key: cord-022607-34hj17sn
authors: Wain‐Hobson, Simon; Vartanian, Jean‐Pierre
title: Editing HBV into oblivion
date: 2004-11-24
journal: Hepatology
DOI: 10.1002/hep.20499
sha: 
doc_id: 22607
cord_uid: 34hj17sn

nan

Hepatitis C virus (HCV) readily sets up persistent infection, and in doing so must evade both innate and adaptive immune responses. Cellular (T-cell) immune responses are thought to play a significant role in determining clinical outcome, with strong and sustained responses typically associated with viral control. 1,2 Cellular immune responses are also potentially involved in immunomediated pathogenesis, both directly and through recruitment or modulation of other inflammatory cells in the liver. Consequently, much recent effort has been expended to attempt to analyze how the virus evades both CD8 ϩ and CD4 ϩ T cells and define the differences between "successful" and "unsuccessful" outcomes (i.e., spontaneous control of viremia vs. chronicity) . Relatively fewer data are available describing how intrahepatic T-cell responses might be linked to pathology.

A general consensus is emerging that in acute infection, regardless of outcome, CD8 ϩ T-cell responses can be readily detected; however, in those cases in which the virus is not controlled, such responses are not sustained at high levels in blood beyond a few weeks. 3 This could be explained partly by viral escape through mutation, as has been elegantly shown in animal models. 4 However, even in cases when epitopes appear to be intact, responses typically are weak or absent in blood ex vivo once chronicity is established. They do not appear to be entirely deleted, because they can be reconstituted in vitro through restimulation with antigen, or even fished out from blood directly using ultrasensitive detection techniques. 5 Thus various groups have proposed that functional alterations in CD8 ϩ T cells may be associated with persistent infection. Those include relatively weak interferon gamma (IFN-␥) production, 6,7 impaired proliferative capacity, 8 and a "stunted" maturation state. 6, 9, 10 It is not clear whether the lack of mature effector cells seen in the circulation is as a result of compartmentalization in the liver, (where they may be deleted) or failure to generate such cells in the first place-potentially through defects in antigen-presenting cells, inhibitory effects of viral gene products (such as core) 11 or failure of CD4 ϩ T-cell help. 1 Things look slightly different in the liver itself. Early studies showed it is possible to clone out CD8 ϩ T cells of diverse specificities from the liver of infected patients. 12, 13 It appears that antigen-specific T cells exist at higher frequencies infected livers-probably a relative increase of approximately 10-fold as a proportion of CD8 ϩ T cells. 14, 15 (It should be noted that enrichment of virusspecific memory T cells-for example, those specific for cytomegalovirus and Epstein-Barr virus-is seen even in normal livers in human and murine models. 16 ) Interestingly, such T cells in both infected and normal livers appear to be activated, as judged by expression of CD69, although this effect is not antigen specific. 15 The questions that emerge then are: Are such T cells fully functional in the intrahepatic environment, and how does such activity relate to disease progression?

The study by Accepezzato et al. is a large-scale analysis of intrahepatic CD8 ϩ T-cell populations using class I tetramers and intracellular cytokine staining to better understand exactly the functionality of the intrahepatic virus-specific T cells. These analyses are technically very demanding because of the very low cell yields available, and previously have only examined very limited patient numbers, compared with nearly 50 in this study. 14, 15 As previously, Accepezzato et al. found low frequencies of virus-specific T cells in blood in most cases but found generally higher overall frequencies in liver. The maturation state in the liver appears to be more advanced than in blood, reflecting accumulation of "effector memory" (CCR7 Ϫ ) cells in the liver. For example, some cells appear to be high in perforin, a situation that is very rarely found in blood among HCV-specific populations. 6, 10 The most interesting results, however, relate to the functionality of the cells. In addition to tetramer staining, the authors stimulated the T cells in vitro with peptide and analyzed the release of IFN-␥ or interleukin 10 (IL-10). IFN-␥ is a classical antiviral cytokine that is believed to be of special relevance in the clearance of infected hepatocytes. 17 IL-10 is a multifunctional cytokine associated with suppression of T helper 1-type responses. In the present study, three secretion patterns seemed to emerge. In some patients, there appeared to be very weak secretion of either cytokine, while in a few patients there appeared to be a dominance of IFN-␥; several other patients exhibited a dominant IL-10 secretory response. Interestingly, the overall proportion of cells secreting IFN-␥ in response to viral peptides correlated positively with the level of intrahepatic inflammation (histological activity index score); conversely, the frequency of IL-10 secreting cells correlated inversely with the histological activity index score.

