d pol binds to phosphatidylinositol-4-phosphate (pi4-p), the localised production of pi4-p increases recruitment of the 3d pol at the expense of cop1. this results in disassembly of the golgi providing membrane vesicles enriched in pi4-p for replication. this role for 3a may not hold for other picornaviruses where replication is not dependent on an active gbf1, and for picornaviruses that express 3a proteins unable to disrupt er to golgi transport [26] [27] [28] [29] . hcv also promotes recruitment of pi4k to replication sites [25 ,30 ] . hcv generates a membranous web with many similarities to the network of spherical vesicles and cm produced by denv. pi4-p lipids are colocalised with hcv replicase protein ns5a, and replication is reduced following pi4kiiib knockdown or overexpression of the sac1 phosphatase that removes phosphate from pi4-p. the hepatitis ns5a protein also binds pi4kiiia and stimulates enzyme activity and knockdown of pi4kiiia prevents formation of the membranous web associated with virus replication [30 ,31 ] . the ns5a protein is anchored to the cytoplasmic face of the membranous web and recruits ns5b and a series of cellular proteins that regulate membrane vesicle formation. these include vesicle-associated membrane protein-associated proteins [32] which bind snares involved in er to golgi transport, and a rab1-gap protein, tbc1d20. ns4b can recruit rab5 and rab5 effectors eea1, rabaptin 5 and rab4 [31 ] suggesting the web may fuse with endosomes [33] . double-membraned vesicles are usually rare in cells, but dmvs are induced during autophagy. this makes it possible that autophagosomes may provide a source for dmvs associated with virus replication. a role for autophagosomes in supporting replication of the coronavirus mouse hepatitis virus (mhv) was first provided by studies where mouse embryonic stem cells lacking crucial autophagy protein atg5 showed a 1000-fold reduction in mhv replication and reduced numbers of dmvs [34] . less clear-cut results come from studies of primary fibroblasts or macrophages where loss of atg5 has little impact on virus replication [35] . vesicles labelled with autophagy marker lc3 are, however, produced during coronavirus infection suggesting that the virus activates autophagy. autophagy may be activated by the nsp6 proteins of cornaviruses, or in the case of the arteriviruses, the equivalent nsp5-7 protein. the nsp6 (nsp5-7) proteins locate to the er where they generate small vesicles enriched in phosphatidylinositol-3-phosphate and early autophagy marker atg5 [36 ] . these vesicles closely resemble cellular organelles called omegasomes that are formed from the er during the initial stages of autophagy [37] , and mature into autophagosomes labelled with autophagy marker atg8/lc3. autophagy is activated during cornavirus infection but this does not mean that all the dmvs generated in the cytoplasm are autophagosomes. most of the dmvs are smaller than autophagosomes and may be formed from invaginations into the er (figure 1 ). vesicles formed from the er during coronavirus infection can also recruit a non-lipidated autophagy marker lc3 (lc3i) by a pathway, that is, paradoxically, independent of autophagy, and linked to the export of er chaperones from the er to endosomes [38 ] . autophagosomes may not therefore play a direct role in the formation of virus-induced dmvs, but may represent a defence against infection. picornaviruses activate autophagy in cell culture models [39] and in some cases inhibition of autophagy reduces replication while activation increases virus yields. the autophagosome marker lc3 colocalises with pv replicase proteins suggesting replication on autophagosomes. translocation of lc3 to vesicles can be induced by expression of pv 2bc alone, but formation of dmvs resembling autophagosomes requires coexpression of 2bc with 3a [16, 39] . as with the coronaviruses, the picornavirus dmvs are approximately one third the diameter of cellular autophagosomes making it difficult to determine if assembly of the replicase complex results in the formation of dmvs directly, or if the dmvs represent modified autophagosomes. virus assembly and replication can also occur in virus factories close to the microtubule organising centre (mtoc) [40] . these inclusions lack cellular membranes and resemble inclusions called aggresomes that form at the mtoc in response to protein aggregation. aggresome inclusions such as lewy and mallory bodies are a pathological hallmark of protein misfolding diseases, and protect cells from the damage associated with protein aggregation. aggresomes and factories share many features in common including recruitment of mitochondria, cellular chaperones and confinement within cages of rearranged vimentin filaments (reviewed in [40, 42, 43] ). many viruses are delivered to the mtoc after entering cells, and in common with protein aggregates, this involves recognition by the microtubule motor protein, dynein. it is possible that viruses may appear foreign or misfolded to cells and stimulate an aggresome response. for the ncldv this may be beneficial and provide a site for replication, for other viruses it may lead to confinement at the mtoc and degradation. delivery of incoming african swine fever virus (asfv) to the mtoc is important for the initiation of replication [41] and replication of both the asfv and the iridovirus frog virus 3 appears to require rearrangement of vimentin [44, 45] . vimentin may provide a scaffold to prevent diffusion of viral components into the cytoplasm. dna and rna are spatially separated within vaccinia virus (vacv) and asfv factories [46 ,47] and individual factories appear to be distinct entities within the cell. although virus factories recruit many host-cell proteins to facilitate their replication, factories also represent effector sites for antiviral activity as vacv and asfv replication sites are targeted by stress granule components and mx proteins respectively [48, 49] . a number of clear similarities between the membrane rearrangements generated by the positive strand rna viruses are beginning to emerge [1, 2] and these are shared between animal and plant viruses. spherules and dmvs differ morphologically but may be formed by similar mechanisms involving ordered assembly of replicase proteins on membrane-bound organelles leading to membrane invagination (figure 1 ). dmvs may be modified spherules where close apposition of er-derived membranes follows initial invagination into the er lumen. for cornaviruses, and possibly picornaviruses, this may result in loss of the pore connecting the spherule to the cytosol and conversion of the spherule into a site for storage of viral rna to suppress innate immune responses to double-stranded rna. the site of spherule formation differs between viruses ( figure 2 ) and is determined by membrane targeting sequences in the nonstructural proteins that recruit the rdrp. this has been demonstrated by the work of miller et al. who could retarget fhv replication complexes from the mitochondria to the er in yeast [50] . recent work has shown that recruitment of rdrp may also involve modification of membrane lipids by arf1-dependent recruitment of pi4kiiib to membranes to generate pi4-p. this mechanism is shared between the enteroviruses of the picornavirus supergroup and the flavivirus hcv [25 ,31 ] . the membrane vesicles generated by picornaviruses are most heterogeneous and varied between subgroups and may be derived from membrane compartments that fragment during infection, rather than be formed for virus replication. it is uncertain which sites of replication generated by plant and animal viruses. the diagram is in two halves. the left represents a plant cell and is surrounded by a cell wall (green). viral replication complexes (vrc) are generated by plant viruses at the er, chloroplast and peroxisomes. tobamovirus vrcs are initiated at the tonoplast. vrcs of many plant viruses can be transported within the cell along the er and between cells using the plasmodesmata. this movement is directed by plant movement proteins (mp); see accompanying article by jeanmarie verchot for details. the right represents an animal cell. alphavirus and nodaviruses form spherules at the endosomal-lysosomal system and mitochondria respectively. coronavirus, arterivirus and flaviviruses form double membrane vesicles (dmvs) from the er and coronaviruses and arteriviruses also form vesicle packets (vps). the membranes used to generate sites of picornavirus replication are unclear but may involve the golgi, er and/or autophagosomes. nucleo-cytoplasmic large dna viruses (ncldv) factories are formed after microtubule-mediated delivery of incoming viruses to the microtubule organising centre next to the nucleus. thereafter they recruit host chaperones, mitochondria and intermediate filaments. membranes house the virus replicase and replication may occur on the cytoplasmic face of the er rather than in vesicles [5] . parallels between plant and animal picornaviruses are therefore difficult to define. the large dna viruses of animals such as poxviruses and other members of the ncldv generate perinuclear inclusions called virus factories that assemble at the mtoc and are maintained by dynein microtubule motor proteins. replication complexes generated in plants often move through cells onboard microtubule motor proteins (figure 2 ), but perinuclear inclusions are not found for plant viruses. as pointed out in the accompanying review by jeanmarie verchot, plants lack a mtoc to concentrate motor cargoes, and replication sites are therefore dispersed throughout the cell. origins of membrane vesicles generated during replication of positive-strand rna viruses organelle-like membrane compartmentalization of positive-strand rna virus replication factories cytoplasmic viral replication complexes cellular origin and ultrastructure of membranes induced during poliovirus infection formation of the poliovirus replication complex requires coupled viral translation, vesicle production, and viral rna synthesis modulation of membrane traffic between endoplasmic reticulum, ergic and golgi to generate compartments for the replication of bacteria and viruses three-dimensional structure of rubella virus factories flock house virus rna replicates on outer mitochondrial membranes in drosophila cells em tomography provides the first 3d reconstructions of membrane invaginations housing the replication complex of (+)strand rna virus composition and three-dimensional architecture of the dengue virus replication and assembly sites em tomography provides a 3d reconstruction of membrane rearrangments induced by dengue virus. the vesicles are connected to a continuous network of endoplasmic reticulum and a pore connects the vesicles to the cytoplasm. some pores lie opposites sites of virus envelopment sars-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum em tomography provides a 3d reconstruction of membrane rearrangemnts induced by sars-coronavirus. the double-membraned vesicles are connected to a continuous network of endoplasmic reticulum poliovirus infection blocks ergic-to-golgi trafficking and induces microtubule-dependent disruption of the golgi complex subversion of cellular autophagosomal machinery by rna viruses the ultrastructure of the developing replication site in foot-andmouth disease virus-infected bhk-38 cells fragmentation of the golgi apparatus provides replication membranes for human rhinovirus 1a remodeling the endoplasmic reticulum by poliovirus infection and by individual viral proteins: an autophagy-like origin for virusinduced vesicles synthesis of semliki forest virus rna requires continuous lipid synthesis complementary transcriptomic, lipidomic, and targeted functional genetic analyses in cultured drosophila cells highlight the role of glycerophospholipid metabolism in flock house virus rna replication hepatitis c virus rna replication is regulated by host geranylgeranylation and fatty acids phospholipid biosynthesis and poliovirus genome replication, two coupled phenomena the human fatty acid synthase: a new therapeutic target for coxsackievirus b3-induced diseases? non-structural proteins 2 and 3 interact to modify host cell membranes during the formation of the arterivirus replication complex formation of the arterivirus replication/transcription complex: a key role for nonstructural protein 3 in the remodeling of intracellular membranes mouse hepatitis coronavirus rna replication depends on gbf1-mediated arf1 activation viral reorganization of the secretory pathway generates distinct organelles for rna replication studies on enterovirus replication show that the 3a nonstructural proten modulates the activity of the arf1 gtpase and its guanine nucleotide exchange factor gbf1. this increases recrutment of srf1 effector protein, phosphatidylinositol-4-kinase-iiib (pi4kiiib) to the cytoplsmic face of the endoplasmic reticulum. formation of phosphatidylinositol-4-phosphate provides a binding site for the viral rna polymerase and inhibition of the pi4kiiib kinase slows replication inhibition of cellular protein secretion by picornaviral 3a proteins differential requirements for copi coats in formation of replication complexes among three genera of picornaviridae effects of foot-and-mouth disease virus nonstructural proteins on the structure and function of the early secretory pathway: 2bc but not 3a blocks endoplasmic reticulum-to-golgi transport inhibition of the secretory pathway by footand-mouth disease virus 2bc protein is reproduced by coexpression of 2b with 2c, and the site of inhibition is determined by the subcellular location of 2c recruitment and activation of a lipid kinase by hepatitis c virus ns5a is essential for integrity of the membranous replication compartment the membrane web generated from the endoplsmic reticulum (er) during hepatitis c replication contains eleveated levels of phosphatidylinositol-4-phosphate. the ns5a protein of hepatitis c locates to er and binds phosphatidylinositol-4-kinase-iii and stimulates kinase activity. knockdown of phosphatidylinositol-4-kinase-iii reduces replication. hepatitus c replication is therefore facilitated by production of phosphatidylinositol-4-phosphate on the cytoplsmic face of the er and this may facilitate recruitment of the rna polymerase roles for endocytic trafficking and phosphatidylinositol 4-kinase iii alpha in hepatitis c virus replication the study shows that the lipid kinase phosphatidylinositol-4-kinase iii alpha is important for hepatitis c replication human vap-b is involved in hepatitis c virus replication through interaction with ns5a and ns5b participation of rab5, an early endosome protein, in hepatitis c virus rna replication machinery coronavirus replication complex formation utilizes components of cellular autophagy virgin hw: coronavirus replication does not require the autophagy gene atg5 coronavirus nsp6 proteins generate autophagosomes from the endoplasmic reticulum via an omegasome intermediate identification of cornavirus proteins able to stimulate starvation independent formation of autophagosomes directly from the endoplasmic reticulum via an omegosome intermediate autophagosome formation from membrane compartments enriched in phosphatidylinositol 3-phosphate and dynamically connected to the endoplasmic reticulum coronaviruses hijack the lc3-i-positive edemosomes, er-derived vesicles exporting short-lived erad regulators, for replication edemosomes are er-derived vesicles that transport er chaperones to endosomes. double-membraned vesicles induced by the murine cornaviruses, mouse hepatitis virus (mhv), recruit edemosome marker edem1 and a non-lipidated lc3 by a pathaway independent of autophagy modification of cellular autophagy protein lc3 by poliovirus a guide to viral inclusions, membrane rearrangements, factories, and viroplasm produced during virus replication aggresomes resemble sites specialized for virus assembly aggresomes and pericentriolar sites of virus assembly: cellular defense or viral design? aggresomes and autophagy generate sites for viral infection vimentin rearrangement during african swine fever virus infection involves retrograde transport along microtubules and phosphorylation of vimentin by calcium calmodulin kinase ii interaction of frog virus 3 with the cytomatrix. iv. phosphorylation of vimentin precedes the reorganization of intermediate filaments around the virus assembly sites colocalization of transcription and translation within cytoplasmic poxvirus factories coordinates viral expression and subjugates host functions demonstrated that individual ncldv factories are compartmentalised and represent separate functional units within the cell regulation of host translational machinery by african swine fever virus formation of antiviral cytoplasmic granules during orthopoxvirus infection inhibition of a large double-stranded dna virus by mxa protein engineered retargeting of viral rna replication complexes to an alternative intracellular membrane the authors would like to thank jeanmarie verchot for helpful discussions during the preparation of this manuscript. tw is supported by bb/e018521 and bb/f012861 grants from bbsrc. cn is supported by bbsrc and defra (se1541). papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest key: cord-008480-p41oae8e authors: o'callaghan, barbara; synguelakis, monique; le gal la salle, gildas; morel, nicolas title: characterization of aminopeptidase n from torpedo marmorata kidney date: 2004-11-12 journal: biol cell doi: 10.1016/s0248-4900(94)80003-0 sha: doc_id: 8480 cord_uid: p41oae8e a major antigen of the brush border membrane of torpedo marmorata kidney was identified and purified by immunoprecipitation. the sequence of its 18 n terminal amino acids was determined and found to be very similar to that of mammalian aminopeptidase n (ec 3.4.11.2). indeed aminopeptidase n activity was efficiently immunoprecipitated by monoclonal antibody 180k1. the purified antigen gives a broad band at 180 kda after sds-gel electrophoresis, which, after treatment by endoglycosidase f, is converted to a thinner band at 140 kda. this antigen is therefore heavily glycosylated. depending on solubilization conditions, both the antigen and peptidase activity were recovered either as a broad peak with a sedimentation coefficient of 18s (2% chaps) or as a single peak of 7.8s (1% chaps plus 0.2 % c(12)e(9)), showing that torpedo aminopeptidase n behaves as an oligomer stabilized by hydrophobic interactions, easily converted into a 160 kda monomer. the antigen is highly concentrated in the apical membrane of proximal tubule epithelial cells (600 gold particles/μm(2) of brush border membrane) whereas no labeling could be detected in other cell types or in other membranes of the same cells (basolatéral membranes, vacuoles or vesicles). monoclonal antibodies prepared here will be useful tools for further functional and structural studies of torpedo kidney aminopeptidase n. aminopeptidase n (ec 3.4.11.2) is an abundant membrane bound peptidase of kidney and intestinal microvilli (for reviews, see [7, 23] ). it hydrolyses the n-terminal amino acid of short peptides, with preference for amino acids with uncharged side chains [23] . while it completes digestion of oligopeptides in the intestine [20] , its function in kidney brush border membrane remains unclear. this enzyme has also been reported in the plasma membrane of various cell types like hepatocytes [1, 25] or myeloid cells [11] . the complete amino acid sequences of human intestinal [21] and rat kidney [28] aminopeptidases n have been determined. northern blot analysis revealed the existence of two human rna transcripts, both encoding for the same protein but under the control of different promoters. however, intestinal and kidney epithelial cells express the same mrnas, which are shorter than the transcripts of myeloid or fibroblastic cells [24] . aminopeptidase n is composed of a single type of subunit, a 140-kda glycosylated polypeptide (for reviews, see [7, 23] ). this subunit possesses a single transmembrane domain near its n terminus while the major part of the enzyme is ectocellular, including the catalytic sites [7, 21, 28] . in the present work, we have characterized the aminopeptidase n from torpedo marmorata kidney and found it to be very similar to the mammalian enzyme. torpedo marmorata were provided by the marine station of areachon (france). crude fractions of kidney membranes were prepared from pooled whole kidneys which had been stored at -80°c for seve .r'al days. the tissue was homogenized (10% w/v) in buffer a (100 mm naci, 20 mm na phosphate buffer, 1 mm edta, 0.5 mm dithiothreitol, 10% glycerol, final ph 7.2) to which 0.5 mm pmsf was added. the homogenate was centrifuged at 500 gm~ for 10 min. membranes were pelleted (40 000 gm~x for 90 rain) from the low speed supematant. balb/c mice were immunized by three intraperitoneal injections (at 15-day intervals) of kidney membrane proteins (about 50/.tg emulsified proteins in freund's adjuvant). three days after a final booster intraperitoneal injection (without adjuvant), immunized spleen cells were fused with the non-secreting myeloma clone p3 x 63-ag8. 653 cells using polyethylene glycol as the fusing agent [4, 9] . hybrids were selected in hypoxanthine, aminopterin and thymidine medium and superuatants from the culture wefts containing hybrid cells were tested for the presence of antibodies binding to the apical membrane of tubular epithelial cells on torpedo kidney frozen sections. 42 clones were selected, some of which were cloned by limiting dilution. crude kidney membranes (2 mg proteinhrd) were solubilized in 1% chaps and 0.2% ci2e9 at 4°c for 90 min and non-solubilized material was pelleted (160 000 gmx for 60 rain). for screening experiments, proteins (about 1 mg/ml) were biotinylated using immunopure nhs-lc-biotin (pierce, 100/zg/mg protein) for 2 h, at 4°c. the reaction was stopped by addition of glycine (150 mm final concentration). then, immunoprecipitation was performed in one step: 0.5 ml of solubilized proteins (1 mg protein/ml), 1 ml of culture supernatant containing monoclonal antibodies, 0.5 ml of a suspension of protein a sepharose-4b (pharmacia, 10 mg dry powder/ml of 200 mm naci, 20 mm tris buffer ph 7.4) and 30/.tl of antimouse igg antibodies raised in rabbit (5 mg ig/ml, biosys)were mixed and incubated overnight at 4°c. beads were recovered by low speed centrifugation (800 g for 5 rain) and washed extensively. bound proteins were eluted by resuspension of the beads in 100 p.i sample buffer (5% sds, 10% fl-mercaptoethanol, 1 m sucrose, 50 mm tris buffer (ph 6.8), 1 mm bromophenol blue), and boiled for 1 rain. proteins were immunoadsorbed as above and eluted from the protein a beads in i00/al sds sample buffer. they were then diluted in 10 volumes of 250 mm na acetate buffer, 20 mm edta, 10 mm fl-mercaptoethanol, 6 mg/ml chaps, final ph 6.5 and incubated at 37°c for 18 h with or without 0.5 u endoglycosidase f/n-glycosidase f (boehringer). after concentration under vacuum, samples were resuspended in sds sample buffer and submitted to gel electrophoresis. electrophoresis was performed in 8-18% linear acrylamide gradient gels according to [10] . proteins in acrylamide gels were either stained with coomassie blue, or electrotransferred onto nitrocellulose as described by [26] . biotinylated proteins on the blots were indirectly visualized using streptavidin-biotinylated peroxidase complex (amersham). the peroxidase substrates were h202 and diaminobenzidine. immunoprecipitation was performed as above but starting from 18 mg solubilized kidney membrane proteins, and using 30 ml of culture supernatant (mab 180k2), 300/11 of rabbit antibody to mouse ig (50 mg ig/ml, biosys), and 9 ml of a suspension of protein a sepharose-4b beads (10 mg dry powder/ml). eluted proteins were dialysed against 10 1 0.05% sds (overnight) and 10 1" 0.025% sds for 4 h (spectrapor 2), and concentrated to 50/11 under vacuum. electrophoresis was performed as above except that 0.1 mm thioglycolate was added to the migration buffer and that a prerun (90 min at 30 ma) was performed [2] . proteins bands were transferred onto 'problott' membranes (applied biosystems) overnight at 30 v in transfer buffer (50 mm boric acid, 50 mm tris-base). the 180 kda band was stained by amidoschwartz and cut out. electroblotted proteins were sequenced by dr jp le caer (institut alfred fessard, cnrs, 91198 gif-sur-yvette) in a 470 a gaz-liquid protein sequencer (abi) using the problott cart.ridge. phenylthiohydantoin amino acids were analysed on a 120a pth analyser (abi, foster city, ca). kidney membrane proteins (2 mg protein/ml) were solubilized for 1 h at 40c in one of the following detergent-containing buffers: 1) 100 mm naci, 20 mm phosphate buffer, 0.5 mm mgci2, 10% glycerol, 2% chaps; ph 7.4; 2)100 mm naci, 20 mm phosphate buffer, 1 mm edta, 10% glycerol, 2% chaps; ph 7.4; 3) 100 mm naci, 20 mm phosphate buffer, 0.5 mm mgci2, 10% glycerol, 1% chaps, 0.2% c12e9; ph 7.4. after eentrifugation (160 000 gmax for 1 h), an aliquot (200 or 400/tl supernatan0 of solubilized proteins was layered on top of 11 ml sucrose gradient. we used 5-30% linear sucrose gradients in the solubilization buffer but with a 10-fold lower detergent concentration. proteins markers: alcohol deshydrogenase (7.4s, boehringer), catalase (11.3s, boehringer), and fl-galactosidase (16s, sigma) were solubilized and submitted to centrifugation in parallel linear sucrose gradients. the 180 kda antigen content in the fractions was estimated after 1/3 to 1/10 dilution in 200/.d transfer buffer (50 mm tris, 0.01% triton x-100, 10% methanol; ph 7.4) and blotting onto nitrocellulose, using a dot-blot apparatus. dots were probed with mab 180ki and antimouse ig antibodies conjugated to peroxidase (institut pasteur productions). intensity of the dot staining was quantified using a scanning densitometer (hoeffer scientific instruments) and compared to that of known amounts of solubilized kidney membrane proteins. enzymatic marker activity was measured as previously described [14, 16] . aminopeptidase n activity was followed at 405 nm using l-alanine p-nitroanilide (sigma) as the substrate in a 50 mm tris phosphate buffer (ph 7.4) [30] . protein content was determined by the method of lowry [12] . kidney pieces were fixed immediately after dissection in 3% paraformaldehyde in 300 mm naci, i00 mm na phosphate buffer (ph 7.4) for several hours. they were then incubated in the saline containing increasing sucrose concentrations (up to 20%) and frozen in isopentane cooled on dry ice. sections (10/.tm thick) were mounted on gelatin coated microscope slides and stored at -80°c. primary antibodies (or culture supernatants) were diluted in 500 mm naci, 20 mm tris buffer (ph 7.4), 0.1% tween 20 and 1% bovine serum albumin (bsa) and allowed to bind overnight. bound antibodies were visualized using fluorescein conjugated antimouse ig antibodies (institut pasteur productions). kidney pieces were immersed in 2.5% glutaraldehyde in 300 mm naci, 40 mm na phosphate buffer (ph 7.0), for 3 h at room temperature. tissue pieces were permeabilized by 0.2% saponin in the same buffered saline for 30 rain and preincubated in 0. by indirect immunofluorescence on frozen kidney sections, 42 clones were selected which produced antibodies that bound to the apical membrane of proximal tubule epithelial cells. each of these 42 culture supernatants was used to immunoprecipitate kidney membrane proteins that had been in some experiments, when kidney membranes were pre-pared in the absence of the protease inhibitor pmsf, an additional 150 kda band was immunoprecipitated by each of the 17 anti-180 kda antibodies (not shown). this 150 kda can be very easily removed from membranes by a single washing step. most probably, it is a proteolytic fragment of the 180 kda antigen bearing the immunogenic regions but lacking the membrane domains of the protein. the 180 kda antigen was immunoprecipitated by monoclonal antibody 180k1, submitted to gel electrophoresis and electroblotted (see materials and methods). the band at 180 kda was cut out and the n-terminal amino acid sequence was determined using automated edman degradation. starting from about 60 pmol of the 180 kda antigen, 40 pmol pth-aminoacids were obtained in each of the successive cycles. the 18 n-terminal amino acid sequence of the 180 kda antigen (table i) turned out to be very similar to the corresponding sequence of aminopeptidase n (ec 3.4.11.2) which has been determined in man [21] , rat [28] , rabbit [3] and pig [21] . this sequence begins with eight hydrophilic table i . n-terminal amino acid sequence of the 180 kda antigen. " for comparison, the n-terminal amino acid sequences of aminopeptidase n from rat kidney [28] and from intestines of man [21] , rabbit [31] and pig [21] are shown. ratkidney akg f y i s k s lg i lg illg human intestine ak g f y i s k s lg i lg illg rabbit intestine akg f y i s ka lg i lg fxlg porcintestine akg f y i s l a lg i agxlxv 4) . immunopreeipitated proteins were directly submitted to sds gel electrophoresis (-) or submitted to endoglycosidase f/n-glycosidase f action (0.5 u at 37°c for 18 h) before electrophoresis. gels were stained with coomassie blue. to check whether the 180 kda antigen was indeed aminopeptidase n, we measured aminopeptidase n activity using alanine p-nitroanilide as the substrate [30] . ~ln the course of immunoprecipitation experiments, enzyme activity was determined both in non-retained sampies (incubation mixture after removal of immunoadsorbed proteins by low speed centrifugation of protein a beads) and in immunoadsorbed protein samples (washed protein a bead suspensions just before sds elution). a significant proportion (42%) of peptidase activity was immunoprecipitated in a single step by monoclonal antibody 180k1 (table ii) . an unrelated monoclonal antibody 14k4 [19] was unable to precipitate any detectable enzyme activity. in the same experiment, the antigen precipitated by antibody 180k1 was visualized as a 180 kda band after sds-gel electrophoresis, which was absent from samples incubated with the control antibody 14k4 (fig 2) . proteins immunoabsorbed by antibodies 180k1 or 14k4, and eluted in sds, were treated by endoglycosidase f (see materials and methods). the 180 kda band was converted to a lower molecular mass polypeptide, at 140 kda (fig 2) . this demonstrates that torpedo zminopeptidase is heavily glycosylated, deglycosylation reducing its apparent molecular mass by about 20%. note, for compar-ison, a small effect of endoglycosidase treatment on the heavy immunoglobulin chain migration (at about 50 kda) in both 180k1 and 14k4 samples. bands at 30 kda most probably correspond to endoglycosidase f (32 kda molecular mass). to investigate whether the torpedo kidney aminopeptidase n was a monomer or an oligomer, we studied the velocity sedimentation in sucrose gradients of both the 180 kda antigen and the aminopeptidase n activity, solubilized in nondenaturing conditions. kidhey membranes were treated by 1% chaps + 0.2% c12e9. solubilized proteins were sedimented in 5-30% linear sucrose gradients at 40000 rpm for 18 h. fractions were collected from the bottom of the gradients and their 180 kda antigen content and peptidase activity were estimated as described in materials and methods. both antigen and activity were recovered as a single peak, with a sedimentation coefficient of 7. 8s (fig 3) . considering that aminopeptidase n is a hydrophilic membrane protein with a single transmembrane domain [7, 21] , we can assume that it binds only a small amount of detergent and behaves as a globular protein in sucrose gradients. taking the approximation of martin and ames [14] , this would give an approximate molecular mass of 160 kda for the 180 kda antigen, which therefore behaves, in these solubilization conditions, as a monomeric protein. then kidney membranes were solubilized by 2% chaps alone, in the presence of 0.5 mm mgc12 (fig 4a) or of 1 mm edta ( fig 4b) . after velocity sedimentation in sucrose gradients containing either mgc12 or edta, a broad peak of antigen was found, with a sedimentation coefficient around 18s. in the presence of mgci 2, the peak of activity was also broad and it did not coincide with that of the antigen ( fig 4a) : it was shifted to lighter fractions. in addition, a second smaller peak of activity was recovered around 9.5s. in the edta containing gradients, the total peptidase activity was much lower than in the mgc12 conditions. it corresponds to edta insensitive peptidase activity. its distribution ( fig 4b) revealed a peak at 9.5s, very similar in amplitude and position to the 9.5s noticed in mgci 2. some activity was also detected in denser fractions. by difference, it was possible to estimate the distribution of edta sensitive peptidase activity, which is similar to that of the antigen (fig 4c) . aminopeptidase n has been shown to be a zinc metalloprotein [7, 23] and inhibition of its activity by the divalent cations chelator edta was not surprising. when solubilized by chaps alone, torpedo aminopeptidase behaves as a large size oligomer. this oligomer is dissociated in the presence of 0.2% ci2eg, and appears therefore to be stabilized by hydrophobic interactions. the presence of dithiothreitol during solubilisation and centrifugation does not modify the sedimentation profile of torpedo aminopeptidase (data not shown), showing that disulfide bonds are not necessary for its oligomerization, as reported for mammalian aminopeptidase [13] . torpedo kidney organization has been schematically described by gfrard [5] . a thin tubular segment, with a ciliated epithelium, connects the glomerule to the proximal convoluted tubule, characterized by its large epithelial cells with their typical brush border apical membrane. the distal tubule follows, with a flat epithelium and epithelial cells devoid of flagella or microvilli. in the large field view presented in figure 5 (upper panel), proximal tubule sections are concentrated on the left whereas distal tubule sections are mainly located on the right. anti-180 kda antibodies labeled very intensely the apical membrane of proximal tubule epithelial cells. no staining of the basolateral membranes was observed, even at higher magnification (lower panel). distal tubules and glomerules were not labeled. in sections of boundary segments of the proximal tubule, the staining was restricted to the apex of the larger epithelial cells. the distribution of the 180 kda antigen was studied at the subeellular level using monoclonal antibodies 180k1 and 180k2 (figs 6 and 7) . these antibodies were chosen because their binding to kidney membranes adsorbed onto nitrocellulose was not affected by incubation of the blots in 3% glutaraldehyde (dot blot experiment not shown). binding of the mabs 180k1 or 180k2 was indirectly visualized using antimouse igg antibodies conjugated to 5 nm gold particles. a general view of the apical portion of a proximal tubule epithelial cell is presented in figure 6 . the apical membrane covers numerous microvilli, about 0.15 #m large and several microns long. this membrane is homogeneously decorated with numerous gold particles. in contrast, no labelling was associated to the basolateral membrane, or to the numerous vesicular or vacuolar membranes located under the brush border. in higher magnification views ( fig 7) the abundance and selectivity of the 180 kda antigen distribution are demonstrated. the density of gold particles associated to the apical membrane (about 600 particles/btm2) was estimated on membrane profiles perpendicular to the planes of the section. gold particles are located extracellularly, at some distance (about 25 nm) from the membrane, except of course on tangential sections (fig 7, top panel) . in some sections of proximal tubules, ciliated cells were found intercalated between epithelial cells (fig 7, lower panel) . no gold particles were associated to their apical membrane, nor to basolateral membranes, in contrast to the microvilli plasma membrane of the adjacent epithelial cells. working on torpedo kidney membranes, we have characterized a major immunogen of the brush border membrane of proximal tubules. in a fusion experiment, we selected hybridoma clones secreting antibodies which bound to these membranes. 40% of these clones produced antibodies which precipitated the same 180 kda antigen, a protein which was only a minor component of kidney membranes. this antigen was highly purified in a single immunoprecipitation step, followed by gel electrophoresis. this purification allowed us to determine the n-terminal amino acid sequence of the antigen, which turned out to be very similar to that of aminopeptidase n from various mammals including man. the first eight amino acids, which probably correspond to the cytoplasmic domain, are identical in all sequences determined; the following amino acids are hydrophobic and some c o n s e r v a t i v e changes are observed. in addition to the 180 kda antigen, monoclonal antibody 180k1 immunoprecipitated efficiently aminopeptidase n activity, showing that the 180 kda antigen carries the activity. mammalian forms of aminopeptidase n are heavily glycosylated membrane proteins composed of a single type of subunit (m r of 160 kda, detergent form [7] ) which possess a single transmembrane domain near its n terminus (see [7, 23] for reviews; [21] ). the torpedo protein was not extracted from membranes at high ionic strength or by alkaline (ph 11) treatment, demonstrating that it is an intrinsic membrane protein. it gave a broad band after sds gel electrophoresis and migrated in these conditions with an apparent molecular mass of 180 kda. when deglycosylated by endoglycosidase f/n-glycosidase f, the antigen gave a thinner band at 140 kda. thus, the torpedo enzyme is also a heavily glycosylated protein; this would explain its high immunogenicity. when solubilized in 2% chaps alone, torpedo aminopeptidase n was recovered in large protein complexes with a sedimentation coefficient around 18s. dithiothreitol was unable to dissociate these complexes (not shown), demonstrating that disulfide bonds were not necessary for the oligomerization of the enzyme, as previously reported [13] . in contrast, low concentrations of ci2e 9 were sufficient to dissociate the oligomers showing that they were stabilized by hydrophobie interactions. in some experiments (not shown), when proteases were not inhibited by pmsf, a 150 kda polypeptide was immunoprecipitated in addition to the 180 kda antigen. this polypeptide, which most probably is a proteolytie fragment of aminopeptidase n, could be eliminated by a simple washing of the crude membranes. this shows that it has lost the transmembrane anchoring domain of the molecule and that it does not remain membrane associated through an interaction with an intact aminopeptidase n monomer, as has been reported for proteolytic digests of porcine [13, 27] and bovine [22] aminopeptidase n. in t o r p e d o kidney, a high density of the antigen (about 600 gold particles/pan 2) was detected in the brush border membrane in proximal tubule epithelia. no gold particles were associated with the apical membrane of other cells, either ciliated cells in proximal tubules or epithelial cells in distal tubules (not shown). no antigen was detected in other membranes of proximal tubule epithelial cells, neither basolateral membranes nor intracellular membranes such as vacuoles or vesicles membranes were labeled. mammalian peptidase n is concentrated in microvillar apical membranes of both kidney proximal tubules and intestinal epithelia. but in enterocytes, enzyme was found associated to purified basolateral membrane fractions, where its specific activity is 20 times lower than that of the brush border membrane fractions [18] . similarly, in madin-darby kidney cells transfected with human aminopeptidase n edna, this enzyme was predominantly recovered in the apical membrane but some of it (about 20%) was associated to basolateral membranes [29] . it was therefore surprising that we could not detect any enzyme in basolateral membranes of torpedo kidney, especially considering the high density of gold particles associated with the apical membrane and the absence of background staining. sorting of aminopeptidase n has been extensively studied in various cell types: hepatocytes [1], enterocytes [15, 17] and kidney epithelial cells [29] . in madin-darby canine kidney cells, aminopeptidase n is directly sorted to the apical plasma membrane [29] . no label was detected in intracellular vesicles in our experiments, suggesting a reduced accessibility of antibodies to these structures. this could also reflect a low turnover of the antigen or possibly that antibodies bind only to mature forms of tl~ enzyme and not to its precursor forms, present in the endoplasmic reticulum and the golgi apparatus [15, 29] . the major part of mammalian aminopeptidase n, including the catalytic domain, is located on the extracellular surface of the epithelium [7, 23] . this is also the case for the torpedo kidney enzyme. indeed, epitopes labelled by antibodies 180k1 and 180k2 are exposed in the extracellular medium since gold particles were on the external side of microvilli membranes, located at some distance (about 25 nm) from the membrane. considering the size of gold particles (5 rim) and that of immunoglobulins g (7 nm), we can estimate that the epitope is about 10 nm outside the plane of the membrane and therefore that a large part of the antigen is localized in the tubular lumen. in conclusion, we have identified the kidney aminopeptidase n from torpedo marmorata. to our knowledge, it is the first characterization of this enzyme from torpedo and from fish in general. monoclonal antibodies prepared here will be useful tools for further functional studies since they bind to the solubilized protein without loss of its enzymatic activity. fine ultrastructural studies will also be possible since these antibodies are able to probe aminopeptidase n in its membrane environment, even after glutaraldehyde fixation of tissues. by its subcellular distribution, its biochemical properties and its n-terminal amino acid sequence, this enzyme closely resembles the mammalian enzymes. biogenesis of the rat hepatocyte plasma membrane in vivo: comparison of the pathways taken by apical and basolateral proteins using subcellular fractionation protein-electroblotting and microsequencing strategies in generating protein data bases from two-dimensional gels rabbit intestinal aminopeptidase n. purification and molecular properties a simple method for polyethylene glycol-promoted hybridization of mouse myeloma cells isolation and characterization of membrane bound arylamidases from human placenta and kidney topology of microvillar membrane hydrolases of kidney and intestine rat intestinal brush border membrane peptidases. i. solubilization, purification and physicochemical properties of five different forms of the enzyme continuous culture of fused cells secreting antibodies of predefined specificity cleavage of structural protein during the assembly of the head of bacteriophage t4 human myeloid plasma membrane glyeoprotein cd13 (gpl50) is identical to aminopeptidase n protein measurement with folin phenol reagent the aminopeptidase from hog intestinal brush border a method for determining the sedimentation behavior of enzymes: application to protein mixtures evidence for the transit of aminopeptidase n through the basolateral membrane before it reaches the brush border of enterocytes l'acetylcholinesttrase des organes 61ectriques des poissons complexes membranalres sorting of endogenous plasma membrane proteins occurs from two sites in cultured human intestinal epithelial cells (caco-2) subcellular fractionation and subeellular localization of aminopeptidase n in rabbit enterocytes immunological identification of a new 14 x 103 m r membranebound protein in torpedo electric organ molecular and cellular basis of digestion complete amino acid sequence of human aminopeptidase n deduced from cloned edna the oligomerie structure of the renal aminopeptidase n from bovine brush border membrane vesicles anchoring and biosynthesis of stalked bursh border membrane proteins: glycosydases and peptidases of erythrocytes and renal tubuli look at (1991) separate promoters control transcription of human aminopeptidase n gene in myeloid and intestinal epithelial cells a human liver aminopeptidase n. the amino acid and carbohydrate contents, and some physical properties of sialie acid containing glycoprotein electrophoretie transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some application on the subunit structure of particulate aminopeptidase from pig kidney amino acid sequence deduced from a edna suggests it encodes the zn-peptidase aminopeptidase n aminopeptidase n is directly sorted to the apical domain in mdck cells human aminopeptidase n is a receptor for human coronavirus 229e the amino acid sequence of the hydrophobic anchor of rabbit intestinal brush border aminopeptidase n we thank drs jp le caer and j rossier (institut alfred fessard, cnrs, 91198 gif-sur-yvette) for amino acid sequencing and dr s o'regan for improving our manuscript. this work was supported by a grant from dret (n ° 92/175). ° key: cord-103812-ls6zgipi authors: norris, rachael p.; terasaki, mark title: gap junction internalization and processing in vivo: a 3d immuno-electron microscopy study date: 2020-06-30 journal: biorxiv doi: 10.1101/2020.06.29.178475 sha: doc_id: 103812 cord_uid: ls6zgipi gap junctions have well-established roles in cell-cell communication by way of forming permeable intercellular channels. less is understood about their internalization, which forms double membrane vesicles containing cytosol and membranes from another cell, called connexosomes or annular gap junctions. here, we systematically studied the fate of connexosomes in intact ovarian follicles. high pressure frozen, serial sectioned tissue was immunogold labeled for connexin 43. within a volume of electron micrographs, every labeled structure was categorized and counted. surface area measurements indicate that large connexosomes undergo fission. subsequent modifications are separation of inner and outer membranes, loss of cx43 from the outer membrane, and outward budding of the modified membranes. we also documented several clear examples of organelle transfer from one cell to another by gap junction internalization. we discuss how connexosome formation and processing may be a novel means for gap junctions to mediate cell-cell communication. gap junctions are arrays of permeable channels between two cells that have well-established roles in intercellular signaling (nielsen et al., 2012) . the basic structural unit is the transmembrane protein connexin. six connexins assemble to form the connexon, which is a pore within the membrane. a connexon in one cell docks head-on with a connexon in a neighboring cell to form a channel between the two cells. large gap junctions may consist of hundreds or thousands of channels packed densely in a patch a few microns in diameter (larsen, 1977) . the current view is that connexons are added to the plasma membrane via small post-golgi vesicles, followed by docking between two cells, but how connexons are removed from the membrane and their subsequent fate are incompletely understood. to turn over the gap junction, cells could undock connexons and then endocytose them in small parcels. instead, connexons remain docked and the gap junction is taken up by one of the two cells (laird et al., 2006; falk et al., 2016) . this was first suggested by electron microscopists who interpreted circular gap junction profiles (first called annular gap junctions) as internalized gap junctions (espey and stutts, 1972; merk et al., 1973) . this interpretation was convincingly corroborated by live imaging of gfp-connexins, which showed formation of vesicles of comparable size (jordan et al., 2001; piehl et al., 2007) . the internalized gap junction structure is now often called a connexosome (laird, 2006) . gap junction internalization is therefore a type of endocytosis, in which the plasma membrane of the neighboring cell remains attached to the endocytosed plasma membrane (heck and devenport, 2017) . gap junction internalization can also be considered to be a form of trogocytosis (joly and hudriser, 2003) in which a portion of the plasma membrane and cytosol of the neighboring cell is transferred to the engulfing cell (see fig. 1a ). there is a possibility that further processing after the initial engulfment is involved in other modes of cell-cell communication (see discussion). internalized gap junctions could likewise play new roles in cell-cell communication. in a previous study (norris et al., 2017) , we addressed several methodological issues that have limited electron microscopic studies of gap junctions in the past. mouse ovarian follicles were high pressure frozen in order to preserve structure better than chemical fixation, which involves diffusion of aldehydes through the cell membrane(s) and cross linking of proteins, during which abnormal processes may occur (murk et al., 2003a) . the frozen tissue was freeze-substituted, embedded in lowicryl, sectioned, then immunolabeled with an antibody to cx43. this allowed us to unambiguously identify gap junctions. serial sections were then imaged to obtain threedimensional information; this distinguished between internalized connexosomes and gap junctions in the process of invagination. likewise, a round profile in a single section could be a vesicle or an invagination, or an apparently empty vesicle could contain intraluminal vesicles; serial sections distinguished these alternatives. here, we investigate the fate of internalized gap junctions by examining cx43 localization. as before, we high pressure froze ovarian follicles and immunolabeled serial sections. we found a surprisingly large number of cx43 labeled structures that we interpret as modifications of the connexosome. mouse ovarian antral follicles were high pressure frozen 30 minutes after exposure to luteinizing hormone (lh). this tissue was used because it has large numbers of cx43 gap junctions (okuma et al, 1996 ; norris et al., 2008 norris et al., , 2017 baena et al., 2020) , which are caused to internalize in response to hormone (larsen et al., 1987) . as in our previous study, the follicles were embedded in lowicryl, serial sectioned, then labeled with primary antibody to cx43 and gold-labeled secondary antibody (norris et al., 2017) . we used a different scanning electron microscope in order to obtain higher resolution images from the sections labeled in our previous study. this provided clearer images of the classic double membrane with a gap structure ( figure 1b -d). we will use the term connexosome to mean a double membrane vesicle completely detached from the plasma membrane in which both limiting membranes were contacting each other and labeled with cx43 throughout the periphery of the vesicle. connexosomes were the most abundant cx43 labeled structure in the cytoplasm, but there were also many other membranous structures that contained cx43 (video 1) which we interpreted to be modifications of the connexosome. we characterized these cx43 structures by classifying and counting them in a defined volume. by doing so, we gained information on the relative abundance of various forms, which could be useful in deducing dynamics. we analyzed two volumes of mural granulosa cells that were each 60 μm x 60 μm in the x/y plane (imaged at 5 nm per pixel), in 73 sections of 60 nm thickness. thus each volume was 15,768 μm 3 . in this volume, there were 86 gap junctions. of these, 75 were a planar patch. the 11 others were present on highly infolded membranes, which we refer to as invaginating gap junctions. there were 132 internalized structures. of the 132 cx43 labeled structures, 76 were connexosomes and 56 appeared to be connexosomes that had undergone processing. direct imaging of living cultured cells has shown that connexosomes are formed by internalization of entire gap junctions, and that connexosomes can undergo fission (piehl et al., 2007; bell et al., 2018) . we examined the high pressure frozen and serial sectioned ovarian follicles for connexosome formation. the areas of structures are measureable in 3d data sets, so we also measured the surface areas of all of the connexin 43 containing membranes because this information could be relevant to connexosome formation ( figure 1e ). the average surface area of the 75 "flat" gap junction plaques in the volume was 1.9 ± 1.1 μm 2 (mean ± sd), with a range of 0.36-6.8 μm 2 . serial sections showed that most were disc shaped, corresponding to a disc of average diameter 1.56 μm. the 11 invaginated gap junctions looked like they could form connexosomes. these were larger than the "flat" gap junctions, with an average surface area of 3.4 ± 1.8 μm 2 with a range of 1.1-6.8 μm 2 (n = 11). connexosomes, on the other hand, had an average surface area of 1.20 ± 0.7 µm 2 , and a range of 0.14-6.8 µm 2 (n=76). the average connexosome area thus was 64% of the average gap junction area (figs. 1e, f). it seems likely that invaginated gap junctions were frozen in the process of internalization and are therefore the source of connexosomes. the surface areas of invaginated gap junctions are significantly larger than connexosomes. four of the 11 invaginated gap junctions contained organelles or vesicles in addition to cytosol. one deeply invaginated gap junction contained a multivesicular endosome and a mitochondrion ( fig. 2a, video 2 ). if such structures were to form connexosomes, it would result in transfer of organelles from one cell to another. we indeed found a connexosome containing a mitochondrion and an apparent endosome (fig. 2b , video 2), and another connexosome enclosing a tubular organelle, possibly er (fig. 2c , video 2). because the entire periphery of the connexosome is gap junction, these organelles must have come from other, previously gap junction-coupled cells. in total, 17 out of 76 connexosomes contained vesicles or organelles in addition to cytosol. of the 132 cx43 labeled internalized structures, 56 appeared to be modified connexosomes. to describe the modifications, we will use the following terms to refer to the membranes and compartments of the unmodified connexosomes. there are an outer and an inner membrane, which are closely apposed because the connexons are docked. the small space between the two membranes originally was the extracellular space. the compartment within the inner membrane came from the cytoplasm of the neighboring cell. the initial modification appears to be fusion of a connexosome with another vesicle. in 7 of the 56 modified connexosomes, a patch of unlabeled outer membrane bulged outward from a labeled inner membrane ( suggesting that different types of vesicles were involved. cx43 labels the inner membrane in areas where the inner and outer membranes have separated. in these regions, the connexons must have become undocked. the inner and outer membranes have become more separated in 7 of the 56 modified connexosomes and small vesicles were present in this enlarged space (fig. 3c ). the small vesicles were generally not labeled with cx43, and the diameters averaged 72 nm. this corresponds closely to the reported diameters of intraluminal vesicles (50-80 nm) found in multivesicular endosomes (murk et al., 2003b; hanson and cashikar, 2012; scott et al., 2014) . the inner membrane is identifiable and labeled while the outer membrane is much less labeled ( fig. 3b -d, video 3). there was a striking example in which the inner and outer membranes were completely separated, and no cx43 label was present in the outer membrane (fig. 3d ). if the connexons had merely become undocked, the amount of label in the outer membrane should be comparable to the amount in the inner membrane. therefore, this is evidence for connexon degradation in the outer membrane. in the remaining 41 of the 56 modified connexosomes, there was an outer membrane similar in diameter to that of unmodified connexosomes that had little to no cx43 labeling. also, instead of a single inner membrane labeled with cx43, there were various different sized vesicles labeled with cx43 ( fig. 4a and b, video 3). the diameters of the cx43 labeled vesicles in modified connexosomes averaged 123 nm, and ranged from 38 nm to 366 nm (fig. 4c ). based on the range of diameters and the presence of cx43, these seem likely to have formed by fission or outward budding (akin to cytokinesis) of the inner connexosome membrane. for comparison, we also measured small vesicles within structures that lacked cx43 labeling (fig. 4d ). unlabeled vesicles were smaller, averaging 56 nm in diameter, with a range of 36 to 70 nm ( fig. 4c ). in addition to subdivision of the inner membrane, some of the modified connexosomes had outer membranes with short tubules extending into the cytosol, (see arrows in fig. 4a and b), as is often seen in endosomes. (klumperman and raposo, 2014 ). luteinizing hormone stimulates the internalization of gap junctions in ovarian granulosa cells (larsen et al., 1987) . we high pressure froze follicles 30 minutes after stimulation and then labeled cx43 by immunogold staining in serial sections (60 nm thick). every cx43 containing structure was categorized and counted in a defined volume of ~30,000 µm 3 . this data allows us to make several novel observations and measurements on connexosome formation and modification. live cell imaging studies of several cultured mammalian cell lines expressing cx43-gfp chimeras showed that either entire gap junctions are internalized, followed by fission (piehl et al., 2007; bell et al., 2018) , or that a small portion of the gap junction center is internalized (falk et al., 2009 ). the 3d data from high pressure frozen tissue allowed us to look for internalization intermediates and also to make the first systematic measurements of gap junction and connexosome areas. most gap junctions were disk shaped on a flattened piece of plasma membrane (n = 75). there were 11 invaginated gap junctions, and their average areas were larger than those of the flat gap junctions. it seems likely that the largest gap junctions begin to invaginate and are the source of connexosomes in this tissue. the internalization of whole invaginated gap junctions should produce correspondingly large connexosomes, but connexosomes as a group are smaller than gap junctions. this is consistent with fission occurring soon after internalization. the simplest connexosome modification was a partial separation of the inner and outer membrane while the rest of the gap junction is intact. the separated outer membrane bulges out, and lacks cx43 while the inner membrane retains it (figures 3b and 3c) . it seems likely that a vesicle has fused with the connexosome, perhaps at a bare patch left over from the internalization process (falk et al., 2009 (falk et al., , 2014 if this vesicle fusion leads to a lowering of ph, it could cause the connexons of the gap junction to undock (falk et al., 2014) . subsequent modifications seem to involve two different processes. one is complete separation of the two membranes with loss of cx43 in the outer membrane but retention in the inner membrane. the other process is the appearance of numerous smaller compartments within the boundary of the former connexosome. small cx43-free vesicles seem likely to derive from outward budding of the outer membrane; they resemble intraluminal vesicles of multivesicular endosomes. outward budding or fission of the inner membrane appears to produce cx43 containing compartments that are somewhat larger than the cx43 free vesicles. there is evidence from previous studies of other tissues for connexosome or connexin degradation by autophagy. autophagosomes engulf internalized gap junctions in the equine hoof wall (leach and oliphant, 1984) , canine ventricular myocardium (hesketh et al., 2010) , hela cells and mouse embryo fibroblasts (lichtenstein et al., 2010; fong et al., 2012) . in mouse liver cells, connexins are degraded by autophagy (bejarano et al., 2012) . however, we did not observe intermediates resembling a phagophore or an autophagosome in our images. our data comes from 30 minutes after application of luteinizing hormone, which might be too early for the final stages of cx43 degradation. another possibility is that in ovarian granulosa cells, connexosomes become something more related to multivesicular endosomes. this conclusion was made by leithe et al (2006) in cultured rat liver epithelial cells that were treated with phorbol ester. their evidence was based on immunolocalization of endosomal markers and cx43 is highly phosphorylated after lh treatment (norris et al., 2008) . if we had not immunogold labeled sections with cx43, a structure as seen in figs. 4a or 4b would likely be identified as a multivesicular endosome. this suggests that in other tissues, some apparent multivesicular endosomes could be modified connexosomes. we propose a sequence of connexosome processing events ( figure 5 ). the initial event is fusion with a vesicle (fig. 5, step 1) . the vesicle fusion adds unlabeled membrane to the outer membrane, and triggers the undocking of connexons, perhaps by lowering the ph (falk et al., 2016) . the connexons of the outer membrane are degraded, perhaps because the cytoplasm of the host cell can recognize that they are undocked (fig. 5, step 2) the uncoupled inner membrane undergoes either fission or outward budding to form various sizes of cx43 containing vesicles (fig 5, step 3) . possible fates of the modified connexosomes are degradation by autophagosome formation or direct fusion with a lysosome (leithe et al., 2006; falk et al., 2014) . however, an alternative possibility is the fusion of the inner vesicles with the modified outer membrane that lacks cx43 (fig. 5, step 4) . this would result in the mixing of membranes from two cells, and the release of cytoplasm from one cell into another. evidence for this happening in other cells is discussed in the next section. what vesicles initially fuse with the connexosomes? many appear to be clear vesicles, which are consistent with endosomes (murk et al., 2003a) , but a significant fraction of vesicles were dark which is characteristic of lysosomes (klumperman and raposo, 2014) . there is evidence that lysosomes are not always degradative and can instead function in secretion and signaling (settembre et al., 2013; perera and zoncu, 2016) . while there is solid evidence for clathrin involvement in connexosome formation (piehl et al., 2007) , an escrt driven process could also help explain some of our observations. escrt machinery for outward budding could become attached and activated on the donor side of the gap junction. there is evidence that escrt machinery can bind to ubiquitinated connexins (auth et al., 2009 ). if the machinery were transferred within the connexosome, it could generate single membrane vesicles by outward budding or fission of the internal membrane after separation of the inner and outer connexosome membranes. the channel properties of connexins are well established for the exchange of small molecules between cells (nielsen et al., 2012) . we discuss here how connexins, by way of forming connexosomes, may also facilitate cytoplasmic and membrane transfer between cells. first of all, our serial section images provide conclusive evidence for transfer of mitochondria and other organelles via connexosomes. a transferred mitochondrion could affect physiological processes of the host cell. in a mouse model for acute lung injury, cx43-dependent mitochondrial transfer from bone marrow derived stroma cells rescued injured lung alveolar epithelial cells (islam et al., 2012) . the authors suggested that mitochondria were transferred in some way by microvesicles; our observations provide a clear cx43-dependent mechanism for transfer. our observations demonstrate only the transfer of an organelle in an enclosed double membrane. if the double membranes were to fuse (or the inner membrane vesicles were to fuse with the outer membrane), this would result in mixing of cytoplasm (e.g. release of mitochondrion in the above example) and membranes ( figure 5 , step 4). it is established that the endosome membrane can fuse with a vesicle within it (bissig and gruenberg, 2014) . this occurs with enveloped viruses such as vesicular stomatitis virus (le blanc et al., 2005) or coronavirus (grove and marsh, 2011) , resulting in the release of nucleocapsids into the cytoplasm. there is now direct evidence that this also occurs with extracellular vesicles (joshi et al., 2020) , resulting in release of cargo to the cytoplasm. membrane mixing involving connexosomes might explain a previous finding in immune cells. macrophages transfer mhc ii bound antigens to dendritic cells in the gut to establish oral tolerance, and this depends on the presence of cx43 (mazzini et al., 2014) . without specifying the role of cx43, the authors raise the possibility that transfer occurs via trogocytosis. in trogocytosis, like connexosome formation, a part of the plasma membrane and cytoplasm of one cell is internalized into another (joly and hudriser, 2003) ; a fusion between outer and inner compartments, followed by budding of the combined membranes would be required to achieve antigen transfer and presentation. we suggest that the mhc ii-antigen complex is transferred from the macrophage via connexosomes followed by fusion of inner and outer membranes to deliver it to the dendritic cell's endosomal system. cell-cell communication by way of connexosome formation and processing may be widespread and warrants further investigation. from this point, we followed the procedure of rubio and wenthold (1997) , with some modifications. samples were freeze-substituted with 2.5% uranyl acetate (electron microscopy sciences) in dry methanol for 32 hours at -90° c in an afs 2 freeze substitution unit (leica biosystems). the temperature was then raised 5° c per hour to -45° c. samples were then rinsed in methanol, and infiltrated with monostep uv light as the temperature in the afs 2 was increased by 5° per hour to 0° c, then held at 0° c for 33 hours more. when samples were removed from the afs 2, they were pink in color and left to polymerize at room temperature until the pink hue was gone two days later. ultrathin sections (60 nm) of lowicryl hm20 embedded follicles were cut on a uc-7 ultramicrotome (leica biosystems) with a diamond knife (diatome, hatfield, pa). the sections were picked up by an automated tape collector on glow-discharged kapton tape (terasaki et al., 2013; kasthuri et al., 2015; baena et al., 2019) . for immunostaining, ribbons of follicle sections on kapton tape were cut to lengths of approximately three inches and attached to a sheet of parafilm with doublesided carbon tape (electron microscopy sciences #77816). sections were rehydrated with 1x pbs (life technologies, grand island, ny) and blocked in 5% normal goat serum (invitrogen, frederick, md) in a solution of 1% bovine serum albumin in pbs. following an overnight incubation at 4°c in primary antibody, sections were rinsed three times for 5 minutes each in pbs, then rinsed once in 1% bsa in pbs. next, secondary antibody diluted at 1:20 was applied to sections for one hour at room temperature. sections were then rinsed with 1x pbs followed by milli-q filtered water and dried overnight. sections were placed back in their original order and post-stained with 5% uranyl acetate in 50:50 methanol: water for 7 minutes, then rinsed generously in water. imaging serial sections of tissue with scanning em immuno-labeled sections on tape were attached to a 10 cm diameter silicon wafer (university wafer, south boston, ma) with double-sided carbon adhesive tape (electron microscopy sciences). wafers were carbon coated (denton, moorestown, nj) and first imaged on a sigma field emission scanning electron microscope (zeiss, thornwood, ny) using a backscatter detector as described in norris et al., 2017 . two volumes of mural granulosa cells were imaged at 5 nm/pixel resolution, with a field of view of 60 square micrometers. original low-resolution images obtained on the zeiss sigma were aligned with the register virtual stack slices macro (fiji), then larger files were aligned and diced for convenient viewing with a custom program (piet, provided by duncan mak and jeff lichtman, harvard university). these files were used to track all cx43-labeled internalized structures. when internalized structures looked complex, they were reimaged using a higher resolution electron microscope as described below. high-resolution images on fei verios 460l higher a. five serial sections through a modified connexosome containing several internal vesicles labeled with cx43. b. five serial sections through a different modified connexosome with internal vesicles labeled with cx43. this vesicle has a darker interior than the vesicle in a. in a. and b., arrows indicate short tubules extending from the outer membranes, as is often seen in endosomes. related video 3 shows all sections through structures in a and b. c. diameters of cx43-labeled internal vesicles are more variable in size and larger than unlabeled internal vesicles. 54 internal vesicles labeled with cx43 were measured within nine modified connexosomes, and 56 unlabeled internal vesicles were measured within nine structures that had no cx43 labeling. d. two sections through a vesicle with unlabeled intraluminal vesicles, and no cx43 labeling, as measured for panel c. scale bars in a,b, and d are 250 nm. proposed events after internalization and fission. as in figure 1 , gap junction proteins are represented by orange rectangles. (1). fusion of a connexosome with a vesicle from the host / recipient cell leads to a local separation of the two membranes (2). the formation of intraluminal vesicles (ilvs) from the outer membrane is also possible. a further modification is the outward budding of the inner membrane that still contains cx43 (3). note that cx43 is decreased or absent in the outer connexosome membrane. while degradation is possible, another possible fate of the modified connexosomes is fusion of the inner vesicles with the limiting membrane of the processed connexosome (4). this would result in release of contents from the other cell and mixing of the plasma membrane from the donor cell with the membrane of the processed connexosome. if some of the donor membrane budded away from the processed connexosome, it could get incorporated into the receiving cell plasma membrane. video 1. multiple structures labeled with cx43 polyclonal antibody. serial sections through an ovarian granulosa cells showing a variety of double membrane vesicles labeled with an antibody to cx43 and 10nm gold. other types of vesicles are also labeled with cx43 (yellow arrows). the scale bar is 250nm. video 2. organelle transfer by gap junction internalization. serial sections through ovarian granulosa cells labeled with anti-cx43 and 10nm gold. corresponding to figure re 2a, the cytoplasm of a cell protruding into another cell is shaded in blue. there is a multivesicular endosome and a mitochondrion protruding into the cell as well. scale bar is 500nm, as in figure 2a . corresponding to figure 2b , a connexosome contains a multivesicular endosome and a mitochondrion. corresponding to figure 2c , another connexosome contains smaller vesicles and other membranes. scale bars are 250nm, as in figures 2c and 2c . video 3. connexosome modifications of the outer and inner membranes. serial sections through ovarian granulosa cells labeled with anti-cx43 and 10nm gold. corresponding to figures 3 and 4 , the full structures of the modified connexosomes in figure 3a -d and in figures 4a and b are shown. scale bars are 250nm. the tsg101 protein binds to connexins and is involved in connexin degradation cellular heterogeneity of the luteinizing hormone receptor and its significance for cyclic gmp signaling in mouse preovulatory follicles. endocrinology serial-section electron microscopy using automated tape-collecting ultramicrotome (atum) autophagy modulates dynamics of connexins at the plasma membrane in a ubiquitin-dependent manner visualization of annular gap junction vesicle processing: the interplay between annular gap junctions and mitochondria alix and the multivesicular endosome: alix in wonderland exchange of cytoplasm between cells of the membrane granulosa in rabbit ovarian follicles gap junction turnover is achieved by the internalization of small endocytic doublemembrane vesicles degradation of endocytosed gap junctions by autophagosomal and endo/lysosomal pathways: a perspective degradation of connexins and gap junctions molecular mechanisms regulating formation, trafficking and processing of annular gap junctions internalized gap junctions are degraded by autophagy the cell biology of receptor-mediated virus entry multivesicular body morphogenesis trans-endocytosis of planar cell polarity complexes during cell division ultrastructure and regulation of lateralized connexin43 in the failing heart mitochondrial transfer from bone-marrow-derived stromal cells to pulmonary alveoli protects against acute lung injury what is trogocytosis and what is its purpose? the origin of annular junctions: a mechanism of gap junction internalization endocytosis of extracellular vesicles and release of their cargo from endosomes saturated reconstruction of a volume of neocortex the complex ultrastructure of the endolysosomal system life cycle of connexins in health and disease structural diversity of gap junctions. a review differential modulation of rat follicle cell gap junction populations at ovulation degradation of annular gap junctions of the equine hoof wall endosome-tocytosol transport of viral nucleocapsids endocytic processing of connexin43 gap junctions: a morphological study autophagy: a pathway that contributes to connexin degradation oral tolerance can be established via gap junction transfer of fed antigens from cxcr1+ macrophages to cd103+ dendritic cells the fine structure of granulosa cell nexuses in rat ovarian follicles influence of aldehyde fixation on the morphology of endosomes and lysosomes: quantitative analysis and electron tomography endosomal compartmentalization in three dimensions: implications for membrane fusion gap junctions luteinizing hormone causes map kinase-dependent phosphorylation and closure of connexin 43 gap junctions in mouse ovarian follicles: one of two paths to meiotic resumption localization of phosphorylated connexin 43 using serial section immunogold electron microscopy colocalization of connexin 43 and connexin 45 but absence of connexin 40 in granulosa cell gap junctions of rat ovary the lysosome as a regulatory hub internalization of large double-membrane intercellular vesicles by a clathrindependent endocytic process glutamate receptors are selectively targeted to postsynaptic sites in neurons endosome maturation, transport and functions signals from the lysosome: a control centre for cellular clearance and energy metabolism stacked endoplasmic reticulum sheets are connected by helicoidal membrane motifs we thank rindy jaffe and matthias falk for critical review of the manuscript and useful discussions. we thank art hand, maya yankova, and maria rubio for technical advice. we thank valentina baena, tracy uliasz, and other members of the jaffe and terasaki labs for technical assistance and help with collecting samples. this work was funded by the fund for science. the authors declare no conflicts of interest. rachael norris contributed to conceptualization; data collection, curation and analysis; visualization, and writing and editing of the original draft. mark terasaki contributed to conceptualization, supervision of the work, funding acquisition, software and resources for data collection; and writing and editing of the original draft. key: cord-035248-m5517zgn authors: stokes, john w.; gannon, whitney d.; sherrill, wren h.; armistead, leslie b.; bacchetta, matthew; rice, todd w.; semler, matthew w.; casey, jonathan d. title: bleeding, thromboembolism, and clinical outcomes in venovenous extracorporeal membrane oxygenation date: 2020-11-09 journal: crit care explor doi: 10.1097/cce.0000000000000267 sha: doc_id: 35248 cord_uid: m5517zgn objectives: bleeding and thromboembolism are common during venovenous extracorporeal membrane oxygenation. the relative frequency of these complications and their impact on clinical outcomes have not been described, and no randomized trials exist to guide anticoagulation strategies in extracorporeal membrane oxygenation. our objective was to examine the relative frequencies of bleeding and thromboembolic events and their associations with survival among a cohort of consecutive patients receiving venovenous extracorporeal membrane oxygenation. design: retrospective cohort study. setting: a single academic medical center. patients: adult patients receiving venovenous extracorporeal membrane oxygenation and anticoagulation. eligibility criteria for this analysis were selected to emulate the population that would be recruited for a randomized trial of anticoagulation strategies during venovenous extracorporeal membrane oxygenation. patients were excluded if they had active bleeding or thromboembolism prior to extracorporeal membrane oxygenation initiation, a history of trauma or surgery in the 7 days prior to extracorporeal membrane oxygenation initiation, an arterial extracorporeal membrane oxygenation cannula, or if they received greater than 48 hours of extracorporeal membrane oxygenation support at another institution interventions: none. measurements and main results: outcomes included bleeding and thromboembolic events, duration of extracorporeal membrane oxygenation support, hospital length of stay, and in-hospital survival among 55 patients receiving venovenous extracorporeal membrane oxygenation. bleeding events occurred in 25 patients (45.5%), and thromboembolism occurred in eight patients (14.5%). bleeding events were associated with longer duration of extracorporeal membrane oxygenation support (p = 0.007) and worse in-hospital survival (p = 0.02). thromboembolic events did not appear to be associated with clinical outcomes. conclusions: in this cohort of patients receiving venovenous extracorporeal membrane oxygenation and anticoagulation, bleeding occurred more frequently than thromboembolism and was associated with worse survival. these results highlight the need for randomized trials to evaluate the safety and efficacy of continuous iv anticoagulation among patients receiving venovenous extracorporeal membrane oxygenation. objectives: bleeding and thromboembolism are common during venovenous extracorporeal membrane oxygenation. the relative frequency of these complications and their impact on clinical outcomes have not been described, and no randomized trials exist to guide anticoagulation strategies in extracorporeal membrane oxygenation. our objective was to examine the relative frequencies of bleeding and thromboembolic events and their associations with survival among a cohort of consecutive patients receiving venovenous extracorporeal membrane oxygenation. design: retrospective cohort study. setting: a single academic medical center. patients: adult patients receiving venovenous extracorporeal membrane oxygenation and anticoagulation. eligibility criteria for this analysis were selected to emulate the population that would be recruited for a randomized trial of anticoagulation strategies during venovenous extracorporeal membrane oxygenation. patients were excluded if they had active bleeding or thromboembolism prior to extracorporeal membrane oxygenation initiation, a history of trauma or surgery in the 7 days prior to extracorporeal membrane oxygenation initiation, an arterial extracorporeal membrane oxygenation cannula, or if they received greater than 48 hours of extracorporeal membrane oxygenation support at another institution interventions: none. measurements and main results: outcomes included bleeding and thromboembolic events, duration of extracorporeal membrane oxygenation support, hospital length of stay, and in-hospital survival among 55 patients receiving venovenous extracorporeal membrane oxygenation. bleeding events occurred in 25 patients (45.5%), and thromboembolism occurred in eight patients (14.5%). bleeding events were associated with longer duration of extracorporeal membrane oxygenation support (p = 0.007) and worse in-hospital survival (p = 0.02). thromboembolic events did not appear to be associated with clinical outcomes. conclusions: in this cohort of patients receiving venovenous extracorporeal membrane oxygenation and anticoagulation, bleeding occurred more frequently than thromboembolism and was associated with worse survival. these results highlight the need for randomized trials to evaluate the safety and efficacy of continuous iv anticoagulation among patients receiving venovenous extracorporeal membrane oxygenation. key words: adult; critical care; extracorporeal membrane oxygenation; hemorrhage; respiratory distress syndrome; thromboembolism b leeding and thromboembolism are common during venovenous extracorporeal membrane oxygenation (ecmo) (1). reported frequencies and associations with clinical outcomes vary and available data are limited by heterogenous study populations (1) (2) (3) . multiple anticoagulation strategies have been proposed to balance the risks of bleeding and thromboembolism during venovenous ecmo (4, 5) , but which strategy is most effective remains unknown. data on the relative frequencies of bleeding and thromboembolism during venovenous ecmo, and their respective associations with survival, are needed to provide preliminary data and inform equipoise for future randomized trials. our objective was to evaluate the frequency and clinical significance of bleeding and thromboembolic events during venovenous ecmo. we hypothesized that bleeding events, but not thromboembolic events, would be associated with worse in-hospital survival. we performed a retrospective cohort study examining data from all patients who received venovenous ecmo at the adult hospital at vanderbilt university medical center between january 1, 2016, and may 10, 2020. prespecified exclusion criteria were used with the goal of including a patient population similar to those who would be included in a randomized trial comparing anticoagulation strategies, a technique known as "target trial emulation" (6) . we excluded patients who had active bleeding or thromboembolism prior to ecmo initiation, experienced trauma or surgery in the 7 days prior to ecmo initiation, received greater than 48 hours of ecmo support at another institution, or received arterial cannulation. the study was approved by the vanderbilt university medical center institutional review board (irb no 200158). we collected the following data from the electronic health record: patient characteristics in the 24 hours prior to ecmo initiation; bleeding and thromboembolic events during venovenous ecmo as previously defined (5); and clinical outcomes, including in-hospital survival, ecmo duration, and hospital length of stay. bleeding events were defined as overt bleeding associated with either a drop in hemoglobin concentration by 2 g/dl or a transfusion of at least two units of packed rbcs in 24 hours, bleeding at any critical site (e.g. intracranial bleeding), or bleeding requiring a procedural intervention (5) . thromboembolic events were defined as cerebral stroke, intracardiac thrombus, acute pump head thrombosis, acute oxygenator failure, pulmonary emboli, or deep vein thrombosis (dvt) (5) . cannula-associated dvts following decannulation did not meet the composite definition for thromboembolic event and were omitted from the survival analysis to limit immortal time bias. continuous variables are presented as median with interquartile range (iqr). categorical variables are summarized as frequencies and percentages. differences between groups were compared using a chi-square test, fisher exact test, or wilcoxon rank-sum test as appropriate. log-rank tests were used to compare time with hospital discharge between groups. all analyses were performed using stata 16.1 (statacorp, college station, tx), and a twosided p value of 0.05 was considered to be statistically significant. no adjustments were made for multiple testing. of the 156 patients who received venovenous ecmo during the study period, 101 met exclusion criteria. a total of 69 patients were excluded for recent trauma or surgery, 13 patients were excluded for active bleeding, 11 patients were excluded for recent thromboembolism, five patients were excluded for receiving ecmo at an another institution for greater than 48 hours, and three patients were excluded for arterial cannula placement while receiving venovenous ecmo. a total of 55 patients were included in the analysis. the median age was 50 years (iqr, 40-60 yr), and 38% were women. according to institutional protocols, all patients received a continuous infusion of unfractionated heparin following ecmo cannulation, titrated to either antifactor xa levels of 0.2-0.4 u/ml or a partial thromboplastin time of 40-60 seconds. a total of 30 bleeding events occurred among 25 patients (45.5%), including eight gastrointestinal bleeds, seven intracranial hemorrhages, four cannula site bleeds, four episodes of hemoptysis, three tracheostomy bleeds, two hemothoraces, and two episodes of epistaxis. of these, six (5 intracranial hemorrhages and 1 gastrointestinal bleed) were considered the primary cause of death. the median time from ecmo cannulation to first bleeding event was 5 days (iqr, 2-7 d). eight patients (14.5%) experienced a thromboembolic event during ecmo, including five deep venous thromboses (dvt), two acute circuit thromboses requiring circuit exchange, and one brachial artery thrombosis. the median time from ecmo cannulation to first thromboembolic event was 6 days (iqr, 2-18 d). no thromboembolic events were considered the primary cause of death. a total of 14 additional cannula-associated dvts were identified on protocolized ultrasound screening following decannulation. baseline characteristics and serum markers of coagulation and thrombocytopenia were similar between groups ( table 1) . anticoagulation monitoring did not vary between groups. rbc transfusion requirement was greater among patients with a bleeding event than patients without a bleeding event (p = 0.002). in univariate analysis, patients who experienced a bleeding event had a longer duration of ecmo support (p = 0.007) and worse inhospital survival compared with patients who did not experience a bleeding event (p = 0.02) ( table 1 and fig. 1) . thromboembolic events did not appear to be associated with any differences in duration of ecmo support, hospital length of stay, or in-hospital survival (table 1 and fig. 1 ). in this retrospective cohort study of patients receiving venovenous ecmo for respiratory failure, all of whom received continuous anticoagulation, nearly half of patients experienced a bleeding event. patients who experienced a bleeding event experienced worse survival than patients who did not experience a bleeding event. in contrast, thromboembolic events were less frequent and did not appear to affect survival. this is the first study to examine the relative impact of bleeding or thromboembolism during venovenous ecmo only. these results should prompt further research to evaluate the safety and efficacy of continuous iv anticoagulation in such patients. several factors may contribute to bleeding and thromboembolism in patients receiving ecmo. the interface of blood and nonbiologic circuit components causes activation of the coagulation system and the consumption and degradation of hemostatic factors (7, 8) . underlying critical illness compounds the risks of bleeding and thromboembolism (7) . continuous anticoagulation during ecmo may increase the risk of bleeding (1), and prior retrospective data suggest a dose-response relationship between anticoagulation and bleeding events (1, 3). conducting venovenous ecmo without continuous systemic anticoagulation has been proposed (9, 10). although confounded by indication bias, recent observational studies suggest that strategies using only prophylactic doses of anticoagulation appear safe in venovenous ecmo (9, 10) . further, a recent systematic review suggested that the rates of thromboembolism and circuit thrombosis among patients who did not receive systemic anticoagulation during venovenous ecmo were comparable with the rates reported among patients treated with systemic anticoagulation (11) . it is possible that avoidance of systemic anticoagulation might improve outcomes for some patients receiving venovenous ecmo. our study has several strengths. by including only patients without a pre-existing indication or contraindication to anticoagulation, the population in our study emulates the population that would be recruited for a randomized trial of anticoagulation strategies during venovenous ecmo. further, we used previously published, objective criteria to define bleeding and thromboembolism. our study has several limitations. the study was conducted at a single center using a retrospective design. although we used structured and prespecified eligibility criteria, selection bias remains possible. the risks of bleeding and thromboembolism may be confounded by severity of illness and immortal time bias. finally, this study was largely conducted prior to the coronavirus disease 2019 (covid-19) pandemic. only one patient in the study cohort experienced respiratory failure as a consequence of covid-19. emerging data describe both hypercoagulability (12) and a higher risk of bleeding for patients receiving venovenous ecmo for covid-19 (13) . it is unknown if the results of this analysis would be different if conducted entirely among a population of patients receiving venovenous ecmo for covid-19. our data include only patients who received anticoagulation and do not inform the risks of thromboembolism among patients receiving venovenous ecmo without anticoagulation or with prophylactic-dose anticoagulation. this purely descriptive univariate analysis does not attempt to account for potential confounders and does not infer a causal relationship between bleeding or thromboembolism and survival. in this cohort of patients receiving venovenous ecmo and anticoagulation, bleeding occurred more frequently than thromboembolism and was associated with worse survival. these results provide preliminary data for a randomized trial examining the safety and efficacy of systemic anticoagulation in select patients receiving venovenous ecmo. drs. stokes and gannon contributed equally to this work. drs. stokes, gannon, sherrill, bacchetta, rice, semler, and casey contributed to conception and design of the study. drs. stokes, gannon, sherrill, and armistead contributed to data acquisition. drs. stokes, gannon, and sherrill contributed to analysis of the data. drs. stokes and gannon drafted the initial article. all authors contributed to the data interpretation and edited the article for important scientific content. all of the authors agree to be accountable for all aspects of the work in regards to accuracy and integrity. bacchetta was supported, in part, by the national institutes of health (nih) jr chair in surgery no 2. dr. rice was supported, in part, by the nih (ul1 rr024975). dr. semler was supported, in part, by the national heart, lung, and blood institute (k23hl143053). dr. casey was supported predictive factors of bleeding events in adults undergoing extracorporeal membrane oxygenation severe respiratory failure, extracorporeal membrane oxygenation, and intracranial hemorrhage anticoagulation practices during venovenous extracorporeal membrane oxygenation for respiratory failure. a systematic review extracorporeal life support organization: elso anticoagulation guideline low-dose versus therapeutic anticoagulation in patients on extracorporeal membrane oxygenation: a pilot randomized trial using big data to emulate a target trial when a randomized trial is not available clinical controversies in anticoagulation monitoring and antithrombin supplementation for ecmo kaplan-meier in-hospital survival curves from time of venovenous extracorporeal membrane oxygenation for patients who did and did not experience a bleeding event and for patients who did and did not experience a thromboembolic event current understanding of how extracorporeal membrane oxygenators activate haemostasis and other blood components feasibility of veno-venous extracorporeal membrane oxygenation without systemic anticoagulation venovenous extracorporeal membrane oxygenation with prophylactic subcutaneous anticoagulation only: an observational study in more than 60 patients thrombosis and bleeding in extracorporeal membrame oxygenation (ecmo) without anticoagulation: a systematic review covid-19: the vasculature unleashed extracorporeal membrane oxygenation for severe acute respiratory distress syndrome associated with covid-19: a retrospective cohort study key: cord-103739-mmkrwj8t authors: snijder, eric j.; limpens, ronald w.a.l.; de wilde, adriaan h.; de jong, anja w. m.; zevenhoven-dobbe, jessika c.; maier, helena j.; faas, frank f.g.a.; koster, abraham j.; bárcena, montserrat title: a unifying structural and functional model of the coronavirus replication organelle: tracking down rna synthesis date: 2020-03-24 journal: biorxiv doi: 10.1101/2020.03.24.005298 sha: doc_id: 103739 cord_uid: mmkrwj8t zoonotic coronavirus (cov) infections, like those responsible for the current sars-cov-2 epidemic, cause grave international public health concern. in infected cells, the cov rna-synthesizing machinery associates with modified endoplasmic reticulum membranes that are transformed into the viral replication organelle (ro). while double-membrane vesicles (dmvs) appear to be a pan-coronavirus ro element, studies to date describe an assortment of additional coronavirus-induced membrane structures. despite much speculation, it remains unclear which ro element(s) accommodate viral rna synthesis. here we provide detailed 2d and 3d analyses of cov ros and show that diverse covs essentially induce the same membrane modifications, including the small open double-membrane spherules (dmss) previously thought to be restricted to gammaand delta-cov infections and proposed as sites of replication. metabolic labelling of newly-synthesized viral rna followed by quantitative em autoradiography revealed abundant viral rna synthesis associated with dmvs in cells infected with the beta-covs mers-cov and sars-cov, and the gamma-cov infectious bronchitis virus. rna synthesis could not be linked to dmss or any other cellular or virus-induced structure. our results provide a unifying model of the cov ro and clearly establish dmvs as the central hub for viral rna synthesis and a potential drug target in coronavirus infection. epidemic, cause grave international public health concern. in infected cells, the cov rna-23 synthesizing machinery associates with modified endoplasmic reticulum membranes that are 24 transformed into the viral replication organelle (ro). while double-membrane vesicles 25 (dmvs) appear to be a pan-coronavirus ro element, studies to date describe an assortment ro [15, 20, 21] , was entirely possible and started to attract 71 attention. notably, dmvs can be also formed in the absence of vrna synthesis by 72 expression of key transmembrane nsps [22] [23] [24] [25] [26] . moreover, several studies suggested a lack of 73 direct correlation between the number of dmvs and the level of cov replication in the 74 infected cell [27, 28] . 75 the interpretation of the cov ro structure and function was further compounded by the 76 discovery of different ro 106 double-membrane spherules (dmss) 107 we first set out to analyse the ultrastructure of mers-cov-infected huh7 cells under sample 108 preparation conditions favourable for autoradiography (see materials and methods) (fig 1, s1 109 video). strikingly, in addition to the dmvs and cm that are well established hallmarks of 110 beta-cov infections, the presence of small spherules, occasionally in large numbers, was 111 readily apparent (fig 1a and 1b) . these spherules were notably similar to the dmss 112 previously described for the gamma-cov ibv [29] . their remarkably regular size of ~80 nm 113 (average diameter 79.8 ± 2.5 nm, n=58), a delimiting double membrane and their electron-114 dense content, made these spherules clearly distinct from other structures, including progeny 115 virions, which had comparable diameter (fig 1c and 1d ). the double-membrane spherules 116 (dmss) generated during ibv infection were previously described as invaginations of the 117 zippered er that remain open to the cytosol [29] . in mers-cov-infected cells, the dmss 118 were connected to the cm from which they seemed to derive ( fig 1e) . clear openings to the 119 cytosol could not be detected for the large majority (~80%, n=54) of the fully reconstructed 120 dmss, which suggests that the original invagination may eventually transform into a sealed 121 compartment. this type of apparently closed dmss were also present, though in a lower 122 proportion (~50%, n=39), in ibv-infected cell samples processed in an identical manner (s1 the 3d architecture of mers-cov-induced ro aligned with previous observations for other 156 cov [15, 29] . no clear openings connecting the interior of the dmvs and the cytosol could 157 be detected. all three types of mers-cov-induced membrane modifications appeared to be 158 interconnected, either directly or indirectly through the er. while dmss were connected to 159 cm, and cm to er, er membranes were often continuous with dmvs (fig 1f, arrowheads) . 160 therefore, like other covs, mers-cov infection appears to induce a network of largely 161 interconnected modified er membranes that, as a whole, can be considered the cov ro. quantification of the autoradiography signal per subcellular structure (see also s1 table) . labelling 214 densities and relative labelling indexes (rli) in different subcellular regions of (c) vero e6 cells 215 infected with sars-cov (moi 10) or (d) huh7 cells infected with mers-cov (moi 5). 216 radioactively-labelled uridine was provided for the indicated periods of time immediately before 217 fixation at 7 hpi and 12 hpi, respectively. control mock-infected cells are excluded from the rli 218 plots, as rli comparisons between conditions require the same number of classes (subcellular 219 regions) and these cells lack ros and virions. budding vesicles (fig 6a-c) .the m and s proteins also localized to the golgi complex, 348 aligning with previous observations for other covs [20, 44, 48, 49] . the mers-cov n 349 protein was found in regions with cm and dmss, though the distribution of signal was 350 homogenous and dmss were not particularly densely labelled (fig 6d) . the presence of the assembly, or the s protein (fig 6e and 6f ). 356 previously, the cm induced by sars-cov and mhv were shown by iem to accumulate 357 viral nsps, while dsrna signal was primarily found inside the dmvs [15, 20] . similarly, 358 nsp3 mapped to the cm induced in mers-cov infection, but also to the dmss to a 359 comparable extent (fig 6g) . our attempts to combine dsrna antibody labelling with thawed 360 cryo-sections were unsuccessful, which made us resort to hpf-fs samples. in these, 361 however, while dmvs were easily detected, the morphology of cm and dmss was less 362 clearly defined. nevertheless, dsrna signal was clearly associated with dmvs, while the 363 dark membranous regions between dmvs that we interpreted as cm and dmss clusters 364 appeared devoid of signal (fig 6h and 6i ). 365 in summary, for the antibodies tested (recognizing n, m, s, nsp3, and dsrna), the labelling 366 pattern in mers-cov-induced dmss closely resembled that of the cm, from which they 367 seem to derive. the absence of labelling for key proteins in virus assembly, like the m and s 368 proteins, strongly suggest that dmss do not represent (spurious) virus assembly events. the comprehensive analysis presented here demonstrates that viruses across different cov 371 genera induce essentially the same type of membrane structures. after somewhat disparate 372 observations [15, 20, 21, 29, 30, 47] , the unifying model that emerges from our study is that our results add to studies that, in the last years and after much speculation, have started to 419 provide experimental evidence that the dmvs induced by +rna viruses are active sites of 420 vrna synthesis [11, [58] [59] [60] . however, it is not clear that dmvs always play the primary role 421 in virus replication that we demonstrate here for cov. for picornaviruses, for example, viruswere freeze-substituted in a leica afs2 system with 0.1% (wt/vol) uranyl acetate as 576 previously described [71] , with the only modification that acetone was replaced by ethanol 577 from the last washing step before lowycril infiltration onwards. cell sections (75 nm thick) 578 were incubated with the primary mouse antibody, then with a bridging rabbit anti-mouse-igg 579 antibody (dako cytomation), and finally with protein a coupled to 15-nm gold particles. 580 after immunolabelling, samples were additionally stained with 7% uranyl acetate and 581 reynold's lead citrate. large mosaic em maps containing dozens of cell profiles were used for the quantitative 608 analysis of the newly-synthesized rna autoradiography signal (see s1 table) . for each 609 cov, different conditions (infected and mock-infected cells, plus different labelling times) 610 were compared using only samples developed after the same period of time. the analysis of 611 the signal in different subcellular regions was carried out using home-built software. areas of 612 4 µm 2 were randomly selected from the mosaic em maps and the autoradiography grains 613 present in those areas were manually assigned to the underlying cellular structures. the 614 abundance of the different types of subcellular structures was estimated through virtual points 615 in a 5×5 lattice superimposed to each selected area, which were also assigned to the different 616 subcellular classes. regularly along the process, the annotated data per condition was split 617 into two random groups and the kendall and spearman coefficients, which measure the 618 concordance between two data sets [73], were calculated. new random regions were added 619 until the average kendall and spearman coefficients resulting from 10 random splits were 620 higher than 0.8 and 0.9, respectively (maximum value, 1). labelling densities and relative 621 labelling indexes (rli) were then calculated from the annotated points [39] . 622 for the analysis of the association of vrna synthesis with each of the different ros motifs, 623 the specific dmvs, dmss and cm included in the analysis were carefully selected. only 624 individual dmvs that were at least one micron away from any other virus-induced membrane 625 modification were included in the analysis. for every grain present in an area of 750 nm 626 radius around each dmv, the distance to the dmv centre was measured. in the case of 627 dmss, which were always part of clusters of virus-induced membrane structures, only dmss 628 in the periphery of these clusters were selected. the quantified signal was limited to sub-629 areas devoid of other ro motifs, which were defined by circular arcs (typically 30 o to 100 o , 630 radius 500 nm) opposite to the ro clusters. cms are irregular structures that appear partially 631 28 or totally surrounded by dmvs. only large cm (> 0.6 µm across) were selected in order to 632 make more apparent (if present) any decay of the autoradiography signal as the distance to 633 the surrounding dmvs increased. for each autoradiography grain, both the distance to the 634 closest cm boundary (d 1 ) and the distance to the opposite cm edge (d 2 ) were measured. the 635 relative distance to the cm edge was then calculated as d 1 /(d 1 +d 2 ) and expressed in 636 percentages. all the measurements in different dmvs, dmss and cm were made using 637 aperio imagescope software (leica) and pooled together into three single data sets. autoradiography is a classic technique that allows the em visualization of a radioactive 3 marker, usually targeting a certain process, and thus reveals the subcellular localization of 4 that process [1, 2] . tritiated uridine, for example, can be used to locate active rna synthesis 5 [3] [4] [5] , as shown also in this study. a clear advantage over the use of alternatives for metabolic 6 labelling of newly-synthesized rna (e.g. br-uridine, br-utp, 5-ethynil uridine) is that the 7 radioactive precursor is chemically identical to the natural substrate. 8 after labelling, the samples are immediately fixed and processed for em. the location of the 9 radioactive marker can then be made apparent by applying a highly-sensitive photographic 10 emulsion (a nuclear emulsion) on top of the cell sections and exposing it for several weeks to 11 months. the beta particles that are emitted as a result of tritium disintegrations generate 12 electrons that get trapped in the silver halide emulsion and create a "latent image". when the 13 emulsion is developed, these negative charges promote the reduction to metallic silver, 14 generating electron-dense grains that are visible by em. in principle, given enough time to 15 accumulate enough radioactive disintegrations, even low levels of the radioactive marker 16 could be detected. in practice, other factors (e.g. background radiation, emulsion aging) set 17 some limits to autoradiography, which is nonetheless a very sensitive technique. 18 the resolution of em autoradiography is limited by the fact that radioactive disintegrations 19 generate beta particles that are emitted in random directions. importantly, the probability of 20 giving rise to signal degreases with the distance from the radioactive source; however, some 21 beta particles may travel up to a few hundred nanometers before striking the photographic 22 emulsion [2] . therefore, it is important to keep in mind that the silver grains may not directly 23 overlay the structure containing the radioactive source. quantitative analyses of the signal 24 2 that take this factor into account, like those presented in this study, become indispensable to 25 maximize the information that autoradiography can provide. as for mer-cov-infected cells (fig 1) . tomographic slices through two regions containing 32 membranous replication factories induced by plus-646 strand rna viruses ultrastructure of the replication sites of positive-strand rna 649 viruses building viral replication organelles close encounters of the membrane types interaction of the innate immune system with positive-656 strand rna virus replication organelles rna virus replication complex parallels form and function of retrovirus capsids. molecular 660 cell three-dimensional 662 analysis of a viral rna replication complex reveals a virus-induced mini-organelle. plos 663 biology composition and three-dimensional architecture of the dengue virus replication and assembly 667 sites template rna 670 length determines the size of replication complex spherules for semliki forest virus three-674 dimensional imaging of the intracellular assembly of a functional viral rna replicase 675 complex the transformation of enterovirus replication structures: a three-dimensional study 679 of single-and double-membrane compartments complex dynamic 682 development of poliovirus membranous replication complexes membrane alterations induced by nonstructural proteins of human norovirus three-690 dimensional architecture and biogenesis of membrane structures associated with hepatitis c 691 virus replication rna replication of 694 mouse hepatitis virus takes place at double-membrane vesicles sars-coronavirus replication is supported by a reticulovesicular network of 698 modified endoplasmic reticulum ultrastructural characterization of arterivirus replication structures: reshaping the 703 endoplasmic reticulum to accommodate viral rna synthesis extensive 707 coronavirus-induced membrane rearrangements are not a determinant of pathogenicity an integrated analysis of 711 membrane remodeling during porcine reproductive and respiratory syndrome virus 712 replication and assembly double-stranded rna is produced by positive-strand rna viruses and dna viruses but not in detectable 717 amounts by negative-strand rna viruses qualitative and quantitative 721 ultrastructural analysis of the membrane rearrangements induced by coronavirus. cellular 722 microbiology mers-coronavirus replication induces severe in vitro cytopathology and is 726 strongly inhibited by cyclosporin a or interferon-alpha treatment severe acute 730 respiratory syndrome coronavirus nonstructural proteins 3, 4, and 6 induce double-membrane 731 vesicles mutations across murine 738 hepatitis virus nsp4 alter virus fitness and membrane modifications expression 742 and cleavage of middle east respiratory syndrome coronavirus nsp3-4 polyprotein induce the formation of double-membrane vesicles that mimic those associated with coronaviral 744 rna replication bronchitis virus nonstructural protein 4 alone induces membrane pairing competitive 751 fitness in coronaviruses is not correlated with size or number of double-membrane vesicles 752 under reduced-temperature growth conditions targeting membrane-bound viral rna synthesis reveals potent inhibition of diverse 757 coronaviruses including the middle east respiratory syndrome virus infectious bronchitis virus generates spherules from zippered endoplasmic reticulum 762 membranes replication organelle comprises double-membrane vesicles and zippered endoplasmic 766 does form meet function in the 769 coronavirus replicative organelle? trends in microbiology isolation of 772 a novel coronavirus from a man with pneumonia in saudi arabia. the new england journal 773 of medicine genomic characterization of a newly discovered coronavirus associated with acute 777 respiratory distress syndrome in humans the new england journal of medicine genomic characterisation and 783 epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding techniques and applications of autoradiography in the light and electron 787 microscope electron microscopy: 789 principles and techniques for biologists virtual nanoscopy: generation of ultra-large high resolution electron microscopy maps relative labelling index: a novel stereological 796 approach to test for non-random immunogold labelling of organelles and membranes on 797 transmission electron microscopy thin sections replication of coronavirus mhv-a59 in sac-cells: 800 determination of the first site of budding of progeny virions ultrastructural characterization of sars coronavirus the intracellular sites of early replication and budding of sars-coronavirus novel 811 contrasting and labeling procedures for correlative microscopy of thawed cryosections coronavirus m proteins accumulate in the golgi complex beyond the site of virion budding proliferative growth of sars 820 coronavirus in vero e6 cells morphogenesis of avian infectious 823 bronchitis virus and a related human virus (strain 229e) ultrastructural 826 characterization of membrane rearrangements induced by porcine epidemic diarrhea virus 827 the e1 glycoprotein of an 830 avian coronavirus is targeted to the cis golgi complex the 838 putative helicase of the coronavirus mouse hepatitis virus is processed from the replicase 839 gene polyprotein and localizes in complexes that are active in viral rna synthesis localization of mouse hepatitis virus nonstructural proteins and rna synthesis indicates a 844 role for late endosomes in viral replication mouse hepatitis virus replicase proteins 847 associate with two distinct populations of intracellular membranes determination of host proteins composing the microenvironment of coronavirus replicase 852 complexes by proximity-labeling. elife the coronavirus nucleocapsid is a 855 multifunctional protein visualizing 859 coronavirus rna synthesis in time by using click chemistry do viruses subvert cholesterol 863 homeostasis to induce host cubic membranes? trends in cell biology cubic membranes: a legend beyond the flatland* of cell membrane organization morphological and 870 biochemical characterization of the membranous hepatitis c virus replication compartment epub 2013/07/26 escaping host factor pi4kb inhibition: enterovirus genomic rna replication in the 875 absence of replication organelles 878 the origin, dynamic morphology, and pi4p-independent formation of 879 encephalomyocarditis virus replication organelles. mbio sars-coronavirus replication/transcription complexes are membrane-protected 884 and need a host factor for activity in vitro early 888 endonuclease-mediated evasion of rna sensing ensures efficient coronavirus replication mechanisms and 892 enzymes involved in sars coronavirus genome expression a new virus isolated from the human respiratory tract reverse genetics system for 898 the avian coronavirus infectious bronchitis virus the coronavirus spike protein is a 902 class i virus fusion protein: structural and functional characterization of the fusion core 903 complex ultrastructure and origin of membrane vesicles associated with the severe 907 acute respiratory syndrome coronavirus replication complex monoclonal 911 antibodies to double-stranded rna as probes of rna structure in crude nucleic acid extracts towards a solution to mers: protective human monoclonal antibodies targeting 916 different domains and functions of the mers-coronavirus spike glycoprotein resolution of a gold latensification-elon 920 ascorbic acid developer for ilford l4 emulsion correlated 923 fluorescence and 3d electron microscopy with high sensitivity and spatial precision a rapid 930 method for assessing the distribution of gold labeling on thin sections dmss (white 33 arrowheads) and zippered er (white arrows). most zippered er consists of long stretches of 34 though branching zippered er, closer to the cm 35 described for beta-cov, was also present (b) virus particles (black arrowheads) budding into 36 the er membranes were often observed. scale bars analysis 39 of previously described samples of cov-infected cells, prepared for em either by hpf(a) or 40 cryo-plunging (b). a targeted search revealed the presence of dmss (white arrowheads) in 41 close association with cm. in comparison with the chemically fixed samples used in this 42 study, the superior ultrastructural preservation of cryo-fixation results in less distorted 43 membranes, but also in a denser cytoplasm and darker cm that makes dms less apparent. 44 (a) example from a mers-cov-infected huh7 cell sars-cov-infected cell (8 hpi) metabolic labelling of newly-synthesized vrna in ibv-infected cells and 48 analysis of the autoradiography signal. vero cells infected with ibv were pre-treated with 49 actinomycin d for 1 hour, then labelled for 30 or 60 min with tritiated uridine a) overview of 51 an ibv-infected vero cell (60 min labelling). the areas containing dmvs and zippered er 52 are outlined in yellow and blue, respectively, and other subcellular structures annotated (n, 53 nucleus; m, mitochondria; au, autophagosome; vcr, virion-containing regions). the 54 autoradiography signal accumulates in areas of virus-induced membrane modifications that 55 often only contain dmvs close-up of the area boxed in black in (a), which contains dmvs the contrast between the densely labelled dmvs and the 58 zippered er and dmss largely lacking signal is apparent and suggests that the 59 autoradiography grains sometimes present on the latter structures arose from radioactive 60 disintegrations in the surrounding active dmvs. (c) in agreement with this possibility, most 61 of the dmss (96%) were devoid of signal, and most of those that contained label where close 62 to an active dmv (n dms = 178). (d) furthermore, the distribution of autoradiography grains 63 around dmss resembled that of a random distribution dmvs proved that these structures are associated with vrna synthesis, as the signal reaches 66 maximum values in the proximity of the dmvs (n dmvs = 51). ((c, d) see materials and 67 methods for the selection criteria and details) electron tomography of the membrane structures induced in mers-cov infection animation illustrating the tomography reconstruction and model presented in fig 1b. the 72 video first shows the tomographic slices (1.2 nm thick) through the reconstructed volume, 73 and then surface-rendered models of the different structures segmented from the tomogram cm (blue) and dmvs (yellow and lilac, outer and inner membranes), er 75 4 (green), and a vesicle (silver) containing virions (pink). the movie highlights the dms 76 association with cm techniques and applications of autoradiography in the light and electron 84 microscope autoradiography & radioautography. electron microscopy: 86 principles and techniques for biologists association of polioviral proteins of the p2 89 genomic region with the viral replication complex and virus-induced membrane synthesis as 90 visualized by electron microscopic immunocytochemistry and autoradiography escaping host factor pi4kb inhibition: enterovirus genomic rna replication in the 94 absence of replication organelles 97 the origin, dynamic morphology, and pi4p-independent formation of 98 encephalomyocarditis virus replication organelles. mbio mers-coronavirus replication induces severe in vitro cytopathology and is 103 strongly inhibited by cyclosporin a or interferon-alpha treatment sars-coronavirus replication is supported by a reticulovesicular network of 108 modified endoplasmic reticulum table 79 data sets and sampling for the quantifications of the autoradiography signal presented 80 in fig 3c and key: cord-253366-03cg831z authors: chakraborty, hirak; bhattacharjya, surajit title: mechanistic insights of host cell fusion of sars-cov-1 and sars-cov-2 from atomic resolution structure and membrane dynamics date: 2020-07-22 journal: biophys chem doi: 10.1016/j.bpc.2020.106438 sha: doc_id: 253366 cord_uid: 03cg831z the emerging and re-emerging viral diseases are continuous threats to the wellbeing of human life. previous outbreaks of severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers had evidenced potential threats of coronaviruses in human health. the recent pandemic due to sars-cov-2 is overwhelming and has been going beyond control. vaccines and antiviral drugs are ungently required to mitigate the pandemic. therefore, it is important to comprehend the mechanistic details of viral infection process. the fusion between host cell and virus being the first step of infection, understanding the fusion mechanism could provide crucial information to intervene the infection process. interestingly, all enveloped viruses contain fusion protein on their envelope that acts as fusion machine. for coronaviruses, the spike or s glycoprotein mediates successful infection through receptor binding and cell fusion. the cell fusion process requires merging of virus and host cell membranes, and that is essentially performed by the s2 domain of the s glycoprotein. in this review, we have discussed cell fusion mechanism of sars-cov-1 from available atomic resolution structures and membrane binding of fusion peptides. we have further discussed about the cell fusion of sars-cov-2 in the context of present pandemic situation. membrane fusion, one of the most fundamental processes for the survival of eukaryotes, occurs when two closely apposed lipid bilayers merge into a continuous single bilayer and the inner contents are mixed with each other. several cellular events such as endocytosis, exocytosis, cellular trafficking, compartmentalization, import of nutrients and export of waste, vesiculation, inter cellular communication, fertilization and many others involve membrane fusion, though they vary vastly in space and time. the fusion of synaptic vesicles is almost 10,000-fold faster than the fusion of vacuoles. despite many diversities all fusion processes follow a common route that includes membrane contact (docking), membrane merger (stalk and transmembrane contact formation), and opening of pore to transfer the intracellular material (pore formation). there are several models that describe the mechanism of membrane fusion, out of which the stalk model is the most accepted one [1] . the continuum model of membrane fusion or stalk model proposes that the fusion starts from a point-like membrane protrusion that transform into an hourglass like connection between two apposed monolayers [2] . this early hemifusion connection is called as the fusion stalk, which further expanded to form the hemifusion diaphragm [3] . the further expansion of the hemifusion diaphragm opens the pore either on the diaphragm or its perimeter [4, 5] . fusion pore can also be formed directly from stalk by avoiding the hemifusion state [6, 7] . though membrane fusion is an integral event for the survival of eukaryotes, enveloped viruses utilize this process to enter the host cell [8, 9] . the emerging and reemerging viral pandemics prompted us to understand the mechanism of fusion for better intervention against viral diseases. the recent worldwide outburst of coronavirus-2 (cov-2) causing severe acute respiratory syndrome (sars), covid-19, has created havoc in terms of morbidity and mortality. middle east severe acute respiratory syndrome (mers) coronavirus and sars coronavirus-1 (sars-cov-1), which belong to the same family, coronaviradae, were confined in a certain part of the world [10] . sars-cov-2 has shown much higher infectivity, and has been spread all over the world. there is no vaccine for mers and sars, and now the whole world is eagerly waiting for covid-19 vaccine. the fusion between the host cell and virus being the first step of infection process, it is important to understand the fusion mechanism. this review discusses the mechanistic insights of the fusion of sars-cov-1 and sars-cov-2 from their atomistic structure, and membrane interaction. enveloped viruses exploit membrane fusion for their entry to the host cell. the enveloped viruses cover their genetic material with the host cell derived lipid bilayer, which houses the fusion protein that is instrumental in the fusion between virus and host cell [11] . the receptor binding domain of the fusion protein finds its interaction partner on the cell surface and the interaction between these two allows the virus to dock on the host cell surface. several viruses such as human immunodeficiency virus and coronavirus fuse at the cell surface to transfer the genetic material in the host cell, whereas viruses like influenza enters the host cell through endocytosis and then fuses with the endosome upon ph trigger [12] . therefore, the viral fusion with the host cell is considered as the first step of viral infection. the fusion protein presents on the viral envelope is called fusion machine as it orchestrates the fusion process between the virus and host cell. interestingly, there is no sequence homology among the fusion proteins of different viruses however, all the fusion proteins share several common features [13] . now we have a decent understanding on the fusion mechanisms aided by three dimensional structures of fusion proteins and peptides of several viruses with the help of nmr spectroscopy, cryo-electron microscopy and x-ray crystallography. in recent years, the fusion proteins are considered as the target for antiviral intervention due to our growing understanding on the proteins and protein complexes that mediate fusion between virus and host cell. the amino acid sequences of fusion proteins are remarkably different among the viruses, but all initiates membrane fusion through trimer formation, and a common pathway of membrane dynamics [13] . the trimeric glycoproteins of class i virus generally contain a signal peptide, a receptor binding domain, a fusion domain, and a cytoplasmic tail (figure 2) . the signal peptide is being cleaved by the signal peptidase but remains in contact with the lipid and subunit of peptidase complex before it is released in the cytosol. the signal peptide of hiv fusion protein, gp160 is known to interact with a calcium binding cytosolic protein, calmodulin [14] . however, the direct role of signal peptide in membrane fusion is not yet clear. the receptor binding domain (rbd) is extremely critical as it provides host tropism and zoonotic transmission of the virus [15] . there are specific receptors on the host cell surfaces, which are being recognized by the receptor binding proteins to dock on the host cell surface. the cd4 receptor binds to the rbd of hiv (gp120) [16] , whereas the rbd of influenza hemagglutinin (ha1) binds to the cell surface sialic acid to anchor on the host cell [17] . the spike protein (s1) of coronaviruses utilizes host angiotensin converting enzyme (ace) to find the host cell for infection [18] . any small alteration in the rbd leads to inefficient binding resulting in reduced infectivity of the virus [19] . interestingly, binding of virus glycoprotein with cell surface proteins other than the specific receptor does not promote entry of the virus to the cell [20] . the interaction between rbd and the cell surface receptor induces dramatic conformational changes in the fusion protein leading to exposure of the fusion domain [21] . [17] . mutations in this stretch of amino acids have been shown to block fusion mediated viral infection for many viruses [22] [23] [24] . interestingly, fusion peptide itself is capable to induce fusion between lipid vesicles, and several putative mechanisms have been proposed on how fusion peptide promotes membrane fusion [19, [25] [26] [27] [28] . the α-helical trimeric heptad region plays a crucial role in inducing fusion by the formation of six helix bundle. the six helix bundle formation, a trademark of class i viral fusion protein, brings two apposing membranes close to each other [18] . the heptad region is further divided into two regions, portions close to the n-terminal (or near the fusion peptide) and the c-terminal (or near transmembrane domain) are termed as fusion peptide proximal region (fppr) and membrane-proximal external region (mper), respectively [29] . the interaction between fppr and mper promotes the formation of six helix bundle. the transmembrane domain is a stretch of 20-25 hydrophobic amino acids that remains anchored to the viral envelope. it is hypothesized that the fusion peptide (partitioned in the host membrane) interacts with the transmembrane domain (anchored in the viral envelope) to facilitate pore formation [30] . residue influenza virus ha peptide into suv at ph 5.0 [38] . in addition, it had been proposed that binding of 8-18 fps of ha would yield required free energy change, 60-120 kcal/mol, for the stabilization of curved membrane lipid membrane which is initially essential for fusion process [38] . insertion of fps into lipid bilayer is known to support stable structures with propensity for stable oligomerizations [41] . the fps of influenza virus ha peptide, hiv-1 gp41 belonging to the type i fusion protein have been serving as an archetypal example for mechanistic studies with model membranes. both ha and hiv-1 gp41 fps assume monomeric -helical structure, determined by solution nmr methods, in detergent micelle (figure 4 ) [44, 45, 55] . a large part (residues ile4-ala15) of helical structure of the fp of hiv-1 gp41 in sds micelle was deduced to be deeply inserted into the micelle core akin to the orientation of transmembrane helices [55] . initial solution nmr and epr studies determined an inverted "v" shaped or boomerang like helical structure of the 20-residue long ha fp in dpc micelle [44] . the non-polar face of the amphipathic helical structure probably be embedded into the monolayer of the lipid bilayer. interestingly, a tightly packed helical hairpin structure of ha fp was determined in dpc micelle by solution nmr. this 23-residue construct of ha fp contains additional three conserved residues trp 21 -tyr 22 -gly 23 at the c-terminus [45] . the helical hairpin structure of ha fp might undergo a structural change to a continuous long helix during fusion pore formation. it is conceivable that virus fusion would require higher order oligomerization or association of the fusion proteins. these oligomeric states are likely to confine fusionsolid state nmr methods showed transition of -helical to -sheet conformations of fps of hiv-1 gp41 and ha in membrane bilayers [50, 58] . structures of fps could significantly be influenced depending on membrane mimetic environments. as stated above, the fp of hiv-1 gp41 assumed helical structure whereas aggregated -sheet conformations were deduced in cholesterol containing lipid bilayers. these observations appear to indicate that multiple conformations of fps may be playing important roles in membrane fusion. although, determination of atomic resolution structures is challenging in lipid bilayers, therefore, structural works utilizing systems of lipid nanodisks or bicelles could be employed for better understating on the effect of membranes [59] [60] [61] [62] . it is worthy to mentioned that host protein is known to be important to aid in membrane coronaviruses (covs) are zoonotic pathogens subdivided, based on genomes and serology, into four different categories alpha, beta, gamma and delta [68, 69] . covs are enveloped viruses with a positive sense single stranded rna as genetic element [68] . covs the trimeric s or spike glycoprotein at the envelop of the covs appears to be determining in host specificity [20, 75] . the s protein of covs including sars-cov-1 and mers helps the virus to bind to the receptor of the host cells and host-virus fusion. the s protein can be divided into two domains s1 and s2 which can be proteolytically cleaved in some covs [76, 77] . the s1 domain of s is largely involve in receptor binding whereas the s2 domain is responsible for host-virus membrane fusion [78] [79] [80] [81] [82] . the full-length s of sars-cov-1 is 1255 amino acid long with several functional domains and potential multiple proteolytic cleavage sites at the boundary of s1 and s2, and also s2' site (figure 7) . cryo-em structures of s proteins from mouse coronavirus (mhv), human coronavirus khu1, sars-cov-1 and mers-cov are reported in the prefusion state [83] [84] [85] . these structures revealed trimeric architecture of the s protein with extensive packing j o u r n a l p r e -p r o o f between s1 and s2 domains. further, the fusogenic components of the s2 domain including hr1, fusion peptides are found to be buried within the core of the trimeric structure, whereas, the receptor binding domain of s1 can be amenable for interactions with the host cell. the s protein is expected to undergo a large conformational change from the latent prefusion state to fusion active state upon receptor binding and priming by host proteases. the x-ray structures of the s2 domain of s protein largely on hr1, hr2 and hr1/hr2 complex revealed canonical trimeric coiled-coil helical structures akin to type i fusion system [79, 80] . the hr1/hr2 complex forms a bundle of trimeric six helix whereby three short helices of hr2 can be found to be tightly packed along the long three helices of hr1. the quaternary association of hr1 and hr2 would bring the fusion peptides and viral tm helix at proximity, presumably facilitate membrane fusion process (figure 3 ). although membrane fusion of sars-cov-1 belongs to the type i fusion system, the large s protein of sars-cov-1 encodes number of regions in the s2 domain with membrane binding and/or membrane fusion activity [86] [87] [88] [89] [90] [91] [92] [93] [94] [95] . the existence of multiple potential fps in the s2 domain of s protein in sars-cov-1 is unique in comparison to type i fusion proteins of hiv and influenza viruses. notably, the single fp, located at the n-terminus, has been known to be responsible for membrane fusion of hiv-1 gp41 and ha influenza viruses [24] [25] [26] . intuitively, membrane fusion mechanism of sras-cov-1 and other covs may be complex involving several distinct stages. the upstream part of the hr1 (residues 892-972) of s2 domain of s protein of sars-cov-1 appears to be involved in membrane fusion (figure 7) . several research groups have identified membrane binding and fusogenic peptides or potential fps from the upstream region (residues 758-890) of hr1 ( table 1) . (table 1) , also termed as internal fusion peptide or ifp2, demonstrating high binding affinity and leakage from model membranes of various compositions [88] . the ifp2 appeared to be binding with negatively charged phospholipids with higher affinity compared to zwitterionic lipids [88] . it may be noteworthy that the ifp1 is located at the c-terminus of a proteolytic cleavage at residues 797/798 at s2' site. this would suggest that after proteolytic priming of s protein at s1/s2 and s2' sites, the ifp1 would remain a part of the fusion protein whereas fp, residues 770-788, will not be covalently bonded to the rest of the s2 domain (figure 7) . in a later study, whittaker and co-workers has defined a yet another segment of residues 798-815 in proximity to the s2'cleavage site as a fusion peptide or ifp1 [93] . mutagenesis studies on the full-length s protein identified residues l803, l804 and f805 that are critically involved in fusion. biochemical analyses with synthetic peptide of ifp1 demonstrated ability of this peptide in membrane fusion. in addition to these fusogenic peptides, a c-terminal segment adjacent to tm or ptm (residues 1185-1202) is known to confer strong membrane partitioning (figure 7) . further, ~170 amino acids long polypeptide between hr1 and hr2 also appeared to be containing peptide motifs with membrane interacting properties and virus-cell fusion inhibiting activity [94] . 873 gaalqipfamqmayrf 888 (ifp2) 88 858 alisgtatagwtfgagaalqipfamqmayr 886 79 j o u r n a l p r e -p r o o f we have determined 3-d structures of fps and ptm of sars-cov-1 in solution of dpc detergent micelle by nmr methods [96, 97] . the atomic resolution structure of the fp ( 770 myktptlkyfggfnfsqil 788 ) revealed a bend helical structure presumably resulting from two gly residues g780 and g781 at the center of the sequence (figure 8) . 1185 lgkyeqyikwpwyvwlgf 1202 ) of s protein has also been determined in dpc micelle. the ptm is a conserved sequence among sars-covs, and also observed in other viruses like hiv-1 and eob. it has been postulated that the ptm segment could be involved in viral membrane fusion process [87] . micelle-bound ptm assumes a fold of helix (residues k1187-y1191)-loop (i1192-k1193)-helix (w1194-f1202) structure whereby the two helices were found to be independently oriented (figure 8) . observations of multiple adjacent fusogenic peptides in the s2 domain prompted us to examine structure and membrane localization of a 64-residue long, residues r758-e821, or lfp (long fusion peptide) in detergent micelle solution [97] . the primary structure of lfp contains fp (residues 770-788) and ifp1 (residues 798-815) with additional residues at the n and c-termini. lfp was over-expressed in e. coli as a fusion protein containing an 81-resiude long prodomain of human furin followed by an asp-pro sequence for formic acid digestion [89, 90] . the construct also contains an additional six residue his-tag at the n-terminus for affinity purification resulting a fusion protein his 6 -prodomain-d-p-lfp. atomic resolution structure, 15 n relaxation and micelle localization were investigated by heteronuclear nmr methods in dpc micelle solution (figures 9, 10) . 3-d structure of lfp demonstrated existence of discretely folded helices connected by several loops (figure 9a ). the cterminal region of lpf defines a long -helix including residues t795-y819 with a kink at residue d812. it may be noted that ifp1, residues 798-815, is included within the c-terminal j o u r n a l p r e -p r o o f helix. the fp segment (residues 770-788) in lfp assumed a helix-loop-helix structure, although, the isolated fp delineated a bend helical structure (figure 8) . interestingly, the nterminal residues r761-q769 of lfp assumed an amphipathic helical conformation ( figure 9a ). the helical structures of lpf were found to be motionally rigid experiencing fast motion in ns-ps time scale. the fusogenic property of the membrane generally depends on the its composition and curvature [100] . protein undergoes oligomerization and may be important for infection process [108, 109] . pandemic due to coronavirus was predicted after sars and mers outbreaks. however, no vaccines or effective drugs were developed for the mitigation of the threats. a new strain of coronavirus called sars-cov-2 or covid-19 is demonstrating a rapid spread j o u r n a l p r e -p r o o f all over the world which was initially found in wuhan, china. sars-cov-2 pandemic, so far, has caused nearly 500,000 deaths globally with an infection of over 11 million people. the high level of infectivity of sars-cov-2 compared to sars-cov-1 could be related to an efficient cell entry of the virus. although, the molecular mechanism of the cell entry process remains unclear, binding of the virus to the host cell receptor is an important step in successful infection. studies have shown that the isolated rbd of sars-cov-2 binds to ace2 with tighter affinity compared to sars-cov-1, indicating potential for higher infectivity [107, [110] [111] [112] [113] . however, paradoxically, experiments with the full-length s protein have evidenced either similar binding affinity or even lower affinity of rbd to human ace2 in comparison to sars-cov-1 [111] [112] [113] . the cryo-em structure of sars-cov-2 showed that the rbd domain could be hidden within the s protein structure for immune evasion [111] . therefore, other factors might be responsible for high infectivity of sars-cov-2. protease cleavage of s protein has been postulated to be one of the factors responsible for high infectivity of sars-cov-2 [112] [113] [114] . an efficient membrane fusion mechanism between sars-cov-2 and host cell could also be responsible for the high level of infection. sequence comparison of s proteins domain between sars-cov-1 and sars-cov-2 indicated high level of conservation both for the s1 and s2 domains [115] . nevertheless, variations can be observed for the fusogenic regions of the s2 domain between two viruses. although, effect of these variations for an efficient membrane fusion remains to be examined. emerging and re-emerging viral infections are continuous threats to human kind. mechanics of membrane fusion point-like protrusion as a prestalk intermediate in membrane fusion pathway on the theory of membrane fusion. the stalk mechanism stalk mechanism of vesicle fusion. intermixing of aqueous contents lipid intermediates in membrane fusion: formation, structure, and decay of hemifusion diaphragm a quantitative model for membrane fusion based on low-energy intermediates energetics of intermediates in membrane fusion: comparison of stalk and inverted micellar intermediate mechanisms the many mechanisms of viral membrane fusion proteins receptor binding and membrane fusion in virus entry: the influenza hemagglutinin epidemiology, genetic recombination, and pathogenesis of coronaviruses role of lipids in virus replication mechanisms of coronavirus cell entry mediated by the viral spike protein structures and mechanisms of viral membrane fusion proteins: multiple variations on a common theme signal peptide fragments of preprolactin and hiv-1 p-gp160 interact with calmodulin recombination, reservoirs, and the modular spike: mechanisms of coronavirus cross-species transmission atomic structure of the ectodomain from hiv-1 gp41 structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid genetic analysis of the sars-coronavirus spike glycoprotein functional domains involved in cell-surface expression and cell-to-cell fusion the sweet spot: defining virus-sialic acid interactions cellular entry of the sars coronavirus receptor-induced conformational changes in the su subunit of the avian sarcoma/leukosis virus a envelope protein: implications for fusion activation studies on the mechanism of membrane fusion: site-specific mutagenesis of the hemagglutinin of influenza virus wild-type and mutant hemagglutinin fusion peptides alter bilayer structure as well as kinetics and activation thermodynamics of stalk and pore formation differently: mechanistic implications a specific point mutant at position 1 of the influenza hemagglutinin fusion peptide displays a hemifusion phenotype hiv-1 fusion peptide decreases bending energy and promotes curved fusion intermediates fusion peptide from influenza hemagglutinin increases membrane surface order: an electron-spin resonance study the gaussian curvature elastic energy of intermediates in membrane fusion energetics of vesicle fusion intermediates: comparison of calculations with observed effects of osmotic and curvature stresses crystal structure of hiv-1 gp41 including both fusion peptide and membrane proximal external regions gp41 transmembrane domain interacts with the fusion peptide: implication in lipid mixing and inhibition of virus-cell fusion fusion peptides and the mechanism of viral fusion viral fusion proteins: multiple regions contribute to membrane fusion physiological and molecular triggers for sars-cov membrane fusion and entry into host cells hypothesis: spring-loaded boomerang mechanism of influenza hemagglutinin-mediated membrane fusion the three lives of viral fusion peptides hiv entry and envelope glycoprotein-mediated fusion the polar region consecutive to the hiv fusion peptide participates in membrane fusion a host-guest system to study structure-function relationships of membrane fusion peptides early and late hiv-1 membrane fusion events are impaired by sphinganinelipidated peptides that target the fusion site sendai virus internal fusion peptide: structural and functional characterization and a plausible mode of viral entry inhibition are fusion pepties good model to study viral cell fusion lipids in biological membrane fusion relationship between the infectivity of influenza virus and the ability of its fusion peptide to perturb bilayers membrane structure and fusiontriggering conformational change of the fusion domain from influenza hemagglutinin the complete influenza hemagglutinin fusion domain adopts a tight helical hairpin arrangement at the lipid:water interface structure and function of the complete internal fusion loop from ebolavirus glycoprotein 2 the amino-terminal region of the fusion peptide of influenza virus hemagglutinin ha2 inserts into sodium dodecyl sulfate micelle with residues 16-18 at the aqueous boundary at acidic ph. oligomerization and the conformational flexibility nmr structure, localization, and vesicle fusion of chikungunya virus fusion peptide interaction of the dengue virus fusion peptide with membranes assessed by nmr: the essential role of the envelope protein trp101 for membrane fusion solid-state nmr spectroscopy of the hiv gp41 membrane fusion protein supports intermolecular antiparallel beta sheet fusion peptide structure in the final six-helix bundle state fully hydrophobic hiv gp41 adopts a hemifusion-like conformation in phospholipid bilayers structurerelated roles for the conservation of the hiv-1 fusion peptide sequence revealed by nuclear magnetic resonance conformation and lipid interaction of the fusion peptide of the paramyxovirus piv5 in anionic and negative-curvature membranes from solid-state nmr a strong correlation between fusogenicity and membrane insertion depth of the hiv fusion peptide structure and dynamics of micelle-associated human immunodeficiency virus gp41 fusion domain ph-dependent self-association of influenza hemagglutinin fusion peptides in lipid bilayers permeabilization and fusion of uncharged lipid vesicles induced by the hiv-1 fusion peptide adopting an extended conformation: dose and sequence effects solid-state nmr spectroscopy of human immunodeficiency virus fusion peptides associated with host-cell-like membranes: 2d correlation spectra and distance measurements support a fully extended conformation and models for specific antiparallel strand registries nanodiscs in membrane biochemistry and biophysics stabilization and structural analysis of a membrane-associated hiapp aggregation intermediate a. ramamoorthy, ph resistant monodispersed polymer-lipid nanodiscs spontaneous lipid nanodisc formation by amphiphilic polymethacrylate copolymers a seminal finding for understanding hiv transmission human prostatic acid phosphatase: structure, function and regulation bacterial curli protein promotes the conversion of pap248-286 into the amyloid sevi: cross-seeding of dissimilar amyloid sequences semen-derived amyloidogenic peptides-key players of hiv infection helical conformation of the sevi precursor peptide pap248-286, a dramatic enhancer of hiv infectivity, promotes lipid aggregation and fusion coronavirus diversity, phylogeny and interspecies jumping epidemiology and clinical presentations of the four human coronaviruses 229e, hku1, nl63, and oc43 detected over 3 years using a novel multiplex real-time pcr method planning for epidemics -the lessons of sars isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus: transmission and phylogenetic evolution broad reception for coronavirus sars-cov and emergent coronaviruses: viral determinants of interspecies transmission coronavirus spike proteins in viral entry and pathogenesis different host cell proteases activate the sars-coronavirus spike-protein for cell-cell and virus-cell fusion evidence that tmprss2 activates the severe acute respiratory syndrome coronavirus spike protein for membrane fusion and reduces viral control by the humoral immune response structure of sars coronavirus spike receptorbinding domain complexed with receptor severe acute respiratory syndrome coronavirus (sars-cov) infection inhibition using spike protein heptad repeat-derived peptides crystal structure of severe acute respiratory syndrome coronavirus spike protein fusion core structure of a proteolytically resistant core from the severe acute respiratory syndrome coronavirus s2 fusion protein bat to human: spike features determining host jump of coronaviruses sars-cov, mers-cov and beynod pre-fusion structure of a human coronavirus spike protein cryo-electron microscopy structure of a coronavirus spike glycoprotein trimer cryo-em structures of mers-cov and sars-cov spike glycoproteins reveal the dynamic receptor binding domains identification and characterization of the putative fusion peptide of the severe acute respiratory syndromeassociated coronavirus spike protein identification of the membraneactive regions of the severe acute respiratory syndrome coronavirus spike membrane glycoprotein using a 16/18-mer peptide scan: implications for the viral fusion mechanism glycoprotein internal membrane-active peptide. biophysical characterization and membrane interaction the aromatic domain of the coronavirus class i viral fusion protein induces membrane permeabilization: putative role during viral entry interaction of a peptide from the pre-transmembrane domain of the severe acute respiratory syndrome coronavirus spike protein with phospholipid membranes structural and dynamic characterization of the interaction of the putative fusion peptide of the s2 sars-cov virus protein with lipid membranes membrane insertion of the three main membranotropic sequences from sars-cov s2 glycoprotein characterization of a highly conserved domain within the severe acute respiratory syndrome coronavirus spike protein s2 domain with characteristics of a viral fusion peptide inhibition of severe acute respiratory syndrome-associated coronavirus infectivity by peptides analogous to the viral spike protein identification of the fusion peptide-containing region in betacoronavirus spike glycoproteins nmr structures and localization of the potential fusion peptides and the pre-transmembrane region of sars-cov: implications in membrane fusion nmr structure and localization of a large fragment of the sars-cov fusion protein: implications in viral cell fusion ph-induced conformational transitions of a molten-globule-like state of the inhibitory prodomain of furin: implications for zymogen activation designed di-heme binding helical transmembrane protein chembiochem membrane composition modulates fusion by altering membrane properties and fusion peptide structure structural and dynamic characterization of the interaction of the putative fusion peptide of the s2 sars-cov virus protein with lipid membranes sars-cov fusion peptides induce membrane surface ordering and curvature membrane cholesterol modulates oligomeric status and peptide-membrane interaction of severe acute respiratory syndrome coronavirus fusion peptide organization of higher-order oligomers of the serotonin(1)(a) receptor explored utilizing homo-fret in live cells excitements and challenges in gpcr oligomerization: molecular insight from fret cryo-electron microscopy structures of the sars-cov spike glycoprotein reveal a prerequisite conformational state for receptor binding structural basis of receptor recognition by sars-cov-2 structural insights of a self-assembling 9-residue peptide from the c-terminal tail of the sars corona virus e-protein in dpc and sds micelles: a combined high and low resolution spectroscopic study self-assembly of a nine-residue amyloid-forming peptide fragment of sars corona virus e-protein: mechanism of self aggregation and amyloid-inhibition of hiapp structure, function, and antigenicity of the sars-cov-2 spike glycoprotein cryo-em structure of the 2019-ncov spike in the prefusion conformation sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor characterization of spike glycoprotein of sars-cov-2 on virus entry and its immune cross-reactivity with sars-cov cell entry mechanisms of sars-cov-2 structural, glycosylation and antigenic variation between 2019 novel coronavirus (2019-ncov) and sars coronavirus (sars-cov) mechanistic insights of host cell fusion of sars-cov-1 and sars-cov-2 from atomic resolution structure and membrane dynamics hirak chakraborty 1,2 and surajit bhattacharjya 3 odisha 768 019, india 2 centre of excellence in natural products and therapeutics key: cord-327765-qdbgkm53 authors: kinnun, jacob j.; bolmatov, dima; lavrentovich, maxim o.; katsaras, john title: lateral heterogeneity and domain formation in cellular membranes date: 2020-09-15 journal: chem phys lipids doi: 10.1016/j.chemphyslip.2020.104976 sha: doc_id: 327765 cord_uid: qdbgkm53 as early as the development of the fluid mosaic model for cellular membranes, researchers began observing the telltale signs of lateral heterogeneity. over the decades this has led to the development of the lipid raft hypothesis and the ensuing controversy that has unfolded. here, we review the physical concepts behind domain formation in lipid membranes, both of their structural and dynamic origins. this, then leads into a discussion of coarse-grained, phenomenological approaches that describe the wide range of phases associated with lipid lateral heterogeneity. we use these physical concepts to describe the interaction between raft-lipid species, such as long-chain saturated lipids, sphingomyelin, and cholesterol with non-raft forming lipids, such as those with short acyl chains or unsaturated fatty acids. while debate has persisted on the biological relevance of lipid domains, recent research, described here, continues to identify biological roles for rafts and new experimental approaches have revealed the existence of lipid domains in living systems. given the recent progress on both the biological and structural aspects of raft formation, the research area of membrane lateral heterogeneity will not only expand, but will continue to produce exciting results. the fluid mosaic model of membranes was proposed by singer and nicolson in 1972, 1 and almost immediately, there were reports showing the existence of membrane lateral heterogeneity 2 . 3 by the late 1970's, it was suggested that lipids could segregate into liquid disordered and liquid ordered domains. 4 over the next decade, research showed that proteins could also co-localize 5 6 7 , 8 and in some cases, preferentially associate with lipids, such as sphingomyelin. 9 in the early 1990's, experiments focusing on cholesterol in model membranes showed the sterol's ability to increase lateral heterogeneity 10 11 and other studies, such as those using detergents to extract biomolecules from natural membranes, soon followed demonstrating protein co-localization with sphingomyelin and cholesterol 12 13 14 15 . 16 eventually, these data led to the hypothesis that sphingomyelin and cholesterol formed liquid ordered domains, called "rafts", in which proteins could associated with 17 . 18 although it was shown that some proteins had a preference for certain lipids, the idea that these would form large domains of functional significance remained controversial. however, recent research has revealed evidence for domain formation, consistent with lipid rafts, in fully functional, living cells 19 . 20 in this review, we discuss the concepts behind lipid domain formation in membranes, the biomolecules that they are made of, and their biological significance. 2 j o u r n a l p r e -p r o o f the propensity for domain formation results from the interaction energy between chemically distinct lipids and proteins. for example, unfavorable interaction energies can result from lipids with different length fatty acid chains, forming for example, different bilayer thicknesses residing near each other. some of these unfavorable interactions can be eliminated or minimized by sequestering lipids of similar length within a domain. to model this, an interaction energy, i.e., the so-called called line tension, λ, is used, and which is defined as: where e b is the total interaction energy at the domain boundary and l b is the length of the domain boundary. 21 note, that the total interaction energy is proportional to the domain boundary length, but the line tension, is not. in general, the greater the line tension, the greater the propensity for domain formation. line tension can be thought of as a string surrounding the domain perimeter that tightens as a function of an increasing unfavorable interaction energy between the the domain and its surroundings. note, that line tension should be considered in relation to the repulsive electrostatic forces that exist between the different lipids. points in phase separation studies. 23, 24 further, experiments have revealed that domain formation is to some extent, dependent on the collective membrane material properties and long-range fluctuations, 25 which will be discussed later. thus, line tension, itself, can vary as a function of system size, under certain circumstances. in addition to domain size having an effect on line tension, it is known that lipid-lipid repulsive interactions -which limit domain formation -also affect line tension. one way to account for lipid-lipid repulsive forces that counteract line tension, is to treat each lipid phase as a density of dipoles. what is important here, is not the absolute dipolar density, but the dipole density difference, ∆m, where lipids of one phase redistribute into the other phase in order to reduce repulsive dipolar interactions. the energy per molecule, e/n , for a circular domain can be written in terms of the opposing line tension and dipolar density difference as: where a 0 is the area per molecule, r is the domain radius, ε is the dielectric constant of the interfacial water, ε 0 is the permittivity of free space, e is euler's number, and δ is the molecular cut-off distance ∼ 0.5nm 26 lipids and membrane proteins have varying intrinsic hydrophobic thicknesses. coexistence of these species within a membrane results in local deformations at boundaries, where lipids splay and tilt to accommodate different thicknesses (see fig. 1 ). however, these deformations have an associated energy cost that results in line tension. energetics of local deformation can be discussed in terms of material properties, such as the splay elastic modulus (b), tilt modulus (k), and the intrinsic curvature (j) between two domains. if we consider two domains with thickness difference, δ, and an average thickness, h 0 , the line tension at the domain boundary can be written as: a feature of increasing importance is the packing defects between membrane lipids. these are introduced when the structure of a lipid is unable to conform to its neighbor. defects can be therefore be introduced as a result of the presence of unsaturated acyl chains or methylated segments that do not pack well with rigid moieties, such as the hydrocarbon rings in sterols, as shown in fig. 2c . akin to material rigidities -as discussed in the previous section -increasing biomolecule rigidity enhances packing mismatch, which increases line tension. in terms of specific theoretical energetics, this contribution to line tension is less 6 j o u r n a l p r e -p r o o f figure 2 : (a) the registration of domains across membrane leaflets maximizes dynamics, is entropically favorable, and is one mechanism for domain coalescence (adapted from haataja et al. 36 where a is the characteristic length of the monolayer that lies between the lipid headgroup diameter and monolayer thickness. 25 an increasing bending rigid difference between ordered and disordered lipid phases increases the value of the logarithm in eq. 4. this repulsive energy increase can result in co-alignment of rigid domains across bilayer leaflets or domains aggregating to more rigid areas of the membrane, such as those with proteins. for the case of domain registration across bilayer leaflets, it has been determined experimentally that domains with similar bending rigidiities can coalesce. 44 it has also been observed that domains are able to coalesce across adjacent bilayers, 45 and membrane undulations are thought to play a similar role 46 36 . 25 moreover, effects of acyl chain packing across bilayer leaflets should not be discounted, as there is evidence that leaflets can influence each other's molecular order. 47 thus the packing between leaflets may influence domain registration and ultimately, domain formation. evidence for lipid-driven lateral heterogeneity in membranes began to appear in lipid mixtures, such as dimyristoylphosphatidylcholine (14 carbon acyl chains) mixed with distearoylphosphatidylcholine (18 carbon acyl chains) 50 51 52 . 53 in general, longer-chained, unsaturated lipids tend to be more ordered, 54 the entropic difference between these two states results in a tension at domain boundaries 37 93 (see fig. 2b ) and adds another possible mechanism for cholesterol increasing domain size through registration. although specific bonding between cholesterol and sphingomyelin may play a major role in cholesterol's ability to increase the size of domains, the effect of non-raft domains should not be discounted. there is research showing that increasing the unsaturated lipid presence of non-raft lipid constituents drives increasing domain size, implying that acyl packing effects may be a big contributor to domain size. it has also been suggested that the "push" mechanism that drives cholesterol from non-raft domains is equally important as the "pull" mechanism, where cholesterol is incorporated into rafts 94 many proteins are relatively rigid and require a hydrophobic surface of sufficient thickness for them to incorporate "properly" into the membrane. differences between the protein's hydrophobic portions and those of the surrounding lipid membrane can result in hydrophobic mismatch, i.e., increased line tension (see fig. 1c early on, some research indicated that certain proteins preferential interact with sphin-gomyelin 14 . 97 since proteins can increase membrane thickness, it is likely that the ordering nature of sphingomyelin reduces membrane tension by deforming the membrane around proteins. also, it has previously been discussed that the nature of sphingomyelin's acyl chains allows for them to be compatible with the rigid cholesterol. since proteins can also be rigid, it is then possible that sphingoymelin is able to interact with proteins in a manner similar to that with cholesterol. it should also be pointed out that proteins, themselves, can form domains on the membrane surface, e.g., scaffolding proteins 97 . 98 often their organization is guided by electrostatics through specific charged amino acids, leading to oligomerization. 99 although some do not penetrate into the bilayer, they do introduce a region of increased local rigidity, which may be sufficient to cause ordered domain registration of apposing bilayer leaflets 25 . where the units are defined such that the coefficients of the quartic terms are unitless. this simple model has three parameters: i.e., κ, r, and µ, with the latter two parameters derived from the thermodynamic potential v [φ] and where κ is the rescaled surface tension. often, the fourth-order derivative term is ignored. this is acceptable as long as κ > 0 in eq. (5). as a result, the fourth-order derivative term becomes irrelevant with respect to the renormalization group and the dominant term becomes the second-order derivative. r is a tunable parameter across the mixing/demixing phase transition, which, in the mean-field, would occur at r = µ 2 /4 or where phase separation takes place. conversely, for r > µ 2 /4, the potential v [φ] is minimized around φ = 0 and we would expect no phase separation, but rather a fluctuating, disordered mixture of the liquid-ordered and liquid-disordered regions with a characteristic correlation length ξ ∼ κ/r. we may also be a somewhat more specific and identify the constant values φ o,d for which the potential is at a minimum in the phase separated regime with r < µ 2 /4. for 0 < r < µ 2 /4, the potential v [φ] has two minima at constant values φ = φ d = 0 and respectively. note that when µ = 0, this transition would have a first-order character and the potential will have a local maximum between the two minima (i.e., a thermodynamic barrier to transitioning between the liquid-disordered and liquid-ordered phases). if µ = 0, the two minima in the free energy become degenerate at φ = 0 at the transition (r = 0) so that there is no thermodynamic barrier and the transition is second-order (continuous). thermal fluctuations change this picture, somewhat, as the distinction between an unstable and a metastable phase becomes ambiguous when thermal fluctuations are taken into account. 100 in lipid vesicles, it is easy to tune across the mixing/demixing transition by varying the temperature. in biological cells, however, the situation is more complicated as it is unclear whether or not the cell membrane is at equilibrium, whereby these phases may not be meaningful. nevertheless, the free energy in eq. (5) is useful as a conceptual tool. we may use it as a basis for constructing the dynamics of the lipid phases by employing an appropriate dynamical model. 101 for instance, it has been recently argued that natural cells tune their membranes such that, they have compositions near criticality (µ = 0 and r ≈ 0). 102, 103 through dynamical processes, such as lipid diffusion, the free energy in eq. (5) trends towards minimization. as the lipid membrane is fluid, the most appropriate model would include the hydrodynamic coupling of the lipids across the membrane. 104 another crucial aspect of the dynamics is the conservation of total lipids, which means that the integrated order parameter, dx φ(x), is a constant (typically zero for equal proportions of liquid-ordered and liquid-disordered regions). excluding the hydrodynamics and thermal fluctuations, the time-dependence of the order parameter is given by: with γ representing mobility of the phase. eq. 6 may be evolved for various initial conditions, for example, to study the evolution of the phase separation of domains in the r < µ 2 /4 region. with such conserved dynamics, we would typically expect lifshitz-slyosov domain evolution, where domains grow as ∼ t 1/3 . 105 however, hydrodynamic effects may modify this scaling in more realistic scenarios. 106 the different phase-separated regions in a lipid vesicle may exhibit different preferred curvatures of the membrane, due to the particular geometry of the constituent lipid molecules. 107 here, the shape of the membrane couples to the dynamics of φ, and we have to combine the free energy in eq. (5) with elastic terms for the membrane and a coupling between φ and membrane curvature. leibler and andelman 108 showed that in the presence of such couplings, the line tension term, κ|∇φ| 2 /2, gets contributions from membrane curvature and can even change signs. when κ is driven to negative values, the quartic derivative term, |∇ 2 φ| 2 /2, must be included in the theory and the free energy, f, is now minimized by spatially modulated configurations. microscopic models also reveal that a coupling to membrane curvature is sufficient to drive the surface tension term negative. 109 by including sign changes in κ, this makes the phase space of the theory in eq. (5) much richer, and the various possibilities present a unified way of thinking about lipid membrane heterogeneity. 110 in the mean-field (ignoring thermal fluctuations), the phase portrait of the model is shown in fig. 4 . figure 4 : schematic of the various phases described by the free energy in eq. (5) for µ = 0. the phase boundaries are given in the single-mode mean-field approximation. 110 we see that when the gradient term κ < 0, it is possible to form an ordered modulated (patterned) phase and a disordered "microemulsion", with a characteristic wave number q 0 . here we have shown possibilities for the phases on the surface of a vesicle, which introduces its own complications due to the finite spherical geometry. for example, the striped modulated phase shown has stripes terminating at the poles. moreover, any of the modulated phases will have defects induced by the spherical topology. when κ < 0, the free energy develops minima with configurations of φ(x) with non-zero fourier modes. in particular, using a simple single-mode approximation, one can show that in the mean-field, the preferred wave number is |q| = q 0 = −κ/2 (see eq. (5) have nanoscopic domains and are more likely to be in the disordered "microemulsion" phase instead of these highly ordered patterned phases. note that we may incorporate dynamics by using an equation such as eq. (6). in the case of a free energy with parameters corresponding to a modulated phase, the dynamics reduce to the so-called "phase field crystal". 115 the dynamics of the modulated phases include interesting cases such as, the formation of a foam after a rapid quench into the modulated phase. 116 such foamy states appear to be relevant for pollen grain patterns, as well. 111 foamy structures may also be observed in synthetic lipid vesicles with specific compositions. 117 if κ is large and negative, then we will be deep in the modulated phase/microemulsion regime and we can expand our free energy around the dominant fourier modes with |q| = q 0 . for a spherical vesicle, fourier modes are inappropriate and one has to expand the field figure 5 : (a) mean-field phase diagram for the modulated phases with a fixed κ < 0 in eq. (5) and a flat and infinite two-dimensional substrate. we see that the cubic term µ tunes between the striped phase (middle), the hexagonal phase (top), and the inverted hexagonal phase (bottom). when r > 0, we also find a microemulsion region, denoted by the yellow. (b) modulated phases on a sphere have a more complex structure, since the pattern has to wrap the sphere 0 = q 0 r times, and has defects. we show the various possible shapes for small values of 0 . note the wide range of shapes, including continuously varying "intermediate" states. in general, on the sphere the free energy landscape becomes much more complicated than for a flat, infinite substrate. the phase diagram is adapted from radja et al. 111 here, we have a rescaled cubic termμ ∝ µ. r is fixed to a negative value ensuring that we are always in a modulated phase. is the location on the spherical membrane in spherical coordinates. we also have the usual spherical harmonics y m (θ), with = 0, 1, 2, . . . the "total angular momentum" mode number and 18 j o u r n a l p r e -p r o o f m = − , − + 1, . . . , the azimuthal mode number. then, we expect that the field has contributions primarily around the spherical harmonic modes with ≈ 0 ≈ q 0 r, with r the vesicle radius. this parameter will strongly influence the kinds of patterned phases that can form, as shown in fig. 5 . we can rewrite the free energy in eq. (5) on the sphere in the following (rescaled) landau-brazovskii 111,118 form: where υ 1 , 2 , 3 m 1 ,m 2 ,m 3 are the so-called gaunt coefficients 119 coming from integrations of products of three spherical harmonics. we see here that the cubic and quartic terms in the thermodynamic potential will couple different spherical harmonic modes. minimizing eq. (7) over the set of coefficients c m yields a rich set of possibilities for modulated phases, some of which are illustrated in fig. 5(b) . a free energy of this type is not only useful for understanding lipid vesicles and pollen grains, but also viral capsid formation. 120 we have spent some time considering the modulated phases (κ, r < 0 in eq. (5)). however, as mentioned previously, lipid heterogeneities in living cells and in many synthetic lipid mixtures are best thought of as disordered, microemulsion phases. the main difficulty of this hypothesis is explaining the origin of the sign change of κ. however, one possibility is that differences in composition between lipid leaflets can induce such a sign change via a mechanism similar to spontaneous curvature. 121 this is plausible as living cells are known to maintain asymmetric lipid compositions on their inner and outer membrane leaflets. 122 in this phase, the order parameter remains, on average, zero ( φ = 0), but exhibits particular fluctuations with dominant contributions at the characteristic wavevectors with |q| = q 0 (or modes = 0 on the sphere), resulting in a structure factor s(q). the structure factor, s(q), for the lipid membrane can be measured via a scattering (neutron or x-ray) experiment. 124 small angle neutron scattering is particularly valuable as it provides better contrast to probe the lateral membrane heterogeneity, even when these heterogeneities are nanoscopic. 125, 126 to get good contrast on the lateral heterogeneity within the membrane, deuterated lipid mixtures may be used to mask one of the liquid phases, for example. a schematic of the idea is shown in fig. 6(a) . here, lipid vesicles are prepared with particular deuteration levels such that, at high temperatures when the lipids remain mixed the sld of the acyl tails match the sld of the surrounding fluid. thus, the lipid vesicles become "invisible" to neutrons, as shown by the flat curves at high temperatures in fig. 6(b) . at lower temperatures, we have the liquid-liquid phase separation (or possibly microemulsion or modulated phase formation) and the slds of the liquid-ordered and liquid-disordered regions will be different, creating contrast for the neutron scattering as shown in fig. 6(a) . in this case, the neutron scattering will come from fluctuations in the lipid composition. therefore, it is a direct probe of our order parameter, φ(x). for scattering from lipid vesicles, the scattering intensity, i, is a probe of the fluctuations |c m | 2 of the spherical harmonic modes of φ(x). for a microemulsion phase, we expect that where r is the vesicle radius and ξ the correlation length. such a phenomenological approach was used successfully to interpret scattering data of lipid vesicles in the presence of melatonin 123 -although a true microemulsion phase could not be established as the scattering data was also consistent with 0 = 0 (a regular phase-separated phase). fits using the microemulsion theory are shown in fig. 6(b) . note, that this theory works well even when compared to a more microscopic model of domain configurations shown in fig. 6(c) . in other words, the simple phenomenological free energy approach presented here provides a conceptual framework for understanding the scattering data and interpreting the wide range of phase behaviors observed for lateral lipid organization in cell membranes. being near a phase transition allows for small changes in the environment to have a large effect on the appearance and size of rafts. one proposed scenario is that rafts may provide a buffering role in stabilizing membrane physiological properties across a range of temperatures. 44 here, high-melting temperature raft lipids diffuse into the disordered phase as temperature increases, maintaining membrane bending rigidity and viscosity. it has also been theorized that cells vary their lipid composition to control the formation of rafts. 150 the fluid mosaic model of the structure of cell membranes lateral phase separation in phospholipid membranes clusters in lipid bilayers and the interpretation of thermal effects in biological membranes domain formation in membranes with quenched protein obstacles: lateral heterogeneity and the connection to universality classes critical size dependence of domain formation observed in coarse-grained simulations of bilayers composed of ternary lipid mixtures fractal boundaries underpin the 2d melting of biomimetic rafts undulations drive domain registration from the two membrane leaflets relating domain size distribution to line tension and molecular dipole density in model cytoplasmic myelin lipid monolayers quantized symmetry of liquid monolayer domains line tension and interaction energies of membrane rafts calculated from lipid splay and tilt domain formation in membranes caused by lipid wetting of protein temperature dependence of structure, bending rigidity, and bilayer interactions of dioleoylphosphatidylcholine bilayers crossover from picosecond collective to single particle dynamics defines the mechanism of lateral lipid diffusion revealing the mechanism of passive transport in lipid bilayers via phonon-mediated nanometre-scale density fluctuations intrinsic curvature in normal and inverted lipid structures and in membranes lipid domain co-localization induced by membrane undulations. biophys cholesterol flip-flop impacts domain registration in plasma membrane models effect of the structure of lipids favoring disordered domain formation on the stability of cholesterol-containing ordered domains (lipid rafts): identification of multiple raft-stabilization mechanisms polyunsaturated fatty acid-cholesterol interactions: domain formation in membranes molecular-level organization of saturated and polyunsaturated fatty acids in a phosphatidylcholine bilayer containing cholesterol all n-3 pufa are not the same: md simulations reveal differences in membrane organization for epa, dha and dpa sphingomyelin analogs with branched nacyl chains: the position of branching dramatically affects acyl chain order and sterol interactions in bilayer membranes undulations, steric interaction and cohesion of fluid membranes mechanical properties of nanoscopic lipid domains long-range interlayer alignment of intralayer domains in stacked lipid bilayers bending rigidities and interdomain forces in membranes with coexisting lipid domains subnanometer structure of an asymmetric model membrane: interleaflet coupling influences domain properties separation of liquid phases in giant vesicles of ternary mixtures of phospholipids and cholesterol domain stability in biomimetic membranes driven by lipid polyunsaturation investigation of phase transitions of lipids and lipid mixtures by sensitivity differential scanning calorimetry miscibility properties of binary phosphatidylcholine mixtures. a calorimetric study dilatometric study of binary mixtures of phosphatidylcholines small-angle neutron scattering study of lipid phase diagrams by the contrast variation method smoothed acyl chain orientational order parameter profiles in dimyristoylphosphatidylcholine-distearoylphosphatidylcholine mixtures: a 2h-nmr study thermotropic phase transitions of pure lipids in model membranes and their modifications by membrane proteins. lipid-protein interactions fluid phase lipid areas and bilayer thicknesses of commonly used phosphatidylcholines as a function of temperature area per lipid and acyl length distributions in fluid phosphatidylcholines determined by 2h nmr spectroscopy lipid bilayer structure bilayer thickness mismatch controls domain size in model membranes cellular lipidomics lipidomics: coming to grips with lipid diversity understanding the diversity of membrane lipid composition raftlike mixtures of sphingomyelin and cholesterol investigated by solid-state 2h nmr spectroscopy hexagonal substructure and hydrogen bonding in liquid-ordered phases containing palmitoyl sphingomyelin cholesterol-sphingomyelin interactions: a molecular dynamics simulation study design and synthesis of sphingomyelin-cholesterol conjugates and their formation of ordered membranes sphingomyelin stereoisomers reveal that homophilic interactions cause nanodomain formation nanoscale membrane domain formation driven by cholesterol functional lipid pairs as building blocks of phase-separated membranes molecular organization in cholesterol-lecithin bilayers by x-ray and electron diffraction measurements combined influence of cholesterol and synthetic amphiphillic peptides upon bilayer thickness in model membranes structure of dipalmitoylphosphatidylcholine/cholesterol bilayer at low and high cholesterol concentrations: molecular dynamics simulation modulation of the bilayer thickness of exocytic pathway membranes by membrane proteins rather than cholesterol elastic deformation of membrane bilayers probed by deuterium nmr relaxation bending rigidity of sopc membranes containing cholesterol entropy-driven tension and bending elasticity in condensedfluid membranes effect of cholesterol on the rigidity of saturated and unsaturated membranes: fluctuation and electrodeformation analysis of giant vesicles lipid miscibility and size increase of vesicles composed of two phosphatidylcholines configurational statistics of acyl chains in polyunsaturated lipid bilayers from deuterium nmr acyl chain conformations in phospholipid bilayers: a comparative study of docosahexaenoic acid and saturated fatty acids docosahexaenoic and eicosapentaenoic acids segregate differently between raft and nonraft domains dha modifies the size and composition of raftlike domains: a solid-state 2h nmr study how polyunsaturated fatty acids modify molecular organization in membranes: insight from nmr studies of model systems cholesterol in bilayers with pufa chains: doping with dmpc or popc results in sterol reorientation and membrane-domain formation deuterium nmr of raft model membranes reveals domain-specific order profiles and compositional distribution lipid lateral diffusion in bilayers with phosphatidylcholine, sphingomyelin and cholesterol: an nmr study of dynamics and lateral phase separation characterization of the ternary mixture of sphingomyelin, popc, and cholesterol: support for an inhomogeneous lipid distribution at high temperatures docosahexaenoic acid promotes micron scale liquid-ordered domains. a comparison study of docosahexaenoic versus oleic acid containing phosphatidylcholine in raft-like mixtures lowering line tension with high cholesterol content induces a transition from macroscopic to nanoscopic phase domains in model biomembranes cholesterol 36 for the interior of polyunsaturated lipid membranes cholesterol hydroxyl group is found to cholesterol is found to presence and role of midplane cholesterol in lipid bilayers containing registered or antiregistered phase domains push-pull mechanism for lipid raft formation push and pull forces in lipid raft formation: the push can be as important as the pull protein-driven lipid domain nucleation in biological membranes caveolins, a family of scaffolding proteins for organizing "preassembled signaling complexes" at the plasma membrane the jip family of mapk scaffold proteins mechanism for targeting the a-kinase anchoring protein akap18δ to the membrane theory of dynamic critical phenomena critical fluctuations in plasma membrane vesicles critical casimir forces in cellular membranes dynamic simulations of multicomponent lipid membranes over long length and time scales the kinetics of precipitation from supersaturated solid solutions dynamic scaling in phase separation kinetics for quasi-two-dimensional membranes lipid polymorphisms and membrane shape ordered and curved meso-structures in membranes and amphiphilic films competition between line tension and curvature stabilizes modulated phase patterns on the surface of giant unilamellar vesicles: a simulation study macroscopic phase separation, modulated phases, and microemulsions: a unified picture of rafts pollen cell wall patterns form from modulated phases firstorder patterning transitions on a sphere as a router to cell morphology feigenson imaging coexisting fluid domains in biomembrane models coupling curvature and line tension modeling elasticity in crystal growth emergence of foams from the breakdown of the phase field crystal model phase diagram of a 4-component lipid mixture: dspc/dopc/popc/chol phase diagrams of multicomponent lipid vesicles: effects of finite size and spherical geometry angular momentum in quantum mechanics orientational phase transitions and the assembly of viral capsids membrane heterogeneity: manifestation of a curvature-induced microemulsion lateral organization, bilayer asymmetry, and inter-leaflet coupling of biological membranes deciphering melatonin-stabilized phase separation in phospholipid bilayers models for randomly distributed nanoscopic domains on spherical vesicles using small-angle neutron scattering to detect nanoscopic lipid domains domains on a sphere: neutron scattering, models, and mathematical formalism phase transition of an isotropic system to a nonuniform state unbiased quantitative proteomics of lipid rafts reveals high specificity for signaling factors enrichment of lck in lipid rafts regulates colocalized fyn activation and the initiation of proximal signals through tcrαβ cholesterol depletion delocalizes phosphatidylinositol bisphosphate and inhibits hormone-stimulated phosphatidylinositol turnover lipid rafts and signal transduction roles for lipid heterogeneity in immunoreceptor signaling hydrophobic mismatch sorts snare proteins into distinct membrane domains protein sorting by lipid phase-like domains supports emergent signaling function in b lymphocyte plasma membranes partitioning of lipidmodified monomeric gfps into membrane microdomains of live cells jessup, w. visualizing lipid structure and raft domains in living cells with two-photon microscopy large-scale fluid/fluid phase separation of proteins and lipids in giant plasma membrane vesicles elucidating membrane structure and protein behavior using giant plasma membrane vesicles helicobacter pylori lipids can form ordered membrane domains (rafts) direct observation of the nanoscale dynamics of membrane lipids in a living cell direct chemical evidence for sphingolipid domains in the plasma membranes of fibroblasts dynamic reorganization and correlation among lipid raft components lipid rafts: contentious only from simplistic standpoints the mystery of membrane organization: composition, regulation and roles of lipid rafts membrane domains beyond the reach of microscopy molecular picture of the transient nature of lipid rafts critical fluctuations in plasma membrane vesicles gratton, e. lipid rafts reconstituted in model membranes characterization of cholesterolsphingomyelin domains and their dynamics in bilayer membranes triton promotes domain formation in lipid raft mixtures liquid general anesthetics lower critical temperatures in plasma membrane vesicles conditions that stabilize membrane domains also antagonize n-alcohol anesthesia docosahexaenoic acid domains: the ultimate non-raft membrane domain membrane lipid raft organization is uniquely modified by n-3 polyunsaturated fatty acids biological and pathophysiological roles of endproducts of dha oxidation permeability of dimyristoyl phosphatidylcholine/dipalmitoyl phosphatidylcholine bilayer membranes with coexisting gel and liquid-crystalline phases line tension at lipid phase boundaries as driving force for hiv fusion peptide-mediated fusion mitochondria do not contain lipid rafts, and lipid rafts do not contain mitochondrial proteins biophysical and biochemical mechanisms by which dietary n-3 polyunsaturated fatty acids from fish oil disrupt membrane lipid rafts membrane raft microdomains mediate lateral assemblies required for hiv-1 infection hiv-1 gag protein can sense the cholesterol and acyl chain environment in model membranes comparative lipidomics analysis of hiv-1 particles and their producer cell membrane in different cell lines target membrane cholesterol modulates single influenza virus membrane fusion efficiency but not rate influenza virus hemagglutinin concentrates in lipid raft microdomains for efficient viral fusion sphingolipid and cholesterol dependence of alphavirus membrane fusion lack of correlation with lipid raft formation in target liposomes tuning membrane phase separation using nonlipid amphiphiles • review article describing recent experimental and theoretical membrane lateral heterogeneity research • describes the static and dynamic physical concepts behind domain formation in model membranes • describes coarse-grained, phenomenological approaches that result in phases associated with lipid lateral heterogeneity key: cord-034898-zjfhpum2 authors: patangi, sanjay orathi; shetty, riyan sukumar; shanmugasundaram, balasubramanian; kasturi, srikanth; raheja, shivangi title: veno-arterial extracorporeal membrane oxygenation: special reference for use in ‘post-cardiotomy cardiogenic shock’ — a review with an indian perspective date: 2020-11-07 journal: indian j thorac cardiovasc surg doi: 10.1007/s12055-020-01051-7 sha: doc_id: 34898 cord_uid: zjfhpum2 the ultimate goals of cardiovascular physiology are to ensure adequate end-organ perfusion to satisfy the local metabolic demand, to maintain homeostasis and achieve ‘milieu intérieur’. cardiogenic shock is a state of pump failure which results in tissue hypoperfusion and its associated complications. there are a wide variety of causes which lead to this deranged physiology, and one such important and common scenario is the post-cardiotomy state which is encountered in cardiac surgical units. veno-arterial extracorporeal membrane oxygenation (va-ecmo) is an important modality of managing post-cardiotomy cardiogenic shock with variable outcomes which would otherwise be universally fatal. va-ecmo is considered as a double-edged sword with the advantages of luxurious perfusion while providing an avenue for the failing heart to recover, but with the problems of anticoagulation, inflammatory and adverse systemic effects. optimal outcomes after va-ecmo are heavily reliant on a multitude of factors and require a multi-disciplinary team to handle them. this article aims to provide an insight into the pathophysiology of va-ecmo, cannulation techniques, commonly encountered problems, monitoring, weaning strategies and ethical considerations along with a literature review of current evidence-based practices. inability to wean off cardio-pulmonary bypass (cpb) is a morbid condition associated with cardiogenic shock secondary to impaired myocardial contractility. once the vicious cycle sets in, vital organ perfusion is compromised, culminating in severe metabolic derangement. the incidence of refractory cardiogenic shock post cardiotomy ranges from 0.5 to 6% [1, 2] with a mortality rate as high as 40% [3] . veno-arterial extracorporeal membrane oxygenation (va-ecmo) has gained popularity over the years as a 'bailout' option after conventional circulatory support methods have proved refractory in the operating room (or)/intensive care unit (icu). va-ecmo facilitates luxurious end-organ perfusion and adequate gas exchange and supports organ functionality allowing time for recovery/ bridge to decision. however, its usage has not been directly linked with early positive outcomes with few articles reporting increased mortality [4] [5] [6] . post-cardiotomy va-ecmo is used in both adult and paediatric populations [4] . the worldwide incidence of instituting post-cardiotomy va-ecmo varies between 0.4 and 3.7% [4] . the extracorporeal life support organization (elso) database states a substantial increase in its use over the last decade. the patient population in which this therapy has been used for dealing with post-cardiotomy cardiogenic shock (pccs) included those with renal insufficiency, prior myocardial infarction, critical left main coronary artery disease, redo-surgery and severe left ventricular (lv) dysfunction [7] . age is not a contraindication for this therapy. va-ecmo has been used to tide over patients with pccs covering the ambit of cardiac surgery and transplantation. cannulation is the first decisive step for a smooth ecmo run that can be either central (atria-aortic) or peripheral (femoral veinfemoral artery/axillary artery) (fig. 1) . a meta-analysis favours the peripheral route due to lesser transfusions, bleeding/ tamponade events and lower mortality [8] . however, these observational studies were based on a small sample size without accounting for confounding factors like the patient pre-initiation condition, effect of lv unloading and the timing of ecmo initiation. notably, the use of temporary mechanical support following pccs is associated with higher vascular complications when the duration exceeds 10 days [9] and incidence of amputation even with distal limb perfusion is 3.2% [10] . offloading the lv reduces the myocardial oxygen demand and allows for quicker myocardial recovery with higher survival rates. the decision on cannulation should be expeditious with minimal blood loss ensuring the myocardium is fully rested to hasten recovery and is fundamental in ensuring survival following pccs. techniques of venting include insertion of an intra-aortic balloon pump (iabp), atrial septostomy, percutaneous ventricular assist device (vad) or direct cannulation of the lv apex [11] . monitoring and maintenance on va-ecmo standard monitoring includes mean arterial pressure (map), central filling pressures, temperature, pulse oximetry and urine output. cardiac output (co) measurement by thermodilution technique is overestimated whereas pulse contour analysis is unreliable due to non-pulsatility [12] . abnormal rhythm leads to ineffective lv ejections resulting in lv distension and myocardial injury. presence of an lv vent prevents distension while reversal to sinus rhythm can be achieved by cardioversion, antiarrhythmics, pacing or ablation [13] . lv pulsatility is assessed by arterial pressure waveform. map is maintained in a range of 65-90 mmhg to ensure vital organ perfusion [14] . temperature monitoring is crucial as ecmo has cooling effects and temperature elevations beyond the 'defined temperature regulated range' signal infection. vascular access, urinary catheter, pneumonia and surgical wound sites are potential sources of infection [15] . hypothermia worsens coagulopathy and platelet dysfunction [16] . transcranial doppler, near-infrared spectroscopy (nirs) and cerebral oximetry monitoring can detect cerebral hypoperfusion and upper body hypoxaemia. nirs helps to identify lower-extremity ischaemia in peripheral arterial cannulation [17] . in va-ecmo, total systemic flow equals the sum of pump flow plus native co. cannulae selected should provide 50-75 ml/kg/min of flow, and when flows are maintained at 80%, it avoids stasis in the pulmonary vasculature [15] . sweep gas flow titration regulates carbon dioxide (co 2 ) levels while oxygenation depends on the fraction of delivered oxygen (o 2 ), oxygenator blood flow and exposed surface area [13] . va-ecmo is not an indication for mechanical ventilation, and patients may be extubated on va-ecmo support [18] . for patients who require mechanical ventilation, there is limited data on optimal ventilation strategies. extrapolating veno-venous ecmo (vv-ecmo) literature, lung protective ventilation is commonly recommended, with 4-6 ml/kg tidal volume, 10 cm h 2 o positive end expiratory pressure (peep) and plateau pressure < 25 cmh 2 o to maintain < 15 cmh 2 o driving pressure (inspiratory pleural pressure-peep) [14] . unfractionated heparin (ufh) remains the mainstay of anticoagulation due to quick onset of action and rapid reversibility. heparin monitoring relies on activated partial thromboplastin time (aptt) and anti-xa activity while activated clotting time (act) is unreliable in low to moderate doses of heparin (7.5 to 50 iu/kg/h) [ 19] . echocardiography enables assessment of biventricular function, aortic valve opening, cannula position, pericardial and pleural effusions and ventricular thrombus formation [12] . mixed venous saturations (scvo 2 ) and lactate levels are monitored for adequacy of tissue perfusion and o 2 delivery to end organs. elevated lactate levels reflect tissue hypoxia and are associated with mortality [20] . plasma free haemoglobin (pfhb) monitoring indicates haemolysis occurring in the ecmo circuit. pfhb levels > 50 mg/dl increase risk of thrombosis due to affinity of the von willebrand factor to platelet glycoprotein gpib [21] . no guidelines exist to decide the optimal time of weaning. recovery of adequate cardiac and respiratory function is a pre-requisite. some authors suggest weaning as early as 48-72 h [22] but rarely beyond 15 days, except in cases of postheart transplantation for resolution of pulmonary hypertension [4] . recovery of cardiac function in the post-cardiotomy setting is limited and rare beyond 7 days [15, 23] . a longer duration of support is associated with increased complications and mortality [23] . weaning is by a multidisciplinary consensus. cardiac index, pulmonary capillary wedge pressure, central venous pressure, pulse pressure > 10 mmhg and map > 60 mmhg with minimal haemodynamic support are indicators of cardiac function recovery [24] . resolution of pulmonary oedema, lung recruitment manoeuvres and clearing of airway secretions by bronchoscopy to ensure partial pressure of o 2 (pao 2 )/fraction of inspired o 2 (fio 2 ) ratio > 200, fio 2 < 60% on the ventilator and fio 2 < 50% on ecmo circuit are desirable [24, 25] . although end-organ recovery to pre-ecmo levels is important [15] , complete recovery from acute tubular necrosis can take weeks; hence, complete resolution of renal function is not mandatory before weaning. the patient can be supported with haemodiafiltration during this period [26] . a weaning trial assesses suitability to separate from the ecmo machine. assessment of right ventricular (rv) function is crucial [26] . ecmo flows are reduced gradually with inotropic and respiratory support whilst being monitored by clinical and echocardiographic variables. cavarocchi et al. [27] described a 4-stage weaning trial using a miniaturized transoesophageal echocardiogram (tee) with 100% positive predictive value. lv ejection fraction (lvef) > 20-25%, aortic velocity time integral (vti) > 10 cm, mitral lateral annulus systolic velocity > 6 cm/s, rv ejection fraction > 24.6%, no lv or rv distension and ability to maintain map with minimal inotropic supports indicate the possibility of a successful wean. assessments of tricuspid regurgitation, tricuspid annular plane systolic excursion or lv filling parameters are not reliable [24] . among clinical variables analysed, pulsatility and lactate clearance predicted a successful wean. however, no threshold was identified [20, 26] . biomarkers such as nterminal fragment of the b-type natriuretic peptide (bnp), troponin i, the mid regional fragment of the proatrial natriuretic peptide, proadrenomedullin and copeptin have poor predictive ability in va-ecmo weaning [28] . fast and slow weaning strategies have been proposed [26, 29] . westrope et al. described a unique technique of pump-controlled retrograde trial off. here, the pump speed is gradually decreased to encourage reversal of flow into the ecmo circuit which creates a controlled arterio-venous (a-v) shunt without a steep drop in systemic vascular resistance [30] . the pulmonary artery catheter is a useful monitoring tool in the weaning/post-weaning phase [27] . levosimendan improves weaning success. it works via non-adrenergic pathways with active metabolites producing effects that last 7-9 days [31] . inhaled nitric oxide improves rv function by reducing pulmonary vascular resistance [32] . total vessel density and perfused vessel density monitoring of sublingual microcirculation have been found to have a good association with aortic vti, lvef parameters during ecmo wean [33] . pccs is an uncommon occurrence with high morbidity and mortality in cardiac surgery. this low co state is refractory to high inotropic and iabp use. in this scenario, va-ecmo is a bridge to decision/recovery. va-ecmo drains blood from the venous system and inputs into the arterial system causing a reduction in rv and lv preload and improving endocardial blood flow by decreasing lv end diastolic pressure. lv afterload is increased in higher map states which distends the lv and leads to pulmonary congestion [34] . the higher flows via ecmo improve macroand micro-circulation [35] . non-pulsatile flow produces atrophic changes in the medial layer of the aorta and reduces vascular contractility [36] . the ecmo oxygenator is made of microporous polypropylene membrane containing hollow fibre bundles. the input gas flows within these bundles while blood passes over it. gas exchange is by diffusion. the o 2 uptake is determined by fio 2 , gradient across membrane fibres, surface area of oxygenator and ecmo flow while sweep gas controls co 2 elimination. as co 2 transfer is six times faster compared to o 2 , failure to clear co 2 indicates oxygenator failure [34] . ischaemic damage due to shunting of pulmonary blood flow, inflammatory activation, collapsed lungs, ischaemiareperfusion damage and passive congestion from lv distension contribute to ecmo-induced lung damage [37] . extubation is recommended to reduce lung damage. however, patients have reduced alveolar ventilation secondary to co 2 clearance by ecmo. to prevent post-extubation atelectasis, it is important to maintain co 2 and ph levels by non-invasive ventilation along with sweep gas flow titration [38] . although va-ecmo provides gas exchange in addition to circulatory support, native lung function is important in peripheral ecmo as myocardial and cerebral oxygen delivery is determined by o 2 content of blood exiting the lv [39] . studies comparing pulsatile with non-pulsatile flows have found maintained cerebral metabolism and autoregulation with both patterns provided map is > 50 mmhg [40] . varying levels of organ damage prior to ecmo improve after establishing adequate flows. effective venous drainage relieves congestion, further improving organ circulation. pulsatile flow enhances end organ recovery and splanchnic circulation compared to non-pulsatile flows although microcirculation is maintained in both patterns [41] . rate of renal recovery is comparable in either flow patterns [40] . there is no difference in clinical outcomes in either pattern provided adequate flows are maintained [41] . lymph flow is dependent on muscle activity and pulsatility. arterial pulsation is a primary determinant of lymphatic drainage in supine patients. non-pulsatile flows lead to peripheral oedema and intestinal congestion [40] . the blood-material interface activates coagulation, fibrinolytic and inflammatory systems releasing proinflammatory mediators that lead to endothelial injury and neutrophil activation affecting other organ systems. mast cell degranulation produces vasoplegia requiring vasoconstrictors to maintain map [15] . major complications can be broadly divided into circuitrelated and patient-related factors ( table 1 ). the commonest mechanical complication is thrombosis within the circuit [42] . thrombosis starts in areas of low flow and increases turbulence. clots on the arterial side of the circuit have a risk of embolization into systemic circulation and should be immediately addressed. clots on the venous side can cause coating of oxygenator and its failure [43] . even when visible clots are absent, microthrombi and fibrin deposits on the oxygenator reduce its efficiency over time due to suboptimal gas exchange. this is more common, but not limited to long ecmo runs. air can enter into the circuit via loosely attached connectors, inadvertently open access ports or tube defects. this can be catastrophic by bringing the pump to a halt. air on the arterial side can embolize as well. in view of the critical nature of the patient subset undergoing ecmo therapy, complications can have a significant impact on outcomes [44] . disseminated intravascular coagulation and acquired von willebrand disease are seen in patients on ecmo due to activation of the coagulation cascade with resultant consumption coagulopathy with a 10-30% incidence of bleeding [45] . the commonest sources of bleed are surgical and cannulation sites. intra-thoracic, intra-abdominal and intra-cranial bleeds can also occur [44] . incidence of reopening for tamponade or haemorrhagic complications can be as high as 40%, paving the way for low-dose heparin protocols during the maintenance phase of ecmo [46] . lower antithrombin iii levels are associated with higher transfusion requirements and mortality rates [47] . replacement of blood components is based on haematocrit (hct), act, prothrombin time (pt) and aptt ratios. point-of-care testing like thromboelastography (teg) and rotational thromboelastometry allows for quick intervention and specific corrections thereby reducing risk of volume overload and inflammatory/immunological activation. the recommended target act is 200-220 s which is reduced to 170-190 s in the event of bleeding. an international normalized ratio (inr) of > 1.5 warrants correction with fresh frozen plasma, and maintaining platelet count of > 50,000/mm 3 is recommended. activated factor vii (viia) is used as a last measure when other modalities have failed. extreme care is exercised when viia is used and a lower dose of 25-50 μg/kg is recommended as opposed to a conventional dose of 50-90 μg/kg to avoid an inadvertent pro-thrombotic state [44] . ufh is the anticoagulant recommended for initiating and maintaining ecmo support as per 2014 elso guidelines [48] . heparin-induced thrombocytopenia (hit) is associated with usage of ufh. hit is an immune-mediated pro-thrombotic condition characterized by antibodies to the heparin-pf4 complex on platelet surfaces which induces thrombosis. it has an incidence of 0.5-5% and mortality rate of 42% [49] . bivalarudin, a direct thrombin inhibitor, is an alternative anticoagulant for hit-positive patients. teg and aptt ratio are used to monitor anticoagulation [50] . the incidence of gastrointestinal (gi) bleeding is 6-13.5% secondary to reduced gut perfusion, decreased gastric ph leading to stress ulcers and a-v malformations in the small bowel as a result of non-pulsatile flows [51] . other contributing factors for gi bleeding include anticoagulation, coagulopathy, thrombocytopenia, platelet dysfunction, acquired von willebrand syndrome and hyperfibrinolysis. gi bleed commonly occurs around the 11th day of ecmo [52] . elderly patients and need of high-volume red blood cell transfusion were associated with higher mortality [53] . elso registry data showed high mortality from gi bleed in contrast to recent studies [51, 52] . no specific guidelines for prevention of gi bleeding are available. a meta-analysis in critically ill patients showed that proton pump inhibitor prophylaxis reduces incidence of gi bleed, albeit with higher risk of ventilatorassociated pneumonia (vap) [54] . elso guidelines recommend correction of coagulation followed by endoscopy and endotherapy. endotherapy using haemospray, fibrin glue, cyanoacrylate, cautery and clips has been used to control gi bleeding successfully [51] . hyperbilirubinaemia and elevated liver enzymes are commonly seen in patients on ecmo and are challenging to manage due to limited therapeutic options. pre-existing liver disease can manifest as acute liver dysfunction on ecmo. when associated with cardiorespiratory problems, passive hepatic congestion can lead to chronic changes and fibrosis [55] . the current concept of 'two hit' ischaemic liver injury happens when a liver primed by such chronic congestion experiences acute hypoperfusion causing a rapid spike in aspartate transaminase (ast) and alanine transaminase (alt) levels followed by hyperbilirubinaemia [56] . hyperbilirubinaemia, an independent predictor of poor outcomes, occurs due to a combination of extracorporeal haemolysis and liver dysfunction [57] . elevated alkaline phosphatase, lactate and bnp are associated with poor outcomes [58] , while elevated ast and alt levels do not seem to be predictive. [61] . in cases where va-ecmo was used as a bridge to heart transplant, pre-existing liver dysfunction with total bilirubin ≥ 120 μmol/l and inr ≥ 3.0 was a predictor of mortality [62] . similarly, a meld unos score > 24 has also been noted to be associated with high mortality [59] . ischaemic hepatitis and liver congestion are self-limiting, and measures which improve cardiac function can restore liver perfusion and aid recovery [55] . molecular adsorbent recirculation system therapy may be considered in the setting of alf as it has been noted to accelerate recovery of liver function and improve survival [63] . acute kidney injury (aki) is seen in 85% of patients undergoing ecmo with complex multifactorial aetiology and pathophysiology with majority of them requiring renal replacement therapy (rrt) [64] . the most common indication for initiating rrt is to achieve fluid balance in patients unresponsive to diuretic therapy. intermittent therapies of rrt are effective in haemodynamically stable patients; however, the most common modality used is continuous rrt (crrt). a large meta-analysis has shown higher mortality when rrt was used sparingly in patients on ecmo [65] . rrt can be provided using either an 'integrated system' or a 'parallel system'. the integrated system could be an 'in-line' haemofilter or an rrt circuit incorporated into the ecmo circuit. however, when an integrated rrt circuit is used, there is a risk of micro-clot formation within the circuit clogging the oxygenator. hence, knowledge of intra-circuit pressures, appropriate modifications in connections of the rrt circuit and additional regional anticoagulation would be necessary. on the other hand, a 'parallel system' involves a separate indwelling vascular catheter to provide rrt, obliviating the need for additional anticoagulation as patients are already anticoagulated for ecmo. close review and readjustment of prescription is crucial as per changing needs of the patient. most studies have shown that use of crrt with ecmo is not associated with increased mortality, and in fact, when used, these subset of patients needed shorter duration of ecmo. data from large ecmo centres have shown that ecmo survivors who have received rrt have similar renal outcomes with no increase in incidence of endstage renal disease in comparison to patients who did not receive rrt [66] . neurological complications in va-ecmo are associated with high mortality and morbidity, with high incidence (7.4-15%) across all age groups [67] . this has been attributed to nonpulsatile flow, low arterial o 2 saturation in the upper half of the body and entrainment of unfiltered thrombi into the systemic circulation [68] . risk factors associated with neurological injury in neonates are low birth weight < 3 kg, gestational age < 34 weeks, pre-ecmo cardio-pulmonary resuscitation (cpr), metabolic acidosis, bicarbonate use and prior ecmo exposure [69] . neonates are more susceptible to intracranial haemorrhage (ich) (1.4%) as opposed to the paediatric group (0.9%) and adults (0.4%) [70] . risk factors in adults include female gender, central cannulation during cardiac surgery, thrombocytopenia, serum creatinine > 2.6 mg/dl, hypercapnia while initiating ecmo, duration of ecmo and use of anticoagulants. incidence of acute ischaemic stroke is 3.6-6% across all age groups which is multifactorial in origin [68] . thrombocytosis at ecmo initiation is a potentially modifiable factor [67] . brain computed tomography scan (fig. 2) is the recommended imaging modality when neurological deficits are identified since magnetic resonance imaging, despite being more sensitive, is contraindicated during ecmo [68] . use of va-ecmo is associated with a higher incidence of electrographic seizures (fig. 3) in neonates (4.9%) and children (3.3%) as compared to adults (0.5%) [70] . nosocomial infection is a major cause of morbidity and mortality on ecmo, with an incidence of 11-64% [71] [72] [73] . the fig. 2 non-contrast computed tomography of the brain showing rightsided intra-parenchymal haemorrhage with peri-haemorrhagic cerebral oedema and midline shift on day 2 of starting va ecmo rate of infection was highest in adults, followed by paediatric and neonatal age groups [71, 72] . it is crucial to practice meticulous infection prevention measures. longer duration of ecmo support is an independent risk factor for infection [73] . bizzarro et al. reported a prevalence of 30.3% in patients on ecmo > 14 days compared to 6.1% in patients with < 7 days support [71] . bloodstream infections and vap were commonly encountered [74] . the median time interval between initiation of ecmo and occurence of a bloodstream infection was 5-8 days [72] . these infections were predominantly caused by gram-positive organisms (coagulase-negative stapylococcus, enterococcus and staphylococcus aureus), candida and pseudomonas species [71, 73] . vascular complications stem from difficult cannulation, low flow states and high use of vasoconstrictors. percutaneous cannulation techniques in peripheral va-ecmo can be associated with posterior vessel wall perforation resulting in inadequate perfusion and subsequent development of a compartment syndrome/retro-peritoneal haematoma [75] . larger-size cannulae (> 20f) usage, female gender and associated peripheral vascular disease are proven risk factors [44] . insertion of a distal perfusion cannula should be considered to augment perfusion [76] . differential hypoxia, north-south/harlequin syndrome, occurs in peripheral va-ecmo when the heart has recovered on the backdrop of a lung still lagging behind. the peripheral ecmo cannot compete with native co, which causes poorly oxygenated blood supply to the upper half of the body while the lower half of the body receives well-oxygenated blood from the circuit [44] . monitoring the patient's arterial saturation in the right upper limb helps in diagnosis. remedial measures include advancement of inferior vena caval cannula and delivery of oxygenated blood into the right atrium by venoarterial-venous ecmo/ hybrid circuit [77] . the incidence of pccs va-ecmo is 2.2% in paediatric cardiac surgery [78] . operative stress and residual lesions added on to a physiologically compromised heart in congenital heart disease can predispose to poor cardiac function post-operatively. there is no consensus on the timing to initiate ecmo in the paediatric population. the indications and contraindications of post-cardiotomy ecmo are listed in table 2 . oxygen extraction ratio (o 2 er) is the ratio between oxygen consumption (vo 2 ) and o 2 delivery (do 2 ). normal o 2 er is 1:5 or 20%, derived from scvo 2 . do 2 reduces in low perfusion states. up to a certain point, tissues maintain aerobic metabolism by extracting o 2 from blood, increasing the central venous extraction and o 2 er. at an o 2 er of 1:2, there is an imbalance between metabolic demand and aerobic metabolism thereby initiating anaerobic metabolism, producing lactate and metabolic acidosis. o 2 consumption in infants and children (5-7 ml/kg/min) is more in comparison to that in fig. 3 an excerpt of continuous eeg monitoring of the patient depicted in fig. 2 showing right hemispheric epileptic activity adults (3-4 ml/kg/min); hence, anaerobic metabolism and organ damage occur earlier [79] . ufh is the mainstay for anticoagulation in children on ecmo. as hit is rare in children [80] , the need for alternative anticoagulants seldom arises. elso guidelines [81] suggest a bolus dose of 50-100 units/kg of ufh before cannulation and an infusion of 10-40 units/kg/h for maintenance. besides act, aptt ratio, teg, antithrombin and activated factor xa levels are used for titration of anticoagulant. transthoracic echocardiography (tte), and catheterbased diagnostic studies aid in detection of postoperative residual lesions which, when addressed, help in weaning from ecmo [82] . the decision to vent the heart is based on tte findings. tte assists in documenting serial ventricular function improvement, identifying pericardial/ pleural effusions, assessing pulmonary hypertension and shunting at atrial or ventricular levels [83] . all inotropes are stopped when complete myocardial rest is indicated. map targets are achieved using vasoconstrictors and vasodilators. although there is limited experience, levosimendan (0.1-0.2 mcg/kg/min for 24 h) can be used during ecls to aid weaning [84] . map, capillary refill time, urine output, lactate levels and scvo 2 trends are reliable monitoring tools. a hct of 35% is targeted if scvo 2 > 75%. in situations of do 2 /vo 2 mismatch/ palliated single-ventricle patients, a higher hct (> 40%) is targetted. gentle ventilation to achieve 5-6 ml/kg tidal volume and a peep of up to 10 mmhg is preferred to avoid barotrauma [81] . ecmo circuitry triggers an inflammatory response and capillary leak, causing fluid to shift out of the intravascular compartment. in the backdrop of intravascular volume depletion secondary to bleeding, patients are predisposed to prerenal aki aggravating a pre-existing hypoxic kidney injury. once volume status is normalized within 48-72 h, the capillary leak subsides and diuresis improves [85] . treatment options for inadequate diuresis are diuretics, peritoneal dialysis (pd) and modified ultrafiltration on the circuit. rrt has not shown to improve mortality in children on ecmo [86] . bowel hypoperfusion secondary to low co, sedative and paralytic agent usage, vasoconstrictive drugs and gut inflammation can predispose to gastric dysmotility and feed intolerance in a child on ecmo [87] . regional gi ischaemia causes hyperlactaemia. withholding enteral feeds translocates gut bacteria thereby increasing the risk of sepsis. starting of trophic feeds once lactate has normalized is a recommended strategy [88] . parenteral nutrition is considered when there is persistent hyperlactataemia or feed intolerance taking on board the risk of infection [87] . severe irreversible brain injury extremely low gestational and weight (< 32 weeks gestation or < 1.5 kg) uncontrollable haemorrhage fig. 4 a-v bridging on ecmo consists of a circuit that runs parallel to the patient with the cannulae in situ but clamped. heparin flush is constantly circulated through the cannulae during the clamped interval to prevent clotting. if there is haemodynamic or respiratory instability, va-ecmo is recommenced by just removing the clamps on the venous and arterial cannulae the timing and sequence of weaning off ecmo is not standardized. once the myocardium shows signs of recovery, organ functions have improved and residual lesions are ruled out or corrected, full ventilatory support is commenced, inotropes started to augment cardiac contractility and ecmo flows are gradually reduced. the left-sided vent, if present, is removed when sustained ejections are seen. on further weaning to minimal ecmo flows (200 ml/min), tte is done. decision is then taken to decannulate the patient. in borderline cases, a-v bridging with a trial period off ecmo (1-4 h) is an option. this consists of a circuit that runs parallel to the patient with the cannulae in situ but clamped (fig. 4) . during a-v bridging, if there is haemodynamic or respiratory instability, va-ecmo is recommenced. if the trial period off ecmo is uneventful, the patient is decannulated [89] . tte plays a pivotal role during various stages of weaning and in the post-wean phase [83] . palliative surgery for univentricular hearts poses special challenges on ecmo. in infants with single ventricular physiology and systemic to pulmonary shunts, the shunt has to be partially occluded to counter pulmonary steal. the alternative is to target higher flows [90] . the success rate for ecls, post glenn and fontan procedures is lower. the reasons are multifactorial, including complex physiology and altered cardiac anatomy, the presence of atrioventricular valve regurgitation, previous surgeries, the need for multiple drainage cannulae and the inability to achieve full flows on ecmo [91] . success after ecmo typically is defined as survival to hospital discharge after a successful wean [26] . for pccs va-ecmo, the average survival rate across all age groups is 46.3% [70] . the indicators of poor outcome have been outlined as advanced age, redo-valve surgery and climbing lactate levels. pre-operative pulmonary hypertension had no bearing on mortality [92] . the 5-year survival rate was 56% for patients discharged from the hospital while 63% for patients surviving at 30 days [93] . discharge from hospital is the most important predictor of successful outcome [2] . lai et al. concluded that a small percentage of patients especially in a high-volume centre would benefit from additional ecmo therapy if their cardiopulmonary function declined. additionally, a higher incidence of infection and need for rrt was reported in this subset [93] . literature per se is nebulous about the quality-of-life predictors after ecmo. studies have shown 15% mortality within 3 months of hospital discharge and this increases to 24% over a 3-year period [94] . on the contrary, studies have depicted 15.7% survival at 18 months without elaborating on the quality of life [95] . the new york heart association (nyha) class is a good tool for ascertaining functional status in ecmo survivors [96] . functionality status of survivors have been diverse, ranging from nyha class i-iv indicating survivors can achieve a reasonable quality of life. chen et al. have reported a higher readmission rate during the first year of follow-up resulting in higher medical expenditure. infections and cardiac events were stated as common causes of mortality/morbidity in the follow-up period [97] . literature has extensively debated on the cost-effectiveness of ecmo. several analyses have deemed ecmo to be an expensive therapy amounting to an average of $92,600 per case in the usa and have recommended package payments to buoy individual institutes delivering this service [98] . in the uk, mechanical support is funded only when it has been advocated as a bridge to transplantation [99] . there has been no such cost analysis done in the indian subcontinent. the average cost of initiating ecmo in india is meagre compared to the west. however, additional costing gets added on to patient maintenance in the icu, which could include rrt, medication, imaging, laboratory and transfusion costs. by advocating a wholesome package module, the implications of cost can be negated with the outcome benefits. from an ethical viewpoint, three perspectives have to be considered: surgeons, the patient's family and financial implications. a surgeon would look forward to successful outcomes, but, when faced with a situation of inability to wean off cpb, it is normal to consider options for recovery even if chances of successful outcome is low. the patient's family should be briefed about the condition and given an opportunity to partake in the decision-making. financial implications of ecmo need be explained as healthcare in india is not funded. though the cost of initiation of ecmo is finite, duration and end-point are difficult to ascertain and costs are impractical to calculate. there are situations where hospitals or other payers like insurance will meet the additional expense, but that is the exception rather than the rule. these nuances should be discussed and documented to enable the family to make an informed decision. counselling and communication is key to supporting the family through this tumultuous phase. va-ecmo has made inroads into the management of pccs in india. bearing its cost implications, it has yet to make a significant impact in the management of these subset of patients. at narayana institute of cardiac sciences (nics), bangalore, india, we on an average perform about 110-115 ecmo runs a year, a majority of which are post-cardiotomy ecmo runs. considering the factors influencing cannulation, our institutional preference is the central route. if the patient presents with pccs, our cannulation sites are the right atrial appendage and the ascending aorta close to the sino-tubular junction. we have observed harlequin syndrome in those patients with high aortic cannulation, and these patients presented with persistent ventricular fibrillation and altered sensorium. this was due to deoxygenated blood from the lv selectively streaming into coronaries and the right innominate artery. this was promptly reversed by changing to a lower cannula position. cannulae are snugged using rubber spigots, following which they are tunnelled sub-xiphoid and chest closed with sternal wires. absolute haemostasis is imperative. a common bleeding point is the aortic cannulation site. for this, 2 rows of purse strings are used that encompass the cannula twice causing a cuff of adventitia to evert around the cannula to stop further bleeding. for lv venting, our strategy is to cannulate the lv apex directly with a separate limb to the inflow circuit (fig. 1) . we keep a dedicated flow sensor on this limb to detect low flow. an obvious disadvantage of this is formation of a lv clot around the cannula inflow. this is prevented by maintaining a higher aptt ratio and using a larger-sized cannula. the decision to vent the left heart is based upon the disappearance of ejections on the arterial trace, presence of spontaneous echo contrast on tee, inadequate flows with obvious signs of lv distention and to prevent harlequin syndrome in peripheral cannulation. we strongly believe in central cannulation for the following reasons: at our institute, the weaning process starts in the icu and the final stage happens in the or under tee guidance. the patient is primed with levosimendan overnight without a loading dose. the ventilator is adjusted for optimal gas exchange. haemodynamic variables are monitored using echocardiography and a pulmonary artery catheter. metabolic stability is monitored by trends in lactate level, base excess in arterial gases and scvo 2 monitoring. anticoagulation is maintained with aptt 45-60 s or act 200-220 s. flows are reduced by 0.5 l/min every 2nd hourly till a flow of 30% is achieved. if a lv vent is in situ, a gated clamp regulates the lv vent flows. flows are reduced in aliquots of 200 ml every second hour till a flow of 500 ml is reached. the patient is idled at this flow till transfer to the or for final wean and decannulation. weaning is aborted at any stage if there is ventricular distension, increase in inotropic levels to maintain haemodynamics, worsening gas exchange or metabolic parameters. we performed a total of 15,480 cardiac surgeries in 2018 and 2019. pccs va-ecmo was instituted in 165 (1.06%) of these patients. our results are broadly outlined in table 3 . bleeding was a significant problem. major transfusions were needed in the initial 24-h period. the transfusion requirements were monitored with 4th hourly clotting screens and teg. the incidence of aki with rrt requirements were comparable across age groups which is similar to published data [65] . our preference for instituting rrt is using a parallel circuit to prevent possible air embolism. rrt in the paediatric patients was provided by pd. a rising trend of total bilirubin was associated with failure to wean and mortality. overall mortality of ecmo patients with limb ischaemia was 87.5%. literature has shown higher incidence of neurological [67] [68] [69] in contrast to our data. the incidence of gi bleed in our cohort was 2.42% which compares favourably with published data [52, 54] . better weaning results were observed between 2018 and 2019 (table 4) a review of the bloodstream infections in patients on pccs va-ecmo revealed a predominance in gramnegative bacilli (gnb) (85%), followed by candida species (10%) and gram-positive organisms (5%). klebsiella species constituted the majority of the gnb bacteraemias. carbapenem resistance was present in 44% of the gramnegative organisms isolated (65% carbapenem resistance in klebsiella species alone). most of the blood culture isolates grew within the first 72 h of incubation. however, 13% of the organisms grew only after 5 days of incubation, highlighting the importance of incubating the blood culture for a minimum of 7 days and monitoring them for growth. pccs va-ecmo has achieved a 'niche' in cardiac surgery as a bridge to decision/recovery. although an expensive and demanding therapy, it is versatile and can save lives if incorporated in a timely fashion. the positive outcomes are driven by the experience of the team handling the case. over time, pccs va-ecmo has achieved a 'standard of care' status. since resource utilization for this therapy is high, a scoring system is warranted to ascertain which subset of patients would benefit and have a positive outcome. venoarterial extracorporeal membrane oxygenation for postcardiotomy shock: risk factors for mortality long-term survival and major outcomes in post-cardiotomy extracorporeal membrane oxygenation for adult patients in cardiogenic shock evolving concepts in diagnosis and management of cardiogenic shock structured review of postcardiotomy extracorporeal membrane oxygenation: part 1-adult patients extracorporeal membrane oxygenation for the treatment of postcardiotomy shock contemporary mechanical circulatory support therapy for postcardiotomy shock postcardiotomy mechanical support: risk factors and outcomes peripheral versus central extracorporeal membrane oxygenation for postcardiotomy shock: multicenter registry, systematic review, and meta-analysis percutaneous cannulation for extracorporeal life support outcomes after peripheral extracorporeal membrane oxygenation therapy for postcardiotomy cardiogenic shock: a single-center experience left ventricular unloading during veno-arterial ecmo: a review of percutaneous and surgical unloading interventions echocardiography for adult patients supported with extracorporeal membrane oxygenation monitoring of the adult patient on venoarterial extracorporeal membrane oxygenation clinical management of venoarterial extracorporeal membrane oxygenation extracorporeal life support: the elso red book. ann arbor: extracorporeal life support organization hypothermia: effects on platelet function and hemostasis cerebral oxygenation as a monitoring parameter for mortality during venoarterial extracorporeal membrane oxygenation awake and fully mobile patients on cardiac extracorporeal life support coagulation monitoring in postcardiotomy ecmo: conventional tests, point-of-care, or both? minerva anestesiol the early dynamic behavior of lactate is linked to mortality in postcardiotomy patients with extracorporeal membrane oxygenation support: a retrospective observational study von willebrand factor, free hemoglobin and thrombosis in ecmo when to discontinue extracorporeal membrane oxygenation for postcardiotomy support duration of extracorporeal membrane oxygenation support and survival in cardiovascular surgery patients how to wean a patient from veno-arterial extracorporeal membrane oxygenation extracorporeal membrane oxygenation management: techniques to liberate from extracorporeal membrane oxygenation and manage post-intensive care unit issues weaning from veno-arterial extra-corporeal membrane oxygenation: which strategy to use? weaning of extracorporeal membrane oxygenation using continuous hemodynamic transesophageal echocardiography usefulness of cardiac biomarkers to predict cardiac recovery in patients on extracorporeal membrane oxygenation support for refractory cardiogenic shock venoarterial extracorporeal membrane oxygenation in cardiogenic shock pump controlled retrograde trial off from va-ecmo levosimendan may improve weaning outcomes in venoarterial ecmo patients pharmacologic approaches to weaning from cardiopulmonary bypass and extracorporeal membrane oxygenation functional evaluation of sublingual microcirculation indicates successful weaning from va-ecmo in cardiogenic shock clinical pearls of venoarterial extracorporeal membrane oxygenation for cardiogenic shock extracorporeal membrane oxygenation, an anesthesiologist's perspective: physiology and principles. part 1 prolonged nonpulsatile left heart bypass diminishes vascular contractility pulmonary complications associated with veno-arterial extra-corporeal membrane oxygenation: a comprehensive review awake" extracorporeal membrane oxygenation (ecmo): pathophysiology, technical considerations, and clinical pioneering flow mixing during peripheral veno-arterial extra corporeal membrane oxygenation -a simulation study physiology of nonpulsatile circulation: acute versus chronic support chronic nonpulsatile blood flow is compatible with normal end-organ function: implications for lvad development clinical management of the extracorporeal membrane oxygenation circuit sources of circuit thrombosis in pediatric extracorporeal membrane oxygenation clinical complications during veno-arterial extracorporeal membrane oxigenation in post-cardiotomy and non post-cardiotomy shock: still the achilles' heel extracorporeal life support organization registry international report 2016 complications of extracorporeal membrane oxygenation for treatment of cardiogenic shock and cardiac arrest: a meta-analysis of 1,866 adult patients anticoagulation and transfusions management in veno-venous extracorporeal membrane oxygenation for acute respiratory distress syndrome: assessment of factors associated with transfusion requirements and mortality recent advances in heparin-induced thrombocytopenia heparin-induced thrombocytopenia during extracorporeal life support: incidence, management and outcomes eacts/eacta guidelines on patient blood management for adult cardiac surgery the role of endoscopy as non-invasive procedure to manage gastrointestinal complications during extracorporeal membrane oxygenation gastrointestinal bleeding in ecmo patients: incidence and outcomes bleeding, transfusion, and mortality on extracorporeal life support: ecls working group on thrombosis and hemostasis efficacy and safety of stress ulcer prophylaxis in critically ill patients: a network metaanalysis of randomized trials impact of dynamic changes of elevated bilirubin on survival in patients on veno-arterial extracorporeal life support for acute circulatory failure current concepts in ischemic hepatitis liver function predicts survival in patients undergoing extracorporeal membrane oxygenation following cardiovascular surgery high serum bilirubin levels, nt-pro-bnp, and lactate predict mortality in long-term, severely ill respiratory ecmo patients extracorporeal life support as a bridge to heart transplantation: importance of organ failure in recipient selection baseline meld-xi score and outcome from veno-arterial extracorporeal membrane oxygenation support for acute decompensated heart failure impact of acute liver failure on mortality during adult ecls five years' experience with a peripheral veno-arterial ecmo for mechanical bridge to heart transplantation extracorporeal membrane oxygenation with multiple-organ failure: can molecular adsorbent recirculating system therapy improve survival? continuous renal replacement therapy during extracorporeal membrane oxygenation: why, when and how? effects of renal replacement therapy in patients receiving extracorporeal membrane oxygenation: a meta-analysis recovery of renal function and survival after continuous renal replacement therapy during extracorporeal membrane oxygenation ischemic and hemorrhagic brain injury during venoarterial-extracorporeal membrane oxygenation acute neurologic complications during extracorporeal membrane oxygenation: a systematic review neurologic complications in neonates supported with extracorporeal membrane oxygenation. an analysis of elso registry data infections acquired during extracorporeal membrane oxygenation in neonates, children, and adults acquired infection during neonatal and pediatric extracorporeal membrane oxygenation nosocomial infections in adult cardiogenic shock patients supported by venoarterial extracorporeal membrane oxygenation epidemiology of infectious complications during extracorporeal membrane oxygenation in children: a single-center experience in 46 runs arterial complications in patients undergoing extracorporeal membrane oxygenation via femoral cannulation thromboembolic events in patients on extracorporeal membrane oxygenation without anticoagulation hypoxia and complications of oxygenation in extracorporeal membrane oxygenation perioperative mechanical circulatory support in children: an analysis of the society of thoracic surgeons congenital heart surgery database physiology of gas exchange during ecmo for respiratory failure heparin-induced thrombocytopenia in paediatrics: clinical characteristics, therapy and outcomes pediatric cardiac failure, extracorporeal life support organization outcomes of pediatric patients undergoing cardiac catheterization while on extracorporeal membrane oxygenation the role of echocardiography in neonates and pediatric patients on extracorporeal membrane oxygenation cardiopulmonary resuscitation in infants and children with cardiac disease: a scientific statement from the the complex relationship of extracorporeal membrane oxygenation and acute kidney injury: causation or association? outcome of pediatric patients treated with extracorporeal life support after cardiac surgery nutritional practices and adequacy in children supported on extracorporeal membrane oxygenation guidelines for the provision and assessment of nutrition support therapy in the pediatric critically ill patient: society of critical care medicine and american society for parenteral and enteral nutrition a comparison of pumpcontrolled retrograde trial off to arterio-venous bridging for weaning from venoarterial extracorporeal membrane oxygenation extracorporeal membrane oxygenation for infant postcardiotomy support: significance of shunt management fontan physiology revisited clinical outcomes in patients after extracorporeal membrane oxygenation support for postcardiotomy cardiogenic shock: a single-centre experience of 92 cases outcomes of venoarterial extracorporeal membrane oxygenation patients requiring multiple episodes of support longterm survival in adults treated with extracorporeal membrane oxygenation for respiratory failure and sepsis long-term prognosis after extracorporeal life support in refractory cardiogenic shock -results from a real-world cohort patients' self-assessed functional status in heart failure by new york heart association class: a prognostic predictor of hospitalizations, quality of life and death long-term outcomes of extracorporeal membrane oxygenation support for postcardiotomy shock financial and clinical outcomes of extracorporeal mechanical support the comparative use of ventricular assist devices: differences between europe and the united states publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations acknowledgements we would like to acknowledge the help and insights provided by dr. julius punnen, dr. varun shetty, dr. rammohan sripad bhat, dr. vijay samuel richard, dr. rangarajan kasturi, dr. radhika manohar, dr. shrinivas hittalamani and dr. anup daniel varghese who are specialists in their fields in writing this document. we would also like to mention the assistance provided by mr. selvakumar, senior key: cord-017566-dvxrwzqw authors: gonzález, maría eugenia; carrasco, luis title: viral proteins that enhance membrane permeability date: 2005 journal: viral membrane proteins: structure, function, and drug design doi: 10.1007/0-387-28146-0_6 sha: doc_id: 17566 cord_uid: dvxrwzqw nan during the infection of cells by animal viruses, membrane permeability is modified at two different steps of the virus life cycle (carrasco, 1995) (figure 6 .1). initially, when the virion enters cells, a number of different-sized molecules are able to co-enter the cytoplasm with the virus particles (fernandez-puentes and otero and carrasco, 1987) . membrane potential is reversibly destroyed, being restored several minutes later. endosomes are involved in the co-entry process, since inhibitors of the proton atpase block early permeabilization even with viruses that do not require endosomal function. a chemiosmotic model has been advanced to explain the molecular basis of early membrane modification by virus particles (carrasco, 1994) . the viral molecules involved are components of virions: glycoproteins when enveloped particles are analyzed or, still unidentified, domains of the structural proteins in the case of naked viruses. attachment of the particle to the cell surface receptor does not alter membrane permeability by itself. inhibitors that hamper virus decapsidation, still allowing virus attachment to the cell surface, block early membrane permeabilization (almela et al., 1991) . at late times of infection, when there is active translation of late viral mrnas, the plasma membrane becomes permeable to small molecules and ions (carrasco, 1978) ( figure 6 .1). different viral molecules may be responsible for this late enhancement of membrane permeability, including viroporins (gonzalez and carrasco, 2003) , glycoproteins, and even proteases (chang et al., 1999; blanco et al., 2003) . this chapter is devoted to reviewing some characteristics of membrane permeabilization by viral proteins. in addition, the methodology used to assay enhanced permeability in animal cells is described. finally, the design of selective viral inhibitors based on the modification of cellular membranes during virus entry or at late times of infection is also discussed. a number of hydrophilic molecules, including some antibiotics, poorly permeate through cellular membranes (contreras et al., 1978; lacal et al., 1980) . this is the case of hygromycin b, anthelmycin, blasticidin s, destomycin a, gougerotin, and edein complex. the aminoglycoside antibiotic hygromycin b (mw 527) is produced by streptomyces hygroscopicus. this hydrophilic molecule is an efficient inhibitor of protein synthesis in cell-free systems but interferes very poorly with translation in intact cells. however, the modification of the plasma membrane by viruses or by other means leads to a rapid blockade of translation (carrasco, 1995) . concentrations of the antibiotic ranging from about 0.1-1 mm are added to the culture medium and protein synthesis is estimated by incubation with radioactive methionine for 1 hr (see figure 6 .2) (gonzalez and carrasco, 2001) . in addition to its simplicity, the hygromycin b test has a number of advantages for assaying changes in membrane permeability. one is its great sensitivity, and another is that this test measures membrane modifications only in cells that are metabolically active. moreover, in cultures where some cells are uninfected, hygromycin b would only enter virus-infected cells that are synthesizing proteins. the hygromycin b test has been applied with success to prokaryotic (lama and carrasco, 1992) and eukaryotic cells, including yeast carrasco, 1995, 1998) and mammalian cells (gatti et al., 1998; gonzalez and carrasco, 2001) . alpha-sarcin is unable to pass through these pores. alpha-sarcin is a protein of 150 amino acid residues, which is produced by aspergillus giganteus (oka et al., 1990) . this protein inhibits translation by modifying ribosomes in an enzymatic manner. thus, a molecule of alpha-sarcin is able to inactivate a great number of ribosomes by hydrolysis of the a4324-g4325 phosphodiester bond in the 28s rrna (chan et al., 1983) . this toxin does not enter mammalian cells because it does not attach to the cell surface and is therefore unable to cross the plasma membrane. however, alpha-sarcin efficiently interferes with protein synthesis in cell-free systems or in cells where the permeability barrier has been destroyed (fernandez-puentes and . alpha-sarcin co-enters cells in conjunction with virus particles, and is liberated to the cytoplasm (otero and carrasco, 1987; liprandi et al., 1997) . in this manner, this toxin irreversibly blocks translation several minutes after virus entry. the molecular basis of the co-entry of macromolecules with virus particles has been analyzed in detail elsewhere (carrasco, 1994 (carrasco, , 1995 . apart from alpha-sarcin, a number of proteins that interfere with translation, many of them of plant origin, have been described (fernandez-puentes and carrasco, 1980; lee et al., 1990) . the release of all these toxins into cells is enhanced by virus particles. not only proteins, but also other macromolecules, including nucleic acids efficiently co-enter with virus particles (cotten et al., 1992) . however, none of these macromolecules passes into cells at late times of infection. apart from the use of translation inhibitors that do not easily permeate into intact cells, a number of assays can be employed to assess modifications in membrane permeability. amongst these assays, we can list the following. cells are preloaded with radioactive uridine and the exit of nucleotides can be monitored after induction of viroporin expression (gonzalez and carrasco, 1998) . unlike with most amino acids, the pool of uridine nucleotides is abundant in the cell interior, thus providing a convenient and sensitive assay for monitoring the exit of molecules from cells. other tests use radioactive glucose derivatives (e.g., 2-deoxyglucose), which cannot be metabolized and accumulate in cells. the analysis of the release of radioactive compounds that are not actively transported into cells leads to the failure to measure enhanced membrane permeability. entry of o-nitrophenyl-␤-d-galactopyranoside (onpg) into the bacterial cells can be determined very simply. this ␤-galactosidase substrate can be incubated with bacterial cells and production of the resulting compound can be followed by determining the absorbance at 420 nm (lama and carrasco, 1992) . there are a number of non-vital dyes employed to characterize cell mortality. it should be noted that the entry of these compounds, in fact, determines a modification in cell membranes, which in some cases does not directly correlate with cell death. trypan blue is a dye widely used for monitoring enhanced membrane permeability. however, this assay is not very sensitive. in addition, trypan blue staining does not discriminate between metabolically active or dead cells, as the hygromycin b tests does. another assay employs the polyamine neurobiotin that needs specific connexin channels to enter mammalian cells (elfgang et al., 1995) . permeabilization of cell membrane increases uptake of this cationic molecule. internalized neurobiotin can be detected in paraformaldehyde-fixed cells, by fluorescence microscopy, using fluorescein isothiocyanate-conjugated streptavidin (gonzalez and carrasco, 1998) . propidium iodide (pi) is a dna-intercalating compound that does not enter intact cells. however, those cells that exhibit increased membrane permeability are able to take up pi, which can be assayed by cell fluorometric analysis (arroyo et al., 1995) . the appearance in the culture medium of cellular enzymes is a clear indicator of cell mortality. this is the case for lactic dehydrogenase and bacterial ␤-galactosidase, present outside the cells (sanderson et al., 1996) . commercial kits to measure this enzymatic activity are available. the release of cellular proteins to the medium occurs at very late times of viral infection, when cells have already died. this alteration takes place at much later times after hygromycin b entry can be detected (blanco et al., 2003) . viroporins are small proteins encoded by viruses that contain a stretch of hydrophobic amino acids (gonzalez and carrasco, 2003) . typically, viroporins are comprised of some 60-120 amino acids. the hydrophobic domain is able to form an amphipathic ␣-helix. the insertion of these proteins into membranes followed by their oligomerization creates a hydrophilic pore. the architecture of this channel is such that the hydrophobic amino acid residues face the phospholipid bilayer while the hydrophilic residues form part of the pore. in addition to this domain, there are other features of viroporin structure, including a second hydrophobic region in some viroporins that also interacts with membranes. this second interaction may further disturb the organization of the lipid bilayer. these proteins may also contain a stretch of basic amino acids that acts in a detergent-like fashion. all these structural features contribute to membrane destabilization. more recently, another domain has been described in some glycoproteins and also viroporins that has the capacity to interact with membranes. this domain is rich in aromatic amino acids and is usually inserted at the interface of the phospholipid bilayer (suarez et al., 2000; sanz et al., 2003) . this type of interaction also leads to membrane destabilization, further enhancing membrane permeability. a number of viroporins from different viruses that infect eukaryotic cells have been reported. this group of proteins includes picornavirus 2b and 3a, alphavirus 6k, retrovirus vpu, paramyxovirus sh, orthomyxovirus m2, reovirus p10, flavivirus p7, phycodnavirus kcv, coronavirus e, and rhabdovirus alpha 10p. a recent review devoted to viroporins discusses the structure and function of a number of proteins of this group (gonzalez and carrasco, 2003) , and so the details of each particular viroporin will not be reviewed in this chapter. the main activity of viroporins is to create pores at biological membranes to permit the passage of ions and small molecules. the cloning and individual expression of viroporin genes has allowed their effects in bacterial and animal cells to be analyzed. thus, the expression of this type of viral gene enhances the permeation of ions and several hydrophilic molecules in or out of cells (carrasco, 1995) . in addition, the purified viroporin molecules open pores in model membranes, providing a system that is amenable to biophysical analysis (fischer and sansom, 2002) . the pore size created by viroporins allows the diffusion of different molecules with a molecular weight below about 1,000 da. the main step affected in animal viruses containing a deleted viroporin gene is the assembly and exit of virions from the infected cells (klimkait et al., 1990; liljestrom et al., 1991; loewy et al., 1995; betakova et al., 2000; watanabe et al., 2001; kuo and masters, 2003) . these genes are not essential for virus replication in culture cells, but the plaque size is much smaller in viroporin-defective viruses. notably, virus entry and gene expression in viroporin-deleted viruses occur as in their wild-type counterparts. an aspect of viroporin function at the molecular level that is still not understood is the link between pore activity and virus budding. in addition to small hydrophobic viral proteins, there are other virus products that promote membrane permeabilization. this occurs with a number of virus glycoproteins (gp) that are known to increase cell membrane permeability, such as the human immunodeficiency virus gp41 (chernomordik et al., 1994; arroyo et al., 1995) , the ebola virus gp (yang et al., 2000) , the cytomegalovirus us9 protein (maidji et al., 1996) , the vaccinia virus a38l protein (sanderson et al., 1996) , rotavirus vp7 and ns4 proteins (charpilienne et al., 1997; newton et al., 1997) , the hepatitis c virus e1 protein (ciccaglione et al., 1998) , and the alphavirus e1 protein (nyfeler et al., 2001; wengler et al., 2003) . the architecture of some viral glycoproteins is such that upon oligomerization, the transmembrane (tm) domains may form a physical pore. in principle, two different regions of a viral fusion glycoprotein could form pores. one such region contains the fusion peptide that would create a pore in the cell membranes upon insertion (skehel and wiley, 1998) , while the tm domain would form a pore in the virion membrane (wild et al., 1994) . moreover, sequences adjacent to the tm region could have motifs designed to destabilize membrane structure (suarez et al., 2000) . entry of enveloped animal viruses leads to early membrane permeabilization, which is mediated by the formation of the two pores (fusion and tm) formed by viral fusion glycoproteins. this early permeabilization induced during the entry of virions requires conformational changes of the fusion glycoproteins. by contrast, after virus replication, newly synthesized glycoproteins may affect membrane permeability when they reach the plasma membrane (figure 6 .3). this modification is achieved only by the tm domain, while the fusion peptide does not participate in this late modification. in viruses that lack the typical viroporin, its function could be replaced by these pore-forming glycoproteins, while for other viruses viroporin activity may be redundant (bour and strebel, 1996) . in the latter case, pore formation may be generated by viral glycoproteins and viroporins (figure 6 .3). we would like to propose the possibility that pore-forming glycoproteins play a key role mainly during virus entry and, in some cases, also during virus budding, while viroporins come into action when viruses need to exit the cell. early membrane permeabilization is always carried out by a virion component. in the case of enveloped viruses, this early event is executed by a structural glycoprotein, which is coupled to the fusion process. an understanding of fusion at the molecular level also requires an explanation of the phenomenon of early membrane permeabilization. we have advanced the idea that viral glycoproteins involved in membrane fusion participate in the dissipation of the chemiosmotic gradient, thus providing the energy to push the nucleocapsid and neighboring macromolecules to the cell interior (carrasco, 1994; irurzun et al., 1997) . fusion glycoproteins do not simply serve to bridge the cellular and the viral membrane, but instead are designed to open pores in both membranes. this pore-opening activity may be necessary to lower membrane potential and to dissipate ionic gradients. several chapters of this book are devoted to the detailed description of the structure and function of these glycoproteins, so we will focus our attention on viral glycoproteins that permeabilize membranes when individually expressed in cells. these membrane active proteins may exhibit this activity later on in the virus life cycle. rotavirus infection provokes a number of alterations in cellular membranes during infection . amongst these alterations, there is an increase in the concentration of cytoplasmic calcium . several rotavirus proteins exhibit membrane-destabilizing activity. the enterotoxin nsp4 induces alterations in membrane permeability (tian et al., 1994) . the individual expression of the non-structural glycoprotein nsp4 has the ability to increase the concentration of cytoplasmic calcium. this increase may be mediated by activation of phospholipase c activity (dong et al., 1997) . rotavirus particles induce the co-entry of protein toxins into cells (cuadras et al., 1997) . at least two structural components possess the ability to permeabilize cells, including vp5 protein and vp7 glycoprotein (charpilienne et al., 1997; irurzun et al., 1997) . infection of lymphocytic human cells by hiv-1 enhances membrane permeability to ions and several compounds (voss et al., 1996; gatti et al., 1998) . there are at least three different hiv-encoded proteins responsible for these alterations: vpu protein, the retroviral protease, and the fusion glycoprotein gp41. apart from the fusion peptide, there are two regions of gp41 that exhibit membrane permeability; one is located at the carboxy terminus (arroyo et al., 1995; comardelle et al., 1997) and another corresponds to the membranespanning domain (arroyo et al., 1995) . the c-terminus of gp41 includes two 20-30 residues, which may form cationic amphipathic ␣-helices, designated as lentivirus lytic peptides 1 and 2 (llp-1 and llp-2). synthetic llp-1 peptide forms pores in planar phospholipid bilayers late early figure 6 .3. participation of pore formation by viral glycoproteins and viroporins in membrane permeability. early membrane permeabilization is coupled to the fusion activity of the corresponding viral glycoprotein. this fusion glycoprotein may create two pores. one is located at the target cell membrane and the other is formed by the tm domain. late membrane permeabilization may be carried out by viroporins or by the tm domains of viral glycoproteins. (chernomordik et al., 1994) , permeabilizes hiv-1 virions to deoxyribonucleoside triphosphates (zhang et al., 1996) , and induces alterations in ion permeability of xenopus oocytes (comardelle et al., 1997) . inducible expression of the hepatitis c virus e1 glycoprotein increases membrane permeability in bacterial cells. the ability of e1 to modify membrane permeability has been mapped to the carboxy terminus of the protein (ciccaglione et al., 1998 (ciccaglione et al., , 2001 . similar permeabilization was found with escherichia coli cells that synthesize semliki forest virus e1 glycoprotein after exposure to low ph (nyfeler et al., 2001) . finally, overexpression of vaccinia virus a38l glycoprotein produces changes in the morphology, permeability, and adhesion of mammalian cells. the potential capacity of a38l protein to form pores at the plasma membrane promotes the entry of calcium ions and pi and the release of lactic dehydrogenase into the culture medium (sanderson et al., 1996) . different approaches have been envisaged for the design of compounds that interfere with virus replication based on modifications in membrane permeability. one such approach makes use of inhibitors of cellular or viral functions that do not permeate easily into intact animal cells. notably, these agents selectively enter into virus-infected cells (carrasco, 1978; benedetto et al., 1980) . most of the inhibitors used thus far interfere with protein synthesis, although compounds that affect other functions could also be employed. entry of these agents leads to the inhibition of translation specifically in virus-infected cells, leading to a profound inhibition of virus growth (contreras et al., 1978; carrasco, 1995; gatti et al., 1998) . although this approach discriminates well between uninfected or virus-infected cells in culture, the high toxicity of the agents thus far assayed has hampered its use in whole animals. perhaps future searches for less toxic compounds would make this approach amenable to application in therapy. in fact, some of the plant toxins that co-enter with virus particles have been described as being antiviral agents (lee et al., 1990) . even compounds such as hygromycin b, which has been used in the veterinary field as an antibacterial agent, could also be used as an antiviral compound for rotavirus infections (liprandi et al., 1997) . the paradigm of an inhibitor of a viral ion channel is amantadine (hay, 1992) (figure 6.4) . this compound has been used as an anti-influenza agent in humans (de clercq, 2001) . the target of amantadine is the influenza-encoded protein m2. residues 27, 30, 31, and 34 of m2 determine the amantadine sensitivity of this ion channel. a drawback of amantadine is the high doses necessary to affect influenza. the search for more effective compounds may provide a more efficacious treatment for this illness. compounds that interfere with the functioning of other viroporins have also been described. this is the case of amiloride derivatives that block hiv-1 vpu activity (ewart et al., 2002) . in this manner, the production of infectious hiv-1 is reduced in the presence of this agent. recently, long alkyl-chain iminosugar derivatives have been found to interfere with the function of the hepatitis c virus p7 protein as an ion channel (griffin et al., 2003; pavlovic et al., 2003) . these compounds exhibited antiviral activity with bovine viral diarrhea virus, which is closely related to the hepatitis c virus (durantel et al., 2001) . much effort has been concentrated recently on the development of antiviral agents that inhibit the fusion step of hiv. binding of hiv gp120 to the cd4 receptor is followed by further interaction of this viral glycoprotein with the coreceptor molecules cxcr4 and ccr5. after this initial interaction, the conformation of the ectodomain of the tm glycoprotein gp41 is profoundly modified. exposure of the fusion peptide at the amino terminus of gp41 triggers its insertion into the target cellular membrane, leading to the fusion of the viral and the cellular plasma membranes. all these steps have been used as targets for anti-hiv therapy (cooley and lewin, 2003) . as regards the fusion step, a variety of peptide mimetic inhibitors have been developed. the pioneering work on peptide t20 has demonstrated that this compound is a potent inhibitor of gp41-induced membrane fusion. t20 exhibits antiviral activity in hiv-infected patients. the detailed mechanism of action of t20 at the molecular level is known. this peptide is homologous with 36 amino acids within the c-terminal heptad repeat region (hr2) of hiv-1 gp41. t20 competitively binds to hr1 and interferes with the formation of the six helix hr1-hr2 bundle complex necessary for membrane fusion. at present there are a great number of peptides that interfere with binding of gp120 or with gp41-induced membrane fusion and that have been tested for their anti-hiv activity and clinical efficacy. in this regard, t1249 is one of the second generation of hr-2 peptide mimetic inhibitors that consists of 39 amino acids. pro 542 is a soluble cd4 receptor (cd4-igg2) that binds to and neutralizes gp120 before virus binding occurs. sch-c is an oxime-piperidine compound that is a coreceptor antagonist. this small molecule acts as an inhibitor of ccr5. md3100 is a non-peptidic, low molecular weight bicyclam compound that prevents interactions between cxcr4 and gp120, blocking signal transduction from cxcr4. future research in this field will provide us with additional antiviral compounds to add to the anti-hiv armory. inhibitors of poliovirus uncoating efficiently block the early membrane permeabilization induced by virus particles membrane permeabilization by different regions of the human immunodeficiency virus type 1 transmembrane glycoprotein gp41 a human virus protein, poliovirus protein 2bc, induces membrane proliferation and blocks the exocytic pathway in the yeast saccharomyces cerevisiae identification of regions of poliovirus 2bc protein that are involved in cytotoxicity inhibition of animal virus production by means of translation inhibitors unable to penetrate normal cells the vaccinia virus a14.5l gene encodes a hydrophobic 53-amino-acid virion membrane protein that enhances virulence in mice and is conserved among vertebrate poxviruses cell killing by hiv-1 protease the human immunodeficiency virus (hiv) type 2 envelope protein is a functional complement to hiv type 1 vpu that enhances particle release of heterologous retroviruses membrane leakiness after viral infection and a new approach to the development of antiviral agents entry of animal viruses and macromolecules into cells modification of membrane permeability by animal viruses the sequence of the nucleotides at the alpha-sarcin cleavage site in rat 28s ribosomal ribonucleic acid membrane permeabilization by small hydrophobic nonstructural proteins of japanese encephalitis virus solubilized and cleaved vp7, the outer glycoprotein of rotavirus, induces permeabilization of cell membrane vesicles an amphipathic peptide from the c-terminal region of the human immunodeficiency virus envelope glycoprotein causes pore formation in membranes mutagenesis of hepatitis c virus e1 protein affects its membrane-permeabilizing activity hepatitis c virus e1 protein induces modification of membrane permeability in e. coli cells a synthetic peptide corresponding to the carboxy terminus of human immunodeficiency virus type 1 transmembrane glycoprotein induces alterations in the ionic permeability of xenopus laevis oocytes inhibition, by selected antibiotics, of protein synthesis in cells growing in tissue cultures hiv-1 cell entry and advances in viral entry inhibitor therapy high-efficiency receptormediated delivery of small and large (48 kilobase) gene constructs using the endosome-disruption activity of defective or chemically inactivated adenovirus particles rotaviruses induce an early membrane permeabilization of ma104 cells and do not require a low intracellular ca 2 ϩ concentration to initiate their replication cycle antiviral drugs: current state of the art rotavirus infection alters na ϩ and k ϩ homeostasis in ma-104 cells the rotavirus enterotoxin nsp4 mobilizes intracellular calcium in human intestinal cells by stimulating phospholipase c-mediated inositol 1,4,5-trisphosphate production study of the mechanism of antiviral action of iminosugar derivatives against bovine viral diarrhea virus specific permeability and selective formation of gap junction channels in connexin-transfected hela cells amiloride derivatives block ion channel activity and enhancement of virus-like particle budding caused by hiv-1 protein vpu viral infection permeabilizes mammalian cells to protein toxins viral ion channels: sructure and function inhibition of hiv type 1 production by hygromycin b the human immunodeficiency virus type 1 vpu protein enhances membrane permeability human immunodeficiency virus type 1 vpu protein affects sindbis virus glycoprotein processing and enhances membrane permeabilization the p7 protein of hepatitis c virus forms an ion channel that is blocked by the antiviral drug the action of adamantanamines against influenza a viruses: inhibition of the m2 ion channel protein entry of semliki forest virus into cells: effects of concanamycin a and nigericin on viral membrane fusion and infection the human immunodeficiency virus type 1-specific protein vpu is required for efficient virus maturation and release the small envelope protein e is not essential for murine coronavirus replication antibiotics that specifically block translation in virus-infected cells expression of poliovirus nonstructural proteins in escherichia coli cells. modification of membrane permeability induced by 2b and 3a poliovirus-mediated entry of pokeweed antiviral protein in vitro mutagenesis of a full-length cdna clone of semliki forest virus: the small 6,000-molecular-weight membrane protein modulates virus release productive penetration of rotavirus in cultured cells induces co-entry of the translation inhibitor alpha-sarcin the 6-kilodalton membrane protein of semliki forest virus is involved in the budding process accessory human cytomegalovirus glycoprotein us9 in the unique short component of the viral genome promotes cell-to-cell transmission of virus in polarized epithelial cells effect of rotavirus infection on intracellular calcium homeostasis in cultured cells rotavirus nonstructural glycoprotein nsp4 alters plasma membrane permeability in mammalian cells expression of semliki forest virus e1 protein in escherichia coli. low ph-induced pore formation complete nucleotide sequence of cdna for the cytotoxin alpha sarcin proteins are cointernalized with virion particles during early infection the hepatitis c virus p7 protein forms an ion channel that is inhibited by long-alkyl-chain iminosugar derivatives overexpression of the vaccinia virus a38l integral membrane protein promotes ca 2 ϩ influx into infected cells interfacial domains in sindbis virus 6k protein. detection and functional characterization coiled coils in both intracellular vesicle and viral membrane fusion membrane interface-interacting sequences within the ectodomain of the human immunodeficiency virus type 1 envelope glycoprotein: putative role during viral fusion the nonstructural glycoprotein of rotavirus affects intracellular calcium levels alteration of intracellular potassium and sodium concentrations correlates with induction of cytopathic effects by human immunodeficiency virus influenza a virus can undergo multiple cycles of replication without m2 ion channel activity entry of alphaviruses at the plasma membrane converts the viral surface proteins into an ion-permeable pore that can be detected by electrophysiological analyses of whole-cell membrane currents peptides corresponding to a predictive alpha-helical domain of human immunodeficiency virus type 1 gp41 are potent inhibitors of virus infection identification of the ebola virus glycoprotein as the main viral determinant of vascular cell cytotoxicity and injury amphipathic domains in the c terminus of the tm protein (gp41) permeabilize hiv-1 virions: a molecular mechanism underlying natural endogenous reverse transcription this work was supported by grants from the fundación para la investigación y prevención del sida en españa (24291), instituto de salud carlos iii (01/0042), and the dgicyt pm99-0002. the authors also acknowledge the fundación ramón areces for an institutional grant awarded to the centro de biología molecular "severo ochoa." key: cord-016938-pk76snuy authors: suh, hwal (matthew) title: collagen fabrication for the cell-based implants in regenerative medicine date: 2008 journal: bioengineering in cell and tissue research doi: 10.1007/978-3-540-75409-1_8 sha: doc_id: 16938 cord_uid: pk76snuy though transplantation of cells, tissue or organ has been regarded as an ideal approach, scarcity of donor is a practical barrier in clinics. current progresses in cell engineering has opened a new era, providing tools for host-regeneration by implanting manipulated cells in forms of cell therapy, which includes delivery of single cells or multicellular structural support of hybridized cells, as a representative individualized treatment method. this chapter mainly concerns on the cellbased implant made of cells and collagen, the main structural protein in extracellular matrix in mammalian tissue, as it has been regarded as a promising method for manufacturing a biologically mimicked artificial tissues. regenerative medicine is to repair, replace and/or modify the disordered or damaged human body, either from a disease and/or an injury, via functional regeneration of the host cells, tissues and organs by placing appropriate cells of optimal quantity into the damaged body and maintaining the cellular functions that provide expected efficacy. outcome of the regenerative medical treatment completely depends on the viability of delivered cells. embedding and planting, referred to as implantation, of devices made of biocompatible materials has been widely applied. metals, ceramics, synthetic and natural polymers are the fundamental biomaterials that are convenient for processing and fabricating into various forms of tissue supporting structures. however, lack of their biological function is too far from the ideal goal for treatment. from this perspective, transplantation which involves transferring procured cells, tissue or organ from a donor and planting into a recipient to replace the damaged lesion has been regarded as an ideal approach that provides biological restoration with recovery of physiological functions. nevertheless, the opportunity for selection of the completely perfect donor for a patient is critically limited in practice. the gene-homogeneity between the donor and recipient, mainly in human leukocyte antigen (hla) types, is the first factor to avoid any post-operative complication oriented from the immune responses. although autologous tissue is the most appropriate if damage or defect is not related to immune disease, applicable size and volume of donor site are extremely limited. allogeneic tissue has been the second choice for transplantation since several immune-suppressive agents have been introduced, but these agents also lead to other immune-depressed diseases by breaking the natural immune homeostasis. xenogeneic tissue and organ transplantation have been suggested by a number of researchers but longevity difference between human and donor animal is a great barrier that cannot be overcome at present. an approach to treat gene-defect oriented congenital disease by delivering correct genes directly to a patient, known as gene therapy, has been introduced as an individualized treatment tool. selected genes hybridized with a carrier vector are injected into a patient, and the vector may infect patient's cells and leave the selected genes within patient's cells. then, the delivered genes may express its genetic characteristics necessary to cure the disease. however, the gene therapy bears several problems: (1) there is no perfect method to deliver genes into appropriate cells, (2) efficiency of gene expression varies, and (3) the gene delivering viral vector may induce unpredictable and unknown complications in body. another individualize treatment tool called cell therapy is planting therapeutic cells. instead of transplanting tissue or organ, therapeutic cells are delivered in forms of either transplantation of natural cells or implantation of artificially manipulated cells. in addition, resource of applicable tissues from which therapeutic cells could be prepared is expanded. conventional transplantation mainly consists of matured tissue or organ, but it is now possible to obtain therapeutic cells from any tissue at any growth stage, including embryonic blastocyst. through in vitro gene modification of cells, it is possible to select only the cells which possess appropriate genes for treatment purposes from outside of the human body. theoretically, in case of autologous cell therapy, patient's cells are harvested through autopsy or procurement, and the treatment gene for correction is transfected into the cells. after culture, only the completely gene-transfected, healthy cells are collected, and the selected cells are further cultured to obtain abundant number of cells to deliver in forms of cell suspension for injection. as the whole procedure is performed in vitro environment, only perfectly modified therapeutic cells are selected. in case of allogeneic cell therapy, donor's hla could be also replaced by the recipient's to escape from the post-operative immune rejection. furthermore, risk of inducing uncontrollable oncogenesis oriented from the mutated cells is avoidable during the cell selecting procedure in vitro [1] . to deliver the selected cells into the selected site in recipient's body, it is necessary to fabricate cell delivery vehicle with biomaterials that supply agents to maintain cell-viability and act as probes for piloting cells toward appropriate site. the cell-based implant is defined as any implant in combination of cells and biomaterials which are intended to repair, modify and/or regenerate human body, through expected regulation and control of cellular functions and/or behaviors after implantation. the cell-based implant consists of artificially manipulated therapeutic cells hybridized with biomaterials in vitro. for cell therapy, each cell is individually hybridized with biomaterials, but in forms of tissue engineered implants, cells are hybridized with scaffold-biomaterials that act as structural support in the same way as the extracellular matrix in tissue does [2] (fig. 8.1 ). substances applied to control biological events of cells may have biological risks. in manipulating cells, various kinds of substances functioning chemically at molecular level are used in cell-modifying procedures. biological agents, such as enzymes in the nucleic acid recombination procedure, cytokines in cell differentiation and proliferation control, and peptides in cell culture media, are regarded as biologic substances, and they must not produce any biological hazard during the manipulation procedures. in gene modification process, to avoid adverse reactions oriented from viral vectors, such as retrovirus, adenovirus and adeno-associate virus, non-viral vectors are designed to avoid biological risks and to manipulate easily. capability of non-viral vectors is mainly dependent on endocytosis mechanism, but the extent of development in an artificial vector that provides efficient endosomal escape to deliver the gene finally into nucleus is still questionable at present. biocompatibility is the primary requirement for any material used in manufacturing implant. in the cell-based implants, the material hybridized with cells shall either biologically or mechanically be compatible with the neighboring cells at the delivered site. for cell therapy, each cell would be hybridized with nano-sized particles that have specific affinity to the treatment target site and self-driving ability. particles shall be dissociated from the delivered cell after landing at the site and be completely excreted without accumulation in the recipient's body and any side effect. size of a tissue cell is about 10 ×10 −5 m (= 10 μm), a virus is 10 ×10 −7 m (= 100 nm), diameter of a dna is 10 ×10 −8 m (= 10 nm), diameter of a hydrogen atom is 10 ×10 −9 m (= 1 nm), and hybridizing substances are smaller than a μm. however, there is no accurate tool to detect these extremely small, nano-scale particles that exist in living body for safety evaluation of a substance at present. the cell-fabricating operator might be exposed to the substances during the whole manipulating procedure, and further investigation is necessary to avoid hazards. otherwise, currently introduced biomaterials for cell encapsulation are designed to protect therapeutic cells from immune rejection while providing the excretion of biologics from the cell, and mostly to manufacture macromolecules with less difficulty to characterize [3] . in producing multicellular structural cell-based implants, so called tissue engineered implants, various biomaterials of either synthetic or natural substance are applied. basically, conventionally available biomaterials can also be adopted as long as the permanent biocompatibility is approved. synthetic polymeric biomaterials are the representatives, and can be non-biodegradable or biodegradable after implantation. non-biodegradable materials are usually intended for use in cell therapy where cell-encapsulation is required to provide and maintain optimal cellular function (e. g., alginate, liposome, etc.) by protecting the cells from host immune reaction, and/or for a tissue engineered implant, which requires the physiological load-bearing compliance (e. g., polyurethane scaffolds for blood vessels, tendons, ligaments, etc.) after implantation. they permanently remain at the planted place in recipient's body. meanwhile, biodegradable materials are usually intended for use in implants which restore the histological structure and replace the cellular function of recipients. they are gradually degraded in recipient's body through hydrolysis or enzymatic function after implantation (e. g., poly l-lactic acid, poly glycolic acid, etc.). in cases of using synthetic polymeric biomaterials, behavior of hybrid cells is mainly dependent on surface characteristics of material. especially, the cellular events such as adhesion, differentiation, proliferation and migration on the non-biological synthetic biomaterial are important for maintaining viability of cells implanted in bio-inert materials. furthermore, an increased regional acidity induced by dissolved acidic component through hydrolysis from the biodegradable materials demonstrates the limitation in mimicking natural biological environment with synthetic biomaterials. natural biomaterials are mainly composed of extracellular matrix (ecm) components (including structural components and biomolecules) originated from autologous, allogeneic and xenogeneic tissues of mammalians. collagen, elastin, chondroitin-6-sulphate and hyaluronic acid are structural components. biomolecules, such as peptides, fibronectin, laminin, vitronectin and fibrin, and cytokines as growth factors and apoptosis signal promoters, are biologically active substances produced by nature. non-mammalian substances such as silk fibroin, crab chitin and chitosan and agar are also included in this criteria. at this scope, it can be recognized that, although any xenogeneic cells are still not permitted, xenogeneic ecm components are permitted for the "trans-" or "im-"plantable biomaterials. for cell-based implants that utilize xenogeneic tissues or their derivatives as biomaterials, secure risk controls shall be applied on sourcing, collecting and handling xenogeneic ecm, on the validation of elimination and/or inactivation of adventitious agents, such as transmissible spongiform encephalopathy (tse) agents in case of using bovine tissue, in products. non-comparable biological superiority of natural ecm components are applied for improving biocompatibility of synthetic polymeric biomaterials as grafting materials onto the surface of material. also, bioactive agents (including biologics, antibiotics, and antimicrobials) and/or synthetic drugs can be medicinal components in biomaterials, and they shall be assessed, in the context of their integration with the cell-based implant, according to pharmaceutical principles. this assessment shall consider the effects of medicinal components on product and vice versa. furthermore, the medicinal components could be an additive to treat the recipient's disease [4] . in mammalians, collagen comprises about 30% of total proteins and exists as a main structural component in ecm and supports anatomical morphology of every tissue and organ. although more than 20 types of them are informed, type i collagen, which has a specific molecule of the super-coiled triple helical peptide chains, is the most abundant in body. in brief, each peptide chain is a specific left-handed helix of 100,000 molecular weight and consists of repeating "-glycine-x-y-" amino acid sequence. as the arginine-glycine-aspartic acid (rgd) sequence is a typical cell adhesive ligand, collagen demonstrates strong cell adhesion property. 3 left-handed helical peptide chains are integrated by intramolecular bindings of hydrogen bonds at glycines to glycines and hydroxyl bonds at hydroxyprolines to hydroxyprolines in each chain to form a right-handed triple helical collagen molecule. in a chain, 1012 amino acids are involved in the formation of a molecule, and each free-end of a chain that is not integrated with the others, is made of 12 -17 amino acids and named as telopeptides. each of these at the c-and n-terminals of molecule binds to another and forms a long linear chain. collagen is easily denatured at temperature over 37 • c through disintegration of the intermolecular bonds, and becomes gelatinized to form a randomly coiled chain with less viscosity than that of collagen. once denatured, gelatin is amorphous and does recover the intermolecular bonds in nature and is more easily digested by metaloprotease than collagen. a type i collagen fibril consists of 5 collagen linear chains with strong intermolecular hydrogen bonds at lysine to lysine in each molecule. in nature, ε-nh 2 of lysines and hydroxylysines at the both extremities of telopeptide is converted to aldehydes by lysineoxidase, and the schiff base formation by binding aldehyde with residual ε-nh 2 or by aldol condensation between aldehyde to aldehyde occur. this strong molecular binding is called as the crosslinking and introduces strong mechanical characteristics to collagen to act as the structural matrix in tissue and provide organ morphology. as hydrogen concentration of body fluid is ph 7.4, collagen is regarded as a weak base substance, and is generally extracted from mammalian tissue. collagen is the hydrophobic protein and is generally insoluble in a neutral solution, but non-crosslinked collagen is soluble in the neutral base solution such as nacl or na 2 hpo 4 . on the other hand, weak acid such as hcl, citric acid and acetate breaks the intermolecular schiff base bonds and the acid soluble collagen molecules are extracted. after extracting acid soluble collagen, non-acid soluble remnants remain, and these are strongly crosslinked collagen fibrils. when additional process using pepsin that digests the linear intermolecular bonds at each telopeptide is performed, telopeptide-free collagen molecules, known as the atelocollagen, can be obtained from remaining collagen fibrils. because telopeptide demonstrates individual gene-characteristics, these atelocollagen molecules are recognized as immune-free substance and are applicable to medical and pharmaceutical purposes. high concentration of bases such as nacl, na 2 so 4 , na 2 hpo 4 aggregate the acid-soluble collagen molecules in solution, and this phenomenon is applied to produce collagen membranes, threads and hollow fibers. however, randomly reconstructed intramolecular and/or intermolecular bonds do not provide sufficient mechanical properties compared to natural collagen in tissue. hence, in order to fabricate collagen with higher mechanical strength, re-crosslinking methods are introduced [5] (fig. 8.2 ). collagen has been regarded as the first candidate for hybridization with cells as it exists in every part of body as the main extracellular matrix component. although implantation of xenogeneic cells is not practically permitted, immunefree atelocollagen is generally extracted from mammalians for medical use. bovine is the most popular resource provided if cows are grown at the officially recognized region that is free from tse. as atelocollagen has no telopeptides that integrate with other atelocollagen molecules, basic technology to produce intramolecular and intermolecular bonds for construction of the extracellular matrix with adequate mechanical properties required by tissue where the artificial cell-based implant is delivered is the recrosslinking method using chemical reagents or physical dynamics. most common chemical reagent adopted for this process is glutaraldehyde that introduces stable covalent nh 2 to nh 2 bindings between the molecules. as amine to amine (nh-nh) bonding exists in every amino acid in molecules, the chemically crosslinked collagen demonstrates high strength, but irregular patterns of the crosslinked fibers and protracted resorption are general disadvantages. furthermore, in case of using glutaraldehyde, complications due to residual aldehydes often conduct calcification that directly leads to the loss of mechanical strength. to overcome these disadvantages, 1-ethyl-3(3-dimethylaminopropyl) carbodiimide (edc), which introduces nh 2 to cooh covalent bonding and has no toxicity, has been applied to the procedure. to avoid any disadvantage arising from the use chemical reagents, cross-linking by physical method is employed. irradiating with gamma or ultraviolet rays produces radicals in the form of unpaired electrons in the nuclei of aromatic residues such as those in tyrosine and phenylalanine, and binding of these radicals results in the collagen cross-linking through random nh 2 to cooh bonding. even though the physical method is safe as it does not involve the use of chemicals, stability of the crosslink is less than that of chemically induced crosslink. collagen can be fabricated into various forms of gel, fiber, membrane with or without pores, and it can even be grafted onto the non-viable metal, ceramic and synthetic biomaterials to introduce biological layer on surface. atelocollagen gel is easily prepared by dispersing in weak acidic solutions. in general, collagen is dispersed in a weak acid solution of ph 6-6.5 and finally adjusted to required ph by adding base for medical purpose. additive substance to ultimate tensile stress of dense membrane in relation to uv irradiation time. each value represents the mean ± sd in five samples, * and †: significantly different compared to the control non-treated group for each type of membrane, * : p < 0.05 and †: p < 0.01. control : nontreated group, uv1/2: uv-irradiated group for 30 mins, uv2: uv-irradiated group for 2 hours, uv4: uv-irradiated group for 4 hours, uv8: uv-irradiated group for 8 hours, ga: 0.625% glutaraldehyde pretreated group for 24 hours (y -axis: maximum tensile stress (kpa)) provide and/or control the biological function of the collagen can be introduced at this stage utilizing the high viscosity property. biochemical property of collagen molecule can be modified by varying the surface electric charge by altering the molecular side chain which leads to the adjustment of hydrophilicity-hydrophobicity balance. succinylization of amines (nh-co(ch 2 ) 2 -cooh) by using anhydrate succinyl acid provides abundant (−) charges, and the succinylated collagen becomes soluble in neutral solution, which finally results in translucent viscous collagen gel with hydrophilicity. meanwhile esterization (-cooch 3 ) of carboxyl group by methanol produces abundant (+) charges on the collagen and provides a favorable hydrophobic niche for protein adhesion. atelocollagen gel is fabricated into fiber form by using an electro spinning. for example, a collagen solution dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (hfp) having concentration over 5% is useful to produce a diameter controlled nanofiber by passing it through a diameter adjustable nozzle under the condition of a high voltage at 25 kv, flow velocity at 2.5m/h, metal collector of 2 cm width with rotating speed at 300 rpm, and distance between the metal collector and spinner of 15 cm, and finally removing organic solute for 48 hours by drying in a vacuum chamber. the produced nanofiber is crosslinked by either edc or uv irradiation later in order to reinforce the fiber strength, and demonstrates the typical triple helical structure of collagen molecule. freeze-drying the gel in vacuum condition is a simple procedure for fabrication of a porous membrane, as lower concentration and higher freezing temperature leads to smaller and bigger pores [6] [7] [8] [9] [10] , (figs. 8.3-8.8). collagen contains much of the cell adhesive rgd sequences in molecule, thus has cell conductive characteristics and is applied either in the independent form, in form of hybridization with biofunctional agents and cells, or, occasionally, in form of copolymer by introduction of synthetic polymers which is mainly aimed to enforce mechanical property. collagen gel is usually applied to cell culture. a cell culture plate whose bottom surface is coated by collagen gel and dried makes it possible for suspended cells to produce a monolayered cell cluster by attaching them to the coated collagen layer. this technique is directly applied to produce skin wound dressing membranes. exposure of dermis to the open air, the grade 3 skin defect, in which the basal epithelial layer that prevents direct contact of inner body to the outer bodily environment and supports epithelium as the bed for keratinocytes is destroyed from burn, and sore spots induced by diabetes mellitus or accident are critical emergency in clinics. they evolve the direct contamination of dermis which usually progresses toward fatal septicemia or skin necrosis. the conventional treatment procedure mainly consists of complete irrigation and debridement of the wound, topical administration of broad spectrum antibacterial and antifungal agents, covering the wound by oily ointment to protect it from air contact, painful daily dressing change, and observation of the auto-regeneration of wound by proliferation of both dermal fibroblasts and epithelial keratinocytes and basement membrane reconstruction. promoting proliferation of these skin cells with protection from air contact until complete healing is the key technology, and collagen membrane has been applied for this. for example, freeze dried atelocollagen in vacuum forms a membrane with random pores, and the pore size into which the cells may proliferate is controlled by the gel concentration and temperature at which it is frozen. freeze dried collagen gel of 1, 2, and 5% in concentration at −20, −40, and −80 • c for 1/2, 1, 2, and 4 hours demonstrated that higher concentration produced less and smaller sized pores. also, the lower temperature and prolonged freezing time decreased viscosity. to produce a collagen membrane with optimal pore size of over 120 μm which permits penetration and proliferation of dermal fibroblast proliferation, the 2% collagen gel freeze dried at −40 • c for 2 hours and crosslinked by edc or uv irradiation for 2 hours was found to be recommendable to use as a dermal cell conductive membrane that provides the appropriate viscosity for initial anchorage, and any inflammatory exudate escapes through pores. a collagen bi-layered membrane is applied to skin in forms of either wound dressing or extracellular structural supporting matrix for artificial skin. laminin, which is a cell adhesive protein and a main component of the basement membrane between dermis and epithelium, is mixed with 2% collagen gel. a porous collagenlaminin membrane is fabricated and freeze-dried as previously described and additional 4% collagen gel is coated on the porous membrane and crosslinking is promoted by edc treatment or uv irradiation. through this procedure, a bi-layered collagen matrix that consists of dense collagen layer overlaid on the prefabricated porous collagen-laminin membrane was produced. in use as a wound dressing, the upper dense layer prohibits direct wound contact with the open air and plays a role as a bed for keratinocyte proliferation, while the porous layer conducts dermal fibroblast proliferation. thus, the painful daily dressing change becomes unnecessary. antibacterial agents encapsulated by hyaluronic acid can be incorporated into the collagen gel and the drug may be released from the collagen membrane to avoid periodic topical drug application. fabrication of the bi-layered collagen membrane is the basic technology applicable to manufacture of artificial skin. in the 1980s, bell introduced a method in fig. 8.9 collagen-laminin hybridized porous membrane for dermis which dermal fibroblasts and collagen gel mixture were cultured at 37 • c, and when the cells were confluent in the gradually shrinked gel, the collagen-fibroblast composite is produced. keratinocytes were overlaid on the composite to mimic anatomy of the dermal cellular structure, but it required about 20 days for the manufacture to be completed and it was therefore not applicable to patients with grade 3 skin wound. on the other hand, green introduced an advanced method in which dermal fibroblasts were cultured to form monolayer on a collagen coated culture plate and then keratinocyte was directly co-cultured on the fibroblast monolayer in a media containing epithelial growth factor. nonetheless, this method still required at least a week for the complete fabrication, and employment of growth factors holds a risk of oncogenesis induction. to produce an artificial skin, autologous dermal fibroblasts of rat were seeded into the edc crosslinked porous collagen-laminin membrane and cultured in minimum essential medium (mem) for 3 days to provide the cell-niche adaptation period. 3-day culture is not enough for complete proliferation of cells into pores, but cells were firmly attached and anchored onto the superficial layer of the porous membrane due to specific cell adhesive characteristics of -rgd-sequences in collagen and laminin. after 4 weeks of coverage onto a grade 3 skin wound, dermis was completely replaced, and basement membrane lined beneath the dense layer as the seeded dermal fibroblasts were cultured in situ. epithelial regeneration upon the dense layer surface was poor and partially occupied by the keratinocytes, but full coverage by epithelium was observed after 6 weeks. this phenomenon can be understood as the porous collagen based membrane can play as a vehicle for dermal autologous cell delivery, and the reconstructed basement membrane, which is a keratinocyte supplying bed in nature, has driven epithelial conduction. later, a collagen-elastin nanofiber was fabricated as previously described, and the extruded fibers were brought onto collagen gel and crosslinked by edc. using nanofiber made it possible to produce a lattice controlled matrix, and uniformly aligned lattices conducted cells to proliferate into the designed lattice pattern [11] [12] [13] [14] [15] [16] [17] (figs. 8.9-8.16 ). collagen gel hybridized with calcium phosphate is applicable as a bone conductive substitute. apatite ( ca 10 (po 4 ) ++ 6 ), a typical inorganic component of skeleton, was synthesized and heated at 980 • c after which apatite crystals were not sintered but degradable by hydrolysis. 1% collagen gel was mixed with the apatite in 12:88 w/v, and the mixture was crosslinked. then the produced apatite-collagen pellet was implanted into rabbit's resected tibiae. 4 weeks after implantation, the resected defect was completely regenerated by host bone with cortical bone continuity and cancellous bone stroma, and after that, typical bone remodeling process followed to produce natural bone. however, mechanical strength of the regenerated area was less than general bone. although collagen based gel is favorable to fabricate cell conductive substitute, weak mechanical property is a barrier for application in the physiological stress bearing tissues, and, to resolve this problem, hybridization of collagen with polymeric biomaterials has been suggested. introducing functional groups on esters, such as polyurethane (pu) and poly lactic-glycolic acid (pl-ga), through treatment with ozone induces surface oxidization that produces carboxyl groups on the surface which react with amines in collagen. as the apatite-collagen provided insufficient strength despite promotion of a favorable osteogenesis, collagen was grafted onto the biodegradable pla membrane to reinforce the strength. pla of m.w. 50,000 was resolved by 99% chloroform solution to produce 9% (w/v) pla solution, and nacl crystals (425 -500 μm) were mixed to the pla solution with adjusting nacl:pla ratio to 9:1 (w/w). the prepared solution was cast into an appropriate shape, and the solvent was allowed to evaporate over 24 hrs to produce a porous membrane after leaching out the nacl particles with distilled water. to induce molecular bindings between the collagen within apatite-collagen composite and the pla membrane surface, pla membrane was oxidized by ozone to produce reactive carboxyl and hydroperoxide groups on the pla surface. after the ozone treatment at 60 v for 60 min, apatite-collagen was delivered onto the ozone treated pla membrane to produce an apatite-collagen grafted pla membrane. the grafted membrane was pressed (1 bar at 37 • c) from vertical direction, and then edc collagen cross-linking was performed to integrate the mixed atelocollagen fibers in the grafted material. the final product was im collagen grafted synthetic polymer is applicable to soft tissue replacement as well. except the air passages such as trachea and bronchus which require almost no repeating dilatation-shrinkage action of compliance, most of the tubular-shaped tissue requires various level of compliance against the mass passing through lumen, as arteries to pulsating blood flow and esophagus to swallowing dietary mass. in general, tubular walls of those tissues consist of multiple-layered smooth muscle cells to resist against pressure at luminal wall exerted by transporting mass, and appropriate flexibility is required. to replace esophagus, polyurethane, a bioinert biomaterial with mechanical advantage of high durability against continuous bending stresses, was employed. an approach is presented for graft copolymerization of type i atelocollagen onto surface of polyurethane (pu) treated with ozone. through surface oxidization by ozone to modify the pu surface, peroxide groups are easily generated. those peroxides are broken down by redox-polymerization and provide active species which initiate graft polymerization by reacting with amines in the collagen molecules. ozone oxidation time and voltage could readily control the amount of peroxide production. maximum concentration of peroxide was about 10.20 ×10 −8 mol/cm 2 when ozone oxidation was performed at 60 v for 30 min. after the reaction of pu by ozone oxidation, type i atelocollagen gel was graft copolymerized onto the pu. all the physical measurements on the collagen grafted surface indicated that the pu surface was effectively covered with type i atelocollagen. interaction of the collagen grafted pu surface with fibroblasts could be greatly enhanced by the surface graft polymerization with type i atelocollagen. attachment and proliferation of fibroblasts on the grafted type i atelocollagen were significantly enhanced, and it is assumed that the atelocollagen matrix supported the initial attachment and growth of cells. in the early stage of proliferation, collagen synthesis in fibroblasts was not activated and remained at a relatively low level due to the grafted type i atelocollagen, increasing only fibroblast differentiation. the mechanical property of tubular tissue was oriented from the alignment of cells that consists of repeated overlayers of longitudinal and circumferential layers and forms wall thickness. regarding this result, a double layered pu tubular scaffold grafted with collagen was fabricated to produce an artificial esophagus. pu purification was performed by diluting pu particles in dmac(dimethyl-acrylamide) solution then precipitating in non-solvent methanol, and drying the precipitate under vacuum for 2 days to remove residual methanol. after purification, pu was dissolved in dmac solution at the final concentration of 13%w/w. glass rod, with diameter of 3 mm and length of 15 cm, was worn with 13% pu solution and then air-dried for 6 hours. this process was repeated twice. after wearing, an end part of the glass rod was cut, and then the rod was worn again in reverse direction by the same process to be uniformly worn. it was dried under vacuum for 12 hours afterward. completely dried sample was swelled in benzene solution for 1 hour and washed with distilled water for 2 hours and ethyl alcohol for 1 hour. after swelling and wearing process, a porous pu tube was separated from glass rod and dried under vacuum. to graft collagen, ozone treatment was performed as described above. the tube was immediately immersed in 1% type i atelocollagen gel, and mohr's salt [feso 4 (nh 4 )2so 4 6h 2 o] was added to the gel to decompose peroxides. then polymerization was allowed to proceed at 35 • c and ph 8.0. type i atelocollagen grafted porous pu tube was dried in vacuum at 25 • c. the tube was dipped in a 2% collagen gel and crosslinked by edc later. by this procedure, a porous collagen-grafted tube with inner diameter of 3 mm, thickness of 0.7 mm covered by dense collagen layer was produced. rabbit's esophageal smooth muscle cells were seeded and cultured in a mechanically stressful environment of 10% strain magnitude and 1 hz frequency. culturing for 18 hours of mechanically stretching condition and 6 hours of stationary condition on every 24-hour period, the cultured cells aligned in the perpendicular direction to the strain direction after 2 days. the collagen grafted pu tube with cell alignment control was subcutaneously implanted in nude mice for 4 weeks, and a histological finding demonstrated that a well-aligned smooth muscle cells reconstructed the tube which is applicable for replacement of esophagus [26] [27] [28] [29] [30] the idea of collagen grafting was also applied to produce a healing-promoting antiadhesive membrane that is particularly necessary in peritoneal surgery to prevent postoperative adhesion. at first, glycolide and d,l-lactide were recrystallized from ethyl acetate and dried under vacuum before use. lactide and glycolide, at a molar ratio of 75:25, were put into a glass ampoule, and mpeg was added for preparing the mpeg-plga block copolymer through ring-opening polymerization. 0.05%(w/w) of stannous octoate added to the solution as a catalyst. the ampoule was evacuated by a vacuum pump, sealed with a torch, and was heated in an oil bath at 130 • c for 12 hrs. after the reaction was complete, resulting polymers were purified by dissolving in methylene chloride and then precipitated in excess methanol. obtained polymers were dried in vacuum. for the preparation of polymer film, 8% solution of mpeg-plga in chloroform was cast on a glass plate, and the solvent was evaporated in a vacuum oven for 2 days. on the other side, a porous collagen-hyaluronic acid (ha-col) membrane was fabricated and crosslinked. 1% of hyaluronic acid (ha) (sodium salt, mw = 120,000 − 150,000) as an aqueous soto prepare ha-col and mpeg-plga bi-layered composite membrane, chloroform was sprayed on the surface of mpeg-plga film, and then cross-linked ha-fn-col membrane was loaded on the slightly dissolved surface of mpeg-plga film. in vitro adhesion test revealed that fibroblasts attached better on ha-col membrane compared to those on mpeg-plga film, plga film or oxidized cellulose film. mpeg-plga film had the lowest cell adhesive property. in confocal microscopic observation, the actin filaments were significantly more polymerized when 50 or 100 μg/cm 3 fibronectin was incorporated on the ha-col membranes. after 7-day culture, fibroblasts penetrated throughout the hafn-col network and the cell density increased whereas very few cells were found attached on surface of the mpeg-plga film. in vivo evaluation by implantation test for 7 days in rabbit's peritoneal wound showed that the composite membrane could protect tissue collagen based biomaterial was also applied as a mesenchymal stem cell (msc) delivery vehicle. liver hepatocyte is a representative stable cell that has tremendous proliferative capacity and ability to differentiate and proliferate when injury or damage occurs in liver. in case of no-proliferation of hepatocytes, oval cells are stimulated to divide and eventually differentiate into mature hepatocytes; therefore, an oval cell is regarded as a compensatory cell in liver injury, and has been concerned to be equivalent to liver stem/progenitor cells. oval cell is oriented from bone marrow mesenchymal cell, and is a facultative bipotential precursor cell that differentiates into hepatocytes or bile duct cells. human mscs harvested from tibial bone marrow and co-cultured with hepatocytes for 3 days in medium with additive hepatocyte growth factor (hgf) produced a confluent mixture of undifferentiated mscs and oval cells. to purify the oval cells from nonparenchymal cells, cell cloning method was applied. characterization of oval cells was performed by a double immunoobserved fiblasts on the peg-plga surface were significantly fewer than on the plga membrane surface. collagen-ha membrane demonstrated high affinity to cell attachment fluorescence method using alpha-fetoprotein (afp) and cytokeratin-19 monoclonal antibody expecting co-expression. isolated human mscs oriented oval cells were seeded on an edc crosslinked porous type i atelocollagen matrix, and cultured in medium containing insulin, dexamethasone, and hydrocortisone as the hormone stimulators and additive cytokines of hgf and epidermal growth factor (egf). for co.) is a commercially available anti-adhesive membrane made by oxidized cellulose 3 weeks, albumin secretion and urea detoxification rate continuously increased either in the hormone additive or the cytokine additive groups, and this was directive knowledge that avoidance of using growth factors in committing msc-oriented oval cell toward hepatocyte can escape from risky induction to liver cancer. the collagen scaffold was able to foster long-term viability and protection of the cells, and this 3-dimensional culture of oval cells is considerable for designing a cell-delivering tool for hepatic disease [33] (figs. 8.33-8.38). asymmetric collagen-fibronectin grafted peg-plga copolymer membrane was appeared to successfully reduce the incidence of postoperative adhesion formation although collagen is a favorable biomaterial to be employed in a wide range of fabrication procedures with cells, there is no established tool to isolate and produce absolute atelocollagen, the immune-free substitute, at present. even a single remaining collagen dimer or trimer may cause immune reactions in future. the resource of collagen, especially for clinical applications is also important, because unknown viruses that produce future unpredictable diseases from xenogeneic extracellular matrix may exist. if single-cell encapsulation by collagen containing signal transduction agents and ligands attracting specific cell adhesive receptors is systemized, commitment of stem cell differentiation and proliferation toward the designated target tissue will be achieved, and this may contribute to the future progress of stem cell-based implant. tissue restoration fundamental concepts for tissue engineering biocompatibility assessment of medical devices and materials extracellular matrix components anti-infection treatment of a transcutaneous device by a collagen-rifampicine composite treatment of collagen for tissue regenerative scaffold reaction monitoring of succinylation of collagen in matrix-assisted desorption/ionization mass spectrometry characterization of uvirradiated dense/porous collagen membranes: morphology, degradation, and mechanical properties characterization of porous collagen/hyaluronic acid caffold modified by 1-ethyl-3(3-dimethylaminopropyl)carbodiimide cross-linking preparation of collagen modified hyaluronan microparticles as antibiotics carrier behavior of fibroblasts on a porous hyaluronic acid incorporated collagen matrix laminin modified infection-preventing collagen membrane containing silver sulfadiazine-hyaluronan microparticles an infection-preventing bilayered collagen membrane containing antibiotic-loaded hyaluronan microparticles: physical and biological properties biological characterization of edc-crosslinked collagen-hyaluronic acid matrix in dermal tissue restoration evaluation of antibiotic-loaded collagen-hyaluronic acid matrix as a skin substitute electrospun nanofibrous membrane for the engineering of cultured skin substitutes biodegradable ceramic-collagen composite implanted in rabbit tibiae behaviors of osteoblasts-like cell (mc3t3-e1) on collagen grafted poly l-lactic acid (plla) membranes with various pore sizes a bone replaceable artificial bone substitute: morphological and physicochemical characterization behavior of osteoblasts on type i atelocollagen grafted ozone oxidized poly l-lactic acid membrane a bone replacable artificial bone substittue: cytotoxicity, cell adhesion, proliferation, and alkaline phosphatase activity a bone replaceable artificial bone substitute: osteoinduction by combining with bone inducing agent interaction of mesenchymal stem cells and osteoblasts for in vitro osteogenesis ex vivo mechanical evaluation of carbonate apatite-collagen grafted porous plla membrane in rabbit calvarial bone type i atelocollagen grafting on polyurethane tube and its mechanical property compliance of surface modified polyurethane tubular scaffold for artificial esophagus type i atelocollagen grafting onto ozone treated polyurethane films: cell attachment, proliferation, and collagen synthesis time-dependent modulation of alignment and differentiation of smooth muscle cells seeded on a porous substrate undergoing cyclic mechanical strain construction of functional soft tissues from premodulated smooth muscle cells using a bioreactor system evaluation of tissue adhesion preventive surface modified natural and synthetic polymeric materials preparation and characterization of biodegradable anti-adhesive membrane for peritoneal wound healing behaviors of isolated rat oval cells in porous collagen scaffold key: cord-285620-oawrnmhy authors: fahimirad, shohreh; fahimirad, zahra; sillanpää, mika title: efficient removal of water bacteria and viruses using electrospun nanofibers date: 2020-08-16 journal: sci total environ doi: 10.1016/j.scitotenv.2020.141673 sha: doc_id: 285620 cord_uid: oawrnmhy abstract pathogenic contamination has been considered as a significant worldwide water quality concern. due to providing promising opportunities for the production of nanocomposite membranes with tailored porosity, adjustable pore size, and scaled-up ability of biomolecules incorporation, electrospinning has become the center of attention. this review intends to provide a detailed summary of the recent advances in the fabrication of antibacterial and antiviral electrospun nanofibers and discuss their application efficiency as a water filtration membrane. the current review attempts to give a functionalist perspective of the fundamental progress in construction strategies of antibacterial and antiviral electrospun nanofibers. the review provides a list of antibacterial and antiviral agents commonly used as water membrane filters and discusses the challenges in the incorporation process. we have thoroughly studied the recent application of functionalized electrospun nanofibers in the water disinfection process, with an emphasis on their efficiency. moreover, different antibacterial and antiviral assay techniques for membranes are discussed, the gaps and limitations are highlighted and promising strategies to overcome barriers are studies. removing bacteria from water supplies which meet the requirement of effective antimicrobial activity, superior filtration flux with acceptable retention potentials (zodrow et al., 2014) . nanofiber membranes, because of their high surface area to volume ratio, nano-sized pores, and high porosity, have been illustrated to improve the efficiency of conventional materials employed for the filtration and separation of particulate materials (aussawasathien et al., 2008) . a number of processing techniques including melt-blown, self-assembly, phase separation, template synthesis, and electrospinning have been employed to prepare nanofibers in recent years. among them, electrospinning is the most promising, efficient method to produce web-like non-woven ultrafine fibers including microfibers (>1 μm) or nanofibers (<1000 nm) from different kinds of polymers. moreover, incorporation of bioactive, antimicrobial and antiviral agents into nanofiber structure is easily possible through the electrospinning process fahimirad and hatami, 2019; faccini et al., 2015) . the present work reviews previous studies on the production and application of electrospun nanofibers as antimicrobial water filtration membranes. the merits and demerits of these novel water microfiltration tools are discussed. moreover, their antibacterial efficiency and disinfection activities are compared with commercial water membrane filters comprehensively. finally, some points are recommended to be noticed as the subsequent future research plans. the objectives of this review were to: (i) introduce the different procedures, which have been applied for incorporation of the various antimicrobial agents into electrospun nanofibers (ii) discuss the different antimicrobial tests used for proving antimicrobial activity of the fabricated electrospun water filters (iii) study the efficiency of the produced antimicrobial electrospun application in the water treatment industry. j o u r n a l p r e -p r o o f the electrospinning approach was invented by cooley in 1900 (cooley, 1900 . this method is easy, cost-effective, uncomplicated, and has the potential for scale-up production. the flexibility in material selection and additive incorporation to obtain appropriate functionality, as well as its considerable capability to produce fibers in the sub-micron range with the high surface-area (up to 40 m 2 g -1 based on the fiber diameter), are prominent privileges of electrospinning process for fabrication of nanofibers. in addition, effective porosity of electrospun nanofibers (almost about 80% with no upper limit) with many small pores, interconnected pore structure directly promote both infiltration rate and contaminant rejection ratio in comparison with conventional materials being used for mf applications (nasreen et al., 2013; wang et al., 2013) . in this process, a prepared solution of polymer is loaded into a syringe and fed at a set flow rate to the spinneret. due to the needle connected to a high voltage power supply under the electric field with a specific voltage, a taylor cone is constructed by elongation of the polymer droplet at the end of the syringe into a characteristic conical shape. enhancing the electrical field causes the formation of a steady jet elongated and whipped consecutively by electrostatic repulsion. the solvent evaporates when electrostatic forces prevail over surface tension and the jet gets finer, so electrospun nanofiber in mf application, such as uniform fiber morphology with controllable pore size, interconnected open pore structure, high porosity, and membrane thickness, turn them to a superior substitute to replace the conventional mf membrane such as the millipore gswp mf membrane with an average pore size of 0.22 mm (wang et al., 2012; barhate and ramakrishna, 2007) . another novel application of electrospun nanofiber in water purification and bacterial rejection is thin-film nanocomposite membrane (tfnc), a major type of reverse osmosis (ro) and nanofiltration (nf) membranes, which compromises of three layers including the first barrier layer of interfacial polymerization, a polyacrylonitrile or poly (vinylidene fluoride) electrospun membrane as the second layer and nonwoven polyethylene terephthalate (pet) as the third layer. the third layer employed as a substructure layer to provide the whole membrane adequate mechanical strength (subramanian and seeram, 2013; yin et al., 2012; fig 2) . high water flux, great solute rejection, minimum membrane fouling, and perfect mechanical persistence are main properties of an ideal tfc membrane and turn it into an excellent candidate for microfiltration and ultrafiltration applications (li and wang, 2010) . sato et al., (2013) fabricated a novel composite fibrous membranes, consisting of an ultra-fine cellulose nanofibrous infused into electrospun polyacrylonitrile (pan, with an average diameter of 0.2 μm a mean diameter of about 30 μm as the barrier layer (40-100 μm in thickness) to provide filtration attributes) nanofibrous scaffold on a melt-blown polyethylene terephthalate (pet, with a mean diameter of 30 μm as the support layer (about 100 μm thick) to sustain mechanical strength) non-woven substrate for water purification. the nanostructure showed a retention rate of 99.9999% for e. coli filtering and the high percent of the ms2 virus, with 30 nm sizes, captured in the electrospun pan scaffold infused with m-ufcns (sato et al., 2013) . recently, taheran et al. (2019) j o u r n a l p r e -p r o o f fabricated a methodical portable water purification instrument using electrospun nanofiber. the device contained three distinct electrospun membranes. the first membrane was made by electrospinning of polyacrylonitrile/chitosan solution at 85:15 mass ratio as an antibacterial membrane, the second membrane was produced from laccase (10 unit g -1 ) immobilized onto pan/biochar 95:05% electrospun mat for removal of micro-pollutants and the third layer was fabricated by electrospinning of pan/biochar at 95:05 ratio as an adsorptive membrane. the applied technology led to approximately 99% removal of microorganisms, 83% of micropollutant removal, and more than 77% of turbidity decline during less than 5 min contact time (taheran et al., 2019) . the important characteristics of a nanofiber mat membrane for application as filters for the separation of contaminations and pathogens from a continuous fluid phase are wetting properties, permeability, porosity, fiber size distribution, and fiber structure. for water filtration, a membrane must be wet-table and surface wetting properties are generally specified by the contact angle. a surface with a low contact angle (below 90 degrees) is considered a hydrophilic surface, while a surface illustrating a high contact angle (over 90 degrees) is referred to as a hydrophobic surface. sessile drop and the captive bubble method are two common techniques used for measuring the nanofibers' contact angle (nuraje et al., 2013) . one of the key parameters in filter design and its performance is porosity. generally, porosity is calculated from the apparent density and bulk density of the membrane. however, other alternative procedures inclusive of image analysis and mercury porosimeter are frequent methods j o u r n a l p r e -p r o o f applied for the evaluation of porosity in the nanofiber membrane (ghasemi-mobarakeh et al., 2007) . electrospun nanofibers are highly porous with interconnected pores in the size range of just a few times the fiber diameter. the small pore size of the nanofibrous membrane introduces a higher retention rate, the interconnected pores leads to better tolerance against fouling and the high porosity defined a higher permeability capability (homaeigohar et al., 2010) . clean water permeability (cwp (l/m2 •h•bar)) illustrates the highest amount of attainable flux dependent on the membrane condition. it can be assayed by calculating the flux at various trans membrane pressures (tmp). the slope of the eventuated curve is regarded as the cwp (bjorge et al., 2009) . the high cwp grants high flux operation to the membranes, introduces the nanofiber mat as an energy-saving membrane, and means that if fouling does not happen, enormous volumes can be treated (daels et al., 2011; he et al., 2018) . the surface charge on membranes is related to affinity corresponding interactions and considered as a significant parameter influencing the disinfection capabilities of the membrane. surface charges can qualify the strength of biomolecular or even pathogen affinity on a material surface. in virus removal, surface charged nanofibers adsorb virus via electrostatic interactivity between the nanofibers and the counter-charges of virus and signify virus remediation improvement (cho et al., 2012) . a series of studies have confirmed that electrostatic attraction between the cationic membrane and the anionic surface of bacteria may lead to morphological defects in consequence of ros generation and cell membrane destruction. indeed, anionic membranes act as powerful non-adhesive site of bacteria attributable to electrostatic repulsion (mukherjee and de, 2018; kolewe et al., 2016) . operating conditions influence the antibacterial activity performance of the nanofibrous membrane. as proved by several experiments, bacterial cells are able to decline their size at higher operating pressure, hence resulting in enhancing permeation through the filter. therefore, less trans membrane pressure (tmp) is usually desired, to retain antibacterial activity during long term application of the membrane. the tmp is described as the mean feed pressure minus the permeate pressure that is essential to push down water through a membrane (mukherjee and de, 2017; . different factors including surface area, surface roughness, pore diameter, zeta potential, and inclusion of biocides or antibacterial agents determine the antimicrobial performance of membrane (rahaman et al., 2014; mukherjee and de, 2018) . accordingly, the employing of electrospun polymeric membranes in bacterial and virus removal from water is performed in two procedures including size exclusion and adsorption (lee et al., 2016) . in most cases, the diameter of water-borne bacteria is more than 0.2 μm. for example, the e. coli size is 0.5-2.0 μm and brevundimonas dimimuta dimension is 0.3-0.9 μm. previous studies have confirmed that using a 0.45 μm pore sized mf leads to a 2 log-4 log bacteria reduction (gómez et al., 2006; ghayeni et al., 1999) . thus, based on the degree of exclusion, the electrospun membrane should have an average pore size of fewer than 0.2 μm. in addition, the narrow pore size distribution is requisite for achieving a high retention rate (ma et al., 2014) . there is a direct relationship between the pore size and the fiber diameter of a porous nonwoven structure. the relationship has been confirmed as the average pore size was approximately 3±1 times the mean fiber diameter, and j o u r n a l p r e -p r o o f the greatest pore size was about 10±2 times the mean fiber diameter. thus pore size of electrospun fiber generally grows with increasing fiber diameters (ma et al., 2011) . various conventionally employed membranes for the application as micro-filters have 0.2 μm theoretical pore sizes. the advantage of electrospun nanofiber membranes in comparison to conventionally used membranes are the simplicity of manufacture, adjustable size of the pores and high porosity (saleem et al., 2020) . in view of the fact that the membrane pore sizes can be controlled by adjusting the electrospinning parameters and besides the fact that the most aquatic bacteria dimensions are more than 0.2 mm, electrospun nanofibers can be designed efficiently with smaller pore dimensions suitable for mf applications (wang and hsiao, 2016) . for instance, accelerating the flow rate raises the pore diameter by enhancing the fiber diameter. moreover, increasing polymer solution concentration and using higher molecular weight polymer increases fiber diameter. employing a secondary ring electrode circling the nozzle cause reducing the fiber deposition and consequently decrease the density of the membrane, the parameter which reduces the pore size. in addition, controlling fiber distribution, post electrospinning modification and using temporary spacers can be utilized for controlling pore size (dong et al., 2015; haider et al., 2018) . as discussed above and based on the size exclusion process, microfiltration larger sized bacteria are substantially seized by the membrane but it is not efficient in separating small sized viruses within 0.01-0.1 µm range size (mi et al., 2014a; barhate and ramakrishna, 2007) . so, rejection of bacteria smaller than membrane pores or viruses needs the incorporation of antiviral or antibacterial agents into the membrane. also, after size-exclusion microbial removal of the membrane, intercepted bacteria can be released and induce membrane biofouling during subsequent filtration. therefore, antimicrobial agents are commonly used to prohibit bacterial j o u r n a l p r e -p r o o f growth and biofoul formation that would decline filter efficiencies (botes and cloete, 2010; wen et al., 2017) . various bioactive agents with different fundamental properties may have consequential impacts on bacteria removal. plus, nanofiltration membranes or ultrafiltration membranes with a positive charge on the surface are able to remove viruses selectively (mukherjee and de, 2017). moreover, incorporating antimicrobial agents into electrospun nanofibers enhance the antimicrobial activity of fabricated nanofibrous membrane (nasreen et al., 2013; park and kim, 2017 ). an ideal bioactive agent incorporated into the functionalized membrane should be nontoxic, water insoluble with no or slight leaching property. also, the functionalization process should not cause adverse influences on the quality and overall performance of the membrane. based on the majority of researches studied in this review, blending and post-modification strategies are two commonly used techniques to incorporate biocide agents into nanofibers aiming for water disinfection application (shalaby et al., 2018; he et al., 2018; makaremi et al., 2016) . blend electrospinning is an easy one-step procedure, mostly used for agents' incorporation into nanofibers (shabafrooz et al., 2014) . using the same solvent, the bioactive agent is dissolved directly into the polymer solution and a homogeneous blended solution of the incorporating agents in the polymer solution is prepared for the electrospinning step (pillay et al., 2013; fahimirad and ajalloueian, 2019). j o u r n a l p r e -p r o o f the agents incorporation into electrospun fibers can be performed after the electrospinning process by physical or chemical treatments. covalent and non-covalent immobilizations are fundamental methods for molecules attached to the fiber surface. non-covalent immobilization is performed by immersion of electrospun mats in a solution compromising the bioactive molecules. by treating with plasma the surface gets activated for subsequent modification using specified ligands like active amine groups. the affinity of incorporated agents to the electrospun nanofiber surface improves by covalent immobilization (wang and windbergs, 2017; kurusu and demarquette, 2019) . some commonly used antimicrobial or antiviral agents in electrospun nanofibers are discussed in this section. silver nanoparticles (agnps) are considered the most efficient nanoparticles for biological applications and the most extensively applied antibacterial agent for water disinfection mukherjee and de, 2018) . agnps are capable to puncture the microorganisms' cell walls, interact with their nucleic acids and attach to their enzymes, which cause the cell membrane destruction and finally growth inhibition. different feasible interactions of ag + ions with various bacterial biomolecules are documented. furthermore, the extended range of antibacterial activities and virulence effects of ag + ions toward several microorganisms (e.g. bacteria, viruses, and fungi) at only a few mg ml -1 are confirmed in previous studies. thus, silver nanoparticles are recognized as potent disinfection agents (lópez-heras et al., 2015) . in water purification, nanosilver materials have been mainly applied to prevent the formation of there are three main methods for agnps incorporation into electrospun nanofibers including 1) blending of prepared synthesized agnps solutions to the polymer solution, 2) agnp synthesis in the polymer solution by employing a precursor, and 3) post-treatments of the electrospun nanofibers for agnp synthesis by reduction of the precursor that has been spun along with the electrospinning solution (fahimirad and ajalloueian, 2019). there are three main approaches to produce iron oxide nanoparticle-nanofiber composites, including (1) electrospinning of solution containing prepared ionps, (2) in-situ synthesizing of ionps during the electrospinning process or in the solution to be electrospun and (3) it has been indicated that due to electrostatic interaction, cunps illustrate antibacterial functions on the bacterial cell through different mechanisms, such as adhesion to the bacterial cell wall, lead to detrimental impacts on protein structure within the cell membrane, denaturation of j o u r n a l p r e -p r o o f proteins in inertial parts of the cell , and adverse effects on phosphorus-and sulfur-containing compounds like dna (raffi et al., 2010) .recently, cunps have gained considerable interest because of their broad-spectrum and acutely effective antibacterial activity with comparatively low cost and high scalability (taner et al., 2011) . recently, zinc oxide (zno) has received much attention due to its non-toxic profile, effective antibacterial activity, adsorptive properties, mechanical, chemical, and thermal stability while encountering diverse environmental conditions (tiwari et al., 2018) . zno particles have illustrated antimicrobial activity against both gram-positive, gram-negative bacteria and even against spores wagner et al., 2016) . zno nps are considered bio-safe, nontoxic, and biocompatible (hameed et al., 2016; farrokhi et al., 2019) . in comparison with bulksized particles, nanoparticles can pass through bacterial cell walls more simply. the release of zn 2+ ions from nps destroy the cell membrane and subsequently enhance cellular internalization of the nanoparticles. it is also confirmed that the antimicrobial function of zno can be ascribed to photocatalytic activity. by receiving uv light which promotes its interaction with bacteria, ros, which has a phototoxic effect on bacteria, will be produced (dimapilis et al., 2018) . journal pre-proof tio 2 is a biocompatible chemical thermally stable compound with high photocatalytic activity and has shown good antimicrobial activities with wide spectrum function against microorganisms (gram-negative and gram-positive bacteria, fungi, and virus). the generation of reactive oxygen species (ros) is the major mechanism of tio 2 . due to its photocatalytic nature, antimicrobial activity of tio 2 nps enhances by exposing uv light on its surface (de dicastillo et al., 2020; levchuk et al., 2018b; levchuk and sillanpää, 2020) . it is proved that lanthanum compounds, such as lanthanum hydroxide (la(oh) 3 ), lanthanum carbonate (la 2 co 3 ), and lanthanum hydroxide (la(oh) 3 ) can attach to phosphate so firmly that they can generate lapo 4 and remove redundant phosphate in a bacterial cell. according to the very significant band to phosphate, nano-lanthanum (la) species represent high effectiveness adsorption and suppress microbial growth by inhibition of the microorganism growth liu et al., 2017) . carbon is the chemical element with atomic number 6 and six electrons situate 1 s 2 , 2 s 2 , and 2p 2 atomic orbital. graphene is a one-atom-thick hexagonal structure consisting of a 2-dimensional bhatnagar et al., 2013) . furthermore, the existence of these functional groups advances the interactions with biomolecules and leads to bacterial death with no intracellular process. go nano-sheets with sharpe edges hurt the bacterial cell membranes, lead to leakage of the intracellular matrix and eventually cause inactivation of bacteria. plus, go generate oxidative stress by producing ros and lead to dna damage and mitochondrial dysfunction (kumar et al., 2019) . in addition, the antiviral activity of go is confirmed by several experiments (ye et al., 2015) . single-walled carbon nanotubes (swnts) are nanometer diameter cylinders fabricated of rolled up graphene sheet in the form of a tube. generally, swcnt length is in the micrometer range and their diameters vary from 0.4 to 2 to 3 nm (eatemadi et al., 2014) . swnts have presented strong and board spectrum antimicrobial activities. the antimicrobial activity of swcnts has been confirmed to be varied by several factors. for instance, longer length nanotubes exhibited superior antimicrobial activity, swcnts having surface groups of -oh and -cooh illustrate more strong antimicrobial activity in comparison with swcnts-nh 2 , also the diameter of nanotubes is an important factor governing their antibacterial effects (dong et al., 2012) . chitosan [poly-(b-1/4)-2-amino-2-deoxy-d-glucopyranose] is quaternary ammonium cations are positively charged polyatomic ions. these ions contain a positively charged nitrogen "head" binding four bonds r including an alkyl group or an aryl group. quaternary ammonium compounds are salts of quaternary ammonium cations (tezel and pavlostathis, 2012) . because of their positively charged sites, they are able to generate electrostatic bonds with the negatively charged sites on bacterial cell walls, resulting in disruption of a cell wall, defect cell membrane permeability and consequently sever leakage of intracellular low-molecular-weight materials (chen et al., 2014) . qacs target bacterial cell membranes. therefore, they illustrate extended-spectrum antimicrobial activity and have been widely employed to construct an antibacterial surface (jennings et al., 2015) . quaternized poly benzalkonium chloride (bac) are some important kinds of qacs (zhu et al., 2018) . easy release of biocides from the membrane improves their exposure rate to bacterial cell. there is a challenging point since the leaching profile of incorporated biocides determines long term bactericidal efficiency of the membrane. leaching of bactericidal agents resulted in the diminution of the membrane antimicrobial performance over time. gradual leaching of the blended biocides during the filtration process not only declines the antibacterial activity, but may j o u r n a l p r e -p r o o f also lead to secondary pollution (fu et al., 2014) . besides chemical contamination and cytotoxicity issues, the continuous release of bactericidal agents causes the development of bacterial resistance due to being exposed to sub-inhibitory concentrations of biocides (sile-yuksel et al., 2014; mukherjee and de, 2018) . thus, there is a challenge to provide process eluding leaching of toxic materials while illustrating rapid pathogens killing ability. nanofiber coatings based methods which promote contact pathogen-killing capacity are promising and can be obtained by chemical modification with tethered biocides functionalities. these strategies may be successful by regarding the right control over the binding quality between the active agent and the underlying biomaterial surface (zhang et al., 2016; bazaka et al., 2015; hilpert et al., 2009) . despite there are numerous researches on application of antibacterial electrospun nanofiber membrane in water filtration, the leaching pattern and durable bactericidal efficiency of the membranes have not been studied comprehensively. this method is generally used for testing the inherent antibacterial performance of fabricated electrospun nanofibers as a membrane. this assay generally consists of qualitative detection and quantitative measurement techniques (zhu et al., 2018) . the inhibitory activity of electrospun nanofibers is assayed by the inhibition zone diameter or agar diffusion method toward the considered bacterial sample, based on the clinical and laboratory standards institute (clsi document m02-a12) (clsi, 2015) . for this reason, 100 μl overnight culture of the tested bacteria (10 6 cfu/ml) is spread across the surface of an appropriate agar plate, the electrospun nanofiber is cut to disk with about 10 mm diameters, sterilized under uv light for 20 min and then incubated on the plates for 18-24 h at 37 °c. then, the area of bacteria growth is detected, and the diameter of the inhibition zone around the electrospun nanofiber is measured. this procedure modifications are also used (santos et al., 2016; jatoi and al mamun, 2020; fig.3 a) . the antibacterial function of the membrane will lead to changes in the bacteria cell morphology. this method is a shaking flask method. briefly, an appropriate amount of sample sterilized nanofiber is weighted, dipped into a flask containing pbs buffer with a cell concentration of 1-4 × 10 5 cfu ml -1 . the flask is incubated with continuous shaking at 37 • c for a determined time. after serial dilutions by the phosphate buffer, the bacterial suspensions are plated in the agar plate. the inoculated plates were incubated at 37 • c for 24 h and the viable bacterial cells are counted by a colony counter (kleyi et al., 2015) . also, the number of bacteria after incubation for a determined time can be indirectly measured by spectrometric optical density at 625 nm . then, the reduction rate is calculated with the following equation: where r is the reduction rate, a is the number of bacteria isolated from the inoculated electrospun nanofibers after defined time contact time, and b is the number of bacteria isolated from the inoculated electrospun nanofibers at zero contact time (yao et al., 2016) . j o u r n a l p r e -p r o o f the aatcc 100 test method quantitatively evaluates the bacteriostatic (growth inhibition) or bactericidal (killing of bacteria) ability of textiles over a 24 hours contact. for this test, firstly a defined weighted of nanofiber is cut (about 3 mg), get sterilized by uv light, then inoculated with 0.1 ml microbial suspension (1-1/5 × 10 5 cfu ml -1 ) and finally overnight incubated at 37 • c. over determined contact period, 10 ml pbs buffer is added to the falcon tubes containing the inoculated treated electrospun nanofiber. after 1 min shaking, 10 µl of the solution is cultured on nutrient agar plates and incubated for 24 h (ardekani et al., 2019). astm e2149 is another antimicrobial standard method used for evaluating the antibacterial function of immobilized antimicrobial nanofibers under dynamic contact conditions. the antibacterial efficiency is evaluated depending on the contact time from several mints to 24 h between the bacterial solution and the sample (ungur and hrůza, 2017) . for both methods, the percentage of growth reduction is calculated with the r equation, mentioned above. in this method, some known dyes are been used to probe if the entrapped bacteria are inactivated by membranes and quantify surviving bacteria, representing an operative, visual and precise antibacterial assay (zhu et al., 2018) . for example, a common dye-based method is detecting the optimal analytical parameters for fluorescence measurements from the dyes syto and propidium iodide (pi). the basis of this approach is the attachment of syto to live-cell and propidium iodide (pi) to dead cells or cells with defected membranes. the optimal analytical parameters are used for measurement fluorescence by evaluating the intensity of emissions at 505-515 nm for syto and 600-610 nm for pi which interpret to quantify of the live cells minimum inhibitory concentration (mic) represents the minimum amount of antibacterial membrane, which could inhibit bacterial growth. in this method, the defined weighted of nanofiber is dissolved in water (or proper dissolvent), 100 µl of this solution is added to the first well and serially diluted by transferring 50 µl of the well pipetted content to the next well containing 50 µl media. thereafter, 50 µl of bacterial cultures (1×10 6 cfu ml -1 ) is poured to each well and plate is incubated at 37 ºc for 24 h. to detect the bacterial growth, resazurin or piodonitrotetrazolium chloride is added to wells. the wells that turned pink (if resazurin used) or purple (if p-iodonitrotetrazolium chloride used) represents the surviving of bacteria, hence no growth inhibition. the nanofiber concentration in the last growth inhibited well is considered as the mic value (nthunya et al., 2017). the bacteria retention test can also be performed with a dead-end filtration module using a vacuum filtration cell, a syringe filter holder 25mm, millipore and a dead-end filtration cell j o u r n a l p r e -p r o o f system (jabur et al., 2016; daels et al., 2011; son et al., 2009) . before the experiments, the membrane cut diameter and sterilized. all pieces of filtration equipment are sterilized with an autoclave method for 20 min at 121 °c. the membrane is fitted into the device. after passing sterile water from the filter, the bacterial suspension is filtered through the membranes using a pressure. in this step there are two different techniques for evaluation of bacterial retention: 1) the filtrate is serially diluted with sterile distilled water and viable counts are assayed by plate counts. the colony count can be facilitated by staining bacterial cells with syto 9 fluorescent dye and using a fluorescence microscope . then the bacterial retention ratio is calculated in terms of lrv (log reduction value) by the equation: lvr= log (c f /c p ) where r= (1-(c p /c f )) × 100 electrochemical disinfection can destroy bacteria and viruses by electroporation and reactive oxygen species (ros) during a short time. electrochemical treatment devices electrochemical disinfection regarded as an effective portable water disinfectant. fabrication of electrospun j o u r n a l p r e -p r o o f porous membrane filter using agents to provide a conducting bed and a strong electric field, facilitate electroporation and production of ros, which signifies the disinfection process (hong et al., 2016; huo et al., 2018) . for testing this ability, an electrochemical filtration device with electrospun nanofiber as a filtration membrane is used. then, a saline solution containing bacterial suspension flows through the nanofiber filter using low voltages at a defined flow rate. the bacterial removal efficiency is calculated by the lvr equation (wen et al., 2017; tan et al., 2018; xie et al., 2020) . in order to determine the deposition of bacteria on a filtrated membrane and the possibility of biofouling, instantly after filtration, the membrane is transferred to an autoclaved beaker filled with pbs buffer and sonicated. the bacteria in the suspended membranes are measured by counting the number of colony cells. moreover, the morphology of trapped bacteria is investigated using sem (xie et al., 2020; makaremi et al., 2016; wen et al., 2017) . different kinds of electrospun nanofibers have been recently fabricated for bacterial removal from water are illustrated in tables 1. usually, evaluation of the antiviral function of nano-filters is carried out using bacteriophage and polypropylene nonwoven textile as support layer, the structure shows excellent retention of bacteria and fine solids, with 240 -400 nm pore size and operating pressure < 2 000 mbar. naked filter is anther novel commercially application of nanofiber in household/bottled water filter with ability to remove 99.9999% of the micro-organic contaminants. nanotrap is another commercial household water filter produced by coway company. astrapool, fluidra has introduced nanofiber based product applied in filtration system for residential pools (http://electrospintech.com/products.html#.xvs_nm0zbiu). liquidity nanotech corporation has created electrospun nanofiber membrane made water purification cartridge with superior flow rate, about a cup per minute, good microbiological retention, 6-log bacteria reduction, 4-log virus reduction and 6-log cyst reduction and simple usage process (https://product.statnano.com/product/1981/liquidity-water-purification-cartridge). pentair company has produced polyethersulfone nanofiber-based cartridge for industrial water purification applications. the cartridge is an absolute barrier to bacteria and viruses: with more than 4-log reduction rate (https://www.directindustry.com/prod/pentair-x-flow/product-71363-1779744.html). the researcher's and industry's attention to research and development of electrospun nanofibrous membranes has been growing because of its simplicity, low-cost, scalable molecules incorporation process on the fabricated non-woven mats, production of membranes with the high surface area.. high surface area to volume ratio, uniform pore size, and high pore interconnectivity and adequate antibacterial property improve the performance of the nanofibrous membrane in water disinfection application (subramanian and seeram, 2013) . however, there are several major concerns to be noticed for the application of electrospun nanofiber in water disinfection. although high surface area and porosity of the electrospun nanofiber are significant advantages, which enhance permeability and selectivity, they also lead to higher mechanical stresses. consequently, the membrane might be compacted or deformed through the filtration process, which causes loss the porosity and subsequently decreases the also, further experimental studies needed to conduct proper control of biomolecules release rate from nanofiber, to ensure a balance between successfully deactivate the bacteria strains and lengthen the period of the function, and minimize contamination. therefore, fabrication of membrane representing inherent self-cleaning, antiviral, and the antibacterial and anti-biofouling feature has gained immense attention for industrial application. recently, focusing on the production of smart antibacterial surfaces has led to a promising "kill−release" strategy. this approach proposed the fabrication of dual-functional antibacterial surfaces by incorporating biocides into non-fouling materials. these membranes are able to maintain their long-term antibacterial activity by killing bacteria attached to their surface and subsequently are potent to release the dead bacteria to reveal a clean surface (wei et al., 2017) . although these smart membranes are applied for biomedical applications, the strategy can be promising for further j o u r n a l p r e -p r o o f designing of novel electrospun nanofiber with these dual functions and strong long-term functional ability in water disinfection. as it is illustrated in tables 1 and 2, despite the significant results obtained from the application of electrospinning in water filtration membrane designing, there are some gaps in this research area. for instance, there are no unanimous standard methods for evaluating the antibacterial or antiviral potential of fabricated electrospun water disinfecting filters. moreover, most of the researchers have used static antibacterial assay approaches that are unable to represent the membrane antibacterial performance under the dynamic water filtration process. moreover, recent related studies have not investigated comprehensibly the long-term antibacterial or antiviral performance of produced nano-membrane in water disinfection. due to extensive endeavors aiming to produce novel smart antibacterial and antiviral membranes and, electrospun nanofibers should be developed rapidly as great candidates for a high effective anti-biofouling membrane for water treatment. the water-stable nanofibers was able to bind to two different viruses and achieved a 3.3 lrv for ppv and a 4.2 lrv. 2d nanostructures for water purification: graphene and beyond zinc oxide nanoparticles for water disinfection electrospun nanomaterials implementing antibacterial inorganic nanophases antimicrobial activity of single-walled carbon nanotubes suspended in different surfactants superhydrophobic modification of pvdf-sio 2 electrospun nanofiber membranes for vacuum membrane distillation carbon nanotubes: properties, synthesis, purification, and medical applications. nanoscale research letters electrospun carbon nanofiber membranes for filtration of nanoparticles from water production of recombinant antimicrobial polymeric protein beta casein-e 50-52 and its antimicrobial synergistic effects assessment with thymol synthesis and therapeutic potential of silver nanomaterials derived from plant extracts. ecotoxicology and environmental safety naturally-derived electrospun wound dressings for target delivery of bio-active agents. international journal of pharmaceutics heavy metal-mediated changes in growth and phytochemicals of edible and medicinal plants. inmedicinal plants and environmental challenges nanocarrier-based antimicrobial phytochemicals recombinant production and antimicrobial assessment of beta casein-ibamp 4 as a novel antimicrobial polymeric protein and its synergistic effects with thymol recent advance in antibacterial activity of nanoparticles contained polyurethane the effect of soft nanoparticles morphologies on thin film composite membrane performance waterborne viruses: a barrier to safe drinking water. plos pathogens water purification by membranes: the role of polymer science bacterial passage through microfiltration membranes in wastewater applications urban wastewater disinfection by filtration technologies. desalination electrospun nanofibrous filtration membrane the antibacterial activity of ta-doped zno nanoparticles. nanoscale research letters sources, behaviour and health risks of antimicrobial resistance genes in wastewaters: a hotspot reservoir a comprehensive review summarizing the effect of electrospinning parameters and potential applications of nanofibers in biomedical and biotechnology in vitro antibacterial activity of zno and nd doped zno nanoparticles against esbl producing escherichia coli and klebsiella pneumoniae. scientific reports high speed water purification and efficient phosphate rejection by active nanofibrous membrane for microbial contamination and regrowth control screening and characterization of surface-tethered cationic peptides for antimicrobial activity silver nanowire-carbon fiber cloth nanocomposites synthesized by uv curing adhesive for electrochemical point-of-use water disinfection antimicrobial nanomaterials as water disinfectant: applications, limitations and future perspectives impact of water quality parameters on bacteria inactivation by low-voltage electroporation: mechanism and control fabrication of electrospun chitosan/nylon 6 nanofibrous membrane toward metal ions removal and antibacterial effect polyacrylonitrile nanofiber mats containing titania/agnp composite nanoparticles for antibacterial applications. materials research express quaternary ammonium compounds: an ironcontaining nanomaterials: synthesis, properties, and environmental applications. rsc advances removal of natural organic matter (nom) from water by ion exchange-a review titanium dioxide-based nanomaterials for photocatalytic water treatment. inadvanced water treatment recent developments in reverse osmosis desalination membranes electrospun gelatin nanofibers loaded with vitamins a and e as antibacterial wound dressing materials antimicrobial nanomaterials for water disinfection and microbial control: potential applications and implications. water research needleless electrospinning: a practical way to mass production of nanofibers a novel point-of-use water treatment method by antimicrobial nanosilver textile material synthesis of antibacterial nanofibrous membrane based on polyacrylonitrile (pan)/chitosan by electrospinning technique for water purification application investigations into the antibacterial behavior of copper nanoparticles against escherichia coli. annals of microbiology control of biofouling on reverse osmosis polyamide membranes modified with biocidal nanoparticles and antifouling polymer brushes fabrication of carbon nanotubes reinforced silica composites with improved rare earth elements adsorption performance a comprehensive review: electrospinning technique for fabrication and surface modification of membranes for water treatment application occurrence, genotoxicity, and carcinogenicity of regulated and emerging disinfection by-products in drinking water: a review and roadmap for research. mutation research/reviews in mutation research recent advances in nanofibrous membranes: production and applications in water treatment and desalination. desalination recent developments in antimicrobial polymers: a review electrospun nanofibers: from filtration membranes to highly specialized tissue engineering scaffolds electrospun nanofibers hybrid composites membranes for highly efficient antibacterial activity evaluation of the physical and chemical characteristics of water on the removal efficiency of rotavirus in drinking water treatment plants and change in induced health risk optimization of wastewater treatment processes using molecular bacteriology advanced oxidation processes for the removal of natural organic matter from drinking water sources: a comprehensive review antibacterial electrospun chitosan/poly (vinyl alcohol) nanofibers containing silver nitrate and titanium dioxide new directions in nanofiltration applications-are nanofibers the right materials as membranes in desalination? electrospun nanofibrous membranes for water purification novel agnws-pan/tpu membrane for point-of-use drinking water electrochemical disinfection synthesis, characterization and antibacterial investigation of silver-copper nanoalloys role of quaternary ammonium compounds on antimicrobial resistance in the environment. antimicrobial resistance in the environment nanotechnology for water purification: electrospun nanofibrous membrane in water and wastewater treatment resazurin assay for assessment of antimicrobial properties of electrospun nanofiber filtration membranes modified polyurethane nanofibers as antibacterial filters for air and water purification. rsc advances nanoparticles composed of zn and zno inhibit peronospora tabacina spore germination in vitro and p. tabacina infectivity on tobacco leaves functional electrospun fibers for the treatment of human skin wounds a novel gravity-driven nanofibrous membrane for point-of-use water disinfection: polydopamine-induced in situ silver incorporation. scientific reports nanofibrous microfiltration membranes capable of removing bacteria, viruses and heavy metal ions electrospun nanofibrous membranes for high flux microfiltration electrospun nanofiber membranes. current opinion in chemical hepatitis e virus genotype 3 strains and a plethora of other viruses detected in raw and still in tap water a review of polymeric membranes and processes for potable water reuse. progress in polymer science smart antibacterial surfaces with switchable bacteria-killing and bacteria-releasing capabilities. acs applied materials & interfaces filtration and electrochemical disinfection performance of pan/pani/agnws-cc composite nanofiber membrane. environmental science & technology guidelines for drinking-water quality -4th ed world health organization world health organization. water, sanitation, hygiene, and waste management for the covid-19 virus: interim guidance prevalence of antibiotic resistance in drinking water treatment and distribution systems swnts-pan/tpu/pani composite electrospun nanofiber membrane for point-of-use efficient electrochemical disinfection: new strategy of cnt disinfection preparation of pan nanofiber web and its antimicrobial and filtration property antiviral activity of graphene oxide: how sharp edged structure and charge matter fabrication of a novel thin-film nanocomposite (tfn) membrane containing mcm-41 silica nanoparticles (nps) for water purification removal and inactivation of waterborne viruses using zerovalent iron. environmental science & technology photo-crosslinked pva/pei electrospun nanofiber membranes: preparation and preliminary evaluation in virus clearance tests. separation and purification technology polyvinylidene fluoride membrane blended with quaternary ammonium compound for enhancing anti-biofouling properties: effects of dosage progress and challenges in photocatalytic disinfection of waterborne viruses: a review to fill current knowledge gaps polyethyleniminemodified chitosan materials for the recovery of la (iii) from leachates of bauxite residue polymeric antimicrobial membranes enabled by nanomaterials for water treatment mitigating biofouling on thin-film composite polyamide membranes using a controlled-release platform key: cord-023200-3caevjvh authors: falanga, annarita; galdiero, massimiliano; morelli, giancarlo; galdiero, stefania title: membranotropic peptides mediating viral entry date: 2018-02-13 journal: pept sci (hoboken) doi: 10.1002/pep2.24040 sha: doc_id: 23200 cord_uid: 3caevjvh the means used by enveloped viruses to bypass cellular membranes are well characterized; however, the mechanisms used by non‐enveloped viruses to deliver their genome inside the cell remain unresolved and poorly defined. the discovery of short, membrane interacting, amphipathic or hydrophobic sequences (known as membranotropic peptides) in both enveloped and non‐enveloped viruses suggests that these small peptides are strongly involved in breaching the host membrane and in the delivery of the viral genome into the host cell. thus, in spite of noticeable differences in entry, this short stretches of membranotropic peptides are probably associated with similar entry‐related events. this review will uncover the intrinsic features of viral membranotropic peptides involved in viral entry of both naked viruses and the ones encircled with a biological membrane with the objective to better elucidate their different functional properties and possible applications in the biomedical field. significant improvements have been achieved in recent years in the understanding of the multiple alternative ways of virus entry into susceptible cells. [1, 2] the strategies employed by viruses to enter cells are different according to the presence or absence of a lipid bilayer surrounding the virus. enveloped viruses present a membrane bilayer while non-enveloped viruses lack this membrane and present on their surface only capsid proteins. the mechanism of cell invasion by the two groups of viruses is rather diverse and the foremost difference is the direct consequence of their distinctive physicochemical state at the interface that occurs at the time of the encounter between the virus and the cell membrane; in particular, two lipid membranes confronting each other in the case of enveloped viruses as opposed to a layer consisting only of proteins that face a lipid layer in the entry of naked viruses. enveloped viruses entry exploits direct fusion with a cellular membrane through the involvement of specialized viral fusion proteins, present on the viral membrane and the consequent transfer of the nucleocapsid into the cytoplasm. [3] on the other hand, the entry of non-enveloped viruses, which lacking the outer viral membrane are unable to take advantage of the cellular mechanism of membrane fusion, involves the activation of viral lytic factors that induce cell membrane rupture. [4] the major features in membrane interaction by enveloped and non-enveloped viruses are reported in figure 1 . notwithstanding the different mechanisms of penetration, the key step is the entry process and the modification of the cell membrane which allows the viral genome to penetrate into the host cell and start the replication cycle in the appropriate cellular compartment. [5, 6] the most critical barriers for viral penetration and replication are the plasma membrane, the cytoplasm, and any other membrane that needs to be crossed in order to have access to the sites where viral replication or assembly take place. the overall picture of the mechanism of viral entry is becoming increasingly complete; in fact, depending on their dimension and structure, they have acquired different strategies to penetrate and take control of cell functions. [3, 4] the molecular details of the interactions at the interface of virus and cell surfaces are quite complex and highly variable, but there is a common idea that only a limited number of pathways allowing viruses to reach the sites of penetration exist, with enveloped and non-enveloped viruses presenting different and unrelated processes, but with general principles driving all fusion events. the main difference between fusion peptides of enveloped viruses and lytic factors of non-enveloped viruses is the fact that fusion peptides promote membrane fusion while lytic factors promote membrane disruption. in this review, we summarize current knowledge on the mechanism of membrane fusion of both enveloped and non-enveloped viruses with a focus on common principles. fusion peptide derived from enveloped viruses and lytic peptides from non-enveloped viruses are described with an effort to find undisclosed differences and similarities. we conclude reporting ground breaking applications of membranotropic peptides (both fusion and lytic peptides) which open a new exiting field of research. enveloped viruses depend on membrane fusion for cell penetration. [7] the two main routes used by enveloped viruses to enter the cell are the endocytic and non-endocytic pathways. [8] most enveloped viruses undergo endocytosis while only few are able to fuse directly to the plasma membrane. [8] viruses that use the endocytic route for cellular internalization are able to escape into the cytosol avoiding lysosomal degradation. therefore, penetration invariably involves membrane fusion mediated by specific viral glycoproteins (catalysts) with the main difference being that viruses using endocytic pathways fuse their envelope with the endosomal membrane from the luminal side. [8] thus, membrane fusion constitutes the essential and ubiquitous mechanism of entry of enveloped viruses, irrespective of their route of entry. [5, 9, 10] viral fusion proceeds through a hemifusion stalk with merging of proximal leaflets, which culminates in the opening and expansion of a pore connecting the two sides of the membrane. [7] the overall process is supported by a catalyst responsible of the lowering of the transition barriers and fusion proteins constitute the catalytic agents fulfilling this function. [11] the entry involves several other critical steps which include cellular receptor or co-receptor binding, internalization, uncoating, and release of viral nucleic acids at the proper site of replication. [7, 12, 13] various factors can mediate an efficient interaction between cells and viruses; for example, cholesterol rich domains are platforms for the entry of many enveloped viruses such as the influenza virus [14, 15] and many viral membranes contain much more cholesterol than the mammalian plasma membranes from which they are derived. [16, 17] viral fusion proteins undergo significant rearrangements from the pre-fusion to the post-fusion conformations which are triggered by either receptor binding, proteolytic cleavage or low endosomal ph, and eventually determine the exposure of previously sequestered hydrophobic peptides, loops, or patches, able to interact with and destabilize one or both the opposing membranes. [6, 7] crystallographic data on pre-and post-fusion structures of viral fusion proteins has resulted in the identification of at least three distinct classes based on their three-dimensional organization and mechanism of fusion ( figure 2 ). [11, 18] class i fusion proteins includes many of the most studied human pathogens such as influenza virus, human immunodeficiency virus (hiv), and ebola virus; the key features is the requirement of a proteolytic cleavage to initiate fusion, [19] activating these proteins which form an extended intermediate on the surface of the virion with the fusion peptide at the n-terminus of the protein engaged in the interaction with the target membrane and forming a link between the two fusing bilayers. the pre-hairpin structure is a unique state in which the fusion protein is simultaneously connecting two distinct membranes, the target membrane through the fusion peptide or patches and its own viral membrane through its transmembrane domain (tm). further conformational changes produce trimers of hairpins with a central a-helical coiled-coil structure. [20, 21] the 6-helix formation is related to the opening of the fusion pore and provides the major driving force for the process. thus, peptides able to prevent 6-helix formation, interfering with refolding of the fusion glycoproteins act as potent viral entry inhibitors. [22, 23] class ii fusion proteins, which include flaviviruses, alphaviruses, and bunyaviruses, are consistently different from those of class i and are mainly composed of b structures. [24] these fusion proteins are often heterodimers or homodimers lying almost flat on the virion surface. [24] they are organized in three globular domains: domain i is charclass iii fusion proteins have a mixed secondary structure with the central a-helical trimeric core similar to class i and two fusion loops located at the tip of an elongated b-sheet similar to class ii fusion proteins; the main representatives of class iii fusion proteins are protein g of vesicular stomatitis virus (vsv), [25, 26] gb protein of herpes simplex virus (hsv), [27] and of epstein-barr virus (ebv), [28] and gp64 from insect-cell baculovirus. [29] the post fusion structure is characterized by the presence of an internal fusion peptide in domain i organized in two hydrophobic fusion loops flanked by a b-sheet domain with a pleckstrin-like fold (domain ii). domain ii is nested with the largely a-helical domain iii, which is composed of trimers that give rise to an elongated, rod-like shape molecule. the a-helical domain is inserted in domain iv which is made of b-sheets and a very long c-terminal extension (domain v). interestingly, the multicomponent herpesvirus fusion machinery requires the presence of gb, gh/gl, and gd and multiple cellular receptors are engaged in the entry pathway in a cascade of molecular interactions. [30] gh/gl and gb are part of the core fusion machinery and need to cooperate in order to trigger the initial lipid destabilization which culminates in the fusion of the two bilayers. [31] [32] [33] despite the fact that it is still debated whether gh is merely a fusion regulator or it plays a more direct role in the fusion process, studies leave little doubt that also the gh/gl complex undergoes dynamic rearrangements during the fusion process. [34] the crystallographic post-fusion structure of gb shows that it is a canonical class iii fusion protein; [27] and several synthetic peptides derived from gb induce the fusion of large unilamellar vesicles and inhibit herpes virus infection. [33, 35, 36] irrespective of their structural differences, the three classes of fusion proteins seem to induce membrane fusion by essentially the same generic mechanism and a common refolding pathway is highly suggestive for the conservation of several phases of the process. the non-enveloped viruses exploit a different mechanism for entry because they lack a membrane which surrounds the protein capsid; as a consequence they require capsid-dependent mechanisms for penetrating the cell membrane or for exiting from the endosome. [37, 38] the entry process is much less known and involves a series of triggers producing conformational and structural rearrangements which end in the exposure and/or release of lytic factors. low ph, receptor interactions, protease cleavage, chaperone-assisted morphological changes, divalent cation chelation, or any combination of these factors which take place at the appropriate site of membrane penetration are essential triggers to produce the activated viral intermediate. [39] the mechanism of membrane disruption involves proteolytic cleavage of the coat protein and programmed exposure/release of small membrane-lytic peptides. in this scenario, viral penetration is mediated by short, membrane interacting, amphipathic and/or hydrophobic sequences present in proteins undergoing a conformational modification which allows the exposure of these domains and their interactions with membranes. [40] similarly to enveloped viruses, capsid proteins seem to be trapped in a metastable state waiting for a trigger to expose their membrane active peptides and the membrane penetrating ability of these sequences is fundamental for entry but at the same time their premature exposure has to be avoided before host cells provide the triggers. flock house virus (fhv) is one of the simplest and most studied non-enveloped viruses. the infection starts with the binding to one or more host cell receptors leading to a receptor-mediated endocytosis mechanism. [41] the receptor binding induces a conformational change that initiates uncoating or viral disassembly once the particle is exposed to low ph within the endocytic pathway blocking this event often inhibits infection. the immature provirion fhv is initially assembled from 180 copies of the coat protein alpha and, subsequently, it undergoes autoproteolytic cleavage to generate the mature infectious virion which contains a large n-terminal fragment, b (363 amino acids), and a small c-terminal fragment, g (44 amino acids). [41] the capsid shell is constituted by the central region of b forms, while the g peptides are the amphipathic helices non-covalently associated with the capsid interior. the g peptides are responsible of the interaction with the membrane and its local disruption which ends in viral entry. these peptides are composed of an n-terminal amphipathic helix separated by a proline-glycine-proline turn from a hydrophobic c-terminal region. the amphipathic g helices are located in the interior of the capsid and when fhv enters cells through receptor mediated endocytosis, the g peptides are exposed and determine the disruption of the membrane with consequent release of the viral genome in the cytosol. [41] this is a dynamic process with g peptides continuously, transiently, and reversibly exposed to the exterior of the capsid; g peptides may not only be exposed but also released from the virus particle which may represent a common paradigm of non-enveloped virus entry. in this dynamic process g peptides may continuously "sample" the environment until they encounter the appropriate cellular trigger; at this point the virus undergoes an irreversible conformational change in which the g amphipathic helices insert into the target membrane, allowing the viral rna to enter the cytoplasm. [40] 3 | m em b ra n otrop i c p ep ti d es many studies are devoted to the comprehension of the mechanism of insertion of fusion peptides, loops, or patches into monolayers inducing nipple formation and curvature in the target cellular membrane through many synergic interactions. the insertion in one leaflet of a closed bilayer will cause the increase of the surface area of the leaflet and the formation of a spontaneous curvature, which is one of the driving forces to reduce the energetic barrier needed for the achievement of fusion. [5] fusion and/or membranotropic domains in viral fusion proteins contain aromatic residues which together with alanines and glycines [6] contribute to the interaction with just one bilayer leaflet. many biophysical and structural techniques using synthetic analogues and model membranes have been used to determine physiologically relevant states during membrane partitioning. [6, 42] the two structural motif widely found in fusion proteins and able to produce membrane curvature are the amphipathic a-helix and tilted peptides. [6, 7] the segregation of hydrophobic and polar residues on the two opposite sides of the amphipathic a-helix is responsible of the superficial insertion into the upper monolayer which modifies the packsimilarly, also tilted peptides are characterized by an asymmetric distribution of hydrophobic residues, which again produces a modification of the organization of the membrane into which they insert. [6] the membrane bound conformation of the influenza virus fusion peptide is characterized by the presence of a hairpin of two tightly packed, antiparallel a-helices; [43, 44] the asymmetric insertion is determined by the fact that hydrophobic residues insert into the proximal membrane leaflet and polar residues project outward. the precise angle of the kink between the two arms depends on the sequence, the ph, and the lipid environment and determines the functional features necessary for activity. as a matter of fact, the compact hairpin structure drives favorable insertion, while its expanded structures promote subsequent membrane destabilization. [45] canonical class ii and class iii fusion peptides correspond to loops which do not undergo conformational changes upon insertion into the target membrane; the interaction clearly involves few hydrophobic residues, and the insertion into the outer leaflet of the membrane is superficial and probably inadequate to destabilize membranes. thus, a cooperative effect attained upon fusion activation is the only explanation for activity. [6] the idea of a single fusion peptide being exclusively responsible for the membrane perturbing activity has been overwhelmed by evidences supporting the concerted action of different membranotropic peptides, [46] which together with the canonical fusion peptide are involved in the modification of membrane curvature. [46] [47] [48] [49] [50] [51] many viral fusion proteins present an additional hydrophobic membrane proximal region at the intersection between the ectodomain and the transmembrane anchor (tm), the so-called mper (or pre-tm); [46] the unusual clustering of aromatic amino acid in these regions prompted the idea of their strong involvement in the fusion process. indeed, peptides corresponding to the pre-tm region partition into membrane interfaces and likely cooperate with fusion peptides and tm domains during apposition of membranes, enhancing the overall hydrophobicity of the environment and contributing to the distortion of the lipid membranes required for fusion. [6] the pre-tm of hsv-1 gh interacts strongly with membranes; [47] the pre-tm of gp47 of foamy virus induces fusion of model membranes; [52] the aromatic domain of the glycoprotein s2 of severe acute respiratory syndrome virus (sars) partitions into lipid membranes and perturbs their integrity; [53] the pre-tm region of the gp2 protein from ebola promotes perturbations of membranes when in a helical structure. [54] hsv fusion involves both gb and gh and several membranotropic sequences are present in both glycoproteins, [48, 55] although the precise role played by each of these regions remains to be elucidated. the two fusion loops at the tip of the domain ii of gb constitute a structural subdomain with the hydrophobic amino acids forming a crest lined on both sides by charged residues; [35] the concerted use of the two peptides produced a significant distortion of the target membrane bilayer, while when they were used separately they presented a lower membrane penetration. [35] the two charged residues represent a novel feature of fusion peptides with the presence of hydrophilic residues on either side favoring insertion. [35] fusion of inner and outer monolayers is clearly involved but not formation of pores, indicating that bilayer perturbation not complemented by leakage is a typical feature of viral fusion peptides which violate the host membrane without compromising its integrity. [35] several gh peptides are able to interact with membranes and play a role in the process. among these, gh625, represents a key achievement in grasping the role of hydrophobic viral peptides. [55] [56] [57] [58] the peptide contains residues critical for interaction such as aromatic residues (tryptophan and tyrosines) which are known for their preferred location at the membrane interface and for their ability to facilitate oligomerization [59] together with numerous hydrophobic residues (glycines, leucines, alanines) which are critical for membrane insertion; [6] at the c-terminus there is an arginine residue which is key for establishing peptide-lipid interactions. gh625 penetrates into membranes from its n-terminal side, and assumes an amphipathic helical conformation. [56] the n-terminal histidine acts as a switch for triggering viral fusion and strongly enhances the fusion activity; [56] the role of the histidine has been reported also for paramyxoviruses [60] and togaviruses. [61] yao et al. [62] in order to investigate how the fusion peptide (fp) and lipids also play a key role in this process, generating membrane curvature thanks to their physico-chemical properties. cholesterol is key as it selectively intercalates into the leaflet of the bilayer favoring its distortion without producing unfavorable hydrophobic/hydrophilic interactions. [16] the secondary structure of the fusion peptide of hiv changes according to the cholesterol content in the membrane, being a-helical in the absence of cholesterol, but shifting to a b conformation with the increase of cholesterol. [63] both the fully a-helical and the fully b-structured peptides are able to insert deeply inside the membrane, while the mixed secondary structures, present at intermediate cholesterol concentrations, are more superficially inserted into lipid bilayers and less effective in inducing membrane fusion. [63] it is likely that different secondary structures and domains with different content of cholesterol might be involved in different stages of fusion. [17] probably, lipid phase discontinuities between liquid ordered and disordered domains containing cholesterol produce membrane defects with exposed hydrophobic surfaces favoring a deeper insertion of fusion peptides, and promoting membrane fusion. [16] in conclusion, the clear view is that membrane fusion is a very complex process involving several domains of the fusion proteins which interact directly or indirectly with biological membranes, and contribute to the merging of the viral envelope and cell membrane. some peptides (as the lytic peptides of nodaviruses, picornaviruses, and