What is the role of intrahepatic virus-specific CD8ϩ T cells in determining acute viral clearance and immunopathology? With regard to acute viral clearance, this is still very unclear and unlikely to be easily addressed in human studies. It would, for example, be very interesting to know whether the early emergence of IL-10 -secreting populations in the liver was associated with failure to initially control the virus. As with many studies, it is very difficult to disentangle cause and effect-IL-10 -secreting cells might reasonably emerge as a consequence of long-term inflammation. The observation that even in these chronically infected patients, several had intrahepatic T-cell populations that expressed either no cytokine or largely IFN-␥ suggests that induction of IL-10 secretion cannot alone explain the propensity of HCV to evade CD8ϩ T cells.

Regarding pathology, IL-10 -secreting CD8ϩ T cells may play an important role in suppression or regulation of inflammation-a feature that actually might represent an appropriate adaptation of T-cell responses to chronic antigen exposure. In this respect, they may be akin to the more classical CD4 ϩ T regulatory cells. Such populations may have anti-self specificities; however, persistent stimulation may induce similar regulatory activity even in CD4 ϩ T cells specific for foreign antigens. A role for CD4 ϩ CD25 ϩ T cells in regulation of peripheral CD8 ϩ T-cell responses has recently been proposed. 18 Why, then, might some patients generate IL-10 -secreting T-cell populations and others not? One question arising from the study is whether the phenotype observed reflects an adaptation only of HCV-specific T cells, or whether it is a feature of the overall infiltrate. HCV-specific T cells appear to represent only a relatively small fraction of the overall intrahepatic CD8 ϩ T-cell response (about 1% in this study, although perhaps slightly higher than this if other epitopes were included). Simply analyzing the overall cytokine preferences of intrahepatic T-cell infiltrates might be very informative. There are suggestions that polymorphisms in chemokine and chemokine receptor genes might influence intrahepatic pathology, so a genetic basis (e.g., in cytokine/receptor genes) for these distinct responses might be relevant. 19 Alternatively, viral factors might lead to diverse outcomes; genotype did not appear to play an obvious role, although this issue is confounded by the fact that some of the peptides used are poorly cross-reactive. Viral mutants (altered peptide ligands) emerging in vivo have been associated with modulation of cytokine secretion of T cells. 20 Finally-looking forward-could the cytokine secretion profiles of intrahepatic T cells be potentially linked to treatment response? This is an intriguing question, given that it is observed that combination therapy for chronic Intrahepatic T-cell decision-making. CD8 ϩ T-cell responses found in blood are usually weak and are low in markers of activation and maturation. In the liver, CD69 expression-indicating recent activation-is observed, and in the present study, perforin expression is also described. Cytokine secretion appears to take one of two main paths: predominantly IFN-␥ or predominantly IL-10. Although the molecular and cellular pathways are not understood in detail, it is likely that IFN-␥ secretion has antiviral activity but is also proinflammatory, while IL-10 is anti-inflammatory. The long-term effect on fibrosis (which was generally fairly mild in the present study) requires further analysis. disease boosts previously weak or undetectable T-cell responses in the periphery. 21, 22 It is still not clear to what extent such responses are reflected in the liver and how they link ultimately with sustained virological responses. Nevertheless, it has been shown that the addition of ribavirin to treatment regimens reduces the IL-10 secretion of recovered antiviral T cells. 22 Thus, it seems plausible that treatment will shift the cytokine balance in the liver, which could influence significantly the overall success of therapy.

One possible message from this study is that T cells in HCV appear to be able to adapt in the face of a persisting virus, and in doing so modulate the intrahepatic environment to one that is less inflammatory (Fig. 1) . The relationship between host and virus in HCV infection is potentially a long-term one. As in human relationships, each partner is capable of making compromises to minimize confrontation. In some cases this behavior modification is more successful than others. It remains to be seen whether-in cases where the relationship is breaking down-we can but watch from the sidelines, or could possibly intervene to re-educate T cells into a more appropriate response. of polymerase errors. Accordingly, their coding capacity is limited by the probability of generating a lethal mutation. Genome sizes range from the 3 kb of hepatitis B virus (HBV) up to the 27 to 32 kb typical of the coronaviruses. The error threshold is that mutation rate just compatible with viable replication. Chemical mutagenesis or the incorporation of ambiguous bases can displace mutation rates beyond the error threshold so resulting in the collapse of information. 1,2 Given this, many have wondered whether nature has not seized upon this singular vulnerability of RNA viruses and retroviruses to "going over the edge."

For nearly 20 years it has been known that negative stranded genomes, particularly those of measles virus, may undergo genetic editing of adenosine in the context of double stranded RNA. Multiple adenosine residues would be deaminated, resulting in inosine. As inosine base pairs as guanosine, adenosine editing generated A3 G hypermutants. 3 Some years later another form of hypermutation cropped up among the classical retroviruses. 4 Massive and monotonous substitution of G for A, involving up to 60% of G residues, was distributed across the entire 10-kb HIV-1 genome. Although the frequency and degree of G3 A hypermutation are most striking for the lentiviral subgroup of retroviruses, which includes human immunodeficiency virus (HIV), elsewhere G3 A hypermutants have been described for only a handful of retroviruses including the "other" human retrovirus, human T-cell leukemia virus (HTLV). The situation took a fascinating turn when Will's group sequenced a couple of subgenomic HBV DNA molecules from the serum of a single patient. 5 The genomes showed signs of extensive G3 A substitution at a frequency typical of HIV G3 A hypermutants. Since then, these two hypermutants have remained the only such examples despite a burgeoning HBV database. The fact that G3 A hypermutation is found among viruses with obligatory reverse transcription steps, notably HBV and the primate lentiviruses, suggests a common mechanism occurring in the cytoplasm.

The conceptual break leading to an understanding of retroviral G3 A hypermutation has come recently in two stunning punches. First, the vif gene is conserved among all the primate lentiviruses, where vif is an abbreviation for "viral infectivity factor." Some established T-cell lines are permissive for the replication of HIV-1 ⌬vif viruses while others are not, suggesting restriction by a host cell protein. Using a subtractive screen Malim's group in London showed that a single gene product, CEM15, was responsible for restricted HIV replication. 6 When screened against the databases, CEM15 proved to be identical to APOBEC3G, which is part of a 7-gene cluster that mapped to chromosome 22q13. 7 What are these genes, denoted APOBEC3A-G? The sequences of all seven show clear amino acid homology to cytidine deaminases, including that of Escherichia coli, but particularly the mammalian enzyme APOBEC1. This name is derived from the fact that the protein is the catalytic subunit of the "apolipoprotein B editing complex" that specifically deaminates cytidine C6666 to uracil (U) in apolipoprotein B messenger RNA (mRNA). 8 Second, a crop of five papers showed that when a HIV-1⌬vif virus was cotransfected along with a human APOBEC3G complementary DNA (cDNA) clone, the molecule was incorporated into budding virions. Upon infection of a susceptible target cell, G3 A hypermutants were recovered with alacrity. As only G3 A substitutions were found, even though reverse transcription results in double strand (ds) DNA formation, this suggested that only one strand was being edited. 9 -13 This was only (bio)logical if the nascent minus DNA strand was being edited, which was rapidly confirmed. All groups showed that viral genomic RNA was not edited.

Deamination of cytidine residues in neosynthesized minus strand DNA yields uracil and occurs post-cDNA synthesis in a manner independent of reverse transcriptase. 14 Now as U base pairs with adenosine, when APOBEC3G-edited minus strand DNA is copied into plus strand DNA by reverse transcriptase, the multiple Us are copied into A. Although referred to as G3 A hypermutants the "action" concerns C residues on the minus DNA strand.