reoviruses) can be generated by an autocatalytic cleavage step of a precursor, whereas others can be generated from the proteolytic activity of cellular enzymes. [64, 65] essentially we can classify lytic peptides in amphipathic a-helices and myristoyl groups; or we can classify them according to their mechanism of membrane action in those causing transient modification of the cellular membrane, pore formation, and total disruption of the limiting membrane. [4] although being clearly different among themselves and with fusion peptides of enveloped viruses, they present notable similarities. incubation of hela cells with various peptides corresponding to the c terminus of the l2 protein of papillomavirus, determines the entrance of propidium iodide into cells. [66] the incubation of the adenovirus internal protein vi with liposomes loaded with a fluorophore, caused the release of the entrapped fluorophore. [67] similarly, incubation of vp5* of rotavirus with liposomes entrapping a fluorophore caused its release, suggesting that vp5* is sufficient to perforate the lipid vesicles. [68, 69] the membrane disrupting g1 peptide of fhv also triggers the release of the fluorophore. [70, 71] as for enveloped virus fusion peptides, the amphipathic a-helix seems to be a key structural motif also for non-enveloped viruses. the n-terminal 21 amino acids of the fhv g peptide form an amphipathic a-helix which is commonly referred to as g1, [4] while the g peptide comprises also a c-terminal region which is commonly considered to play a supporting role for the correct positioning of g1. the g peptide assumes a random coil conformation in solution, while it adopts a kinked helical conformation in model membranes; similarly to other membranotropic peptides also viral lytic factors seem to be able to adopt a membrane-active conformation when interacting with the lipid bilayer. [38, 72] the g1 peptide is able to spontaneously partition into lipid bilayers and increases the membrane permeability of liposomes; [67, [69] [70] [71] 73] in particular, it mediates liposome lysis through the insertion only into the outer leaflet of the lipid bilayer, and locating parallel to the membrane surface, with the hydrophobic face of the helix packed against the membrane surface. [70, 72] a concentration dependent membrane leakage process [74] similar to other non-enveloped viruses such as poliovirus vp4 [75] and reovirus l1n [76] is observed. similarly to the influenza [77] and hiv gp41 [78] fusion peptides from enveloped viruses, the amphipathic region of g peptide presents a kinked helical structure in solution. the rigid, boomerang like structure assumed by the influenza fusion peptide in lipid environment is required to promote membrane fusion; [79] in fact, abolishing the kink in the structure or making it flexible eliminates membrane fusion [79] probably for the failure of the fusion peptide to insert deep into the lipid bilayer and pack against the hydrocarbon moieties. at neutral ph the g peptide is able to cause membrane disruption; while at low ph it is only able to alter its location relative to the capsid, but does not increase its membrane interacting ability. [74] surprisingly, thanks to a kinked structure and a tight alignment of the hydrophobic residues on one side of the peptide at low ph similar to influenza virus, fhv g peptide shows localized perturbation of lipid arrangements with no proof of pore formation. [38, 70] while membrane destabilization requires the simple insertion of the fusion peptide into the outer leaflet of the lipid bilayer, leakage needs both a deeper insertion and an interaction between peptides inside the membrane. the amphipathic region of g peptide oligomerizes in bilayers [71] with a low content of cholesterol, which being more fluid would promote association between peptides. [74] the presence of the g c-terminus is absolutely necessary for virus entry; [80] as a matter of fact, truncations, or point mutations in the c-terminal region of g determine a disordering of the pentameric bundle formed by the n-terminal amphipathic helices of g in fhv particles and hamper in vitro and in vivo membrane lysis. the pentameric bundles constitute the viral "membrane attack module," and an essential function of the g c-terminal region is to maintain this module in its correct conformation [4] (figure 3 ). the g and g1 peptides cause a similar localized disorder of the target bilayer and the presence of the cterminal hydrophobic helical region in full-length g make the peptide effective at concentrations achievable in the context of viral infections. the kinked helical structure seems to be a common trait of enveloped and non-enveloped viruses and differences in length and angularity of the helices as well as differences in the amino acid content may cause variations in the mechanism of interaction with the bilayer; the presence of this motif in viral proteins may signal a membrane associated role for this component during a certain step of the viral life cycle which adds to eventual other roles. adenovirus requires acidic ph for exposure of the amphipathic helix contained in its protein vi and disrupts endosomal membranes to release its nucleocapsid. [67] mutations in the amphipathic helix reduce infection and endosome escape, supporting the view that both the hydrophobic character and a-helical structure are key to allow maximal membrane disruption. the n-terminal amphipathic helix of protein vi, as fhv g peptides, lies parallel to model membranes with the hydrophilic face interacting with the phospholipid head groups, and probably causes disruption of the bilayer by introducing positive curvature in figure 3 schematic representation of fhv capsid (a). an expanded view of the crystallographic structure (pdb: 4ftb) of one subunit (a protein) showing the location of the amphipathic region of g peptide in yellow (b). schematic representation of a protein, which undergoes auto cleavage during maturation producing b and g (c) with relative sequence of g peptide membranes. [73] mutations in the amphipathic region of protein vi, which prevent insertion into the membranes, severely affects membrane penetration and cellular entry. [81] proteins vp1 and vp4 play a fundamental role in membrane penetration by poliovirus [82] with the exposure of amphipathic a-helical nterminal approximately 30 amino acid region of vp1 being necessary for liposome binding; [83] while the n-terminus of poliovirus vp4 contains a hydrophobic myristoyl (c14acyl) group for insertion into membranes. [84] following receptor binding, the amphipathic a-helix within the n-terminal portion of vp1, is exposed and probably forms pores to transfer the genome to the cytoplasm. interestingly, vp4 also performs a role in membrane penetration and the hypothesis is that vp1 primarily function is to secure the particle to the limiting membrane, while vp4 participates directly in pore formation. mammalian orthoreovirus protein l1 contains a small hydrophobic peptide (l1n) with a myristoyl group at its n-terminus. [85] disruption of cellular membranes, requires the complete dissociation of l1n peptides from the particle; auto-cleavage of l1 during reovirus entry generates l1n peptides that are linked to an n-terminal myristoyl group. after lipid association, the l1n peptide changes conformation from an extended to a b-strand rich secondary structure. [85] l1n is able to generate size-selective pores in erythrocyte or liposomal membranes. it is likely that the b-hairpins in l1n associate with the membranes forming a b-barrel pore. [85] probably, membrane disruption caused by l1n is similar to that of b-barrel toxins. [86] it is highly probable that a future more detailed knowledge of the mechanism used by lytic peptides of non-enveloped viruses will lead to the discovery of more common features between enveloped and nonenveloped membranotropic peptides. membranotropic peptides thanks to their adaptness to interact with the membrane, are opening the way for numerous applications. [87, 88] their ability to bind lipid membranes is correlated to their simultaneous hydrophobic and amphipathic nature, while their insertion into the bilayer is due to their capability to change conformation according to the environment; moreover, they are able to penetrate deep into the hydrophobic core but do not span the bilayer in a pore-like manner; on the contrary, they tend to self-associate at the interface between the membrane and the aqueous compartments. below are reported main applications. one of the main applications of fusion peptides of enveloped viruses is as inhibitors of viral penetration. their ability to directly interact with the hydrophobic surfaces present on cell membranes and/or fusion proteins allows them to interfere with virus entry. [89, 90] the inhibition mechanism is still unclear but it is likely that inhibition of infectivity is correlated to inactive aggregates formed between the fusogenic stretches present in the viral protein and in the peptides. in particular, the formation of aggregates is related to their ability to oligomerize or to mimic the mode of binding of their original domains in their partner protein; thus, stabilizing a pre-fusion intermediate and preventing merging of the bilayers. [55, [91] [92] [93] self-oligomerization of fusion peptides has been proposed to be responsible of inhibition by several groups. [94, 95] hiv fusion peptides form structurally defined oligomeric complexes which have been considered responsible of inhibition; [49, 96] moreover, mutants of the native sequence with a lower helical content and tendency to self-associate into b-sheets are able to inhibit membrane fusion with different magnitude and at various stages. [97] virip is a peptide designed to target gp41 fusion peptide and thus block hiv-1 infection; it has undergone clinical studies and was demonstrated to be as active as peptides targeting the coiled-coil. [98, 99] its clinical evaluation represents the proof of concept that membranotropic sequences could inhibit viral replication in infected individuals and may have potential clinical effectiveness. moreover, the knowledge that several domains are implicated in the fusion mechanism and may interfere with the intramolecular interactions between the several domains, clearly demonstrates that they all represent potential targets for the design of entry inhibitors. [100] membranotropic peptides are emerging as delivery vectors. [101] [102] [103] until now, the most widely used delivery vectors are cationic cell penetrating peptides (cpps), which enter essentially by endocytosis causing the entrapment of the cargo into endosomes with only minor quantities of the cargo able to reach the target where to exert the biological function. on the contrary, membranotropic peptides are internalized by direct penetration of the membrane and thus determine immediate bioavailability of the delivered molecule. fusion membranotropic peptides are particularly noteworthy because they can physically interfere with the membrane hydrophobic interior forming bulges that protrude from the membrane and ease contacts between fusing bilayers; in particular, they are able to translocate molecules through the plasma membrane directly into the cell, promoting lipid-membrane reorganizing processes, and causing local and temporary membrane destabilization with subsequent reorganization, circumventing the endosomal entrapment by favoring the escape from the endosome. [57, 104] the internalization mechanism is also related to the toxicity of the internalized drug and the development of resistance. the gh625 is able to directly translocate across the membrane bilayer and to transport several cargos such as quantum dots, [105] liposomes, [106] dendrimers, [107, 108] nanoparticles; [109] it is also able to cross the bbb in vivo. [110, 111] mpg is an amphipathic peptide composed of the fusion peptide of hiv-1 associated to an hydrophilic domain with positively charged residues derived from the nuclear localization sequence (nls) of simian virus 40 (sv40) large t antigen (pkkkrkv), through a spacer (wsq). [112, 113] in vitro mpg is able to deliver both sirna and dna after just 1 h. [114] the principal internalization mechanism was shown to be independent of the endosomal pathway and to involve the study of the internalization of g peptide derived from fhv [40] revealed that this is mediated by relatively high cell surface adsorption leading to enhanced macropinocytic uptake and cytosolic distribution and also revealed a higher efficiency of internalization compared with tat. [115] influenza virus fusion peptide has been used for increasing transfection efficiency, its ph-dependent fusogenic and endosomolytic activities are able to enhance lysosomal degradation before the contents of the endosomes are delivered to lysosomes. [116] the development of industrial applications in drug delivery is probably one of the most exciting and fastest growing fields, with the possibility of these peptides to pass through the bbb and become an important player in the fight against all pathologies correlated to neurosciences. scientists envisage also a possible use of viral membranotropic peptides as an alternative to classical antibiotics in order to combat the antibiotic resistance problem. [117] antimicrobial peptides (amps) are widely exploited and represent attractive candidates for the development of anti-infective agents; [117] [118] [119] recently, attention has been devoted also to the exploitation of membranotropic peptides derived from viral fusion proteins as antibacterial drugs. in fact, some amps with helical structure seem to share high sequence (preference for alanines and glycines) and structure (amphipathic a-helix) similarity with fusion peptides and suggest a convergent evolution correlated to their ability to disturb lipid bilayers. [120] the fusion domain of influenza virus was evaluated for its antibacterial activity; analysis showed that the amidation of the c-terminus is a key factor to render the fusion peptide an antibacterial peptide and optimization of the amphiphilic balance can improve efficacy. [120, 121] the antibacterial activity of viral membranotropic peptides is not yet widely evaluated and much work is still open in this field; in particular, their mechanism of perturbation of membrane bilayers may allow the design of novel sequences with the ability to denature the membrane bilayer of bacteria which will add to their many roles. development of effective vaccines against viruses is another worldwide concern. a potent vaccine needs to be able to induce both [122] in vivo prime-boost immunization enhanced humoral and cellular immune responses, suggesting the promising application of membranotropic peptides as vaccine candidates in future (table 1) . the membrane entry of enveloped and non-enveloped viruses employs fundamentally different mechanisms, although common themes have emerged in the entry process. this similarity is essentially represented by the presence/exposure of small membranotropic peptides which cause membrane disruption and/or promote membrane fusion. entry involves membrane fusion versus perforation, but cellular triggering factors and structural intermediates appear to share some similarities. interestingly there is also some similarity with the mechanism used by bacterial toxins to cross biological membranes in order to reach the cytosol; in fact, many toxins, undergo conformational changes which allow them to initiate the translocation process. [86, 123] how exactly both enveloped and non-enveloped viruses overcome host cell membrane barriers to deliver their genomes remains an intriguing problem. comprehensive structural and biochemical studies on enveloped viruses have brought to the conclusion that a unifying mechanism for host cell entry exists; where a membranotropic fusion loop, peptide, or patches catalyze fusion of the two membranes. in contrast, interaction of non-enveloped viruses with host cells during entry is less defined; while membrane active peptides have been discovered as necessary elements for entry in several well-studied non-enveloped virus capsids. in conclusion, it is now evident that the success of membranotropic peptides further stimulates challenging research on the unraveling of the many roles and applications that could be developed for both enveloped virus fusion peptides and small lytic peptides in nonenveloped viruses; membranotropic peptides are attracting increasing attention from the scientific community and their future will be dictated by the progresses in their industrial applications. http://orcid.org/0000-0002-7849-7024 entry of enveloped viruses into host cells: membrane fusion biophysical modulation of peptide-membrane interactions author biographies annarita falanga received the degree in food science and technology at the university of naples he earned his phd in virology from the university of cambridge (uk) in 1998. he was appointed lecturer in microbiology in 1994 at the department of animal health of the faculty of veterinary medicine of the university of naples 'federico ii'. he moved to the faculty of medicine of the second university of naples where he was appointed associate professor and then full professor of microbiology giancarlo morelli is a full professor of chemistry at the university of naples 'federico ii she carried out research activities at the columbia university of new york in 1996-1997. in 1999 she earned the position of assistant professor of inorganic chemistry at the university of naples and since 2015 she is associate professor. since 2012, she is adjunct professor at loyola university chicago. research activities focus on antimicrobial peptides and drug delivery mechanisms. how to cite this article membranotropic peptides mediating viral entry key: cord-013223-f43hks44 authors: chronopoulos, antonios; kalluri, raghu title: emerging role of bacterial extracellular vesicles in cancer date: 2020-10-15 journal: oncogene doi: 10.1038/s41388-020-01509-3 sha: doc_id: 13223 cord_uid: f43hks44 shedding of microbial extracellular vesicles constitutes a universal mechanism for inter-kingdom and intra-kingdom communication that is conserved among prokaryotic and eukaryotic microbes. in this review we delineate fundamental aspects of bacterial extracellular vesicles (bevs) including their biogenesis, cargo composition, and interactions with host cells. we critically examine the evidence that bevs from the host gut microbiome can enter the circulatory system to disseminate to distant organs and tissues. the potential involvement of bevs in carcinogenesis is evaluated and future research ideas explored. we further discuss the potential of bevs in microbiome-based liquid biopsies for cancer diagnostics and bioengineering strategies for cancer therapy. extracellular vesicles (evs) are released by all three domains of life-eukaryotes, bacteria, and archaea-and represent a universal, evolutionarily conserved mechanism for intercellular communication [1] . our growing appreciation of the functional significance of the human microbiota in health and disease has triggered a marked interest in microbial-derived evs and their functional role in intrakingdom and inter-kingdom communication, transfer of nutrients within microbial communities, delivery of virulence factors and toxins, horizontal gene transfer, and modulation of host immunity [2] [3] [4] [5] [6] [7] [8] [9] . in recent years, the advent of next generation sequencing technologies combined with large scale initiatives like the nih-funded human microbiome project and the eufunded metagenomics of the human intestinal tract consortium has expanded our understanding of the human microbiome and helped shape an evolving view of the human body as a "supraorganism" that harbors trillions of bacterial and human cells in~1:1 ratio [2, [10] [11] [12] . these large consortia have revealed staggering microbial gene diversity in the human microbiome as well as tremendous inter-individual variability in the microbial species inhabiting the human body [2, 13] . interestingly, while the human genome project has revealed a total number of 20,000 genes for the human genome [14] , currently accepted estimations from analyzing the bacterial communities in the gut have yielded more than 2000,000 bacterial genes vastly outnumbering the number of genes contributed by the human genome [2, 10] . a healthy human host is inhabited by more than 1000 microbial species [2] . most of those belong to a few dominant taxonomic groups, or phyla (actinobacteria, bacteroidetes, firmicutes, proteobacteria, cyanobacteria, fusobacteria) that gives some level of consistency across individuals at the higher taxonomic levels; however, the relative proportions of those phyla, as well as the individual microbial species and genes can vary dramatically across individuals, even in the case of monozygotic twins [15, 16] . individual humans may be 99.9% identical to one another in terms of their host genome (with 0.1% of the variation being mostly snps and small indels) but completely different (up to 90%) in their microbiomethe collective genome of one's microbial communities [17] . the relationship of these complex microbial communities (bacteria, fungi, viruses, and archaea) with their human host can be either symbiotic (mutually beneficial), commensal (neutral co-existence), or potentially pathogenic [18] . accumulating evidence has revealed microbe-host interactions can critically influence host health by manipulating host immune responses, nutrient metabolism, maintenance of the intestinal barrier, and protection from pathogen colonization [9, [19] [20] [21] . similarly, disruption of the gut microbiome (a term known as microbial dysbiosis) has been critically implicated in a range of human diseases including cancer, mental health, and cardiovascular and immune disorders [22] [23] [24] . the intersectionality of microbiome research and evs owing to advancements in both fields has emerged as promising research endeavor. the increasing appreciation that microbiota-derived ev can enter the systemic circulation and be detected in human body fluids is likely to stimulate completely new areas of investigation in microbiome research, biomarkers and liquid biopsies, bev-based therapeutics, onco-immunology, as well as fundamental microbial ev biology. bacterial cells communicate with their host and other bacteria through direct contacts and secretion of soluble products, such as metabolites (e.g., short-chain fatty acids), lipoglycans, quorum sensing peptides, nucleic acids, proteins, and membrane vesicles, also known as bacterial extracellular vesicles (bevs) [25, 26] . both pathogenic and commensal bacteria secrete bevsspherical membrane-enveloped particles ranging in size from 20-400 nm that disseminate part of the biological content of the parent bacterium into the extracellular milieu [6, 8] . detailed proteomic and biochemical analyses have shown that bevs carry diverse cargo including membrane-bound and periplasmic proteins, enzymes (such as autolysins) and toxins, polysaccharides, nucleic acids (dna and rna), and peptidoglycan [6, 27] . however, there does not appear to be a single mechanism modulating bev export, nor universal markers for bev cargo [28] . several lines of evidence suggest that bevs are heterogeneous in their structure, size, density, and molecular cargo composition, and this heterogeneity stems from the different biogenesis routes, the unique membrane envelope structure of the parental bacterium they originate from as well as the genetic background of the producing strain and the growth conditions [8] . mounting evidence shows bevs can follow different formation routes, which can lead to distinct bev subtypes with different molecular cargo and thus potentially different biological function [8, 29] . gram-negative bacteria follow two main pathways for vesicle formation. the first formation route involves blebbing of the outer membrane of the bacterial envelope, generating outer-membrane vesicles (omvs); and the second pathway entails explosive cell lysis forming outer-inner membrane vesicles (oimvs) and explosive outer-membrane vesicles (eomvs) [8, 26] . gram-positive bacteria produce cytoplasmic membrane vesicles (cmvs) through endolysin-triggered bubbling cell death [8, 29] . generally, omvs are the archetypal bevs and most heavily studied. the membrane blebbing process giving rise to omvs generally occurs through a disruption of crosslinks between the outer membrane and the underlying peptidoglycan cell wall layer. regardless of their biogenesis route, we collectively refer to all those vesicle subtypes as bevs. furthermore, gram-negative and gram-positive bacteria have a distinctly different cell envelope architecture, which is reflected in the membrane composition of the bevs they produce [8, 29] . the cell wall of gram-negative bacteria consists of a thin layer of peptidoglycan (a polymer-like mesh made of sugars and amino acids) in the periplasmic space between two membrane bilayers; the inner (or cytoplasmic) and outer membrane. the outer membrane contains lipopolysaccharides (lps; also known as endotoxin) on its outer leaflet and various membrane-bound proteins and channels such as porins that facilitate non-vesiclemediated transport. in contrast, gram-positive bacteria completely lack an outer membrane but have a much thicker peptidoglycan cell wall, which is linked to the underlying cytoplasmic membrane via lipoteichoic acids (lta) [8, 29] . mirroring this envelope architecture, gram-negative bevs consist of an outer membrane with an interior leaflet of phospholipids and an exterior leaflet of lps, which is known to engage toll-like receptor 4 (tlr4) [8, 26] . gramnegative bevs are typically enriched in various outermembrane proteins, such as ompa and encapsulate periplasmic luminal components. however, the presence of cytoplasmic cargo (dna, rna, virulence factors) is debated and likely contingent on the specific biogenesis route of the three different gram-negative bev subtypes (omv, oimvs, and eomvs). previous reports that have found nucleic acids and cytoplasmic proteins in omvs might not have been bona fide omvs, formed through controlled blebbing of the outer membrane as this mechanism does not give direct access to cytoplasmic cargo. instead, it is the biogenesis mechanism of the double bilayered oimv and/or the single bilayered-eomv subtypes that enables direct access to and encapsulation of cytoplasmic cargo. indeed, endolysin-mediated degradation of the peptidoglycan cell wall (a key process in explosive cell lysis) results in cell envelope fragments that can recircularize and enclose released chromosomal and plasmid dna and other nucleic acids or cytoplasmic components. gram-positive bev, also known as cmvs as previously mentioned contain both membrane and cytoplasmic components and show lta on their surface that can engage the toll-like receptor 2 (tlr2) [26, 29] . a number of other environmental factors can also affect the rate of vesicle production and even bias toward certain biogenesis routes. some of these include media composition, temperature, growth phase, iron and oxygen availability, exposure to antibiotics, and genotoxic stress [8, 28] . the localization of chromosomal dna in bevs from various gram-negative pathogenic bacteria (pseudomonas aeruoginosa, porphyromonas gingivalis, salmonella typhimurim) is mostly surface-associated (or extraluminal) with smaller amounts located in the intraluminal space [30] . sequencing of the intraluminal bev dna has been found to be enriched in specific regions of the bacterial chromosome involved in virulence, stress response, antibiotic resistance, and metabolism. it remains speculative at this point, whether surface-associated versus intraluminal bev dna serves different functions. it is not unreasonable to assume a potential role for external dna in biofilm formation versus a role for internal bev dna in intercellular communication and horizontal gene transfer of antibiotic resistance or virulence genes [30] . in addition to potentially modulating the innate immune response via or more cytosolic dna sensors, the possibility that pathogenic bev-derived dna can be transferred and detected in the nucleus of non-phagocytic cells (e.g. epithelial cells) [30] , raises the intriguing possibility that bacterial genetic material could be transferred to human somatic cells and integrated into the host genome. integration of bacterial dna sequences has been in fact detected more frequently in human cancer cells versus normal cells, especially in gastrointestinal (gi)-related tumors with close proximity to the gut microbiome, suggesting a potential role of bacterial dna in carcinogenesis [31] . bevs contain numerous microbe-associated or pathogenassociated molecular patterns (mamps/pamps) including lps, lipoproteins, peptidoglycan, and bacterial nucleic acids. the mamp content of bevs enables them to engage with host pattern recognition receptors (prr) in immune cells as well as nonimmune cells (e.g., epithelial cells at mucosal surfaces) to promote host pathology, immune tolerance, or confer protective immunity [31] . the diverse immunomodulatory effects of bevs depend largely on the specific parental bacterium and its relationship with the host. for instance, bevs from pathogenic bacteria have the potential to exacerbate infection by dampening immune responses [32, 33] , or trigger an overexaggerated immune reaction resulting in sepsis [5, 34] . in contrast, bevs from symbiotic or commensal bacterial species in the gi tract promote maturation and immunological tolerance to confer protection from colitis or sepsis [35, 36] . depending on the localization of prrs and the specific route of bev uptake, the recognition of bev-associated mamps/pamp can occur in various host cellular compartments, including the plasma membrane, endosomes, and cytoplasm [37] (fig. 1) . cell surface members of the tlr family, namely tlr2 and tlr4, recognize extraluminal bev ligands such as lps and lta molecules, peptidoglycan, and lipoarabinomannan [38] [39] [40] [41] [42] . the cytosolic receptors nucleotide-binding oligomerization domain-containing protein 1 (nod1) and 2 (nod2) are directly involved in sensing peptidoglycans (key component of bacterial cell wall) present in bevs secreted from pathogenic or commensal bacteria [43] [44] [45] [46] . nod1/2 are key components of innate immunity and critical in host defense against bacterial infections and modulation of inflammatory responses. more recently nod1/ 2 have been involved in maintaining intestinal homeostasis and microbiota balance [47, 48] . intraluminal bev nucleic acids can be detected via dna-sensing and rna-sensing receptors. following endocytosis, bev rna cargo can be sensed through endosomal tlrs including tlr3, tlr7, tlr8, and tlr13. similarly, rnas delivered into the cytoplasm following fusion of bevs with the host cell plasma membrane can activate cytosolic rna sensors such as rig-i-like receptors [5] . in a similar way, bev dna cargo can be sensed through the endosomal tlr9 or the cytosolic dnasensing cyclic gmp-amp synthase-stimulator of interferon genes pathway, although direct evidence for the latter is lacking [40, 49, 50] . various routes of bev endocytosis have been described in host cells including macropinocytosis, lipid-raft-dependent and lipid-raft-independent endocytosis, as well as dynamin-, caveolin-, and clarithindependent entry [6] . the utilization of different paths of uptake may reflect the size heterogeneity of bevs and the size selectivity of each route of endocytosis. in general, prr activation triggers the activation of kinases and transcription factors, that lead to the production of cytokines and chemokines resulting in the recruitment of immune cells and upregulation of co-stimulatory molecules commonly involved in adaptive immunity. tlrs for instance, signal via the adapter proteins myeloid differentiation primary response 88 or tir-domain-containing adapter-inducing interferon-β, leading to downstream activation and nuclear translocation of transcription factors including nuclear factor kappa-lightchain-enhancer of activated b cells, interferon-regulatory factor, and activator protein-1 to induce production of proinflammatory cytokines and type i interferons [5, 51] . there exists a growing consensus that bevs from the resident microbiota can enter the systemic circulation [26, [52] [53] [54] [55] [56] [57] . bevs released by bacteria in the gut lumen can cross the epithelial barrier to gain access into the underlying submucosa enabling them to interact with various resident immune cell populations (dendritic cells, neutrophils and macrophages) as well as potentially disseminate more widely around the body via the systemic or lymphatic circulation to reach distant tissues and organs or even the brain (fig. 2) . the presence of systemic circulating bevs was recently reported by tulkens et al. in the plasma of patients with altered intestinal barrier function [52] . of note, a number of communicable and noncommunicable diseases as well as lifestyle factors can trigger gut microbial dysbiosis thereby altering intestinal permeability, including obesity, diabetes, antibiotic use, diet and caloric restriction, or sleep deprivation. in the aforementioned study, patients with hiv, inflammatory bowel disease, or intestinal mucositis were found to have elevated circulating lps-positive bevs relative to healthy controls. the level of circulating bevs correlated positively, but modestly, with the plasma levels of zonulin-a biomarker of epithelial barrier integrity-that phosphorylates zo-1 proteins in epithelial and endothelial cells leading to tight junction disassembly. while these bevs are likely to be originating by gut bacteria, the authors did not fully prove it leaving the possibility of contributions from microbial niches in other body sites. simulating compromised tight junction integrity using an in vitro colitis model (caco-2 epithelial monolayer challenged with dss) also resulted in paracellular translocation of bevs. the same group by tulkens et al. most recently reported a detailed protocol for recovering bevs with high specificity from human body fluids, including blood plasma and stool through the sequential implementation of sizeexclusion chromatography and density-gradient ultracentrifugation [26] . the underlying physical property that distinguishes bevs from host-derived eukaryotic evs (eevs) in blood plasma is their differential buoyant density. when run on a density gradient, eevs float typically at 1.083-1.111 g/ml while bevs are slightly heavier at 1.133-1.201 g/ml. this allows for label-free isolation of bevs from eevs, although subsequent biochemical characterization is typically needed. this was the first study that provided a preliminary characterization of bevs in human body fluids with ompa and lps being the markers for bev identification. however, a low yield of bev in blood plasma (~10 6 bev/ml) was typically noted. interestingly, plasma contained bevs from both gram-negative (tlr4 reporter assay) and gram-positive bacteria (tlr2 reporter assay) and immunogold electron microscopy also identified the presence of both single-bilayered and double-bilayered bevs, the latter being indicative of oimvs, which typically contain cytoplasmic cargo and nucleic acids. the presence of chromosomal dna in circulating oimvs was not pursued further and 16s rrna sequencing data are lacking. implementation of rigorous experimental controls is imperative for samples with low microbial biomass that are inherently prone to misinterpretation due to contaminating molecules from commonly used laboratory reagents in dna extraction kits and library preparation [58, 59] . while these studies leave the impression that entry of bevs in the circulatory system occurs only under certain conditions of compromised gut epithelial barrier integrity, this phenomenon may be common even in the steady state of healthy individuals. indeed, a recent study looked into the biodistribution of fluorescently labeled bevs (derived from the major human gut commensal bacteria bacteroides thetaiotaomicron) following oral administration in mice under normal healthy conditions [53] . while the majority of the labeled bevs remained in the lumen of the gi tract, a small population could enter the circulatory or lymphatic systems via the gi tract. some of the bevs were found to accumulate in the liver, suggesting that they can transmigrate through the intestinal epithelium and enter the hepatobiliary system through the portal vein. a portion of the bevs could also be detected in the heart and lungs suggesting bevs can cross several host cellular barriers including epithelial barrier and the lymphatic-vascular endothelium to enter the bloodstream and disseminate systemically. even if the gut epithelial barrier is not compromised leading to increased paracellular transport, bevs could use other mechanisms to enter the circulatory system, such as active trans-cellular migration across the intestinal epithelium [60] . in fact, active trans-cellular migration across epithelial monolayers has been observed for eukaryotic exosomes (e.g., endocytosis, mvb formation, and exocytosis across the other side of the layer) and has even been suggested as a possible mechanism for breaching the blood-brain barrier [61] . luminal antigens and bevs may also be captured by dendritic cells (from the underlying lamina propria) and transported through the intestinal epithelium or via the assistance of mucus-secreting goblet cells [56, [62] [63] [64] . in addition, intestinal m-cells (specialized epithelial cells of the mucosa-associated lymphoid tissue) could be involved in the translocation of luminal bevs to the submucosa and systemic circulation [65] . macrophages infected with pathogenic bacteria are known to release exosomes with bev-associated components that can elicit pro-inflammatory responses. the uptake, intracellular trafficking, and processing of bev cargo by the intestinal epithelium needs to be further investigated to examine the possibility of de novo secretion of host epithelial-cellderived exosomes carrying luminal bev-associated cargo; this would represent a mechanism of transferring luminal antigens and bevs directly to antigen-presenting cells in the submucosa. circumstantial evidence for this exist from a study in which exosomes recovered from the basolateral compartment of bev-treated epithelial cells were found to encapsulate bev antigens as well as from a co-culture model in which exposure of dendritic cells to conditioned media from bev-stimulated epithelial cells resulted in polarization to a mixed t h 2-type and t h 17-type response [6, [66] [67] [68] . a case for the existence of blood-derived bevs has also been reported in a transgenic mouse model of alzheimer's disease [55] . metagenomic sequencing of the blood-derived bev dna revealed taxonomical diversity that reflected the diversity of the intestinal microbiota as well as a distinct bev-associated microbial landscape relative to wild-type controls. blood-derived bevs could represent an alternative to fecal sampling for profiling the gut microbiome and evaluating pathogenic variations in the intestinal microbiota (dysbiosis) in the context of neurodegenerative diseases. a few studies have also reported the presence of bacterial nucleic acids in the brain [69] . in view of the presence of bevs in the bloodstream and their ability to cross boundary epithelial layers, it is interesting to speculate that a fraction of the circulating bevs might gain access to the brain through the blood-brain barrier or alternatively be produced by brain-resident bacteria. the gut microbiome is highly dynamic and impacted by a plethora of environmental factors such as diet, exercise, sleep habits, and medications. the presence of bevs in systemic circulation through their translocation from gi tract or other microbial niches has challenged our notion that blood is a sterile compartment and has put bevs in the spotlight as long-range "hormonal-like" mediators of interkingdom communication. future investigations are expected to delve deeper into the cross-talk between the resident microbiota and distant organs mediated by systemically circulating bevs, as this would further expand our understanding of how the human microbiome can regulate tissue and organ homeostasis in health and disease. microbial dysbiosis is a major contributing factor in oncogenesis and tumor progression for a number of gi-tractrelated malignancies, including gastric, colorectal, liver, and pancreatic cancer, and might even influence the treatment response to chemotherapy and immunotherapy [33, [70] [71] [72] [73] [74] [75] [76] [77] [78] [79] . the mechanisms by which bacteria affect carcinogenesis and tumor progression are largely unknown. some of the tumor-promoting mechanisms could be direct such as by inducing genomic instability or indirect by generating a proinflammatory tumor microenvironment (tme) and suppression of immunosurveillance [72, 73] . although bevs have been implicated in the pathogenesis of inflammatory disease [6, 80] , their role in cancer remains unknown. it is also uncertain whether the tumor-promoting or tumorinhibiting effects of the inter-kingdom cross-talk between the intestinal or intra-tumoral bacteria and the host cells in the tme is mediated through secreted microbial metabolites such as scfas or bevs. it is a tantalizing possibility that the gut microbiome may be implicated in all carcinomas including those that are ostensibly remote from the gi lumen (or its drainage) through the immunomodulatory action of systemically circulating bevs on recipient cells in distant organs, or even participating in the formation of pre-metastastic niches. some indirect preliminary evidence toward this direction came out from a seminal retrospective pan-cancer examination of whole-genome sequencing datasets in the tcga (33 cancer types from 10,000 treatment-naïve patients totaling 17,000 samples) for microbial reads that found unique microbial signatures in tissue and blood that could discriminate between and within most major types of cancer, including tumors distally from the gi tract [57] . the authors used various computational approaches, including independently trained machine learning models to filter, normalize, and classify microbial sequences. after stringent filtering approaches to remove potential contamination that discarded up to 92.3% of total sequencing reads, a total of 0.9% of the total reads could be mapped to sequences of microbial origin and resolved to a particular genus. the tcga-derived blood-borne microbial dna (mbdna) signatures could discriminate within and between most cancer types even in low-grade tumor stages and in cancers lacking genomic alterations. apart from the retrospective bioinformatic analysis, the group further validated the mbdna models while benchmarking against plasma-based cell-free tumor dna (ctdna) assays in a separate cohort, which included a group of healthy controls and a group of individuals with advanced-stage cancer, including prostate cancer, lung cancer, and melanoma. remarkably, plasmabased cell-free mbdna retained strong generalizable discrimination between healthy controls and grouped patients with cancer with specificity and sensitivity exceeding 90% while also retaining high discriminatory capacity in pairwise comparisons between the three individual types of cancer and their respective healthy controls, except for melanoma. this study raises the possibility of using microbial-based liquid biopsies for early detection of certain cancers and tumors lacking known mutational drivers. such a microbiome-based oncology diagnostic tool might provide distinct advantages over regular ctdna assays in predicting and stratifying patients based on differential survival and treatment response. this is because oncogenesis can proceed at variable rates in hosts with similar cancer mutational landscapes but with host microbial factors playing a much bigger role. in fact, pancreatic tumors from long-term survivors have a high quantity and quality of neoantigens [81] , that exhibit homology to infectious disease-derived peptides, suggesting a neoantigen molecular mimicry with microbial epitopes, and demonstrating host microbial factors can be predictive of patient outcomes. this was corroborated with a recent study showing that the tumor microbiome in pdac patients is predictive of long versus short-term survival irrespective of the genomic composition of the tumor [74] . it remains undetermined whether the observed microbial nucleic acids in the blood came from live or lysed bacteria or bevs. the presence of live bacteria in blood is improbable as it would likely cause some degree of bacteremia but conclusive data could be made available through culturomics, rather than metagenomics, to identify the existence of otherwise hard-to-culture live blood-borne bacteria. the most likely scenario, given the results obtained from the plasma-derived, cell-free dna validation cohort as well as the accumulating evidence arguing for the presence of dna-containing circulating bevs, that at least a significant portion of those nucleic acids is associated with bevs. more work will be needed to determine whether this is the case, and whether the dna-containing microbial evs are driving cancer or are merely passengers. to date there is no mechanistic study investigating how bevs can impact oncogenesis and tumor progression, and their role is likely to be nuanced and context-dependent. previous studies of bevs in infectious diseases could provide hints for their putative role in cancer. for instance, microbial dysbiosis in cancer could trigger the systemic release of microbiota-derived bevs that could act as tumorpromoting entities by invoking tolerogenic immune reprogramming of the tme. this could be achieved through different routes by bevs driving suppressive cellular monocytic differentiation in a tlr-dependent manner, to indirectly elicit t-cell anergy [73] . the distal action of systemic gut-derived circulating bevs might also be the missing link between conditions associated with persistent disturbances in gut microbiota and metastatic dissemination of pre-established tumors. bev-associated mamps could interact with host cells in distant organs to initiate proinflammatory signaling and trigger alterations in the myeloid landscape to foster pre-metastatic niches for future colonization. from a therapeutic standpoint one could speculate that systemic administration of commensal healthy host-derived bevs directly to tumor-bearing hosts could represent a superior alternative to fecal microbiota transplantation that is currently being pursued in clinical trials [82] . bevs possess several intrinsic properties that have made them appealing candidates for vaccine development against infectious pathogens. bevs exhibit high stability to a wide range of temperatures and treatments and are nonreplicative in nature and thus safe, and carry many of the same immunogenic surface-and membrane-associated components of their parental bacterium [6] . depending on the originating species, bevs can stimulate both humoral and cell-mediated immunity and together with their nanoparticulate nature, provide them with their own adjuvanticity, as they are able to enhance t-cell response to antigens. for instance, the clinically approved omv-based 4cmenb vaccine containing three highly immunogenic proteins confers broad protective antibody responses against different neisseria meningitidis (n. menigitidis) serogroup b isolates [83] . notably, the same omv-based vaccine that protects against n. meningitidis also confers a level of cross-protection against neisseria gonorrhoeae likely due to the genetic and antigenic similarity shared between the two pathogens [84, 85] . the endotoxicity of bevs can be easily manipulated through genetic engineering techniques. one could speculate that bevs from certain commensal bacteria may have therapeutic value. in the future, we expect to see surging interest in the potential of bevs as cancer immunotherapeutic agents to elicit durable antitumor immune responses or alternatively as personalized or universal cancer vaccines. the potential of bevs in cancer immunotherapy was highlighted by a recent report showing that systemic intravenous administration of gram-negative bevs from the genetically modified escherichia coli (e. coli) msbb −/− strain (endotoxin-free) has a selective tropism for tumor tissue (potentially through the epr effect) and a remarkably capability of inducing long-term antitumor immune responses through the production of cytokines cxcl10 and interferon-γ that can fully eradicate established tumors without notable adverse effects [86] . similar antitumor effects were also observed for the grampositive bevs derived from lactobacillus acidophilus (l. acidophilus) and staphylococcus aureus (s. aureus). there is also an enormous potential in using genetic engineering techniques to modify bacteria and subsequently purify recombinant bevs for use as cancer vaccines. bevbased cancer vaccines decorated with multiple heterologous tumor antigens on their surface and/or immunostimulatory bacterial dna (cpg motifs) for targeted delivery to antigen-presenting cells hold immense potential for eliciting a strong durable antitumor immune response potentially in conjunction to ctla-4 and anti-pd1 immunotherapies. conflict of interest md anderson cancer center and rk hold patents in the area of exosome biology and are licensed to codiak biosciences, inc. md anderson cancer center and rk are stock equity holders in codiak biosciences, inc. rk is a consultant and scientific adviser for codiak biosciences, inc. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. the biology, function, and biomedical applications of exosomes current understanding of the human microbiome membrane vesicle release in bacteria, eukaryotes, and archaea: a conserved yet underappreciated aspect of microbial life virulence and immunomodulatory roles of bacterial outer membrane vesicles extracellular vesicle rna: a universal mediator of microbial communication? immune modulation by bacterial outer membrane vesicles outer-membrane vesicles from gram-negative bacteria: biogenesis and functions types and origins of bacterial membrane vesicles host-and microbiota-derived extracellular vesicles, immune function, and disease development human microbiome project c. structure, function and diversity of the healthy human microbiome a human gut microbial gene catalogue established by metagenomic sequencing are we really vastly outnumbered? revisiting the ratio of bacterial to host cells in humans the landscape of genetic content in the gut and oral human microbiome international human genome sequencing c. finishing the euchromatic sequence of the human genome organismal, genetic, and transcriptional variation in the deeply sequenced gut microbiomes of identical twins a core gut microbiome in obese and lean twins defining the human microbiome the nih human microbiome project microbe-derived extracellular vesicles as a smart drug delivery system the role of the microbiome in human health and disease: an introduction for clinicians intestinal barrier and gut microbiota: shaping our immune responses throughout life. tissue barriers the influence of the gut microbiome on cancer, immunity, and cancer immunotherapy microbial translocation is a cause of systemic immune activation in chronic hiv infection the microbiome in cancer immunotherapy: diagnostic tools and therapeutic strategies inter-kingdom signalling: communication between bacteria and their hosts analyzing bacterial extracellular vesicles in human body fluids by orthogonal biophysical separation and biochemical characterization global proteomic profiling of native outer membrane vesicles derived from escherichia coli environmentally controlled bacterial vesicle-mediated export through the wall: extracellular vesicles in gram-positive bacteria, mycobacteria and fungi bacterial membrane vesicles transport their dna cargo into host cells bacteria-human somatic cell lateral gene transfer is enriched in cancer samples neisserial outer membrane vesicles bind the coinhibitory receptor carcinoembryonic antigen-related cellular adhesion molecule 1 and suppress cd4+ t lymphocyte function helicobacter pylori and gastrointestinal tract adenocarcinomas circulating bacterial membrane vesicles cause sepsis in rats outer membrane vesicles of a human commensal mediate immune regulation and disease protection extracellular vesicles derived from gut microbiota, especially akkermansia muciniphila, protect the progression of dextran sulfate sodium-induced colitis the role of pattern-recognition receptors in innate immunity: update on toll-like receptors toll-like receptor 4 signaling licenses the cytosolic transport of lipopolysaccharide from bacterial outer membrane vesicles pseudomonas aeruginosa outer membrane vesicles modulate host immune responses by targeting the toll-like receptor 4 signaling pathway differential responses of pattern recognition receptors to outer membrane vesicles of three periodontal pathogens mycobacteria release active a. chronopoulos, r. kalluri membrane vesicles that modulate immune responses in a tlr2-dependent manner in mice bacterial membrane vesicles mediate the release of mycobacterium tuberculosis lipoglycans and lipoproteins from infected macrophages membrane vesicles from pseudomonas aeruginosa activate the noncanonical inflammasome through caspase-5 in human monocytes outer membrane vesicles from probiotic and commensal escherichia coli activate nod1-mediated immune responses in intestinal epithelial cells bacterial membrane vesicles deliver peptidoglycan to nod1 in epithelial cells aggregatibacter actinomycetemcomitans outer membrane vesicles are internalized in human host cells and trigger nod1-and nod2-dependent nf-kappab activation outer membrane vesicles prime and activate macrophage inflammasomes and cytokine secretion in vitro and in vivo gene-microbiota interactions contribute to the pathogenesis of inflammatory bowel disease dna sensing by the cgas-sting pathway in health and disease b cell activation by outer membrane vesicles-a novel virulence mechanism toll-like receptor signalling increased levels of systemic lps-positive bacterial extracellular vesicles in patients with intestinal barrier dysfunction the uptake, trafficking, and biodistribution of bacteroides thetaiotaomicron generated outer membrane vesicles fantastic voyage: the journey of intestinal microbiota-derived microvesicles through the body metagenome analysis of bodily microbiota in a mouse model of alzheimer disease using bacteria-derived membrane vesicles in blood the healthy human blood microbiome: fact or fiction? microbiome analyses of blood and tissues suggest cancer diagnostic approach comparison of placenta samples with contamination controls does not provide evidence for a distinct placenta microbiota reagent and laboratory contamination can critically impact sequence-based microbiome analyses elucidation of exosome migration across the blood-brain barrier model in vitro tumor-derived extracellular vesicles breach the intact blood-brain barrier via transcytosis goblet cells deliver luminal antigen to cd103+ dendritic cells in the small intestine dendritic cells express tight junction proteins and penetrate gut epithelial monolayers to sample bacteria cx3cr1-mediated dendritic cell access to the intestinal lumen and bacterial clearance peyer's patch dendritic cells sample antigens by extending dendrites through m cell-specific transcellular pores exosomes derived from m. bovis bcg infected macrophages activate antigen-specific cd4+ and cd8+ t cells in vitro and in vivo exosomes secreted by bacterially infected macrophages are proinflammatory vibrio cholerae o395 outer membrane vesicles modulate intestinal epithelial cells in a nod1 protein-dependent manner and induce dendritic cell-mediated th2/th17 cell responses 16s rrna next generation sequencing analysis shows bacteria in alzheimer's post-mortem brain the fungal mycobiome promotes pancreatic oncogenesis via activation of mbl fusobacterium nucleatum in colorectal carcinoma tissue and patient prognosis mutational signature in colorectal cancer caused by genotoxic pks(+) e. coli the pancreatic cancer microbiome promotes oncogenesis by induction of innate and adaptive immune suppression tumor microbiome diversity and composition influence pancreatic cancer outcomes the human gut bacterial genotoxin colibactin alkylates dna gut microbiome modulates response to anti-pd-1 immunotherapy in melanoma patients potential role of intratumor bacteria in mediating tumor resistance to the chemotherapeutic drug gemcitabine gut microbiome influences efficacy of pd-1-based immunotherapy against epithelial tumors versatile effects of bacterium-released membrane vesicles on mammalian cells and infectious/inflammatory diseases identification of unique neoantigen qualities in long-term survivors of pancreatic cancer modulating the microbiome to improve therapeutic response in cancer immunogenicity and safety of an investigational multicomponent, recombinant, meningococcal serogroup b vaccine (4cmenb) administered concomitantly with routine infant and child vaccinations: results of two randomised trials effectiveness of a group b outer membrane vesicle meningococcal vaccine against gonorrhoea in new zealand: a retrospective case-control study exploitation of neisseria meningitidis group b omv vaccines against n. gonorrhoeae to inform the development and deployment of effective gonorrhea vaccines bacterial outer membrane vesicles suppress tumor by interferongamma-mediated antitumor response acute-on-chronic liver failure: definition, diagnosis, and clinical characteristics key: cord-011888-3mvzkff6 authors: moore, john p.; robling, kristyn; romero, cristian; kiper, keturah; dachavaram, soma shekar; crooks, peter a.; hestekin, jamie a. title: oxone(®)-mediated tempo-oxidized cellulose nanomaterial ultrafiltration and dialysis mixed-matrix hollow fiber membranes date: 2020-06-15 journal: polymers (basel) doi: 10.3390/polym12061348 sha: doc_id: 11888 cord_uid: 3mvzkff6 recent exploration of cellulose nanomaterials has resulted in the creation of oxone(®)-mediated tempo-oxidized cellulose nanomaterials (oto-cnms). these materials, when incorporated into a polymer matrix, have properties showing increased flux, decreased membrane resistance, and improved clearance, making them an ideal material for dialysis. this study is the first to focus on the implementation of oto-cnms into hollow fiber membranes and a comparison of these membranes for ultrafiltration and dialysis. ultrafiltration and dialysis were performed using bovine serum albumin (bsa), lysozyme, and urea to analyze various properties of each hollow fiber membrane type. the results presented in this study provide the first quantitative evaluation of the clearance and sieving characteristics of oxone(®)-mediated tempo-oxidized cellulose-nanomaterial-doped cellulose triacetate mixed-matrix hemodialyzers. while the cellulose nanomaterials increased flux (10–30%) in ultrafiltration mode, this was offset by increased removal of albumin. however, in dialysis mode, these materials drastically increased the mass transfer of components (50–100%), which could lead to significantly lower dialysis times for patients. this change in the performance between the two different modes is most likely due to the increased porosity of the cellulose nanomaterials. according to the centers for disease control and prevention (cdc), chronic kidney disease is the 14th leading cause of death in the united states [1] . there are an estimated eight million american adults with reported diagnoses of kidney disease [2] . the cdc found that, in 2016, over 50,000 americans died from kidney disease [3, 4] . recent research has shown renal replacement therapy is essential in treating covid-19 [5] . currently, the most common treatment technique is to filter the blood through hemodialysis or peritoneal dialysis. dialysis does not cure the disease, like kidney transplantation. still, it can remove contaminants from the blood, like the kidneys, to maintain the health of the individual for a significant period. the majority of patients will receive treatment three times per week, for 3 to 6 h. each treatment is based on how much blood can be filtered out of the body at a specific time, as well as the performance of the membranes. hemodialysis machines operate on the principle of diffusion [6] . blood is pumped out of the body and passed through hollow fiber modules. the membranes are made of a semi-permeable material, usually cellulose or polysulfone, that has a pore size that is too small to allow blood cells and intermediary for nanocellulose production yields two forms of oxone ® -mediated tempo-oxidized cellulose nanomaterials (oto-cnms), form i and form ii [20] . form i is partially oxidized, and form ii is fully oxidized. they were both found to have a novel crystalline structure [21] . neither form i or form ii has been tested within polymer-based filtration studies. figure 1 shows the general chemical structure of form i compared to form ii. the production of form i and form ii was also found to be an efficient method of crystalline nanocellulose production. utilizing the procedure from moore et al., oto-cnms form i and form ii were synthesized and used in conjunction with cellulose triacetate (cta) from acros organics (fair lawn, nj, usa), n-methyl pyrrolidone (nmp) from vwr (radnor, pa, usa), and deionized water to create novel membranes for filtration [21] . the proteins used for ultrafiltration and dialysis characterization were bovine serum albumin (bsa), lysozyme, and urea supplied from vwr. these are common proteins found in the blood. bsa was chosen to represent large proteins in the blood; lysozyme was selected to represent middle molecular weight proteins in the blood, and urea was chosen because it is the primary waste product that is excreted from the kidneys. all membranes were housed in polyvinyl chloride pipes (pvc) supplied from lowe's (fayetteville, ar, usa) using underwater epoxy resin. ethanol for cleaning was also provided through vwr. four different solutions were created for membrane casting. the first, the control, was created using only cta and nmp. the casting solution was made using 10 wt % cta and 90 wt % nmp. these were mixed in a bottle and placed on a bottle roller for 5-7 days to allow for the complete incorporation of the cta. the second solution, form i, was created using cta, nmp, and oto-cnm form i. the casting solution was made using 9 wt % cta, 90 wt % nmp, and 1 wt % form i. to incorporate the form i nanocrystals, nmp and form i were blended in a 500-ml beaker at 6000 rpms for 5 min in a water bath. then, the solution was placed in a qsonica (newtown, ct, usa) sonicator at 500 watts at 20 khz for 5 min with 20-s intervals of sonication followed by 10 s of no sonication. after sonication, the oto-cnms were fully dispersed in solution. the solution was then run through a coarse filter using vacuum filtration to remove any large chunks. after filtration, it was added to a roller bottle with the cta and placed on a bottle roller for 5-7 days. this process was repeated with the other two types of solutions but with adjusted concentrations of oto-cnm form i and form ii. the third solution, 50/50, was prepared using cta, nmp, and form i and form ii. the solution was made using 9 wt % cta, 90 wt % nmp, 0.5 wt % form i, and 0.5 wt % form ii. the same mixing procedure was followed that was used for form i. the fourth solution, form ii, was prepared using cta, nmp, and form ii. the solution was made with 9 wt % cta, 90 wt % nmp, and 1 wt % form ii. this solution was mixed with the same procedure as form i and 50/50. all compositions of oto-cnm/cta membranes based on wt % for form i to form ii can be found illustrated in supplemental table s1. membranes were created with non-solvent-phase-induced separation hollow fiber casting. a water bath at 35 • c was filled so the fibers would be fully immersed as they traveled the length of the bath. a bore solution containing 15 wt % nmp and 85 wt % deionized water was utilized. a spinneret was used to extrude the hollow fibers. it was set to obtain a 200-µm thickness of the membranes. the polymer solutions were passed through the outer layer of the spinneret at a pressure of 25 psi, and the bore was passed through the spindle at a pressure of 1-2 psi to create a hollow tube. the spinneret was set at a 5-cm height above the water level to obtain a consistent time spent passing into the bath. as the solutions passed through the water bath, phase inversion occurred. the fibers were collected in a roll at a rate of 1.7 m per minute until casting was complete. immediately after completion, the fibers were placed in a hot water bath at 87-89 • c for 3 min. this heat treatment sets the membranes to strengthen them and ensure high quality. after heat treatment, they were placed in a container filled with room temperature deionized water for storage until further use. no membrane was stored more than one month before use. for each dialysis run, a new module had to be created. for each module, pvc pipes were assembled in the fashion shown in figure 2 to create complete hemodialysis modules for testing. each module contained 20 fibers. the fibers were inserted into the pvc apparatus and glued into place with underwater epoxy resin. this glue was used to seal the ends of the device, so no leaking would occur during testing. about 3 ml of glue was used for each end. after the glue fully solidified, the ends were cut to create an even surface, and threaded caps were added to the module to create the completed module. these were stored in a room temperature deionized water bath until ready for testing (up to a month). polymers 2020, 12, x for peer review 4 of 14 was used to extrude the hollow fibers. it was set to obtain a 200-µ m thickness of the membranes. the polymer solutions were passed through the outer layer of the spinneret at a pressure of 25 psi, and the bore was passed through the spindle at a pressure of 1-2 psi to create a hollow tube. the spinneret was set at a 5-cm height above the water level to obtain a consistent time spent passing into the bath. as the solutions passed through the water bath, phase inversion occurred. the fibers were collected in a roll at a rate of 1.7 m per minute until casting was complete. immediately after completion, the fibers were placed in a hot water bath at 87-89 °c for 3 min. this heat treatment sets the membranes to strengthen them and ensure high quality. after heat treatment, they were placed in a container filled with room temperature deionized water for storage until further use. no membrane was stored more than one month before use. for each dialysis run, a new module had to be created. for each module, pvc pipes were assembled in the fashion shown in figure 2 to create complete hemodialysis modules for testing. each module contained 20 fibers. the fibers were inserted into the pvc apparatus and glued into place with underwater epoxy resin. this glue was used to seal the ends of the device, so no leaking would occur during testing. about 3 ml of glue was used for each end. after the glue fully solidified, the ends were cut to create an even surface, and threaded caps were added to the module to create the completed module. these were stored in a room temperature deionized water bath until ready for testing (up to a month). dialysis and ultrafiltration module testing began with apparatus assembly. the filtration apparatus allowed solutions to pass through the membrane during ultrafiltration and two fluids to pass concurrently during dialysis. the transmembrane pressures were kept consistent with the use of pressure dampers. once the module was connected, water was passed through the dialysate side (outside) and the feed side (inside) of the membranes. the system was run for one hour prior to testing at 1-2 psi to stabilize the membrane. the dialysate side was run at a flow rate of 300 ml/min, and the feed side was run at a flow rate of 200 ml/min. the transmembrane pressure was kept consistent at 1 psi. during the hour of stabilization, the feed solutions were prepared. for bsa and lysozyme experiments, the concentration was 1 mg/ml; specifically, 800 mg of protein was dissolved into 800 ml of water. for the urea experiments, the concentration was 2 mg/ml; specifically, 1600 mg of urea was dissolved into 800 ml of water. once the pure water stabilization was finished, the setup was switched over to ultrafiltration, which involved turning off the dialysate. the feed was maintained at a flow rate of 200 ml/min. three water samples were collected from the permeate (dialysate side) at 1 psi transmembrane pressure. each sample was collected for 90 s and then weighed for flux calculation. after three samples were collected at the 5, 10, and 15 min marks, the dialysis and ultrafiltration module testing began with apparatus assembly. the filtration apparatus allowed solutions to pass through the membrane during ultrafiltration and two fluids to pass concurrently during dialysis. the transmembrane pressures were kept consistent with the use of pressure dampers. once the module was connected, water was passed through the dialysate side (outside) and the feed side (inside) of the membranes. the system was run for one hour prior to testing at 1-2 psi to stabilize the membrane. the dialysate side was run at a flow rate of 300 ml/min, and the feed side was run at a flow rate of 200 ml/min. the transmembrane pressure was kept consistent at 1 psi. during the hour of stabilization, the feed solutions were prepared. for bsa and lysozyme experiments, the concentration was 1 mg/ml; specifically, 800 mg of protein was dissolved into 800 ml of water. for the urea experiments, the concentration was 2 mg/ml; specifically, 1600 mg of urea was dissolved into 800 ml of water. once the pure water stabilization was finished, the setup was switched polymers 2020, 12, 1348 5 of 13 over to ultrafiltration, which involved turning off the dialysate. the feed was maintained at a flow rate of 200 ml/min. three water samples were collected from the permeate (dialysate side) at 1 psi transmembrane pressure. each sample was collected for 90 s and then weighed for flux calculation. after three samples were collected at the 5, 10, and 15 min marks, the feed solution was switched to the desired experimental solution to obtain ultrafiltration data. three more samples were collected and weighed at the 5, 10, and 15 min marks. after ultrafiltration, the dialysate was turned back on to run dialysis with the desired component. three flow rates were used to model different rates of dialysis. the first flow rate was 200 ml/min for the feed and 300 ml/min for the dialysate. feed-in, feed-out, and dialysate-out samples were collected to measure mass transfer. the second flow rate was 300 ml/min for the feed and 500 ml/min for the dialysate. samples were gathered in the same manner as for the first flow rate. the third flow rate was 400 ml/min for the feed and 500 ml/min for the dialysate. samples were again collected in the same manner. after samples were collected, the machine was turned off, and the module was disconnected. a 70% ethanol/water mixture was run through the apparatus for 30 min at 500 ml/min to clean and sterilize the system after each use. the protein samples collected during dialysis and ultrafiltration were analyzed for their concentration using a biotek epoch multi-volume spectrophotometer (winooski, vt, usa). a uv-transparent 96-well plate was utilized. samples were pipetted in triplicate in 100-µl increments into the well plate. the absorbance was run at 280 nm for the bsa and lysozyme samples. the urea samples were prepared with a quantichrom urea assay kit provided from vwr and run at 520 nm in a standard polystyrene 96-well plate, both supplied through vwr. scanning electron microscope (sem) images were taken using a philips xl30 environmental scanning electron microscope machine (north billerica, ma, usa) to characterize the membrane morphology. they were taken at 65× and 150× magnification at 10 kv. cross-sections of the fibers were prepared using a freeze-cracking method. a dialysis membrane must be able to operate in ultrafiltration mode (water removal) as well as dialysis mode. this first section will define some of the terms commonly used in dialysis as well as analyzing ultrafiltration performance. one of the first terms used, k uf, is the pure water flux of the membrane at a constant pressure, where q uf is the permeate flow rate, a is the membrane area, and p is the pressure on the inlet or outlet of the donating stream [9] . the ultrafiltration mode sieving coefficient (s o ) of each membrane was determined using equation (2), where c f is the concentration of the filtrate and c p is the concentration of the permeate [9] . it must be noted that two types of sieving coefficients will be used in this paper. the sieving coefficient, as described in equation (2), is the ultrafiltration sieving coefficient and refers to how much of a component passes through the membrane under a constant pressure, in this case, 1 psi. an element with 0% rejection has a sieving coefficient of 1, and a component with a 100% rejection has a sieving coefficient of 0. three components of interest-bsa, lysozyme, and urea-were run in ultrafiltration mode to find their sieving coefficients ( figure 3 ). supplementary table s2 shows the sieving coefficients in table form with standard deviation. polymers 2020, 12, x for peer review 6 of 14 sieving coefficient data shows many things about the characteristics of these membranes. first, as would be desired in a dialysis membrane, the control and all modified membranes have urea sieving coefficients near or at unity, ensuring smooth passage of components like urea and salts. second, the sieving coefficients for the middle molecules range from 0.68 to 0.88. as lysozyme is in the molecular weight range of uremic toxins, it shows that molecules within this size range can pass through the membrane. finally, the sieving coefficient for bsa under these conditions ranged from 0.13 to 0.31, with the 50/50 membrane having a sieving coefficient significantly higher than any of the other materials. excessive loss of bsa may make the 50/50 membrane unsuitable for dialysis. to better understand how the additives were affecting the active membrane area, equations were used to estimate their pore sizes. a general set of equations used for calculated pore sizes are given below in equations (3) and (4) equation (4) is a hydrodynamic model that uses the values calculated from the sieving coefficient, where λ is the ratio of the solute radius (rs) to the pore radius (rp) [12] . the pore radius was determined by the minimization of the sum of the squared residuals between the model (equation (4)) and the data for bsa. the values obtained for pure water flux (kuf pure water), as well as pore size (rp) along with the area (a) in meters squared, hollow fiber radius (r) in microns, and hollow fiber thickness (t) in microns of the test membranes as measured through sem are displayed in table 1 . when bsa was used to calculate pore radius, the data suggests that each of the modified membranes had a bigger pore radius than the control with 50/50 showing the most significant increase. the bsa numbers reported were used to estimate pore radius, because bsa is assumed to be spherical while lysozyme is not [26] [27] [28] . the flux data of these membranes show that the control and form ii are about the same, with about a 10% increase for 50/50 and a 30% increase for form i. after analyzing all of this data, we conclude that, for pure components in water, it appears from sieving coefficient data shows many things about the characteristics of these membranes. first, as would be desired in a dialysis membrane, the control and all modified membranes have urea sieving coefficients near or at unity, ensuring smooth passage of components like urea and salts. second, the sieving coefficients for the middle molecules range from 0.68 to 0.88. as lysozyme is in the molecular weight range of uremic toxins, it shows that molecules within this size range can pass through the membrane. finally, the sieving coefficient for bsa under these conditions ranged from 0.13 to 0.31, with the 50/50 membrane having a sieving coefficient significantly higher than any of the other materials. excessive loss of bsa may make the 50/50 membrane unsuitable for dialysis. to better understand how the additives were affecting the active membrane area, equations were used to estimate their pore sizes. a general set of equations used for calculated pore sizes are given below in equations (3) and (4) equation (4) is a hydrodynamic model that uses the values calculated from the sieving coefficient, where λ is the ratio of the solute radius (r s ) to the pore radius (r p ) [12] . the pore radius was determined by the minimization of the sum of the squared residuals between the model (equation (4)) and the data for bsa. the values obtained for pure water flux (k uf pure water ), as well as pore size (r p ) along with the area (a) in meters squared, hollow fiber radius (r) in microns, and hollow fiber thickness (t) in microns of the test membranes as measured through sem are displayed in table 1 . when bsa was used to calculate pore radius, the data suggests that each of the modified membranes had a bigger pore radius than the control with 50/50 showing the most significant increase. the bsa numbers reported were used to estimate pore radius, because bsa is assumed to be spherical while lysozyme is not [26] [27] [28] . the flux data of these membranes show that the control and form ii are about the same, with about a 10% increase for 50/50 and a 30% increase for form i. after analyzing all of this data, we conclude that, for pure components in water, it appears from ultrafiltration data that form i modified is the best candidate, due to its high flux and relatively high rejections of bsa. cellulose triacetate membranes are used in dialysis to remove urea, and the rate at which urea is removed is still a major limiting factor in dialysis treatment time. while urea is not considered the most significant component to remove for a patient's health, it is a good indicator of dialysis membrane performance. (k oa ) is the mass transfer area coefficient, otherwise known as the theoretical clearance at infinite blood and dialysate flow rates. it can be used as a representative term to describe treatment capabilities, as well as to determine theoretical treatment times for patients [29] . (k oa ) is defined using fick's law, equation (5) [30, 31] . (∆c m ) represents the change in concentration across the membrane, and (n) represents the solute transport rate in mg/min. the solute transport rate (n) was calculated using equation (6), where (q f ) represents the flow rate, subscript (f ) represents feed, subscript (d) represents dialysate, subscript (o) represents out, and subscript (i) represents in. (a) represents the area, and (∆c m ) represents the log mean concentration difference, which was calculated using equation (7) . subsequently, the clearance (k) can be determined from equation (8). a series of triplicate dialysis experiments were performed with bsa, lysozyme, and urea, as shown in table 2 . as can be seen by looking at urea, the clearance (k experimental ) is over 100% higher with form i as compared to the control. higher clearance values are also seen for form ii and the 50/50 mix. an increase in clearance was also seen with lysozyme (desirable) and bsa (undesirable) in the modified materials compared to the control. for bsa, the diffusion was calculated based on the sieving coefficient (sc in table 2 ), and it ranged from 1×10 −2 to 8×10 −3 . the value 8×10 −3 is comparable with the membrane performance of commercial membranes, while the other sieving coefficient values fall slightly outside [11] . however, another study suggests that, for high cut-off membranes, it may fall inside the range, which allows a sieving coefficient to go as high as 0.2 with a protein loss of fewer than 23 grams per session [12] . the process of membrane fabrication depends on the polymeric materials used and their specific interactions with the solvents upon phase separation [32, 33] . this is never more evident than when observing the membranes through a scanning electron microscope (sem). a cross-sectional sem of each of the four types of membranes is shown in figure 4 . while key parameters of membrane casting, such as spinneret size, bath temperature, uptake speed, drop height, etc., were kept consistent in this experiment, closer inspection with sem revealed a dramatic change in the internal pore morphologies. compared to the control (figure 4a) , each of the modified material membranes (figure 4b-d) showed alterations to the support material that increased porosity caused by the cellulose nanoparticle additive. a similar effect was demonstrated by bai et al. in the production of pvdf composite membranes using unmodified cellulose nanomaterials, leading to larger finger-like pores and more pores in the bottom surface. as a result, the cellulose nanomaterial composite membranes were improved, i.e., in pure water flux, mechanical properties, and maintained solute rejection [34] . it has been shown that the support structures of membranes can lead to dramatically different separation performances under different operating conditions [35, 36] . more importantly, the improvement in the dialysis performance of these membranes can be explained by looking at the backing, i.e., the macroporous support structure. manickam and mccutcheon derive a theory for transport with pressure-driven versus diffusion-based processes [37] . their approach shows that backing is a significant source of resistance in a diffusion-based process, while it is not in a pressure-driven process. thus, more porous, spongy backings would be predicted to lead to a better dialysis transport rate while not necessarily affecting the ultrafiltration rate. the membrane performance of commercial membranes, while the other sieving coefficient values fall slightly outside [11] . however, another study suggests that, for high cut-off membranes, it may fall inside the range, which allows a sieving coefficient to go as high as 0.2 with a protein loss of fewer than 23 grams per session [12] . the process of membrane fabrication depends on the polymeric materials used and their specific interactions with the solvents upon phase separation [32, 33] . this is never more evident than when observing the membranes through a scanning electron microscope (sem). a cross-sectional sem of each of the four types of membranes is shown in figure 4 . while key parameters of membrane casting, such as spinneret size, bath temperature, uptake speed, drop height, etc., were kept consistent in this experiment, closer inspection with sem revealed a dramatic change in the internal pore morphologies. compared to the control (figure 4a) while the experiments were conducted with a total volume similar to a dialysis system, the membrane area was approximately an order of magnitude smaller. therefore, calculations were utilized to determine how these membranes would act if a typical dialysis membrane area were used [38] . the theoretical urea clearance was calculated by simultaneously solving fick's law, the log mean concentration difference, and a mass balance to express the ratio k/q f as a function of two dimensionless parameters, neither of which involves solute concentration. this is shown in equation (9) , where z is the ratio of the feed flow rate to the dialysate flow rate and r = k oa a/q f [39, 40] . the clearance values (k theoretical ) determined from the above dimensionless expression based on the experimental data can be found in table 3 . from the theoretical clearance values, predicted treatment time was calculated following the us national kidney foundation (kt/v) target. in this equation, a 70-kg person, at 60% water weight, must reach a kt/v of [6] . governmental standards are set on this premise to ensure patients are successfully treated during their time at the hospital [41] . it was found that form i had the most drastically decreased treatment time, at a 26% reduction. form i showed a decrease in treatment time, while the control showed the typical treatment time (approximately four hours on average). furthermore, the membrane can also be observed as a function of size, as shown in figure 5 , where the modified membranes show the ability to maintain appropriate treatment times at smaller sizes without a significant loss in performance. in other words, in the control, we see a 25% loss in performance in the control when moving from a 1-m 2 membrane to one half that size, and, in the modified sample, oto-cnm form i mixed-matrix membrane, only an 11% loss in performance. this effect is exacerbated at smaller membrane sizes. some significant conclusions can be drawn from this data. first, the theoretical treatment time for the 1-m 2 membrane area is in the range of what has been previously reported [42] . this suggests that these membranes could make effective dialysis membranes. second, the differences in clearance from the control versus the modified membranes suggest a smaller membrane can be used to achieve the same results seen in the unmodified membranes. it is important to note that results may seem more significant in the smaller test modules than when compared at the one-meter square membrane area. this is because both sets of solutions are using the same volumetric flow rates. since dialysis is an unsteady process, larger membrane areas have the capacity for high urea removal rates in the beginning, returning the blood at almost 0 urea. this makes the flow rate a major limiting step in the removal of urea. however, when using a smaller module, the membrane can be more fully utilized, and the gradients affecting transfer can remain larger. to fully realize the potential of these new membranes, less membrane area would need to be used in the same operating conditions as traditional hemofiltration membranes. smaller modules in the same operating conditions allow more effective and efficient treatment with less blood out of the patient. furthermore, it has been shown that less blood being removed from the body increases the health of the dialysis patient [43, 44] . from the theoretical clearance values, predicted treatment time was calculated following the us national kidney foundation (kt/v) target. in this equation, a 70-kg person, at 60% water weight, must reach a kt/v of [6] . governmental standards are set on this premise to ensure patients are successfully treated during their time at the hospital [41] . it was found that form i had the most figure 5 . dimensionless analysis of the time of clinical operation completion (kt/v=1.2) as observed against membrane surface area, a (m 2 ), for the control, form i, 50/50, and form ii samples show membrane performance at different active membrane areas under the same conditions, i.e., a 300ml/min feed flow rate and 500 ml/min dialysate flow rate using the same assumptions utilized in table 3 . 2) as observed against membrane surface area, a (m 2 ), for the control, form i, 50/50, and form ii samples show membrane performance at different active membrane areas under the same conditions, i.e., a 300 ml/min feed flow rate and 500 ml/min dialysate flow rate using the same assumptions utilized in table 3 . in this study on the implementation of two derivatives of cellulose nanomaterials into hollow fiber membranes for ultrafiltration and dialysis, oto-cnm form i and form ii mixed-matrix membranes have been quantitatively evaluated for the first time by determining model molecule selectivity (bsa, urea, and lysozyme), as well as transport rates (flux and diffusion rates). each of the three membranes made from cellulose nanomaterials showed improvements when compared to the control (cellulose triacetate). all showed an increase in flux, mass transfer area coefficient, and urea clearance. the differences were more significant when in dialysis mode, which suggests that the porous, spongy backing layer reduced the resistance in the diffusion-based process. these membranes have characteristics that fall within the range of commercial dialysis membranes, indicating that they could be an attractive candidate for future hemofiltration membrane development. further analysis of dialysis membranes like these will/could show the possibility of the use of less membrane area and shorter treatment times, leading to better patient health. table s1 : the compositions of oto-cnm/cta membranes based on wt % for form i to form ii; supplemental table s2 : membrane sieving coefficient information. cardiovascular disease in patients with chronic kidney disease chronic kidney disease and the risks of death, cardiovascular events, and hospitalization behavioral counseling to promote a healthful diet and physical activity for cardiovascular disease prevention in adults without cardiovascular risk factors: us preventive services task force recommendation statement severe covid-19: a review of recent progress with a look toward the future. front. public health hemodialysis: prescription and assessment of adequacy. renal urology news membranes for hemodialysis the future of hemodialysis membranes transport characteristics of asymmetric cellulose triacetate hemodialysis membranes preparation and preliminary dialysis performance research of polyvinylidene fluoride hollow fiber membranes requirements and pitfalls of dialyzer sieving coefficients comparisons membrane innovation: closer to native kidneys second generation nanoporous silicon nitride membranes for high toxin clearance and small format hemodialysis clinical implications of hemodialysis membrane biocompatibility importance of development of dialysis membranes for purification of small, middle, high molecular weight and protein-bound uremic toxins: the asymmetric cellulose triacetate (solacea™-h) nanomaterials for membrane fouling control: accomplishments and challenges tempo-oxidized cellulose nanofibrils dispersed in organic solvents thermal stabilization of tempo-oxidized cellulose mediated micron cellulose oxidation procedure: preparation of two nano tempo-cellulose forms oxone®-mediated tempo-oxidized cellulose nanomaterials form i and form ii a critical review of recent advances in hemodialysis membranes hemocompatibility and guidelines for future development simulating nephron ion transport function using activated wafer electrodeionization high-flux and low-flux membranes: efficacy in hemodialysis effect of dialysis dose and membrane flux in maintenance hemodialysis application note: viscosity measurement of a model protein solution of bsa light scattering of bovine serum albumin solutions: extension of the hard particle model to allow for electrostatic repulsion charge interaction and temperature effects on the solution structure of lysozyme as revealed by small-angle x-ray scattering cellulose triacetate: another membrane for continuous renal replacement therapy membrane filtration: a problem solving approach with matlab linear solutions of fick's law influence of non-solvent chemistry on polybenzimidazole hollow fiber membrane preparation cellulose nanocrystal-blended polyethersulfone membranes for enhanced removal of natural organic matter and alleviation of membrane fouling preparation and characterization of poly (vinylidene fluoride) composite membranes blended with nano-crystalline cellulose novel dual-layer hollow fiber membranes applied for forward osmosis process enhanced antifouling performance of pvdf ultrafiltration membrane by blending zinc oxide with support of graphene oxide nanoparticle understanding mass transfer through asymmetric membranes during forward osmosis: a historical perspective and critical review on measuring structural parameter with semi-empirical models and characterization approaches a non-dimensional analysis of hemodialysis mathematical modeling of membrane filtration porosity and pore size determination in polysulfone hollow fibers impact of blood and dialysate flow and surface on performance of new polysulfone hemodialysis dialyzers handbook of biomedical instrumentation hemodialysis system. in modelling and control of dialysis systems this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license characterization facility for the image support, as well as emily e. thompson for final grammatical corrections. it should be noted that four authors-john p. moore ii, soma shekar dachavaram, peter a. crooks, and jamie a. hestekin-have started an llc looking to market and develop cellulose materials such as the ones presented in this text. (a) membrane area, m 2 (c f ) the concentration of the filtrate mg/ml (c p ) the concentration of the permeate mg/ml (k) clearance, ml/min (k experimental ) experimental clearance, ml/min (k oa ) is the mass transfer area coefficient ml/(m 2 *min) (k theoretical ) determined clearance values, ml/min (k uf ) pure water flux of the membrane at constant pressure (l/hr/m 2 /psi) (m w ) molecular weight, daltons (n) represents the solute transport rate mg/min (p) pressure, psi (q) represents the flow rate, ml/min (q uf ) permeate flow rate, ml/min (r) hollow fiber radius, µm (r p ) pore radius, µm (r s ) theoretical solute radius, µm r = k oa a /q f , dimensionless (s o ) sieving coefficient, dimensionless subscript (f ) represents the feed subscript (d) represents dialysate subscript (o) represents out subscript (i) represents in (t) hollow fiber thickness, µm (λ) the ratio of the solute radius to the pore radius, dimensionless (z) ratio of the blood flow rate, dimensionless key: cord-279463-bli8hwda authors: lipp, joachim; dobberstein, bernhard title: the membrane-spanning segment of invariant chain (iγ) contains a potentially cleavable signal sequence date: 1986-09-26 journal: cell doi: 10.1016/0092-8674(86)90710-5 sha: doc_id: 279463 cord_uid: bli8hwda abstract the human invariant chain (iγ) of class ii histocompatibility antigens spans the membrane of the endoplasmic reticulum once. it exposes a small amino-terminal domain on the cytoplasmic side and a carboxyterminal, glycosylated domain on the exoplasmic side of the membrane. when the exoplasmic domain of iγ is replaced by the cytoplasmic protein chloramphenicol acetyltransferase (cat), cat becomes the exoplasmic, glycosylated domain of the resulting membrane protein iγcat∗. deletion of the hydrophilic cytoplasmic domain from iγcat gives rise to a secreted protein from which an amino-terminal segment is cleaved, most likely by signal peptidase. we conclude that the membrane-spanning region of iγ contains a signal sequence in its amino-terminal half and that hydrophilic residues at the amino-terminal end of a signal sequence can determine cleavage by signal peptidase. the human invariant chain (ly) of class ii histocompatibility antigens spans the membrane of the endoplasmic reticulum once. it exposes a small amino-terminal domain on the cytoplasmic side and a carboxyterminal, glycosylated domain on the exoplasmic side of the membrane. when the exoplasmic domain of ly is replaced by the cytoplasmic protein chloramphenicol acetyltransferase (cat), cat becomes the exoplasmic, glycosylated domain of the resulting membrane protein i$at*. deletion of the hydrophilic cytoplasmic domain from l$xt gives rise to a secreted protein from which an amino-terminal segment is cleaved, most likely by signal peptidase. we conclude that the membranespanning region of ly contains a signal sequence in its amino-terminal half and that hydrophilic residues at the amino-terminal end of a signal sequence can determine cleavage by signal peptldase. translocation of proteins across the membrane of the endoplasmic reticulum (er) requires signal sequences and specific receptors that recognize them (see recent reviews by hortsch and meyer, 1984; walter et al., 1984; rapoport and wiedmann, 1985; wickner and lodish, 1985) . signal sequences have been found at the amino-terminal end of precursors for secretory and transmembrane proteins. in many cases they are cleaved during their translocation across the membrane by a specific protease (signal peptidase). signal sequences are quite variable in length, ranging from 16 to more than 50 amino acid residues (von heijne, 1983) . they all have a central core of hydrophobic amino acid residues, and most of them have a positively charged amino-terminal segment (von heijne, 1985) . signal sequences on nascent polypeptides are recognized by the signal recognition particle (srp), a ribonucleoprotein complex that mediates the interaction with the membrane by the selective binding to docking protein (or srp receptor) (walter et al., 1981b; meyer et al., 1982; gilmore et al., 1982) . membrane proteins are also inserted into the er membrane by an srp-mediated mechanism (anderson et al., 1983; rottier et al., 1985; spiess and lodish, 1986; lipp and dobberstein, 1986) . those spanning the membrane once have either the carboxyl terminus (type i membrane proteins) or the amino terminus (type ii membrane proteins) exposed on the cytoplasmic side. membrane insertion of type i membrane proteins most likely proceeds in a manner very similar to that of secretory proteins (lingappa et al., 1978) . type i membrane proteins are usually synthesized with a cleavable signal sequence and, in contrast to secretory proteins, are held in the membrane by a "stop transfer" sequence. examples of type i membrane proteins are the vesicular stomatitis virus g protein and class i and class ii histocompatibility antigens (lingappa et al., 1978; dobberstein et al., 1979) . of the type ii membrane proteins so far investigated, all are synthesized without a cleavable signal sequence. the neuraminidase of influenzavirus (60s et al., 1984) , the invariant chain (ii or ly) of class ii histocompatibility antigens (claesson et al., 1983; strubin et al., 1984; long, 1985; lipp and dobberstein, 1986) , the transferrin receptor (schneider et al., 1984) , and the asialoglycoprotein receptor (chiacchia and drickamer, 1984; holland et al., 1984; spiess and lodish, 1986 ) all belong to this class of membrane proteins. some steps in their membrane insertion must be similar to that of secretory and type i membrane proteins, as an srp-and docking protein-dependent membrane insertion has been demonstrated for some of them (spiess and lodish, 1986; lipp and dobberstein, 1986) . membrane insertion might occur in a loop-like fashion as this scheme can most easily explain how the different membrane topologies of membrane proteins are achieved (engelman and steitz, 1981) . as type ii membrane proteins contain only a single stretch of hydrophobic amino acid residues, this might function as a signal for membrane insertion as well as a membrane anchor (markoff et al., 1984; spiess and lodish, 1986) . to identify and characterize this sequence, we tested membrane insertion of the human invariant chain (ly) and several deletion and fusion proteins derived from it in a cell-free membrane insertion system. ly is a typical type ii membrane protein (claesson et al., 1983; strubin et al., 1984; lipp and dobberstein, 1986) . it exposes 30 amino-terminal residues on the cytoplasmic side, spans the membrane between residues 30 and 60, and exposes a large carboxy-terminal domain on the exoplasmic side. this domain has two sites for the addition of n-linked carbohydrate units. membrane insertion of ly requires srp and docking protein (lipp and dobberstein, 1986) . as the amino-terminal, cytoplasmic domain is hydrophilic and shows no resemblance to a signal sequence, it has been proposed that the membrane-spanning region, or part of it, functions as an internal, uncleavable signal sequence (dobberstein et al., 1983; claesson et al., 1983; lipp and dobberstein, 1986) . we demonstrate here that the membrane-spanning region of ly is composed of a potentially cleavable signal sequence fused to part of a membrane anchor, which together with the cytoplasmic domain determine the orientation of ly in the er membrane. deletion of the cytoplasmic domain exposes the signal sequence at the amino terminus of the membrane-spanning region, resulting in cleavage of this otherwise uncleaved signal. claesson et al., 1993) . ply. the complete ly coding and all of its 3' noncoding sequence was cloned behind the t5 promoter (p) in the pds5 expression vector. plycat, the portion downstream of the pstl site in ply was replaced by the chloramphenicol acetyltransferase (cat) gene resulting in an in-frame fusion protein. pan-lycat, the segment between the sau3a and sstll sites of plycat coding for the cytoplasmic domain was deleted. a new atg initiation codon right in front of the membrane-spanning segment is provided by the vector (see figure 4a ). the regions coding for protein are boxed. the membrane-spanning region of ly is indicated by loops; the hydrophilic domains by dots. cat-derived sequences are indicated by slanted lines. the position of n-linked glycosylation sites in ly and the potential n-linked glycosylation site in cat protein are indicated by an asterisk. relevant cleavage sites for restriction endonucleases are also indicated. protein segments that perform a particular function can be identified by their deletion or addition to unrelated proteins. we used this approach to localize and characterize the region in ly that is responsible for membrane insertion. deletions and fusions were made at the dna level after cloning of ly cdna into an expression vector. messenger rna was transcribed from these plasmids and translated in a cell-free system. the resulting proteins were tested for their ability to insert into microsomal membranes (blobel and dobberstein, 1975; stueber et al., 1984) . plasmids ply, plycat, and pan-lycat we have shown previously that cdna sequences cloned behind the strong t5 promoter in pds5 can be transcribed very efficiently by e. coli rna polymerase (stueber et al., 1985) . when transcription is performed in the presence of the cap analog 7mgpppa, the resulting mrna can be translated efficiently in eukaryotic cell-free systems. we have observed, however, that a stretch of gc residues at the 5' end of a cdna negatively affects expression of the resulting rna (unpublished observation). the ly cdna construct (py-2) had been gc tailed and was inserted into the pstl site of pbr322 (claesson et al., 1983) . we deleted the 5' gc tail and cloned ly cdna, or part of it, into the polylinker site of pds5 or p6/5r (see experimental procedures for details). ply contains the entire l-y coding region behind the t5 promoter ( figure 1 ). plycat is an in-frame fusion between the 5' region of ly encoding the cytoplas, mic, membrane-spanning segment plus 12 amino acids of the exoplasmic portion of ly and the gene encoding the cytoplasmic protein chloramphenicol acetyltransferase (cat). the cat protein contains one potential site for the addition of n-linked oligosaccharide 36 amino acid residues downstream of its original initiator methionine. in an-iycat, the entire hydrophilic, cytoplasmic segment from ly was deleted. the new initiator methionine is provided by the vector and is located in front of the hydrophobic segment. in vitro translation and membrane insertion of ly when ply was transcribed by e. coli rna polymerase and the resulting mrna translated in the wheat germ cell-free system, a single polypeptide species of 27 kd was obtained ( figure 2 , lane 1). this is the expected molecular weight for nonglycosylated ly (claesson et al., 1983) . when rough microsomes (rm), derived from dog pancreas, were added to the translation system, a higher molecular weight species of 33 kd appeared. this increase of 6 kd in molecular weight is consistent with the addition of two oligosaccharides to the two n-glycosylation sites. the 33 kd form ly* was reduced in molecular weight by about 2 kd when proteinase k was used to remove the cytoplasmically exposed domain (figure 2 , lanes 2 and 3). when protease digestion was performed in the presence of the detergent np 40, ly' was digested. these data suggest that ly' is integrated into the membrane and exposes 20-30 amino acid residues on the cytoplasmic side and a 30 kd domain on the exoplasmic side of the membrane. the identity of ly and its glycosylated form was confirmed by immunoprecipitation with antibodies raised against the amino-terminal 72 (anti-iyn) or against the carboxy-terminal 144 (anti-iyc) residues of ly. as shown in figure 2 , lanes 5, 6, 8, and 9, these antibodies recognize glycosylated and nonglycosylated forms of ly. no protein could be precipitated with anti-iyn antibody when the cytoplasmic domain was removed from membrane-integrated ly' by protease digestion (figure 2, lane 7) . as the antibody is directed against the amino-terminal portion of ly, the data directly demonstrate that the amino terminus is located on the cytoplasmic side and is accessible to the protease. with anti-iyc antibody, the processed form of ly is readily detectable, demonstrating an exoplasmic location of the carboxy-terminal portion of ly ( figure 2 , lane 10). membrane insertion of iycat an analysis of membrane insertion was performed for ly-cat and cat as described above for ly. cat was expressed from pds5. iycat was synthesized in the absence of microsomal membranes as a 34 kd protein ( figure 3 , lane 1) and in the presence of microsomal membranes as a 37 kd protein called lycat* ( figure 3 , lane 2). 12 3 4 in vitro translation and membrane insertion of ly ply was transcribed in the presence of the cap analog 7mgpppa by e. coli rna polymerase. the resulting mrna was translated in the wheat germ cell-free system in the absence (lanes 1,5. and 8) or presence (lanes 2, 3, 4, 6, 7, 9. and 10) of rm. the membrane topology of ly was determined by treatment with proteinase k (pk) (lanes 3, 7, and 10) or pk and the detergent np40 (lane 4). proteins were separated by sds-page and visualized by autoradiography. lanes 1-4 show total protein synthesized. samples characterized in lanes 5-7 were immunoprecipitated with an antibody raised against the amino-terminal 72 amino acid residues of ly (anti-iyn); in lanes 8-10, with an antibody against the carboxy-terminal portion of ly (anti-iyc). the increase in molecular weight is consistent with the addition of one n-linked oligosaccharide to the cat-derived portion. there is one potential site for n-linked glycosylation in the cat protein. after protease digestion in the presence of microsomal membranes, lycat* is reduced in molecular weight by about 2 kd, suggesting that it exposes 20-30 amino acid residues on the cytoplasmic side ( figure 3 , lanes 2 and 3). cat protein obtained after transcription-translation from pds5 is not modified by the added microsomes. as expected, no shift in molecular weight can be seen (figure 3 , lanes 5 and 6). cat protein was very resistant to protease digestion even in the presence of np40 (figure 3, lanes 7 and 8). i-&at, in contrast, was very sensitive to added protease. this might reflect a difference in conformation between the free cat protein and the cat-derived portion in iycat. the location of cat outside of the membrane vesicles can be demonstrated by sedimenting the membranes by centrifugation. cat protein is then found in the supernatant (data not shown). we conclude from the data obtained with ircat and cat that the signal for membrane insertion must be located within the first 72 amino acid residues of ly. to localize this signal more precisely, we deleted the first 30 residues of iycat. catinsertion of k&at and cat protein rna derived from plycat or pds5 was translated in the wheat germ cell-free system in the absence or the presence of rm. membrane insertion was tested by treatment with proteinase k (pk) and np40. addition of rm, pk, and np40 is indicated at the bottom of each lane. of an+cat in all secretory proteins the cleavable signal for membrane translocation is located at the amino-terminal end of the precursor polypeptide. the main feature of this signal appears to be its hydrophobicity. in ly the only hydrophobic stretch of amino acid residues that resembles a signal sequence is located in the membrane spanning region about 30 amino acid residues away from the amino-terminal initiator methionine. we asked whether removal of the 30 amino-terminal residues in iycat would affect its membrane insertion and topology. the cytoplasmic domain of iycat was deleted and the initiator methionine was placed in front of the membranespanning segment. the amino-terminal sequences of ly-cat and an-i$at as deduced from the dna sequences are shown in figure 4a . when rna derived from panlycat was translated in the wheat germ cell-free system, a single polypeptide of 29 kd was synthesized, an-iycat ( figure 46 , lane 1). this was, as expected, about 3 kd smaller than the iycat protein ( figure 46 , lane 1). in the presence of microsomes, two new protein bands appeared, one about 1 kd smaller and one 2 kd larger than an-ircat both of these forms were resistant to proteinase k, indicating that they were inserted into or translocated across microsomal membranes (figure 48 , lanes 3 and 4). we suspected that the smaller molecular weight form was generated by signal peptidase cleavage without concomitant glycosylation and that the larger molecular weight form was glycosylated and cleaved by signal peptidase. these possibilities were tested. from pan-lycat was translated in the wheat germ cell-free system in the absence or presence of rm. membrane insertion and topology was tested by treatment with proteinase k (pk) and np40. components were added as indicated below the lanes. iycat translated in the wheat germ cell-free system is shown for comparison. processed and glycoeylated to detect the signal peptide cleavage of a glycosylated protein on a polyacrylamide gel it is necessary to block its glycosylation, but still allow membrane insertion to occur. addition of n-linked oligosaccharides onto nascent polypeptides can be blocked by including synthetic acceptor peptides in an in vitro membrane insertion assay (bause, 1983; lau et al., 1983) . iycat and an-iycat were translated in the presence of microsomes with and without the acceptor peptide asn-leu-thr. the size of iycat synthesized in the presence of rm and acceptor peptide was indistinguishable from that made in the absence of rm. when proteinase k was used to digest its cytoplasmically exposed domain, the size was reduced by about 2-3 kd ( figure 5a ). we can conclude that nonglycosylated iycat synthesized in the presence of rm and acceptor peptide is inserted into the membrane in the same way as its glycosylated form and that no signal sequence is cleaved during membrane translocation ( figure 5a figure 58, lanes 2 and 3) . an-iycat'was also found to be protected against exogenous proteinase k ( figure 56, lane 4) . this suggested to us that the larger form was glycosylated and proteolytically processed and that an-iycat' was generated by a proteolytic cleavage, most likely by signal peptidase. to determine the site of cleavage in the proteolytically processed forms of an-ircat, the positions of leucine in the amino-terminal regions of an-itcat and membraneinserted an-lrcat*' were determined. an-i$at was translated in the absence or presence of rm with [sh]leutine as label. as an-iycat is essentially the only protein synthesized from pan-lycat-derived mrna, the complete translation mixture was subjected to automated edman degradation. as seen in figure 6a , leucine residues are found at the positions 3, 10, 13, 14, and 15, as predicted from the sequence deduced from py-2 cdna (claesson et al., 1983) . the initiator methionine is probably removed during or shortly after translation (kozak, 1983) . the positions of leucine residues in the membranetranslocated forms of an-i$at were similarly determined. as rm in the in vitro assay do not translocate all chains, some cytoplasmic forms remained (see inserts in figures 6a and 6b ). leucine residues were found at positions 1,2,3, and 13 ( figure 66) . larger peaks at positions 3 and 10 are consistent with the presence of some unprocessed an-iycat (see insert in figure 6b ). taking into account the size reduction of about 1 kd by the processing 13, 14, and 15 in authentic an-i$at, we conclude that processing has occurred between amino acid residues 12 and 13 ( figures 6b and 6c ). proteolytically processed an-lycat is translocated into the lumen of microsomal vesicles with the proteolytic removal of 12 of the 30 hydrophobic amino acid residues in the membrane-spanning region of an-iycat, the question arose as to whether the processed protein was still anchored in the membrane or whether it was now released into the lumen of the microsomal vesicles as is the case for secretory proteins. we used the extractability with carbonate as a criterion for membrane integration. treatment of rm with carbonate at ph 11 releases proteins that are not integrated into the lipid bilayer as well as proteins present in the lumen of microsomal vesicles. an+cat was translated in the presence of rm. membranes were isolated by centrifugation through a sucrose cushion and resuspended in carbonate buffer. solubilized components were then separated from membranes by centrifugation. proteins in the membrane pellet and supernatant were analyzed by sds-page and autoradiography. membrane-spanning proteins, ly and iycat, and the secretory protein, mouse granulocyte-macrophage colony stimulating factor (gm-csf), were used as control (gough et al., 1985) . as is shown in figure 7 , ly" and i-&at*, as expected for membrane-spanning proteins, were found in the membrane fraction. both an-i$at' and the gm-csf' were found essentially in the soluble, carbonatereleased fraction. thus an-iycat' is released after the proteolytic processing into the lumen of the microsomal vesicles. proteolytic processing, as described above for an-iycat, was also obtained for an+, a protein that lacks the amino-terminal 30 residues of ly (data not shown). our results show that the membrane-spanning segment of the type ii membrane protein ly contains a potentially cleavable signal sequence. this signal sequence is located in the amino-terminal half of the membrane-spanning segment, and it is cleaved when the preceding cytoplasmic domain is removed. all properties known to identify a signal sequence and a cleavage by signal peptidase can be demonstrated. to restrict vertical mobility of the membranespanning segment. (6) an-itcat, during its initial stage of membrane insertion, also spans the membrane with its hydrophobic segment. however, as no charged amino acid residues are present at the extreme amino-terminal end, the hydrophobic segment has some freedom to change its topology across the membrane. part of the hydrophobic segment might now be pulled into the lumen of the er membrane, and a former cryptic site for signal peptidase cleavage might become accessible to the active center of signal peptidase. first, the cleavage occurs concomitant with insertion into the er membrane as is typical for cleavable signal sequences of presecretory proteins (blobel and dobberstein, 1975) . second, the cleaved segment is located at the aminoterminal end of the deletion protein an-iycat. it is 13 amino acid residues long and composed entirely of hydrophobic or uncharged residues. signal sequences can vary in length from about 15 to over 60 residues. the only structural element identified so far for a signal sequence is its hydrophobic core, usually 8-12 residues long. it is followed by a more polar region 5-7 residues long, which is thought to define the cleavage site for signal peptidase. thus, a "minimal" signal sequence would be composed of an 8 residue hydrophobic core followed by a 5 residue region conferring cleavage specificity (von heijne, 1983 (von heijne, , 1985 . the segment cleaved from protein an-iycat would be consistent with such a minimal length signal sequence. finally, the amino acid residues around the cleavage site in membrane-translocated an-lycat*' are consistent with cleavage by signal peptidase. based on a sequence comparison of 78 eukaryotic signal sequences, von heijne found that only small neutral residues are found at the site of cleavage (-1 position) and that only small neutral and uncharged ones are found at the -3 position, that is 3 amino acid residues in front of the signal peptidase cleavage site (von heijne, 1983) . in the segment cleaved from an-i$at, threonine, a small neutral amino acid, is found at the -1 position, and leucine, an uncharged amino acid, at the -3 position. both of these residues fulfill the above described criteria for a signal peptidase cleavage site. thus, place (rm) and time of cleavage (cotranslational), hydrophobic character of the cleaved segment, and property of the cleavage site demonstrate that an-i$at contains a signal sequence at its amino terminus which is cleaved upon membrane insertion by signal peptidase. how can we possibly explain how the deletion of the cytoplasmic, hydrophilic segment from iycat reveals a cleavable signal sequence in a formerly membranespanning region? to us the most plausible explanation is that the position of the hydrophobic segment in the membrane is different in iycat and an-i$at signal peptidase is known to be an integral membrane protein not exposed on the cytoplasmic side of rm (jackson and blobel, 1977; lively and walsh, 1983; evans et al., 1986) . as in many secretory proteins, the cleavage site for signal peptidase is surrounded on either side by 1 or even 2 charged amino acid residues. it is reasonable to assume that the active center of this enzyme is located close to the exoplasmic side of the er membrane, not within the membrane. we propose that the removal of the cytoplasmic, hydrophilic segment from iycat allows the hydrophobic segment to shift its position within the er membrane. most likely it positions itself more toward the exoplasmic side. hence, a potential signal peptidase cleavage site becomes accessible to the active center of signal peptidase (see figure 8b ). it has been noted previously in type i membrane proteins that a deletion of the charged amino acid residues flanking the membrane-spanning region does not affect the overall topology (zuninga and hood, 1986; cutler et al., 1986) . in the case of es glycoprotein of semliki forest virus, it has been shown that mutation of the basic amino acid residues at the cytoplasmic side of the membranespanning segment reduces the stability of the mutant protein in the membrane (cutler et al., 1986) . when the membrane-spanning regions of type i and type ii membrane proteins are compared, no obvious structural difference can be found. in both types of membrane proteins these regions comprise a stretch of 20 to 30 hydrophobic amino acid residues that is flanked on the cytoplasmic side by positively charged amino acid residues. in type i membrane proteins the segment spanning the membrane does not appear to participate in the initial stage of membrane insertion. type i membrane proteins usually have cleavable signal sequences that initiate the membrane translocation of the amino-terminal half of the protein. the membrane-spanning region, in its position close to the carboxy-terminal end, seems only to function in anchoring the protein in the membrane. yost et al. placed the membrane-spanning segment of the murine surface immunoglobulin heavy chain close to the amino-terminal end of a fusion protein (yost et al., 1983) . in this position the segment did not provide the signal function for membrane insertion. as, however, a hydrophilic segment of about 40 amino acid residues precedes the membrane-spanning segment, the question still remains as to whether a membrane-spanning region from a type i membrane protein, when placed into the appropriate surrounding, can also initiate translocation across the er membrane. it is well conceivable that certain hydrophilic sequences preceding a hydrophobic segment play a crucial role in exposing a potential signal for membrane insertion. up to now no special structural features, besides hydrophobicity, are known to be crucial for the function of a signal sequence. a common step has been proposed for the early stage of membrane insertion of secretory and membrane proteins (dobberstein et al., 1983; spiess and lodish, 1986; lipp and dobberstein, 1986) . this was based largely on the finding that both of these types of proteins require srp and docking protein for their membrane insertion. here, we show that a type ii membrane protein can be converted into a secretory protein by removal of the cytoplasmic segment. this directly demonstrates that the signal for membrane insertion of these two types of proteins can be the same. further deletion into the carboxy-terminal half of the ly hydrophobic segment is required to elucidate whether the cleaved signal sequence contains all the information for membrane insertion. it is conceivable that the functional signal sequence extends over the cleaved signal sequence into the adjacent hydrophobic part. for some secretory protein it has been observed that the cleavable signal sequence is not sufficient for membrane insertion. in the case of staphylococcal protein a, sequences of the amino-terminal part of the mature protein are required for membrane insertion and correct processing (abrahmsen et al., 1985) . srp can arrest elongation of presecretory and type ii membrane proteins after 70 or even more amino acid residues have been polymerized (walter and blobel, 1981a; meyer et al., 1982; lipp and dobberstein, 1986; lipp et al., unpublished data) . these domains are then inserted into the er membrane by a yet unknown mechanism. as the amino terminus of a type ii membrane protein has to remain on the cytoplasmic side, the formation of a loop during membrane insertion has been proposed. in the case of a secretory protein, signal peptidase would be able to act as soon as the loop appears on the exoplasmic side. an initial interaction of basic residues in a signal sequence with the phosphates of the membrane lipids was originally proposed by lnouye for the lipoprotein of e. coli (inouye et al., 1977) . our results rule out an essential role of these basic residues in er membrane insertion. the an-iycat protein does not contain any charged amino acid residues preceding the hydrophobic segment. it is nevertheless translocated across the er membrane and processed. the rules that define the cleavage site for signal peptidase in presecretory proteins are not yet fully understood. von heijne points out that the type of amino acids at the -1 and -3 position in front of the site of cleavage are im-portant in assigning a cleavage site. here we show that sequences at the very beginning of a signal sequence can also influence cleavage by signal peptidase. in the case of ly, these charged residues can prevent cleavage by signal peptidase. the variability in the length and in the amount of charged amino acid residues at the amino terminus of asignal sequence has not as yet been explained. mutation and deletion experiments have clearly shown that charged residues are not essential for membrane insertion. in the light of our findings, we propose that the charged amino acids at the amino terminus of signal sequences function in the alignment of signal sequences in the er membrane such that signal peptidase can cleave at a very specific site with high fidelity. our prediction is that removal of charged residues from the amino-terminal end of the signal sequences can lead to an altered or less specific signal peptidase cleavage. wheat germ was obtained from general mills, california. the acceptor peptide benzoyl-asn-leuthr-n-methylamide was a generous gift from e. bause, cologne. standard molecular cloning techniques, as described by maniatis et al. (1962) were used. the cdna clone py-2, containing the entire coding region of the human invariant chain cloned into the pstl site of pbr322, was obtained from p. a. peterson's laboratory, uppsala, sweden (claesson et al., 1963) . the expression plasmids pds5, pds6, and pds5/3 have been described previously (stueber et al., 1964) . they allow efficient transcription by e. coli rna polymerase of cdnas cloned behind the strong t5 promoter p25. figures 9a and 9b summarize the construction of the fusion and deletion plasmids described below. plycat py-2 was digested with pstl, and the 317 bp fragment containing the 5' end and the 660 bp fragment containing the 3' end of the ly coding region were isolated. the 317 bp fragment coding for the ly cytoplasmic domain, the membrane-spanning segment and 12 amino acid residues of the exoplasmic domain, was cleaved by sauda to remove the sgc tail. the 234 bp sau3a-pstl fragment was isolated and cloned into bamhiipstl-cut pds5. this results in an in-frame fusion of the 5' end of ly to the cat gene. ph initial attempts to clone the completely coding region into pds5 failed. when expressed, this region is probably lethal to the bacterium. to repress transcription from the t5 promoter/operator (p/o) in bacteria, we cloned the lac i repressor between the b/a gene and the t5 p/o. this plasmid is called pfllycat. for the construction of ply, prlycat was linearized by pstl and the 660 bp pstl fragment, coding for the carboxy-terminal domain of it, was ligated into this site. transformants containing the 660 bp fragment were screened for expression of immunoprecipitable ly chain after in vitro transcription-translation. to delete the cytoplasmic domain from ircat, the 950 bp sstll-xbal fragment from plycat was isolated and ligated at the xbal site of bamhllxbal cut p6/5r. the protruding ends at the bamhl and the sstll sites were blunted with sl nuclease and ligated. as a result, a new atg initiation codon is placed just in front of the membrane-spanning segment of ly. the construction was confirmed by dna and amino acid sequence analyses (see figure 4a ). p6lsr to repress transcription from the t5 promoter the lac i gene was inserted between the b/a gene and the t5 p/o region of pds5/3 (stueber et al., 1984) . against ly domains to raise antibodies against the amino-and the carboxy-terminal domains of ly, fusion proteins of b-galactosidase and parts of ly were produced in bacteria and used as antigens to raise antibodies in rabbits. from a pstl digest of w-2, the 317 bp fragment coding for the aminoterminal 72 amino acids of ly and the 860 bp fragment coding for the exoplasmic carboxy-terminal domain of ly were isolated. each of the fragments was inserted into the pstl site of the bacterial expression vector pex1 (stanley and luzio, 1984) . fusion proteins expressed in nfl bacteria were separated on preparative sds-polyacrylamide gels (7% acrylamide; laemmli, 1970) . protein bands were visualized by koac precipitation, and fusion proteins were eluted from gel slices. two rabbits were immunized with each of the two fusion proteins. antibodies against the amino terminus of ly (anti-iyn) and its carboxyl terminus (anti-iyc) were obtained. they reacted with authentic ly chains synthesized by human raji cells (data not shown). lmmunoprecipitations after translation and posttranslational assays, antigens in a 25 vi aliquot were solubilized by adding nonidet-p40 (np40) to 0.5%. then 1 ~1 of either anti-iyn or anti+c antiserum was added and the mixtures incubated for 15 min at 4oc. forty microliters of a 1:l slurry of protein a-sepharose (equilibrated in 0.2% np40,lo mm tris-hci [ph 7.51, 150 mm naci, and 2 mm edta) was added to each sample, and incubation continued for 60 min at 4°c. beads were sedimented by centrifugation and washed three times with 0.2% np40, 10 mm tris-hci (ph 7.5), 150 mm naci, and 2 mm edta, twice with 0.2% np40,lo mm tris-hci (ph 7.5), 500 mm naci, and 2 mm edta, and once with 10 mm tris-hci (ph 7.5). sample buffer for sds-page was added to the sedimented beads, and antigens were analyzed by sds-page and fluorography. in vitro transcription and translation plasmids were transcribed in vitro by e. coli rna polymerase, and the resulting mrna was translated in a wheat germ cell-free system as described by stueber et al. (1984) . to test for membrane translocation, rough microsomes from dog pancreas were included in the translation (blobel and dobberstein, 1975) . glycosylation onto asparagine residues was blocked by the addition of the acceptor peptide benzoylasn-leuthr-n-methylamide to a final concentration of 30 pm (lau et al., 1983; bause, 1983) . assays to test translocation of in vitro-synthesized proteins across, or their insertion into, the er membrane, accessibility to proteinase k was used. a 10 pl aliquot of a translation mixture containing rough microsomes was incubated for 10 min at 25oc with either 0.3 mg/ml of proteinase k or 0.3 mg/ml of proteinase k and 0.5% np40. further proteolysis was stopped by the addition of phenylmethylsulfonyl fluoride (pmsf) to 0.1 mglml, and the sample was further characterized by sds-page (laemmli, 1970) and fluorography or, where indicated in the figure, by immunoprecipitation. to remove secretory and peripheral membrane proteins, rough microsomes were subjected to a carbonate wash with 0.1 m na&os, ph 11 (fujiki et al., 1982) . peptide; h. gausepohl for performing automated amino acid analysis: m. t. haeuptle, i. ibrahimi, and d. meyer for critical reading of the manuscript, and annie steiner for expert typing. this work was supported by grant do 199/4-z from the deutsche forschungsgemeinschaft. the costs of publication of this article were defrayed in part by the payment of page charges. this article must therefore be hereby marked "advertisement" in accordance with 18 usc. section 1734 solely to indicate this fact. received april 23, 1986; revised july 1, 1986. multiple mechanisms of protein insertion into and across membranes a stop transfer sequence confers predictable transmembrane orientation to a previously secreted protein in cell-free systems clonal variation in cell surface display of an h-2 protein lacking a cytoplasmic tail we thank p a. peterson and l. claesson, uppsala, for plasmid py-2; e. bause, cologne, for the acceptor abrahamsen, l., moks, t., nilsson, b., hellman, u., and uhlen, m. (1985) . analysis of signals for secretion in the staphylococcal protein a gene. embo j. 4, 3901-3906. adams, g. a., and rose, j. k. (1985) . structural requirements for a membrane-spanning domain for protein anchoring and cell surface transport.cell 47, looi-1015.anderson, d. j., mostov, k. e., and blob& g. (1983) . mechanisms of integration of de novo-synthesized polypeptides into membranes: signal recognition particle is required for integration into microsomal membranes of calcium atpase and of lens mp26 but not of cytochrome be. proc. natl. acad. sci. usa 80, 7249-7253. bause, e. (1983) . structural requirements of n-glycosylation of proteins. biochem. j. 209, 331-336. blobel, g., and dobberstein, b. (1975) lingappa, v. r., katz, f. n., lodish, h. f., and blobel, g. (1978) . a signal sequence for the insertion of a transmembrane glycoprotein. similarities to the signals of secretory proteins in primary structure and function. j. biol. chem. 253, 8667-8670.lipp, j., and dobberstein, b. (1986). signal recognition particle-dependent membrane insertion of mouse invariant chain: a membrane spanning protein with a cytoplasmically exposed amino-terminus. j. cell biol. 702, 2169 -2175 . long, e. 0. (1985 . in search of a function for the invariant chain associated with la antigens. surv. immunol. res. 4, 27-34. lively, m. o., and walsh, k. a. (1983) spiess. m., and lodish. h. f. (1986) . an internal signal sequence: the asialoglycoprotein receptor membrane anchor. cell 44, 177-165. stanley, k. k., and luzio, j. p (1984) . construction of a new family of high efficiency bacterial expression vectors: identification of cdna clones coding for human liver proteins. embo j. 3, 1429 -1434 . strubin, m., mach, b., and long, e. 0. (1984 . the complete sequence of the mrna for the hla-dr associated invariant chain reveals a polypeptide with an unusual transmembrane polarity. embo j. 3,869~872. key: cord-000884-zq8kqf6h authors: shen, hsin-hui; lithgow, trevor; martin, lisandra l. title: reconstitution of membrane proteins into model membranes: seeking better ways to retain protein activities date: 2013-01-14 journal: int j mol sci doi: 10.3390/ijms14011589 sha: doc_id: 884 cord_uid: zq8kqf6h the function of any given biological membrane is determined largely by the specific set of integral membrane proteins embedded in it, and the peripheral membrane proteins attached to the membrane surface. the activity of these proteins, in turn, can be modulated by the phospholipid composition of the membrane. the reconstitution of membrane proteins into a model membrane allows investigation of individual features and activities of a given cell membrane component. however, the activity of membrane proteins is often difficult to sustain following reconstitution, since the composition of the model phospholipid bilayer differs from that of the native cell membrane. this review will discuss the reconstitution of membrane protein activities in four different types of model membrane—monolayers, supported lipid bilayers, liposomes and nanodiscs, comparing their advantages in membrane protein reconstitution. variation in the surrounding model environments for these four different types of membrane layer can affect the three-dimensional structure of reconstituted proteins and may possibly lead to loss of the proteins activity. we also discuss examples where the same membrane proteins have been successfully reconstituted into two or more model membrane systems with comparison of the observed activity in each system. understanding of the behavioral changes for proteins in model membrane systems after membrane reconstitution is often a prerequisite to protein research. it is essential to find better solutions for retaining membrane protein activities for measurement and characterization in vitro. the cell membrane separates intracellular components from the outside environment and is constituted by various phospholipids, cholesterol, glycolipids and proteins. integral membrane proteins have at least one polypeptide segment spanning the membrane bilayer whereas peripheral membrane proteins are temporarily attached to the lipid bilayer or to integral membrane proteins by various interactions such as hydrophobic, electrostatic and other types of non-covalent interactions. membrane proteins work as a selective filter to regulate molecules entering cells and also serve in communicating with the surrounding environment. thus, membrane proteins play an essential role in the physiological functions needed for cell survival. the functional activities of membrane proteins are modulated by the structure of the surrounding lipids molecules in the membrane [1, 2] ; thus the composition of the lipid bilayer can affect the inter-or intra-molecular interactions between the lipid bilayer and membrane proteins [3] . investigating membrane proteins in vivo is difficult because the membrane proteins are associated with a complex mixture of other proteins, and are prone to aggregation in solution [4] . it is still a major challenge at this stage to extract information needed in vivo to address specific questions in the function of the cell membrane. to simplify cell membrane systems, model membranes such as monolayers, bilayers, liposomes and nanodiscs have been developed, enabling detailed investigation of membrane protein structure in lipid membranes. model membrane environments more closely resemble the natural lipid bilayer than alternatives such as detergents. however, many features of phospholipid structure need to be considered and optimized in the creation of a suitable model membrane. for example, the hydrophobicity of the lipid chain defined by the lengths of the fatty acid chains, is an important parameter for retaining protein activity. other factors affecting the reconstituted membrane protein activity are the chemical properties of the lipid head groups which control membrane hydrophilicity. both parameters are crucial in stabilizing membrane protein structure. there are a number of approaches used to create a model membrane in order to mimic properties of the native cell membrane, and we will review these various approaches for reconstituting membrane proteins into different types of model membrane-monolayers [5] , supported planar lipid bilayer [6] and liposomes [7] as shown in figure 1a -c. we will also discuss the emerging technology of nanodiscs [8] ( figure 1d ). nanodiscs are a new class of model membrane, with attractive properties that address shortcomings of other approaches in the study of membrane proteins. the first section gives a brief summary of each method and a comparison of their strengths and weaknesses. in the following section, we describe four case studies and will compare the protein activity changes when the membrane proteins are reconstituted into different model membranes. in these case studies, we demonstrate how protein activities are modulated by the lipid environment and discuss how this environment helps to retain protein activities. and black in b represent water and a substrate respectively. nanodiscs contain membrane scaffold proteins, shown in green. one of the most common approaches to study the membrane protein structure and activity uses a langmuir monolayer at the air-water interface. this method has been extensively used for more than a century [9, 10] . reconstitution of membrane proteins helps obtain further information on their organization and structure in the langmuir membrane [11, 12] . it is a simple method to create a phospholipid monolayer at an air-water interface. basically, a desired amount of lipid or lipid mixtures are dissolved in organic solvents such as chloroform or chloroform/ethanol mixtures, followed by spreading the lipid/solvent mixtures on the water surface. by evaporating out the solvent, the phospholipid molecules self-assemble vertically as a monolayer film at the air-water interface, with their hydrophilic head groups immersed in the water and their hydrophobic tail pointed to the air as shown in figure 1a [13] . a major advantage of using the langmuir monolayer system is that parameters such as thickness, surface pressure, molecular area and subphase thickness can be well controlled [10] . more advanced characterization techniques, such as π-a isotherm uv-vis adsorption, x-ray reflectivity, ellipsometry and rheology, have been developed to gain detailed information on the binding of proteins onto the phospholipid monolayer and to monitor enzyme activities when binding to the monolayer [14] . however, a limitation of langmuir monolayers is that the lack of a layer comparing to the natural cell structure (bilayer) and the high surface tension of water that can cause protein denaturation. despite this limitation, there are several successful studies using this approach. two types of membrane proteins in monolayer model membrane system will be briefly described below: rhodopsin [15, 16] , bacteriorhodopsin [17, 18] and the aliphatic peptide gramicidin [19, 20] have been successfully reconstituted and studied in monolayers at the air-water interface. in order to obtain information on the secondary structure and orientation, the protein layer can be investigated in situ at the air-water interface by either polarization modulation infrared reflection absorption spectroscopy (pm-irras) or x-ray reflectivity in combination with surface pressure-area isotherms [21] . the study of gramicidin is an example of such an approach, and while gramicidin is unfolded at high molecular area (low pressure), it is refolded upon compression and retains its precise structure and orientation. likewise, for both rhodopsin and bacteriorhodopsin, the secondary structures measured in monolayers are indistinguishable from that in native membranes when appropriate conditions are used. while some experiments have suggested that spreading of rhodopsin in certain conditions (>5 m/n) leads to denaturation [21] , bacteriorhodopsin, in contrast, is very stable in most testing conditions (compression and temperature change). the different properties of the protein are probably due to the ability of baceriorhodopsin to form a stable two-dimensional crystalline structure at the air-water interface [21] . phospholipid monolayers are simple model membrane systems that are perfectly suited to study the binding of peripheral proteins onto a membrane surface. peripheral membrane proteins spontaneously bind onto phospholipid monolayers at the air-water interface by injecting themselves into the subphase underneath the lipid monolayer. in most cases, useful information can be obtained by measuring the binding of peripheral proteins onto the monolayer. for example, the kinetics and dynamics of adsorption of myristoylated and nonmyristoylated recoverin onto phospholipid monolayers have been investigated using surface pressure isotherm described in figure 2 [21] . the curve can be fitted with stretched exponential which can convert into the rate of adsorption of myristoylated and nonmyristoylated which is 0.028 s −1 and 0.0048 s −1 , respectively. this indicates that the adsorption of myristoylated recoverin is six times faster than nonmyristoylated recoverin. reconstituting enzymes into the langmuir monolayers at the air-water interface has been found to be a very useful approach to understand the hydrolysis of membrane phospholipids. for example, the interfacial recognition and adsorption of phospholipases a2 (pla2) and phospholipases c (plc) to the phospholipid membrane interface are poorly understood. by using this approach, it appears that both pla2 and plc are active at the monolayer model membrane, indicating that the kinetics of phospholipid hydrolysis at the air-water interface can be monitored by biophysical characterization techniques in situ such as pm-irras and infrared reflection adsorption spectroscopy [22] . moreover, it has been found that in the presence of calcium, phospholipid hydrolysis by pla2 resulted in the production of calcium-palmitate complexes. this suggests that calcium is necessary for pla2 secretion. the formation of a supported lipid bilayer on a solid substrate was reported by tamm and mcconnell in 1985 as a new model membrane system to study the physical properties of biological membranes and their constituent lipid and protein molecules [23, 24] . supported planar lipid bilayers are prepared by several methods [25, 26] . vesicle fusion is the simplest method for supported bilayer formation and the fusion mechanism on a hydrophilic support is well understood [27, 28] . essentially, the bilayer is prepared by the fusion of small unilamellar vesicles on solid supports such as sio 2 , glass and modified gold surface by van der waals, electrostatic, hydration and steric forces. the supported lipid bilayer has polar hydrophilic headgroups facing the aqueous surroundings and two hydrophobic tails that face the interior of the membrane which more closely resembles biological membranes than the langmuir monolayer. the supported lipid bilayer can confer many key functions to biological membranes. however, one side of the hydrophilic head group is still tightly attached to the solid support and this may, in some cases, affect the fluidity of the model membrane. this matters, since integral membrane proteins may not diffuse in the plane of the membrane. furthermore the orientation of membrane proteins cannot be controlled in the supported planer lipid bilayer. to alleviate some of these problems, a new tethered polymer-supported planar lipid bilayer system was developed to investigate the reconstitution of integral membrane proteins in a laterally mobile form into the supported lipid bilayer [29] . wagner and tamm [30] have successfully designed a supported lipid bilayer on a polyethyleneglycol cushion shown in figure 3 . the polymer cushion minimizes the interactions of the proteins with the substrate and the polymer. it also provides a soft support and, for increased stability, covalent linkage of the membranes to the supporting quartz or glass substrates. in low polyethyleneglycol concentration regimes, the bilayers were assembled with high lateral lipid diffusion coefficients (0.8-1.2 × 10 −8 cm 2 /s). cytochrome b5 and annexin v were used to test the polyethyleneglycol cushion system. two populations of laterally mobile proteins were observed in the polyethyleneglycol cushion-supported bilayers. approximately a quarter of cytochrome b5 diffused with a diffusion coefficient of 0.8-1.2 × 10 −8 cm 2 /s, and more than half of the cytochrome b5 diffused with a diffusion coefficient of ~2 × 10 −10 cm 2 /s. similar results were found in the annexin v system. annexin v diffused with two populations with diffusion coefficients of 3 × 10 −9 cm 2 /s and 4 × 10 −10 cm 2 /s. the new polymer-supported lipid bilayer system has increased the mobile fraction and retained the full lateral mobility of both cytochromes b5 and annexin when integrated or bound to the supported lipid. although polymer cushions allow for successful integration of small membrane proteins into bilayers, further challenges stem from studies with large transmembrane proteins. polymer cushions cannot provide large transmembrane proteins with good solvent accessibility, or enough space for the motion; required for the activity. while several types of polymer cushions have been developed, including polymethyl methacrylate diblock polymer cushions [31] , poly(ethylene imine) [32, 33] cushions and poly(ethylene glycol) tethered lipopolymers [30] , these cushions are mostly limited to a thickness of up to 10 nm. a recent development of a maleic anhydride copolymer thin film has film thickness up to 60 nm [34] . the hydrophilic polymer-cushioned supported lipid bilayers provide a higher mobility and homogeneous distribution of the incorporated beta-amyloid precursor protein cleaving enzyme (bace) on the bilayer surface, and enhances the enzymatic activity of bace (increased from 8% to 16%). even so, the activity of the incorporated bace remains significantly lower (16%) than that of the native enzyme (100%). another important classic category of membrane proteins are the transporters of ions and small molecules. studies of how ion channels regulate the transport of substrates [7] are important for fundamental biology. however, it is challenging to incorporate ion channels in supported lipid bilayers due to leakage or instability issues. detailed studies of ion channel conduction or gating require considerable period of time (possibly >1 h), and it is difficult to set up a stable and electrically quiet environment for the ion channel in planar lipid bilayer. a better alternative has proven to be reconstitution of ion channels into proteoliposomes. lipid vesicles, also known as liposomes, consist of a self-closed lipid bilayer. they have been widely used for more than 30 years to reconstitute the membrane proteins in unilamellar phospholipid vesicles. liposomes are relatively easy to construct by procedures such as extrusion method or ultrasonication, with reverse-phase evaporation. furthermore, giant vesicles of unilamellar or multilamellar nature can be "micro-manipulated" under an optical microscope. reconstitution of membrane proteins in liposomes usually requires detergents wherein purified membrane proteins are solubilized in detergent, then mixed with the desired phospholipid vesicles forming an isotropic solution of mixed phospholipid-protein-detergent micelles. the detergent can then be removed slowly by dialysis, gel filtration or biobead adsorption. when the detergent concentration reaches a critical level, the protein will spontaneously associate with the phospholipid membrane to form biologically active liposomes, called proteoliposomes. however, it has been a hard feat to control the final orientation of protein in the proteoliposomes [35] , as well as the amount of protein inserted due to the limited area available. in many cases, disorientation of the protein causes aggregation. despite these difficulties, there have been many successful cases of membrane proteins reconstituted in the proteoliposomes, and we describe two examples below. several integral membrane proteins have been successfully reconstituted into proteoliposomes such as rhodopsin [36] , g proteins [37, 38] , proapoptotic bcl-2 proteins and t-bid [39] , phosphocholine cytidylyltransferase (ct) [40] and p protein kinase c (pkc) [41] . however, these studies also found that the resulting protein activities are sensitive to the membrane curvature of the liposomes. this indicates that different phospholipids can cause considerable curvature stress changes in the liposomes [42] . specifically, the curvature stress has been suggested to modulate the free energy and folding of the integral membrane proteins [43] . sometimes the activity of different enzymes is modulated by the same driving force of the membrane curvature, but there may also be variation of activity through different mechanisms. for example, the activities of both ct [40] and pkc [41] are enhanced by increasing the negative curvature strain of the membrane. the activity of ct appears to be directly coupled with the membrane curvature, in contrast, the activity of pkc does not have a direct relationship with the curvature strain and enzymatic activity [41] . the activity of pkc is instead modulated by nonlamellar-forming lipids via a less direct mechanism. liposomes have been commonly used for reconstituting different types of transporters to allow for the free diffusion of solution or catalysis of obligatory co-transporters. a large number of functional membrane proteins have been successfully reconstituted into liposomes but only a few examples will be discussed here. the reconstitution of colicin ia and e1 in either soybean phospholipids or e. coli phospholipids show that there is channel formation in the liposomes but there are unspecific channels allowing passage of ions, such as rubidium, sodium, chlorine, potassium or phosphate but not of sugars [7, 44] . an example of the reconstitution of selective transport comes from the d-glucose transporter, purified from human erythrocytes and extracted from detergents followed by incorporation into proteoliposomes. with incorporation of the d-glucose transporter, the proteoliposomes become permeable to d-glucose but not to l-glucose. the transport was inhibited by cytochalasin b which is a potent inhibitor of d-glucose transporter [45, 46] . several types of atp-dependent ion transporters such as ca 2+ /mg 2+ -atpase, na + /k + -atpase, and h + /k + -atpase have been reconstituted into proteoliposomes [47] . upon addition of atp, ions are observed to be transported inwards and can form a complex. the single-channel property of channels incorporated into proteoliposomes can be investigated using the well-known patch-clamp method [48] . channel activity is monitored following excision of the patch from the proteoliposomes. ion-channel reconstitution makes possible the investigation of the influence of membrane lipid composition on channel function. the kinetic investigation of these channels under physiological conditions has been discussed elsewhere [47] . another up-to-date method is using organic solvent or oil mixed with water that creates water-in-oil (w/o) microdroplets coated by phospholipid. the hydrophilic head group immerses in the water and the hydrophobic tail locates in the oil/organic solvent phase. the application of the water-in-oil system could cover a wide range of applications from monolayer, planer lipid bilayer and liposomes. funakoshi et al. [49] and maglia et al. [50] used a planer lipid bilayer formed by two microdroplets driven to come in contact to reconstitute ion channels in the bilayer. this method is extremely simple and reproducible. recently, the water-in-oil microdroplets are extended to form liposomes by using droplet-transfer method invented by yoshikawa [51] . by using this approach, it is possible to modulate the lipid compositions of outer and inner leaflets and furthermore to orient a reconstituted membrane protein in liposomes [52] . nanodiscs offer a solution to some of the challenges described in the previous sections. the first attempt to reconstitute membrane proteins in the phospholipid bilayers using nanodisc technology was initiated by sligar's group a decade ago [8] . the nanodisc is a self-assembly of phospholipids and a membrane scaffold protein derived from human serum apolipoprotein a1. the detergent, cholate, can be used to solubilize phospholipids and membrane scaffold proteins into a micelle mixture. following detergent removal with dialysis or bio-beads adsorbent, a nanodisc self-assembles. the phospholipid associates as a bilayer domain while the membrane scaffold protein wraps around the edges of the discoidal structure in a belt-like configuration ( figure 1d ). it is possible to modify the diameter of the bilayer disc by genetically engineering the apolipoprotein a1 by changing the number of amphipathic helices. by this approach, the diameter of nanodiscs can be made anywhere from 9.8 to 17 nm, and therefore accommodate a range of membrane proteins. the ratio of phospholipid: membrane scaffold protein is precisely defined which helps engineer the different size of membrane proteins in the nanodiscs. detailed formation of different types of nanodiscs has been described elsewhere [53, 54] . the great advantage of using nanodiscs is keeping the membrane proteins in aqueous solution, in native-like phospholipid bilayer environment that is soluble, stable, monodisperse and detergent-free. most important, it isolates proteins or complexes as individual particles in monomeric or oligomeric states for analysis by techniques that range from activity assays to electron microscopy. since 2003, there have been more than 100 membrane proteins reconstituted into nanodiscs [54] , ranging from signaling receptors to transport machines. we will discuss the applications separately below. nanodiscs have been used to analyze the influence of binding substrate on monodisperse receptors which are isolated from the cell-surface membrane. those receptors include g protein-coupled receptors (gpcr) [55, 56] , cholera toxin receptor ganglioside g m1 , bacterial chemoreceptor [57] and epidermal growth factor receptor. introduced into nanodiscs, the receptors stay in monodispersed, controllable, predefined oligomeric states in which it is possible to characterize the oligomeric status. for example, two different gpcr proteins, the beta-adrenergic receptor (β 2 ar) and rhodopsin [58] have been extensively studied using nanodiscs. β 2 ar was one of the first receptors assembled into nanodiscs which was found to be functionally active (54% of starting activity recovered) and shown coupling to its g-protein. rhodopsin is a light-activated gpcr present in the photoreceptor cells of the retina and transducin is an important g-protein naturally expressed in retina rods and cones. assembly of functional rhodopsin into nanodiscs was found to activate transducin with high efficiency and to isolate the high affinity of transducin-metharhodopsin ii complex. this provides strong evidence that the monomeric state of rhodopsin can activate and interact with the transducin. a dimeric rhodopsin nanodisc was separated for monomeric forms using sucrose density gradients. even with two rhodopsins in the nanodiscs, interaction with a single transducin molecule was observed and found to activate the transductin with high efficiency [56] . numerous membrane associated enzymes have been incorporated into nanodiscs. cytochrome p450 (cyp) enzymes have been extensively studied, including cyp2b4 [59] , cyp6b1 [8] , cyp73a5 [60] and cyp19 [61, 62] . this system has provided a means for studying the extensive collection of membrane bound cytochromes p450 with the same biochemical and biophysical tools that have been previously limited to use with the soluble p450s. for example, the cytochrome p450 3a4 (cyp3a4) is a membrane-bound protein which is a human hepatic drug-metabolizing enzyme. most studies of the ligand binding by cyp34a are carried out in the presence of detergents below their critical micelle concentrations [63, 64] but are compared by the propensity of cyp34a to aggregate. even in studies attempting to use liposomes, cyp3a4 is unlikely to exist in its native state because the detergent concentrations are much higher than the phospholipid concentrations. as a result, the understanding of the structure and composition of cyp3a4 in the lipid phase was limited and the membrane effect on cyp3a4 ligand binding behavior is unclear. nanodiscs have been utilized to study cyp3a4 which displays monophasic reduction kinetics. with a high lipid-protein ratio, cyp3a4 is captured as a monomer. however, at lower lipid ratio, cy3a4 self-associates and heterogeneous behaviors are induced. the nanodiscs prohibit self-association in this case as there is only one cyp3a4 per nanodisc and show significant improvement in homogeneity and stability. this opens up new possibilities for detailed analysis of equilibrium and steady-state kinetic characteristics of catalytic mechanisms of cyp enzymes [63] . the sec translocon is a membrane-embedded protein assembly that drives protein translocation into or across membranes. the core translocon is formed from a trimeric arrangement of secy, sece and secg [65] . the secyeg promoter has 15 transmembrane helices sitting in the phospholipid membrane. the oligomerization of secyeg has been proposed to be necessary to proper function. researchers were successful in reconstituting sec into membrane vesicles in 1990 and have had great success in characterizing several partial reactions of secyeg functions [65] . reconstituting a single secyeg into a nanodisc with different types of lipids [66] suggests that the acidic lipids can stabilize the secyeg channel in the nanodisc bilayer and trigger dissociation of the seca dimer. a model has been proposed by alami et al. [66] , suggesting that the dissociation of the seca dimer provoked by the secyeg complex is followed by activation of the seca atpase. furthermore, dalal et al. [67] , using the nanodisc technology, have also shown that only the secy dimer together with acidic lipids supports the activation of the seca translocation atpase. recently, a high resolution single-particle cryo-em structure of single secyeg complexes in nanodiscs, bound to translating ribosomes was first solved at subnanometer resolution [68] . it allows the secyeg complex to be investigated in a natural lipid bilayer environment and identifies the ribosome-lipid interactions. wu et al. [69] also used surface plasmon resonance to investigate the competitive binding of ribosomes and seca. the data suggest that both ribosomes and seca can interact simultaneously with secyeg complex during membrane protein insertion, but seca competes with ribosome when it binds to the secyeg complex. in the previous section, we have shown that membrane proteins can be assembled into four different types of model membrane and the activities of some of the membrane proteins can be retained, allowing their physicochemical properties to be studied. but is there a model membrane system that is the best for membrane-protein reconstitution? the reconstitution of the same membrane protein into different model membranes has been compared and, in this section, we list four membrane proteins with varying activities in different model membranes. ganglioside g m1 is a naturally occurring native receptor that binds to cholera toxin via hydrogen bonds [70] . it is an excellent receptor for studying lipid-receptor interaction. several different approaches to reconstituting the glycolipid receptor g m1 in model membranes have enabled the measurement of binding of its interaction partner cholera toxin. in liposomes and supported lipid bilayer systems, the ganglioside g m1 is free to diffuse across long distances and exhibits a non-uniform lateral distribution, i.e., self-aggregation, even at low incorporation ratios. therefore the binding activity of ganglioside g m1 with cholera toxin b is restricted [71] . investigations of ganglioside g m1 incorporated into nanodiscs found reduced protein aggregation. bricarello et al. [72] found that the reconstitution of a low concentration of ganglioside g m1 in nanodiscs, shows binding of cholera toxin with a significantly higher affinity than in liposomes or supported lipid bilayers. this is due to the interaction of ganglioside g m1 with the headgroup region of the disc which reduces the oligomerization, thereby causing a potential effect on the affinity of toxin binding. thus, nanodisc technology restricts the ganglioside g m1 oligomerization by controlling the number of ganglioside g m1 monomer isolated by each nanodisc. borch et al. [73] have also used sensor chip-based surface plasmon resonance (spr) technology to measure the detailed kinetic binding of the interaction between soluble molecules and membrane receptors inserted in the bilayer of nanodiscs. the corresponding spr sensorgrams are displayed in figure 4 . overall, the change of the sensorgram indicates that the spr sensorchip is immobilized with histidine-modified nanodisc or the cholera toxin b bound to the nanodiscs. the sensorgrams in both figure 4a ,b shows the binding of nanodiscs (576 ru) on the antibody immobilization surface on the sensor chip. by injecting the cholera toxin b over two flow cells presented in figure 4a and 4b, the spr sensorgrams can detect the interaction of the cholera toxin b with nanodisc with or without the existence of g m1 . it has been revealed that the captured 2% g m1 -nanodiscs bound 238 ru of the cholera toxin b without binding to the capturing nanodiscs without g m1 ( figure 4b ). the measured kinetic values of the interaction are in agreement with those reported by previous studies on the interaction of the cholera toxin with the g m1 receptor embedded in different membrane systems. this, therefore, serves as a proof of concept that nanodiscs can be employed in kinetic spr studies. the nucleus envelope is composed of two bilayers (the outer nuclear membrane and inner nuclear membrane) and contains abundant ion channels, through which ions and small molecules diffuse between the cytoplasm, nucleoplasm and perinuclear (i.e., intermembrane) space. the nuclear ionic channels represent a ubiquitous structure in the nuclei in a wide range of cells, although little is known about its functional properties. to characterize nuclear ionic channels, guihard et al. [74] attempted to reconstitute nuclear envelope vesicles derived from the canine liver nuclei into a planar lipid bilayer and giant proteoliposomes. they found that the success rate of nuclear envelope fusion into planar lipid bilayers was extremely low although cardiac nuclear ionic channels were successfully incorporated into planar lipid bilayers. the detection of the nuclear ionic channels activity was not possible. such a low efficiency can be explained by the clustering of nuclear envelope vesicles, and the low density of single vesicles, as well as the presence of residual chromatin and/or nuclear proteins (histones or lamins) which would prevent fusion events with the bilayer. another approach is reconstituting nuclear envelope vesicles into giant proteolipsosmes and detecting the single ion channel by the patch-clamp technique [49] . large conductance, voltage-gated, k + and cl − selective nuclear ionic channels are characterized and plotted as a current-voltage relationship presented in figure 5a ,b respectively. it has been found that under asymmetrical 150/50 mm kcl conditions, the zero current potential for unitary currents is at 322 mv ( figure 5a ). calculated from the goldman-hodgkin-katz (ghk) flux equation, a p k +/p cl − ratio is 9.4. this value indicates the k + selectivity for this channel. in figure 5b , the cl − selective nuclear ionic channel yields a positive zero current potential of +27.3 mv, with a p cl −/p k + ratio of 80, indicative of a high cl − selectivity over k + . this suggests super fusion of the channel under asymmetrical (150/50 mm) kcl conditions. the current-voltage relationship curves indicate that the nuclear ion channels can be functionally characterized by incorporating the proteins into the giant proteoliposomes where it is possible to retain their channel activity. furthermore, the measured activities are consistent with those described for native nuclear ion channels. p-glycoprotein, the most extensively studied atp-binding cassette transporter, has been implicated in the phenomenon of multidrug-resistance in tumor cells and has been suggested to play a significant role in drug absorption and deposition. how p-glycoprotein interacts with its substrates is still unknown. functional studies are limited because of the difficulty of obtaining large quantities of stable p-glycoprotein. besides that, no atpase activity of p-glycoprotein solubilized in detergent could be detected. when p-glycoprotein is reconstituted into proteolipsomes, it has detectable atpase activity; however, the whole complex is very unstable. heikal et al. [75] have further found that p-glycoprotein reconstituted in the proteoliposomes has a half-life of less than one day. in 2009, ritchie et al. [76] performed a detailed study of drug-stimulated atpase kinase activity of p-glycoprotein using the nanodisc technology. the p-glycoprotein protein was reconstituted into both msp1e3d1 disc and liposomes in order to compare its atpase kinase activities. the results described in figure 6 demonstrate that p-glycoprotein is functionally active when reconstituted into the nanodiscs (close squares). comparing to the atpase kinase activity of p-glycoprotein reconstitution in lipsosomes (close circles), there is a twofold increase in the maximum atpase activity in the nanodiscs. this could be due to the uniform orientations of p-glycoprotein in the nanodiscs while there are two possible orientations in liposomes. these data not only show that p-glycoprotein is functionally active when reconstituted into the nanodiscs, but that it also exhibits higher specific activity than the current standard reconstitution system. figure 6 . comparsions of the atpase activity of p-glycoprotein in nanodiscs (square) and proteoliposomes (circle). open symbols: basal activity in the absence of drug; filled-in symbols: activity in the presence of nicardipine [76] . atp-binding cassette transporters utilize the energy of atp hydrolysis to transport a wide range of substrates across cellular membranes and for non-transport-related processes such as translation of rna and dna repair [77] . a member of the atp-binding cassette super family, the maltose transporter malfgk2 from e. coli, together with the substrate-binding protein male, is one of the best-characterized atp-binding cassette binding cassette transporters suitable for various reconstitution techniques. bao and fuong have reported the reconstitution of the maltose transporter in nanodiscs, in detergent and in proteoliposomes. the atpase activity of the malfgk2 complex in various environments is shown in figure 7 . the data presented in the first column of figure 7 show that the basal atpase activity for assembly in the nanodiscs and detergent (~700 nmol/min/mg) is 10-fold higher than in proteoliposomes because of the decrease in the activation energy barrier of the transporter [78] in detergent micelles and nanodiscs. however, in the presence of male, the rate of atp hydrolysis increases in all assembly conditions. this is because male captures maltose and delivers the sugar to the transporter. note that the basal atpase activity assembly in the nanodiscs dramatically increases from 700 to 2300 nmol/min/mg. the maltose alone has no effect on the basal atpase activity in the nanodiscs and detergent. however, in nanodisc and detergent, an inhibition of the atpase activity was observed in the presence of both maltose and male in the nanodiscs. this is because that maltose reduces the binding affinity of the male-malfgk 2 complex, which therefore has reduced the atpase activity. in proteoliposomes, the atpase activity (~40 nmol/min/mg) shows a further 10-fold increase in the presence of both maltose and mele in the figure. the author used another type of male mutant which binds maltose with higher affinity. this male mutant, in contrast, shows a reduction of the atpase activity in proteoliposomes which has the same effect as the nanodiscs and detergents. overall, proteoliposomes have shown a low basal atpase activity because the lipid stabilized the transporter. however, the nanodiscs have been shown to be a better medium than proteoliposomes for studying the atp hydrolysis ability of atp-binding cassette transporters. this review summarizes and compares the most up-to-date methods for reconstituting membrane proteins into model membranes. there is no superior method for reconstituting membrane proteins in the model membrane; instead two or more model membranes should be considered, depending on the particular needs of the system and the proteins of interest. in general, systems based on lipid bilayers supported on a solid substrate are still the most favored and well-developed of the methods to study membrane proteins in the bilayer. this approach allows detailed study of the fundamental properties of biological membranes and is practical to reproduce the bilayer system. on the other hand, the proteoliposome is more suitable for ion channel reconstitution in the bilayer, as well as for combination with the patch-clamp method to detect the ionic selectivity of the channel. finally the self-assembled nanodiscs system provides a robust and common means for rendering these targets soluble in aqueous media while providing a native-like bilayer environment that maintains functional activities. nanodisc technology offers another way to prepare monodisperse samples of membrane proteins in the bilayer environment, and it is emerging as the favored approach in studies concerning membrane protein complexes. biochemical and functional characterization of the membrane association and membrane permeabilizing activity of the severe acute respiratory syndrome coronavirus envelope protein lipid-protein interactions in human erythrocyte-membrane acetylcholinesterase. modulation of enzyme activity by lipids correlation between the effect of the anti-neoplastic ether lipid 1-o-octadecyl-2-o-methyl-glycero-3-phosphocholine on the membrane and the activity of protein kinase calpha weak dependence of mobility of membrane protein aggregates on aggregate size supports a viscous model of retardation of diffusion structure and phase transitions in langmuir monolayers allogeneic stimulation of cytotoxic t cells by supported planar membranes effect of colicins ia and e1 on ion permeability of liposomes direct solubilization of heterologously expressed membrane proteins by incorporation into nanoscale lipid bilayers surface enhanced raman scattering of a lipid langmuir monolayer at the air-water interface lipid monolayers: why use half a membrane to characterize protein-membrane interactions? langmuir monolayer of artificial pulmonary surfactant mixtures with an amphiphilic peptide at the air/water interface: comparison of new preparations with surfactant polymyxin b-lipid interactions in langmuir-blodgett monolayers of escherichia coli lipids: a thermodynamic and atomic force microscopy study langmuir balance investigation of superoxide dismutase interactions with mixed-lipid monolayers modern physicochemical research on langmuir monolayers structure of rhodopsin in monolayers at the air-water interface: a pm-irras and x-ray reflectivity study formation, structure, and spectrophotometry of air-water interface films containing rhodopsin proton transport by bacteriorhodopsin in planar membranes assembled from air-water interface films structural and spectroscopic characteristics of bacteriorhodopsin in air-water interface films spectroscopic and structural properties of valine gramicidin a in monolayers at the air-water interface effects of gramicidin-a on the adsorption of phospholipids to the air-water interface organization, structure and activity of proteins in monolayers monitoring of phospholipid monolayer hydrolysis by phospholipase a2 by use of polarization-modulated fourier transform infrared spectroscopy supported planar membranes in studies of cell-cell recognition in the immune system supported phospholipid bilayers formation of high-resistance supported lipid bilayer on the surface of a silicon substrate with microelectrodes supported lipid bilayer self-spreading on a nanostructured silicon surface simulations of temperature dependence of the formation of a supported lipid bilayer via vesicle adsorption simulations of lipid vesicle rupture induced by an adjacent supported lipid bilayer patch membrane lateral mobility obstructed by polymer-tethered lipids studied at the single molecule level tethered polymer-supported planar lipid bilayers for reconstitution of integral membrane proteins: silane-polyethyleneglycol-lipid as a cushion and covalent linker reversible activation of diblock copolymer monolayers at the interface by ph modulation, 1: lateral chain density and conformation polymer-cushioned bilayers. i. a structural study of various preparation methods using neutron reflectometry polymer-cushioned bilayers. ii. an investigation of interaction forces and fusion using the surface forces apparatus controlled enhancement of transmembrane enzyme activity in polymer cushioned supported bilayer membranes orientation and reactivity of nadh kinase in proteoliposomes modulation of rhodopsin function by properties of the membrane bilayer role of lipid polymorphism in g protein-membrane interactions: nonlamellar-prone phospholipids and peripheral protein binding to membranes influence of the membrane lipid structure on signal processing via g protein-coupled receptors the apoptotic protein tbid promotes leakage by altering membrane curvature modulation of ctp:phosphocholine cytidylyltransferase by membrane curvature elastic stress the role of membrane biophysical properties in the regulation of protein kinase c activity membrane lipid polymorphism: relationship to bilayer properties and protein function elastic coupling of integral membrane protein stability to lipid bilayer forces reconstitution of colicin e1 into dimyristoylphosphatidylcholine membrane vesicles the permeability of bilayer lipid membranes on the incorporation of erythrocyte membrane extracts and the identification of the monosaccharide transport proteins binding of cytochalasin b to human erythrocyte glucose transporter conformational dynamics of na+/k+-and h+/k+-atpase probed by voltage clamp fluorometry the extracellular patch clamp: a method for resolving currents through individual open channels in biological membranes lipid bilayer formation by contacting monolayers in a microfluidic device for membrane protein analysis droplet networks with incorporated protein diodes show collective properties cell-sized liposomes and droplets: real-world modeling of living cells oriented reconstitution of a membrane protein in a giant unilamellar vesicle: experimental verification with the potassium channel kcsa phospholipid phase transitions in homogeneous nanometer scale bilayer discs membrane protein assembly into nanodiscs functional reconstitution of beta2-adrenergic receptors utilizing self-assembling nanodisc technology transducin activation by nanoscale lipid bilayers containing one and two rhodopsins using nanodiscs to create water-soluble transmembrane chemoreceptors inserted in lipid bilayers atomic-force microscopy: rhodopsin dimers in native disc membranes single-molecule height measurements on microsomal cytochrome p450 in nanometer-scale phospholipid bilayer disks co-incorporation of heterologously expressed arabidopsis cytochrome p450 and p450 reductase into soluble nanoscale lipid bilayers the critical iron-oxygen intermediate in human aromatase the ferrous-oxy complex of human aromatase kinetics of dithionite-dependent reduction of cytochrome p450 3a4: heterogeneity of the enzyme caused by its oligomerization ligand binding to cytochrome p450 3a4 in phospholipid bilayer nanodiscs the effect of model membranes the atpase activity of seca is regulated by acidic phospholipids, secy, and the leader and mature domains of precursor proteins nanodiscs unravel the interaction between the secyeg channel and its cytosolic partner seca two copies of the secy channel and acidic lipids are necessary to activate the seca translocation atpase cryo-em structure of the ribosome-secye complex in the membrane environment competitive binding of the seca atpase and ribosomes to the secyeg translocon crystal structure of cholera toxin b-pentamer bound to receptor gm1 pentasaccharide self-aggregation-an intrinsic property of g(m1) in lipid bilayers ganglioside embedded in reconstituted lipoprotein binds cholera toxin with elevated affinity nanodiscs for immobilization of lipid bilayers and membrane receptors: kinetic analysis of cholera toxin binding to a glycolipid receptor patch-clamp study of liver nuclear ionic channels reconstituted into giant proteoliposomes the stabilisation of purified, reconstituted p-glycoprotein by freeze drying with disaccharides chapter 11-reconstitution of membrane proteins in phospholipid bilayer nanodiscs abc transporters, mechanisms and biology: an overview discovery of an auto-regulation mechanism for the maltose abc transporter malfgk2 the authors gratefully acknowledge financial support from the australian research council (arc). hhs is an arc super science fellow and tl is an arc federation fellow. tl and lm were awarded the arc super science fellowships and grant (fs110200015). we thank victoria hewitt for her critical reading of the manuscript. key: cord-028738-ing07qma authors: roy, nimisha; kashyap, jyoti; verma, deepti; tyagi, rakesh k.; prabhakar, amit title: prototype of a smart microfluidic platform for the evaluation of sars-cov-2 pathogenesis, along with estimation of the effectiveness of potential drug candidates and antigen–antibody interactions in convalescent plasma therapy date: 2020-07-08 journal: trans indian natl doi: 10.1007/s41403-020-00148-0 sha: doc_id: 28738 cord_uid: ing07qma originating in china during december 2019, the novel corona-virus, sars-cov-2, has created mayhem worldwide in a very short time. the outbreak has been so rapid and widespread that the only option to treat the patients was administering drugs already available in the market like chloroquine/hydroxychloroquine (an antimalarial drug) and remedesivir. a large number of patients have been cured but the attribution to survival by these drugs has been controversial. till date, we do not have any specific drug or vaccine available for covid-19 and the pandemic seems to be far from over. to handle the current challenges posed by the outbreak effectively, we need to employ innovative interdisciplinary approaches. organ-on-chip (ooc), particularly lung-on-chip, is one such approach which combines the potential of microfluidics, cell culture and molecular biology into a single miniaturised platform. the device is realized to be capable of simulating in-vivo physiological responses of an organ. in the current study, an ooc, which is a multichannel 3d cell culture microfluidic device, is made via soft lithography technique, using polydimethylsiloxane-polymer and diverse polymeric porous/semipermeable membranes. several polymer membranes i.e. pdms, polyvinylidene fluoride (pvdf), nitrocellulose, polyester etc., integrated into the microdevices, were efficiently explored to realize their better cell-adhesion and viability property. we also propose for the application of a simple, smart and cost-effective lung-on-chip platform to study the sars-cov-2 pathogenesis in humans, drug toxicity testing and provide insights into antigen–antibody interactions. this platform will enable us to study multiple phenomena at a micro-level generating more reliable data and a better understanding of the underlying mechanisms of sars-cov-2 infection and pathogenesis. on march 11, 2020, the who declared covid-19 a pandemic considering the global apocalyptic effects of this disease. although the scourge of this pandemic did not spare any nation, the top ten among most severely affected countries includes usa, brazil, russia, united kingdom, italy, spain, india, germany, france and peru. as per the may 31, 2020 situation report of who, the total number of cases and deaths worldwide are 5, 934,936 and 367,166, respectively. presently, no drug or vaccine for this disease is available. however, several potential drug and vaccine candidates are in various stages of clinical trials. nevertheless, considering the lengthy procedures of conventional drug discovery pipeline it would take substantial time before these drugs reach the market. the rate at which covid-19 spread across the globe has been alarming, and based on the initial information about the pathogenesis the only option was to administer existing drugs approved for other infectious diseases; however, their usage for covid-19 has been controversial because of the associated side effects. as the pandemic continues to spread, some important queries are imminent. firstly, can existing drugs be repurposed for effective treatment of covid-19? if so, how their efficacy can be evaluated rapidly? secondly, what alternative options can be employed to reduce the time of clinical trials by rapid drug testing and approval? the current need is a screening platform that can provide reliable results in less time. the organ-on-chip technology (ooc) is one such alternative. though the field is still in its infancy, it has tremendous potential to revamp the conventional drug discovery pipeline. the ooc technology emerged with the pioneering work by huh et al. at the wyss institute at the harvard university in 2013 (huh et al. 2011) in which they described a fabrication protocol for organ-on-chip using polydimethylsiloxane (pdms). since the inception of the concept, enormous efforts have gone into converting this proof-of-concept into actual working devices which are currently being globally used by researchers with some of the breakthrough results (zhu et al. 2010; neuzil 2012) . various startups have been set up as spin-offs from various research institutions across the globe with huge market potential and substantial revenue-earning. in india, however, this promising platform has not been harnessed. in the current pandemic situation, this technology may prove to be a valuable tool for not only for performing drug toxicity testing but may also be used for analysing the sars-cov-2 pathogenesis in human. oocs are a designed network of microfluidic channels intended for mimicking the smallest physiological and biochemical functional units of various organs. the chip dimensions are in the range of a few centimetres with microchannels network in the range of micrometres. these oocs provide significant advantages over the traditional 2d cell culture (fig. 1 ) in petri-plates like: (i) closely mimicking the cell's natural microenvironment or niche, (ii) minimal requirements of reagents, (iii) options for flexible design to suit experiment needs and (iv) high throughput efficiency. initially, the primary goal for developing the ooc was to expedite the conventional drug discovery pipeline (wikswo 2014) . in the current drug discovery and development paradigm, it takes about 10 years for a single drug to reach the market. about 90% of the drugs fail at various stages of clinical trial (kola and landis 2004) . this culminates into severe loss in terms of money and time, and sometimes volunteer's life. in addition, the present animal testing is deemed inhumane with organisations such as peta protesting against it. there have also been many incidents where a novel drug performed excellently in animal models but subsequently failed to produce the same expected response in humans. sometimes, this too resulted in life-threatening repercussions to the human clinical trial candidates (suntharalingam et al. 2006; strooper 2014) . opposed to all the above-mentioned drawbacks, ooc being an enclosed 3d cell culture platform mimics the smallest functional unit of an organ hence it provides reliable results as they are capable of generating in-vivo like environment on a chip in a comparatively lesser amount of time. one of the most important advantages is in its ability to recreate the cell microenvironment by precisely controlling the flow rate of fluids using pumping devices, such as syringe pumps. some of the other pertinent advantages include reduced risk of contamination, resolution up to the single-cell level to study cellular morphology, mechanisms with the scope of parallelisation to study cellular reactions simultaneously. figure 1 represents the comparison between 2 and 3d cell culture platforms (halldorsson et al. 2015) . although it would be too early to say that these chips would replace animal testing, they will surely provide a platform to study diseases at the molecular level and for rapid drug screening. this fig. 1 comparative presentation of a 2d, and b 3d cell culture platfroms would be a boon to the society in urgent situations such as in current pandemic (fukumoto and narasaiah 2013) . our group has been working on organ-on-chip technology that is primarily focussing on lung/liver-on-chip devices and searching for alternate ways of fabrication and application of versatile materials to lower the cost. in the liver-on-chip device, we tried to mimic the hepatic sinusoids which are the site for mixing of the oxygen-rich blood coming from the hepatic artery and the nutrient-rich blood from the portal vein. further, we performed experiments in the search for the most optimal materials, by replacing the commonly used porous pdms membrane that will be cell life-supporting, economical and easily accessible membrane to serve as an integral part of oocs. we conducted a comparative study of cell adhesion on various easily available membranes to examine parameters of cell adhesion and viability properties. we also performed static cultures in the chip to assess for cell viability inside the chip. for this purpose, we conducted studies with human liver cell line hepg2 as they are a good representative model of hepatocytes. although the ultimate aim of developing this platform is to assess the drug toxicity on the hepatocytes the protocol can be easily adapted to fabricate other organs too. we illustrate the application of 'lung-on-chip', a microengineered biomimetic platform to get a deeper insight into the infectious mechanism of the virus on the lung epithelial cells along with the evaluation of the efficacy of drug/ vaccine and their toxicity testing. our fabrication protocol takes inspiration from the protocol mentioned by huh et al. but we have introduced some major changes in fabrication and materials to lower the cost of developing the chip. in addition, the bonding of membranes with upper and lower channel layers has been customized with a novel membraneprocessing method. this 'lung-on-chip' platform is basically a 3-dimensional alveolar-capillary interface (fig. 2) , which is the site of gaseous exchange between blood and alveolus (the functional unit of lungs). under normal conditions, the alveolar membrane and the capillary boundary are close and hence optimal exchange of gases will occur to support life. patients with a severe infection of covid-19 develop acute respiratory distress syndrome (ards). in this syndrome, the alveolus sacs are filled with fluid and hence the interface between oxygen and blood capillary wall thickens. as a result of the fluid accumulation, the amount of oxygen in the alveoli decreases leading to less oxygen being diffused into the blood-stream. since there is a decline in the amount of oxygen reaching the organs, the patient dies slowly due to multiple organ failure. studies have shown that such a microfluidic platform can be effectively utilised to study ards (viola et al. 2019) . recent studies (chen et al. 2020; shen et al. 2020 ) have suggested that the plasma from the patients, who have recovered from covid-19, can be used to treat patients based on successful results of this therapy during sars, mers and ebola outbreak. the plasma of recovered patients develops antibody against the virus and these can be transfused to other patient's body to help fight the infection. however, the fda has only allowed it to be used as an experimental therapy for only a few critically ill patients (tanne 2020). one of the major issue associated with this therapy is the risk of mild to severe allergic responses that could even be life-threatening (roback and guarner 2020) . utilising this platform we can also study the mechanism of convalescent plasma therapy on a micro-scale by introducing antibody-rich plasma into a virally infested chip. the results so obtained may be of immense importance for designing future therapies. in addition, there is immense flexibility in fig. 2 illustration showing a lung alveoli and blood capillary interface, and b the proposed lung-on-chip platform aiming to recapitulate this alveoli-capillary interface, as it is the place where the exchange of gases takes place the design of ooc devices, it can be easily tailored to the needs of a single cell type culture or multiple cell type coculture on the same chip (yeo et al. 2011 ). while designing the 'lung-on-chip' device, the critical consideration is that the device assembly must resemble the alveolar-capillary interface as is found in the human lung. besides, a mechanism to induce the stretching of the membrane should be possible as is the case with the alveolar membranes during breathing. the device consists of three layers: an upper channel layer, lower channel and the side vacuum channels. a 3d microfluidic device replicating the alveolar-capillary interface has been described in fig. 3 . it consists of two channels separated by a porous membrane. in the original practice, a pdms membrane was used; however, we have also explored the possibilities of other membranes easily available in the laboratory i.e. polyvinylidene fluoride (pvdf), nitrocellulose, polyester etc. the width of channels is selected after considering the average diameter of the human lung-alveolus. initially, the device uv-mask were designed using auto-cad and printed on transparency-sheet using the high-resolution printer. further, the master-moulds of su-8 polymer were fabricated using the photolithography technique. later, the upper and the lower channels were fabricated separately using pdms polymer, via the general soft-lithography technique. afterwards, the upper and lower channels were bonded together using oxygen plasma, after proper alignment under the microscope, keeping the membrane sandwiched in between the two channels. the microchannel network (fig. 3 ) and its respective geometry and purpose are be defined below: (i) the upper channel: it is lined with the alveolar lung cells, and oxygen/air-flow is introduced through the channel. this channel has a greater depth as compared to the lower channel. (ii) the porous membrane: it is the membranous-interface, from where the diffusion of nutrients and waste material takes place, as well as, it is also the site for the cell-cell interactions. in general, this membrane needs to be very thin and stretchable. the porous membrane is present between the upper and lower cell culture channels. (iii) the lower channel: it represents the endothelial lining of the blood vessels that exchanges oxygen from alveolus in return of carbon dioxide. (iv) side vacuum channels are for inducing relaxation and contraction as experienced by the human lung. the above-mentioned fabrication process has resulted in the development of an efficient device with appropriate geometry and dimension, having ideal microchannel bonding strength. both upper and lower channel is made of pdms (polydimethylsiloxane) which is a transparent biocompatible polymer, along with polymer membrane, which is observed to be permeable to life-supporting gases. once the device was fabricated after soft-lithography and bonding; the cells were seeded and immobilised inside the channels at 80% confluence. the cells were regularly observed under the phase-contrast microscope to assess for their normal morphology and health (fig. 4) . initial results of staining and microscopy observations have shown excellent cell-viability inside the channels through live-dead cell staining. in this section, we describe the overall methods used and intermediate results we obtained till now. we describe our work plan and timeline under the sub-heading 'timeline envisaged' for the future work through an illustrative diagram. human hepatocellular carcinoma (hepg2) cells were procured from the national cell repository (ncss, pune, india) and were cultured and maintained according to the atcc guidelines. cells were maintained in dmem (sigma-aldrich, germany) supplemented with 10% fetal bovine serum (fbs) (pan biotech, germany), 100 µg/ml of penicillin, streptomycin, and 0.25 μg/ml amphotericin. cells were grown and maintained under conditions of 5% co 2 with 95% atmospheric air at 37 °c temperature in a humidified incubator. for propidium iodide (pi) and hoechst staining cells were seeded in 35 mm plate on both membranes (nitrocellulose and polyester) and control plate and allowed to grow to 60-70% of confluency. after 24 h, the cells were stained with a blue fluorescent nuclear dye hoechst 33258 (sigma-aldrich, 0.25 µg/ml) and red fluorescent dye pi (thermo fisher scientific, 4 µm) for dead cells and incubated for a minimum of 2 h at 37 °c. subsequently, the cells were rinsed with pbs, and fresh media was added. cells were observed for live-dead cells under a nikon upright fluorescence microscope with water immersion objectives (model evolution fig. 4 fluorescence images of hepg2 cells cultured on both nitrocellulose and polyester membrane and stained via pi/ hoechst dual-staining process performed for 1 h at 370 °c. both nitrocellulose and polyester membranes exhibit cell adhesivity and viability similar to control. cells were visualized for live/dead cells using live-cell imaging. hoechst fluorescence images indicate that all cells have a normal healthy and intact nuclei; pi fluorescence images represent dead cells vf, media cybernetics, usa) microscope, and images were taken at 40 × magnification. in our experimental study, we envisage examining some of the cost-effective and commonly available synthetic biopolymer membranes as a substitute to the porous/semipermeable pdms polymer membrane that works similar to it in our ooc device and exhibits better cell-adhesion and viability. these membranes are also promising because they are chemically and mechanically stable, and biologically inert. in addition, its porous structure allows it for the exchange of biomolecules. in our study, we analysed the effect of nitrocellulose and the polyester membrane. these membranes were tested for its ability to support cell viability and adhesivity through dual staining with pi and hoechst for live/dead cell nuclei using hepg2 cells as a cell model for hepatocytes followed by live-cell microscopy. strikingly, we observed that both the membranes were efficient for adhesion of cells, and cells remained viable even after 24 h. in both membranes, high cell viability was maintained similarly to the control as shown in fig. 4 . hoechst staining revealed that all cells have intact and evenly shaped nuclei. in terms of the pi staining, images displayed non-apoptotic changes in cells. this outcome confirms that both membranes are suitable for cell adhesivity and viability and might serve as promising candidates for the porous/semi-permeable polymeric membranes in our occ device. based on the results obtained during this study and based on available literature we can assert that these membranes will also serve as excellent substrates for the cells lines used for the lung-on-chip device. li et al. (2013) performed a study concluding that nitrocellulose membrane displays excellent cytocompatibility of different cell lines on nitrocellulose membrane for tissue culture. due to its affinity towards membrane proteins, nitrocellulose membrane provides essential support for cell adhesion. apart from being porous, this membrane can be rendered transparent enabling easy visibility of cells under microscopes. hanke et al. (2012) in their elegant study demonstrated that excellent and immediate cell adhesion on both sides of the permeable polyester membrane by simple surface enhancement techniques of dielectrophoresis and electrostatic forces enabling a quick cell adhesion, even against gravity. therefore, both these membrane can effectively be utilised for our intended lung-on-chip platform. the technology innovation supports advanced in-depth insights concerning the covid-19 and lung-cell interactions, requisite biomarker and transcription of various factors that need to be studied including enzyme-linked immunosorbent assay (elisa) for il-6 and il-8, mucus secretion, cell surface p-gp expression, sodium fluorescein permeation etc. the major goals of our study have been illustrated in fig. 5 . there is immense flexibility in the plan of the proposed microfluidic device, as it can be customized, according to the specific needs of individual cell types and cell cocultures, and can be implemented on the same chip. the reported 'lung-on-chip' devices closely mimic the cell's natural microenvironment; for example, by continuous culture perfusion or by creating chemical gradients. further, there are possibilities of analysing low numbers of cells or single cells in high temporal and/or spatial resolution via automation, parallelization, on-chip analysis, as well as schematic for our objective of the proposed study. the major goals include: a studying antigen-antibody interaction, and b screening of the potential drug candidates for toxicity, which will aid in c speeding of drug development. d prediction of any outcomes that may arise as a result of drug treatment. further, it will be possible to e study disease mechanisms at micro levels which would give us useful insights in fighting the disease. f other drugs that are available in the market and show potential to fight covid-19 can be tested and repurposed accordingly direct coupling to downstream analytical chemistry platforms. one of the major issue associated with drugs is hepatotoxicity, and hence by combining this platform with the liver-on-chip platform already developed by us, we can get a deeper insight into the pharmacokinetics of the drug. this research documented herein requires an interdisciplinary approach as it requires the expertise of researchers working in the domain of microfluidics, mammalian cell culture and virology. to fabricate the chip using photolithography, appropriate consumable fabrication-polymer materials i.e. biocompatible polymer poly-dimethyl siloxane (pdms) and su-8 photoresist; however, other low-cost biocompatible polymers may also be explored. the basic laminar hood is essential to ensure the contamination-free fabrication of the cell-impregnated chip. the essential equipment set-up exclusively used for producing such contaminationfree micro-devices, includes desiccator, centrifugal mixer, spin-coater, uv-curer, hot plate, hot air oven, plasma system, etc. in addition, while performing dynamic cell culturing, syringe-pumps and peristaltic-pumps would be required for continuous controlled flow of fluids at precise and very low flow rate. the entire chip can be moved from one place to another without any concern, ensuring that the surface remains contamination-free. for this purpose, the device needs to be packed suitably and kept in an appropriate thermally controlled atmosphere to maintain the cell viability. to mimic the alveolar-capillary interface, calu-3 (cultured in dulbecco's modified eagle's medium: f-12 supplemented with 10% fbs, 1% l-glutamine, 1% penicillin and 1% non-essential amino acids) and egm-2mv human lung microvascular endothelial cell line will be required, along with other reagents such as the specific culture media, buffers, antibiotics, antimycotic solutions, pbs, fbs, trypsin. the types of equipment present in a standard mammalian cell culture lab are sufficient to transfer and incubate cells inside the device. to study the disease model and pathophysiology of covid-19, the cells would have to be infested with the isolated virus. as this is potentially hazardous, therefore, bsl-3 and bsl-3 plus labs would be required to perform these studies. the government of india has approved 126 labs, with bsl-3 and bsl-3 plus, across the country for covid-19 testing and conducting research. because the devices are portable, these can easily be transferred to distant laboratories for carrying out the requisite tests. figure 6 explains the entire work plan in a nutshell. the proposed study can be divided into three main stages: 1. design, fabrication and rapid prototyping of the device (15-30 days): the devices are proposed to be fabricated via the soft-lithography process. to begin with, the channels would be designed using the autocad software. the mask would then be printed on transparent sheets with the help of high-resolution inkjet printer and would be left to dry in sterile conditions and cut in the appropriate size. device fabrication will be done using optimised cost-effective photolithography process which does not necessarily require a clean-room facility. all the steps are performed under a laminar hood. (i) su-8 master fabrication: the first step in the fabrication is developing a master using su-8 polymer, which is an epoxy-based negative photoresist. substrate surface preparation: the glass slides (75 × 25 mm) would be used as the substrate for developing the su-8 master. initially, the glass slides will first be cleaned through acetone boiling using sonicator followed with detergent and di water. further, wiped with ipa to ensure perfect cleaning. thermal treatment will be given via heating the slide at 200 °c for 5 min on a hotplate spin coating: since our device consists of two channels of varying depths, hence for each channel su-8 mask will be required. as the thickness of the su-8 layer varies with the rpm, hence, the final thickness of the microstructure will be defined by adjusting the rotational speed of spin coater chuck. pre-bake: this is done on a hotplate at two different temperatures. first, at 65 °c to create slow evaporation of the solvent resulting in a uniform coating and enhanced adhesion to the substrate. the second cycle consists of gradually heating the substrate with the su-8 layer up to 95 °c, to densify the su-8. exposure using uv-aligner: placing the su-8 polymer-coated glass-slide, beneath a predesigned uv-mask, and exposed with uv-aligner, for a defined period to suitably cross-link the polymer. post-bake: this step is accountable for increasing the degree of cross-linking in the su-8 irradiated areas (due to the photoacid generated in the uv exposure step) making it resistant to the action of solvents in the developing step. the temperature is applied gradually to reach two specific temperatures: 65 °c followed by 95 °c. development of the su-8 microstructure: for the final su-8 microstructure, it is necessary to remove the non-polymerized su-8. to achieve this, the microstructures are immersed in a container with su-8 developer from microchem while continuously shaking the container to give a rigorous wash to the master mould. rinse and dry: once all the non-polymerised su-8 is dissolved in the developer, it is wiped with a clean wipe and rinsed with a stream of di water. passivation of the su-8 master: to make the su-8 master more durable and resistant to peel off, the silanization process is necessary. to do this, the su-8 mould is placed in the vacuum desiccator and few drops of aptes (silanizing agent) is placed, in a small open container using a disposable dropper. further, a vacuum environment is quickly created and the lid of the desiccator was sealed. the vacuum pump is switched off and the setup kept stand like this for an hour so that a passivation monolayer is formed on the su-8 mould. the process is always done inside the fume hood. (ii) pdms polymer casting and rapid prototyping: for making the upper and lower channel, first the pdms elastomer was mixed with few drops of curing agent in the ratio 10:1 (wt/wt) or 9:1 (v/v) and vigorous mixing is done manually or via mixers for around 10 min. once the mixing is completed, it is kept inside desiccator to remove all the bubble formed as a result of vigorous mixing. the duration of this degasification may range from 15 min to 1 h fig. 6 schematic diagram for the overall plan of the proposed work, illustrating the major planned steps in the entire study. cells from petri dish is seeded inside the device channels (both upper and lower) at specified cell density (cells/ml). the device is then incubated overnight. to check cell viability, cells are stained with fluorescent dyes like hoechst and pi. further to perform studies under dynamic conditions, the platform is connected with pumps keepin the floe rates very precise and slow. the media is changed routinely and all assays are performed under controlled flow conditioned depending upon the number of bubbles. while degasification is in progress, the hot plate is maintained at 150 °c. the temporary boundary is made around the su-8 master using aluminium foil, ensuring for no leakage and the bubble-free pdms prepolymer mixture were poured/cast in it. pouring/ casting should not start from the region where the microstructures are present. the complete pdms casting arrangement is placed on the hotplate, which is maintained at 150 °c for 10 min and the mould is removed from the hotplate. the foil boundary is removed and the pdms layer is gently peeled off, and inlets and outlets holes were created at preplanned places using 1.5 mm hole puncher. the membrane to be used in between the channels is incised precisely by observing under a microscope. the width of the membrane should be slightly greater than the channel width and length equal to the length of the channel. the membrane is treated with corona treater for 1 min by doing sweeping motions and it is placed on top of the bottom pdms layer under a microscope. the top pdms layer is treated with corona treater for 1 min and it is placed on top of the membrane under a microscope to ensure proper alignment. the whole assembly is kept inside the oven at 60 °c overnight (7-8 h). further, the assembly is taken out and brought at room temperature, and with a sharp scalpel, the edges of the device are trimmed and cleaned. finally, it is kept in a clean closed box. 2. cell seeding and culturing inside the channels (15-30 days): the cells will be cultured following the standard protocol given by the manufacturer following all measures to avoid contamination. a confluency of 70-80% is required before seeding cells inside the device. the cell seeding density (cells/ml) is always done according to the volume of the channels and the days of continuous incubation of cells in the device. the counting of cells before seeding is done using a haemocytometer. before seeding the cells inside the channels, sterility of the channels should be ensured to avoid contamination. to do this, 70% ethanol (v/v) is perfused for 10 min followed by washing with autoclaved di water. further, the device is placed under uv light for 5-10 min. once the device is sterilised completely, the cells are seeded as done routinely. the pipet tip the placed on the inlet of the channel and the suspension is dispensed swiftly. the device with the seeded cells is placed inside the incubator for 24 h. the cells are visualized under the microscope for cell adhesion. if the cells have adhered properly, the inlet/outlet ports of the top channel are closed and the device is reversed to seed cells inside the lower channel via repeating the same step. to ensure a long term contamination-free health cell culture, the media may be changed every/alternate day depending on confluency. 3. immunological staining for cell viability and functionality testing (15-20 days): (i) staining with hoechst and pi: to observe live/dead cells, the cells are stained with hoescht and pi dye using the abovementioned process applied in the intermediate result section and subsequently observed under the microscope. (ii) enzyme-linked immunosorbent assay (elisa) for il-6 and il-8: the media collected from the chips will be stored at − 80 °c until experimenting. after thawing them, the levels of secreted il-6 and il-8 will be analyzed according to the manufacturer's instructions, using commercial human il-6 and il-8 elisa kits to find the corresponding absorbance (shrestha et al. 2020 ). (iii) immunofluorescence staining of intercellular junctions: at room temperature, the cells are fixed with 4% volume/volume (v/v) paraformaldehyde in pbs after washing three times with pbs. it is incubated for 15 min and washed with pbs twice. follow permeabilization of the cells with 0.3% (v/v) triton x-100 for 10 min and blocking with 2% (w/v) bovine serum albumin (bsa) in pbs for 2 h. for staining of endothelial junctions, primary ve-cadherin antibody is diluted in bsa solution at a dilution of 1:100, and introduced into the lower channel. the mixture is then incubated at 4 °c overnight. antibody solution is removed by flushing the channels with d-pbs three times. fluorescently labelled goat anti-rabbit antibody is diluted in 1:200 bsa solution and introduced into the lower channel. it is incubated at room temperature in the dark for 1 h. antibody solution is removed by flushing the channels with d-pbs three times. the device is now ready for visualization of endothelial adherens junctions using fluorescence microscopy. the whole process is repeated with fluorescently labelled anti-occludin antibody in the upper microchannel to visualize tight-junction formation in the epithelial cells (huh et al. 2011). 4 . drug efficacy and toxicity testing (10-15 days): to study the drug efficacy/toxicity of various drug candidates, the primary requirement is to infest the chip with the isolated virus under bsl-3 laboratory. once the chip is infested, the appropriate dosage of the drug would be given and drug toxicity study would be performed using mts assay kit according to the manufacturer's protocol. 5. studying the mechanism of convalescent plasma therapy: to study the underlying mechanism and the associated immune responses-like variations in cytokine activity at the cellular level, we will introduce plasma containing the antibody inside the infested channels. the proposed 'lung-on-chip' platform is a simple yet innovative device containing a defined network of microfluidic channels lined by living human cells on the microchannel wall. these may replicate the smallest functional units of organs and organ-level physiology. the protocol followed here can be customised for other tissues or organs, as well, to study the effect of the drug on multiple organs. it is a rapid and low-cost alternative to the lengthy conventional drug testing pipeline that involves animal testing which would undoubtedly be an advantageous study tool in pandemic situations calling for urgent measures. we are in the initial experimental stages with very promising results. with further experimentation and validation, this platform would help tackle a resolve several issues associated with various aspects of covid-19 resulting in designing therapies that are effective and elicit negligible side effects. convalescent plasma as a potential therapy for covid-19 lessons from a failed γ-secretase alzheimer trial human lung on a chip: innovative approach for understanding disease processes and effective drug testing advantages and challenges of microfluidic cell culture in polydimethylsiloxane devices generating cell co-cultures by rapid cell adhesion on opposite sides of polyester membranes can the pharmaceutical industry reduce attrition rates? use of nitrocellulose membranes as a scaffold in cell culture revisiting lab-on-a-chip technology for drug discovery convalescent plasma to treat covid-19: possibilities and challenges treatment of 5 critically ill patients with covid-19 with convalescent plasma a rapidly prototyped lung-on-a-chip model using 3d-printed molds cytokine storm in a phase 1 trial of the anti-cd28 monoclonal antibody tgn1412 covid-19: fda approves use of convalescent plasma to treat critically ill patients microphysiological systems modeling acute respiratory distress syndrome that capture mechanical forceinduced injury-inflammation-repair the relevance and potential roles of microphysiological systems in biology and medicine world health organization (2020) coronavirus disease 2019 (covid-19): situation report, 132. world health organization cultured human airway epithelial cells (calu-3): a model of human respiratory function, structure, and inflammatory responses publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord-289541-y7lewk1t authors: zhang, li-zhi title: fabrication of a lithium chloride solution based composite supported liquid membrane and its moisture permeation analysis date: 2006-05-01 journal: j memb sci doi: 10.1016/j.memsci.2005.09.035 sha: doc_id: 289541 cord_uid: y7lewk1t a novel composite supported liquid membrane has been prepared for ventilation air moisture recovery. the membrane is composed of three layers: two hydrophobic protective layers and a sandwiched hydrophilic support layer in which licl solution is immobilized to facilitate water vapor transfer. a test is conducted to measure the moisture permeation rate through the composite membrane. various resistances in the cell and in the composite membrane are clarified. linear equilibrium relations between humidity, temperature, and licl concentration in the liquid solution layer are obtained to aid in the model set-up. it has been found that the mean moisture permeation rate through the composite membrane is around 1.14 × 10(−4) kg m(−2) s(−1), almost two times higher than that through a solid hydrophilic cellulose acetate membrane with comparative thickness. further, the supported liquid layer only accounts for 12% of the total moisture transfer resistance in the cell, indicating that there is much potential for further performance improvement. people's concern on indoor air quality has greatly deepened since the outbreak of the severe acute respiratory syndrome epidemic (sars) that devastated south china and some other parts of the world in the spring of 2003. increased fresh air ventilation is the most efficient and necessary way to have a better indoor air quality, either in eliminating disgusting odors, diluting vocs, or decreasing the possibility of being infected by viruses. increased ventilation rates usually lead to high-energy consumption in air-conditioning. this is because the relative humidity in an occupied building must be controlled to within 40-60% for health and comfort reasons. it has been proved that ventilation air constitutes about 68% of the total moisture load in most commercial buildings [1] . as a consequence, treatment of the latent load from the ventilation air is a difficult and imminent task for hvac engineers, especially in hot and humid climates. * tel.: +86 20 87114268; fax: +86 20 87114268. e-mail address: lzzhang@scut.edu.cn. to save energy in treating fresh air, various techniques for independent air dehumidification have been proposed [2, 3] . among those drawn much attention is one that involves membrane technology-the so called membrane based total heat recovery (mthr) [4] [5] [6] [7] . mthr is an air-to-air heat exchanger where the two incoming streams (fresh air and exhaust air) exchanges heat and moisture simultaneously through membranes. the fresh air represents outdoor air (fresh air intake for a hvac system). the exhaust air represents stale room air that would normally be exhausted to the outside. the core material of an mthr ventilator are vapor-permeable membranes, therefore both heat and moisture are transferred between these two air streams when they flow through the unit. thus, heat and humidity would be recovered from the exhaust stream in winter and excess heat and moisture would be transferred to the exhaust in order to cool and dehumidify the incoming air in summer. in this way, large quantities of energy in hvac could be saved. hydrophilic polymer membranes that are permeable to vapor, but impermeable to air, have been considered for mthr ventilators. nafion [8, 9] , regenerated cellulose [10] , cellulose triacetate [11] , sulfonated poly(phenylene oxide) [12] , polyether-polyurethane [13] , siloxane-amido copolymer [14] , polystyrene-sulfonate [15] , polyvinylidene fluoride and polyethersulfone [16] , and cellophane [17] are good choices since they have already been used in air dehumidification applications which have similar moisture transfer mechanisms to moisture recovery. however, moisture diffusion coefficients in such polymer membranes are usually very low, in the order of 10 −12 to 10 −13 m 2 s −1 [11, 18] , while mthr ventilators only have limited transmembrane vapor partial pressure difference, consequently performances are quite limited currently. in contrast to solid membranes, moisture diffusion in liquid membranes (∼10 −9 m 2 s −1 [19, 20] , diffusion coefficients) is several orders higher than that in solid membranes. due to this reason and the inherent high selectivity, in recent years, there has been much effort in progressing the researches of supported liquid membranes (slm) in various fields: air dehumidification [21] , so 2 /co 2 separation [22] , h 2 s/ch 4 separation [23] , wastewater treatment [24] , metal ions concentration (uphill transport) [25] , separation of isomeric amines between two organic phases [26] , to name but a few. to improve the performances of mthr ventilators, in this study, a novel membrane, a composite slm, which employs licl liquid solution immobilized in a porous support membrane to facilitate the transport of moisture, is prepared. to protect the slm, two hydrophobic polyvinylidene fluoride (pvdf) layers are formed on both surfaces of the slm. the concept is shown in fig. 1 . the sweep represents exhaust air. moisture permeation through this membrane is of great interest. moisture transfer characteristics will be the focus of this study. three types of commercial membrane were obtained from a supplier. very hydrophilic cellulose acetate (ca) membranes with nominal pore diameter 0.22 m a thickness 50-70 m are used as the support media to immobilize licl solution. two hydrophobic pvdf membranes (equal nominal pore diameter 0.15 m, thickness 45 m) are used as the protective layer. crystals of licl·h 2 o with laboratory class purity is used as the solute. before the preparation of composite membrane, each membrane is experimented and observed for their basic microstructures. figs. 2 and 3 show the scanning electron photomicrograph (sem) graphs of the ca membrane and pvdf membrane, respectively. under room temperature, well-stirred licl solution with 35% mass fraction is first prepared in a closed glass container. vacuum degassing is applied for 2 h for the three membranes, after which, the ca membrane is dipped into the licl solution. after 24 h, the ca membrane is moved from the solution and placed onto a clean glass plate which is cleaned by alcohol. surplus licl solution on surfaces of ca membrane is blotted off with paper tissue. to be sure that no ionic liquid is removed from the membrane pores, the cleaning procedure is very gentle. at this stage, pvc glue is brushed on one surface of the two pvdf membranes, and at the same time on both surfaces of the ca membrane. after a few seconds, the two pvdf membranes are glued to the ca membrane and are pressed together gently for a few seconds. the prepared composite membrane is placed in a constant-temperature-constant-humidity chamber for another 24 h, before experiment is performed. for comparison, a composite membrane with no licl solution immobilized in the ca membrane is also made with the same procedure. the cross-section sem views of the two composite membranes are shown in figs. 4 and 5, respectively. to prevent the microstructure being destroyed by knife crushing when preparing cross-section samples, the membranes are first frozen in liquid nitrogen before they are broken off to see the cross-sections. as seen from fig. 4 , there are some gaps between different layers. some big cavities in the support layers are also observed, which are presumed to be imperfections in membrane fabrications. however, they have no adverse effects for this study because during operation, they will be filled with liquid solution. in the preparation process, some thickness of the ca membrane is dissolved by the glue, resulting in a lesser support layer thickness than raw material. fig. 5 with licl solution shows that the ca layer and pvdf layer connect to each other very closely and have a dense and continuum interface. there are more big cavities in the support layer. the reason behind this may be that with licl solution soaked, the wetted molecular chains in ca membrane structure become more flexible and they will swell and expand to two sides. the boundaries between different layers are pressed together and linked to each other closely. the final ca layer thickness is 52 m. the membrane module is a circular cell having an exchange area of 176.7 cm 2 . it is composed of two parts: the lower chamber and the cap, as shown in fig. 6 . when testing, the flat sheet membrane is placed on the lower chamber inside which saturated salt solution is contained. the cap is then covered on the membrane surface and forms a sandwiched structure. the membrane and the inner surface of the cap form a cone-shaped cavity. the air is supplied through the air slits in the cap. it is introduced through two diametrically positioned inlets (symmetrically placed) into a circular-shaped channel at the perimeter, from where the air is distributed over the membrane surface through the circular air slit. the air flows inward radially, until it exits the cap outlet in the center. the cap is designed that a constant axial air velocity is realized. when flowing across the membrane, the air stream exchanges moisture with the salt solution through the composite slm, and is humidified. this test uses nacl solution since it can ensure outlet humidity not saturated, for the protection of rh sensors. the whole experimental set-up is shown in fig. 7 . the cell is supplied with clean and humidified air from an air supply unit. the supply air flows from a compressed air bottle and is divided into two streams. one of them is humidified through a bubbler immersed in a bottle of distilled water, and then re-mixed with the other dry air stream. the humidity of the mixed air stream is controlled by adjusting the proportions of air mixing. the air flow rates are controlled by two air pumps/controllers at the inlet and outlet of the cell. the humidities and temperatures to and from the cell are measured by the built-in rh and temperature sensors, which are installed in the pumps/controllers. a detailed description of the test procedure is given in [27] . in the test, the vapor evaporation is slow, and the cell is well conductive. therefore, only moisture transfer is considered, by neglecting thermal influences. moisture transfer from the saturated nacl solution to air stream above membrane, is depicted in fig. 8 . variations of air humidity along the transfer path are shown in fig. 9 to simplify the mass transfer model. there are totally five resistances that can be clarified: resistance in the lower chamber air gap (1-2); resistance in the first protective layer l1 (2-3); resistance in the supported liquid membrane l2 (3) (4) ; resistance in the other protective hydrophobic layer l3 (4) (5) ; and resistance in the air stream h d (5) (6) , as demonstrated in fig. 10 . the moisture permeation rate from the solution surface to the air stream can be summarized by where r tot is the total resistance from the solution surface to air stream and ω is the humidity difference between the solution in lower chamber and air stream above membrane. where subscripts l and d represent solution in lower chamber and air in air duct, respectively. the mean moisture permeation rate across the whole membrane surface in the cell is calculated by where ω lm is the logarithmic mean humidity difference between the solution surface and air stream, and it is calculated by where subscripts o and i represent outlet and inlet of air stream, respectively. the total resistance is comprised of five parts as where r l , r 1 , r 2 , r 3 , r d are resistances in air gap, in the first protective layer, in the liquid membrane layer, in the second protective layer, and in air stream, respectively. moisture resistance below the membrane can be represented by the vapor diffusion distance from solution surface to the membrane lower surface. where l is the height of air gap (m), ρ a the dry air density (kg m −3 ), and d va is the vapor diffusivity in dry air (m 2 s −1 ). moisture transfer resistance (m 2 s kg −1 ) in the composite membrane comprises three layers: where where δ i is membrane thickness in ith layer (m) and d ei is the equivalent diffusivity of moisture in ith membrane, i = 1-3. the two protective layers on both sides of the liquid membrane are highly hydrophobic. the established theory of gas diffusion in such membranes considers three mechanisms: poisseuille flow, ordinary molecular diffusion, and knudsen diffusion, or a combination of them. the governing quantity that provides a guideline in determining which mechanism is operative in a given pore under given operating conditions is the ratio of the pore size to the mean free path λ, which is calculated for a species i using the following expression [28] : where σ i is the molecular collision diameter (m), 2.641 and 3.711å for water vapor and air, respectively [19] , k b the boltzmann constant, 1.38 × 10 −23 j k −1 , p m the mean total pressure within the membrane pores (pa), and t is the absolute temperature (k). for gaseous mixtures of two components, the mean free path and the collision diameters are different from the corresponding quantities for the pure component. the following relationship can be applied for vapor-air mixtures [28] : under room temperature and atmospheric pressure, calculated λ for air is 0.07 m; while under vacuum conditions, mean free path for air may be several microns to several meters. knudsen number, where d p is mean pore diameter (m). when kn ≥ 10, the knudsen flow is dominant, the poisseuille mechanism may be neglected [28] . actually, in most cases for hvac industry with microporous membranes, knudsen number is larger than 10, and poisseuille flow can be neglected, then the flow is considered to be combined knudsen and ordinary diffusion. ordinary diffusion coefficient of water vapor molecule in air is expressed by ref. [29] d 0 = c a t 1.75 where c a = 3.203 × 10 −4 . the terms v v and v a are molecular diffusion volumes and are calculated by summing the atomic contributions: v a = 20.1, and v v = 12.7 [19] . m v and m a are molecular weights of vapor and air in kg mol −1 . m is 0.018 kg mol −1 for water vapor and 0.029 kg mol −1 for air, respectively. knudsen diffusion coefficient [29] where r is gas constant, 8.314 j mol −1 k −1 . the effective diffusivity of combined knudsen and ordinary flow is [29] moisture flux (kg m −2 s −1 ) is expressed by where ω is humidity difference between the two sides of l1 or l3. consequently, water transfer in liquid membrane [22] : where d wl is water diffusivity in liquid membrane (m 2 s −1 ) and c w is the difference of water concentration in liquid membrane solution (kg m −3 ) between the two sides of liquid membrane. water vapor partial pressure, temperature, and licl solution concentration are governed by a thermodynamic equation [30] log where in this equation p v , is in kpa, t in k, and m is molality of the electrolyte (mol licl/kg water). water concentration in solution is where ρ sol is solution density (kg m −3 ), and it is calculated by the following equation [30] : where ρ w is pure water density at temperature t, and ρ i are given below [31] : in moist air, water vapor partial pressure is calculated by ref. [7] p v = ωp ω + 0.622 (25) humidity ratio in ambient air is in the range of 0.005-0.035 kg/kg, therefore the above equation can be simplified to p v = 1.608ωp (26) as can be seen, the relations between the air humidity and water concentration in solution are rather complicated and need iterations to find solution. calculations of thermodynamic equilibrium chart of licl solution with eqs. (18)(24) found that under isothermal conditions, a linear equation similar to henry's sorption law can be used to express the water concentration in the solution as where k p is called the henry coefficient (kg m −3 pa −1 ) and c w0 is a constant (kg m −3 ). table 1 lists the curve regressed values of k p and c w0 under different temperatures. this table gives the following correlations to estimate k v and c w0 from temperature: table 1 values of k p and c w0 for licl solution the moisture diffusion resistance in the liquid membrane layer can be expressed by where the equivalent diffusion coefficient of vapor in liquid membrane is convective moisture resistance on air stream side is where k is convective mass transfer coefficient (m s −1 ). convective mass transport in the cell has been investigated by the author and co-workers previously [32] and is expressed in terms of a correlation by where sh, re, and sc are sherwood number, reynolds number, and schmidt number, respectively. they are defined as where v is the kinematic viscosity of air (m 2 s −1 ), r the radial coordinate (m), r 0 the radius of the cell (m), and u a is the air velocity (m s −1 ) in radial direction. moisture conservation in air stream is represented by a onedimensional steady-state equation: boundary conditions: ref. [20] . others are from manufacturer's specifications. mean moisture permeability across the whole membrane surface (kg m −2 s −1 )/(kg/kg), is calculated by where a c is the cross-section area of air duct (m 2 ) and a t is the transfer area of membrane in the cell (m 2 ). the permeability pe here represents moisture transfer rate (kg s −1 ) for unit area of membrane under unit transmembrane humidity difference (kg vapor/kg dry air). it reflects the performance of membrane. dimensionless radius table 2 lists the values of operating conditions and system configurations. for each test, several minutes are needed for the system to become steady state. after outlet rh reaches steady state, water vapor permeability can be calculated with eq. (33). this is the experimental data. the outlet rh can also be predicted with eq. (30) . this is the model prediction. in calculations, the cell radius is divided into 50 grids. fig. 11 plots the relative humidity of outlet air under different air flow rates. the model predictions are also plotted in the figure. they are in agreement. maximum difference is 3%. fig. 12 shows the distributions of equivalent air relative humidity on both surfaces of liquid membrane and in air stream, along cell radius. they have lower values at inlet and higher values at outlet, indicating moisture emits continuously from salt solution in lower chamber to air stream. fig. 13 plots the distributions of equilibrium mass fraction of licl in liquid on both surfaces of liquid membrane, along cell radius. before the test, liquid membrane has a uniform mass fraction of 35%. during the operation, new equilibrium between liquid membrane and surrounding air relative humidity has been set-up. as a result, licl concentration in the liquid membrane re-distributed and forms a non-uniform mass fraction field. under the gradients of licl (or water) concentrations, moisture is transferred from the lower gap to the air stream. the liquid solution layer also acts as a barrier to air transfer since little air is dissolved in licl solution. fig. 14 shows the local vapor emission rate along cell radius. as seen, the emission rate exhibits a non-uniform distribution on membrane surface. it decreases from 3 × 10 4 kg m −2 s −1 at air inlet to 2.4 × 10 −5 kg m −2 s −1 at air outlet. the mean moisture emission rate is 1.14 × 10 −4 kg m −2 s −1 , which is two times higher than the performance of ca membranes in our previous study with the same system (3.8 × 10 −5 kg m −2 s −1 ) [32] . the resulted mean permeability is 0.043 (kg m −2 s −1 )/(kg/kg). with models just proposed, various resistances in moisture transfer can be estimated. fig. 15 shows the percentages of various resistances to total resistance, 35.9 m 2 s kg −1 . as seen, the fig. 15 . percentages of various resistances to total moisture transfer resistance, v = 10 l min −1 . current cell fluid dynamics has a relatively larger convective moisture transfer resistance, accounting for 23% of the total resistance. the two protective layers account for 28% of the total resistance each. the air gap diffusion resistance amounts to less than 10% of the total resistance. the supported liquid layer, licl solution layer, only accounts for 12% of the total resistance. in real applications in mthr ventilators, only membrane resistance and convective resistance are considered. as a result, to further improve performances in future, the resistances in protective layers and flow channels should be lowered as a priority. one efficient measure may be to lower the thickness of protective layers-to a dozen micrometers, for instance. using turbulent flow arrangement such as cross-corrugated parallel plates may be the good way to lower resistance in flow channels. a composite supported liquid membrane for moisture recovery has been developed. the supported liquid layer and the two protective layers are in good contact, from sem observations. the microstructure and the performances are good, though there are some undesired cavities formed in the support layer. the measured water vapor permeation rates are two times higher than a hydrophilic solid membrane with comparative thickness. various resistances in the composite membrane and in the test cell have been clarified. it is found that the liquid layer only accounts for 12% of the total resistance, therefore there should be much potential for further performance improvement in future, by decreasing other resistances. that is the direction currently being pursued. dehumidification equipment advances energy wheel effectiveness. part i. development of dimensionless groups performance comparisons of desiccant wheels for air dehumidification and enthalpy recovery membrane modules for building ventilation heat and mass transfer in a membrane-based energy recovery ventilator effectiveness correlations for heat and moisture transfer processes in an enthalpy exchanger with membrane cores membrane-based enthalpy exchanger: material considerations and clarification of moisture resistance water transport in ionic polymers water transport properties of nafion membranes. part i. single-tube membrane module for air drying removal of water vapor and vocs from nitrogen in a hydrophilic hollow fiber gel membrane permeator permeation of water vapor through cellulose triacetate membranes in hollow fiber form studies on the sulfonation of poly(phenylene oxide) (ppo) and permeation behavior of gases and water vapor through sulfonated ppo membranes. ii. permeation behavior of gases and water vapor through sulfonated ppo membranes interaction of polyetherpolyurethane with water vapor and water-methane separation selectivity hollow fiber air drying water transport across polystyrenesulfonate/alumina composite membranes membrane porosity and hydrophilic membrane based dehumidification performance temperature influence on moisture transfer through synthetic films water vapor permeability and diffusivity through methylcellulose edible films diffusion-mass transfer in fluid systems on the application of a membrane air-liquid contactor for air dehumidification dehumidification of air by a hygroscopic liquid membrane supported on surface of a hydrophobic microporous membrane liquid membranes for flue gas desulfurization hydrogen sulfide separation from gas streams using salt hydrate chemical absorbents and immobilized liquid membranes equilibrium and mass transfer characteristics of 2-chlorophenol removal from aqueous solution by liquid membrane instability mechanisms of supported liquid membranes supported liquid membranes using ionic liquids: study of stability and transport mechanisms evaluation of moisture diffusivity in hydrophilic polymer membranes: a new approach pervaporation and vacuum membrane distillation processes: modelling and experiments mass transfer of hcl and h 2 o across hydrophobic membrane during membrane distillation thermodynamic properties of aqueous electrolyte solutions. 1. vapor pressure of aqueous solutions of licl, libr, and lii properties of aqueous solutions of lithium and calcium chlorides: formulations for use in air conditioning equipment design laminar fluid flow and mass transfer in a standard field and laboratory emission cell (flec) this project 50306005 is supported by national natural science foundation of china. the author also acknowledges help from graduate students-miss zhang mingrui and mr. xu xueli in doing experiments. knudsen number l height of air gap (m) m molality of electrolyte (mol licl/kg water) m molecule weight (kg mol −1 ) p partial pressure (pa) key: cord-323319-u5hfkjv8 authors: xu, mengchang; zhou, wenhu; chen, xuncai; zhou, ying; he, binsheng; tan, songwen title: analysis of the biodegradation performance and biofouling in a halophilic mbbr-mbr to improve the treatment of disinfected saline wastewater date: 2020-10-22 journal: chemosphere doi: 10.1016/j.chemosphere.2020.128716 sha: doc_id: 323319 cord_uid: u5hfkjv8 disinfectant-containing wastewaters have been generated from many places, including marine industries. the synthetic naclo-containing wastewaters have been effectively treated in a saline mbbr-mbr (moving bed biofilm reactor & membrane bioreactor) system containing marine microorganisms. a low concentration of nacl (below 100 mg/l) is not enough to kill the microorganisms, but can affect their bioactivity and induce membrane biofouling. a linear relationship has been obtained for the half-life of membrane biofouling as a function of the naclo concentration (10-100 mg/l): [half-life] = 25－0.12×[naclo concentration]. the cod and nh(3)-n removals are the highest at a salinity of 30 g/l for the marine bioreactors. the behaviour of the typical biofoulants, measured real-timely by fluorescence spectroscopy, can indicate the levels of membrane biofouling and microbial activity, responding to the naclo and nacl influences. based on the behavior of biofoulants, this work has also proposed a novel strategy of biofoulants monitoring for membrane antifouling, where antifouling responses can be carried out when the concentration of biofoulants significantly increases. eps and smp are key biofoulants that require to be monitored for membrane antifouling. degraded to be smaller molecules with less biofouling tendency (tan and li, 2016) . following 68 the above antifouling strategies, however, a proper monitoring system is required to warn us 69 j o u r n a l p r e -p r o o f about any biofouling situations in mbr for timely antifouling responses (flemming, 2003) . 70 transmembrane pressure (tmp), read from a pressure gauge, has been widely used as an 71 indicator in practice for real-time monitoring, where a high tmp over 52 kpa means a significant 72 membrane fouling (liang et al., 2007) . however, the increase of tmp lags behind the changes in respectively, as shown in figure 5a . as a novel strategy of biofoulants monitoring for membrane 299 antifouling, antifouling responses could be carried out when the detected fluorescent intensity 300 significantly increased. as shown in figure 5b , the intensity of biofoulants significantly 301 increased at the 10 th day and the 20 th day responding to the environmental shocks. as soon as the 302 warnings observed, the naclo and nacl shocks were removed for antifouling when the novel 303 strategy was used. subsequently, the intensity of biofoulants gradually decreased. the tmp then 304 had a slower raising rate, showing a smaller tendency of membrane fouling. the antifouling 305 response was carried out when the intensity of biofoulants suddenly increased. the recovery of 306 bioactivity of bioreactors took about 2 days (from 10 th day to 14 th day; from 20 th day to 24 th day). overall, using the novel strategy of biofoulants monitoring the membrane biofouling rate was 308 approximately half of the rate when the conventional strategy of tmp monitoring was used. it is 309 suggested that the spectroscopic monitoring of membrane biofoulants has anti-biofouling 310 advantage in the saline mbbr-mbr system for the naclo-containing wastewater treatment. the naclo-containing saline wastewaters were effectively treated. the mbr was not sensitive 314 to low naclo concentrations under 5 mg/l. a linear relationship was noticed for the half-life of 315 membrane biofouling as a function of the naclo concentration. the synergistic detrimental 316 effect from naclo and nacl was found to be higher than their toxicities simply acting alone. the behaviour of the typical biofoulants, measured real-timely by fluorescence spectroscopy, manual of diagnostic tests for aquatic animals 2009. 332 methods for disinfection of aquaculture establishments correlation of eps content in activated sludge at different sludge retention times with membrane 335 fouling phenomena enhanced membrane biofouling potential by on-line chemical cleaning in 337 membrane bioreactor cleaning and disinfection for community facilities: interim recommendations for u.s. 339 community facilities with suspected/confirmed coronavirus disease anaerobic membrane bioreactors for antibiotic wastewater treatment: performance and membrane 343 fouling issues gb 28233-2011: safety and hygienic standard for disinfection by sodium 345 hypochlorite generator water and wastewater monitoring methods 349 membrane fouling reduction and improvement of sludge characteristics by bioflocculant addition in 350 submerged membrane bioreactor water sprinkler transforms into "disinfection vehicle long-term operation of 354 submerged membrane bioreactor (mbr) for the treatment of synthetic wastewater containing styrene 355 as volatile organic compound (voc): effect of hydraulic retention time (hrt) role and levels of real-time monitoring for successful anti-fouling strategies -an 358 overview influence of elevated ph shocks on the performance 360 of a submerged anaerobic membrane bioreactor effects of high sodium-chloride concentrations on activated-362 sludge treatment can 364 membrane bioreactor be a smart option for water treatment simultaneous removal of organic matter and salt ions from saline wastewater 366 in bioelectrochemical systems response of biological waste treatment systems to changes in salt 368 concentrations the effect of salt on the performance and 370 characteristics of a combined anaerobic-aerobic biological process for the treatment of synthetic 371 wastewaters containing reactive black 5 analytical methods for soluble microbial products (smp) and 373 extracellular polymers (ecp) in wastewater treatment systems: a review biofouling behavior and performance of forward 375 osmosis membranes with bioinspired surface modification in osmotic membrane bioreactor soluble microbial products in membrane bioreactor operation: 378 behaviors, characteristics, and fouling potential a critical 380 review of extracellular polymeric substances (epss) in membrane bioreactors: characteristics, roles in 381 membrane fouling and control strategies observations of fouling biofilm formation applicability of an integrated moving sponge biocarrier-osmotic membrane bioreactor md system 386 for saline wastewater treatment using highly salt-tolerant microorganisms effect of high salinity on activated sludge characteristics and 389 membrane permeability in an immersed membrane bioreactor effect of high salinity on activated sludge characteristics and 392 membrane permeability in an immersed membrane bioreactor novel external extractive membrane 395 bioreactor (embr) using electrospun polydimethylsiloxane/polymethyl methacrylate membrane for 396 phenol-laden saline wastewater desalination and reuse of high-salinity shale gas produced water: drivers, technologies, and future 399 directions protein, cell and bacterial fouling resistance of 401 polypeptoid-modified surfaces: effect of side-chain chemistry cultivation of activated sludge using sea 403 mud as seed to treat industrial phenolic wastewater with high salinity real-time monitoring of biofoulants in a membrane 406 bioreactor during saline wastewater treatment for anti-fouling strategies behaviour of fouling-related components in an enhanced membrane bioreactor 409 using marine activated sludge a critical review on saline wastewater treatment by 411 membrane bioreactor (mbr) from a microbial perspective membrane fouling from 413 ammonia recovery analyzed by atr-ftir imaging review on the state of 415 science on membrane bioreactors for municipal wastewater treatment concentration 417 and detection of sars coronavirus in sewage from xiao tang shan hospital and the 309th hospital of the 418 chinese people's liberation army impact of temperature seasonal change on sludge characteristics understanding ultrafiltration 423 membrane fouling by soluble microbial product and effluent organic matter using fluorescence 424 excitation-emission matrix coupled with parallel factor analysis removal and degradation 426 mechanisms of sulfonamide antibiotics in a new integrated aerobic submerged membrane bioreactor 427 system probing the radical chemistry in uv/persulfate-based 429 saline wastewater treatment: kinetics modeling and byproducts identification key: cord-338827-1moy43hr authors: stillwell, william title: membrane transport date: 2016-07-15 journal: an introduction to biological membranes doi: 10.1016/b978-0-444-63772-7.00019-1 sha: doc_id: 338827 cord_uid: 1moy43hr life depends on a membrane's ability to precisely control the level of solutes in the aqueous compartments, inside and outside, bathing the membrane. the membrane determines what solutes enter and leave a cell. transmembrane transport is controlled by complex interactions between membrane lipids, proteins, and carbohydrates. how the membrane accomplishes these tasks is the topic of this chapter. life depends on a membrane's ability to precisely control the level of solutes in the aqueous compartments, inside and outside, bathing the membrane. the membrane determines what solutes enter and leave a cell. transmembrane transport is controlled by complex interactions between membrane lipids, proteins, and carbohydrates. how the membrane accomplishes these tasks is the topic of chapter 19. a biological membrane is semipermeable, meaning it is permeable to some molecules, most notably water, while being very impermeable to most solutes (various biochemicals and salts) found in the bathing solution. this very important concept of unequal transmembrane distribution and, hence, permeability between water and other solutes came out of the pioneering work of charles overton in the 1890s (see chapter 2) . how does a biological membrane accomplish semipermeability? the barrier to solute movement is largely provided by the membrane's hydrophobic core, a very thin (w40 å thick), oily layer. the inherent permeability of this core varies from membrane to membrane. generally, the more tightly packed the lipids comprising the bilayer, the lower its permeability will be. lipid bilayers are very impermeable to most solutes because of their tight packing. fig. 19 .1 depicts the membrane permeability of a variety of common solutes [1] . note the data are presented as a log scale of solute permeability (p in cm/s) and ranges from na þ ¼ 10 à12 cm/s to water ¼ 0.2 â 10 à2 cm/s, spanning almost 10 orders of magnitude! lipid bilayer permeability is not a constant but instead is affected by environmental factors. for example, luvs (large unilamellar veicles) made from dppc (16:0, 16:0 pc) have a sharp phase transition temperature, t m , of 41.3 c. at temperatures well below t m , the luvs are in the tightly packed gel state and permeability is extremely low. at temperatures well above t m , the luvs are in the loosely packed liquid disordered state (l d , also called the liquid crystalline state) and permeability is high. however, maximum permeability is not found in the l d state, but rather at the t m [2] . as the luvs are heated from the gel state and approach the t m , domains of l d start to form in the gel state. solutes can then pass more readily through the newly formed l d domains than the gel domains resulting in an increase in permeability. at t m there is a maximum amount of coexisting gel and l d state domains that exhibit extremely porous domain boundaries. it is through these boundaries that most permeability occurs. as the temperature is further increased, the luvs pass into the l d state and the interface boundaries disappear, reducing permeability to that observed for the single-component l d state. thus, maximum permeability is observed at the t m . the tendency for solutes to move from a region of higher concentration to one of lower concentration was first defined in 1855 by the physiologist adolf fick (fig. 19.2 ). his work is summarized in what is now the very well-known fick's laws of diffusion [3] . the laws apply to both free solution and diffusion across membranes. fick developed his laws by measuring concentrations and fluxes of salt diffusing between two reservoirs through connecting tubes of water. fick's first law describes diffusion as: diffusion rate ¼ àda dc dx where d ¼ diffusion coefficient (bigger molecules have lower ds); a ¼ cross-sectional area over which diffusion occurs; dc/dx is the solute concentration gradient (diffusion occurs from a region of higher concentration to one of lower concentration). the relationship between a solute's molecular weight and its diffusion coefficient is shown in table 19 .1. large solutes have low diffusion coefficients and therefore diffuse more slowly than small solutes. the diffusion rate for a particular solute under physiological conditions is a constant and cannot be increased. this defines the theoretical limit for an enzymatic reaction rate and also limits the size of a cell. if a solute starts at the center of a bacterial cell, it takes about 10 à3 s to diffuse to the plasma membrane. for this reason, typical cells are microscopic (see chapter 1) . at about 3.3 pounds and the size of a cantaloupe, the largest cell on earth today is the ostrich egg. however a fossilized dinosaur egg in the american museum of natural history in new york is about figure 19 .1 log of the permeability (p in cm/s) across lipid bilayer membranes for common solutes ranging from na þ (10 à12 cm/s) to water (0.2 â 10 à2 cm/s). this range spans almost 10 orders of magnitude [1] . the size of basketball. since an egg's only function is to store nutrients for a developing embryo, its size is many orders of magnitude larger than a normal cell. osmosis is a special type of diffusion, namely the diffusion of water across a semipermeable membrane. water readily crosses a membrane down its potential gradient from high to low potential ( fig. 19 .3) [4] . osmotic pressure is the force required to prevent water movement across the semipermeable membrane. net water movement continues until its potential reaches zero. an early application of the basic principles of osmosis came from the pioneering work on hemolysis of red blood cells by william hewson in the 1770s (see chapter 2) . it has also been discussed that mlvs (multilamellar vesicles, liposomes) behave as almost perfect osmometers, swelling in hypotonic solutions and shrinking in hypertonic solutions (see chapter 3) [5, 6] . liposome swelling and shrinking can be easily followed by changes in absorbance due to light scattering using a simple spectrophotometer. therefore, osmosis has been investigated for many years using common and inexpensive methodologies and a lot is known about the process. membranes are rarely, if ever, perfectly semipermeable. deviation from ideality is defined by a reflection coefficient (s). for an ideal semipermeable membrane where a solute is totally movement of solutes across membranes can be divided into two basic types: passive diffusion and active transport [7] . passive diffusion requires no additional energy source other than what is found in the solute's electrochemical (concentration) gradient and results in the solute reaching equilibrium across the membrane. passive diffusion can be either simple passive diffusion where the solute crosses the membrane anywhere by simply dissolving into and diffusing through the lipid bilayer, or facilitated passive diffusion where the solute crosses the membrane at specific locations where diffusion is assisted by solute-specific facilitators or carriers. active transport requires additional energy, often in the form of atp, and results in a nonequilibrium, net accumulation (uptake) of the solute on one side of the membrane. the basic types of membrane transport, simple passive diffusion, facilitated diffusion (by channels and carriers) and active transport are summarized in fig. 19 .4 [8] . there are countless different examples of each type of membrane transport process [7] . only a few representative examples will be discussed here. even simple passive diffusion requires energy to cross a bilayer membrane. in order to cross a membrane, the solute must first lose its waters of hydration, diffuse across the membrane, and then regain its waters on the opposite side. the limiting step involves the energy required to lose the waters of hydration. table 19 .2 shows the relationship between the waters of hydration (proportional to the number of eoh groups on a homologous series figure 19.3 osmosis and osmotic pressure. water is placed in a u-shaped tube where each of the tube arms is separated by a semipermeable membrane with pores of a size that water can easily pass through but a solute cannot. upon addition of the solute to the tube's right arm, water diffuses from left to right (high water potential to low). the column of water in the tube's right arm (the one containing the solute) rises until the extra weight of the column equals the osmotic pressure caused by the solute. a pump could then be used to counter the osmotic pressure whereupon the solution columns in the right and left arms of the tube are made the same. the pump pressure required to equalize the height of the two columns is the osmotic pressure [4] . note a small amount of the solute leaks from right to left since no filter is perfect. of solutes) and the activation energy for transmembrane diffusion. as the number of waters of hydration increases from glycol < glycerol < erythritol, the activation energy for diffusion also increases. the activation energy compares very well with the energy of hydration. however, water diffusion does not fit this model. water permeability is just too high. several possibilities have been suggested to account for the abnormally high membrane permeability of water: 1. water is very small and so it just dissolves in bilayers better than larger solutes. 