Upon this vibrant stage, Turelli et al. have come forth with an intriguing study of the effect of APOBEC3G expression on HBV replication. 15 They assayed core-associated HBV DNA resulting from transfection of human hepatoma Huh7 cells with a HBV-producing plasmid. When cotransfected with human APOBEC3G, HBV DNA synthesis was strongly curtailed. Important controls showed that APOBEC3G was incorporated into the core particles yet did not affect hepatitis B c antigen (HBcAg) production. However, three findings suggest that HBV does not parallel the HIV hypermutation paradigm. Firstly, G3 A hypermutated HBV DNA was not found despite searching. Secondly, core-associated HBV RNA was reduced more than 10-fold, suggesting that the block in HBV DNA synthesis results primarily from an inhibition of viral pregenomic RNA packaging. Finally and remarkably, serine substitutions of functionally critical cysteine residues in APOBEC3G failed to abrogate the antiviral activity for HBV but did so in the HIV control-so controls are useful! For the purist, a negative result, the inability to find hypermutated HBV DNA-remains just that. However, the antiviral activity of the APOBEC3G serine mutants and reduced RNA packaging represent positive results, which are challenging, to say the least. Certainly the lack of hypermutants is coherent if the cytosine deamination activity of APOBEC3G is not involved in HBV restriction.

The vexing point is that the experiments were driven with single strand (ss) DNA cytosine deamination as the working hypothesis, even though HBV G3 A hypermutants do exist, albeit rarely. Commenting on the Science paper in the form of a "technical comment," Rösler et al. identified bona fide G3 A hypermutants at low frequency in an analogous transfection protocol, albeit using the widely known cell line HepG2. 16 To complicate matters, they failed to identify hyermutants using Huh7 cells, which led them to postulate a cell line effect. Commenting on Rösler et al., Turelli et al. refuted this idea because they could achieve APOBEC3G restriction of HIV using Huh-7 as a transfection support. 17 The latter comment is especially interesting in that it shows that HBV replication can be restricted by APOBEC3F. 17 Now, among the APOBEC3 cluster of gene products, only APOBEC3F and 3G can restrict HIV replication. Each has a subtle sequence bias in the way it deaminates DNA. APOBEC3F shows a preference for cytidine in the context of TpC, while APOBEC3G prefers the CpC. Interestingly, the two naturally hypermutated subgenomes showed an overall bias for TpC, indicating that they were probably edited more by APOBEC3F than by APOBEC3G. 5 This nicely fits with the new finding. As for HIV, the same two APOBEC3 members are involved in HBV restriction.

At the low resolution of whole-liver mRNA profiling, only APOBEC3C is strongly expressed, while APOBEC3F and APOBEC3G are expressed at borderline levels (http://genecards.bcgsc.bc.ca/). By contrast, immune cells express copious amounts of most APO-BEC3 molecules. Hence, APOBEC profiling of liver tissue might well reflect circulating lymphoid cells. If hepatocytes expressed little or no APOBEC3 molecules this would help explain the dearth of naturally observed HBV G3 A hypermutants in the databases.

How can one square HBV restriction by APOBEC3G when there is little expression of APOBEC3G in the normal liver? Perhaps the mRNA profiling is too macroscopic, too low-resolution to be of much use. Alternatively, the inflammatory response to HBV might upregulate APOBEC3G. Certainly the PKCa/␤I / MEK / ERK pathway has been shown control basal levels of APOBEC3G mRNA in some T-cell lines, 18 which consequently declined when cells were treated with inhibitors or arrested in the G 0 state of the cell cycle by serum starvation. Alternatively, given the expression of most APOBEC3 molecules in lymphoid tissue, rare and abortive infection of CD4 ϩ and CD8 ϩ T lymphocytes by HBV is another working hypothesis. 19 Could APOBEC3G function by simply binding to C residues in HBV genomic RNA so precluding it from becoming packaged? But if so, why should this not occur for HIV replication? We do not know. Yet the parallel with human APOBEC1 is striking: When expressed alone in E. coli, it is highly mutagenic for DNA, 20 yet if incorporated into an editing complex it edits a single C residue in the apolipoprotein B mRNA. Could it be that just beneath the plasma membrane APOBEC3G acts nonspecifically as a ssDNA cytosine deaminase, whereas deep down in the endoplasmic reticulum where HBcAg particles are assembled, it is part of a multiprotein complex that modulates the activity of the APOBEC3G subunit?

An analysis of primate APOBEC3G gene sequences indicates that they are evolving under positive selection although selection is present in lineages for which there is no natural simian immunodeficiency virus (SIV), such as orangutans and macaques. 21 These primates are of Asian origin, whereas all naturally occurring SIVs are found in equatorial Africa. HBV has arguably been in primates for a longer period of time than has SIV-the presence of HBV-like viruses in gibbon and orangutan are cases in point. 22, 23 The absence of HBV and SIV in the macaque lineage suggests that selection on APOBEC3G is probably unrelated to retroviruses. The lack of restriction of single mouse homologue of APOBEC3G on murine retroviral vectors 24 suggests that a blanket interpretation of these molecules as part of innate antiretroviral immunity is too simplistic, at least for the moment.