2. due to its size, water can readily enter very small statistical pores (w4.2 å in diameter). statistical pores result from the simultaneous lateral movement of adjacent membrane phospholipids in opposite directions. statistical pores have only a fleeting existence and cannot be isolated or imaged. 3. passage down water chains. 4. water can be carried down kinks in acyl chains that result from acyl chain melting (see lipid melting in chapter 9). 5. water may rapidly cross membranes through nonlamellar regions (eg, micelles, cubic or h ii phasedsee chapter 10). 6. high water permeability will occur at regions of packing defect (eg, surface of integral membrane proteins, boundary between membrane domains). 7. through pores or channels used to conduct ions. 8. through specific water channels known as aquaporins (see below, chapter 19, section 3.5). the only molecules that can cross a membrane by simple passive diffusion are water, small noncharged solutes, and gasses. charged or large solutes are virtually excluded from membranes and so require more than just simple passive diffusion to cross a membrane. facilitated diffusion (also known as carrier-mediated diffusion) is, like simple passive diffusion, dependent on the inherent energy in a solute gradient. no additional energy is required to transport the solute and the final solute distribution reaches equilibrium across the membrane. facilitated diffusion, unlike simple passive diffusion, requires a highly specific transmembrane integral protein or carrier to assist in the solute's membrane passage. facilitators come in two basic types: carriers and gated channels. facilitated diffusion exhibits michaelis-menton saturation kinetics ( fig. 19 .5, part a, right), indicating the carrier has an enzyme-like active site. like enzymes, facilitated diffusion carriers exhibit saturation kinetics and recognize their solute with exquisite precision, easily distinguishing chemically similar isomers like d-glucose from l-glucose. fig. 19 .5 (part a) compares simple passive diffusion to facilitated diffusion. the figure is not to scale, however, as facilitated diffusion is orders of magnitude faster than simple passive diffusion. a well-studied example of a facilitated diffusion carrier is the glucose transporter, or glut [9] . from the activation energies for transmembrane simple passive diffusion of glycol, glycerol and erythritol presented in table 19 .2, it can be estimated that the activation energy for glucose should be well over 100 kj/mol, but instead it is only 16 kj/mol. this large discrepancy is attributed to the presence of a glucose-facilitated diffusion carrier. fig. 19 .6 demonstrates the mode of action of one of these transporters, glut-1, from the erythrocyte [10] . gluts occur in nearly all cells and are particularly abundant in cells lining the small intestine. gluts are but one example in a superfamily of transport facilitators. gluts are integral membrane proteins whose membrane-spanning region is composed of 12 a-helices. gluts function through a typical membrane transport mechanism [10] . glucose binds to the membrane outer surface site causing a conformational change associated with transport across the membrane. at the inner side of the membrane, glucose is released into the internal aqueous solution ( fig. 19 in virtually all organisms there exists a wide variety of ion channels, the most widely distributed being potassium channels [11] . there are four basic classes of potassium channels, all of which provide essential membrane-associated functions including setting and shaping action potentials and hormone secretion: 1. calcium-activated potassium channel 2. inwardly rectifying potassium channel 3. tandem pore domain potassium channel 4. voltage-gated potassium channel potassium channels are composed of four protein subunits that can be the same (homotetramer) or closely related (heterotetramer). all potassium channel subunits have a distinctive pore-loop structure that sits at the top of the channel and is responsible for potassium selectivity [12] . this is often referred to as a selectivity or filter loop. the selectivity filter strips the waters of hydration from the potassium ion, allowing it into the channel. farther down the structure is a 10-å-diameter, transmembrane, water-filled central channel that conducts potassium across the membrane. elucidating the three-dimensional structure of this important integral membrane protein by x-ray crystallography ( fig. 19 .7) [12] was a seminal accomplishment in the field of membrane biophysics. for this figure 19 .7 three-dimensional structure of the potassium channel [12] . the channel itself is the clear opening in the center of the structure and a single k þ is shown in the center of the channel. work from 1998, rod mackinnon ( fig. 19 .8) of rockefeller university was awarded the 2003 nobel prize in chemistry. until the potassium channel work, just obtaining the structure of nonewater-soluble proteins was next to impossible. mackinnon's work elucidated not only the structure of the potassium channel but also its molecular mechanism. it has served as a blueprint for determining the structure of other membrane proteins and has greatly stimulated interest in the field. in some ways, na þ channels [13] parallel the action of k þ channels. they are both facilitated diffusion carriers that conduct the cation down the ion's electrochemical gradient. in excitable cells such as neurons, myocytes, and some glia, na þ channels are responsible for the rising phase of action potentials (see chapter 18) . therefore agents that block na þ channels also block nerve conduction and so are deadly neurotoxins. there are two basic types of na þ channels: voltage-gated and ligand-gated. the opening of a na þ channel has a selectivity filter that attracts na þ . from there the na þ ions flow into a constricted part of the channel that is about 3e5 å wide. this is just large enough to allow the passage of a single na þ with one attached water. since the larger k þ cannot squeeze through, the channel is selective for na þ . of particular interest are two extremely potent biological toxins, tetrodotoxin (ttx) and saxitoxin (stx) (fig. 19 .9, [14] ), that, in seafood, have killed and injured many humans. both toxins shut down na þ channels by binding from the extracellular side. ttx is encountered primarily in puffer fish but also in porcupine fish, ocean sunfish, and triggerfish. ttx (fig. 19.9 , left) is a potent neurotoxin that blocks na þ channels while having no effect on k þ channels. puffer fish is the second most poisonous vertebrate in the world, trailing only the golden poison frog that is endemic to the rain forests on the pacific coast of colombia. in some parts of the world puffer fish are considered to be a delicacy but 19. membrane transport must be prepared by chefs who really know their business, as a slight error can be fatal. puffer poisoning usually results from consumption of incorrectly prepared puffer soup, and ttx has no known antidote! saxitoxin (stx, fig. 19 .9, right) is a na þ channeleblocking neurotoxin produced by some marine dinoflagellates that can accumulate in shellfish during toxic algal blooms known as red tide. saxitoxin is one of the most potent natural toxins, and it has been estimated that a single contaminated mussel has enough stx to kill 50 humans! stx's toxicity has not escaped the keen eye of the united states military, which has weaponized the toxin and given it the designation tz. the driving force for transmembrane solute movement by simple or passive diffusion is determined by the free energy change, dg. is the solute concentration on the right side of a membrane; [s o ] is the solute concentration on the left side of a membrane; r is the gas constant; t is the temperature in k; z is the charge of the solute; f is the faraday; dj is the transmembrane electrical potential. solute movement will continue until dg ¼ 0. if dg is negative, solute movement is left to right (it is favorable as drawn). if dg is positive, solute movement is right to left (it is unfavorable in the left-to-right direction) or energy must be added for the solute to go from left to right. the equation has two parts; a transmembrane chemical gradient and a transmembrane electrical gradient (dj). the net movement of a solute is therefore determined by a combination of the solute's chemical gradient and an electrical gradient inherent to the cell. if the solute has no charge, z ¼ 0 (as is the case for glucose) and the right hand part of the equation (zfdj) drops out. therefore, the final equilibrium distribution of glucose across the membrane will have the internal glucose concentration equal to the external glucose concentration and is independent of dj, the electrical potential. at equilibrium for a noncharged solute, the situation for a charged solute like k þ is more complicated. the net dg is determined by both the chemical gradient àâ s 0 o ã ½s o á and electrical gradient (dj). the dj results from the sum of all charged solutes on both sides of the membrane, not just k þ . therefore even if the k þ concentration is higher inside the cell than outside (the chemical gradient is unfavorable for k þ uptake), the dj may be in the correct direction (negative interior) and of sufficient magnitude to drive k þ uptake against its chemical gradient. aquaporins are also known as water channels and are considered to be "the plumbing system for cells" [15, 16] . for decades it was assumed that water simply leaked through biological membranes by numerous processes described above (chapter 19, section 2). however, these methods of water permeability could not come close to explaining the rapid movement of water across some cells. although it had been predicted that water pores must exist in very leaky cells, it was not until 1992 that peter agre (fig. 19 .10) at johns hopkins university identified a specific transmembrane water pore that was later called aquaporin-1. for this accomplishment agre shared the 2003 nobel prize in chemistry with rod mackinnon for his work on the potassium channel. aquaporins are usually specific for water permeability and exclude the passage of other solutes. a type of aquaporin known as aqua-glyceroporins can also conduct some very small uncharged solutes such as glycerol, co 2 , ammonia, and urea across the membrane. however, all aquaporins are impermeable to charged solutes. water molecules traverse the aquaporin channel in single file (fig. 19.11 ) [17] . a characteristic of all living membranes is the formation and maintenance of transmembrane gradients of all solutes including salts, biochemicals, macromolecules, and even water. in living cells, large gradients of na þ and k þ are particularly important. typical cell concentrations are: cell interior: 400 mmol/l k þ , 50 mmol/l na þ cell exterior: 20 mmol/l k þ , 440 mmol/l na þ figure 19.10 peter agre, 1949e. living cells will also have a dj from à30 to à200 mv (negative interior) resulting from the uneven distribution of all ionic solutes including na þ and k þ . the chemical and electrical gradients are maintained far from equilibrium by a multitude of active transport systems. active transport requires a form of energy (often atp) to drive the movement of solutes against their electrochemical gradient, resulting in a nonequilibrium distribution of the solute across the membrane. a number of nonexclusive and overlapping terms are commonly used to describe the different types of active transport. some of these are depicted in fig. 19 .12 [18] . figure 19.12 basic types of active transport [18] . cell membrane cell membrane figure 19.11 aquaporin. water molecules pass through the aquaporin channel in single file. primary active transport is also called direct active transport or uniport. it involves using energy (usually atp) to directly pump a solute across a membrane against its electrochemical gradient. the most studied example of primary active transport is the plasma membrane na þ ,k þ -atpase discussed below (chapter 19, section 4.2). other familiar examples of primary active transport are the redox h þ -gradient generating system of mitochondria (see chapter 18), the light-driven h þ -gradient generating system of photosynthetic thylakoid membranes, and the atp-driven acid (h þ ) pump found in the epithelial lining of the stomach. there are four basic types of atp-utilizing primary active transport systems (table 19 .3). arguably the most important active transport protein is the plasma membrane-bound na þ ,k þ -atpase. this single enzyme accounts for one-third of human energy expenditure and is often referred to as the "pacemaker for metabolism." as a result the na þ ,k þ -atpase has been extensively studied for more than 50 years. the enzyme was discovered in 1957 by jens skou (fig. 19.13 ) who, 40 years later, was awarded the 1997 nobel prize in chemistry. as is often the case in biochemistry, a serendipitous discovery of a natural product from the jungles of africa has been instrumental in unraveling the enzyme's mechanism of action. the compound is ouabain (fig. 19.14) , a cardiac glycoside first discovered in a poison added to the tip of somali tribesmen's hunting arrows. in fact the name ouabain comes from the somali word waabaayo that means "arrow poison." the sources of ouabain are ripe seeds and bark of certain african plants and ouabain is potent enough to kill a hippopotamus with a single arrow. for decades after its discovery, ouabain was routinely used to treat atrial fibrillation and congestive heart failure in humans. more recently, ouabain has been replaced by digoxin, a structurally related, but more lipophilic cardiac glycoside. there are several important observations about na þ ,k þ -atpase that had to be factored in before a mechanism of action could be proposed. these include: 1. na þ ,k þ -atpase is an example of active antiport and primary active transport. 2. na þ ,k þ -atpase is inhibited by ouabain, a cardiac glycoside. 3. ouabain binds to the outer surface of na þ ,k þ -atpase and blocks k þ transport into the cell. 4. na þ binds better from the inside. 5. k þ binds better from the outside. 6. atp phosphorylates an aspartic acid on the enzyme from the inside. 7. phosphorylation is related to na þ transport. 8. dephosphorylation is related to k þ transport. 9. dephosphorylation is inhibited by ouabain. 10. three na þ ions are pumped out of the cell as two k þ ions are pumped in, driven by hydrolysis of one atp. 11. na þ ,k þ -atpase is electrogenic. mechanism of na þ ,k þ -atpase [19] is based on toggling back and forth between two conformational states of the enzyme, enz-1 and enz-2 ( fig. 19.15 ). three na þ s bind from the inside to na þ ,k þ -atpase in one conformation (enz-1). this becomes phosphorylated by atp causing a conformation change producing enz-2wp. enz-2wp does not bind na þ , but does bind two k þ ions. therefore, three na þ ions are released to the outside and two k þ ions are bound from the outside, generating enz-2wp (2k þ ). upon hydrolysis of wp, na þ ,k þ -atpase (enz ii) reverts back to the original enz-1 conformation that releases two k þ ions and binds three na þ ions from the inside. ouabain blocks the dephosphorylation step. secondary active transport (also known as cotransport) systems are composed of two separate functions. the energy-dependent movement of an ion (eg, h þ , na þ , or k þ ) generates an electrochemical gradient of the ion across the membrane. this ion gradient is coupled to the movement of a solute in either the same direction (symport) or in the opposite direction (antiport, see fig. 19 .12, [18] ). movement of the pumped ion down its electrochemical gradient is by facilitated diffusion. the purpose of both types of co-transport is to use the energy in an electrochemical gradient to drive the movement of another solute against its gradient. an example of symport is the sglt1 (sodium-glucose transport protein-1) in the intestinal epithelium [20] . sglt1 uses the energy in a downhill transmembrane movement of na þ to transport glucose across the apical membrane against an uphill glucose gradient so that the sugar can be transported into the bloodstream. the secondary active symport system for lactose uptake in escherichia coli is shown in fig. 19 .16 [21] . lactose uptake is driven through a channel by a h þ gradient generated by the bacterial electron transport system [22] . the free energy equation for transport described above can be rearranges for cases employing h þ gradients (see chapter 18) to: where dm h þ is the proton motive force; dj is the transmembrane electrical potential; r is the gas constant; t is the temperature in k; n is the solute charge (þ1 for protons); f is the faraday; dph is the transmembrane ph gradient. it is the force on an h þ (called the proton motive force) that drives lactose uptake. note that the ability to take up lactose is a combination of the electrical gradient and the ph gradient. although lactose uptake is directly coupled to h þ transmembrane movement, it is possible to take up lactose even if the ph gradient is zero (ie, if the dj is sufficiently large). over 50 years ago, peter mitchell (see chapter 18, fig. 18 .26) recognized the importance of what he termed "vectorial metabolism" [23, 24] . water-soluble enzymes convert substrate to product without any directionality. mitchell proposed that many enzymes are integral membrane proteins that have a specific transmembrane orientation. when these enzymes convert substrate to product they do so in one direction only. this enzymatic conversion is therefore unidirectional, or "vectorial." mitchell expanded this basic concept into his now famous "chemiosmotic hypothesis" for atp synthesis in oxidative phosphorylation (chapter 18) [25, 26] . for this revolutionary idea mitchell was awarded the 1997 nobel prize in chemistry. vectorial metabolism has been used to describe the mechanism for several membrane transport systems. for example, it has been reported in some cases the uptake of glucose into a cell may be faster if the external source of glucose is sucrose rather than free glucose. through a vectorial transmembrane reaction, membrane-bound sucrase may convert external sucrose into internal glucose plus fructose more rapidly than the direct transport of free glucose through its transport system. mitchell defined one type of vectorial transport as group translocation, the best example being the pts (phosphotransferase system) discovered by saul roseman in 1964. pts is a multicomponent active transport system that uses the energy of intracellular phosphoenol pyruvate (pep) to take up extracellular sugars in bacteria. transported sugars include glucose, mannose, fructose, and cellobiose. components of the system include both plasma membrane and cytosolic enzymes. pep is a high-energy phosphorylated compound (dg of hydrolysis is à61.9 kj/mol) that drives the system. the high-energy phosphoryl group is transferred through an enzyme bucket brigade from pep to figure 19 .16 lactose transport system in escherichia coli [21] . uptake of lactose is coupled to the movement of an h þ down its electrochemical gradient. this is an example of active transport, co-transport, and active. glucose producing glucose-6-phosphate in several steps (pep / ei / hpr / eiia / eiib / eiic / glucose-6-phosphate). the sequence is depicted in more detail in fig. 19 .17 [27] . hpr stands for heat-stable protein that carries the high-energy wp from ei (enzyme-i) to eiia. eiia is specific for glucose and transfers wp to eiib that sits next to the membrane where it takes glucose from the transmembrane eiic and phosphorylates it producing glucose-6-phosphate. although it is glucose that is being transported across the membrane, it never actually appears inside the cell as free glucose but rather as glucose-6-phosphate. free glucose could leak back out of the cell via a glucose transporter, but glucose-6-phosphate is trapped inside the cell where it can rapidly be metabolized through glycolysis. group translocation is defined by a transported solute appearing in a different form immediately after crossing the membrane. the term ionophore means "ion bearer." ionophores are small, lipid-soluble molecules, usually of microbial origin, whose function is to conduct ions across membranes [28, 29] . they are facilitated diffusion carriers that transport ions down their electrochemical gradient. ionophores can be divided into two basic classes: channel formers and mobile carriers (fig. 19.18 ) [30] . channel formers are long lasting, stationary structures that allow many ions at a time to rapidly flow across a membrane. mobile carriers bind to an ion on one side of a membrane, dissolve in and cross the membrane bilayer and release the ion on the other side. they can only carry one ion at a time. four representative ionophores will be discussed: the k þ ionophore valinomycin, the proton ionophore 2,4-dinitrophenol, synthetic crown ethers, and the channel-forming ionophore nystatin (fig. 19.19 ). superficially valinomycin resembles a cyclic peptide (fig. 19.19) . however, upon closer examination the ionophore is actually a 12-unit (dodeca) depsipeptide where amino acid peptide bonds are alternated with amino alcohol ester bonds. therefore the linkages that figure 19 .17 the bacterial pts system for glucose transport [27] . 19 . membrane transport hold the molecule together alternate between nitrogen esters (peptide bonds) and oxygen esters. the units that comprise valinomycin are d-and l-valine (hence the name "valinomycin"), hydroxyvaleric acid and l-lactic acid. the circular structure is a macrocyclic molecule with the 12 carbonyl oxygens facing the inside of the ring where they chelate a single k þ . the outside surface of valinomycin is coated with nine hydrophobic side chains of d-and l-valine and l-hydroxyvaleric acid. the polar interior of valinomycin precisely fits one k þ . the binding constant for k þ -valinomycin is 10 6 while na þ -valinomycin is only 10. this emphasizes the high selectivity valinomycin has for k þ over na þ . valinomycin, figure 19.19 representative examples of ionophores: the k þ ionophore valinomycin, the proton ionophore 2,4-dinitrophenol, the synthetic crown ether 18-crown-6, and the channel forming ionophore nystatin. therefore, has an oily surface that readily dissolves in a membrane lipid bilayer, carrying k þ across the membrane down its electrochemical gradient. valinomycin was first recognized as a potassium ionophore by bernard pressman in the early 1960's [31, 32] . he reported that valinomycin, a known antibiotic, stimulated k þ uptake and h þ efflux from mitochondria. many studies showed that valinomycin dissipates essential transmembrane electrochemical gradients causing tremendous metabolic upheaval in many organisms including microorganisms. it is for this reason that valinomycin was recognized as an antibiotic long before it was identified as an ionophore. currently several ionophores are added to animal feed as antibiotics and growth enhancing additives [33] . recently valinomycin has been reported to be the most potent agent against sars-cov (severe acute respiratory-syndrome coronavirus), a severe form of pneumonia first identified in 2003 [34] . fig. 19.19 ) is considered to be the classic uncoupler of oxidative phosphorylation (see chapter 18) . it is a synthetic lipid-soluble proton ionophore that dissipates proton gradients across bioenergetic membranes (mitochondrial inner, thylakoid, bacterial plasma). an uncoupler is therefore an h þ -facilitated diffusion carrier. elucidating the role of dnp in uncoupling oxidative phosphorylation was an essential component in support of peter mitchell's chemiosmotic hypothesis [25] . electron movement from nadh or fadh 2 to o 2 via the mitochondrial electron transport system generates a considerable amount of electrical energy that is partially captured as a transmembrane ph gradient (see chapter 18) . the movement of h þ s back across the membrane, driven by the electrochemical gradient, is through a channel in the f 1 atpase (an f-type primary active transport system discussed above, (chapter 19, section 4.1)) that is coupled to atp synthesis. dnp short-circuits the h þ gradient before it can pass through the f 1 atpase, thus uncoupling electron transport, the energy source for the h þ gradient, from atp synthesis. therefore, in the presence of dnp, electron transport continues, even at an accelerated rate, but atp production is diminished. the energy that should have been converted to chemical energy in the form of atp is then released as excess heat. this combination of properties led to the medical application of dnp to treat obesity from 1933 to 1938 [35] . upon addition of dnp: • the patient became weak due to low atp levels. • breathing increased due to increased electron transport to rescue atp production. • metabolic rate increased. • body temperature increased due to inability to trap electrical energy as chemical energy in the form of atp, releasing heat. • body weight decreased due to increased respiration burning more stored fat. dnp was indeed a successful weight loss drug. two of the early proponents of dnp use as a diet drug, cutting and tainter at stanford university, estimated that more than 100,000 people in the united states had tested the drug during its first year in use [35] . dnp, however, did have one disturbing side effectddeath! fatality was not caused by a lack of atp, but rather by a dangerous increase in body temperature (hyperthermia). in humans, 20e50 mg/kg of dnp can be lethal. although general use of dnp in the united states was discontinued in 1938, it is still employed in other countries and by bodybuilders to eliminate fat before competitions. crown ethers are a family of synthetic ionophores that are generally similar in function to the natural product valinomycin [36] . the first crown ether was synthesized by charles pederson (fig. 19.20) while working at dupont in 1967. for this work pedersen was co-awarded the 1987 nobel prize in chemistry. crown ethers are cyclic compounds composed of several ether groups. the most common crown ethers are oligomers of ethylene oxide with repeating units of (ech 2 ch 2 oe) n where n ¼ 4 (tetramer), n ¼ 5 (pentamer), or n ¼ 6 (hexamer). crown ethers are given structural names, x-crown-y, where x is the total number of atoms in the ring and y is the number of these atoms that are oxygen. crown refers to the crown-like shape the molecule takes. crown ether oxygens form complexes with specific cations that depend on the number of atoms in the ring. for example, 18-crown-6 ( fig. 19.19 ) has high affinity for k þ , 15-crown-5 for na þ , and 12-crown-4 for li þ . like valinomycin, the exterior of the ring is hydrophobic, allowing crown ethers to dissolve in the membrane lipid bilayer while carrying the sequestered cation down its electrochemical gradient. it is now possible to tailor make crown ethers of different sizes that can encase a variety of catalysts for phase transfer into the bilayer hydrophobic interior where they can be used to catalyze reactions inside the membrane. nystatin (fig. 19.19 ) is a channel-forming ionophore that creates a hydrophobic pore across a membrane [37, 38] . channel-forming ionophores allow for the rapid facilitated diffusion of various ions that depend on the dimensions of the pore. nystatin, like other channel-forming ionophores (eg, amphotericin b and natamycin), is a commonly used antifungal agent. finding medications that can selectively attack fungi in the presence of figure 19 .20 charles pedersen, 1904e1989. normal animal cells presents a difficult challenge since both cell types are eukaryotic. bacteria, being prokaryotes, are sufficiently different to present a variety of anti-bacterial approaches not amenable to fungi. however, fungi do have an achilles heel. fungal plasma membranes have as their dominant sterol ergosterol, not the animal sterol cholesterol (see chapter 5) . nystatin binds preferentially to ergosterol, thus targeting fungi in the presence of animal cells. when present at sufficient levels, nystatin complexes with ergosterol and forms transmembrane channels that lead to k þ leakage and death of the fungus. nystatin is a polyene antifungal ionophore that is effective against many molds and yeast including candida. a major use of nystatin is as a prophylaxis for aids patients who are at risk for fungal infections. gap junctions are a common structural feature of many animal plasma membranes [39, 40] . in plants similar structures are known as plasmodesmata. gap junctions were introduced earlier in chapter 11 (see fig. 11 .6). gap junctions represent a primitive type of intercellular communication that allows transmembrane passage of small solutes like ions, sugars, amino acids, and nucleotides while preventing migration of organelles and large polymers like proteins and nucleic acids. gap junctions connect the cytoplasms of two adjacent cells through nonselective channels. connections through adjacent cells are at locations where the gap between cells is only 2e3 nm. this small gap is where the term "gap junction" originated. gap junctions are normally clustered from a few to over a 1000 in select regions of a cell plasma membrane. early experiments involved injecting fluorescent dyes, initially fluorescein (molecular weight 300), into a cell and observing the dye movement into adjacent cells with a fluorescence microscope [41, 42] . currently lucifer yellow has become the fluorescent dye of choice for gap junction studies, replacing fluorescein. at first, the dye only appeared in the initially labeled cell. with time, however, the dye was observed to spread to adjacent cells through what appeared to be points on the plasma membrane. these points were later recognized as gap junctions. by varying the size of the fluorescent dye, it was shown that there was an upper size limit for dye diffusion. solutes had to have a molecular weight of less than w1200 to cross from one cell to another [41] . although gap junctions were obviously channels that connected the cytoplasms of adjacent cells, it was years before their structure, shown in fig. 19 .21, was determined [43, 44] . each channel in a gap junction is made up of 12 proteins called connexins. six hexagonally arranged connexins are associated with each of the adjacent cell plasma membranes that the gap junction spans. each set of six connexins is called a connexon and forms half of the gap junction channel. therefore, one gap junction channel is composed of 2 aligned connexons and 12 connexins. each connexin has a diameter of about 7 nm and the hollow center formed between the 6 connexins (the channel) is about 3 nm in diameter. gap junctions allow adjacent cells to be in constant electrical and chemical communication with one another. of particular importance is the rapid transmission of small second messengers, such as inositol triphosphate (ip 3 ) and ca 2þ . it appears that all cells in the liver are interconnected through gap junctions. this presents a possible dilemma. if even a single cell is damaged, deleterious effects may be rapidly spread throughout the entire liver. preventing this is one important function of ca 2þ . extracellular ca 2þ is w10 à3 mol/l while intracellular levels are maintained at w10 à6 mol/l. if a cell is damaged, ca 2þ rushes in, dramatically increasing intracellular ca 2þ . gap junction channels close if intracellular ca 2þ reaches 10 à3 mol/l, thus preventing the spread of damage. gap junctions are particularly important in cardiac muscle as the electrical signals for contraction are passed efficiently through these channels [45] . as would be expected, malfunctions of gap junctions lead to a number of human disorders including demyelinating neurodegenerative diseases, skin disorders, cataracts, and even some types of deafness. there are several other ways that solutes, including large macromolecules, can cross membranes. these methods include receptor-mediated endocytosis (rme, discussed in chapter 17), phagocytosis, pinocytosis, exocytosis, and membrane blebbing. these methods involve large sections of a membrane containing many lipids and proteins. two similar transport processes that have been known for a long time are pinocytosis and phagocytosis [46] . both involve nonspecific uptake (endocytosis) of many things from water and ions through to large macromolecules and, for phagocytosis, even whole cells. pinocytosis is greek for "cell drinking" and involves the plasma membrane invaginating a volume of extracellular fluid and anything it contains including water, salts, biochemicals and even soluble macromolecules. phagocytosis is greek for "cell eating" and involves the plasma membrane invaginating large insoluble solids. figure 19.21 gap junction [43] . six connexins form a connexon and one connexon from each cell unite to form a gap junction. pinocytosis is a form of endocytosis involving fluids containing many solutes. in humans, this process occurs in cells lining the small intestine and is used primarily for absorption of fat droplets. in endocytosis the cell plasma membrane extends and folds around desired extracellular material, forming a pouch that pinches off creating an internalized vesicle ( fig. 19.22, [19e47] ). the invaginated pinocytosis vesicles are much smaller than those generated by phagocytosis. the vesicles eventually fuse with the lysosome whereupon the vesicle contents are digested. pinocytosis involves a considerable investment of cellular energy in the form of atp and so is many 1000 times less efficient than rme (see chapter 17) . also, in sharp contrast to rme, pinocytosis is nonspecific for the substances it accumulates. pinocytosis is not a recent discovery as it was first observed decades before the other transport systems discussed in chapter 19. its discovery is attributed to warren lewis in 1929. phagocytosis is a type of endocytosis that involves uptake of large solid particles, often >0.5 mm [47] . the particles are aggregates of macromolecules, parts of other cells, and even whole microorganisms and, in contrast to pinocytosis (shown in fig. 19.22 ), phagocytosis has surface proteins that specifically recognize and bind to the solid particles. fig. 19 .23 [48] depicts events in phagacytosis. phagocytosis is a routine process that ameba and ciliated protozoa use to obtain food. in humans, phagocytosis is restricted to specialized cells called phagocytes that include white blood cell neutrophils and figure 19.22 pinocytosis, a type of endocytosis. an invagination of the plasma membrane encapsulates many water-soluble solutes ranging in size from salts to macromolecules. 19 . membrane transport macrophages. as with pinocytosis, phagocytosis generates intracellular vesicles called phagosomes that have sequestered solid particles they transport to the lysosome for digestion. phagocytosis is a major mechanism used by the immune system to remove pathogens and cell debris. in fact, very early studies of the immune system led elie metchnikoff to discover phagocytosis in 1882. for this work metchnikoff shared the 1908 nobel prize in medicine with paul ehrlich. exocytosis is the process by which cells excrete waste and other large molecules from the cytoplasm to the cell exterior [49] and therefore is the opposite of endocytosis. exocytosis generates vesicles referred to as secretory or transport vesicles (chapter 17). in exocytosis, intracellular (secretory) vesicles fuse with the plasma membrane and release their aqueous sequestered contents to the outside at the same time that the vesicular membrane hydrophobic components (mostly lipids and proteins) are added to the plasma membrane ( fig. 19 .24, [50] ). steady state composition of the plasma membrane results from a balance between endocytosis and exocytosis. the resultant process of plasma membrane recycling is amazingly fast. for example, pancreatic secretory cells recycles an amount of membrane equal to the whole surface of the cell in w90 min. even faster are macrophages that can recycle contents of their plasma membrane in only 30 min. before approaching the plasma membrane for fusion, exocytosis vesicles had a prior life that is considered in chapter 17. the vesicles must first dock with the plasma membrane, a process that keeps the two membranes separated by <5e10 nm. during docking, complex molecular rearrangements occur to prepare the membranes for fusion. the process of vesicle fusion and release of aqueous compartment components is driven by snare proteins (see chapters 10 and 17) [51, 52] . blebbing of the plasma membrane is a morphological feature of cells undergoing late stage apoptosis (programmed cell death, see chapter 24) [53] . a bleb is an irregular bulge in the plasma membrane of a cell caused by localized decoupling of the cytoskeleton from the plasma membrane. the bulge eventually blebs off from the parent plasma membrane taking part of the cytoplasm with it. it is clear in fig. 19 .25 [54] that the plasma membrane of an apoptotic cell is highly disintegrated and has lost the integrity required to maintain essential transmembrane gradients. blebbing is also involved in some normal cell processes, including cell locomotion and cell division. carefully controlled solute movement into and out of cells is an essential feature of life. there are many ways solutes are transported across the thin (w40 å) membrane hydrophobic barrier. transport is divided into passive diffusion and active transport. a biological membrane is semipermeable, being permeable to some molecules, most notably water (osmosis), while being very impermeable to most solutes that require some form of transporter. passive diffusion (simple and facilitated) only requires the energy inherent in the solute's electrochemical gradient and results in its equilibrium across the membrane. in contrast, active transport requires additional energy (ie, atp), and results in a nonequilibrium, net accumulation of the solute. passive transport can involve simple diffusion or facilitated carriers including ionophores and channels. active transport comes in many, often complex forms. examples of active transport include primary active transport (uniport), secondary active transport (co-transport, antiport), and group translocation. besides the multitude of transport systems, transport can be accomplished by gap junctions, receptor mediated endocytosis, phagocytosis, pinocytosis, exocytosis, and apoptotic membrane blebbing. chapter 20 will discuss bioactive lipids, highly specialized lipids that are functional at very low levels. discussed bioactive lipids include ceramides, diacylglycerol, eicosanoids, steroid hormones, and phosphatidic acid. principles of membrane transport effect of the gel to liquid crystalline phase transition on the osmotic behaviour of phosphatidylcholine liposomes fick's laws of diffusion water systems: aqua technology for the 21st century osmotic properties and water permeability of phospholipids liquid crystals osmotic behaviour and permeability properties of liposomes (review) membrane transport: a practical approach figure 12.4. comparison of passive and active transport the glut4 glucose transporter transporte de glucosa: glut y sglt seminarios de biología celular y molecular e usmp filial norte potassium channels. methods and protocols the structure of the potassium channel: molecular basis of k þ conduction and selectivity resurgence of sodium channel research differences in saxitoxin and tetrodotoxin binding revealed by mutagenesis of the na þ channel outer vestibule aquaporins in kidney pathophysiology what are aquaporins for? the royal swedish academy of sciences. the nobel prize in chemistry: peter agre, roderick mackinnon. 2003. nobelprize.org. the official web site of the nobel prize biological membranes and transport. solute transport across membranes. figure 10-21 atpase: structure, mechanism, and regulation renal na þ -glucose cotransporters chapter 9 e signal transduction. a. energy transduction: uses of atp nutrient transport by ruminal bacteria: a review group-translocation: a consequence of enzyme-catalysed group-transfer coupling of metabolism and transport by enzymic translocation of substrates through membranes chemiosmotic hypothesis of oxidative phosphorylation proton current flow in mitochondrial systems enzyme i of the phosphoenolpyruvate: sugar phosphotransferase system. proteopedia, weizmann institute of science in israel biological applications of ionophores structural aspects of ionophore function principles of membrane transport. figure 11.5 mechanism of action of valinomycin on mitochondria induced active transport of ions in mitochondria the role of enteric antibiotics in livestock production. canberry (australia): avcare limited deciphering the biosynthetic codes for the potent anti-sars-cov cyclodepsipeptide valinomycin in streptomyces tsusimaensis atcc 15141 use of dinitrophenol in obesity and related conditions: a progress report synthesis and molecular recognition studies of crown ethers probing the structureàfunction relationship of polyene macrolides: engineered biosynthesis of soluble nystatin analogues revealing the orientation of nystatin and amphotericin b in lipidic multilayers by uv-vis linear dichroism hexagonal array of subunits in intracellular junctions of the mouse heart and liver gap junctions: molecular basis of cell communication in health and disease. benos d, series editor. current topics in membranes and transport, series ed size limit of molecules permeating the junctional membrane channels cell-to-cell diffusion of fluorescent dyes in paired ventricular cells chapter: channels and transporters. molecular pathogenesis of cholestasis. madame curie bioscience database a quantitative analysis of connexin-specific permeability differences of gap junctions expressed in hela transfectants and xenopus oocytes gap junctions in cardiovascular disease mechanisms of phagocytosis in macrophages phagocytosis of microbes: complexity in action phagocytosis of bacteria and bacterial pathogenicity the molecular machinery of synaptic vesicle exocytosis cell and cell structure membrane fusion: grappling with snare and sm proteins synaptic vesicles really do kiss and run membrane blebbing during apoptosis results from caspase-mediated activation of rock1 human development, bio 380f key: cord-030961-5gzc7193 authors: wang, jiajun; lapinski, nicole; zhang, xiaohui; jagota, anand title: adhesive contact between cylindrical (ebola) and spherical (sars-cov-2) viral particles and a cell membrane date: 2020-08-28 journal: mech soft mater doi: 10.1007/s42558-020-00026-3 sha: doc_id: 30961 cord_uid: 5gzc7193 a critical event during the process of cell infection by a viral particle is attachment, which is driven by adhesive interactions and resisted by bending and tension. the biophysics of this process has been studied extensively, but the additional role of externally applied force or displacement has generally been neglected. in this work, we study the adhesive force-displacement response of viral particles against a cell membrane. we have built two models: one in which the viral particle is cylindrical (say, representative of a filamentous virus such as ebola) and another in which it is spherical (such as sars-cov-2 and zika). our interest is in initial adhesion, in which case deformations are small, and the mathematical model for the system can be simplified considerably. the parameters that characterize the process combine into two dimensionless groups that represent normalized membrane bending stiffness and tension. in the limit where bending dominates, for sufficiently large values of normalized bending stiffness, there is no adhesion between viral particles and the cell membrane without applied force. (the zero external force contact width and pull-off force are both zero.) for large values of normalized membrane tension, the adhesion between virus and cell membrane is weak but stable. (the contact width at zero external force has a small value.) our results for pull-off force and zero force contact width help to quantify conditions that could aid the development of therapies based on denying the virus entry into the cell by blocking its initial adhesion. electronic supplementary material: the online version of this article (10.1007/s42558-020-00026-3) contains supplementary material, which is available to authorized users. viral infection is one of the major public health issues in the world. from one of the most lethal viruses, ebola virus, to the novel coronavirus (sars-cov-2) causing the present pandemic, countless people have died and been sickened from virus infection and complications. moreover, this is an ongoing concern because there will always be new viruses or mutated ones. it is therefore important to develop an understanding of how virus particles infect our body's cells. one of the vital moments during infection is the internalization of the virus particle, often by hijacking the normal physiological adhesive function of the receptors on the surface of the cell membrane. uptake of most virus particles into a host cell is mediated by the receptor-dependent adhesive interaction between cell-surface and virus-surface molecules [1] [2] [3] . this initiates endocytosis, which comprises physiochemical interactions between the virus particle and cell membrane and several kinetic processes. during this process, the adhesive forces tend to bend and pull against tension to wrap around the viral particle [4, 5] . because blocking this adhesion is a prime therapeutic target, it is important to electronic supplementary material the online version of this article (https://doi.org/10.1007/s42558-020-00026-3) contains supplementary material, which is available to authorized users. understand the mechanics of initial virus adhesion to the cell. in particular, we aim to connect adhesion properties (measured, for example, by single-molecule force spectroscopy) and the behavior of the whole virus-cell membrane interaction. the mechanics and biophysics of this problem have been approached in a number of ways ranging from all-atom molecular dynamics simulations that contain atomistic structural detail to continuum models that contain only a few parameters that characterize virus, membrane, and adhesive properties [6, 7] . the former provides tremendous descriptive and predictive power but are, by construction, specific to a particular system. the latter allow investigation of more general principles at the cost of specificity. there is a significant literature on continuum models of adhesion between particles (biological or inorganic) and membranes. (see reference [8] for a review.) shanahan developed a model for compliant particle adhesion onto a rigid surface [9] . long et al. modeled aspects of the fusion of synaptic vesicles with the plasma membrane, which is related to the problem of soft vesicles attaching onto a rigid surface [10] . a similar model about kinetics of virus binding and fusion was developed by chou [11] . zhang et al. studied nanoparticle cellular endocytosis by considering parameters such as ligand density [5] . an important subclass of models, to which our work belongs, shares the feature that the total free energy is written as the sum of three contributions: from bending, tension, and adhesion. several of the models are based on the theory that the marginal reduction of the free energy during the adhesion process balances the marginal cost of bending the membrane [12] [13] [14] . several studies consider the process of virus particle wrapping and engulfment by the cell membrane [15, 16] . seifert and lipowsky (1990) studied the adhesion of vesicles and found that there is a transition between a bound and free state based on competition between bending and adhesion energies [12, 13] . other studies have considered the combined effects of bending, tension, and adhesion, as the particle is wrapped partially to fully, sometimes representing the membrane as a continuum plate [8, 14, 17] . although this problem of adhesion has been well-developed, much less attention has been paid to the contact mechanics of viruses onto a flexible membrane. this is of interest because such analyses can be used to interpret in detail atomic force microscopy (afm) force-spectroscopy measurements of force-deflection response used to characterize the adhesion process [18] , particularly to interpret the force-spectroscopy studies on virus-host cell interactions [19] [20] [21] . it is also of interest because virus attachment often occurs under external load, which is accounted for in a contact mechanics model but not in most of the literature cited above. in this work, we create a continuum model for the small-deflection adhesive contact mechanics of virus particle attachment onto the host cell membrane in terms of the principal biophysical properties of the virus, membrane, and their interaction. we seek equilibrium states by minimizing the total free energy of the virus-membrane system. in particular, we seek to describe the force-deflection and contact area-deflection relationships. these results also help to retrieve conditions for lack of adhesion, pull-off force, and contact area between the virus particle and cell membrane. the common physical basis for the model is to assume that, for given applied deflection, equilibrium states are achieved by a balance of adhesion energy of cell surface receptors, which drives adhesion, against the cost of the associated deformation, represented by bending and tension energies of the cell membrane [17, 22] . we show that the solution is governed by two dimensionless material parameters that can be identified as a normalized bending stiffness and tension. (these parameters have previously been identified) [8, 13] . in particular, quantities of interest such as the equilibrium contact size (if any) or force required to remove the virus from the membrane depend (in dimensionless form) only on these two parameters. the analysis is carried out for 2d cylindrical and 3d axisymmetric virus models representing, for example, a filamentous virus like ebola or a nominally spherical virus like sars-cov-2. in the following sections, we first outline how the problem is posed for both cylindrical and axisymmetric models. in later sections, we present the results of the model. the supporting information contains details of derivations. we now describe in outline the continuum models for adhesive contact between the virus and cell membrane, driven by adhesion and external displacement or force, and resisted by tension and elastic bending. figure 1 sketches the geometry of both cylindrical (i.e., 2d) and spherical (i.e., axisymmetric) models. aspects of the 2d model have been previously studied by mkrtchyan et al. [23] , but without external force or displacement. for both the 2d and axisymmetric cases, we consider the case where the viral particles are stiff compared to the cell membrane to which they attach. all the parameters and non-dimensional parameters used for the models are summarized in table 1 . the cell membrane, represented in fig. 1 by the blue line, can be split into two parts, one that contacts the virus particle and a second one that is out-of-contact. the virus particles in the 2d and axisymmetric models are assumed to be cylindrical and spherical, respectively. because the virus is represented by a rigid cylinder, the region of the membrane in contact has a circular cross-section in 2d, ignoring end effects because the radius of the virus is quite small compared to its length [24] . for example, the ebola virus has diameter of about 80 nm, whereas its length can reach 1-2 μm. in the axisymmetric model, the virus particle is assumed to be spherical, and its region of contact with the membrane is a spherical cap. for example, the novel coronavirus (sars-cov-2) is nominally spherical with a diameter of around 120 nm. since the diameters of both types of viruses are much smaller in size than the host cell but large compared to the thickness of the membrane (~4 nm), we assume that the membrane of the host cell is originally flat and deforms when in contact with the virus (blue line, fig. 1 ). the width of contact section in the 2d model is 2a, and the host cell membrane is supported some distance l = a + b away from the center of the virus attachment region. for nominally spherical viruses (such as hiv, zika virus and sars-cov-2), the contact region is circular with radius, a. the membrane of the host cell is supported some radial distance l away from the axis of symmetry. as in the 2d model, the membrane is assumed to be flat in its stress-free state; it deforms when in contact with the virus particle. for both 2d and axisymmetric models, the interaction between the viral particle and the cell membrane is driven by adhesive interactions and externally applied force or displacement. attachment of the viral particle to the cell membrane is resisted by energy required to bend the cell membrane and by the tension it is under. we presume that the tension is set by some effect such as osmotic pressure and is not constitutively linked to the deformation, i.e., it holds a constant isotropic value. in our models, the parameters that govern the adhesive contact mechanics are (more in table 1 ) bending rigid κ, tension σ, adhesion free energy per receptor β, binding receptor density ρ, and the radius of the virus, r. these parameters are associated with contributions to the energy of the system: bending, tension, and adhesion (u bending , u tension , and u adhesion , respectively [5, 14] . there is additionally the potential of the external force or an applied external displacement. in the work presented here, we carry out the contact mechanics calculation under displacement control, so it enters as a parameter. thus, total energy is the bending and tension energies are each the sums of contributions from two regions, region i where the membrane is in contact with the virus and region ii where it is free of applied lateral loads. we consider special limiting cases in which either tension or bending dominates over the other. calculating the energies in eq. (1) requires knowledge of the contact region and the shape of the membrane outside the contact region. this is governed by the helfrich hamiltonian [14, 25] which generally results in nonlinear euler-lagrange governing equations [7, 10, 14, 26] . however, since in this work we are interested primarily in conditions close to the no-adhesion case, we can take advantage of a major simplification in the shape equation for small deformations. we can additionally neglect the fig. 1 the geometry of the two models. (upper left) sketch of a stiff cylindrical (i.e., 2d) virus particle attaching onto a flexible membrane; (upper right) sketch of a stiff spherical virus particle attaching onto a flexible axisymmetric membrane; (lower left) mechanical model of 2d virus particle attachment driven by adhesion and external force or displacement and resisted by membrane bending and tension; (lower right) mechanical model of spherical virus particle attachment driven by adhesion and external force or displacement and resisted by membrane bending and tension coefficient of gaussian curvature in the helfrich hamiltonian because the processes we study involve no change in topology. with these two simplifications, the governing equation simplifies to a linear differential equation [14] : typical values are tension σ~μn/m and bending rigidity κ~10-100 k b t [5, 7, 25] , where k b is boltzmann's constant. in the following, we will use specific forms of eq. (2a) for plane and axisymmetric problems. the ratio ffiffiffiffiffiffiffiffi κ=σ p represents a length scale. if the size of the virus is large compared to this length scale, then tension dominates. conversely, if the virus is small compared to this length, then bending does. for the viral particles we wish to consider, using typical values given in table 1 , we estimate this ratio to be about 50 nm, which means that both tension and bending energies can be important. a normalized version of eq. (2a) is where α ¼ κ 2ρβr 2 is a normalized bending stiffness, γ ¼ σ 2ρβ is normalized tension, and ρβ, as the product of number density of surface ligand-receptor pairs and the adhesion energy of one such pair, is the specific (per unit area) adhesion energy of the interface. thus, in normalized form, this problem is governed by two dimensionless material parameters, α and γ. versions of these two parameters are well known in the literature [8, 13, 14] . in the 2d model, the membrane in region i conforms to the cylindrical virus particle so that the normalized deflection w(x) in region i is governed by the circular shape of the virus cross-section. the deflection w(x) in region ii is governed by the 1d version of eq. (2a) [17] κ normalizing eq. (3) as defined in table 1 , we get bending stiffness normalized by adhesion α ¼ κ to better understand the behavior, we focus most of our attention on the two limits in which either tension or bending dominate. (we can ignore bending if α γ ≪1; we can ignore tension if α γ ≫1.) in the axisymmetric model, the deflection w(r) in region i is constrained by contact of the membrane to the surface of the viral particle. the problem is axisymmetric, and so, deflection w(r) is a function of radial position only. in region ii, it is governed by the differential equation [17] : where r is the radial distance from the lower pole. again, we focus on the two limits in which either tension or bending dominate. also, for the majority of the discussion in the remainder of this manuscript when we refer, for example, to tension, we mean normalized tension, since the discussion is almost exclusively of solutions to the normalized dimensionless governing equations and parameters. in normalized variables, the state of the system is described by five dimensionless variables (under displacement control): δ; a; l; α; γ, i.e., the normalized displacement of the virus, contact width/radius, size of the membrane patch, normalized bending stiffness, and tension. we find that the effect of l is weak, and we hold it fixed in this work at l ¼ 5, thus reducing to four the number of dimensionless variables. in region i, the deflection is specified by the fixed shape of the virus, and δ; a. in region ii, the deflection of the membrane is given by solving eqs. (4) or (6) subject to clamped boundary conditions where the membrane is held fixed and continuity and smoothness of deflection at the intersection between the two regions. for example, in the axisymmetric spherical virus case where bending dominates, the deflection in region ii is given by the solution of eq. (6) and is where the constants c 1 , c 2 , c 3 , c 4 are obtained by the boundary and continuity conditions (see si for details). with the solution for deflection known, the various contributions to energy can be computed in normalized form. again, for the same case, where the first term represents adhesion energy (negative in value), the second term the bending energy inside the contact region, and the third term the bending energy in region ii. in general, the total energy associated with a particular configuration of the system can be written as we now impose two equilibrium conditions. the first enforces the condition that for other fixed parameters, the contact radius adjusts to satisfy the condition that when this condition can be satisfied, it provides a solution a * δ; α; γ à á , eliminating the explicit dependence of total energy on contact width so that u total ¼ u total δ; α; γ à á . (this condition can sometimes be satisfied exactly; on other occasions, it is computed numerically.) next, we apply the condition that the force is conjugate to the applied deflection so that in this way, for all the different conditions studied, we obtain force-deflection f δ; α; γ à á and contact width-deflection a δ; α; γ à á relations. a special point in the force-deflection trace is the maximum or pull-off force, f max α; γ ð þ. similarly, an important point in the contact width-deflection trace is contact width corresponding to zero force, a 0 α; γ ð þ. both these quantities depend only on the two materials parameters. for the sake of brevity, those results are presented in si. the discussion just presented to obtain the solution in each of the cases studied is captured by the algorithm described below: we first present results for the cylindrical virus (fig. 2 ). figure 2 a and b show results in the bending-dominated limit and fig. 2c , d the tension-dominated limit. the tension-dominated limit can also be solved exactly without the small-deflection assumption. for consistency, in the main text, we present results for small deflections only. the exact result for this specific case is provided in supporting information s1.1. positive values of force represent tension. in the bending-dominated limit, the force-deflection relation is approximately (but not exactly) linear. its most interesting feature is a maximum in the force (for α < 1), which corresponds (fig. 2b) to a reduction in contact width down to 0. with increasing bending stiffness α, the peak force reduces. at the critical value of α = 1, indicated by the dashed vertical lines, the pull-off force reduces down to 0. that is, there is no adhesion between the virus and cell membrane in the absence of external force. this result is consistent with conclusions obtained by models based only on the balance of bending and adhesion. for higher values of bending stiffness, the contact reduces to 0 while still under compressive load. subsequently, the force-deflection behavior follows that of a cylinder in line contact with the membrane. of course, in reality, true line contact is not possible due to the finite thickness of the membrane. figure 2 c shows the force-displacement results in the tension-dominated limit for a range of γ. the force-deflection response is nearly linear again, with slope that increases with increasing tension. interestingly, the force for all values of γ is identical at a positive (tensile) value for zero indentation. as a consequence, indentation depth at zero force is negative and increases with decreasing γ. the contact radius as a function of indentation depth is shown in fig. 2d . unlike the bending-dominated case, even for large γ, the contact width is quite substantial for considerable tensile extension. this is unlike the bending-dominated case in which there is no force-free adhesion if bending stiffness exceeds a critical value. this means that for the tension-dominated limit of the 2d model, there is not a critical value at which adhesion is blocked-see also fig. s1 in supporting information. figure 3 shows results for the spherical virus adhesion. figures 3 a and b show the force-deflection and contact radiusdeflection results in the bending-dominated limit. like in the 2d model, for α > 1, the contact reduces to 0 while deflection is still negative. for this range of bending stiffness, contact radius at zero force is 0 as is the pull-off force (see si). figure 3 c shows force-deflection results for the tension-dominated limit. the results here are strikingly different from the 2d model in that the force jumps to 0, corresponding to a jump to 0 in the normalized contact radius, fig. 3d . the pull-off force reduces with increasing tension, but only mildly so. we developed and studied adhesive contact between a stiff viral particle and a cell membrane. our model is based on a continuum description: minimization of energy that is composed of contributions from bending, tension, adhesion, and external force or deflection. we considered cylindrical (filamentous) and spherical viruses, representative of two typical viral shapes such as ebola and sars-cov-2. the principal results of the analyses are normalized force-deflection and contact width/radius versus deflection as a function of two dimensionless materials parameters, α and γ, denoting normalized bending stiffness and tension, respectively. we paid attention to limits in which bending or tension dominate the other. in both the bending-dominated cases, a striking result is that for sufficiently stiff membranes, there is zero pull-off force and contact radius at zero force also vanishes. for larger stiffness, contact vanishes even under compressive indentation. the tensiondominated case is qualitatively different for the 2d and axisymmetric shapes. in the tension-dominated limit of the cylindrical model, the contact width a decreases very slowly with increasing deflection. in the axisymmetric geometry, in contrast, there is a welldefined pull-off force that corresponds to contact shrinking to 0. in our model, force is applied for two reasons, (a) because under physiological conditions, viral particles often adhere to endothelial and epithelial cells where they are subjected to fluid forces. although these forces are generally in shear with respect to the contact, for simplicity, we chose to apply normal forces as that preserves the symmetry of the problem. (b) one of the applications of our model is to help interpret nanoindentation experiments in which a small functionalized bead is pressed into contact with a cell surface and retracted. we believe that the force-displacement results can be related directly to the atomic force microscopy (afm) force-displacement measurements [27] [28] . our continuum model is a simple representation, attempting to capture many important details in only a few physical parameters. however, our model is suitable for application to interpretation of nanoindentation and force-spectroscopy experiments. it also advances understanding of the biomechanics of filamentous and spherical virus-adhesive contact mechanics to cell membranes. the biological context is, of course, more complicated. for example, in both the sars-cov-2 and ebola cases, adhesive receptors are elongated structures: the so-called spike [29] and tim [30] receptors, respectively. the continuum approach we have used represents these by an adhesion energy density. in so doing, important features, such as their discrete nature, can no longer be addressed [31, 32] . also, receptors have often to protrude through a glycocalyx layer in order to overcome electrostatic repulsion to adhere, and all these details are lumped into the continuum parameters of our model. in our model, we chose to support the membrane at some distance from the contact axis. this represents a characteristic distance between points where the cytoskeleton attaches to the membrane. another way to account for the cytoskeleton would be to represent it by a foundation. a simple way would be to use a winkler foundation in which case the governing equation for the membrane shape (eq. 2) would change to : ð12a; bþ this introduces a new dimensionless parameter, ϕ, with associated characteristic length: very approximately, the distance in our work can be thought of as representing this length. adhesion and fusion efficiencies of human immunodeficiency virus type 1 (hiv-1) surface proteins multiple receptors involved in human rhinovirus attachment to live cells influenza virus binds its host cell using multiple dynamic interactions how does a virus bud? physical principles of nanoparticle cellular endocytosis the computational route from bilayer membranes to vesicle fusion fluid lipid membranes: from differential geometry to curvature stresses wrapping of nanoparticles by membranes adhesion of a liquid-filled spherical membrane adhesion energy can regulate vesicle fusion and stabilize partially fused states stochastic entry of enveloped viruses: fusion versus endocytosis adhesion of vesicles adhesion of vesicles and membranes elastic deformation of a fluid membrane upon colloid binding adhesion and wrapping in colloid− vesicle complexes kinetics of particle wrapping by a vesicle theory of plates and shells life at the nanoscale -atomic force microscopy of live cells biomechanical characterization of tim protein-mediated ebola virus-host cell adhesion entropy-driven tension and bending elasticity in condensed-fluid membranes adhesion of cylindrical colloids to the surface of a membrane biomechanical characterization of tim protein-mediated ebola virus-host cell adhesion elastic properties of lipid bilayers: theory and possible experiments shape transformations of vesicles: phase diagram for spontaneous-curvature and bilayer-coupling models frequency spectrum of the flicker phenomenon in erythrocytes cell surface area regulation and membrane tension membrane tension and membrane fusion sars-cov-2 infects t lymphocytes through its spike protein-mediated membrane fusion characterizing functional domains for tim-mediated enveloped virus entry detailed mechanics of membrane-membrane adhesion and separation. i. continuum of molecular cross-bridges detailed mechanics of membrane-membrane adhesion and separation. ii. discrete kinetically trapped molecular cross-bridges acknowledgments the authors would like to thank the reviewers for their helpful comments.funding this work was supported by the nih grant 1 r15 ai133634-01a1 and by nsf grant 1804117. key: cord-333757-h12aozg2 authors: modis, yorgo title: class ii fusion proteins date: 2013-07-10 journal: viral entry into host cells doi: 10.1007/978-1-4614-7651-1_8 sha: doc_id: 333757 cord_uid: h12aozg2 enveloped viruses rely on fusion proteins in their envelope to fuse the viral membrane to the host-cell membrane. this key step in viral entry delivers the viral genome into the cytoplasm for replication. although class ii fusion proteins are genetically and structurally unrelated to class i fusion proteins, they use the same physical principles and topology as other fusion proteins to drive membrane fusion. exposure of a fusion loop first allows it to insert into the host-cell membrane. conserved hydrophobic residues in the fusion loop act as an anchor, which penetrates only partway into the outer bilayer leaflet of the host-cell membrane. subsequent folding back of the fusion protein on itself directs the c-terminal viral transmembrane anchor towards the fusion loop. this fold-back forces the host-cell membrane (held by the fusion loop) and the viral membrane (held by the c-terminal transmembrane anchor) against each other, resulting in membrane fusion. in class ii fusion proteins, the fold-back is triggered by the reduced ph of an endosome, and is accompanied by the assembly of fusion protein monomers into trimers. the fold-back occurs by domain rearrangement rather than by an extensive refolding of secondary structure, but this domain rearrangement and the assembly of monomers into trimers together bury a large surface area. the energy that is thus released exerts a bending force on the apposed viral and cellular membranes, causing them to bend towards each other and, eventually, to fuse. enveloped viruses acquire a lipid bilayer membrane when they bud across the plasma membrane or the membrane of the endoplasmic reticulum (er) during assembly of the virion. 1, 2 during infection, the viral membrane must be fused to the host-cell membrane to deliver the viral genome into the cytoplasm for replication (fig. 1) . the fusion of the viral and host-cell membranes is therefore the central molecular event during the entry of viral entry into host cells, edited by stefan pöhlmann and graham simmons. ©2013 landes bioscience and springer science+business media. enveloped viruses into cells. adjacent membranes do not fuse spontaneously; membrane fusion requires considerable energy (on the order of 100 kj mol -1 or 40 kt). 3, 4 envelope proteins anchored in the viral membrane provide this energy in the form of a conformational rearrangement that bends the apposed membranes towards each other, inducing them to fuse. [5] [6] [7] most 'fusion proteins' (or their cleavage products) also effect cellular attachment of the virus prior to the membrane fusion event by binding to a receptor on the cell surface, except the paramyxo-and alphaviruses, in which a second envelope protein binds the receptor. fusion proteins of enveloped viruses fall into two structural classes. the influenza virus haemagglutinin (ha) is the prototype of class i fusion proteins, 8 which encompass those of other orthomyxo-and paramyxoviruses such as measles virus, retroviruses such as human immunodeficiency virus (hiv), filoviruses such as ebola virus, and coronaviruses such as sars (see . class ii fusion proteins are a structurally and evolutionarily distinct class of proteins found in flaviviridae, such as dengue, yellow fever, and west nile viruses, and on alphaviruses, such as semliki forest and sindbis viruses. hepatitis c has a similar genomic organization to the flaviviruses, and therefore most likely relies on a class ii fusion protein as well. crystal structures of several class i and class ii fusion proteins before [9] [10] [11] [12] [13] [14] [15] and after 5, [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] their fusogenic conformational rearrangements have provided us with a detailed molecular understanding of the fusion mechanism ( table 1 ). the structures show that, despite the absence of similarities in the protein folds of the two classes, fusion proteins from both classes use the same physical principles and general topology to drive membrane fusion. first the fusion protein inserts a hydrophobic fusion anchor partway into the outer bilayer leaflet of the host-cell membrane. the fusion anchor is either an n-terminal peptide, 30 as in influenza and hiv, 31 or an internal loop, as in sars coronavirus, 32 avian sarcoma leucosis virus 33 and all class ii enveloped viruses. 34 second, the fusion protein folds back on itself, directing the (c-terminal) viral transmembrane anchor towards the fusion anchor. this fold-back forces the host-cell membrane (held by the fusion anchor) and the viral membrane (held by the c-terminal transmembrane anchor) against each other, resulting in fusion of the two membranes. in this chapter, i describe our current picture of how class ii fusion proteins drive viral membrane fusion, based on the structural and biochemical data available to date. three-dimensional structures of eight class ii fusion proteins in their native, or prefusion states, 10, [12] [13] [14] 35, [93] [94] [95] 97 that is, the conformation that they adopt on the surface of a mature virus particle, have been determined at near atomic resolution. figure 2 shows the three-domain structures of e 13 and e1, 12 the fusion proteins of dengue virus (a representative flavivirus) and of semliki forest virus (an alphavirus), respectively. the two proteins share a common molecular architecture, despite a lack of significant sequence similarity. domain i, an eight-stranded -barrel, organizes the structure. two long insertions between pairs of consecutive -strands in domain i form the elongated domain ii, which bears the fusion anchor, a fusion loop in class ii proteins, at its tip (figs. 2, 4) . domain ii contains twelve -strands and two -helices. domain iii is an igc-like module, with ten -strands. domain iii contains most of the antigenic sites on e, as well as most of the structural determinants of virulence and tropism. 10 this observation, and the widespread occurrence of immunoglobulin modules in cell-adhesion proteins, suggest that domain iii participates in attachment to a cellular receptor. 10 indeed, positively charged patches on the surface of domain iii in dengue virus have been suggested to promote attachment by binding heparan sulfate on the cell surface. 36 both e1 and e have one or more glycosylation sites. these glycans can aid viral attachment to the cell surface, in as found in the mature virus particle. 48 the fusion loop in a-c is marked with an asterisk. a second subunit of e, forming the dimer found on the viral surface and in solution, is shown in light gray. d) view rotated 90° relative to c, with the second subunit omitted for clarity. e) crystal structure of sfv e1 in the prefusion conformation, 12 as found in the mature virus particle. 50 the fusion loop is marked with an asterisk. f) view rotated 90° relative to e. the case of dengue virus by binding to the lectin dc-sign. 37, 38 as expected from their differ only in the length and structure of surface-exposed loops, some of which have been implicated in receptor binding. 10, 39, 40 despite these hints on the basis of cellular attachment, however, a cellular receptor that specifically recognizes an envelope protein on a class ii enveloped virus has yet to be conclusively identified, although candidate receptors for dengue virus type 1 41 and west nile virus 42 were recently suggested. it is important to note that all the crystal structures of fusion proteins determined so far, from both classes and regardless of their conformational state, lack the c-terminal viral membrane anchor. this anchor consists of one or two transmembrane helices, and has been intentionally omitted in constructs targeted for crystallization to facilitate expression and handling, and to promote crystallization. the crystallized species are therefore referred to as soluble fragments of the ectodomains of the full-length fusion protein. furthermore, all available crystal structures of class ii fusion proteins lack the 'stem' region, 43 a 30-55 amino acid linker between domain iii and the c-terminal transmembrane anchor (figs. 2a-b, 3). as i will discuss below, the stem region plays a key role in the final stages of membrane fusion. its function is analogous to that of the 'outer helix' in class i fusion proteins. 8 both class i and class ii fusion proteins rely on a proteolytic cleavage event to become primed to respond to the environmental conditions appropriate for fusion. these conditions are usually the acidic ph of an endosome ( fig. 1 ), but for some class i enveloped viruses, such as hiv, coreceptor binding is required instead. in contrast to class i fusion proteins, however, class ii fusion proteins rely on a priming proteolytic cleavage that does not cleave the fusion protein itself. instead, class ii proteins associate with a second, 'protector' protein, called m (for membrane protein) in flaviviruses or e2 in alphaviruses. the protector protein is cleaved by furin when immature virus particles assembled in the er reach the trans-golgi network. 44 the cleavage produces mature virus particles, which are then released from the host cell by exocytosis. the cleavage of the protector protein releases a conformational constraint on the fusion protein, which allows it to adopt its mature conformation (described above) in a large rearrangement on the viral surface. in the mature conformation, the fusion protein is primed to respond to acidic ph and induce membrane fusion with a further conformational rearrangement (described below). structures from electron cryomicroscopy of both immature 45, 46 and mature 47-50 flavivirus and alphavirus particles, provide a detailed picture of the rearrangement that accompanies maturation in these viruses. alphaviruses retain the t = 4 icosahedral packing of their envelope proteins, but domains that form spikes on the immature virion swing in towards the threefold symmetry axis, during maturation. 46, 50 the rearrangement is more dramatic in flaviviruses, in which the fusion protein e breaks the t = 3 icosahedral symmetry of the immature virion 45 to adopt an unusual icosahedral herringbone pattern in the mature virion. 47, 51 in both alphaviruses and flaviviruses, the fusion proteins form dimers in the mature virion albeit in different configurations. 10, 12 the key feature of the maturation process in both genera, however, is that cleavage of the protector protein allows the fusion loop to reposition itself so that it is poised to insert into the host-cell membrane in response to acidification of the solute in the endosome. mature virus particles are sequence identities (≥ 37%), flaviviral e proteins have very similar overall structures, and therefore infectious, 44, 52 unlike immature virions, 53, 54 which are insensitive to ph. the fusion loop is shielded from the viral surface in mature virions by e-e dimer contacts in flaviviruses, or by protein e2 in alphaviruses (figs. 3a,b, 5a). the three-dimensional structures of four class ii fusion proteins in their postfusion states 29,55,56,94 reveal striking differences from the prefusion forms (fig. 3) , and suggest a 13 and b) two sfv e1 12 molecules in the prefusion conformation as found on the viral surface, viewed perpendicular to the viral membrane. the fusion loop is buried, either in the dimer interface (a), or under e2 (b). the outer (proximal) bilayer leaflets of the cellular and viral membranes are shown to scale as solid rectangles. the thin outer layer within each leaflet represents the polar headgroup layer, and the thicker inner layer represents the hydrocarbon layer. the stem-anchor segments are absent from the crystal structure, but are represented here schematically as rods in the viral membrane. c,d) upon acidification of the solute in the endosome, the domain ii rotates 15-30° about a hinge in the domain ii-domain i interface. this exposes the fusion loop, which then inserts into the host cell membrane. the postfusion, trimeric structures of den e 29 (e) and sfv e1 (f). 55 after insertion of their fusion loops into the target membrane, the fusion proteins form trimers and fold back on themselves, bringing the fusion loops close to the c-terminal transmembrane anchors. molecular mechanism for membrane fusion (see below and fig. 5 ). like class i fusion proteins, flaviviral e proteins and alphaviral e1 proteins are both homotrimers in their postfusion conformations. class ii proteins form trimers from monomers on the viral surface, while class i proteins are trimeric in their prefusion state. 8 however, a comparison of the pre-and postfusion states of influenza ha-the only example in its class where both structures are known for the same protein-shows that, as in class ii fusion proteins, nearly all of the trimer contacts in the postfusion state are formed during the fusogenic conformational rearrangement. unlike influenza ha, which undergoes extensive refolding during membrane fusion, the three domains of class ii fusion proteins retain most of their folded structures (fig. 3) . instead, the domains undergo major rearrangements in their relative orientations, through flexion of the interdomain linkers. domain iii undergoes the most significant displacement in the fusion transition. it rotates by about 70°, and its center of mass shifts by 30-40 å towards domain ii. this folding-over brings the c-terminus of domain iii about 40 å closer to the fusion loop, at tip of domain ii (fig. 3) . domain ii rotates 15-30° with respect to domain i about a hinge region 13 in which mutations affect the ph threshold of fusion in various flaviviruses. [57] [58] [59] [60] [61] [62] these conformational rearrangements position the end of domain iii-and the beginning of the stem region that links domain iii to the c-terminal viral transmembrane anchor-towards the fusion loop ( fig. 3e-f) . 29, 55 a deep channel extends from the c-terminus of the crystallized fragment along the intersubunit contact between domains ii to the fusion loops, in both the dengue and semliki forest virus postfusion trimer structures. in the full-length fusion proteins, it is thought that the stem binds in this channel in an extended, but mainly -helical conformation. 29, 48 this proposed stem conformation places the viral transmembrane anchor in the immediate vicinity of the fusion loop, just as in the postfusion conformation of class i viral fusion proteins. the fusion transition in class ii viral proteins is irreversible. the refoldings just described may impart irreversibility by contributing a high barrier to initiation of trimerization and an even higher barrier to dissociation of postfusion trimers once they have formed. moreover, many new polar and nonpolar contacts are formed during the fusion transition, in several different areas along the threefold axis of the trimer. the total surface buried is 13,000-15,000 å 2,29,55 nearly four times more than is buried in the prefusion dimer. the stem, which is missing from currently available crystal structures, most likely forms additional contacts with the core trimer structure. the stem does indeed promote trimer assembly even in the absence of liposomes. 43 the process of viral membrane fusion in both class i and class ii enveloped viruses begins with the exposure of a fusion anchor, and its subsequent insertion into the host-cell membrane. fusion anchors from both viral classes vary in length but are in general rich in glycines and hydrophobic residues, particularly aromatic residues such as trp or phe. sequence conservation is poor between fusion proteins of both classes. the fusion anchor in class i fusion proteins-the 'fusion peptide'-is a region of approximately 20 residues at or near the n-terminus of the envelope protein. the crystal structure of the parainfluenza virus 5 fusion (f) protein in its prefusion form reveals the fusion peptide wedged between two subunits of the protein, in a partly extended, partly -sheet and partly -helical conformation. 15 structural studies on influenza ha in its postfusion conformation using nmr and other spectroscopic techniques show that the fusion peptide is mostly -helical in character and that its structure changes only subtly as it inserts partway into the outer leaflet of the host-cell lipid bilayer. 63, 64 none of the currently available postfusion class i protein crystal structures contain information on the fusion peptide. the recently determined crystal structures of class ii fusion proteins in pre 10,12-14 and postfusion 29, 55, 56 conformations offer the first direct views of fusion anchors-in this case, the fusion loops-as they insert into a target membrane (fig. 4) . like the class i fusion peptide, the class ii fusion loop penetrates only partway into the hydrocarbon layer of the target membrane. exposed carbonyls and charged residues prevent the fusion loop from penetrating further than 6 å. 29, 56 in flaviviruses, the fusion loop adopts a tightly folded conformation, which is stabilized by a disulfide bond (fig. 4a) . the structure of the fusion loop is essentially identical in the pre-and postfusion conformations of the protein, suggesting that membrane insertion has no effect on the structure of the fusion loop. during the fusion transition, three hydrophobic residues in the fusion loop (trp, leu, and phe) become exposed on the molecular surface. three fusion loops end up in close proximity at the tip of the trimer in the postfusion conformation, where they form a crater-like surface with a hydrophobic rim (fig. 3e) . electron cryomicroscopy 29 and mutagenesis studies 34 confirm that these hydrophobic, mostly aromatic residues on the crater rim insert into the host-cell membrane, acting as an 'aromatic anchor' for the fusion protein. the concave shape of the crater is thought to be important in generating distortions or perturbations in the host-cell membrane, 29 which are required for fusion. 65 in alphaviruses, the fusion loop is also rich in aromatic and other hydrophobic residues. unlike flaviviral fusion loops, however, alphaviviral fusion loops do not form trimer contacts (fig. 3f) . indeed, in the postfusion structure of the semliki forest virus e1 trimer, the fusion loops have high temperature factors and exhibit a high degree of flexibility despite the presence of two disulfide bonds. thus, the structures of the fusion loops are poorly defined, but each fusion loop seems to adopt a very different conformation (fig. 4b,c) . 55 the fusion loops in the postfusion semliki forest virus e1 structure form quite polar surfaces, with many mainchain carbonyls and some polar or charged sidechains exposed on the surface. this suggests that, in contrast to flaviviral fusion loops, alphaviral fusion loops either change their conformation upon membrane insertion to shield polar groups from the membrane, or the fusion loops only interact with the polar headgroups of the lipids, and do not penetrate into the hydrocarbon layer. semliki forest virus e1 trimers form irregular clusters, or 'rosettes' of about 40-60 trimers through contacts between fusion loops in adjacent trimers. 55 this is reminiscent of influenza virus ha, which aggregates into rosettes through interactions between the fusion peptide, at low ph and after proteolytic activation. 66 this fusion loop/peptide clustering may provide a mechanism for the direct coupling of several e1/ha trimers to work in concert around a single fusion site (see below). combined with previous knowledge, the structures of the fusion proteins from class ii viruses in their postfusion states 29, 55, 56 have led to a much better understanding of how conformational changes in the proteins drive membrane fusion. the structures confirm two major principles of membrane fusion machineries: (1) the fusion protein must insert an anchor into each of the two membranes to be fused, and (2) the protein folds back on itself in a thermodynamically favorable conformational rearrangement that drives membrane fusion by forcing the two anchors into close proximity. in the current model, viral membrane fusion proceeds as follows (fig. 5) . first, receptor binding by an envelope protein, which in flaviviruses is also the fusion protein, leads to clathrin-mediated endocytosis of the virus (figs. 1, 5a ). when the virus reaches endosomal compartments the low ph of the lumen (ph 6) causes an initial conformational rearrangement that leads to the exposure of the previously buried fusion loop 48, 50 at the tip of domain ii. in flaviviruses, domains i and ii flex relative to each other by 30°. 29 this hinge motion causes domain ii, and therefore the fusion loop, to swing away from the viral surface and towards the host-cell membrane (fig. 5b) . indeed, mutations at the domain i-domain ii interface in various flaviviruses alter the ph threshold of fusion. 13, [57] [58] [59] [60] [61] [62] as domain ii swings away from the viral surface, constraints imposed by the tight packing of e on the viral surface are released, allowing e to diffuse freely in the plane of the viral membrane. the stem may also be able to extend away from the membrane at this stage. in alphaviruses, constraints are released in response to low ph by the dissociation of the protector (and receptor-binding) protein e2. this exposes the fusion loop and allows domain ii of e1 to swing towards the nearest threefold symmetry axis in the virus particle in a 15° hinge motion relative to domain i, leading to the formation of trimer contacts with adjacent e1 molecules. 46 the second key step in the fusion process is insertion of the exposed fusion loop into the host-cell membrane (fig. 5c) . alphaviral e1 has already formed some trimer contacts at this stage, but flaviviral e proteins probably insert their fusion loops as monomers. membrane insertion probably catalyzes trimerization of the fusion loops, 67 by lateral rearrangement of e monomers. this trimeric prefusion intermediate (fig. 5c ) bridges host-cell and viral membranes, anchored by its fusion loops in the former and by the figure 5 . proposed fusion mechanism for fusion mediated by class ii fusion proteins. a) the virus binds to a receptor on the cell surface. in flaviviruses, the fusion protein e binds the receptor, while in alphaviruses, the 'protector' protein e2 binds the receptor. following attachment, the virus is internalized to an endosome. b) acidic ph in the endosome causes domain ii to hinge outward from the virion surface, exposing the fusion loop, and allowing e monomers to rearrange laterally in the plane of the membrane. c) the fusion loop inserts into the hydrocarbon layer of the host-cell membrane, promoting trimer formation. d) formation of trimer contacts spreads from the fusion loop at the tip of the trimer, to the base of the trimer. the protein folds back on itself, directing the fusion loop towards the c-terminal transmembrane anchor. energy release by this refolding bends the apposed membranes. e) creation of additional trimer contacts between the stem-anchor and domain ii leads first to hemifusion and then (f) to formation of a lipidic fusion pore. viral transmembrane anchors in the latter. this proposed intermediate is analogous to the 'prehairpin' intermediate postulated for class i viral fusion mechanisms. 68 upon insertion of the fusion loops into the host-cell membrane, formation of trimer contacts spreads from the fusion loops at the trimer tip to domain i at the trimer base. domain ii shifts and rotates, folding the stem and c-terminal anchor back towards the fusion loop (fig. 5d) , and burying additional protein surfaces. free energy released by this refolding drives the two membranes to bend towards each other, 5-7 as the c-terminal anchor is forced closer to the fusion loop, forming apposing nipples in the membranes (fig. 5d) . 3 fusion-loop insertion may induce positive bilayer curvature, which would stabilize the lateral surfaces of the nipples. the concave shape of the crater-like surface formed by the fusion loops at the trimer tip may also have a destabilizing effect on the membrane, as has been postulated for fusion peptides in class i fusion proteins. 65 based on the energy required to deform lipid bilayers, it seems likely that a ring of trimers refolding in concert is needed to provide sufficient energy to form nipples in the membranes. 3, 4 it is unclear exactly how many trimers are needed to drive membrane fusion in class ii viruses, nor how their conformational changes are coupled. in the case of influenza, fusion requires the concerted action of at least three ha trimers, 69 and is more likely driven by rings of 6-8 trimers. 70 the clustering of fusion loops may provide a mechanism for the direct coupling of several e1 trimers to work in concert around a single fusion site in alphaviruses, but such clustering has not been observed in flaviviruses. it is possible that coupling occurs via the membrane: only when several trimers fold back in concert can they overcome the resistance of the membrane to deformation and reach their final, most stable postfusion conformation (figs. 5d-f). as the fusion transition proceeds, the stem zippers up onto the core of the trimer, along a channel that spans domain ii, at the intersubunit contact regions (figs. 3, 5d-f) . the zippering up of the stem onto the domain ii forces the fusion loop and the viral transmembrane anchor closer and closer, until the proximal leaflets of the two membranes fuse to form a 'hemifusion stalk' (fig. 5e ). hemifusion is thought to be an essential intermediate of membrane fusion. 3, 4, 71 (fig. 5e ) illustrates the need for shallow penetration of the viral fusion anchor into the host-cell membrane: assuming several trimers do in fact act in concert around a single fusion site, fusion anchors from different trimers would collide if they inserted beyond the outer (proximal) lipid bilayer leaflet. this constraint on the length of the fusion anchor holds true for both class i fusion peptides and class ii fusion loops. hemifusion stalks can 'flicker' open into narrow fusion pores. 71 in order to prevent the transient fusion pores from closing, the stem must complete its zippering up onto the core of the trimer, and the c-terminal transmembrane anchor must migrate into the pore (fig. 5f) . indeed, the transition from hemifusion stalk to full fusion pore appears to require that the viral transmembrane anchor span the membrane completely, in all biological membrane fusion systems. thus, the replacement of the c-terminal transmembrane anchor of influenza ha with a glycosylphophatidylinositol (gpi) lipid anchor, [72] [73] [74] or with a half-length protein -helical anchor, 75 stalls the fusion reaction at the stage of hemifusion. other viral fusion proteins and cellular snare fusion proteins also require at least one transmembrane anchor. [76] [77] [78] [79] [80] [81] [82] [83] upon completion of fusion, the trimer has reached the conformation seen in the postfusion crystal structures. 29, 55, 56 the stems (not present in the structures) are docked along the surface of domains ii, and the fusion loops and transmembrane anchors lie next to each other in the fused membrane (fig. 5f ). some class ii fusion proteins, including those of alphaviruses, can only fuse membranes containing cholesterol and sphingolipids. 84 the structural basis for this requirement is still not well understood. several mutations in different regions of the semliki forest virus fusion protein e1 lower its dependence on cholesterol and/or sphingolipids for membrane fusion. 85, 86 it is unclear, however, whether the lower dependence on cholesterol of these mutants is due to an apparent destabilization of the e1 homotrimer, 87 or to the different physical properties of membranes lacking cholesterol and sphingolipids. in flaviviruses, cholesterol facilitates fusion, but neither cholesterol nor sphingolipids are essential for fusion. 88 many class ii viruses, especially the flaviviruses, represent important human pathogens such as dengue, hepatitis c, yellow fever, west nile, japanese encephalitis and tick-borne encephalitis viruses. 89 for most of these viruses, there are no specific treatments for infection, their control by vaccination has proved elusive, 89 and the number of infections is on the rise. recently determined three-dimensional structures of class ii fusion proteins suggest new strategies for inhibiting viral entry by blocking membrane fusion. one such strategy stems for the discovery in dengue virus e of a long, tapering channel lined with hydrophobic side chains. 13 in the crystal structure, the channel is occupied by a molecule of the detergent n-octyl--d-glucoside. in the absence of detergent, a -hairpin covering the channel swings towards the protein, and closes up the channel. 13 the location of this 'ligand-binding pocket' at the domain i-domain ii interface coincides with that of mutations affecting the ph threshold of fusion in various flaviviruses. [57] [58] [59] [60] [61] [62] most of these mutations involve side chains lining the ligand-binding pocket. the postfusion structure of dengue virus e shows that this region acts as a hinge between domains i and ii during the fusogenic conformational rearrangement (see above). 29 the opening up of a ligand-binding pocket just at the locus of a hinge suggests that compounds tightly inserted at this position might hinder the conformational changes required for membrane fusion (fig. 6a) . the mechanism of action of such compounds might resemble that of some of the well-studied antipicornaviral compounds, which block a concerted structural transition in the icosahedral assembly. 90 alternatively, small molecules that pry open the -hairpin on binding in the pocket may inhibit infection by facilitating the low-ph conformational change, causing premature triggering. knowledge of the structure of the binding pocket with a bound ligand will guide efforts to design derivative ligands with higher affinities for use as inhibitors of flaviviral membrane fusion. the postfusion structures of dengue 29 and semliki forest 55 viruses suggest a second possible strategy for fusion inhibition, related to an approach successful in developing an hiv antiviral compound. 91 peptides corresponding to the stem region of the gp41 fusion protein inhibit hiv entry by binding to the trimeric, n-terminal 'inner core' of the protein and interfering with the folding back against it of the stem and c-terminal viral transmembrane anchor. the way in which the stem is likely to fold back in class ii viral fusion proteins (figs. 3, 5d-f) suggests that an analogous strategy may be successful with class ii viruses. peptides derived from stem sequences could block completion of the fusogenic conformational change, by competing with the stem for interaction with surfaces on domain ii, at the trimer interface (fig. 6b) . stem-like peptides or peptidomimetic compounds could thus inhibit viral membrane fusion in class ii enveloped viruses by preventing the final folding back of the fusion protein that is required to drive the viral and host-cell membranes together to fuse. all viral membrane fusion proteins use the same physical principles and topology to drive membrane fusion. class ii fusion proteins are structurally and evolutionarily distinct class of proteins found in flaviviridae, such as dengue, yellow fever, and west nile viruses, and on alphaviruses, such as semliki forest and sindbis viruses. unlike class i fusion proteins such as influenza ha, which undergoes extensive refolding during membrane fusion, the three domains of class ii fusion proteins retain most of their folded structures. instead, the domains undergo major rearrangements in their relative orientations, through flexion of the interdomain linkers. class ii fusion proteins rely on a hydrophobic fusion loop to anchor themselves in the target cellular membrane. like the class i fusion peptide, the class ii fusion loop penetrates only partway into the hydrocarbon layer of the target membrane. class ii fusion proteins drive membrane fusion in a foldback rearrangement of a trimeric protein assembly. crystal structures of class ii envelope proteins have suggested two specific strategies for fusion inhibition, with hydrophobic small molecules and "stem"-like peptides or peptidomimetics, respectively. a) the discovery of a ligand-binding pocket at the interface between domains i and ii in dengue virus e, 13 just at the locus of a hinge motion required for fusion, suggests that compounds inserted in the pocket might hinder the hinge motion and hence inhibit the fusion transition. this approach would block the first step in the fusion mechanism ( fig. 5a-b) . b) peptides corresponding to the stem region of the fusion protein may inhibit viral entry by binding to the trimeric core of the protein in its postfusion conformation, 29, 55 and interfering with the folding back against it of the fusion protein's own stem. an analogous strategy has been successful with hiv gp41. 91, 92 this approach would block the last step in the fusion mechanism ( fig. 5e-f) . flaviviridae: the viruses and their replication togaviridae: the viruses and their replication a quantitative model for membrane fusion based on low-energy intermediates a mechanism of protein-mediated fusion: coupling between refolding of the influenza hemagglutinin and lipid rearrangements structural basis for paramyxovirus-mediated membrane fusion evidence that the transition of hiv-1 gp41 into a six-helix bundle, not the bundle configuration, induces membrane fusion membrane fusion machines of paramyxoviruses: capture of intermediates of fusion receptor binding and membrane fusion in virus entry: the influenza hemagglutinin structure of the haemagglutinin membrane glycoprotein of influenza virus at 3 a resolution the envelope glycoprotein from tick-borne encephalitis virus at 2 a resolution structure of the haemagglutinin-esterase-fusion glycoprotein of influenza c virus the fusion glycoprotein shell of semliki forest virus: an icosahedral assembly primed for fusogenic activation at endosomal ph a ligand-binding pocket in the dengue virus envelope glycoprotein variable surface epitopes in the crystal structure of dengue virus type 3 envelope glycoprotein structure of the parainfluenza virus 5 f protein in its metastable, prefusion conformation structure of influenza haemagglutinin at the ph of membrane fusion retrovirus envelope domain at 1.7 angstrom resolution core structure of gp41 from the hiv envelope glycoprotein atomic structure of a thermostable subdomain of hiv-1 gp41 atomic structure of the ectodomain from hiv-1 gp41 crystal structure of the simian immunodeficiency virus (siv) gp41 core: conserved helical interactions underlie the broad inhibitory activity of gp41 peptides three-dimensional solution structure of the 44 kda ectodomain of siv gp41 crystal structure of the ebola virus membrane fusion subunit, gp2, from the envelope glycoprotein ectodomain crystal structure of human t cell leukemia virus type 1 gp21 ectodomain crystallized as a maltose-binding protein chimera reveals structural evolution of retroviral transmembrane proteins n-and c-terminal residues combine in the fusion-ph influenza hemagglutinin ha(2) subunit to form an n cap that terminates the triple-stranded coiled coil structural characterization of the human respiratory syncytial virus fusion protein core crystal structure of severe acute respiratory syndrome coronavirus spike protein fusion core structural basis for coronavirus-mediated membrane fusion. crystal structure of mouse hepatitis virus spike protein fusion core structure of the dengue virus envelope protein after membrane fusion purification of the fusion protein of sendai virus: analysis of the nh2-terminal sequence generated during precursor activation detection of a fusion peptide sequence in the transmembrane protein of human immunodeficiency virus structure of a proteolytically resistant core from the severe acute respiratory syndrome coronavirus s2 fusion protein structure and membrane interaction of the internal fusion peptide of avian sarcoma leukosis virus mutational evidence for an internal fusion peptide in flavivirus envelope protein e dengue virus infectivity depends on envelope protein binding to target cell heparan sulfate dendritic-cell-specific icam3-grabbing nonintegrin is essential for the productive infection of human dendritic cells by mosquito-cell-derived dengue viruses dc-sign (cd209) mediates dengue virus infection of human dendritic cells monoclonal antibodies that bind to domain iii of dengue virus e glycoprotein are the most efficient blockers of virus adsorption to vero cells an external loop region of domain iii of dengue virus type 2 envelope protein is involved in serotype-specific binding to mosquito but not mammalian cells serotype-specific entry of dengue virus into liver cells: identification of the 37-kilodalton/67-kilodalton high-affinity laminin receptor as a dengue virus serotype 1 receptor interaction of west nile virus with alpha v beta 3 integrin mediates virus entry into cells mapping of functional elements in the stem-anchor region of tick-borne encephalitis virus envelope protein e proteolytic activation of tick-borne encephalitis virus by furin structures of immature flavivirus particles the first step: activation of the semliki forest virus spike protein precursor causes a localized conformational change in the trimeric spike structure of dengue virus: implications for flavivirus organization, maturation, and fusion visualization of membrane protein domains by cryo-electron microscopy of dengue virus structure of west nile virus cryo-electron microscopy reveals the functional organization of an enveloped virus, semliki forest virus placement of the structural proteins in sindbis virus cleavage of protein prm is necessary for infection of bhk-21 cells by tick-borne encephalitis virus fusion activity of flaviviruses: comparison of mature and immature (prm-containing) tick-borne encephalitis virions the murray valley encephalitis virus prm protein confers acid resistance to virus particles and alters the expression of epitopes within the r2 domain of e glycoprotein conformational change and protein-protein interactions of the fusion protein of semliki forest virus structure of a flavivirus envelope glycoprotein in its low-ph-induced membrane fusion conformation nucleotide changes responsible for loss of neuroinvasiveness in japanese encephalitis virus neutralization-resistant mutants mutations in the envelope protein of japanese encephalitis virus affect entry into cultured cells and virulence in mice changes in the dengue virus major envelope protein on passaging and their localization on the three-dimensional structure of the protein epitopes on the dengue 1 virus envelope protein recognized by neutralizing igm monoclonal antibodies attenuation of murray valley encephalitis virus by site-directed mutagenesis of the hinge and putative receptor-binding regions of the envelope protein single mutation in the flavivirus envelope protein hinge region increases neurovirulence for mice and monkeys but decreases viscerotropism for monkeys: relevance to development and safety testing of live, attenuated vaccines structure of an analog of fusion peptide from hemagglutinin membrane structure and fusion-triggering conformational change of the fusion domain from influenza hemagglutinin structure and function of membrane fusion peptides studies on the structure of the influenza virus haemagglutinin at the ph of membrane fusion membrane interactions of the tick-borne encephalitis virus fusion protein e at low ph hiv entry and its inhibition membrane fusion mediated by the influenza virus hemagglutinin requires the concerted action of at least three hemagglutinin trimers dilation of the influenza hemagglutinin fusion pore revealed by the kinetics of individual cell-cell fusion events hemifusion between cells expressing hemagglutinin of influenza virus and planar membranes can precede the formation of fusion pores that subsequently fully enlarge lipid-anchored influenza hemagglutinin promotes hemifusion, not complete fusion gpi-anchored influenza hemagglutinin induces hemifusion to both red blood cell and planar bilayer membranes meta-stability of the hemifusion intermediate induced by glycosylphosphatidylinositol-anchored influenza hemagglutinin the transmembrane domain of influenza hemagglutinin exhibits a stringent length requirement to support the hemifusion to fusion transition the caenorhabditis elegans unc-64 locus encodes a syntaxin that interacts genetically with synaptobrevin close is not enough: snare-dependent membrane fusion requires an active mechanism that transduces force to membrane anchors mutations within the putative membrane-spanning domain of the simian immunodeficiency virus transmembrane glycoprotein define the minimal requirements for fusion, incorporation, and infectivity deletion of the cytoplasmic tail of the fusion protein of the paramyxovirus simian virus 5 affects fusion pore enlargement role of the cytoplasmic tail of ecotropic moloney murine leukemia virus env protein in fusion pore formation the role of the cytoplasmic tail region of influenza virus hemagglutinin in formation and growth of fusion pores truncation of the cooh-terminal region of the paramyxovirus sv5 fusion protein leads to hemifusion but not complete fusion functional analysis of the cytoplasmic tail of moloney murine leukemia virus envelope protein membrane fusion of semliki forest virus requires sphingolipids in the target membrane a single point mutation controls the cholesterol dependence of semliki forest virus entry and exit a conserved histidine in the ij loop of the semliki forest virus e1 protein plays an important role in membrane fusion novel mutations that control the sphingolipid and cholesterol dependence of the semliki forest virus fusion protein involvement of lipids in different steps of the flavivirus fusion mechanism the site of attachment in human rhinovirus 14 for antiviral agents that inhibit uncoating inhibiting hiv-1 entry with fusion inhibitors potent suppression of hiv-1 replication in humans by t-20, a peptide inhibitor of gp41-mediated virus entry crystal structure of west nile virus envelope glycoprotein reveals viral surface epitopes crystal structure of dengue virus type 1 envelope protein in the postfusion conformation and its implications for membrane fusion glycoprotein organization of chikungunya virus particles revealed by x-ray crystallography structural changes of envelope proteins during alphavirus fusion structural insights into the neutralization mechanism of a higher primate antibody against dengue virus crystal structure of the japanese encephalitis virus envelope protein this chapter was originally written in 2006 and was updated in 2012 to add recent relevant advances. key: cord-272666-3uidpr79 authors: doyle, nicole; neuman, benjamin w.; simpson, jennifer; hawes, philippa c.; mantell, judith; verkade, paul; alrashedi, hasan; maier, helena j. title: infectious bronchitis virus nonstructural protein 4 alone induces membrane pairing date: 2018-09-06 journal: viruses doi: 10.3390/v10090477 sha: doc_id: 272666 cord_uid: 3uidpr79 positive-strand rna viruses, such as coronaviruses, induce cellular membrane rearrangements during replication to form replication organelles allowing for efficient viral rna synthesis. infectious bronchitis virus (ibv), a pathogenic avian gammacoronavirus of significant importance to the global poultry industry, has been shown to induce the formation of double membrane vesicles (dmvs), zippered endoplasmic reticulum (zer) and tethered vesicles, known as spherules. these membrane rearrangements are virally induced; however, it remains unclear which viral proteins are responsible. in this study, membrane rearrangements induced when expressing viral non-structural proteins (nsps) from two different strains of ibv were compared. three non-structural transmembrane proteins, nsp3, nsp4, and nsp6, were expressed in cells singularly or in combination and the effects on cellular membranes investigated using electron microscopy and electron tomography. in contrast to previously studied coronaviruses, ibv nsp4 alone is necessary and sufficient to induce membrane pairing; however, expression of the transmembrane proteins together was not sufficient to fully recapitulate dmvs. this indicates that although nsp4 is able to singularly induce membrane pairing, further viral or host factors are required in order to fully assemble ibv replicative structures. this study highlights further differences in the mechanism of membrane rearrangements between members of the coronavirus family. viruses rely on their host cell to provide most of what they need to replicate and in order to do this, they hijack many cellular processes. a well-studied example is the ability of positive-sense single-stranded rna viruses (+rna) to induce cellular membrane rearrangements upon expression of viral proteins [1, 2] . this reorganization of cellular membranes is a critical step in the viral replication cycle since these areas of restructured membranes act as a site for assembly of all components required for viral rna synthesis as well as offer protection from detection by the host antiviral defenses [3, 4] . although the structures of these membranes are relatively well-understood, the mechanisms behind their formation, and particularly the viral and host proteins involved, are often not. the precise structure of virally induced membrane rearrangements varies between viruses [5, 6] , but viruses generally cause proliferation of membranes, forming structures, such as convoluted membranes (cm), as well as distinct types of vesicles. most common are double membrane vesicles (dmvs), which are discrete from the cytoplasm and are produced by viruses, such as poliovirus [7, 8] , hepatitis c virus [9, 10] , human norovirus [11] , and recently the equine torovirus, berne virus [12] . spherules, which are invaginated vesicles with a channel connecting them to the cytoplasm, have been found in semliki forest virus [13] , some flaviviruses [14] [15] [16] [17] , as well as brome mosaic virus (bmv), which is able to induce their formation with the expression of just one viral protein [18] . an important +rna virus family, the coronaviruses, include pathogens of both animal and human importance, such as severe acute respiratory syndrome coronavirus (sars-cov), middle east respiratory syndrome coronavirus (mers-cov), mouse hepatitis virus (mhv), porcine epidemic diarrhea virus (pedv), and infectious bronchitis virus (ibv). within this subfamily of viruses, we see variations in membrane rearrangements formed. dmvs and cm are found in cells infected with the alphaand betacoronaviruses, such as sars-cov, mers-cov, and mhv [19] [20] [21] [22] [23] [24] . in the case of the gammacoronavirus ibv, although dmvs are found, the virus induces little cm and instead induces membrane zippering to form zippered endoplasmic reticulum (zer) as well as double membrane spherules, which are found tethered to the zer [25] , producing a much more defined structure when compared to cm. subsequent to this discovery, mers-cov infection has also been shown to produce small circular structures similar in appearance to the spherules seen in ibv infection but less distinct [26] . the coronaviral proteins involved in the production of membrane rearrangements have been recently investigated with the three transmembrane non-structural proteins (nsps) nsp3, 4, and 6, which are the focus of these studies. nsps 3, 4, and 6 from different coronaviruses are accepted as functional homologues, although amino acid sequence conservation is low (ranging from 13.4 to 25.9% amino acid homology for nsps 3, 4, and 6 between ibv strain beaur and mhv strain a59). these proteins do, however, have conserved secondary structure and conserved domains, including enzymatic domains in nsp3, transmembrane domains in all three proteins, and cytoplasmic endo-domains in nsps 4 and 6. for a detailed review of the domain organization and known functions of nsps 3, 4, and 6, see [27] . nsp4 of mhv has been shown to be important for the normal function and stability of dmvs, where mutations in nsp4 resulted in attenuated virus and impairment of dmv formation [28] [29] [30] . in addition, nsp3 has been shown to localize to dmvs and cm in sars-cov-infected cells [31] . in a related group of viruses, the arteriviruses, expression of two nsps (nsps2 and 3) was able to produce dmvs [32] [33] [34] . these nsps of the arterivirus are considered functional homologs to coronavirus nsp3 and 4 [35] . upon co-expression of nsp3 and 4 from mhv, both proteins located to areas of curved membranes from where they were shown to be able to recruit nsp2 and 6; however, nsp3 and 4 alone were not able to induce the formation of dmvs [36, 37] . following on from this, it was shown that co-expression of sars-cov nsp3 and 4 induced membrane pairing and with the addition of nsp6 the formation of dmv-like structures [38] . in a subsequent study by others, it was shown that expression of only nsp3 and 4 from either mers-cov or sars-cov was able to induce dmv formation, and furthermore, addition of nsp6 made no difference to their shape or size, and did not induce the spherule-like structures seen following infection with whole virus [26] . interestingly, however, a small molecule inhibitor, k22, has been shown to inhibit the replication of several coronaviruses in vitro. in hcov-229e, k22 impaired dmv formation, while k22 resistance was associated with mutations in nsp6, emphasizing a role for nsp6 in dmv formation [39] . ibv is a pathogen of poultry, causing significant economic losses to the poultry industry worldwide as well as animal welfare problems. various strains of ibv cause disease that varies in severity from mild respiratory problems to virulent strains that can cause nephropathology and reproductive organ pathology. in this study, we compared the membrane rearrangements induced by viral proteins from two different strains of ibv, the pathogenic m41 and the apathogenic beaur. these strains were chosen because beaur and other strains of ibv induce dmv, zer, and spherule formation; however, m41 produces a low spherule phenotype when compared with other strains of the virus [40] . as the role in membrane rearrangements for nsp3 and 4 is well-established for several nidoviruses and considering that nsp6 may also play some role, here we investigated the role that these three nsps play in the formation of ibv membrane rearrangements. avian df1 cells were maintained in dmem (sigma aldrich, gillingham, uk) supplemented with 10% fcs (sigma aldrich, gillingham, uk). ibv strains beaur and m41-ck (here referred to as m41) have been described previously [41, 42] . plasmids expressing tagged nsps derived from either the apathogenic strain beaur or the pathogenic strain m41 were generated to produce pegfp-n1-m41 nsp3, pmcherry-n1-beaur nsp4, pmcherry-n1-m41 nsp4, pcdna3.1(-)-beaur nsp6-3xflag, and pcdna3.1(-)-m41 nsp6-3xflag. rna was extracted from virus-infected cells using an rnaeasy kit (qiagen, hilden, germany) following the manufacturer's protocol. rna was reverse transcribed using superscript iii (fisher scientific, loughborough, uk) and a random primer following the manufacturer's protocol. pcr was carried out on cdna using primers specific for each gene, including flanking restriction sites. pcr products were digested and ligated into pegfp-n1 (takara bio europe, saint-germain-en-laye, france) or pmcherry-n1 (takara bio) using xhoi and bamhi restriction sites. plasmid pcdna3.1(-) was modified by insertion of a 3xflag motif between the kpni and hindiii sites to generate pcdna3.1(-)-3xflag. the pcr products were then ligated into this backbone using the xhoi and bamhi restriction sites. plasmid sequences were verified using sanger sequencing. the er marker plasmid pyfp-er was kindly provided by dalan bailey. df1 cells seeded into six-well plates were transfected with pegfp-n1-m41 nsp3, pmcherry-n1-beaur nsp4, pmcherry-n1-m41 nsp4, pcdna3.1(-)-beaur nsp6-3xflag or pcdna3.1(-)-m41 nsp6-3xflag, pegfp-c2, pmchery-n1, or pcdna3.1(-)-beaur nsp7-3xflag using lipofectamine 2000 (fisher scientific). cells were transfected with a total of 1000 ng plasmid with a dna:lipofectamine 2000 ratio of 1:3 following the manufacturer's instructions. after 24 h, cells were lysed in cell lysis buffer (25 mm tris-hcl (ph 7.4), 150 mm nacl, 1 mm edta, 1% v/v triton-x100, 5% v/v glycerol, 1× halt protease inhibitor complex (fisher scientific). cell lysates were heated with 4× sample buffer (bio-rad laboratories, watford, uk) and separated on 4-20% tgx gels (bio-rad). proteins were transferred to a nitrocellulose membrane and blocked in 5% milk in pbs-t. membranes were incubated with primary antibodies to detect gfp (biolegend, london, uk), mcherry (abcam, cambridge, uk), or flag (m2; sigma aldrich, gillingham, uk). after 1 h, membranes were washed with pbs-t and incubated with irdye conjugated secondary antibodies (li-cor, cambridge, uk). membranes were imaged using an odyssey clx infrared imaging system (li-cor). df1 cells seeded onto glass coverslips were transfected with pegfp-n1-m41 nsp3, pmcherry-n1-beaur nsp4, pmcherry-n1-m41 nsp4, pcdna3.1(-)-beaur nsp6-3xflag, and pcdna3.1(-)-m41 nsp6-3xflag alone or in combination using lipofectamine 2000. cells were transfected with a total of 500 ng plasmid with a dna:lipofectamine 2000 ratio of 1:2 following the manufacturer's instructions. after 24 h, cells were fixed for 20 min in 4% paraformaldehyde in pbs at room temperature. cells were then permeabilized in 0.1% triton x-100 in pbs for 10 min and blocked in 0.5% bsa in pbs for 1 h. primary anti-flag m2 antibody (sigma aldrich) and anti-pdi antibody (enzo life sciences, exeter, uk) were diluted in blocking buffer and cells incubated for 1 h. after three washes in pbs, alexa fluor conjugated secondary antibodies (fisher scientific) were diluted 1/500 and cells incubated for 1 h. after a further three washes in pbs, nuclei were strained using topro3 (fisher scientific) or dapi (sigma aldrich) and coverslips mounted with vectashield (vector laboratories, peterborough, uk). cells were visualized using a leica sp5 confocal microscope (leica microsystems, milton keynes, uk). quantitation of transfected cells was performed manually on three randomly selected fields of view. df1 cells in six-well plates were either infected with beaur and incubated for 1 h at 37 • c when fresh 1× bes medium (mem, 0.3% tryptose phosphate broth, 0.2% bovine serum albumin, 20 mm n,n-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (bes), 0.21% sodium bicarbonate, 2 mm l-glutamine, 250 u/ml nystatin, 100 u/ml penicillin, and 100 u/ml streptomycin) was added, or were transfected with plasmids as described above. at 24 hpi, cells were washed once in 0.9% saline and scraped into the saline buffer. cells were pelleted at 500× g for 5 min at 4 • c and 500 µl 2% glutaraldehyde in 0.1 m sodium cacodylate was added to the pellet. df1 cells were then rinsed three times in 0.1 m sodium cacodylate and incubated in 1% osmium tetroxide for 2 h. after three washes in water, cells were incubated in 2% uranium acetate aqueous for 2 h at 4 • c. cells were dehydrated in increasing concentrations of acetone and then embedded in agar 100 resin (agar scientific, stansted, uk). sections approximately 50 to 60 nm in thickness were cut and stained with 2% uranyl acetate to enhance contrast. data was recorded at 80 kv on a phillips cm20 (amsterdam, netherlands) with a charge-coupled device (ccd) camera. cell sections used here each contained a single visible nucleus, with intact nuclear and plasma membranes. alternatively, df1 cells were seeded onto thermanox coverslips (fisher scientific) and either infected with beaur and incubated for 1 h at 37 • c, after which time fresh 1× bes medium was added, or cells were transfected with plasmids as described above. after 24 h, cells were fixed in 2% glutaraldehyde for 1 h, incubated in 1% aqueous osmium tetroxide solution for 1 h, then dehydrated in increasing concentrations of ethanol. cells were embedded into agar 100 resin and sections of 80 nm were cut, collected on hexagonal 200 thin bar copper grids, and stained with 2% uranyl acetate and lead citrate. data was recorded on a fei tecnai 12 tem (fei, cambridge, uk) used at 100 kv with a tvips f214 digital camera. df1 cells seeded onto thermanox coverslips were transfected and processed as before. sections 250 or 300 nm thick were cut from the resin-embedded blocks and collected on 50 mesh copper hexagonal grids coated in formvar or pioloform-coated copper slot grids. ten or 15 nm gold particles were applied to the grids to serve as fiducial markers for subsequent alignments. data was recorded on a jeol 2100 f tem (jeol, welwyn garden city, uk) used at 200 kv with a tvips f416 digital camera, or on a tecnai 20 tem (fei) used at 200 kv with a fei 4 k × 4 k eagle ccd camera. samples were mounted in a jeol high angle tilt holder or a fischione double tilt tomography holder, respectively. a single axis tilt series was collected using serial em or fei software. each single axis tilt series was collected over 100 • to 130 • in increments of between 1 • and 2.5 • and subsequently aligned and reconstructed in imod [43] . our previous studies have shown that ibv is able to induce diverse membrane rearrangements in vero cells, primary chicken kidney cells (ckcs) and tracheal organ cultures (tocs). these membrane rearrangements include dmvs, zer, and spherules [25, 40] . in order to further characterize membrane rearrangements induced by ibv, we analyzed the membrane rearrangements induced by beaur in df1s. unlike primary ckcs, df1s are a continuous avian cell line that are more easily transfected and are therefore used throughout this study. although the spike protein of beaur has increased tropism to allow for virus entry into additional cell lines, including df1 cells, m41 is not adapted to infect these cells [44, 45] . df1 cells were infected with beaur, fixed after 24 h, processed for em, and imaged. consistent with previous work, dmvs, zer, and spherules were all seen in ibv-infected df1 cells ( figure 1 ). beaur. df1 cells were infected with beaur for 24 h, fixed, and processed for electron microscopy (em). viral particles are indicated with arrowheads, double membrane vesicles (dmvs) with asterisks, and zippered endoplasmic reticulum (zer) and associated spherules with arrows. scale bar represents 500 nm. other viruses in the nidovirales order have been shown to require expression of only two or three nsps to induce membrane rearrangements similar to those seen under virus infection conditions [26, 34, 38] . to begin to understand the roles of ibv nsps in rearranging cellular membranes, nsps4 and 6 from apathogenic beaur and nsps3, 4, and 6 from pathogenic m41 were tagged with fluorescent or epitope tags. it was not possible to generate a plasmid expressing nsp3 from beaur due to presumed toxic sequences, as has been found for this region in other coronaviruses [46] [47] [48] . df1 cells were transfected with these plasmids and after 24 h cells were lysed and proteins separated by sds-page and detected by western blot. all fusions proteins were found to be intact with bands detectable at the predicted molecular weights (figure 2a) , although an additional 49 kda band was present in nsp3-gfp expressing cells, presumably due to a cleavage event within nsp3. it was also noted that nsp6-3xflag from m41 migrated at a higher molecular weight than nsp6-3xflag from beaur, most likely due to differences in post-translational modification. subsequently, df1 cells were transfected with these plasmids and after 24 h cells were fixed, labelled with an anti-flag antibody, and visualized by confocal microscopy. all three nsps showed reticular cytoplasmic labelling consistent with localization to the er (figure 2b ), as has been observed previously [26, 37, [49] [50] [51] [52] [53] . in addition to er localization, nsp4 was found in both small and large puncta in cells where the level of nsp4 expression was higher (comparison shown in figure 2b ). nsp6 was also found in small cytoplasmic puncta when expressed alone (figure 2b ). to confirm er localization, df1 cells were transfected with either the plasmid expressing nsp3-gfp alone or plasmids expressing nsp4 or 6 together with pyfp-er, as indicated. after 24 h, cells were fixed and labelled with anti-flag-and nsp3-expressing cells with anti-protein disulphide isomerase (pdi), a resident er protein. colocalization between yfp-er or pdi and nsp3, 4, and 6 was observed, confirming that these proteins localize to the er (figure 2c ). cells were transfected with plasmids expressing tagged nsps, as indicated, or empty vectors or nsp7-3xflag as controls. cell lysates were separated by sds-page and proteins detected by western blot. from left to right, nsp3-gfp detected using anti-gfp, nsp4-mcherry detected using anti-mcherry, and nsp6-3xflag detected using anti-flag, as labelled. molecular weight markers are shown on the left and asterisks indicate the nsp bands on each blot. (b) df1 cells were transfected with plasmids expressing nsp4-mcherry and nsp6-3xflag from beaur, and nsp3-egfp, nsp4-mcherry, and nsp6-3xflag from m41. after 24 h, cells were fixed with 4% paraformaldehyde and imaged. nsp3 (green), nsp4 (red), and nsp6 (blue) were imaged as labelled. nuclei were stained with topro3 (grey) and scale bars indicate 10 µm. (c) df1 cells were transfected with plasmids expressing nsp4-mcherry and nsp6-3xflag from beaur, and nsp3-egfp, nsp4-mcherry, and nsp6-3xflag from m41 together with yfp-er. after 24 h, cells were fixed with 4% paraformaldehyde and imaged. nsp3 (green) and nsp4 and nsp6 (red) were imaged along with markers for the er; pdi (red) or yfp-er (yellow) as indicated. nuclei were stained with dapi (grey) and scale bar represents 10 µm. next, to understand whether co-expression of these proteins results in changes in their localization, df1 cells were transfected with combinations of the plasmids. after 24 h, cells were fixed and labelled with an anti-flag antibody. upon co-expression of some combinations of these viral proteins, this staining pattern changed. expression of nsp3 with nsp4 resulted in both proteins localizing to cytoplasmic puncta, although some signals for both proteins also remained in the er (figure 3 ). co-expression of nsp3 with nsp6, or nsp4 with nsp6, did not result in relocalization of either protein, with nsp3 remaining er-associated, nsp4 remaining both er-associated and localized in cytoplasmic puncta, and nsp6 remaining both er-localized and in cytoplasmic puncta (figure 3) . interestingly, co-expression of nsps3, 4, and 6 resulted in relocalization of all three proteins to cytoplasmic puncta, some containing nsp3 and 4, some nsp6 only, and some puncta containing nsp3, 4, and 6 ( figure 3 ). nsps4 and 6 derived from either beaur or m41 exhibited the same pattern of localization. this demonstrates that co-expression of ibv nsps in the absence of any other viral components can result in their relocalization within the cell, presumably as a result of protein-protein interactions and potentially associated with rearrangement of cellular membranes. figure 3 . co-expression of ibv non-structural proteins results in their relocalization from the er to cytoplasmic foci. df1 cells were transfected with plasmids expressing nsp4-mcherry and nsp6-3xflag from beaur, and nsps3-egfp, nsp4-mcherry, and nsp6-3xflag from m41 in pairs or in a combination of three, as indicated. solid arrows indicate areas of nsp3 and 4 colocalization, open arrows indicate areas of nsp3, 4, and 6 colocalization. nuclei were strained with topro3 (grey) and scale bar represents 10 µm. to further understand the ability of ibv nsps3, 4, and 6 to rearrange cellular membranes, proteins were expressed in cells and analyzed by electron microscopy (em). initially, to assist with subsequent analysis by em, the percentage of total cells in figures 2b and 3 that were expressing the nsps of interest, as well as the percentage of cells expressing other combinations of nsps, was quantified (table s1 ). df1 cells were transfected with tagged nsp3, 4, and 6 derived from beaur or m41 alone and in combination. after 24 h, cells were chemically fixed, embedded in resin, and visualized using an electron microscope. a phenotype common to all transfected cells was small, tight whorl-like structures which stained more strongly than other structures (figure 4a ). these were considered an artefact of transfection. transfection of cells with empty pegfp-n1, pmcherry-n1, or pcdna3.1(-)-3xflag did not result in changes to cellular membranes (figure 4a ). different types of membrane structures were observed in the transfected cell samples that were absent from mock treated cells, including paired membranes, disordered or piled membranes, and dmv-like structures. nsp4 in other coronaviruses has been shown to be important in membrane modifications, particularly in the formation of conventional dmvs [29, 30] . initially, the effect of expression of nsp4 in df1 cells was investigated. interestingly, it was observed that expression of beaur nsp4 alone was capable of forming paired membranes. this the first time this has been observed for any coronavirus nsp4. these paired membranes were observed both as very large areas of extensive accumulations or as small regions of shorter sections of paired membranes. the paired membranes were tightly apposed, often connected to the er, were largely free of ribosomes, and strongly resembled ibv-induced zer (figure 4b) , although the electron density often surrounding ibv-induced zer was missing here and no spherules were present. transfection of m41 nsp4 also induced membrane pairing (figure 4b) with an appearance comparable to that of beaur nsp4-induced paired membranes. for cells transfected with the beaur nsp4 expression vector, 31 out of 235 cell sections (13%, percentage of total cells not transfected cells) contained piled membranes, and 3 out of 108 (3%) sections for m41, significant to p < 0.00001 by a fisher's exact test. it has previously been shown for other coronaviruses that membrane pairing requires co-expression of nsps3 and 4 or that co-expression of these proteins results in dmv accumulation [26] . therefore, the effect of co-expression of nsp3 with nsp4 was investigated. firstly, the effect of expression of nsp3 alone on cellular membranes was determined. although over 200 cells were examined from multiple experiments, expression of nsp3 was found to have no striking phenotype with cellular membranes appearing unchanged in the presence of nsp3 derived from m41 when compared with untransfected cells. furthermore, surprisingly, expression of nsp3 with nsp4 had no effect on the membrane pairing ability of nsp4 (figure 4c) , with membrane rearrangements appearing comparable to cells expressing nsp4 alone, i.e., paired membranes connected to the er and lacking ribosomes, found covering both large and smaller areas of the cytoplasm. specifically, the numerous dmv-like structures observed in cells expressing nsp3 and 4 from either mers-cov or sars-cov were not observed here [26, 38] . overall, this data confirms that ibv nsp4 alone is the main driving factor in membrane pairing and co-expression of nsp3 does not alter this function. coronavirus nsp6 has previously been linked to autophagy induction when expressed alone [49] . nsp6 derived from sars-cov has also been shown to induce single membrane vesicle accumulation and microtubule organizing center vesiculation [38] . therefore, the cellular membrane rearrangements induced by expression of ibv nsp6 were analyzed. in cells expressing nsp6 alone from either beaur or m41, large areas of tangled single membranes, which appear to be derived from the er, were observed ( figure 5 ). these piled, disordered membranes strongly resemble the disordered membrane bodies seen previously upon expression of sars-cov nsp3 [38] . to determine the effect of expression of nsp6 with other nsps on the formation of disordered membranes or any other structures, samples transfected with plasmids expressing nsp6 and either nsp4 or nsp3 were analyzed. in cells co-expressing nsp3 and 6, it was observed that cells expressing beaur nsp6 formed disordered membranes while those expressing m41 nsp6 did not. co-expression of nsp4 and nsp6 produced the paired membranes associated with nsp4 expression (for both beaur and m41 nsp4). disordered membranes were only found in cells co-expressing beaur nsp6 but none when co-expressing m41 nsp6. this indicates that while nsp6 from either beaur or m41 can induce the formation of disordered membranes when expressed singly, co-expression of nsp6 with either nsp3 or 4 disrupts this mechanism and to a greater extent in m41. finally, the membrane rearrangements induced by co-expression of ibv nsps3, 4, and 6 were investigated by electron microscopy to determine whether co-expression of all three transmembrane nsps could result in the formation of structures comparable to replication organelles in ibv-infected cells. the major phenotype observed following co-expression of all three nsps was the paired membranes induced by expression of nsp4 alone ( figure 6 ). when nsp4 and nsp6 derived from beaur were expressed with m41 nsp3, a very limited number of dmv-like structures was observed (3 in 329 cell sections). in cells co-expressing nsp3, 4, and 6 derived from m41, no dmv-like vesicles were found in 489 cell sections with only nsp4-associated paired membranes being detected. in neither combination were the spherules usually found during virus infection observed. therefore, although co-expression of ibv nsps 3, 4, and 6 may be sufficient for formation of dmvs, this does not seem to be a very efficient process compared with dmv formation by nsp3 and 4 from the betacoronaviruses studied previously [26, 38] and nsp6 is unlikely to be the additional nsp required for ibv dmv formation. in order to further understand the paired membranes induced by expression of ibv nsp4, electron tomography (et) was used to visualize membrane rearrangements in three dimensions. in addition, et was used to confirm that, unlike for other coronaviruses [26, 38] , co-expression of ibv nsp3 and 4 does not result in the formation of dmvs. df1 cells were transfected with plasmids expressing either beaur nsp4 or beaur nsp4 with m41 nsp3. after 24 h, cells were fixed and processed for et. the paired membranes produced by nsp4 expression (indicated by arrows) were found to form sheet-like structures with sections of paired membranes dilating in several places (arrowheads) (figure 7a , video s1). a comparison with cells expressing nsp3 and 4 showed there is no noticeable difference between the areas of paired membranes induced upon expression of these nsps (figure 7b, video s2) . therefore, expression of ibv nsp4 alone results in the formation of paired er membranes. addition of nsp3 does not alter the membrane structures induced with no formation of either dmvs, as seen for other covs or spherules. induction of host cell membrane rearrangements is a tool used by many +rna viruses, such as coronaviruses [1, 2] . these membrane rearrangements vary between the different members of the family, with the alpha and betacoronaviruses inducing convoluted membranes and dmvs and the gammacoronavirus ibv inducing zippered er, spherules, and dmvs [19] [20] [21] [22] [23] 25, 26] . the formation of these membrane rearrangements is, however, a well-conserved mechanism used by these viruses in order to provide a site for viral rna synthesis. although the pool of knowledge about these structures has been growing, the mechanisms behind their formation remain largely unclear. some light has been shed in recent years on the specific viral proteins involved in the formation of these structures; however, these studies were lacking in ibv. in this study, we looked at the involvement of nsps3, 4, and 6, which have all been implicated in the formation of membrane rearrangements. as transmembrane proteins, these are likely candidates in reordering the host cell membranes to the advantage of the virus. we showed firstly that df1 cells are a suitable cell type to use for studying ibv membrane rearrangements in addition to those already tested [40] . in order to assess the involvement of nsps 3, 4, and 6 in virus-induced membrane rearrangements, plasmids expressing gfp, mcherry, or 3xflag fusion proteins were generated. to confirm expression of full-length fusion proteins, western blots were performed using antibodies against the tags. for all the constructs, full-length nsp fusion proteins were detected. however, in cells expressing nsp3-gfp, an additional 49 kda band was seen indicating that as well as full-length protein, a cleavage product corresponding to the c-terminus of nsp3 plus gfp was also being produced. next, we expressed nsps alone or in combination in df1 cells to assess their ability to rearrange cellular membranes. when expressed alone, all three nsps had a reticular, cytoplasmic localization consistent with previous observations that these nsps localize to the er [26, 37, [49] [50] [51] [52] [53] , although nsp4 and nsp6 in addition had a punctate localization with nsp4 in particular forming large foci in some cells. er localization was subsequently confirmed by colocalization of the three nsps with er markers. when nsps3 and 4 were co-expressed, both proteins localized to large and small cytoplasmic puncta with some protein also remaining in the er. this suggests that nsp3 and 4 are able to interact with one another, again consistent with previous findings for other coronaviruses [26, 37] , resulting in nsp3 moving into the nsp4-containing puncta. co-expression of nsp3 and 6 or nsp4 and 6 did not result in alteration of their cellular localization. however, when nsp3, 4, and 6 were co-expressed, nsp3 and 4 colocalized as seen before but some puncta now also contained nsp6, although some puncta contained only nsp3 and 4 or nsp6 alone. this suggests that, as seen in other coronaviruses, nsp3 and 4 together, but not alone, are able to direct nsp6 into the nsp3/4 puncta [36, 37] . subsequently, em was used to identify changes to the structure of cellular membranes upon expression of these three proteins. surprisingly, expression of nsp3 did not induce any notable phenotype. expression of nsp3 from either sars-cov or mers-cov results in the production of disordered membrane bodies likely derived from the er [26, 38] . it is not clear why nsp3 derived from ibv behaves so markedly differently from nsp3s expressed by other coronaviruses. however, the previously studied nsp3s have all been derived from betacoronaviruses so nsp3 from gammacoronaviruses, including ibv, may function somewhat differently. indeed, an amino acid sequence comparison between nsp3 sequences from beaur and the betacoronavirus mhv a59 shows only 13.4% homology and 25.9% similarity. therefore, although these are accepted as functional homologs, there is scope for these proteins to behave differently from one another. furthermore, given that we have previously demonstrated that ibv-induced membrane rearrangements are distinct from those induced by alphaand betacoronaviruses [25] , differences in the mechanism of their formation might reasonably be expected. interestingly, expression of nsp6 alone induced membrane proliferation and the formation of disordered membranes similar to the disordered membrane bodies (dmbs) induced by sars-cov and mers-cov nsp3 [26, 38] . expression of nsp6 alone did not appear to induce microtubule organizing center vesiculation as seen upon expression of sars-cov nsp6 [38] and the presence of autophagosomes was also not apparent [49, 54] , although this is likely due to differences in experimental approaches, namely the use of em in this study compared to immunofluorescence of whole cells used previously [54] . therefore, ibv nsp6 also appears to function somewhat differently to nsp6 from sars-cov in its ability to rearrange membranes. the most striking phenotype came upon expression of nsp4; expression of nsp4 alone was sufficient to induce areas of paired membranes. furthermore, et demonstrated that these are sheet-like areas of paired er membranes, highly similar to zer in ibv-infected cells. it was noted that the paired membranes, although resembling zer in infected cells, lacked the electron density often surrounding the membranes [25] . this reflects the lack of the other viral proteins making up the replication complex, which, presumably, accumulate on the cytoplasmic surface of the zer. nsp4-induced paired membranes were observed as both small regions throughout the cytoplasm and also in extensive areas of paired membranes. these two phenotypes potentially reflect the different localizations observed by confocal microscopy with some cells containing nsp4 localized only to the er and some cells containing large cytoplasmic puncta corresponding to the large areas of paired membranes. use of correlative light electron microscopy (clem) in the future would confirm this. attempts were made to confirm the nsp4 homotypic interaction by co-immunoprecipitation; however, this was not successful. it has previously been shown for mhv that nsp4 can self-associate [37] , although earlier attempts to demonstrate the interaction in sars-cov failed [55, 56] , highlighting that detection of this interaction can be challenging. however, it is likely that self-interaction between nsp4 proteins located in both membranes of the er zippers the two er membranes together to generate the paired membranes seen, although it cannot be ruled out that instead an interaction with one or more cellular proteins is required. significantly, this is the first time for any coronavirus that, regardless of mechanism, a membrane pairing function for nsp4 alone has been described. surprisingly, addition of nsp3 did not alter the membrane rearrangements induced by nsp4 alone. previous work by others has shown that for other related coronaviruses and arteriviruses, membrane pairing requires the expression of nsp3 and 4 (or their homologs) [36] [37] [38] 57] . in addition to this, however, co-expression of nsp3 and 4 for other coronaviruses resulted in the formation of numerous dmv-like structures [26, 38] . despite extensive searching and the use of electron tomography to gain three-dimensional information, we were not able to detect any dmvs in cells expressing nsp3 and 4. the reason for this difference is not clear. here, we used separate plasmids to express nsp3 and 4 but this strategy was also used in previous work and when compared with a cleavable nsp3-4 precursor did not yield different results [26] . therefore, the protein expression strategy is unlikely to be the reason that dmvs were not formed. it is possible that the presence of the shorter nsp3 fragment detected by western blot prevented the formation of dmvs. however, full-length nsp3 was also present and therefore should have been capable of inducing dmvs in combination with nsp4. in addition, dmvs were not detectable in cells expressing either nsp3 from m41 and nsp4 from beaur or cells expressing nsp3 and 4 from m41, indicating that the use of proteins from different virus strains was not the reason for the lack of dmvs. indeed, nsp3 relocalized to both beaur and m41 nsp4-containing foci suggesting that m41 nsp3 is capable of interacting with both nsp4 proteins. again, attempts were made to confirm interaction between nsp3 and nsp4 by co-immunoprecipitation, but this was not successful. interactions between full-length or the c-terminus of nsp3 and nsp4 from other coronaviruses have been shown previously [37, 55] . interestingly, sakai et al. showed that just two amino acid residues in nsp4 are necessary for the interaction with nsp3; however, these residues are only conserved in betacoronaviruses, not in alphaor gammacoronaviruses [58] , so it is likely that the mechanism of any nsp3/nsp4 interaction is different in ibv. overall, the data indicates that dmv formation by ibv requires the presence of additional viral protein(s), either to direct an interaction between nsp3 and nsp4 if it cannot occur directly or because dmv formation is via another mechanism. co-expression of nsps 3, 4, and 6 did appear to result in the formation of a very small number of dmv-like structures. however, these were significantly less numerous and less easily identifiable than those observed by oudshoorn et al. [26] . therefore, nsp6 does not appear to be the ibv protein required, in addition to nsp3 and 4, to induce dmvs and other viral proteins must play a role. throughout this study, we were unable to detect spherules associated with ibv infection, although we did identify membranes highly similar to zer. in our previous work, we demonstrated that m41 virus has a low spherule phenotype and the region of the genome from the 5 end to nsp13 was responsible for this [40] . unfortunately, we were unable to clone nsp3 from beaur due to toxicity problems in escherichia coli. it was also not possible to clone nsp3 from two further strains of ibv. as the nsp3 used in this study was derived from m41, it is possible that this is the reason that spherules were not detected under any conditions. nsp3 from beaur and m41 are highly related with 90.5% amino acid homology and 95.2% similarity with the majority of the differences occurring within the non-functional papain-like protease 1 domain. despite that fact, it cannot be ruled out that these differences are sufficient to prevent spherule formation. in future, cloning the c-terminal part of nsp3 from beaur, as other groups have done for mhv [36] , may provide further insight into the role of nsp3 in membrane modifications. an alternative explanation for the lack of spherules could be that the precise molar ratio of nsps to one another, as well as the presence of cleavage intermediates, generated as a result of expression via a polyprotein during virus infection is critical for spherule formation. in that case, the expression approach taken here of transfecting multiple plasmids into cells would not result in the correct ratio of proteins or presence of cleavage intermediates, thereby preventing spherule formation. however, oudshoorn et al. were also unable to identify cms and spherule-like structures when combinations of nsps were expressed either from separate plasmids or as a polyprotein [26] . instead, it is more likely that additional viral proteins are required for spherule formation. this is not necessarily surprising. for alphaviruses, spherules are only formed in the presence of all nsps and although they are able to form in the absence of rna, the length of rna present directly affects the size of the spherule produced [59, 60] . furthermore, in the case of flock house virus, spherules only form when rna synthesis is actively occurring [61] . therefore, spherule formation by ibv may require expression of additional nsps, including those required for rna synthesis, as well as an rna template. alternatively, it may require expression of additional nsps that direct interaction with cellular proteins that facilitate changes to the membrane. the mechanisms behind the formation of virus-induced membrane rearrangements required for replication organelle formation are doubtlessly complex. although we have identified a clear role for ibv nsp4 in membrane pairing and the formation of zippered er, numerous questions remain and further differences between ibv and members of the betacoronavirus sub-family have been highlighted. the identity of the ibv proteins required for both spherule and dmv formation remain unknown and further study is required to complete our understanding of the critical stage of the virus replication cycle. supplementary materials: the following are available online at http://www.mdpi.com/1999-4915/10/9/477/s1, table s1 : cells expressing each nsp compared to the total number of cells counted, video s1: electron tomographic reconstruction of paired membranes in an nsp4-expressing cell, video s2: electron tomographic reconstruction of paired membranes in an nsp3 and nsp4-expressing cell. modification of intracellular membrane structures for virus replication virus factories, double membrane vesicles and viroplasm generated in animal cells organelle-like membrane compartmentalization of positive-strand rna virus replication factories the hepatitis c virus-induced membranous web and associated nuclear transport machinery limit access of pattern recognition receptors to viral replication sites ultrastructure of the replication sites of positive-strand rna viruses architecture and biogenesis of plus-strand rna virus replication factories structural and functional characterization of the poliovirus replication complex complex dynamic development of poliovirus membranous replication complexes identification of the hepatitis c virus rna replication complex in huh-7 cells harboring subgenomic replicons sequential biogenesis of host cell membrane rearrangements induced by hepatitis c virus infection membrane alterations induced by nonstructural proteins of human norovirus ultrastructural characterization of membranous torovirus replication factories biogenesis of the semliki forest virus rna replication complex composition and three-dimensional architecture of the dengue virus replication and assembly sites the endoplasmic reticulum provides the membrane platform for biogenesis of the flavivirus replication complex 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 ultrastructural characterization of zika virus replication factories a positive-strand rna virus replication complex parallels form and function of retrovirus capsids ultrastructure and origin of membrane vesicles associated with the severe acute respiratory syndrome coronavirus replication complex rna replication of mouse hepatitis virus takes place at double-membrane vesicles qualitative and quantitative ultrastructural analysis of the membrane rearrangements induced by coronavirus mers-coronavirus replication induces severe in vitro cytopathology and is strongly inhibited by cyclosporin a or interferon-α treatment ultrastructural characterization of membrane rearrangements induced by porcine epidemic diarrhea virus infection infectious bronchitis virus generates spherules from zippered endoplasmic reticulum membranes 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 bioinformatics and functional analyses of coronavirus nonstructural proteins involved in the formation of replicative organelles mutation in murine coronavirus replication protein nsp4 alters assembly of double membrane vesicles murine hepatitis virus nonstructural protein 4 regulates virus-induced membrane modifications and replication complex function mutations across murine hepatitis virus nsp4 alter virus fitness and membrane modifications sars-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum formation of the arterivirus replication/transcription complex: a key role for nonstructural protein 3 in the remodeling of intracellular membranes open reading frame 1a-encoded subunits of the arterivirus replicase induce endoplasmic reticulum-derived double-membrane vesicles which carry the viral replication complex non-structural proteins 2 and 3 interact to modify host cell membranes during the formation of the arterivirus replication complex evolving the largest rna virus genome membrane rearrangements mediated by coronavirus nonstructural proteins 3 and 4. virology mobility and interactions of coronavirus nonstructural protein 4 severe acute respiratory syndrome coronavirus nonstructural proteins 3, 4, and 6 induce double-membrane vesicles targeting membrane-bound viral rna synthesis reveals potent inhibition of diverse coronaviruses including the middle east respiratory syndrome virus extensive coronavirus-induced membrane rearrangements are not a determinant of pathogenicity reverse genetics system for the avian coronavirus infectious bronchitis virus taxonomic studies on strains of avian infectious bronchitis virus using neutralisation tests in tracheal organ cultures computer visualization of three-dimensional image data using imod selection of and recombination between minor variants lead to the adaptation of an avian coronavirus to primate cells contributions of the s2 spike ectodomain to attachment and host range of infectious bronchitis virus engineering the largest rna virus genome as an infectious bacterial artificial chromosome strategy for systematic assembly of large rna and dna genomes: transmissible gastroenteritis virus model systematic assembly of a full-length infectious cdna of mouse hepatitis virus strain a59 coronavirus nsp6 proteins generate autophagosomes from the endoplasmic reticulum via an omegasome intermediate detection of nonstructural protein 6 in murine coronavirus-infected cells and analysis of the transmembrane topology by using bioinformatics and molecular approaches membrane topology of murine coronavirus replicase nonstructural protein 3 topology and membrane anchoring of the coronavirus replication complex: not all hydrophobic domains of nsp3 and nsp6 are membrane spanning localization and membrane topology of coronavirus nonstructural protein 4: involvement of the early secretory pathway in replication visualizing the autophagy pathway in avian cells and its application to studying infectious bronchitis virus analysis of protein-protein interactions and involvement of viral proteins in sars-cov replication analysis of intraviral protein-protein interactions of the sars coronavirus orfeome biogenesis and architecture of arterivirus replication organelles two-amino acids change in the nsp4 of sars coronavirus abolishes viral replication template rna length determines the size of replication complex spherules for semliki forest virus partially uncleaved alphavirus replicase forms spherule structures in the presence and absence of rna template nodavirus-induced membrane rearrangement in replication complex assembly requires replicase protein a, rna templates, and polymerase activity the authors would like to thank paul britton and pip beard for helpful discussions. the authors declare no conflict of interest. the funding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results. key: cord-303238-us3dybue authors: kanjanahaluethai, amornrat; chen, zhongbin; jukneliene, dalia; baker, susan c. title: membrane topology of murine coronavirus replicase nonstructural protein 3 date: 2007-05-01 journal: virology doi: 10.1016/j.virol.2006.12.009 sha: doc_id: 303238 cord_uid: us3dybue mouse hepatitis virus (mhv) is a member of the family coronaviridae. these positive strand rna viruses encode a replicase polyprotein that is processed into 16 nonstructural proteins (nsps). the nsps assemble with membranes to generate double membrane vesicles, which are the sites of viral rna synthesis. mhv nsp3 contains multiple domains including two papain-like protease domains, plp1 and plp2, and a predicted transmembrane (tm) domain. in this study, we determined the membrane topology of nsp3-tm and showed that tm-mediated tethering of plp2 is important for processing at cleavage site 3. biochemical analysis revealed that nsp3 is an integral membrane protein that is inserted into endoplasmic reticulum (er) membranes co-translationally and glycosylated at asparagine-2357. proteinase k digestion experiments indicate that the tm domain of nsp3 has 4 membrane-spanning helices. we show that nsp3-tm is sufficient to mediate er membrane association of a cytosolic protein. this study is the first detailed analysis of the topology and function of the coronavirus nsp3 tm domain. proteolytic processing of a replicase polyprotein and the generation of a membrane-associated replication complex are common themes in studies analyzing the replication of positive strand rna viruses. for the majority of coronaviruses, the proteolytic processing of the viral replicase polyprotein is mediated by three distinct viral proteinases to generate 16 replicase products [reviewed in (ziebuhr, 2005) ]. both coronavirus and the related arterivirus replicase products have been shown to assemble with cellular membranes to generate double-membrane vesicles that are the sites of viral rna synthesis (goldsmith et al., 2004; gosert et al., 2002; pedersen et al., 1999; snijder et al., 2006) . the goal of our research is to characterize the coronavirus replicase proteolytic processing cascade and identify factors that mediate membrane-association of the replication complex. our model system is the replication of mhv, one of the prototype coronaviruses. mhv is a ∼31.5-kilobase (kb) positivestrand rnavirus that replicates in the cytoplasm of infected cells. the 5′-most 22 kb of the mhv genomic rna contains two large open reading frames (orfs), termed orf1a and orf1b (lee et al., 1991) . during translation of the genomic rna, the two orfs are joined via a ribosomal frameshifting mechanism to produce a polyprotein of ∼800 kilodaltons (kda) in size (brierley et al., 1987; lee et al., 1991) . this polyprotein is the viral rnadependent rna polymerase, termed the replicase. the mhv replicase is processed by three distinct proteases contained within orf1a of the replicase polyprotein. two of the proteases are papain-like cysteine proteases, termed plp1 and plp2. the third protease domain is distantly related to the picornavirus family of 3c-like proteases, and is termed 3clpro. these three proteases process the replicase polyprotein to produce intermediates and products that function in the replication of genomic rna and the synthesis of a nested set of subgenomic mrnas [reviewed in (brian and baric, 2005; sawicki and sawicki, 2005) ]. studies have shown that the replicase products alone are sufficient to mediate viral rna synthesis , and to generate the double-membrane structures that serve as the sites for viral rna synthesis (pedersen et al., 1999) . however, the role of proteolytic processing in regulating the assembly and function of the mhv replication complex is not yet understood. therefore, we wanted to identify regions of the replicase polyprotein that may direct or regulate proteolytic processing and/or membrane association. the focus of this study was to identify the regions of mhv nsp3 that direct membrane association and plp2 activity. previously, we showed the mhv plp2 can act in trans to process cleavage site 3 (cs3) at the nsp3/nsp4 junction (kanjanahaluethai and baker, 2000; kanjanahaluethai et al., 2003) . other coronaviruses, such as the human coronavirus 229e, have been shown to utilize plp2 (also termed pl2pro) to process sites both upstream and downstream of the catalytic domain (ziebuhr et al., 2001) . the papain-like protease (plpro) encoded by the coronavirus that causes severe acute respiratory syndrome (sars-cov) processes three sites in the replicase polyprotein (harcourt et al., 2004) , and has recently been shown to have de-ubiquitinating activity (barretto et al., 2005; lindner et al., 2005) . the crystal structure of this enzyme has been resolved and is currently being targeted for anti-viral drug development (ratia et al., 2006) . a better understanding of the coronavirus papain-like proteases may facilitate anti-viral drug development for sars and also other recently identified human coronavirus infections caused by nl63 (van der hoek et al., 2004) and hku1 (woo et al., 2005) , which can cause pneumonia and respiratory tract infections in children and the elderly. the aims of this study were to determine the topology of mhv nsp3 and to identify the regions in nsp3 required for plp2 activity. we used a trans-cleavage assay to determine if an expressed plp2 domain was sufficient for the recognition and processing of a substrate containing cs3. we found that constructs containing plp2 and the downstream putative tm domain were able to efficiently process the substrate. bioinformatic analysis of the nsp3-tm domain indicated the presence of putative membrane-spanning helices and consensus sites for n-linked glycosylation. site-directed mutagenesis of the asparagine residues and analysis of endoglycosidase h (endo h) sensitivity revealed that asparagine-2357 in nsp3-tm is glycosylated. to investigate the topology of nsp3-tm, we tested for sensitivity to digestion with proteinase k and found that at least two lumenal domains are protected, consistent with 4 membranespanning regions in nsp3-tm. we showed that nsp3-tm alone is sufficient to confer membrane association to a normally cytosolic protein, enhanced green fluorescent protein (egfp), and that a predicted multi-spanning tm domain is conserved in nsp3 of all coronaviruses. thus, the nsp3-tm domain is important for membrane association of the replicase and tethering the plp2 domain for viral polyprotein processing activity. the mhv nsp3-tm domain is important for plp2 processing at cleavage site 3 analysis of sars-cov plpro activity showed that the nsp3 hydrophobic domain downstream of plpro is essential for processing at cs3 (harcourt et al., 2004) . to determine if a similar domain is important for mhv plp2 activity, we generated a series of plp2 c-terminal deletion constructs and tested each construct for expression and processing activity. plp2 expression constructs were generated by polymerase chain reaction (pcr) amplification and cloning of the amplified region into pcdna3.1/v5-his, as described in materials and methods. to determine if each clone was expressing a protein of the expected size, we analyzed the products after t7-mediated expression and immunoprecipitation (fuerst et al., 1986; kanjanahaluethai and baker, 2000) . we detected the expected series of truncated plp2 proteins that ranged in size from ∼96 kda to ∼ 50 kda (fig. 1b, lanes 1-6) . to determine if these plp2 products were sufficient to mediate processing of cs3, we tested each construct in the trans-cleavage assay by co-transfection with the substrate (fig. 1c, lanes 7-12) . we found that only two of the plp2 expression products, pplp2-2485 and pplp2-2390, were able to efficiently process the substrate and produce the 44-kda cleavage product, nsp4 (fig. 1c, lanes 7 and 8). these two constructs encompass all or a major part of the predicted nsp3-tm domain indicating that membrane tethering of plp2 is important for recognition and processing at cs3. thus, both sars-cov plpro (harcourt et al., 2004) and mhv plp2 require the downstream tm domain for recognition and processing at the nsp3/nsp4 junction. bioinformatic analysis of nsp3-tm predicts a series of putative membrane-spanning sequences previously, we showed that mhv nsp3 is indeed an integral membrane protein, but the role of the tm in mediating this membrane association was not investigated (gosert et al., 2002) . initial bioinformatic analysis indicated two transmembrane helices in nsp3 (ziebuhr et al., 2001) . to extend these studies of membrane association of coronavirus replicase products, we analyzed the amino acid sequence of mhv-jhm nsp3 (from glycine-833 to glycine-2840) for probability of transmembrane helices using the five different programs designed to search for putative membrane-spanning sequences: phobius, tmhmm, hmmtop, sosui and tmpred (fig. 2) . interestingly, each program generated a unique prediction for the topology of nsp3 (fig. 2c ). the number of predicted membrane-spanning domains varied from three (phobius) to seven (tmpred). however, since both the n-and c-termini of nsp3 are cleaved in the cytosol, the number of membrane-spanning helices must be either two [as previously predicted (ziebuhr et al., 2001) ], four or six. to better understand the topology of the nsp3-tm domain, we performed membrane-association, fractionation and proteinase k protection experiments. to determine if the nsp3-tm is indeed required for membrane association, we performed in vitro transcription and translation of the plp2 expression constructs in the absence or presence of canine microsomal membrane (cmm) and assayed for membrane association. the newly translated proteins were metabolically radiolabeled with [ 35 s]-translabel, subjected to centrifugation to separate the membrane-associated pelleted fraction from the soluble fraction. protein products of both fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page), and visualized by autoradiography (fig. 3) . the percentage of total proteins detected in soluble and pelleted fractions was quantitated by phosphoimaging analysis. in the absence of cmm, the translated protein products were detected predominantly in the soluble fraction (fig. 3a) . in contrast, when cmms were added to the mixture, protein products that included all or part of nsp3-tm (plp2-2485, -2390 and -2258) were detected predominantly in the pelleted fraction, consistent with membrane association (fig. 3b ). to determine if the membrane association occurred co-translationally or post-translationally, we added the cmms after termination of translation, and assessed membrane association. previous studies with hepatitis c virus revealed post-translational insertion of the ns5a replicase protein (brass et al., 2002) . we found that the translation product of plp2-2485 is inserted co-translationally, with no detectable post-translational insertion into membranes (fig. 3c ). these results indicate that mhv nsp3 membrane insertion likely occurs via the normal, signal recognition particle (srp)-dependent er translocation mechanism, and not via a tail-anchoring or post-translational mechanism. to distinguish between membrane association versus integral membrane insertion, membrane extraction experiments were performed. protein products expressed from pplp2-2485, pplp2-2390 and pplp2-2258 constructs were translated in vitro in the presence of cmm and the pelleted fraction was subsequently subjected to differential extraction methods as indicated in fig. 4 . as expected, treatment with nte buffer alone had no effect and the proteins pelleted with membranes, whereas treatment with detergent triton x-100 disrupted the membranes, and the proteins were detected in the soluble fraction (fig. 4a) . treatments with urea, nacl or sodium carbonate, ph 11.5 have been shown to disrupt the association between peripheral, membrane-associated proteins, but such treatments do not disrupt integral membrane proteins (bordier, 1981) . we tested for disruption of plp2-tm proteins and found that majority of the proteins remained in the pelleted membrane fraction, consistent with integral membrane proteins (fig. 4b ). we noted that the protein product expressed from pplp2-2258, which contains only one or two predicted membrane spanning helices, was dissociated by treatment with high ph, but that the protein products expressed from constructs that extended further into the tm were not disrupted from the membranes by these treatments, consistent with the characteristics of integral membrane proteins. bioinformatic analysis also revealed two consensus sequences for n-linked glycosylation [the consensus being nx(t/s), where x is any amino acid (helenius and aebi, 2001) ] in nsp3-tm. if one or more of the predicted sequences was indeed a transmembrane helix, then the sites for n-linked glycosylation could be lumenal, and subject to modification. to determine if either of the putative sites was modified by glycosylation, we the amino acid numbering is according to ncbi accession number nc006852. conserved domains previously identified by comparative sequence analysis (ziebuhr et al., 2001) and the recently described adp-ribose-1ʺ-phosphatase (adrp) domain (saikatendu et al., 2005) are indicated. two putative sites for n-linked glycosylation were predicted at asparagine residues 2281 and 2357. (b) the diagram shows the results of an analysis of the nsp3 for predicted transmembrane helices using tmhmm program. the predicted tm domain designates the boundaries of the putative membrane-spanning regions a, b, c, d, and e. (c) sequence of the plp2-tm (amino acid residues 1610 to 2485) were analyzed for the prediction of transmembrane domains using phobius, tmhmm, hmmtop, sosui and tmpred programs. subjected the proteins expressed from wild-type pplp2-cen (kanjanahaluethai and baker, 2000) and two asparagine-toalanine substitution mutants to endo h treatment (fig. 5) . expressed proteins were radiolabeled with [ 35 s]-translabel, lysates were prepared, and subjected to immunoprecipitation with anti-v5 antibody as described in materials and methods. the plp2-cen protein was either untreated, or treated with endo h for 16 h, then analyzed for mobility by electrophoresis through a 5% polyacrylamide gel. we found that the untreated wild-type protein migrated at approximately 110 kda (lane 1). however, after digestion with endo h, the protein migrated at 108 kda (lane 2), consistent with the loss of one n-linked modification. the plp2-cen-n2357a protein with an asparagine-to-alanine substitution migrated more quickly than the wild type or n2281a protein, indicating that n2357 is the site modified by n-linked glycosylation (compare lanes 3-5). furthermore, proteins generated by endo h treatment of the pplp2-cen-n2357a and pplp2-cen-n2281a migrated more quickly than wild type plp2-cen (lanes 6-8). these results indicate that nsp3 is glycosylated at asparagine-2357. overall, these experiments demonstrate that nsp3-tm does have transmembrane and lumen sequences that can tether the plp2 domain to intracellular membranes. to further investigate the topology of nsp3-tm, we tested for sensitivity to proteinase k digestion. cytosolic domains are sensitive to proteinase k, whereas transmembrane and lumenal domains are protected from digestion with proteinase k. analysis of plp2-2485 revealed two major fragments of 28 kda and 10 kda protected from proteinase k digestion (fig. 6, lane 2) . we noted the predicted size of the tm domain from k2227-y2469 is 28.0 kda, and the predicted size of the k2227-i2308 (a and b helices) is 9.6 kda. analysis of plp2-2390 revealed two protected fragments of 19 kda and 10 kda, respectively (fig. 6, lane 4) . the predicted size of the k2227-s2390 fragment is 18.8 kda. by combining the results of the integral membrane assays (figs. 3 and 4) , the glycosylation assay (fig. 5) , and the proteinase k sensitivity assay (fig. 6a ), we were able to generate a topology model for nsp3-tm that is consistent with all the data (figs. 6b and c). our results indicate that nsp3-tm has four membrane-spanning sequences and two lumenal domains (fig. 6b ) and that the 10 kda fragment likely represents the proteinase k resistant fragment generated by the first two membrane-spanning sequences. our model is most similar to the predicted models generated by the tmhmm and sosui programs with the exception that the fourth predicted membrane-spanning sequence (which is the only domain not consistent in these two predictions) is not a membrane-spanning sequence, but instead remains lumenal, and the final membranespanning domain is in the reverse orientation. our results, and the results of others (hugle et al., 2001; miller et al., 2006) , demonstrate the importance of experimental validation of bioinformatics predictions of membrane-spanning sequences. for example, in the case of dengue virus type 2 nonstructural protein 4b, miller and co-workers found that two computerpredicted transmembrane helices were in fact lumenal and one was glycosylated (miller et al., 2006) . in addition, we note the value of using multiple programs to better estimate the complexity of the bioinformatic prediction. the differences in the predicted models could then be tested experimentally. here, we provide an initial model of nsp3-tm that should be refined by further experimentation. similar studies should also be performed to determine the topology of nsp4 and nsp6, the other coronavirus replicase products with multiple predicted transmembrane helices. finally, to determine if the tm domain of mhv nsp3 was sufficient to confer membrane association to a cytosolic protein, we appended nsp3-tm to egfp and determined the localization of the fusion protein using confocal microscopy (fig. 7) . egfp normally is distributed throughout the cell (fig. 7a) . we found that appending the mhv nsp3-tm sequence to egfp fig. 4 . membrane extraction experiments of the plp2 expressed protein products treated with triton x-100, urea, nacl or sodium carbonate solution ph 11.5. in vitro transcription and translation reactions of pplp2-2485, pplp2-2390 or pplp2-2258 were performed in the presence of cmm. subsequently, reaction mixtures were centrifuged to sediment cmm containing associated plp2 protein. the supernatant were removed and the pellets were resuspended in nte buffer or 0.5% triton x-100 (a), 4 m urea, 1 m nacl or 100 mm sodium carbonate solution ph 11.5 (b) and incubated for 20 min at 4°c. subsequently, membrane sedimentation analyses were performed as described under materials and methods. soluble (s) and pellet (p) fractions were applied in equivalent amounts and subjected to 12% sds-page analysis. quantitation was performed by phosphoimaging and values expressed in % were given at the bottom and depicted as bars. was sufficient to tether it to membranes, as shown by the intense, perinuclear localized signal (fig. 7b ). to determine if the nsp3-tm domain is retained in the er membranes or is transported through the medial golgi, we radiolabeled the efgp-nsp3tm protein in transfected cells, immunoprecipitated the protein and subjected the immunoprecipitated products to endo h. we found that efgp-nsp3tm is sensitive to treatment with endo h, indicating that the protein is retained in the er and does not pass through the medial golgi. thus, the nsp3-tm domain is sufficient to confer membrane-localization and retention in the er. these studies are in agreement with our previous findings showing that the tm domain of sars-cov nsp3 (previously termed the hd) confers membrane association of egfp (harcourt et al., 2004) . in summary, using biochemical fractionation and proteinase k protection assays, we show that nsp3-tm likely has four membrane-spanning domains, and that lumenal residue asparagine-2357 is modified by glycosylation. furthermore, we found the region nsp3-tm domain is required for efficient mhv plp2 process activity at cleavage site 3 in the polyprotein. why does mhv plp2 require membrane association for proteolytic processing of the plp2 cleavage site? one possible explanation is that membrane-tethering brings plp2 into close proximity with a membrane-associated substrate. it is also possible that the nsp3-tm membrane tether is important for anchoring the replicase complex to intracellular membranes. bioinformatic analysis of the nsp3 of 10 other coronaviruses revealed that the membrane-spanning features of nsp3-tm are conserved in all viruses (fig. 8 , analysis using the tmhmm program is shown as an example), even though the amino acid identity is relatively low (18-32% identity within nsp3). therefore, the tm domain is likely to be important for both plp2 activity and assembly of the replication complex. further studies will be required to determine the precise topology of the tm domain in other coronaviruses, and if the lumenal sequences in nsp3-tm play any role in interacting with host factors during viral replication. hela cells expressing the mhv receptor, hela-mhvr cells (gallagher, 1996) were used for all transfection experiments. the cells were grown in dulbecco's modified eagle's medium supplemented with 10% fetal bovine serum (invitrogen, carlsbad, ca), 0.5% penicillin/streptomycin, 2% glutamine, and 5 mm sodium n-2-hydroxyethylpiperazine-n′-2ethanesulfonic acid, ph 7.4. recombinant plasmid dna constructs expressing the plp2 coding region were generated using specific primers (listed in table 1 ) to amplify the designated region from the parental plasmid pplp2-cen (kanjanahaluethai and baker, 2000) . the region of interest was generated by pcr amplification using la-taq polymerase according to the manufacturer's instructions (clontech, palo alto, ca). the amplified region was then digested with restriction enzymes bamhi and xhoi and ligated into the corresponding sites in the pcdna3.1/v5-his expression vector (stratagene, la jolla, ca) using t4 ligase (new england biolabs). the ligated dna product was transformed into xl-1 blue competent cells according to the manufacturer's instructions (stratagene), except that the bacteria were grown at 25°c. hela-mhvr cells were infected with a recombinant vaccinia virus expressing the bacteriophage t7 polymerase (vtf7-3) at a multiplicity of infection of 10. then, infected cells were co-transfected with recombinant plasmid dnas encoding the mhv-jhm indicated protease domain and the substrate using lipofectamine according to manufacturer's instruction as previously described (fuerst et al., 1986; kanjanahaluethai and baker, 2000) . newly synthesized proteins were metabolically labeled with 50 μci/ml [ 35 s]-translabel (icn, costa mesa, ca) from 5.5 to 10.5 h post-infection (hpi). to harvest the cells, radioactive labeled cells were washed with phosphate buffered saline (pbs), and cell lysates were prepared by scraping the cells in lysis buffer a [4% sds, 3% dtt, 40% glycerol and 0.065 m tris, ph 6.8 (schiller et al., 1998) ]. the lysates were either used directly for immunoprecipitation assays or stored at − 70°c for future studies. radiolabeled cell lysate was diluted in 1.0 ml ripa buffer [0.5% triton x-100, 0.1% sds, 300 mm nacl, 4 mm edta, and 50 mm tris-hcl, ph 7.4 (schiller et al., 1998) ] and subjected to immunoprecipitation with anti-v5 monoclonal antibody (invitrogen) and protein-a sepharose beads (amersham biosciences, piscataway, nj). the immunoprecipitated products were eluted with 2× laemmli sample buffer, incubated at 30°c for 30 min, and analyzed by electrophoresis on a 5.0-12.5% gradient polyacrylamide gel containing 0.1% sds. following electrophoresis, the gel was fixed in 25% methanol-10% acetic acid, enhanced with amplify (amersham biosciences) for 60 min, dried, and exposed to kodak x-ray film at − 70°c. site-directed mutagenesis of putative glycosylation sites in mhv-jhm nsp3-tm domain plasmid dna pplp2-cen which encompasses mhv-jhm gene 1 amino acid residues 1525-2485 (kanjanahaluethai and baker, 2000) was subjected to site-directed mutagenesis at positions 7055 and 7056 for pplp2-n2281a and positions 7283 and 7284 for pplp2-n2357a using synthetic oligonucleotides with mismatches encoding specific nucleotides changes as shown in table 1 . mutagenesis was performed according to the manufacturer's instructions (quickchange site-directed mutagenesis; stratagene), and as previously described (kanjanahaluethai and baker, 2000) . mutations were confirmed by dna sequence analysis. the tnt t7-coupled reticulocyte lysate system (promega, madison, wi) was used according to the manufacturer's instructions. the recombinant plasmid dna encoding the designated plp2 region was linearized by digestion with pmei. in vitro transcription and translation was performed for 90 min at 30°c in the presence of 0.8 μci of [ 35 s]-translabel per ml in a volume of 25 μl. where indicated, 1.0 μl of cmm (promega) was added prior to the incubation. for analysis of membrane association, the products of in vitro transcription and translation were centrifuged at 14,000 rpm for 10 min. the supernatant was removed, the pellet that may contain aggregated or membrane-associated protein was suspended in 2× laemmli sample buffer, heated at 95°c for 5 min, and both fractions were analyzed by sds-page and subjected to autoradiography. protection of translation products by microsomal membranes was examined by digestion with proteinase k (hugle et al., 2001) . following translation, reaction mixtures (after incubation with rnase a) were adjusted to 0.5 mg/ml of proteinase k (roche, indianapolis, in) and incubated for 30 min on ice. proteinase k digestion was terminated by addition of phenylmethylsulfonyl fluoride to 1 mg/ml and incubation was continued for 5 min on ice. a portion of each reaction mixture (generally 4 μl) was mixed with 40 μl of 2× laemmli sample buffer, heated at 95°c for 5 min, analyzed by 15% sds-page and subjected to autoradiography. for endo h treatment, lysates from vtf7.3-infected and pplp2-tm transfected cells were prepared and subjected to immunoprecipitation as described above. protein-a sepharoseantibody-antigen complexes were washed once in ripa buffer and endo h treatment was performed as suggested by the manufacturer (roche). briefly, the complexes were resuspended in 20 μl of 50 mm sodium phosphate buffer, ph 6.0, and incubated in the presence or absence of a final concentration of 1 unit/μl of endo h for 16 h at 37°c. following the incubation, 25 μl of 2× laemmli sample buffer was added to each sample, mixed, and incubated for 30 min at 37°c. the sepharose beads were pelleted by a brief, high-speed spin in a microfuge, and the supernatant loaded directly for analysis by 12% sds-page and subjected to autoradiography. for membrane extraction experiments, the pellets from 15 μl in vitro transcription-translation reactions performed in the presence of cmm were resuspended in 40 μl nte buffer, 0.5% triton x-100, 4 m urea, 1 m nacl, or 100 mm sodium carbonate (ph 11.5) and incubated for 20 min at 4°c (hugle et al., 2001) . subsequently, supernatant and pellet fractions were separated by centrifugation at 14,000 rpm for 10 min, and analyzed by sds-page and autoradiography. quantitation was performed by using phosphoimaging analysis. the putative transmembrane domain 1 region of mhv-jhm nsp3 (nt 6896 to 7648) was pcr amplified from the pplp2-cen (kanjanahaluethai and baker, 2000) with primers b410 and b411 (table 1) , cloned into the mammalian expression vector for egfp, pegfp-c1 (bd biosciences), and designated pegfp-nsp3tm. the plasmid dna encoding egfp or egfp-nsp3tm was transfected into hela-mhvr cells in 8well chamber culture slides with lipofectamine according to manufacturer's instructions, for 48 h. expression of egfp and egfp-nsp3tm fusion protein products was detected by confocal microscopy (zeiss lsm 510 lazer-scanning confocal microscope). egfp-nsp3tm was amplified by primers zcp1 and zcp2 (table 1) using pegfp-nsp3tm as template, and then cloned into bamhi and xbai sites of pcdna3.1/v5-hisb (invitrogen) to generate the construct of pcdna3.1-egfp-nsp3tm. the plasmid dna was expressed via the vaccinia virus-t7 expression system, proteins radiolabeled with 35 stranslabel, cell lysates were subjected to immunoprecipitation with anti-v5 antibody, and products were either incubated with endo h or buffer alone, and analyzed by electrophoresis on 10% sds-page. the papain-like protease of severe acute respiratory syndrome coronavirus has deubiquitinating activity phase separation of integral membrane proteins in triton x-114 solution an amino-terminal amphipathic alpha-helix mediates membrane association of the hepatitis c virus nonstructural protein 5a coronavirus genome structure and replication an efficient ribosomal frame-shifting signal in the polymerase-encoding region of the coronavirus ibv eukaryotic transientexpression system based on recombinant vaccinia virus that synthesizes bacteriophage t7 rna polymerase murine coronavirus membrane fusion is blocked by modification of thiols buried within the spike protein rna replication of mouse hepatitis virus takes place at double-membrane vesicles identification of severe acute respiratory syndrome coronavirus replicase products and characterization of papain-like protease activity intracellular functions of n-linked glycans the hepatitis c virus nonstructural protein 4b is an integral endoplasmic reticulum membrane protein identification of mouse hepatitis virus papain-like proteinase 2 activity identification of the murine coronavirus mp1 cleavage site recognized by papain-like proteinase 2 the complete sequence (22 kilobases) of murine coronavirus gene 1 encoding the putative proteases and rna polymerase the papain-like protease from the severe acute respiratory syndrome coronavirus is a deubiquitinating enzyme subcellular localization and membrane topology of the dengue virus type 2 non-structural protein 4b open reading frame 1a-encoded subunits of the arterivirus replicase induce endoplasmic reticulum-derived double-membrane vesicles which carry the viral replication complex severe acute respiratory syndrome coronavirus papain-like protease: structure of a viral deubiquitinating enzyme structural basis of severe acute respiratory syndrome coronavirus adp-ribose-1ʺ-phosphate dephosphorylation by a conserved domain of nsp3 coronavirus transcription: a perspective processing of the coronavirus mhv-jhm polymerase polyprotein: identification of precursors and proteolytic products spanning 400 kilodaltons of orf1a ultrastructure and origin of membrane vesicles associated with the severe acute respiratory syndrome coronavirus replication complex viral replicase gene products suffice for coronavirus discontinuous transcription identification of a new human coronavirus characterization and complete genome sequence of a novel coronavirus, coronavirus hku1, from patients with pneumonia the coronavirus replicase the autocatalytic release of a putative rna virus transcription factor from its polyprotein precursor involves two paralogous papain-like proteases that cleave the same peptide bond we thank nicole kreuziger, kari severson and ami knop ullrich for excellent technical assistance, and dr. alexander gorbalenya, leiden university medical center for useful suggestions during the course of this work. this research was supported by public health service research grants ai 45798 and hhsn2662040035c. key: cord-319754-5isw53wl authors: balgoma, david; gil-de-gómez, luis; montero, olimpio title: lipidomics issues on human positive ssrna virus infection: an update date: 2020-08-31 journal: metabolites doi: 10.3390/metabo10090356 sha: doc_id: 319754 cord_uid: 5isw53wl the pathogenic mechanisms underlying the biology and biochemistry of viral infections are known to depend on the lipid metabolism of infected cells. from a lipidomics viewpoint, there are a variety of mechanisms involving virus infection that encompass virus entry, the disturbance of host cell lipid metabolism, and the role played by diverse lipids in regard to the infection effectiveness. all these aspects have currently been tackled separately as independent issues and focused on the function of proteins. here, we review the role of cholesterol and other lipids in ssrna+ infection. the ongoing covid-19 pandemic is developing (july 2020) worldwide with devastating global consequences, both for social organization and healthcare systems. covid-19 illness is brought about by infection with the severe acute respiratory syndrome coronavirus sars-cov-2 [1, 2] , which is an enveloped positive single-stranded rna virus (ssrna+) [3] . the most abundant studies related to human diseases induced by ssrna-positive viruses refer to picornaviridae, coronaviridae, and flaviviridae [4] . this impact in a short time span has brought the biology and biochemistry of viral infection mechanisms to reach momentum. the infection mechanisms have been described for diverse unrelated viral families [5] , with the majority of them being dna viruses. within picornaviridae, coronaviridae, and flaviviridae, rhino and poliovirus (picornaviridae), sars-cov, middle east respiratory syndrome coronavirus (mers-cov), hepatitis c virus (hcv), west nile virus (wnv) and dengue virus (denv) fall within the viruses whose life cycle biology is better known. nonetheless, knowledge regarding virus entry mechanisms and other related features of the virus life cycle has been gained from the research on the influenza virus from the orthomysoviridae family and the human immunodeficiency virus from the retroviridae family. consequently, these and other unrelated viruses will be also considered in this review from the point of view of the different aspects that affect the lipidomics of the viral infection. all ssrna+ viruses initially infect mammal cells through the interaction of virus proteins with any given host cell protein. further fusion of the virus and host cell membranes is required for the viral genetic material to get into the cell. once inside the cell, the genomic and subgenomic viral rnas are translated into the virus proteins; these then lead the virus replication, which is a process that involves modulation of the host cell lipid metabolism [3, 5, 6] . consequently, along with other features, current lipid studies about the aforementioned virus infection focus their research on membrane fusion and modulation of the lipid metabolism of the host cell. these two processes are considered separated disciplines of the infection. the fight against the virus infection encompasses primarily the inhibition of the binding of the viral spike protein to the host cell's receptor protein. consequently, most of the current research focuses on the role played by viral proteins but the lipid environment, where the proteins carry out their function and regulation, is considered secondarily [7] . nevertheless, improving the knowledge on how the lipids are involved in the mechanisms of infection may provide clues to develop treatments and better counteract the virus-induced pathology [3] . to fill this gap, here, we review the main aspects regarding the lipidome regulation of the viral infection mechanism by ssrna+ viruses. the initial step in virus infection is the binding of any viral structural glycoprotein to a receptor of the host cell. the spike protein accounts for such function in coronaviruses (covs) and other enveloped viruses. after the virus is attached to the host cell protein, the process of membrane fusion starts to get the viral genome into the host cell. this process implies viral envelope and host cell membrane fusion, for which an energetically cost-effective barrier must be overcome. for example, in coronavirus, membrane fusion is driven by the fusion peptide (class i), which is localized within the spike protein (s protein) and becomes active after cleavage of the s protein at specific sites by host proteases or ph-dependent mechanisms [4, 6, 8] . a different mechanism of attachment and endocytosis drives the virus entry in the case of hcv. this mechanism is more complex than that of coronaviruses and involves interaction of the virus envelope e1 and e2 proteins (class ii fusion loop) with several host cell proteins [9] [10] [11] . however, a membrane fusion-driven pore is also required in hcv to deliver the viral genetic material into the host cell cytoplasm. two main mechanisms of membrane fusion have been described: viral endocytosis by host cell membrane (endocytic pathway), and both viral and host cell plasma membrane fusion (non-endocytic pathway). after docking of the virus to the attachment factor or the receptor on the host cell surface, the virus may internalize its genomic material or the entire particle [12] [13] [14] . the non-endocytic pathway encompasses the direct delivery of the genetic material through a pore formed in the cell membrane by the induction of viral proteins at neutral ph. this pathway is typical of non-enveloped viruses. the endocytic pathway is more complex and harnesses the host cell endocytosis machinery for the virus internalization. three main ways have been described in the endocytic pathway, namely: the clathrin-mediated endocytosis (cme), the caveolae-mediated endocytosis (cavme), and the macropycnocytosis. the best-known endocytic mechanism is the clathrin-mediated endocytosis. the cme is used by small to intermediate-sized viruses. this mechanism uses vesicles coated by the protein clathrin, which forms a polyhedral lattice that surrounds the cell membrane-derived vesicle where the virus is internalized into the cell cytoplasm through the early endosomes. clathrin coating is coordinated by the adaptor protein (ap-2) and other adaptors; it is less commonly ap-independent. the protein dynamin is involved in regulating the clathrin-coated vesicle (ccv) formation as well as its scission from the membrane. some viruses proceed to membrane fusion at this stage for releasing their genome into the cytoplasm. the early endosomes have a ph of about 6.0 to 6.5; therefore, it is considered that membrane fusion is not strictly ph-dependent. other viruses need a lower ph for the membrane fusion to be effective; thus, it is considered ph-dependent. a further step leading to endosome maturation to become late endosomes with a ph of about 5 has to proceed before the membrane fusion takes place and the genetic material is delivered to the cytoplasm. sequential acidification of the virus proteins from the early to late endosomes has also been suggested through the self-organized endosomal network. maturation of the early endosomes to late endosomes and trafficking between them is controled by the rab proteins, which are members of the ras superfamily of small g proteins. subsets of rab proteins differ between the early and late endosomes, and the rab subset change is accompanied for by formation of the phosphoinositide pi(3,5)p 2 from the precursor pi(3)p. regarding lipid composition, early endosome membrane lipids are primarily composed of unsaturated and short alkyl chains, whereas long and saturated alkyl chains, such as in gangliosides, are predominant in the membrane lipids of late endosmes. membrane fusion in some viruses requires a further step in which late endosomes are fused with lysosomes, this step giving rise to the late endosome/lysosome pathway. cholesterol depletion driven by its synthesis inhibition or extracting agents as methyl-β-cyclodextran (mβcd) is used to assess whether the virus entry takes place through the caveolae/raft endocytosis. this pathway in less known and encompasses the formation of initial endocytic vesicles enriched in cholesterol from lipid-rafts, with complex signaling routes that involve the activity of tyrosine kinases and phosphatases. thereafter, the cargo is transported to the endoplasmic reticulum (er) through early and late endosomes. most of the viruses using this endocytic pathway have different gangliosides as receptors, mainly gm1, which has a high concentration in caveolae. polyomavirus, which are non-enveloped dna viruses that replicate in the nucleus, use preferently this endocytic pathway, but picornaviruses and the coronavirus hcov-229e have also been reported to internalize through the caveolae-mediated endocytosis [15] . macropinocytosis is a phagocytic-like mechanism of virus entry that is currently utilized by the cell to internalized fluids; it is dependent on actin and implies the actin cytoskeleton rearrangement to enable internalization of the virus particle [14] . macropinocytic vacuoles (macropinosomes) are formed after membrane ruffles fold to reach at its end the membrane again, and the vacuole is closed through self-membrane fusion. these vacuoles containing the viral particle may traffick afterwards through the early and late endosome network. macropinocytosis is common to large-sized viruses. however, recent work [16] has shown that ebola virus (ebov) may use a macropinocytosis-like process to entry the host cell in a clathrin, caveolae, and dynamin-independent manner, but dependent of actin and a lipid raft. conversely, this virus may use as well an endocytic pathway that is dependent on clathrin, caveolae, and dynamin. which endocytic route is used by this virus depends on the host cell type. description of the current methodologies used to study the entry route by viruses can be found in reference [14] . some viruses may use different entry mechanisms, this feature being likely dependent upon the membrane lipid composition of the host cell they infect as well as the particular cell surface factor attachment used. cme is the entry route currently used by hcv, hiv-1, ebov, rotaviruses, and some coronaviruses, even though other routes can also be used as for ebov (see above). a reaction between clathrin and actin seems to be necessary for the effective entry of these viruses. regulation by microtubules of the cme has been reported for flaviviruses. denv, wnv, and semliki forest virus (sfv, alphavirus family, togaviridae) have been found to depend on early endosomes (rab5 protein marker) for entry but not late endosomes (rab7 protein marker), which means that they do not have strict low ph requirements or depend on different acidification mechanisms for membrane fusion. conversely, influenza avian virus (iav) needs both early and late endosomes to entry, thus reflecting low ph dependence for membrane fusion. marburg virus (marv) may use for internalization a cme through the endo/lysosomal pathway. coronaviruses differ in their internalization mechanism among strains. thus, while hcov-229e is known to use the cav-me route, sars-covs use an endocytic pathway that is clathrin-and caveolae-independent but receptor and ph-sensitive, with lipid rafts playing an essential role [17] . this endocytic mechanism implies internalization of the receptor protein angiotensin-convering enzyme 2 (ace2) along with the spike protein into the early endosomes, but the receptor is afterward recycled to the membrane via lysosomes. nonetheless, previous studies showed that sars-cov could enter through a ph-independent direct membrane fusion as it could infect cells that do not express ace2, such as enterocytes and hepatocytes [18] . recent research on the virus sars-cov-2 points to ph-independent direct cell and viral membrane fusion, which is a process that is driven by the subunit s2 of the spike protein after cleavage by the cellular serine protease tmprss2 [19] . on the contrary, the infectious bronchitis virus (ibv), a gamma-coronavirus, was reported to use the cme pathway to entry, with vesicle scission being mediated by gtpase dynamin 1, and a dependence on low ph and lipid raft localization of the receptor. tracking of the virus trip inside the cell was followed by using diverse inhibitors, cholesterol sequestering agents, and virus particles labeled with fluorescent markers. membrane fusion takes place at the late endosome/lysosome step of the endocytic pathway, with deep rearrangement of the host cell cytoskeleton being induced by the endosomal viral cargo [15] . accordingly, viruses may sequester on their own profit the diverse endocytic pathways that are currently used by the host cell, but variability of the proteins and even the general mechanisms may also exist as a consequence of virus specifity. membrane fusion has been described to proceed through the catalytic action of three different types of fusion peptides or fusion loops of class i, ii, or iii. these proteins afford the free energy necessary to overcome through conformational changes the kinetic barrier due to repulsive hydration strength. most of the knowledge on the viral and host membrane fusion has been gained from the influenza virus and its type i fusion peptide hemagglutinin. a detailed description of the three fusion peptide-guided mechanisms involved in membrane fusion has been previously reviewed in [20] [21] [22] . bringing the viral and the host membranes closer enough (c.a. 20 å) for inducing the membrane fusion is a process that entails membrane curvature and changes in the lipid bilayer phase. they are driven by the insertion of a hydrophobic region of the fusion peptide, which requires dehydration of the inter-membrane space. nonetheless, from experiments with no-protein fusogens, such as polyethilen glycol, it seems that membrane curvature stabilization is not a key player in membrane pore opening. the calculated displacement of lipids in the outer leaflet of the host membrane accounts for no more than 10% of the membrane area (about 3500 å 2 ), which does not represent a substantial energetic demand [21] . this energetic burden has been demonstrated to be afforded by the cooperation of three fusion peptides in influenza virus membrane fusion [23] , whereas two adjacent trimers of the fusion protein are required in west nile virus [24] . this result points to the fact that the viral membrane curvature may not actually impose a constraint for proceeding to the hemifusion step and the formation of a steep curvature stalk, where the outer leafleats are merged. by the mesurement of electron density profiles through x-ray reflectivity in stalks formed from bilayers in a lamellar state with different lipid compositions, aeffner et al. [25] determined that the inter-bilayer separation should attain 9.0 ± 0.5 å in order to facilitate dehydration and promote stalk formation. these authors also found that increasing the relative proportion of nonbilayer-forming, cone-shaped lipids, such as glycerophosphoethanolamine or cholesterol, favored the stalk formation by reducing the hydration energy barrier and, possibly, by contributing with their intrinsic negative curvature. as well, the energy required for dehydration was, in this study, found to decrease with the length of the acyl chains of the glycerophospholipids. however, the hemifusion stalk stage was not detected by gui et al. [26] using fluorescence and electron microscopy. the results of this study show that such a stage might be an unstable intermediate that is quickly resolved toward the postfusion stage. contrarily, localized point-like contacts were abundantly visualized in this study, where the dimples formed in the target membrane, about 5 nm wide, were drawn toward the virus surface. they were able to detect up to well-resolved four types of virus-target membrane contacts at ph 5.5 and 5.25 using liposomes of dioleylglycerophosphocholine, dopc, with 20% cholesterol. at the lowest ph, a tight contact of the two membranes through an extended length of about 100 nm (catalogued by the authors as type iii) was the predominant interaction, whose abundance was increased by about 3-fold in cholesterol-containing liposomes in comparison to only dopc liposomes. using synthetic peptides that resemble the fusion peptide hemagglutinin and electron spin resonance (esr), ge and freed [27] found that the most relevant effect of the synthetic fusion peptides was the induction of highly ordered membrane domains, which came motivated by virtue of electrostatic interactions between the peptide and negatively charged phospholipid headgroups. a similar effect was reported for two putative fusion peptides enclosed in the spike glycoprotein of sars-cov-1. it was found in this study that the inner water content in the lipid bilayer was dropped by the insertion of the fusion peptide as a consequence of increased lipid packing, but only in membranes containing negatively charged lipids, whereas the water content was only slightly altered in zwitterionic dipalmitoylglycerophosphocholine (dppc) liposomes [28] . additionally, the fusion peptides created opposing curvature stresses in the highly bended membranes containing nonbilayer-forming phospholipids. however, previous studies had pointed out that interaction with the lipid headgroups is not an essential factor in reaching the membrane hemifusion state [21, 29] . in sars-cov, the possibility of existing two fusion peptides that act in coordination has been suggested [7] ; one of the peptides would promote the dehydration process, while the other one would act in modifying/disturbing the lipid organization within the target membrane [26, 28, 30] . hence, the catalytic role of the fusion peptide(s) is likely to tackle three properties of the target membrane in the virus entry machinery: (i) dehydration of the intermembrane space for the fusing membranes coming into the required proximity, (ii) to promote negative curvature to form the hemifusion stalk, and (iii) to alter the lipid packing density, which will be generated in the highly curved local dimples of the stalk [22, 28] . the effectiveness of these three processes is likely to depend upon the membrane lipid composition. further research is devoted to this issue, and new clues are expected to come from electron and fluorescence microscopy [31] . since the dominant phospholipid in the outer leaflet of most membranes is the bilayer-forming, positive charged diacylglycerophosphosphocholine (pc), the idea was raised that the viral docking to the receptor on the target cell and, consequently, the membrane fusion were likely to take place at specific microdomains with particular lipid composition, the so-called lipid rafts [27, [32] [33] [34] [35] [36] . a special characteristic of the lipid rafts is the high content of cholesterol [37] [38] [39] . even though a high content of sphingolipids and gangliosides is also a defining characteristic of lipid rafts (figure 1 ), direct in vivo visualization still remains unresolved [39] . metabolites 2020, 10, x for peer review 5 of 22 altered in zwitterionic dipalmitoylglycerophosphocholine (dppc) liposomes [28] . additionally, the fusion peptides created opposing curvature stresses in the highly bended membranes containing nonbilayer-forming phospholipids. however, previous studies had pointed out that interaction with the lipid headgroups is not an essential factor in reaching the membrane hemifusion state [21, 29] . in sars-cov, the possibility of existing two fusion peptides that act in coordination has been suggested [7] ; one of the peptides would promote the dehydration process, while the other one would act in modifying/disturbing the lipid organization within the target membrane [26, 28, 30] . hence, the catalytic role of the fusion peptide(s) is likely to tackle three properties of the target membrane in the virus entry machinery: (i) dehydration of the intermembrane space for the fusing membranes coming into the required proximity, (ii) to promote negative curvature to form the hemifusion stalk, and (iii) to alter the lipid packing density, which will be generated in the highly curved local dimples of the stalk [22, 28] . the effectiveness of these three processes is likely to depend upon the membrane lipid composition. further research is devoted to this issue, and new clues are expected to come from electron and fluorescence microscopy [31] . since the dominant phospholipid in the outer leaflet of most membranes is the bilayer-forming, positive charged diacylglycerophosphosphocholine (pc), the idea was raised that the viral docking to the receptor on the target cell and, consequently, the membrane fusion were likely to take place at specific microdomains with particular lipid composition, the so-called lipid rafts [27, [32] [33] [34] [35] [36] . a special characteristic of the lipid rafts is the high content of cholesterol [37] [38] [39] . even though a high content of sphingolipids and gangliosides is also a defining characteristic of lipid rafts (figure 1 ), direct in vivo visualization still remains unresolved [39] . an unexplored possibility is that rafts do not have a permanent localized existence, but they arise under the induction of certain proteins such as the hydrophobic insert of the viral fusion peptide or the fusion loop. this fact might be also responsible for bringing negatively charged lipids from the inner leaflet of the bilayer to its outer leaflet by flip-flop mechanisms. this hypothesis would explain the promotion of virus entry by the interaction of the fusion peptide with the negatively charged phospholipid headgroups [25, 27] as well as the kinetics of the membrane fusion [25] . a number of studies have shown that the hemifusion step and pore widening are sped up after increasing the relative concentration of cholesterol in the bilayer composition, whereas either the depletion of cholesterol in the cell culture medium or the inhibition of cholesterol synthesis by an unexplored possibility is that rafts do not have a permanent localized existence, but they arise under the induction of certain proteins such as the hydrophobic insert of the viral fusion peptide or the fusion loop. this fact might be also responsible for bringing negatively charged lipids from the inner leaflet of the bilayer to its outer leaflet by flip-flop mechanisms. this hypothesis would explain the promotion of virus entry by the interaction of the fusion peptide with the negatively charged phospholipid headgroups [25, 27] as well as the kinetics of the membrane fusion [25] . a number of studies have shown that the hemifusion step and pore widening are sped up after increasing the relative concentration of cholesterol in the bilayer composition, whereas either the depletion of cholesterol in the cell culture medium or the inhibition of cholesterol synthesis by statins was able to halt the viral infection at the virus entry step [26] [27] [28] 40, 41] . the effect of cholesterol on promoting membrane merging has also been observed for bis-(monoacylglycero)-phosphate (bmp) [26] . this particular phospholipid was shown to be strictly necessary for dengue virus (denv) entry even at low endosomal ph [42] . as pointed out above, the exact role played by cholesterol is not known in detail, but its intrinsic negative curvature seems to be an essential characteristic in promoting the stalk formation during virus entry. however, a recent study shows that the cholesterol action is likely to involve a direct influence on the oligomeric state of the fusion peptide after insertion into the host cell membrane, as well as on the effects of the fusion peptide on the membrane reorganization and dynamics [43] . in another recent study, a new lipid-label-free methodology was used to measure the kinetics of influenza virus infection [44] . according to the results of this study, cholesterol is able to augment the efficiency of membrane fusion in a receptor binding-independent manner. nevertheless, the rate of membrane fusion was not altered. these results led the authors to conclude that the positive effect of cholesterol in membrane lipid mixing is related to its capability to induce negative curvature. since membrane mixing was achieved in this latter study without binding of the spike protein of the influenza virus to the host cell receptor, the catalytic effect of the fusion peptide might proceed in an independent way in this virus. cleavage of the spike protein in sars-cov-1 does not seem to be also necessary for the fusion peptide to become fusogenic, but rearrangement of disulfide bridges in the s1 peptide after receptor binding are likely involved in the conformational changes driving the fusion mechanism [43, 45] . contrary to these latter results, which point to the fact that membrane fusion is independent of viral protein attachment to its receptor, guo et al. reported lipid raft-dependent viral protein binding with the suppression of viral infection if the lipid rafts were disrupted with cholesterol drug-induced depletion; lipid rafts, as recognized by the caveolin-1 marker, were the membrane domain where structural proteins of the infectious bronchitis virus (ibv) co-localized but the nonstructural proteins did not [35] . the question regarding whether the lipid-raft domains may serve as platforms to concentrate the proteins required for viral entry and, even though some evidence exists, to activate signaling pathways inside the host cell still remains unsolved. sphingomyelins (sms) are also common lipids found in lipid rafts, which contribute to make these membrane microdomains detergent-resistant [34] . the structure of a representative of this lipid class is illustrated in figure 2 . the ganglioside gm1, a sphingolipid, is used as a marker of lipid rafts [34] . sphingolipids (sls) promote to an extent higher than chol the liquid-ordered phase in the outer leaflet of the membrane bilayer because of the long saturated acyl chains they currently contain (the r group in figure 2 may extend to a length of up to 26 c), in addition to their capability to form intermolecular hydrogen bonds [46] . a relevant function of the lipid rafts has been suggested to be the connection between the events outside the cell with the pathways inside the cell, thus acting as 'signaling platforms'. with the aim of this function to be properly accomplished, the lipid rafts would act as concentrators of specific transmembrane proteins, mainly receptors, whose compatibility with the membrane phase would determine their selectivity. thus, sls would account for a role in connecting the outer leaflet with the inner leaflet through their long saturated acyl chains. regarding virus entry, research has been primarily focused toward the role played by cholesterol, but a number of studies have also enlightened the sm influence on this early step of viral infection. the displacement of cholesterol by sms and the other way round has been demonstrated, with the bilayer liquid-ordered phase being preferentially determined by the interaction between sm and cholesterol. this interaction would be controlled to a certain extent by the intracellular actin meshwork, which would also be responsible for the compartmentalization of the membrane into lipid-specific domains [47] . furthermore, the actin role is possibly extended to the routing of the viral genomic material toward the replication place inside the host cell. the hydrolysis of sm by sphingomyelinases to render the corresponding ceramide in specific membrane domains is proposed to regulate the dynamics of cholesterol in the cell membrane, the effect of such regulation being the progressive disassembly of cholesterol from the liquid-ordered phase and its displacement. since the interaction of ceramides with cholesterol has been suggested to be an apoptotic regulator, it can be expected that viral proteins would act in recruiting cholesterol to displace the ceramide and to avoid the programmed cell death. this fact is added to the other characteristics conferred by cholesterol to the membrane mechanical properties discussed above. to study the influence of ceramide on membrane fusion during semliki forest virus (sfv, alphavirus family, togaviridae) infection, ceramide analogs have been used [48] . according to this experiment, in which cholesterol-containing pc plus pe liposomes were used, the roles played by the 3-hydroxyl group and the 4,5-trans carbon-carbon double bond of the sphingosine backbone ( figure 2 ) were found to be essential in the fusion process. in additon, ceramide was the simplest sl to accomplish this significant contribution in mediating the fusion, independently of the length of the acyl chain. more recently, a ca 2+ -dependent pathway of infection by the rubella virus (ruv, rubivirus family, togaviridae) was demonstrated to proceed through direct binding of the fusion loop in the viral e1 protein to sm/cholesterol-enriched membranes [49] . however, the treatment of host cells with sphingomyelinase proved that sm is exclusively required for viral entry but is not required for the further steps of viral replication. sm in the host cell membrane and acid sphingomyelinase (asmase) activity have also been shown to be required by the ebola virus (ebov), a negative single-stranded rna virus belonging to the filoviridae family, to get into the host cell. the asmase activity renders ceramide that provoques raft enlargement and membrane invagination [50] . this study also showed that the virus was able to recruit both sm and asmase to the raft where the viral attachment was happening. conversely, bovine herpesvirus 1 (bohv-1, herpesviridae family) seems to require sm in the virus envelope but does not in the host cell [51] . the role played by ceramides is contradictory as they may enhance or inhibit virus replication, but this sl action seems to be related to the viral replication phase rather than to the internalization phase [52] [53] [54] . in virus using the endocytic pathway, similar to the influenza virus or the ebola virus, it has been shown that activity of glucosylceramidase (gba) is required for viral entry and membrane fusion through the regulation of endocytosis, but in a virus-dependent manner. it was also shown that trafficking of the epidermal growth factor (egf) to late endosomes was impaired in gba-knockout cells, which negatively affects the virus entry through spoiling the endocytic pathway [55] . indeed, co-clustering of the ha attachment factor and egf in submicrometer domains that overlap partially has been reported recently [56] . accordingly, there is evidence that sls have a function in enveloped ssrna viruses at the early stage of infection that accounts for the viral entry modulation, but further research is still necessary to unveil the exact mechanisms of sl reactions. metabolites 2020, 10, x for peer review 7 of 22 displace the ceramide and to avoid the programmed cell death. this fact is added to the other characteristics conferred by cholesterol to the membrane mechanical properties discussed above. to study the influence of ceramide on membrane fusion during semliki forest virus (sfv, alphavirus family, togaviridae) infection, ceramide analogs have been used [48] . according to this experiment, in which cholesterol-containing pc plus pe liposomes were used, the roles played by the 3-hydroxyl group and the 4,5-trans carbon-carbon double bond of the sphingosine backbone ( figure 2 ) were found to be essential in the fusion process. in additon, ceramide was the simplest sl to accomplish this significant contribution in mediating the fusion, independently of the length of the acyl chain. more recently, a ca 2+ -dependent pathway of infection by the rubella virus (ruv, rubivirus family, togaviridae) was demonstrated to proceed through direct binding of the fusion loop in the viral e1 protein to sm/cholesterol-enriched membranes [49] . however, the treatment of host cells with sphingomyelinase proved that sm is exclusively required for viral entry but is not required for the further steps of viral replication. sm in the host cell membrane and acid sphingomyelinase (asmase) activity have also been shown to be required by the ebola virus (ebov), a negative single-stranded rna virus belonging to the filoviridae family, to get into the host cell. the asmase activity renders ceramide that provoques raft enlargement and membrane invagination [50] . this study also showed that the virus was able to recruit both sm and asmase to the raft where the viral attachment was happening. conversely, bovine herpesvirus 1 (bohv-1, herpesviridae family) seems to require sm in the virus envelope but does not in the host cell [51] . the role played by ceramides is contradictory as they may enhance or inhibit virus replication, but this sl action seems to be related to the viral replication phase rather than to the internalization phase [52] [53] [54] . in virus using the endocytic pathway, similar to the influenza virus or the ebola virus, it has been shown that activity of glucosylceramidase (gba) is required for viral entry and membrane fusion through the regulation of endocytosis, but in a virus-dependent manner. it was also shown that trafficking of the epidermal growth factor (egf) to late endosomes was impaired in gba-knockout cells, which negatively affects the virus entry through spoiling the endocytic pathway [55] . indeed, co-clustering of the ha attachment factor and egf in submicrometer domains that overlap partially has been reported recently [56] . accordingly, there is evidence that sls have a function in enveloped ssrna viruses at the early stage of infection that accounts for the viral entry modulation, but further research is still necessary to unveil the exact mechanisms of sl reactions. some covs (hcov-oc43 and hcovhku1), as well as influenza a virus (whose fusion loop is hemagglutinin, ha) and other non-related viruses (i.e., non-enveloped simian virus 40 sv-40, of polyomavirus family), use the sialoglycan moiety (9-o-acetyl-sialic acid) of gangliosides or glycoproteins located in membrane lipid rafts as receptors for the spike protein. the amino acid trp90 in the domain a of the hcov-oc43 s protein was shown to be essential for receptor binding. however, despite the fact that binding to 9-o-acetyl-sialic acid is required for membrane fusion, further interaction of the virus protein with other host membrane sialoglycans or proteins is also necessary to induce the conformational changes leading to membrane fusion [57, 58] . conversely, formation of the complex sv40 protein with the host cell ganglioside gm1 was found to be enough to induce the membrane curvature and invaginations required for membrane fusion [59] . as already discussed above, some studies have depicted the possibility that interaction of the fusion peptide or fusion loop with negatively charged phospholipids on the host membrane might be required for an efficient membrane fusion [25] . in this regard, phosphatidylserine (ps) contained in the virus envelope has been demonstrated to serve after externalization as a virus co-receptor through the t cell immunoglobulin mucin domain 1 (tim-1) receptor in ebov and other viruses, even in an indispensable fashion [60] [61] [62] [63] . in the study of nanbo et al. [63] , flipping of ps from the inner leaflet to the outer leaflet of the cell membrane for virion adquisition and incorporation to its envelope is proposed as a previous step to tim1 binding. in herpes simplex virus (hsv), phospholipid scramblase-1 (plscr1), after activation by hsv exposure, flips both ps and akt to the outside of the membrane in a ca 2+ -dependent mechanism. ps is restored to the inner leaflet 2 to 4 h after infection to avoid apoptotic triggering [62] , suggesting a different role for ps in relation to the tim-1 ps receptor. however, the function of tim-1 as an essential receptor for hav has been disputed [64] due to the finding that quasi-enveloped ha virions (ehav) were able to infect tim1-knockout vero cells to a similar extent to naked hav. hence, the authors proposed tim1 to be an accessory attachment factor by binding ps on the hav envelope rather than an essential virus protein receptor. in spite of these contradictory data, ps seems to act in any way in virus attachment and entry in certain virus families, at least contributing to the process efficiency, but the exact role may depend on every virus or it may be complementary to other factors. a phospholipid currently associated to the inner leaflet of the lipid rafts is phosphatidylinositol (pi), which is a negatively charged phospholipid with important and versatil signaling functions ( figure 2 ) [65, 66] . abundant data suggest that a derivative of pi, the phosphatidylinositol 4,5-biphosphate (pip2), accumulates preferently in liquid-disordered phases (l d ) [7] , where the cholesterol content is presumed to be low, and interplays with ps, which is rather localized in liquid-ordered phases (l o ). pis play an essential role also in endosome maturation, which is a requisite for efficient virus infection of those using the endosomal pathway [56, 66] . during hiv infection, pip2 has been proposed to coordinate the actin cytoskeleton changes required for efficient virus entry in cd4+ t cells [67] ; after virus attachment to the host cell receptor, pip2 is recruited to the binding membrane microdomain, and in this way, pip2 controls the protein reactions, leading to actin polymerization. as well in hiv-1, the requisite of pip2 accumulation for the virus gag protein to be properly anchored and stabilized in the inner leaflet of the cell plasma membrane has been pointed out [68, 69] . two isoforms, α and γ, of the phosphatidylinositol-4-phosphate 5-kinase family type 1 (pip5k1) have recently been shown to participate in gag stabilization by pip2 through targeting the gag precursor pr55 gag to the cell plasma membrane [70] . as commented above, interaction with the headgroup of negatively charged phospholipids such as ps or pi may also contribute to the dehydration process in the formation of the hemifusion stalk, with this contribution happening by promotion of the inverted hexagonal phase in the lipid bilayer and binding of ca 2+ [25] . in in vitro experiments with cos-7 cells and multilamellar vesicles (mlvs), unspecific binding of the marburg virus (marv) mvp40 protein to pip, pip2, and even pip3 species present in the mlvs, both in the presence or absence of ps, has been reported. in this study, it was also found that with increasing ps concentration, the association of mvp40 to mlvs rose up to a threshold. furthermore, the addition of sphingosine with the aim to reduce the negative charge load in the inner leaftet of the cos-7 cells led to a decrease in the binding level. these facts suggest that the electronic density, rather than the specific lipid species, is a determinant factor for binding [70] . activation of the pi3k pathway for signaling is one of the most relevant features taking place for both entry and budding during infection by a number of viruses [58, [71] [72] [73] . pi3k converts pip2 into phosphatidylinositol 3,4,5-triphosphate (pip3). in addition to stabilizing proteins or serving as a binding factor, pip2 has been shown to collaborate with akt through the signaling pathway pi3k/akt on avoiding apoptotic events, and in this way, keeping the host cell metabolically active for virus replication and budding [71] [72] [73] . all these results clearly bring evidence that the lipid environment surrounding proteins involved in virus infection has a relevant function in the virus entry mechanism. different lipids are essential for virus docking to the cell receptor either serving directly as (co)-receptors or providing the appropriate environment (lipid rafts) for the necessary reactions (e.g., membrane curvature). in addition, the virus, through specific protein conformational changes, takes advantage of several cell signaling pathways controlled by diverse membrane lipids. this process allows the virus to govern the cell metabolism following endocytosis of the viral genetic molecules. after the virus or its genome gets inside the infected cell, ssrna+ viruses and other enveloped ones that replicate in the cytoplasm manage the cell metabolism to develop the replication scaffold, this membrane structure bolstering the so-called 'virus factory' [5, 58, [74] [75] [76] [77] [78] [79] [80] . there is consensus on that the functions of these structures are (i) to compartmentalize the diverse processes involved in viral genome replication, its envelopment, and structural protein assembly; (ii) to increase virion concentration during budding before infecting naïve cells; and (iii) to create a protected environment to escape the innate immune recognition of the viral components. virus replication imposes an extra-energetic expenditure to the cell metabolism. hence, cell central metabolism is orchestated by viral proteins to redirect toward the generation of enough energy and metabolites that are required for virus replication. in particular, building the scaffold demands a high rate of new lipid synthesis. therefore, the lipid metabolism is hijacked by the virus proteins for the de novo synthesis of fatty acids in order to generate the scaffold membranes, the replication complexes (rcs), as well as for energy production in the β-oxidation pathway in the mitochondria. concurrently, the cell metabolism needs to be kept above a threshold level to avoid exhaustion of the host cell. full understanding of the mechanisms and related factors involved in virus-host interaction is a requisite for developing efficient antiviral infection therapies. the scaffold structure raised for building the viral factory varies between different virus in their morphology and possibly lipid composition. flaviviruses develop a so-called 'membranous network' (mn) in a spherule/invagination type, while coronavirus does through a quarter-like type delimited by 'double membrane vesicles' (dmvs). nonetheless, hcv (flaviviridae) uses dmvs instead [77] ; hence, this morphological separation may have exceptions or be somewhat diffuse. an extended review of the different virus family-related morphologies of the mns as well as diverse factors influencing their formation can be found in [22, 75, 76] . it should be remarked that the exact lipid composition of the rcs' membranes is not known in detail yet, although there is evidence that their lipid profile differs from that of the organelles from which they are generated. the enrichment of typical lipids such as cholesterol, sphingomyelins, and glycosphingolipids in the lipid rafts seems to be a common feature of these mns. the rcs' membranes may be originated from the endoplasmic reticulum (er) in the perinuclear area, as for example in sars-cov and faviviridae [75, 78, 80] , from the golgi, giving rise to cytopathic vesicles (cpvs) as in togaviridae and picornaviridae [75] , from mitochondria (nodaviridae) [79] , or from the cell plasma membrane (cpvs in alphaviruses) [75] . however, vesicle trafficking between the er and the golgi organelles may contribute to an undefinition in this regard. mns, and in particular dmvs, are connected to the cytosol through a pore, which is believed to serve as the gate to the replication scaffold for the requiered metabolites, in particular nucleotides. this pore-mediated gate has not been detected up to date in sars-cov's dmvs, which raises the concern of how the required metabolites get inside the rcs. there is evidence from a number of studies that dmvs are the site of replication, but it has also been shown that dmvs can be developed irrespective of whether rna replication takes place by the sole action of the viral proteins, at least for hcv [81, 82] . viral nonstructural proteins nsp3, nsp4, and nsp6 are involved in dmv development in sars-cov-1 in a time-dependent manner and correlating with rna replication. timecourse events have been shown to run with the initial formation of single membrane vesicles (smvs) during the first 2-4 h after cell infection. these futher evolve to dmvs 16 h after infection, and they ultimately turn into multimembraneous vesicles (mmvs) close to the cis-golgi at the budding stage 36-48 h after infection, this latter transformation being coincident with the formation of vesicle packets [75, 78, 79, 83] . in hcv, ns5a seems to be enough for dmv formation, but the collaboration of ns3-5b is required for completing efficient dmvs, whereas ns4b is likely responsible for inducing the formation of smvs [77, 80, 82] . even though particular hints can be likely associated to every particular virus, there are common features shared by all ssrna+ viruses regarding rcs' structure and buildup. enveloped viruses such as ssrna+ viruses have a membrane lipid whose profile is different to that of the original organelle membrane when the envelope is created. since the viral membrane is known to be enriched in cholesterol, sphingolipids, and phospholipids with saturated acyl chains, the dmv is believed to be also primarily composed of such classes of lipids. an unusual sphingolipid, dehydrosphingomyelin, along with ps and plasmalogens of pe were reported in the hiv envelope [84] . a role for sphingomyelin-to-ceramide conversion has been proposed in wnv budding, as its envelope was found to be highly enriched in sphingomyelin [85] . more recently, using multi-color super-resolution microscopy and mass spectrometry analysis, a substantial increase in pip2 (from 11% to 51%) and pip3 (from 0.01% to 0.13%) was reported in the hiv membrane as compared with the plasma membrane of the host cell [69] ; this fact is related to the recruitment of gag protein for efficient membrane fusion as aforementioned ( figure 1) . however, the most striking and known lipid-related factor associated to the mns' development is the pi4kiii signaling pathway. the pi4pα isoform, which is mainly expressed in the er, has been shown to be a key factor for hcv replication, whereas the pi4kiiiβ is found in the golgi and is required by picornaviruses and some hcv strains [75] . this enzyme interacts with the viral protein ns5a, and disrupting this interaction prevents virus replication. the product of the pi4k enzyme is pip4; enrichment in this pi has been shown to act in different processes regarding virus replication: membrane curvature, directly or indirectly through recluting cholesterol [86] , glycosphingolipid transport to the rcs by the action of the fapp2 protein [87] , and protein concentration. however, conversely to these studies, it has been shown that currently used inhibitors of pi4kiiiα, enviroxime and bf738735, actually exert their inhibition against pi3k [88] . thus, this result points out a genomic dependence on the pi kinases in hcv; otherwise, the action on pi3k is required only at the entry stage (see above). enviroxime-like inhibitors have been shown to halt enterovirus replication through the action against pi4kβ [89] . the de novo lipid synthesis has also been evidenced for wnv, from the flaviviridae family as hcv, to proceed in a pi4p-independent fashion and, concurrently, it is not related to pi4kiii signaling [90] . there is no clear evidence on the fact that the pi4k signaling pathway has a relevant function in mns' development. hence, while pi4kiiiβ was shown to be important for sars-cov's dmv formation [91] , another study did not find its metabolite, pi4p, within the host factors involved in sars-cov replication, and the authors attibute to pi4p a function rather in virus entry. however, the authors of this latter study acknowledge that sirna methodology may provide false negatives [92, 93] . since dmvs are not common in healthy cells but they can be observed during authophagy, it has been suggested that sars-cov and other coronaviruses use the autophagy pathway for development of the dmvs; indeed, it has been shown that nsp6 in mhv or the equivalent nsp5-7 in arteriviruses, which hits the er, can activate such a pathway [79, 94] . nonetheless, dmvs are smaller than autophagosomes, and hence, they might be rather endoplasmic reticulum derived vesicles (edesomes) enriched in pi3p and not follow exactly the same synthetic route [94] . further work on coronaviruses and autopahgy found that only the lc3-i protein, the microtubule-associated proteins 1a/1b light chain 3b, is localized on the replication membranes, but the active protein lipidated with phosphatidylethanolamine lc3-ii inserted into the autophagosome membrane is absent. accordingly, present knowledge on coronaviruses in regard to autophagy suggests that they take benefit of the autophagocytic components but do not develop autophagosomes per se [95] . the autophatocytic pathway has also been associated to the start of hcv infection, but it seems not to be necessary for the infection to go on [82] . later on, it was shown that autophagy was key in rna replication at the onset of hcv infection [96] , but the virus life cycle can go ahead afterward without the autophagy system intervention. further work has shown that hcv, and possibly denv, uses the autophagy system to evade the innate immune system [97] . using immortalized human hepatocytes defective of the autophagy-related proteins either beclin (bcn1) or atg7, it was shown in the latter study that disruption of the autophagy machinery elicites activation of the interferon signaling pathway and leads to apoptosis of the infected cells. triggering of the autophagy pathways takes place after binding of the virus to the cell surface via the downregulation of mtor and inactivation of akt signaling [95] . conflicting results have been reported for the induction of autophagy by hcv in regard to the unfolded protein response (upr) [95] . recent work [98] has bound the induction of autophagy by hcv to golgi membrane fragmentation to render vesicles that colocalize with the hcv replicons. the immunity-related gtpase m protein (irgm) mediates the phosphorylation of the early autophagy initiator ulk1 as well as the golgi membrane fragmentation in response to hcv infection. the protein lc3 has also been detected in the replication membranes of the hiv-1, and the association of lc3-ii with gag-derived proteins seems to be a requisite for the efficient maturation of the gag subunit p24 [14, 99] . members of the picornaviridae family, non-enveloped viruses, have been reported to subvert the autophagosome pathway as a means to exit the infected cell without membrane lysis; support for this spreading mechanism comes from the finding of numerous extracelular vesicles that are enriched in phosphatidylserine phospholipids [14] . the best studied virus regarding autophagy is the dengue virus (denv). even though it was initially suggested that the denv replication complexes are developed from autophagosomes, further work pointed out that the replication of denv took place on invaginations arising from the endoplasmic reticulum (er), while autophagy was rather used by denv to modify the lipid metabolism in a way that is known as lipophagy [100, 101] . lipophagy was first shown to be an active way to get energy under starvation [102] through the association of autophagic components with lipid droplets (lds). recently, lipophagy has been demonstrated to regulate the fatty acid availability for the β-oxidation through contact sites between the mitochondria and the er [103] . regarding virus-associated hijacking of the cell lipid metabolism, heaton and randall [100] early showed that increased β-oxidation and the depletion of triglycerides was concurrent with and necessary for denv replication. then, these features were linked to the action of autophagy through the association with lipid droplets. a recent study by zhang et al. [104] has found that aup1, a type iii protein with signals for lds and er, plays a relevant role in lipophagy induced by denv and other flaviviruses such as wnv. unmodified aup1 is required for lipophagy triggering. a 10-fold increase in the content of diacylglycerophosphocholines (pcs) was measured in this study in infected cells containing unmodified aup1, this increase being concomitant with a depletion of triacylglycerols and cholesterol esters, whereas the contents of free fatty acids and unesterified cholesterol rose. conversely, smaller lds, but not a reduction of their abundance, were observed in aup1-knocked-out cells. thus, these data point to an augmented consumption of lds in the infected cells. this study unveils the mechanism that leads to the commented results; after the denv protein ns4a associates with aup1, the complex is relocalized from lds to autophagosomes, where the acyltransferase domain of aup1 is activated for the generation of phospholipids. this process was found to be dependent on the aup1 ubiquitylation status, with ns4a inhibiting the ubiquitylation of aup1. similar to viral entry, cholesterol has been found to be also relevant in the rcs' membranes [79, 82] . up to a c.a. 9-fold enrichment of cholesterol was found in hcv-developed dmvs as compared to its content in the er membranes from which dmvs were originated [77] . a key protein in cholesterol metabolism associated to non-vesicular transport is the oxysterol-binding protein (osbp). this protein has been described to transport cholesterol to pi4p-enriched membranes, which would agree with its collaboration in delivering cholesterol to dmvs with an abundant content of this pi [77] . the ceramide transfer protein (cert) and the four-phosphate adaptor protein 2 (fapp2) are known to undergo a similar fate in hcv infection [82] . an important protein involved in cellular lipid homeostasis is the sterol regulatory element binding protein (srebp), a bhlh-zip transcription factor with three isoforms; srebp1c regulates the expression of fatty acid (fa) biosynthesis genes [105, 106] , whereas srebp2 transactivates genes implied in cholesterol biosynthesis, intracellular lipid transport, and lipoprotein import [107] . a recent study shows that the inhibition of srebp with the retinoid derivative and rar-α agonist am580 prevents mers-cov infection by avoiding the formation of functional dmvs [105] . in this study, the lipid metabolism was the most affected pathway, with sterol biosynthesis being strengthened at expense of the glycerophospholipid metabolic pathways. fast activation of the lipid biosynthesis enzymes acetyl-coa carboxylase (acc), fatty acid synthase (fas), and hmg-coa synthase (hmgcs) was observed in such study, whose activity was partially blocked by am580 inhibition of srebp enzymes. promotion of lipid biosyntheis after infection had already been pointed out for hcv in an elegant proteomics and lipidomics study [108] . hcv infection elicites changes in the proteome of host cells that resembled the warburg effect described in cancer cells toward lactate production and the support of continuous glycolysis; concurrently, the up-regulation of citrate synthase (cs) and other lipogenic enzymes 24 h after infection was interpreted by the authors of the latter study as indicative of re-routing of the tricarboxylic acid (tca) cycle for cytosolic accumulation of citrate, which would be used in fa synthesis. the up-regulation of peroxisomal and mitochondrial fa oxidation pathways is concurrent with the other metabolic changes. an increase in pro-apoptotic ceramides was observed in the latter study as well; two possible interpretations were attributed to this finding, either a cytopathic effect after cell cycle arrest over time enough to complete virus offspring or a defense response of the host cell to avoid infection spread. blocking cholesterol suitability for the membraneous network or endosomes used for the virus replication and internalization has been demonstrated to inhibit the virus life cycle in a number of unrelated viruses. disruption of the srebp pathway restrains the andes virus (andv), an ssrnavirus, internalization, although it does not bind to the cell surface receptor [109] . in addition to srebp2, other components of this pathway were found to be necessary. the dependence of viral entry on the sterol regulatory element binding protein cleavage activating protein (scap) and the site 1 protease (s1p) was evidenced in cells null for these proteins. thus, in the study of petersen et al. [109] , the virus was not internalized in cells lacking s1p, this result pointing out that a complete cholesterol biosynthesis pathway is required. infectivity was also reduced 10-fold when the cells were treated with methyl-β-cyclodextrin (mβcd), a cholesterol sequestering agent, and comparable results were obtained after cell treatment with mevastatin or the s1p inhibitor pf-429242. however, the s1p dependence of virus infectivity does not seem to affect other viruses, thus this route being likely selective for hantaviruses [110] . in this study, the genetic or pharmacological disruption of the srebp pathway at the site of the regulatory element membrane-bound transcription factor peptidase/site 1 protesase (mbtps1/s1p) dramatically reduced viral infection, which is a feature that confirms the essential dependence of hantavirus on the high membrane cholesterol content for membrane fusion and effective infection. the down-regulation of sterol synthesis at the gene level after infection was found to be controlled by an interferon regulatory loop, in which a type i interferon-dependent mechanism down-regulates the expression of srebp2 [111] , this result showing a link between the innate immune response and cholesterol biosynthesis after viral infection. this type i interferon response toward cholesterol synthesis down-regulation was dependent on the mevalonate-isoprenoid branch as a supply of mevalonate completely blocked the cholesterol synthesis, whereas a supply of cholesterol did not. additionally, in the presence of geranylgeraniol, the type i interferon inhibition of sterol biosynthesis was severely diminished. further research has shown that interferon may regulate the sterol synthesis pathway in multiple forms through micrornas [112] . in particular, mir-342-5p was found to hit multiple srebp-independent targets of the mevalonate-sterol synthesis pathway after viral infection. the type i interferon response was also observed in regard to the impairment of the formation of double membrane structures induced by arteriviruses as replication sites [113] . host cell fight against viral infection by a reduction of cholesterol availability has been also pointed out to come from the antiviral effector protein interferon-inducible transmembrane protein 3 (ifitm3). this protein interacts with vesicle-membrane-protein-associated protein a (vapa), impeding its association with the oxysterol binding protein (osbp), and consequently, altering the normal function of osbp. as a result of the ifitm3 action, virus release into the cytosol is blocked by the accumulation of cholesterol in multivesicular bodies and endosomes. this effect restrains the membrane fusion of the intraluminal vesicles and that of the multivesicular bodies, which is a requisite for virus budding and release to the cytosol [114] . the viral accesory protein of hiv nef competes with the cholesterol transporter abca1 to prime the transport of cholesterol to lipid rafts as a viral strategy to raise the replication membranes, thus overcoming the antiviral properties of abca1 [115] . the replication of rabies virus (rabv), an ssrna virus, is halted by the action of viperin (virus inhibitory protein, endoplasmic reticulum-associated, ifn-inducible) in raw264.7 cells. this protein is induced by the rabv, ifv, hiv, or hcv infection through promotion of the innate immune response bound to the tlr4 signaling pathway. the inhibitory activity of viperin on virus budding is related to its capability to substantially drop the contents of cholesterol and sphingomyelin in the replication membranes [116] , thus pointing out the relevance of the membrane lipid composition for efficient virus replication. the induction of viperin has also been proven for hcv and ifav [111] . however, viperin does not intervene in the inhibition of arterivirus-induced double membrane formation [113] . remodeling of the lipid metabolism by virus infection may leave signals at the organism level even some years after healing. the metabolome profile of patients undergoing sars-cov-1 infection during the outbreak of 2002-2003 was assessed 12 years after overcoming the pathology [117] . an outstanding result of this study regarding disturbed lipid metabolism was the elevation of phosphatidylinositol (pi) and lysophosphatidylinositol (lpi) species concentrations in serum, which in turn correlated positively with the levels of very low-density lipoproteins (vldl); higher concentrations of products of the phospholipase a 2 (pla 2 ) such as lysophospholipids (lppls) and free arachidonic acid (aa) were also found in patients as compared to healthy voluntiers, with a correlation between the level of aa and the ratio of lpi(18:0) to total 18:0-pis being observed as well. these results show a potential high sensitivity of sars-cov patients to pla 2 activity. in the general context, the metabolome of these patients pointed to hyperlipidemia, cardiovascular abnormalities, and glucose metabolism alteration as a delayed efffect of the viral infection. nonetheless, the authors acknowledge that some of the related metabolic disturbations are likely owed to the pharmacological treatment. high levels of pla 2 group iid (pla2g2d) in lungs of middle-aged mice as compared to young mice had previously been associated to a fatal or worse outcome [118] . the authors of this study conclude that the negative influence of this enzyme in sars-cov infection was to increase the concentration of anti-inflammatory lipid mediators, mainly protaglandin d 2 (pgd 2 ), which impaired the efficient function of the immune system [119] . in the recent sars-cov-2 outbreak (covid-19), mortality has mostly affected aged people above 60 years old, thus showing an age-related fatality as for sars-cov-1 and mers-cov [120] . using a lipidomics approach, the effect of hcov-229e and mers-cov infection on the host cell lipid profile was recently investigated in cell culture [121] . the main conclusions of this study agree with the raised content of aa and lppls through plase activity, which indicates that the possible virus-induced activation of cpla 2 favors virus replication as a factor required for dmvs' formation. in this study, linoleic acid (la) or aa supplementation to the culture cells suppressed replication, which is a result that may be interpreted as a demonstration of the perturbation of the la/aa axis of the lipid metabolism. in the covid-19 outbreak, it has been suggested that increasing the levels of vitamin d could help fighting against the sars-cov infection [122] . this suggestion is based on the fact that 25-hydroxyvitamin d3 was found to protect huh7 cells against mers-cov [105] . vitamin d is a lipid-related compound belonging to the group of fat-soluble secosteroids, with the most important form in humans being vitamin d3 (cholecalciferol) [123] . in a recent study, high doses of vitamin d have shown protective effects against denv infection through regulation of the toll-like receptor expression as well as the modulation of pro-inflammatory cytokines release, which suggests that its action is focused toward the immune system modulation rather than to lipid metabolism [124] . however, evidence on the beneficial effects of vitamin d uptake is still poor, and more studies are devoted to this issue. lipids, as components of membranes, are related to viroporins, which are specific viral proteins that are known to create ion channels for ion trafficking [125] [126] [127] . the effect on cell metabolism of diverse viroporins differs among them, but there is evidence that they are closely related to viral pathogenity [125] . viroporins may play a relevant role during virus infection, as they are involved in membrane permeability and calcium homeostasis. their participation in the development of vacuoles from the er during the dmvs' formation has been suggested, but data on this issue are still scarce. the regulation of ca 2+ flux by viroporins might favor the membrane fusion through the interaction of this cation with the phospholipid headgroups and concurrently facilitate the required dehydratation reaction. viroporins are not required for virus replication with the exception of rotaviruses and picornaviruses; thus, whether this function is exerted through the ion channels or another property of viroporins remains an issue still unknown [125] . the lipid composition of the membrane may influence the viroporin activity, leading to different versions of ion channels, which depends on the electric charge that the phospholipids confer to the membrane and curvature [127] . a viroporin from rotavirus, nsp4, was shown to co-localize with the autophagy marker protein lc3 in membranes accomodating virus replication; this viroporin is implicated in the sequestering of autophagy for the transport of proteins from the er to the replication sites [128] . further research is necessary to understand the role played by viroporins in virus infection in order to consider them as potential therapeutic targets. remodeling of the virus-induced host cell lipid metabolism is a remarkable feature of the viral infection that affects viral entry, replication of the genomic material, and the releasing of progeny. a comperhensive view of the process is illustrated in figure 3 . the main actors are well known to be cholesterol, sphingolipids, and pis, but other lipid species and their related pathways such as the la/aa axis are also relevant. how to target the lipid metabolism in a safe manner to avoid virus infection or reduce its pathogenity is a promising therapeutic tool, but it demands improving the knowledge on the actual pathways that are affected over the virus life cycle. the exact mechanism through which the enzyme inhibitors act on the key enzymes of the lipid metabolism is additionally required to develop more efficient and safe therapeutic drugs. since the lipid metabolism is essential for proper cell function, selective drugs targeting the virus or exclusively the infected cells have to be used to avoid harmful side effects. a new coronavirus associated with human respiratory disease in china world health organization. who director-general's opening remarks at the media briefing on covid-19 an overview of their replication and pathogenesis the virus-host interplay: biogenesis of +rna replication complexes virus factories: associations of cell organelles for viral replication and morphogenesis virus entry: open sesame the role of cholesterol in membrane fusion world health organization. who director-general's opening remarks at the media briefing on covid-19 an overview of their replication and pathogenesis the virus-host interplay: biogenesis of +rna replication complexes virus factories: associations of cell organelles for viral replication and morphogenesis virus entry: open sesame the role of cholesterol in membrane fusion identification of the fusion peptide-containing region in betacoronavirus spike glycoproteins structural characterization of the hcov-229e fusion core hepatitis c virus entry. in hepatitis c virus: from molecular virology to antiviral therapy how hepatitis c virus invades hepatocytes: the mystery of viral entry endocytosis of influenza viruses. microbes infect virus entry by endocytosis viral journeys on the intracellular highways infectious bronchitis virus entry mainly depends on clathrin mediated endocytosis and requires classical endosomal/lysosomal system single virus tracking of ebola virus entry through lipid rafts in living host cells sars coronavirus entry into host cells through a novel clathrin-and caveolae-independent endocytic pathway tissue distribution of ace2 protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor hepatitis c virus infection: host-virus interaction and mechanisms of viral persistence cell entry of enveloped viruses viral membrane fusion new biophysical approaches reveal the dynamics and mechanics of type i viral fusion machinery and their interplay with membranes influenza-virus membrane fusion by cooperative fold-back of stochastically induced hemagglutinin intermediates sequential conformational rearrangements in flavivirus membrane fusion energetics of stalk intermediates in membrane fusion are controlled by lipid composition visualization and sequencing of membrane remodeling leading to influenza virus fusion two conserved residues are important for inducing highly ordered membrane domains by the transmembrane domain of influenza hemagglutinin sars-cov fusion peptides induce membrane surface ordering and curvature direct measurement of polyethylene glycol induced depletion attraction between lipid bilayers nmr structures and localization of the potential fusion peptides and the pre-transmembrane region of sars-cov: implications in membrane fusion fluorescence microscopy of the hiv-1 envelope lipid rafts are involved in sars-cov entry into vero e6 cells coronavirus and influenza virus proteolytic priming takes place in tetraspanin-enriched membrane microdomains the important role of lipid raft-mediated attachment in the infection of cultured cells by coronavirus infectious bronchitis virus beaudette strain influenza virus assembly and lipid raft microdomains: a role for the cytoplasmic tails of the spike glycoproteins influenza virus hemagglutinin concentrates in lipid raft microdomains for efficient viral fusion membrane lipids: where they are and how they behave the mystery of membrane organization: composition, regulation and roles of lipid rafts lipid rafts: controversies resolved, mysteries remain cholesterol promotes hemifusion and pore widening in membrane fusion induced by influenza hemagglutinin the hemifusion structure induced by influenza virus haemagglutinin is determined by physical properties of the target membranes dengue virus ensures its fusion in late endosomes using compartment-specific lipids membrane cholesterol modulates oligomeric status and peptide-membrane interaction of severe acute respiratory syndrome coronavirus fusion peptide target membrane cholesterol modulates single influenza virus membrane fusion efficiency but not rate the molecular biology of coronaviruses cholesterol interactions with ceramide and sphingomyelin sphingolipid-dependent fusion of semliki forest virus with cholesterol-containing liposomes requires both the 3-hydroxyl group and the double bond of the sphingolipid backbone both sphingomyelin and cholesterol in the host cell membrane are essential for rubella virus entry ebolavirus requires acid sphingomyelinase activity and plasma membrane sphingomyelin for infection role of sphingomyelin in alphaherpesvirus entry ceramide suppresses influenza a virus replication in vitro rhinoviruses infect human epithelial cells via ceramide-enriched membrane platforms differential utilisation of ceramide during replication of the flaviviruses west nile and dengue virus glucosylceramidase maintains influenza virus infection by regulating endocytosis influenza a viruses use multivalent sialic acid clusters for cell binding and receptor activation structural basis for human coronavirus attachment to sialic acid receptors lipid interactions during virus entry and infection gm1 structure determines sv40-induced membrane invagination and infection role of the phosphatidylserine receptor tim-1 in enveloped-virus entry tim-family proteins promote infection of multiple enveloped viruses through virion-associated phosphatidylserine ebola virus requires a host scramblase for externalization of phosphatidylserine on the surface of viral particles herpes simplex viruses activate phospholipid scramblase to redistribute phosphatidylserines and akt to the outer leaflet of the plasma membrane and promote viral entry tim1 (havcr1): an essential "receptor" or an "accessory attachment factor" for hepatitis a virus? pip5k-driven ptdins(4,5)p2 synthesis: regulation and cellular functions function and dysfunction of the pi system in membrane trafficking pip2: choreographer of actin-adaptor proteins in the hiv-1 dance synchronized hiv assembly by tunable pip2 changes reveals pip2 requirement for stable gag anchoring quantification of phosphoinositides reveals strong enrichment of pip2 in hiv-1 compared to producer cell membranes investigation of the lipid binding properties of the marburg virus matrix protein vp40 type i phosphatidylinositol-4-phosphate 5-kinase α and γ play a key role in targeting hiv-1 pr55gag to the plasma membrane make yourself at home: viral hijacking of the pi3k/akt signaling pathway hepatitis b spliced protein (hbsp) suppresses fas-mediated hepatocyte apoptosis via activation of pi3k/akt signaling multifaceted roles for lipids in viral infection ultrastructure of the replication sites of positive-strand rna viruses does form meet function in the coronavirus replicative organelle? morphological and biochemical characterization of the membranous hepatitis c virus replication compartment sars-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum virus factories, double membrane vesicles and viroplasm generated in animal cells three-dimensional architecture and biogenesis of membrane structures associated with hepatitis c virus replication expression of hepatitis c virus proteins induces distinct membrane alterations including a candidate viral replication complex mechanisms of cellular membrane reorganization to support hepatitis c virus replication. viruses severe acute respiratory syndrome coronavirus nonstructural proteins 3, 4, and 6 induce double-membrane vesicles the hiv lipidome: a raft with an unusual composition the composition of west nile virus lipid envelope unveils a role of sphingolipid metabolism in flavivirus biogenesis oxysterol-binding protein is a phosphatidylinositol 4-kinase effector required for hcv replication membrane integrity and cholesterol trafficking modulation of hepatitis c virus genome replication by glycosphingolipids and four-phosphate adaptor protein 2 pi4kiii inhibitor enviroxime impedes the replication of the hepatitis c virus by inhibiting pi3 kinases phosphatidylinositol 4-kinase iii beta is a target of enviroxime-like compounds for antipoliovirus activity west nile virus replication requires fatty acid synthesis but is independent on phosphatidylinositol-4-phosphate lipids phosphatidylinositol 4-kinase iiiβ is required for severe acute respiratory syndrome coronavirus spike-mediated cell entry a kinome-wide small interfering rna screen identifies proviral and antiviral host factors in severe acute respiratory syndrome coronavirus replication, including double-stranded rna-activated protein kinase and early secretory pathway proteins host factors in coronavirus replication. in roles of host gene and non-coding rna expression in virus infection current topics in microbiology and immunology divergent roles of autophagy in virus infection the autophagy machinery is required to initiate hepatitis c virus replication knockdown of autophagy enhances the innate immune response in hepatitis c virus-infected hepatocytes hepatitis c virus triggers golgi fragmentation and autophagy through the immunity-related gtpase m. proc. natl. acad. sci autophagy during viral infection-a double-edged sword dengue virus-induced autophagy regulates lipid metabolism dengue virus and autophagy autophagy regulates lipid metabolism autophagy regulates fatty acid availability for oxidative phosphorylation through mitochondria-endoplasmic reticulum contact sites flaviviruses exploit the lipid droplet protein aup1 to trigger lipophagy and drive virus production activators of the complete program of cholesterol and fatty acid synthesis in the liver common fatty markers in diseases with dysregulated lipogenesis coronavirus nsp6 proteins generate autophagosomes from the endoplasmic reticulum via an omegasome intermediate srebp-dependent lipidomic reprogramming as a broad-spectrum antiviral target the major cellular sterol regulatory pathway is required for andes virus infection haploid genetic screen reveals a profound and direct dependence on cholesterol for hantavirus membrane fusion host defense against viral infection involves interferon mediated down-regulation of sterol biosynthesis an interferon regulated microrna provides broad cell-intrinsic antiviral immunity through multihit host-directed targeting of the sterol pathway antiviral innate immune response interferes with the formation of replication-associated membrane structures induced by a positive-strand rna virus the antiviral effector ifitm3 disrupts intracellular cholesterol homeostasis to block viral entry hiv-1 nef mobilizes lipid rafts in macrophages through a pathway that competes with abca1-dependent cholesterol efflux viperin inhibits rabies virus replication via reduced cholesterol and sphingomyelin and is regulated upstream by tlr4 temporal proteome and lipidome profiles reveal hepatitis c virus-associated reprogramming of hepatocellular metabolism and bioenergetics critical role of phospholipase a2 group iid in age-related susceptibility to severe acute respiratory syndrome-cov infection virus-induced inflammasome activation is suppressed by prostaglandin d2/dp1 signaling the trinity of covid-19: immunity, inflammation and intervention characterization of the lipidomic profile of human coronavirus-infected cells: implications for lipid metabolism remodeling upon coronavirus replication. viruses evidence that vitamin d supplementation could reduce risk of influenza and covid-19 infections and deaths sunlight and vitamin d for bone health and prevention of autoimmune diseases, cancers, and cardiovascular disease effect of high doses of vitamin d supplementation on dengue virus replication, toll-like receptor expression, and cytokine profiles on dendritic cells viroporins: structure and biological functions viral membrane channels: role and function in the virus life cycle autophagy hijacked through viroporin-activated calcium/calmodulin-dependent kinase kinase-signaling is required for rotavirus replication this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors thank m. s. crespo for helpful reading of the manuscript. the authors declare no conflict of interest. the authors thank m. s. crespo for helpful reading of the manuscript. the authors declare no conflict of interest. key: cord-314402-kjzkk51t authors: zou, guijin; liu, yue; gao, huajian title: eml webinar overview: simulation-assisted discovery of membrane targeting nanomedicine date: 2020-06-08 journal: extreme mech lett doi: 10.1016/j.eml.2020.100817 sha: doc_id: 314402 cord_uid: kjzkk51t the covid-19 pandemic has brought infectious diseases again to the forefront of global public health concerns. in this eml webinar (gao, 2020), we discuss some recent work on simulation-assisted discovery of membrane targeting nanomedicine to counter increasing antimicrobial resistance and potential application of similar ideas to the current pandemic. a recent report led by the world health organization (who) warned that 10 million people worldwide could die of bacterial infections each year by 2050. to avert the crisis, membrane targeting antibiotics are drawing increasing attention due to their intrinsic advantage of low resistance development. in collaboration with a number of experimental groups, we show examples of simulation-assisted discovery of molecular agents capable of selectively penetrating and aggregating in bacterial lipid membranes, causing membrane permeability/rupture. through systematic all-atom molecular dynamics simulations and free energy analysis, we demonstrate that the membrane activity of the molecular agents correlates with their ability to enter, perturb and permeabilize the lipid bilayers. further study on different cell membranes demonstrates that the selectivity results from the presence of cholesterol in mammalian but not in bacterial membranes, as the cholesterol can condense the hydrophobic region of membrane, preventing the penetration of the molecular agents. following the molecular penetration, we establish a continuum theory and derive the energetic driving force for the domain aggregation and pore growth on lipid membrane. we show that the energy barrier to membrane pore formation can be significantly lowered through molecular aggregation on a large domain with intrinsic curvature and a sharp interface. the theory is consistent with experimental observations and validated with coarse-grained molecular dynamics simulations of molecular domain aggregation leading to pore formation in a lipid membrane. the mechanistic modelling and simulation provide some fundamental principles on how molecular antimicrobials interact with bacterial membranes and damage them through domain aggregation and pore formation. for treating viral infections and cancer therapy, we discuss potential sizeand lipid-type-based selectivity principles for developing membrane active nanomedicine. these studies suggest a general simulation-assisted platform to accelerate discovery and innovation in nanomedicine against infectious diseases. eml webinar speakers are updated at https://imechanica.org/node/24132 one of the most important public health issues that the world faces today is the threat of global infectious disease outbreaks. in the last several decades, newly identified and re-emerging virus related infectious diseases including human influenza, hiv/aids, severe acute respiratory syndrome (sars), dengue, malaria, ebola, middle east respiratory syndrome (mers) have demonstrated serious epidemic potential with devastating loss of life and wealth. the covid-19 pandemic has brought infectious diseases again to the forefront of global public health concerns. as of jun. 4, 2020, more than 6 million people had been infected and hundreds of thousands had died worldwide [1] . in addition to the virus, bacterium is another major human pathogen that are responsible for a wide range of lifethreatening infections [2] . antibiotics are widely prescribed and can effectively kill or inhibit the growth of metabolically active bacteria [3, 4] . however, in recent years, the increasing prevalence of antibiotic resistant strains and the ability of bacterial cells to convert to a quiescent subpopulation (often referred to as "persisters" [5] ) that exhibits a high level of tolerance to most antibiotics [6] [7] [8] [9] [10] have caused clinical challenges that may eventually lead to a crisis level shortage of effective antibiotics for treating bacterial infections. a recent report led by the world health organization (who) warned that 10 million people worldwide could die of bacterial infections each year by 2050 [11] . consequently, there is an urgent societal need to develop novel antibiotics with unconventional antimicrobial strategies. antimicrobial chemotherapy seeks to eradicate the infecting pathogen from its host in the shortest possible treatment period. most conventional antibiotics targeting biosynthetic processes that occur in actively growing bacteria, including the biosynthesis of proteins, rna, dna, peptidoglycan and folic acid [12] , fail to meet the challenges related to the mutation, resistance and persisters [6] [7] [8] [9] [10] . on the other hand, the bacterial membrane, as an essential structural and functional component of biological organisms, providing selective permeability for cellular homeostasis and metabolic energy transduction even if the bacteria are in the dormant state, has attracted increasing attention as targets for new generation antibiotics, as membrane-active agents are inherently effective on sleeping bacterial persisters and also difficult for the bacterial to acquire resistance [13] . viruses, especially the rna viruses such as the influenza and coronaviruses, are highly susceptible to mutations [14] . since the outbreak of sars-cov-2 in january, abundant mutations have been reported in just a few months [15] [16] [17] [18] , and membrane active nanomedicine might be a promising but underexploited approach to effectively counter viral mutations. in this webinar, we discuss some examples of simulation-assisted discovery of molecular agents which are promising candidates for a new generation of membrane-active antibiotics and potential application of similar ideas to the current pandemic. as shown in fig.1 , the webinar covered the antimicrobial mechanisms of synthetic antibiotics in attachment, penetration and aggregation in the cell membrane, leading to membrane distortion, pore formation and rupture. some derived theory and physical insights can also be applied to some antiviral agents that seem effective for a broad range of viruses by disrupting the lipid envelop [19] , which may help discover, design and fabricate membrane targeting antiviral agents to combat covid-19. the study suggested a simulation-assisted platform to accelerate discovery and innovation in nanomedicine. j o u r n a l p r e -p r o o f fig.1 interactions between membrane targeting antibiotic agents and bacterial membrane, including membrane attachment and penetration, diffusion and aggregation within the membrane, and membrane deformation, distortion, pore formation and rupture. staphylococcus aureus is an opportunistic human pathogen carried by approximately one third of human populations, and infections by s. aureus remain a major cause of death [20] . to demonstrate how simulation can assist the discovery of membrane-active antibiotics, we started from all-atom molecular dynamics (md) simulations of the interactions between a class of retinoids (fig. 2a ) and the lipid bilayer membrane of s. aureus. a previously established lipid bilayer composed of dopc/dopg at a 7:3 ratio was adopted to model the negatively charged s. aureus membrane [21] . four synthetic retinoids (fig. 2a) were systematically investigated, among which cd437 and cd1530 were identified as potential antibiotics through a biological screening assay [22] , while adarotene and adapalene were selected because of their similar structures as cd437 and cd1530. the simulations showed that the carboxylic acid and the phenolic groups of cd437, cd1530 and adarotene help anchor these retinoids to the surface of the membrane bilayer through binding with hydrophilic lipid heads, followed by penetration into the bilayer and becoming embedded in the outer membrane leaflet, inducing substantial perturbations to the membrane. in comparison, adapalene, a molecule of similar structure, cannot penetrate into the bilayer as the hydrophobic methoxy group does not bind to the lipid heads ( fig. 2b ). free energy profiles associated with the membrane penetration of these molecular agents were calculated based on steered molecular dynamics and umbrella sampling [23] [24] [25] . the free energy mapping shows that membrane penetration by cd437, cd1530 and adarotene is energetically favourable with barriers on the order of thermal fluctuation while adapalene exhibits a high energy barrier and an unfavourable transfer energy against membrane penetration (fig. 2c ). in comparison with cd437 and cd1530, adarotene shows higher energy barrier and smaller transfer energy, hence less favourable for membrane penetration. these results are consistent with the biomembrane-mimicking giant unilamellar vesicles (guvs) experiments (fig. 2d) , where domain aggregation and pore development are observed on the surfaces of the guvs exposed to cd437, cd1537 and adarotene, while the guvs exposed to adapalene remain intact. further in vivo and in vitro experiments indicated that cd437 and cd1530 are potential new membrane-active antibiotics with fast bacteria killing rate and low resistant development, while adarotene and adapalene exhibit lower and no antimicrobial activities, respectively [26] . the discovery of this class of retinoids as new antibiotics demonstrated the powerful roles of molecular simulations. the simulations and free energy mapping revealed atomistic insights on how molecular agents attach on and penetrate into the bacterial membranes, and the excellent agreement with experiments indicated that modelling and simulation can effectively capture whether a given compound is a potential candidate for membrane-active antibiotics. one of the major challenges in developing membrane targeting agents is to ensure their selectivity for pathogen since those of low membrane selectivity are typically toxic to mammals [5] . despite many common features among all biological membranes, the lipid compositions of bacterial and mammalian cell membranes are quite different. specifically, unlike the negatively charged s. aureus membrane, the outer leaflet of mammalian cell membrane is neutrally charged, with cholesterol ranging from 20 to 50 mol% [27] [28] [29] . this intrinsic difference between bacterial and mammalian cell membranes provides a basis for developing membrane targeting antibiotics. taking a clinically approved anthelmintic drug bithionol (fig. 3a) as an example, we found that the simulations can also help capture membrane selectivity. all-atom md simulations show that bithionol can be recruited to the bacterial membrane surface, penetrates into the membrane and embeds in the outer leaflet of the lipid bilayer. in contrast, bithionol fails to penetrate mammalian mimetic lipid bilayers with 7:3 popc/cholesterol ratio [27, 28] (fig. 3b) . the free energy mapping confirms that penetration of bithionol into the mammalian mimetic membrane is energetically unfavourable (fig. 3c) . moreover, the presence of cholesterol leads to a more ordered alignment of the membrane lipids, condenses the hydrophobic region of the membrane and decreases membrane fluidity. as a result, the energy barrier and transfer energy associated with membrane penetration increases monotonically with the percentages of cholesterol, suggesting that cholesterol plays a key role in membrane selectivity [30] . the guvs experiments on the effects of bithionol on different lipid bilayers are in agreement with the simulations: bacterial mimetic guvs exposed to biothionol undergo domain aggregation, pore development and rupture, while no such effects are observed on mammalian mimetic guvs. cell-based assays further demonstrate that bithionol indeed exhibits bactericidal activity and low levels of toxicity to mammalian cells [30] . our modelling and simulation demonstrated that the selectivity of bithionol for bacterial membranes correlates with its ability to penetrate and embed in bacterial-mimic lipid bilayers, but not in cholesterolrich mammalian-mimic lipid bilayers. this then provided a fundamental insight into the molecular mechanisms by which membrane-active molecular agents selectively disrupt bacterial over mammalian membranes . 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 the above studies demonstrated that modelling and simulation can predict the ability of molecular agents to selectively penetrate into lipid membranes of pathogen, providing a theoretical basis to understand membrane activity and selectivity of membrane active nanomedicine. however, we also found that, while some molecular agents (such as pq401) can penetrate into the mammalian-mimetic membrane, they do not disrupt mammalian-mimetic guvs and red blood cells [31] . this observation suggests that penetration is only the first and necessary step, but not sufficient for the final rupture of the lipid membrane. more detailed experimental observations on the evolutions of guvs exposed to membrane-active compounds indicate that the entire interaction process leading to the final rupture of lipid membranes include further intricately coupled physical processes such as diffusion, domain aggregation, deformation, pore formation and growth (figs.1, 2d, 3d) , which raise a number of open questions. for example, what is the driving mechanism for domain aggregation? why is there a characteristic domain size? why does domain aggregation facilitate pore formation? what are the effects of homogeneous vs interfacial nucleation of pores? how does the spontaneous curvature of domain affect pore formation? how does the vesicle size affect pore formation? how to predict the energy barrier and time scales for pore formation? in this webinar, we highlighted some selected questions related to the characteristic domain size and pore formation and growth. regarding domain aggregation, consider an inclusion domain of inserted molecular agents with area in an infinite membrane . the domain adopts an axisymmetric shape while inducing an intrinsic curvature 0 . the membrane has bending modulus and is under remote tension σ . when the aggregated domain incorporates dispersed molecules, a mixing energy per unit area δ is induced, which includes the interaction energy between the dispersed molecules and lipids as well as the entropy change during the aggregation process. for simplicity we ignore the line tension on the domain interface. we find that if − 0 2 /2 < δ < 0, the domain has an optimal size. specifically, when − 0 2 /2 ≪ δ < 0, the domain size can be written as * ≈ − 8 δ σ 0 2 . thus, we conclude that a negative mixing energy drives domain growth while the intrinsic curvature and surface tension limits its size. we also modeled the pore (denoted by ) growth in the presence of an inclusion domain. apart from the bending energy and in the inclusion and the lipid membrane, the system energy also includes three line tensions, namely − on the lipid-inclusion interface, − on the lipid-pore interface and − on the inclusion-pore interface. we find that both the bending energy and the lipid-inclusion line tension contribute a negative energy variation during the pore growth process, thereby facilitating the pore growth. following this discovery, it is also shown that smaller vesicles have lower energy barrier for rupture since their high curvature lead to higher bending energy, a conclusion in line with the experimental observations [31] . such size effect can potentially be utilized to understand the recently developed antiviral agent by cho et al. [19] with a strong size-based selectivity. they showed experimentally that this antiviral agent can selectively rupture smaller liposomes and have a broadspectrum efficacy against a wide range of viruses. given that sars-cov-2 possesses a similar size, this antiviral agent may also be a potential candidate for treating covid-19. we also probed the influence of domain size on pore formation. with numerical solutions, we find that larger domains exhibit lower energy barrier for pore growth than smaller ones. as the interfacial pore grows, the domain shape remains almost unperturbed in large domains. yet in smaller domains, either the shape of the pore or the domain can be distorted with substantial increase in the energy cost. in this webinar, we summarized some recent studies with a long term goal to establish a simulationassisted discovery platform for membrane active nanomedicine capable of selectively penetrating and aggregating in bacterial/viral lipid membranes, causing membrane permeability/rupture. our multiscale modelling and simulation studies demonstrated that the membrane activity of molecular agents correlates with their ability to enter, perturb and permeabilize the lipid bilayers. further study on different cell membranes revealed the molecular mechanism of cholesterol-based selectivity. following the molecular penetration, we established a continuum theory and derived the energetic driving force for domain aggregation and pore growth on lipid membranes. we showed that there exists an optimal domain size and that the energy barrier to membrane pore formation can be significantly lowered through molecular aggregation on a large domain with intrinsic curvature and a sharp interface. the curvature effect of lipid membrane derived from the theory may have important implications to develop membrane targeting antiviral agents with size-based selectivity. the current drug discovery process for antibiotics using biological screening methods (such as c. elegans survival [22] ) are usually expensive, time consuming and inefficient. moreover, it has been difficult to identify the underlying mechanisms of positive hits in the screening and improve them subsequently. here, we showed that mechanistic modelling and simulations can help reveal some fundamental principles on how molecular antimicrobials interact with bacterial membranes and damage them through domain aggregation and pore formation. our study highlights a promising approach to establishing a simulation-assisted platform to accelerate the screening, assessment, design and optimization of membrane targeting nanomedicine at reduced cost against infectious diseases and, more importantly, to effectively counter increasing challenges with pathogen resistance/mutation. this work was supported by the u.s. national science foundation (grant cmmi-1562904) and a start-up grant from the nanyang technological university and institute of high performance computing, a*star, singapore. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 targeting non-multiplying organisms as a way to develop novel antimicrobials non-inherited antibiotic resistance targeting bacterial membrane function: an underexploited mechanism for treating persistent infections persister cells clinical practice guidelines by the infectious diseases society of america for the treatment of methicillin-resistant staphylococcus aureus infections in adults and children metabolite-enabled eradication of bacterial persisters by aminoglycosides activated clpp kills persisters and eradicates a chronic biofilm infection novel antibody-antibiotic conjugate eliminates intracellular s. aureus secretary-general, o.f. the, u. nations, no time to wait: securing the future from drug-resistant infections report to the secretary-general of the united nations april 2019 summary of recommendations and key messages summary of iacg recommendations a exploiting current understanding of antibiotic action for discovery of new drugs growth of mycobacterium tuberculosis biofilms containing free mycolic acids and harbouring drug-tolerant bacteria the evolution and emergence of rna viruses global spread of sars-cov-2 subtype with spike protein mutation d614g is shaped by human genomic variations that regulate expression of tmprss2 and mx1 genes phylogenetic network analysis of sars-cov-2 genomes we shouldn't worry when a virus mutates during disease outbreaks a genomic perspective on the origin and emergence of sars-cov-2 therapeutic treatment of zika virus infection using a brain-penetrating antiviral peptide methicillin-resistant staphylococcus aureus process of inducing pores in membranes by melittin whole animal automated platform for drug discovery against multi-drug resistant staphylococcus aureus steered molecular dynamics and mechanical functions of proteins the weighted histogram analysis method for free-energy calculations on biomolecules. i. the method free weighted histogram analysis implementation including robust error and autocorrelation estimates a new class of synthetic retinoid antibiotics effective against bacterial persisters complementary biophysical tools to investigate lipid specificity in the interaction between bioactive molecules and the plasma membrane: a review microsecond molecular dynamics simulations of lipid mixing understanding the diversity of membrane lipid composition a selective membrane-targeting repurposed antibiotic with activity against persistent methicillin-resistant staphylococcus aureus the neutrally charged diarylurea compound pq401 kills antibiotic resistant and antibiotic tolerant staphylococcus aureus, mbio key: cord-341378-pw60qx7c authors: armstrong, john; niemann, heiner; smeekens, sjef; rottier, peter; warren, graham title: sequence and topology of a model intracellular membrane protein, e1 glycoprotein, from a coronavirus date: 1984 journal: nature doi: 10.1038/308751a0 sha: doc_id: 341378 cord_uid: pw60qx7c in the eukaryotic cell, both secreted and plasma membrane proteins are synthesized at the endoplasmic reticulum, then transported, via the golgi complex, to the cell surface(1–4). each of the compartments of this transport pathway carries out particular metabolic functions(5–8), and therefore presumably contains a distinct complement of membrane proteins. thus, mechanisms must exist for localizing such proteins to their respective destinations. however, a major obstacle to the study of such mechanisms is that the isolation and detailed analysis of such internal membrane proteins pose formidable technical problems. we have therefore used the e1 glycoprotein from coronavirus mhv-a59 as a viral model for this class of protein. here we present the primary structure of the protein, determined by analysis of cdna clones prepared from viral mrna. in combination with a previous study of its assembly into the endoplasmic reticulum membrane(9), the sequence reveals several unusual features of the protein which may be related to its intracellular localization. in the eukaryotic cell, both secreted and plasma membrane proteins are synthesized at the endoplasmic reticulum, then transported, via the golgi complex, to the ceu surface 1 -4. each of the compartments of this transport pathway carries out particular metabofic functions 5-8, and therefore presumably contains a distinct complement of membrane proteins. thus, mechanisms must exist for locafizing such proteins to their respective destinations. however, a major obstacle to the study of such mechanisms is that the isolation and detailed analysis of such internal membrane proteins pose formidable technical problems. we have therefore used the el glycoprotein from coronavirus mhv • a59 as a viral model for this class of protein. here we present the primary structure of the protein, determined by analysis of edna clones prepared from viral mrna. in combination with a previous stu'!} of its assembly into the endoplasmic reticulum membrane , the sequence reveals several unusual features of the protein which may be related to its intracellular localization. the coronaviruses are a diverse class of enveloped rna viruses of considerable medical and agricultural significance; they also provide a model for the study of persistent viral infections (see ref. 10 for review). in contrast to many enveloped viruses, the corona virus mouse hepatitis virus (mhv) a59 buds inside the cell, into the lumen of the endoplasmic reticulum 11 the assembled virion then appears to travel, via the golgi complex, to the cell surface. of the two viral membrane proteins, the smaller one, el, is necessary for formation of the envelope, and is restricted to internal cell membranes; apparent?; it only reaches the cell surface as part of the budded virion 12 • 3 . thus, the el glycoprotein is potentially a convenient model for studying those features of a membrane protein that determine its arrest at a particular destination on the membrane transport pathway. the mrnas of mhv-a59 form a 'nested set': the seven rnas share the 3' region of the positive-stranded genome, but extend to different lengths towards the 5' end 15 -18 • from each rna, only the 5' gene is translated 19 ' 20 . in addition, a noncoding 'leader' sequence of approximately 70 bases, from the 5' end of the genome, is common to the mrnas 18 • 21 • 22 . the e1 gene is second from the 3' end and is therefore translated from the second smallest mrna, rna 6 (refs 19, 20) . the sequence of the 3' -terminal gene, encoding the viral nucleocapsid protein, has been determined previously 23 • 24 • copy dna clones spanning the e1 gene were prepared by two methods 23 -25 and sequenced in the vectors m13mp8 (ref. two versions were found, in two different clones, for the sequence immediately upstream from the e1 initiator codon. the shorter one is shown in fig. 1 ; in the second clone, an additional copy of the pentanucleotide a tct a was found between nucleotides 65 and 66, making the sequence similar to that of the region adjacent to the nucleocapsid gene of another strain of mhv 29 . this difference could represent a mutation; alternatively, it may reflect heterogeneity in the normal mrna population. indirect support for the latter possibility comes from the observation that a rnase-t 1 oligonucleotide from this region of rna 6, corresponding to the shorter sequence, was recovered in markedly lower yield than those from the rest of the molecule 30 • this site represents the point of fusion between the 5' leader sequence and the coding portion of the rna. the fusion is thought to occur by 'jumping' of the viral rna polymerase to particular sites on its genome-length, negativestranded template; the resumption of transcription then produces each of the subgenomic mrnas 22 " 31 ' 32 • thus, it seems possible that the polymerase may jump to more than one point on the template for each mrna, generating variable numbers of the repeated pentanucleotide aucua in the resulting transcript. figure 1 shows the amino acid sequence encoded by the e1 gene. the predicted molecular weight of the protein is 26,000, slightly higher than that observed by gel electrophoresis 19 • 33 but consistent with the unusual electrophoretic behaviour of this 33 , and other, hydrophobic proteins. several features of the protein, when assembled into membranes in the virus 33 , or in vitro 9 , are reflected in the sequence. first, in contrast to the majority of membrane proteins, el is known to lack a cleaved 'signal peptide' 9 : the n-terminal region of the sequence contains no good candidate for a cleava3e site 34 • second, the n-terminal region bears 0-linked sugars 5 ' 36 , which, uniquely among viral proteins so far studied, are the only known post-translational modification to el. assuming that the terminal met is removed 37 , then-terminal sequence is ser-ser-thr-thr, which is identical to the 0-glycosylated amino terminus of m-type glycophorin a (ref. 38 ). the 0-linked sugars of el are themselves identical to those found in glycophorin 39 . third, most of the protein is resistant to proteolysis when assembled in the membrane. only 2.5 kilodaltons of polypeptide from the nterminus are cleavable on the luminal side of the membrane (or outside the virion) and 1.5 kilodaltons from the c-terminus from the cytoplasmic (or intra-virion) side 9 , suggesting that the protein is largely buried in the membrane. in the sequence, a run of 22 uncharged residues from positions 26 to 4 7 represents a potential membrane-spanning region; residues 1-25 correspond to the portion removable by protease. a further sequence of uncharged residues, positions 57-106, is sufficiently long to cross the membrane twice more. if this region is divided in two, and each half plotted as an a-helical 'wheel', all the polar side chains of both sections cluster within 140°. thus, a plausible conformation for this region is two hairpinned helices in the membrane, with adjacent polar faces (fig. 2a) . there are no other long hydrophobic sequences, implying that the region from residues 107 to -190 is either folded in the membrane to neutralize charges, or, more likely, is adjacent to the membrane but resistant to proteolysis. these features are summarized in fig. 2b . which, if any, of these various features might be responsible for the protein's intracellular localization? we do not know, for example, whether the protein has an active 'signal' causing its arrest on the transport pathway, or, alternatively, if it lacks a signal for onward transport; nor do we know whether a sorting process might operate on one or the other side of the membrane. the availability of a edna clone for the protein presents the opportunity to investigate these questions by allowing expression of the cloned dna and in vitro mutagenesis. this approach has already been applied to two other viral glycoproteins, to investigate the importance of their cytoplasmic domains for transport to the cell surface, yielding opposite conclusions 40 • 41 • an intrinsic problem with the method, however, is the difficulty of distinguishing specific effects due to alterations at the site of mutagenesis, from a general structural disruption of the molecule. in this respect the e1 protein may be advantageous in that it provides the possibility of creating a more 'active' phenotype in the mutated molecule: specifically, particular alterations to the protein may result in its transport to the cell surface. we thank willy spaan for communicating results before publication, g. heisterberg-moutsis (g. b. f. braunschweig) for help with oligonucleotide synthesis, ben van der zeijst for discussion and annie steiner for preparing the manuscript. j.a. was supported by fellowships from the royal society and the outside (lumenl fig. 2 a, distribution of polar side chains in the hydrophobic regions of the el sequence. residues (1), 57-81 (2) and 82-106 (3) are plotted as a-helices and viewed endon. polar side chains are boxed: proposed hydrophilic faces of helices 2 and 3 are indicated. b, possible topologies of the el protein across the membrane. arrows indicate sites accessible to protease; broken arrows represent inefficient pro-teolysis9. european molecular biology organization, h.n. by the deutsche forschungsgemeinschaft, sfb4 7 (virologie), teilprojekt b3, and p.r. by a short-term embo fellowship. some of these results have been presented in preliminary form elsewhere 24 • 25 • proc. natn. acad. sc< u.s.a molecular biology and pathogenesis of coronaviruses molecular biology and pathogenesis of coronaviruses key: cord-298019-gf2asni1 authors: galdiero, stefania; falanga, annarita; morelli, giancarlo; galdiero, massimiliano title: gh625: a milestone in understanding the many roles of membranotropic peptides date: 2014-10-12 journal: biochim biophys acta biomembr doi: 10.1016/j.bbamem.2014.10.006 sha: doc_id: 298019 cord_uid: gf2asni1 here, we review the current knowledge about viral derived membranotropic peptides, and we discuss how they may be used for many therapeutic applications. while they have been initially discovered in viral fusion proteins and have been involved in the mechanism of viral entry, it is now clear that their features and their mode of interaction with membrane bilayers can be exploited to design viral inhibitors as well as to favor delivery of cargos across the cell membrane and across the blood–brain barrier. the peptide gh625 has been extensively used for all these purposes and provides a significant contribution to the field. we describe the roles of this sequence in order to close the gap between the many functions that are now emerging for membranotropic peptides. over the past few decades peptides have progressively achieved increased value in drug design and pharmaceutical delivery. moreover, great interest has been dedicated to the identification of peptides as drug candidates. the number of peptides in the pharmaceutical industry is continuously growing and about 10% of the entire drug market is represented by peptide based drugs [1, 2] . bioactive peptides can be derived from natural sources or can be discovered through rational engineering, high-throughput screening, or structure-based design starting from defined protein regions [3] . among the many peptides playing a relevant role in biology, some show a high propensity for binding to lipid membranes due to their simultaneous hydrophobic and amphipathic nature. this class of hydrophobic peptides is characterized by the presence of unusual conspicuous amounts of alanine and glycine residues and sometimes also prolines. such a degree of ala/gly content is uncommon for hydrophobic domains such as signal sequences and transmembrane anchors; in fact, their presence may account for the intrinsic conformational flexibility which is a typical feature of membrane interacting peptides. also aromatic residues are generally present and dominate the interactions that take place at this unique physical-chemical environment of the water-membrane interface [4] . the favorable interactions of aromatic side chains with phospholipid moieties located at the membrane interface contribute to the insertion of the peptide into the bilayer. amphipathicity is a key feature of these peptides. the term amphipathicity generally refers to molecules with both hydrophilic and hydrophobic faces [5] . peptides can be amphipathic in their primary structure or secondary structure. primary amphipathic peptides correspond to the sequential assembly of a domain of hydrophobic residues with a domain of hydrophilic residues divided by a spacer domain; while secondary amphipathic peptides are generated by the conformational state which allows positioning of hydrophobic and hydrophilic residues on opposite sides of the same molecule. in particular, amphipathic, hydrophobic peptides present one face with large and aromatic residues and the other with small residues such as ala/gly. this distribution of amino acid residues facilitates the membrane interaction and peptide insertion into the bilayer [6] . conformational polymorphism plays a key role; in fact, the ability to shift from random to α/β conformations as a consequence of membrane composition and peptide concentration has emerged as a common structural pattern for this class of peptides [6] . there are several types of membrane active peptides which can be roughly divided in antimicrobial peptides [7] , viral peptides [8] and cell penetrating peptides [9] . although very different in primary sequence one from the other, it may be hypothesized that their common physical features could result in a shared mechanism of action and essentially determines the many roles that they can play in nature. among the hydrophobic peptides with a propensity for membrane binding, characterized by a high interfacial hydrophobicity or amphipathicity, the ones derived from enveloped virus glycoproteins are attracting considerable attention. these peptides can interfere with enveloped virus entry by direct physical interaction with the hydrophobic surfaces present on membranes and/or fusion proteins and are, thus, critical for both fusion and entry. viral glycoproteins undergo conformational changes as a consequence of either low endosomal ph or receptor binding which leads to the exposure of hydrophobic peptides, loops or patches, which then interact with and destabilize one or both the opposing membranes. crystallographic data on the post-fusion structures of viral fusion proteins have allowed the identification and characterization of three different classes [10, 11] . class i fusion proteins are characterized by trimers of hairpins with a central α-helical coiled-coil structure and have been identified in orthomyxoviruses, paramyxoviruses, retroviruses, filoviruses and coronaviruses [12] [13] [14] [15] [16] . class ii fusion proteins are present on viral envelopes as pre-fusion dimers which convert into post-fusion trimers of hairpins composed of β structures and have main representatives in the flaviviridae and togaviridae families [17, 18] . class iii fusion proteins are characterized by a central α-helical trimeric core similar to class i and two fusion loops located at the tip of an elongated β-sheet similar to class ii fusion proteins and members are present in herpesviridae and rhabdoviridae families [19] . despite several differences in the mechanism of entry elicited by the three classes of fusion glycoproteins, they all induce membrane fusion in a similar manner through the formation of an analogous hairpin structure which allows fusion peptides to insert into cell membranes and to drive membrane destabilization. thus, during the viral entry process, the hydrophobic surfaces that become exposed are characterized by somewhat variable but at the same time detailed physical characteristics which include the size, shape and secondary structure of exposed hydrophobic patches, as well as the nature of the neighboring polar or charged residues. the wimley-white interfacial hydrophobicity scale (wwihs) is an experimentally-determined free energy scale that calculates the propensity of individual amino acids in peptide sequences to partition from water into a phosphatidylcholine interface [20, 21] and has been effectively used to identify fusion peptides in viral glycoproteins. the hydropathy analysis allows calculating a hydrophobicity score along the sequence of a protein, identifying segments with a propensity to interact with membrane interfaces. wwihs values are calculated assuming random coil peptides partitioned into the bilayer interface; the values are minimum possible values and the δg may get more favorable if peptide binding also promotes an increase in secondary structure [22, 23] . the interfacial helical hydrophobic moment (ihhm) is a further physico-chemical factor that is important for membrane interactions and secondary structure formation of peptides bound to membrane interfaces. the ihhm describes the degree to which a peptide sequence would have segregated hydrophobic and hydrophilic faces if it folded into an α-helix [24] . a peptide with a large ihhm can interact strongly with membranes as a helix due to partitioning-folding coupling [22, 23] even with a wwihs score that is not positive overall. the presence of the fusion peptide within the ectodomain exposed to the aqueous phase is a feature shared by all viral fusion proteins and constitutes an absolute requirement for their fusogenic activity. fusion peptides are typically 20-30 residues long and potentially fold into amphipathic helices and are rich in glycines and alanines, providing them a high degree of conformational flexibility. thus, their structure is polymorphic and strongly dependent on the environment. the fusion peptide of influenza virus, for example, has been observed in random coil, α-helical and β-sheet conformations in different environments [25] (fig. 1) . it has been proposed that all three forms have some physiological relevance; the peptide may be unstructured in solution on the way to the target membrane; it may be helical at low concentrations but may self-associate in β-sheets at higher concentrations in the membrane interface. the fusion peptide of hiv also undergoes conformational transitions; it adopts α helical or β sheet structures depending on concentration, lipids and ionic conditions [26] . the helical form of the influenza fusion peptide is probably a key determinant to promote fusion. the structure of the peptide in membrane shows a kink which separates the n terminal and the short c terminal helices which together form a boomerang shaped structure [27] . both helical arms are amphipathic with bulky hydrophobic residues facing the membrane interior. the conserved n-terminal glycine residue is critical for fusion and for the correct structure of the peptide inside the membrane; in fact, when it is mutated to a valine the n-terminal helix is partially unwound and the fusion peptide is inactive [28] . actually, there are numerous studies demonstrating that a delicate balance between α and β structures, is essential for membrane fusion and is influenced by environmental conditions such as ph, ionic strength, peptide sequence, presence or absence of divalent cations, cholesterol content and also by the lipid/peptide ratio. for instance, studies performed on the ebola fusion peptide, show that the conformational transition from an α-helix to a β-sheet is induced by a change in the peptide to lipid ratio in the membrane [29] . at low peptide concentration in lipids, it is essentially an α-helix; while as the local peptide concentration increases in the membrane, the proportion of α-helix drops off in favor of a mainly antiparallel β-sheet structure. this concentration dependent effect on peptide conformation might be of biological relevance [29] . the functional meaning of the conformational polymorphism is unclear, although it is believed to be fundamental to enable backbone reorientation of the fusion protein; therefore, the ability to insert at various levels might be required for the evolving of final stages of the fusion cascade [30] . independently from the principal conformational organization, the degree of insertion plays a key role for inducing membrane fusion. it has also been hypothesized that [31] fusion domains first assemble as β-sheets on the surface of the membrane and later convert into α-helices to complete fusion. aromatic residues are generally present in fusion peptides and may help in overcoming the energy cost of peptide bond partitioning into membranes. the interactions with phospholipid moieties located at the membrane interfaces may also help in stabilizing the insertion into just one leaflet of the bilayer. the initial interaction with the external leaflet is thought to generate elastic stresses which drive to bilayer fusion, helping to overcome the hydration repulsion forces between approaching bilayers by orienting the poorly solvated face toward the external medium [32] . the asymmetric insertion into one membrane monolayer may promote expansion of the polar head region and determine a curvature stress onto the overall lipid bilayer; the created bulges that protrude from the membrane can facilitate the formation of lipid contacts between fusing bilayers [33] . particular attention has also been devoted to the effect of additional membranotropic sequences on the overall fusogenicity. the presence of additional fusogenic sequences was evidenced in sendai f1 [34] , measles f1 [35] , sars-cov s2 [36] , hepatitic c virus e1 and e2 [37] , dengue e [38, 39] and herpes virus gb and gh [40] [41] [42] . the idea that a single fusion peptide is the solely responsible for the complete membrane fusion event has been substituted by the assumption that a concerted action of different membranotropic regions is necessary for membrane interacting/perturbing activity. as a matter of fact, also membrane proximal regions (pre-tm) play a key role in fusion [41, [43] [44] [45] . the pre-tm domains are particularly rich in aromatic residues which enable them to insert into the membrane interface. effective therapeutics against enveloped viruses are still scarcely represented. a few drugs have been developed against hiv, influenza virus, hepatitis virus and a few other viruses but they are still not ideal and in some cases have proved to induce resistance [46] . as a consequence, for most enveloped viruses, there are no effective therapies and entry inhibitors represent an interesting and underutilized target. peptides with a propensity for membrane binding can also interfere with enveloped virus entry by direct physical interaction with the hydrophobic surfaces present on cell membranes and/or fusion proteins. as recently reviewed by badani et al. [46] , there are many peptide inhibitors that are somewhat hydrophobic and/or amphipathic with a propensity to bind to bilayer membrane interfaces and other hydrophobic surfaces. it is not known whether membrane binding directly affects viral fusion, or whether interaction with the fusion protein itself is an absolute requirement for entry inhibition. it is widely accepted that membrane binding of an inhibitory peptide will greatly increase the effective concentration of the peptide close to the fusion protein, indicating that the interaction with membrane and the interaction with the fusion protein may be effectively coupled [47] . as a matter of fact, the potential of numerous fusion peptides and/or membranotropic peptides derived from proteins of enveloped viruses as entry inhibitors has been widely described in literature [48] [49] [50] [51] . the accepted view is that the inhibition of infectivity may be due to the formation of inactive aggregates between the fusogenic stretches present in both the viral protein and the synthetic peptides. these aggregates are formed as a consequence of their ability to oligomerize or to mimic the modes of binding of their original domains in their partner protein. it has been hypothesized that they stabilize a pre-fusion intermediate and prevent merging of the bilayers. it is now evident that several domains are essential for membrane fusion and thus peptides involved in the fusion mechanism may all interfere with the intramolecular interactions between the several domains and may represent interesting targets for the design of entry inhibitors. thus, membrane physical properties could be critically important to the events that drive viral entry and it is conceivable that peptides interfere with the function of viral fusion proteins by changing the physical chemistry of the membrane itself by direct interaction. many laboratories are working to unravel the mechanism of action of viral membranotropic peptides and several hypotheses have been proposed. many studies suggest that multiple mechanisms may take place simultaneously. indirect ways in which interfacial binding peptides can affect viral entry have also been hypothesized. selfoligomerization of membrane embedded fusion peptides has been proposed to be responsible of inhibition [52, 53] . the most studied case is that of hiv, where the inhibition has been attributed to the formation of structurally defined oligomeric complexes [54, 55] ; while mutants with a lower helical content and tendency to self-associate into β-sheets [56] are able to inhibit membrane fusion at various stages. it is interesting to note that there are clinical studies on a peptide called virip, which is designed as an inhibitor of the hiv fusion peptide [57, 58] . this sequence was able to block hiv-1 infection by targeting gp41 fusion peptide [58] and optimized versions of this sequence proved to be as potent as inhibitors targeting the coiled coil sequences and moreover were devoided of cellular toxicity. a 10-day monotherapy clinical trial enrolling 18 hiv-1 infected patients [57] showed that the drug can be well tolerated by patients and reduces their plasma viral load. its identification and clinical evaluation represent the first proof of concept that membranotropic sequences could suppress viral replication in infected individuals and have potential clinical effectiveness. the hypothesis that peptide entry inhibitors act by a physicalchemical interaction with hydrophobic surfaces exposed during the fusion process suggests that this novel approach may be a general rule; moreover, instead of focussing on the structure-based design, it would be possible to design novel hydrophobic/amphipathic inhibitors which could be easily made protease resistant by the introduction of nonnatural or d-amino acids. the membrane bilayer represents a semi-permeable barrier, defining the interior of an individual cell; its existence confers cells their potential to survive and function properly. nevertheless, crossing of the cellular membranes remains one of the major obstacles for the proper delivery of therapeutics [59, 60] . the lipophilic nature of biological membranes restricts the direct intracellular delivery of most compounds; whereas small molecules and ions can diffuse across the bilayer, larger molecules are generally excluded from simple diffusion into the cell. the differing hydrophobicity/hydrophilicity of the lipid membrane renders the transfer across this barrier extremely difficult due to differences in solubility. notwithstanding the therapeutic potential of a number of novel molecules, their pharmaco-distribution properties hamper the possibility to reach the stage of pharmaceutical preparations and stimulate industrial interest; in fact, these molecules need to be delivered intracellularly to exert their therapeutic action inside the cytoplasm or onto individual organelles. it is, thus, evident that the therapeutic potential of a drug is largely dependent on the development of delivery tools able to selectively and efficiently carry it to target cells with minimal toxicity. the translocation across the membrane is by far less well understood than the binding step. there is a significant similarity in the physico-chemical parameters between membrane partitioning peptides and membrane translocating peptides [61] . a novel intriguing hypothesis is that hydrophobic peptides that partition into membranes may also be able to cross cell membranes and enter cells. therefore, these peptides may also cross endothelial layers in vivo, including the blood-brain barrier [62, 63] . delivery across cellular membranes involves several membrane reorganization processes such as transient permeabilization of the cell membrane, which are similar to the ones involved in the entry of viruses. membrane fusion and its disruption are related processes, although leakage and fusion capacities of peptides do not always correlate and the features/activities of membranotropic peptides may depend on particular environmental and temporal conditions. since not all membranotropic peptides are able to cross the membrane bilayer, it is essential to identify structural characteristics of hydrophobic peptides know to enter the cell membrane to highlight any feature that is involved in the penetration which may help in the design of novel delivery tools. thus, an important feature to consider is the structural requirements for cellular uptake and the ability of membranotropic peptides to interact with the cell surface and lipid moieties of the cell membrane. a very complete review describing the binding and translocation of membrane active peptides has been recently published [64] which highlights the fact that peptide translocation is not coupled with dye flux. graded dye flux would occur concomitant with peptide translocation, which would explain incomplete dye release; whereas all-or-none flux would occur with peptides unable to translocate, and therefore these peptides accumulate on the membrane until a rupture point is reached, resulting in complete dye release [64] . cell-penetrating peptides (cpps) have been widely used due to their capability to transport several kinds of macromolecules across the membrane bilayer in vitro and in vivo [65] [66] [67] . cpps are short and usually basic amino acid rich peptides originating from proteins that are able to cross biological barriers, such as the viral tat protein. although the uptake mechanism of cpps is still debated, it seems to involve mainly the endocytic pathway, trapping the conjugated cargo in endosomes eventually ending in lysosomes where common enzymatic degradation mechanisms take place, therefore leading to a limited delivery of therapeutic agents to the intracellular target. hydrophobic peptides that efficiently traverse biological membranes, promoting lipid-membrane reorganizing processes, such as fusion or pore formation and involving temporary membrane destabilization and subsequent reorganization [8, 68] , may be able to circumvent the endosomal entrapment either favoring the escape from the endosome or by translocating a cargo through the plasma membrane directly into the cytosol. this idea has been exploited to design the drug delivery tool called mpg. mpg is an amphipathic peptide whose primary sequence is composed of the hydrophobic amino acids of the hiv-1 fusion peptide (galflgflgaagstmga) associated to a hydrophilic domain derived from the nuclear localization sequence (nls) of simian virus 40 (sv40) large t antigen (pkkkrkv). these hydrophilic and hydrophobic segments are separated by a three amino-acid spacer (wsq) [69, 70] . this peptide exploits the known properties of the glycine-rich hiv fusion peptide essential for membrane fusion activity and the nls of the sv40 large t antigen to improve the nuclear addressing of the peptide [71, 72] . at the moment this is the only viral fusion peptide that has been widely exploited for applications in drug delivery. a milestone in understanding the role of hydrophobic viral peptides is represented by the sequence "gh625" derived from glycoprotein h of herpes simplex virus type i. herpes simplex virus (hsv) is an important human pathogen, responsible for significant morbidity and mortality worldwide and is characterized by a complex multi-component entry machinery. hsv enters host cells by fusion of the viral envelope with either the plasma membrane or an endosomal membrane, and the entry pathway is likely determined by both virus and host cell factors and involves multiple viral glycoproteins and cellular receptors in a cascade of molecular interactions [73] [74] [75] [76] . the envelope glycoproteins gh/gl, gb and gd are all essential for the entry process and their expression is able to induce the fusion of cellular membranes in a virus-free system [77, 78] . both gh/gl and gb constitute the core fusion machinery and cooperate to induce the initial lipid destabilization that ends in fusion [79] and both gb and gh contain several membranotropic sequences [40] [41] [42] 49, 50, [80] [81] [82] . although it has recently become available the crystal structure of the gh-gl complex [83] , it is still debated whether gh is merely a fusion regulator or it plays a more direct role in the fusion process and many studies suggest that the gh-gl complex may undergo dynamic rearrangements [77, 84] . in particular, some peptides derived from the gh ectodomain block virus entry, while others have the ability to bind and disrupt model membranes. gb is considered a canonical class iii fusion protein and has been demonstrated to be involved in virus attachment, penetration and cell-to-cell spread. the crystal structure of gb is a trimer in which multiple contacts between protomers throughout the molecule contribute to its stability [85] . it has been hypothesized [86] that gb refolds similarly to class i fusion proteins and that the packing of the c-terminal arm against the coiled-coil provides the driving force for gb refolding from the prefusion to the post-fusion conformation. the gb structure corresponds to a post fusion conformation and it is now widely accepted that gb undergoes conformational changes upon variations of ph in order to bring about fusion [87] [88] [89] [90] . several synthetic gb peptides induced the fusion of large unilamellar vesicles and inhibited herpes virus infection [42, 91] . when the crystal structures of gb and of gh/gl were not yet available, we reported the identification of several sequences in gh and gb with the ability to interact with the membrane and among these sequences there was also the canonical fusion peptide of gb [40, 42, 80, 92] . although it is not yet well understood the role played by the other membranotropic sequences and in particular by the glycoprotein gh in the whole fusion process, it is now widely accepted that several regions in the fusion glycoproteins are involved in the local destabilization of the membrane bilayer which ends in the fusion of the viral envelope with the host cell membrane. gh625 was first selected and characterized by our group in 2005 [40] and still is the hsv-1 peptide with the highest fusion capability and the most widely studied. it was initially identified using the wwih scale and subsequent works allowed determining the many applications of this sequence from membrane fusion, to viral inhibition and drug delivery [63, 93, 94] . the twenty residue peptide gh625 (from aa 625 to aa 644) is a membrane-perturbing domain, (fig. 2) which interacts with biological membranes and is implicated in the merging of the viral envelope and the cellular membrane [42, 50] . the peptide contains residues crucial for its capacity to interact and destabilize target lipid membranes. it is rich in hydrophobic residues including glycines, leucines, alanines, and aromatic residues such as tryptophan and tyrosines, which are known to be located preferentially at the membrane interface. the peptidelipid interactions are initiated by the arginine residue located at the c-terminus; in fact, when the arginine is mutated, the fusogenic activity of the peptide is strongly impaired. the hydrophobic domain is also crucial for its insertion into the membrane and further supports the view that hydrophobic interactions between fusion proteins and cellmembrane phospholipids initiate membrane perturbation in the early stages of viral fusion. the many biophysical experiments performed on gh625 have shown that the peptide interacts with model membranes, penetrates the bilayer from its n-terminal side, has a tryptophan residue buried inside the bilayer, and adopts a helical conformation with its hydrophobic residues on one face of the helix and polar or charged residues on the opposite face [40, 49, 50] . the analysis of peptides with longer and shorter sequences derived from this region and of their interactions with membranes clearly demonstrated that the activity of this region depends on the amino acid sequence and on its length. the presence of a histidine residue at the n-terminus of the native sequence strongly increases the fusion activity [50] . the importance of a single histidine residue as a switch for triggering viral fusion was also reported for other viruses [95] such as paramyxoviruses, therefore supporting the importance and specificity of the histidine moiety in activating fusion. furthermore, a conserved histidine in one of the fusion loops of semliki forest virus e1 protein was found to be fundamental [96] . the histidine in gh625 both helps the initial interactions with the membrane and the oligomerization process [50] . this hypothesis is further supported by the fact that the histidine is located at the n-terminus and correct configuration of the n-terminal fusion peptide appears to be crucial for the fusogenic function of several fusion proteins as well as its location in the membrane core after peptide-bilayer interaction. the addition of one histidine at the n-terminus of gh625 is sufficient to make the peptide approximatively 8-fold more active. in particular, the addition of any other residue at the n-terminus impaired the fusion ability of the sequence (data not published). gh625 strongly interacts and spontaneously penetrates the lipid-phase and inserts into membranes with a α-helical structure [50, 81, 82] . both the tryptophan and tyrosine are on the same side of the helix in the three-dimensional structure, forming an amphiphilic helix in which one side is constituted by aromatic and hydrophobic residues, whereas the other side is formed by hydrophilic or small residues. the interaction between the aromatic ring of tryptophan and the side chain of tyrosine is important for maintenance of structural stability during the interaction with the membrane. an amphipathic α-helix is believed to be an important feature of membranotropic peptides playing a crucial role for mediating lipid-protein interactions during the binding of proteins to membranes and once bound, the hydrophobic face of the amphipathic peptide would allow the peptide to enter the membrane interior, thereby triggering local fusion of the gh625 has the ability to penetrate deep into the bilayer as a helix without causing significant bilayer perturbations which may help explaining its ability to perform several different roles. gh625 showed a significant inhibitory effect and this effect appears conditioned by its ability to partition into membranes and aggregate within them. since the peptide self-associates in aqueous and lipid solutions, it is possible that it binds to its counterpart in the gh protein. gh625 may also interact with the host cell membrane, therefore its ability to moderately inhibit viral entry when cells are treated first, is dependent on the possibility that the virus will find a modified cell membrane still exhibiting on its surface the peptide [48] . moreover, gh625 does not have any activity in virus preincubation experiments, indicating that an eventual binding partner site on the pre-fusion gh protein is probably hidden and not available to interactions with free peptides, as also demonstrated by the analysis of the gh crystallographic structure. while the n-terminal histidine residue was proven to be fundamental for the interaction with the membrane bilayer and for translocation across the membrane, the absence of this residue induced similar levels of viral inhibition when compared with the full length peptide. the substitution of leu627 with a valine residue does not alter the hydrophobicity of the peptide, and does not influence its infectivity inhibition properties while its substitution with a polar residue (serine) substantially reduces its inhibitory activity [49] . recently, poly(amide)-based dendrimers functionalized at their termini with gh625 were shown to inhibit both hsv-1 and hsv-2 at a very early stage of the entry process, most likely through an interaction with the viral envelope glycoproteins; thus, preventing the virus from coming into close contact with cellular membranes, a prerequisite for viral internalization [97] . the 50% inhibitory concentration was 100 and 300 nm against hsv-1 and hsv-2 respectively, with no evidence of cell toxicity at these concentrations, indicating that the functionalization of a dendrimer with a membranotropic peptide represents a promising strategy for inhibition of viruses of the herpesviridae family. the multivalent display of gh625 on the dendrimer scaffold results in an almost six fold increase of antiviral activity for hsv-1 and two fold for hsv-2 in comparison to the activity of the dendrimer itself, and more than 100-fold increase in the activity of the unsupported peptide. the cytotoxicity profile measured by the mtt assay showed that the peptidodendrimer is not toxic to vero cells up to the highest concentration investigated in antiviral testing, while some toxicity was observed for the unfunctionalized dendrimer, especially at higher concentrations, demonstrating another advantage of the peptide functionalization [97] . any inhibitory activity was excluded when the compounds were added at a post-entry step and also when cells were pre-treated with the dendrimer derivatives, indicating that both the peptidodendrimer and the dendrimer are not able to interfere with viral replication once the virus has gained access to the cellular milieu. the peptidodendrimer might sterically hinder the gh relative domain, either in a pre-fusogenic or in an intermediate conformation, preventing a complete and functional interaction between gh and the membrane to fuse. the mechanism of inhibition may involve binding to gh itself through oligomerization of the gh625 domain present on the glycoprotein or interaction with other glycoproteins present on the virion envelope, such as gb or gd. the modification of a dendrimer scaffold with membranotropic peptides represents an attractive strategy for the design of a new class of antiviral drugs that exert their effect, coupling the intrinsic anti-viral properties of the dendrimer with the activity of membranotropic peptides and have the potential of being developed as multifunctionalized scaffolds to provide a therapeutic molecule to directly deliver to its target [97] . the inhibition of membrane fusion represents an attractive target for drug design and although further studies are needed to better define the exact mechanism of inhibition by hydrophobic peptides and the specific nature or location of their interactions with viral targets, the data obtained for gh625 suggest that hydrophobic domains play a significant role in membrane fusion and provide an alternative approach to the development of viral peptide inhibitors outside of the classical inhibitory heptad repeat regions. gh625 cellular uptake is associated with its hydrophobic and amphipathic characters which provide the necessary ability to interact with membrane lipids and to form a transient helical structure that temporarily affects membrane organization, thereby facilitating insertion into the membrane and translocation [98] . compared to tat peptide (a positively charged cpp) which mainly exploits the endocytic pathway, gh625 crosses membrane bilayers mainly through a translocation mechanism. a one amino acid shorter version of this fusogenic peptide was also found to improve the endosomal release of dna/lipofectamine lipoplexes and transgene expression up to 30-fold in human cell lines [99] . it has been recently demonstrated that gh625 is able to traverse the membrane bilayer and to transport into the cytosol several compounds, such as qds [98] , liposomes [100] , nps [62] , dendrimers [101] , and proteins [102] . examples of using gh625 as an intracellular delivery enhancer are provided in the remaining part of the paragraph (fig. 3) . qds are fluorescent probes under intense research and development for broad applications in molecular, cellular and in vivo imaging [103] . although considerable success has been achieved in using qds for labeling fixed cells and for imaging cell membrane proteins, only limited progress has been made for molecular imaging inside living cells because of their insufficient ability to traverse cell membranes. several authors have recently reported on the functionalization of qds with positively charged cpps and established that the main route of entrance is via endosomal uptake, therefore, escape from the endosomal system is of paramount importance [104] [105] [106] . gh625-qd internalization was demonstrated to be highly successful and to involve the endocytic pathway only to a minor extent [98] . liposomal aggregates have also attracted great attention due to their success as in vivo carriers of drugs [107] . to enhance the antitumor efficacy of liposomal drugs, the efforts of many research groups are directed toward the improvement of cellular internalization of liposomes through the addition of surface ligands and cell penetrating peptides. liposomes decorated with gh625 and loaded with doxorubicin (dox) [100] , were able to penetrate inside living hela cells. the results obtained suggest that the functionalization of liposomes with gh625 could affect the uptake mechanism of liposomes and their intracellular distribution and dox release. this evidence could be useful in the design of carriers for a controlled delivery and release of dox in order to avoid side effects associated to dox itself. dendrimers [108, 109] also represent a very promising tool for drug delivery, combining the advantageous features of nanoparticles (ideal size as in vivo carriers, multivalency), of polymeric materials (low cost, tunable properties, biocompatibility) and of small molecules (monodispersity and detailed control of their properties) [109, 110] . their surface modification by means of conjugation or adsorption of a biospecific ligand, may allow their delivery to specific sites and modulation of drug release minimizing toxic effects and increasing intracellular bioavailability [111] . thus, the dendrimeric scaffolds may be a promising tool for an efficient drug delivery engine. little information is available on the mechanism of dendrimer uptake and intracellular trafficking [112] . studies performed on pamam dendrimers [113] and pamam dendrimers functionalized with the tat [114] indicate that endocytosis mechanisms contribute to the internalization and intracellular trafficking and that adding the tat failed to enhance delivery efficiency. the attachment of gh625 to the termini of a poly(amide)-based dendrimer allows the conjugate to penetrate into the cellular matrix, whereas the unfunctionalized dendrimer is excluded from translocation. the peptide-functionalized dendrimer is rapidly taken into the cells mainly through a non-active translocation mechanism [101] . the combination of the benefits of dendrimers and peptides chemistry could be useful for the development of a selective carrier which could cross the membrane and be efficiently internalized into the cellular targets. many therapeutic drugs are excluded from entering the brain, due to their lack of transport through the blood-brain-barrier (bbb) [115] . the development of new strategies for enhancing drug delivery to the brain is fundamental in diagnostics and therapeutics of central nervous diseases (cns). most strategies to transport drugs inside the cns cause disruption of the anatomical texture of the bbb, therefore impairing its natural function; as a consequence, effective delivery approaches should be cautiously assessed considering their impact on the overall protective function of the bbb [116] . targeted delivery of a therapeutic cargo to the intended site of action in the brain appears to be one of the most promising non-invasive approach to overcome the bbb, combining the advantages of brain targeting, high incorporation capacity, reduction of side effects and circumvention of the multidrug efflux system [117] [118] [119] [120] . polystyrene nanoparticles (nps) decorated on their surface by gh625 showed that the uptake of nps with gh625 by brain endothelial cells was greater than that of the nps without the peptide and functionalized nps were free to move intracellularly [62] . most importantly, gh625 decreased np intracellular accumulation as large aggregates and enhanced the np bbb crossing. the surface functionalization with gh625 may change nps fate and provides a good strategy for the design of promising carriers to deliver drugs across the bbb for the treatment of brain diseases. whether multifunctional nanosystems, designed and tested in vitro, are able to properly work in vivo into mammalian hosts, is not fully granted. to address this issue, in vivo studies are necessary, thus validating design strategies and facilitating optimization and further functionalization. although numerous studies showed that gh625 is an efficient carrier for bioactive cargoes in vitro [121] , these results did not guarantee that it can be developed into a useful pharmaceutical delivery platform. the ability of gh625 to cross the bbb in vivo was also recently evaluated [122] . gh625 was administered in vivo to rats and its presence in the liver and in the brain was detected. within 3.5 h from its i.v. administration, gh625 can be found beyond the bbb in proximity fig. 3 . the many applications of gh625 to drug delivery. confocal microscopy images showing the internalization of gh625 functionalized: a) proteins [102] ; b) liposomes [100] ; c) qdots [98] ; d) dendrimers [101] . e) scanning electron microscopy images of functionalized polystyrene nanoparticles [62] . of cell neurites. gh625 has no toxic effect in vivo, since it does not affect brain maximal oxidative capacity and mitochondrial respiration rate. the data suggest that gh625, for its ability to cross the bbb, represents a novel nano-carrier system for drug delivery to the central nervous system. these results open new possibilities for direct delivery of drugs into patients in the context of theranostics and might address the treatment of several human diseases. other peptides have been proposed as a drug delivery system; it was demonstrated that tat was able to enter tissues in vivo in mice [123] ; antp was able to activate endogenous t cells in mice [124] . these peptides are highly positively charged, and absorptive-mediated transcytosis has been proposed for their transport across the bbb. a bradykinin analogue has also been reported to increase the penetration of small molecules by transitory opening of the bbb [125] . gh625 is the first viral membranotropic peptide which was shown to be a potential delivery system for macromolecules in vivo; these results coupled with previous in vitro data support the view that gh625 enters the bbb without involving endocytic processes. hence, the eventual cargo may be immediately and completely available [122] . the presence of multiple metabolic barriers may restrict the application of such peptide-based ligand for targeted drug delivery in vivo. peptides alone or conjugated on the surface of nanocarriers are subject to proteolysis in the blood after systemic administration. in addition, the bbb is also a metabolic barrier due to the presence of various enzymes in brain capillary endothelial cells. gh625 starts to be degraded after 1 h of incubation but the intact peptide is still present after 3.5 h of incubation and thus holds the potential for extending brain targeting efficiency due to its resistance to proteolysis for 3.5 h [122] . it is still under-recognized that some amino acid sequences in virtue of their specific features can play many different roles in nature. membranotropic viral peptides derived from fusion glycoproteins are widely studied especially for their ability to fuse membranes but there are many literature data also describing other roles besides membrane fusion. gh625 is an example of how these sequences can be employed for completely different purposes: fusion of membranes, viral inhibition and drug delivery. till now, gh625 is the only membranotropic peptide that has been extensively used for many applications and among them as a drug delivery system for the brain. in the development of new therapies to treat brain pathologies, the bbb represents a major obstacle against the use of potential drugs for treating disorders of the cns due to the impermeable nature of the cell membranes of this compartment to several molecules [115, 126] . the data reported on the in vivo application of gh625 for brain delivery, support the novel view that synthetic peptides derived from viral membranotropic sequences can be used successfully to deliver biologically active substances inside the bbb. the exact molecular mechanism of gh625 entry remains to be established but it appears to be a general feature of membranotropic peptides, which may be used for mediating delivery to virtually any tissue and in particular across the bbb, conveying a wide variety of cargoes with intact bioactivity into virtually any tissue or organ. other sequences have been found to be useful for drug delivery which happens to have the same features of the viral fusion peptide, indicating that it may be possible to design novel sequences with pre-determined characteristics which can be useful to treat many diseases. however, still much work has to be done on this type of peptides; we should not forget that their mechanism of perturbation of membrane bilayers may also allow the design of new membranotropic peptides with the ability to denature the membrane bilayer of bacteria and thus we may add to their many roles also the antibacterial activity which may represent an alternative to classical antibiotics in order to combat the antibiotic resistance problem. much of the vast literature on membranotropic peptides is devoted to single activities of these sequences; yet compelling structure-function relationship studies bridging the gap among all these activities are necessary. the future of peptide-based drugs synthetic therapeutic peptides: science and market therapeutic peptides: technological advances driving peptides into development the preference of tryptophan for membrane interfaces use of hydrophobic moment plot methodology to aid the identification of oblique orientated α-helices antimicrobial peptides and viral fusion peptides: how different they are? antimicrobial peptides: promising compounds against pathogenic microorganisms membrane fusion and fission: enveloped viruses recent progress of cellpenetrating peptides as new carriers for intracellular cargo delivery mechanism of membrane fusion by viral envelope proteins viral membrane fusion structure of the haemagglutinin membrane glycoprotein of influenza virus at 3 a resolution retrovirus envelope domain at 1.7 angstrom resolution crystal structure of the ebola virus membrane fusion subunit, gp2, from the envelope glycoprotein ectodomain structure of the parainfluenza virus 5 f protein in its metastable, prefusion conformation structural basis for coronavirus-mediated membrane fusion. crystal structure of mouse hepatitis virus spike protein fusion core the envelope glycoprotein from tick-borne encephalitis virus at 2 å resolution the fusion glycoprotein shell of semliki forest virus: an icosahedral assembly primed for fusogenic activation at endosomal ph class iii viral membrane fusion proteins hydrophobic interactions of peptides with membrane interfaces experimentally determined hydrophobicity scale for proteins at membrane interfaces folding of β-sheet membrane proteins: a hydrophobic hexapeptide model folding of amphipathic α-helices on membranes: energetics of helix formation by melittin amphipathic helix motif: classes and properties ph-dependent self-association of influenza hemagglutinin fusion peptides in lipid bilayers conformational transitions of membrane-bound hiv-1 fusion peptide membrane structure and fusiontriggering conformational change of the fusion domain from influenza hemagglutinin structure and function of membrane fusion peptides structure and orientation study of ebola fusion peptide inserted in lipid membrane models the three lives of viral fusion peptides fusion activity of hiv gp41 fusion domain is related to its secondary structure and depth of membrane insertion in a cholesterol-dependent fashion lipid tail protrusion in simulations predicts fusogenic activity of influenza fusion peptide mutants and conformational models protein-driven membrane stresses in fusion and fission paramyxovirus f1 protein has two fusion peptides: implications for the mechanism of membrane fusion participation of two fusion peptides in measles virus-induced membrane fusion: emerging similarity with other paramyxoviruses identification of the membrane-active regions of hepatitis c virus p7 protein: biophysical characterization of the loop region the membraneactive regions of the hepatitis c virus e1 and e2 envelope glycoproteins the membrane-active regions of the dengue virus proteins c and e hydrophobic segment of dengue virus c protein. interaction with model membranes fusogenic domains in herpes simplex virus type 1 glycoprotein h evidence for a role of the membrane-proximal region of herpes simplex virus type 1 glycoprotein h in membrane fusion and virus inhibition the identification and characterization of fusogenic domains in herpes virus glycoprotein b molecules interfacial pre-transmembrane domains in viral proteins promoting membrane fusion and fission viral fusion proteins: multiple regions contribute to membrane fusion interaction of a peptide derived from glycoprotein gp36 of feline immunodeficiency virus and its lipoylated analogue with phospholipid membranes peptide entry inhibitors of enveloped viruses: the importance of interfacial hydrophobicity a synthetic peptide corresponding to a conserved heptad repeat domain is a potent inhibitor of sendai virus-cell fusion: an emerging similarity with functional domains of other viruses peptides containing membrane-interacting motifs inhibit herpes simplex virus type 1 infectivity analysis of a membrane interacting region of herpes simplex virus type 1 glycoprotein h the presence of a single n-terminal histidine residue enhances the fusogenic properties of a membranotropic peptide derived from herpes simplex virus type 1 glycoprotein h computational identification of self-inhibitory peptides from envelope proteins self-association of glutamic acid-rich fusion peptide analogs of influenza hemagglutinin in the membrane-mimic environments: effects of positional difference of glutamic acids on side chain ionization constant and intra-and inter-peptide interactions deduced from nmr and gel electrophoresis measurements oligomerization of fusogenic peptides promotes membrane fusion by enhancing membrane destabilization fusion peptides derived from the hiv type 1 glycoprotein 41 associate within phospholipid membranes and inhibit cell-cell fusion. structure-function study a synthetic all d-amino acid peptide corresponding to the n-terminal sequence of hiv-1 gp41 recognizes the wildtype fusion peptide in the membrane and inhibits hiv-1 envelope glycoproteinmediated cell fusion thermodynamics of fusion peptide-membrane interactions short-term monotherapy in hiv-infected patients with a virus entry inhibitor against the gp41 fusion peptide discovery and optimization of a natural hiv-1 entry inhibitor targeting the gp41 fusion peptide multifunctional nanocarriers endocytic mechanisms for targeted drug delivery direct cytosolic delivery of polar cargo to cells by spontaneous membranetranslocating peptides shuttle-mediated nanoparticle delivery to the blood-brain barrier exploitation of viral properties for intracellular delivery membrane-active peptides: binding, translocation, and flux in lipid vesicles twenty years of cell-penetrating peptides: from molecular mechanisms to therapeutics truncated hiv-1 tat protein basic domain rapidly translocates through the plasma membrane generation of endosomolytic reagents by branching of cell-penetrating peptides intracellular delivery: exploiting viral membranotropic peptides a novel potent strategy for gene delivery using a single peptide vector as a carrier a new peptide vector for efficient delivery of oligonucleotides into mammalian cells detection of a fusion peptide sequence in the transmembrane protein of human immunodeficiency virus in vitro mutagenesis of a putative dna binding domain of sv40 large-t nectin-2-mediated entry of a syncytial strain of herpes simplex virus via ph-independent fusion with the plasma membrane of chinese hamster ovary cells structure-function analysis of herpes simplex virus glycoprotein b with fusion-from-without activity entry of herpes simplex virus 1 and other alphaherpesviruses via the paired immunoglobulin-like type 2 receptor alpha glycoprotein d receptor-dependent, low-ph-independent endocytic entry of herpes simplex virus type 1 fusing structure and function: a structural view of the herpesvirus entry machinery glycoproteins gb, gd, and ghgl of herpes simplex virus type 1 are necessary and sufficient to mediate membrane fusion in a cos cell transfection system herpes simplex virus glycoproteins gb and gh function in fusion between the virion envelope and the outer nuclear membrane biophysical characterization and membrane interaction of the two fusion loops of glycoprotein b from herpes simplex type i virus role of membranotropic sequences from herpes simplex virus type i glycoproteins b and h in the fusion process structure and orientation of the gh625-644 membrane interacting region of herpes simplex virus type 1 in a membrane mimetic system crystal structure of the conserved herpesvirus fusion regulator complex gh-gl crystal structure of the epstein-barr virus (ebv) glycoprotein h/glycoprotein l (gh/gl) complex crystal structure of glycoprotein b from herpes simplex virus 1 residues within the c-terminal arm of the herpes simplex virus 1 glycoprotein b ectodomain contribute to its refolding during the fusion step of virus entry conformational modifications of gb from herpes simplex virus type 1 analyzed by synthetic peptides structure of a trimeric variant of the epstein-barr virus glycoprotein b crystal structure of the low-ph form of the vesicular stomatitis virus glycoprotein g the postfusion structure of baculovirus gp64 supports a unified view of viral fusion machines analysis of synthetic peptides from heptad-repeat domains of herpes simplex virus type 1 glycoproteins h and b multiple peptides homologous to herpes simplex virus type 1 glycoprotein b inhibit viral infection peptide inhibitors against herpes simplex virus infections lipid composition modulates the interaction of peptides deriving from herpes simplex virus type i glycoproteins b and h with biomembranes a histidine switch in hemagglutinin-neuraminidase triggers paramyxovirus-cell membrane fusion a conserved histidine in the ij loop of the semliki forest virus e1 protein plays an important role in membrane fusion dendrimers functionalized with membrane-interacting peptides for viral inhibition a peptide derived from herpes simplex virus type 1 glycoprotein h: membrane translocation and applications to the delivery of quantum dots a fusogenic segment of glycoprotein h from herpes simplex virus enhances transfection efficiency of cationic liposomes clickable functionalization of liposomes with the gh625 peptide from herpes simplex virus type i for intracellular drug delivery dendrimer functionalization with a membrane-interacting domain of herpes simplex virus type 1: towards intracellular delivery pedone, gh625 is a viral derived peptide for effective delivery of intrinsically disordered proteins probing cellular events, one quantum dot at a time intracellular delivery of quantum dot-protein cargos mediated by cell penetrating peptides intracellular trafficking and unpacking of sirna/quantum dot-pei complexes modified with and without cell penetrating peptide: confocal and flow cytometric fret analysis spatiotemporal multicolor labeling of individual cells using peptide-functionalized quantum dots and mixed delivery techniques the benefits and challenges associated with the use of drug delivery systems in cancer therapy dendrimers and dendrons: concepts, synthesis, applications designing dendrimers for biological applications dendrimers and dendritic polymers in drug delivery surface modifications of nanocarriers for effective intracellular delivery of anti-hiv drugs crossing cellular barriers using dendrimer nanotechnologies comparison of the endocytic properties of linear and branched peis, and cationic pamam dendrimers in b16f10 melanoma cells tat-conjugated pamam dendrimers as delivery agents for antisense and sirna oligonucleotides current approaches to enhance cns delivery of drugs across the brain barriers astrocyte-endothelial interactions at the blood-brain barrier nanoparticle-mediated targeted delivery of antiretrovirals to the brain nanobiotechnology-based strategies for crossing the blood-brain barrier biomaterial-based technologies for brain anti-cancer therapeutics and imaging receptor-mediated delivery of magnetic nanoparticles across the blood-brain barrier review of a viral peptide nanosystem for intracellular delivery the peptide gh625 enters into neuron and astrocyte cell lines and crosses the blood brain barrier in rats tat-mediated delivery of heterologous proteins into cells introduction of exogenous antigens into the mhc class i processing and presentation pathway by drosophila antennapedia homeodomain primes cytotoxic t cells in vivo facilitation of drug entry into the cns via transient permeation of blood brain barrier: laboratory and preliminary clinical evidence from bradykinin receptor agonist, cereport getting into the brain: approaches to enhance brain drug delivery the authors thank luca de luca for excellent technical assistance. key: cord-104279-choywmwd authors: nan title: membrane protein sorting in the yeast secretory pathway: evidence that the vacuole may be the default compartment date: 1992-10-01 journal: j cell biol doi: nan sha: doc_id: 104279 cord_uid: choywmwd the targeting signals of two yeast integral membrane dipeptidyl aminopeptidases (dpaps), dpap b and dpap a, which reside in the vacuole and the golgi apparatus, respectively, were analyzed. no single domain of dpap b is required for delivery to the vacuolar membrane, because removal or replacement of either the cytoplasmic, transmembrane, or lumenal domain did not affect the protein's transport to the vacuole. dpap a was localized by indirect immunofluorescence to non-vacuolar, punctate structures characteristic of the yeast golgi apparatus. the 118-amino acid cytoplasmic domain of dpap a is sufficient for retention of the protein in these structures, since replacement of the cytoplasmic domain of dpap b with that of dpap a resulted in an immunolocalization pattern indistinguishable from that of wild type dpap a. overproduction of dpap a resulted in its mislocalization to the vacuole, because cells expressing high levels of dpap a exhibited vacuolar as well as golgi staining. deletion of 22 residues of the dpap a cytoplasmic domain resulted in mislocalization of the mutant protein to the vacuole. thus, the cytoplasmic domain of dpap a is both necessary and sufficient for golgi retention, and removal of the retention signal, or saturation of the retention apparatus by overproducing dpap a, resulted in transport to the vacuole. like wild type dpap b, the delivery of mutant membrane proteins to the vacuole was unaffected in the secretory vesicle-blocked sec1 mutant; thus, transport to the vacuole was not via the plasma membrane followed by endocytosis. these data are consistent with a model in which membrane proteins are delivered to the vacuole along a default pathway. ar~y proteins that reside in the organelles of the secretory pathway of eukaryotic cells have targeting information that directs retention in or sorting to the appropriate compartment (pfeffer and rothman, 1987) . in the absence of retention or sorting signals, soluble proteins of the secretory pathway are secreted; thus, the default pathway for these proteins is secretion (burgess and kelly, 1987; pelham, 1989) . in saccharomyces cerevisiae, mutations in the targeting signal of the soluble vacuolar protein, carboxypeptidase y (vails et al., 1990) , or the retention signal of the soluble er protein, bip (hardwick et al., 1990) , result in the secretion of these proteins. likewise, the flow of membrane proteins to the cell surface of nonpolarized mammalian cells is apparently by default, because mutations that disrupt the retention of er or golgi-retained membrane proteins (machamer et al., 1987; machamer, 1991; jackson et al., 1990) or the sorting of a lysosomal membrane protein (williams and fukuda, 1990) result in localization to the plasma membrane. little is known regarding membrane protein sorting in s. cerevisiae, although a previous study suggested that the cell surface is the default compartment for membrane proteins (fuller et al., 1989b) . in this paper, we characterize the targeting of two membrane proteins of the yeast secretory pathway, dap dipeptidyl aminopeptidases (dpaps) ~ dpap a and dpap b of the golgi apparatus and vacuole, respectively. the biogenesis of two membrane proteins of the yeast vacuole, dpap b (see fig. 1 a), and alkaline phosphatase (alp), has been characterized (klionsky and emr, 1989; roberts et al., 1989) . both dpap b and alp are type ii membrane glycoproteins (nomenclature of singer, 1990) , consisting of nh2-terminal cytoplasmic domains of approximately 30 amino acids, single hydrophobic membrane anchors, and cooh-terminal lumenal catalytic domains. these proteins transit the early compartments of the secretory pathway (i.e., er and golgi), but not the later compartments (i.e., secretory vesicles), indicating that the proteins do not transiently reside at the plasma membrane before delivery to the vacuole. the localization signals of these two proteins have not been identified, although the lumenal domain of alp has been shown to be unnecessary for vacuolar targeting (klionsky and emr, 1990) . the biosynthesis of several membrane proteins that reside this paper is dedicated to the memory of david merrill stevens. in the yeast golgi apparatus has also been examined. in yeast, three membrane-bound proteases, kex2p, kexlp, and dpap a (see fig. 1 a) process the mating pheromone a-factor precursor polypeptide as it traverses the secretory pathway (bussey, 1988; fuller et al., 1988) . the biosynthetic pathways of kex2p and kexlp have been characterized (fuller et al., 1989a,b; cooper and bussey, 1989) , and kex2p has been shown to function in a late golgi compartment (julius et al., 1984; graham and emr, 1991) . kex2p has been localized by indirect immunofluorescence to three to six punctate structures per cell that exhibit a somewhat random distribution within the cytoplasmic compartment (franzusoff et al., 1991; redding et al., 1991) . thus, the golgi apparatus of yeast is not localized to a perinuclear location, as in mammalian cells, but rather is dispersed throughout the cell. both kexlp (a. cooper and h. bussey, manuscript submitted) and dpap a (see below) have been immunolocalized to punctate bodies that are similar to those containing kex2p in size, abundance, and distribution. in this paper, a combined gene fusion and mutational analysis was used to show that no single domain of dpap b is required for vacuolar localization. furthermore, both overproduction of dpap a and a mutation in the cytoplasmic domain of dpap a resulted in mislocalization of this protein to the vacuole. finally, the fusion proteins analyzed in this work are shown to be transported directly to the vacuole from the golgi, and not to the plasma membrane. the bacterial strain mc1061 (casadaban and cohen, 1980) was used for all subcloning steps. oligonucleotide-directed mutagenesis was carded out in strain cj236 (kunkel et al., 1987) . the yeast strains used were jhry20-1aaia3 (mata, dap2a: : h1s3, stel3a : :leu2, ura3-52, leu2-3, 1eu2412, his3-a200, pep4-3; roberts et al., 1989) , sey2012zs (match, dap2:: leu2, emr et al., 1983) , cjry25-6b (mata dap2a : :leu2 mnn9 ura3-52 leu2-3 leu2412, and sey-5016 ( mata, s e cl-l , dap 2 : : le u2 , ura3-5 2 , le u2-3, leu2412) . yeast cultures were grown in yepd or minimal (sd) medium supplemented with the appropriate nutrients as previously described (sherman et al., 1982) . for the simultaneous induction of the gal/ promoter and the secl4 secretion defect, ceils were grown to log phase in minimal media plus raltinose and then harvested and resuspended in yep-raitinose. after a 1-h incubation at 25~ galactose was added directly to the cultures and at the same time the cultures were shifted to 34~ after 2 h, the cells were fixed immediately and prepared for indirect immunofluorescence as described below. oligonucleotides for mutagenesis were prepared by the university of oregon biotechnology laboratory on an applied biosystems 380b dna synthesizer (foster city, ca) as described (ito et al., 1982) . tran35s-label and zymolyase 100t were from icn biomedicals (irvine, ca), endo h was from boehringer marmheim (indianapolis, in), ultra-pure sds was from bdh biochemicals (san francisco, ca), glusulase was from dupont pharmaceuticals (wilmington, de), and all antibodies (except anti-dpap b, anti-dpap a, anti-alp, and anti-vat2p antibodies) used for indirect immunofluorescence were from jackson immunoresearch (west grove, pa), cappel products (malvern, pa), or promega biotech (madison, wi). all other reagents were from sigma chemical co. (st. louis, mo). dpap and invertase assays were performed as previously described (gildstein and lampen, 1975; roberts et al., 1991) . restriction endonuclease digests and ligations were performed as recommended by the suppliers. plasmid purification, agarose gel electrophoresis, fill-in reactions of sticky-ended dna fragments using t4 dna polymerase, and dna-mediated transformation of escherichia coli were done according to standard procedures (maniatis et al., 1982) . lithium acetate transformations of yeast were performed as described (jto et al., 1983) . a disruption of the chromosomal ste/3 locus was constructed by onestep gene disruption (l~thstein, 1983) , using the plasmid pslk349 (kindly provided by dr. george sprague). psl349 consists of pbr322 containing a 7.2 kbp bamhi ste/3 fragment, from which a 1.6-kbp bcli fragment within the coding region of the dpap a lumenal domain (c. a. flanagan, d. a. barnes, m. c. flessel, and j. thorner, manuscript submitted for publication) was replaced by the 2.9-kbp bglii leu2 fragment. to create a strain lacking both dpap a and dpap b, a disruption of the chromosomal dap2 locus with the his3 gene was made using the plasmid pgp6, which contains the 1.2-kbp ecori-bamhi his3 fragment (sikorski and hieter, 1989) in place of the l3-kbp bsteii-kpni portion of the coding region of dap2 (roberts et al., 1989) . the c~-factor signal sequence was fused to the lumenal domain of dpap b as follows: a sail linker was inserted at the hincll site of plasmid p771, which contains a portion of the 5' region of the mfcd gene (kurjan and herskowitz, 1982) , including 70 bp of non-coding region (not including the uas) and 66 bp of coding region, including the signal sequence, signal peptidase cleavage site (waters et al., 1988) , and 3 nh2-terminal residues of pro-a-factor, fused to the kre/gene (boone et ai., 1990) . the acci site at position +140 of the dap2 gene was changed to a sali site using oligonucleotide-directed mutagenesis (kunkel et al., 1987) . mutagenesis of dap2 was performed using the vector pcjr27, which contains the 3.3-kbp bamhi-hindiii da/'2 fragment in the plasmid ks + (stratagene, san diego, ca). the 2.7-kbp sali-hindui fragment, encoding the lumenal domain of dpap b, was inserted into the sali-hindiii sites of p771/sali, fusing the coding regions of mfal and dap2 in frame, c~fss-b was placed under the control of the ga/_j promoter (johnston and davis, 1984) by inserting the 2.9-kbp bamhi-hindiii fragment from this plasmid into the bamhi-hindiii sites of pc jr52, which contains the 822-bp ecori-bamhi ga/_,/ promoter fragment inserted into ecori-bamhi sites of the cen plasmid pseyc68 ca modified version of pseyc58; emr et al., 1983) . the resulting fusion protein consists of the nh2-terminal 22 residues of prepro-afactor and two residues arising from linker sequences fused to residue 49 of the lumenal domain of dpap b (fig. l b ; amino acid sequence nh2-mrfpsiftavlfaassala-apvgrphh . . . ). the bb-inv fusion protein expression vectors, pcjr13 and pc jr15 (2p and cen plasmids, respectively), were constructed by inserting the 0.6-kbp bamhi-acci fragment (acci blunt ended) into the invertase fusion vectors psey304 (a derivative of the 2/z plasmid psey303; emr et al., 1986) and the cen plasmid pseyc306 (johnson et al., 1987) at the bamhi and hin-diii sites (hindlij blunt ended). the resulting fusion protein contains the 48 nh2-terminal residues of dpap b fused to residue three of cytoplasmic invertase ( fig. 1 b) . bb-inv was placed under the control of the gal/promoter by cutting pcjr15 with ecori and hiediii and ligating in the 822-bp gal/fragment (johnston and davis, 1984) . this fused the 3' end of the gal/fragment at nucleotide -92 of the dap2 sequence. vectors encoding a20-bb and a27-bb were constructed as follows. oligonucleotide-directed mutagenesis (kunkel et al., 1987) was used to create 60-and 81-bp deletions in dap2 (a20-bb and a27-bb, respectively) in pcjr27. the ~3.2kbp bamhi-hindiii mutant dap2 fragments were inserted into the bamhi-hindiii sites of the cen plasmid pseyc58, creating pmnh1 (620-bb) and pc jr45 (a27-bb). &20-bb and a27-bb were over-produced by replacing the dap2 promoter with the gal/promoter as follows. the hindiii site at -92 of dap2 was blunt ended and religated, creating an nhei site. the ~3-kbp nhei-hindiii fragments of pc jr43 and pc jr44 were inserted at the xbai-hindiii sites of pc jr52, creating pc jr56 and pc jr54, respectively. the a20 and a27 deletions removed residues 2-21 and 2-27 of dpap b, changing the nh2-terr~nal acid sequence of dpap b from nh2-meg-geeeveripdelfdtkkkhlldklirv30 to nh2-mhlldklirv and nh2-mirv, respectively ( fig. 1 b) . the 2pt plasmid encoding dpap a, pcjr46, was constructed as follows: the 5.9-kbp xbai-bamhi fragment, containing the stf_j3 gene (c. a. flanagan, d. a. barnes, m. c. flessel, andj. thorner, manuscript submitted for publication) was isolated from the plasmid p13-3 (julius et al., 1983) and inserted into puc13. the 5.9-kbp sali-bami-ii fragment from this vector was inserted into the xhoi-bglii sites of the 2# plasmid pckr201 (c. raymond and t. stevens, unpublished results) . the cen plasmid pcjr78 was made by inserting the 4.5-kbp eagi-pvuii ste/3 fragment into the eaglecorv sites of prs316 (sikorski and hieter, 1989) . the plasmid pc jr64, encoding the fusion protein aa-b, was constructed by inserting the 2.5-kbp bamhi-mlui (mlui blunt ended) fragment from p13-3, encoding the stf.j3 promoter and the cytoplasmic and transmembrane domains of dpap a, and the 2.7-kbp sall-hindlii fragment of dap2 (sail blunt ended), encoding the lumenal domain of dpap b, into the bamhi-hindlii sites of pseyc58. the 5.2-kbp bamhi-hindlii fragment of the resulting vector (pcjr41) was cloned into the bamhi-hindlii sites of the 2# plasmid, psey18 (emr et al., 1986) . the resulting plasmid encodes aa-b, which consists of the nh2-terminal 150 residues of dpap a to residue 47 of dpap b (fig. 1 c) . for the construction of the fusion protein a-bb, xbai sites were created in both the dap2 and ste/3 genes just upstream of the coding regions of the transmembrane domains of dpap b and dpap a, respectively, using oligonucleotide-directed mutagenesis (kunkel et al., 1987) . nucleotides 69 and 70 of the dap2 gene were changed from gt to tc (roberts et al., 1989) , and nucleotides 342-344 of ste/3 were changed from gcc to aga (with the a of the initiation codon as 1) (c. a. flanagan, d. a. barnes, m. c. flessel, and j. thorner, manuscript submitted for publication). a 1.1kbp saci-xbai fragment (encoding the ste1