While no cellular function has been ascribed to APOBEC3G and its immediate paralogues, once in place some of them represent formidable barriers to retroviral infection. Retroviruses have either to avoid cells in which APOBEC3 molecules are abundantly expressed or escalate and overcome the obstacle via the acquisition of some novel gene product. Otherwise their genomes will be edited beyond the error threshold and into oblivion. The lentiviral vif gene fits the latter scenario.

The paper by Turelli et al. shows that there is far more to APOBEC3 genes than initially thought. These are exciting times with much to be done. It will be fascinating to see how the picture develops.

Different clinical behaviours of acute HCV infection are associated with different vigor of the anti-viral T cell response

Analysis of a successful immune response against hepatitis C virus

CTL responses are induced during acute HCV infection but are not sustained

The outcome of hepatitis C virus infection is predicted by escape mutations in epitopes targeted by cytotoxic T lymphocytes

Ultra-sensitive class I tetramer analysis reveals previously undetectable populations of antiviral CD8ϩ T cells

Sustained dysfunction of antiviral CD8ϩ T lymphocytes after infection with hepatitis C virus

Determinants of viral clearance and persistence during acute hepatitis C virus infection

Impaired effector function of hepatitis C virus-specific

CD8ϩ T cells in chronic hepatitis C virus infection

Memory CD8ϩ T cells vary in differentiation phenotype in different persistent virus infections

Pervasive influence of hepatitis C virus on the phenotype of antiviral CD8ϩ T cells

Suppression of host immune response by the core protein of hepatitis C virus: possible implications for hepatitis C virus persistence

HLA class I-restricted cytotoxic T lymphocytes specific for hepatitis C virus. Identification of multiple epitopes and characterization of patterns of cytokine release

Liver derived CTL in HCV infection; breadth and specificity of responses in a cohort of patients with chronic infection

Quantitative analysis of HCV-specific CD8ϩ T cells in peripheral blood and liver using peptide-MHC tetramers

Direct ex vivo comparison of the breadth and specificity of the T cells in the liver and peripheral blood of patients with chronic HCV infection

Virus-specific CD8 ϩ T lymphocytes within the normal human liver

Viral clearance without destruction of infected cells during acute HBV infection

Suppression of HCV-specific T cells without differential hierarchy demonstrated ex vivo in persistent HCV infection

Association of genetic variants of the chemokine receptor CCR5 and its ligands, RANTES and MCP-2, with outcome of HCV infection

Immunobiology of hepatitis C virus (HCV) infection: the role of CD4 T cells in HCV infection

The dynamics of T-lymphocyte responses during combination therapy for chronic hepatitis C virus infection

Hepatitis C virus-specific T-cell reactivity during interferon and ribavirin treatment in chronic hepatitis C

The broad-spectrum antiviral ribonucleoside ribavirin is an RNA virus mutagen

Lethal mutagenesis of HIV with mutagenic nucleoside analogs

Biased hypermutation and other genetic changes in defective measles virus in human brain infections

Wain-Hobson S. Selection, recombination, and G3 A hypermutation of human immunodeficiency virus type 1 genomes

Naturally occurring hepatitis B virus genomes bearing the hallmarks of retroviral G3 A hypermutation

Isolation of a human gene that inhibits HIV-1 infection and is suppressed by the viral Vif protein

An anthropoid-specific locus of orphan C to U RNA-editing enzymes on chromosome 22

Molecular cloning of an apolipoprotein B messenger RNA editing protein

Hypermutation of HIV-1 DNA in the absence of the Vif protein

The cytidine deaminase CEM15 induces hypermutation in newly synthesized HIV-1 DNA

Broad antiretroviral defence by human APOBEC3G through lethal editing of nascent reverse transcripts

DNA deamination mediates innate immunity to retroviral infection

Species-specific exclusion of APOBEC3G from HIV-1 virions by Vif

APOBEC3G is a single stranded DNA cytidine deaminase and functions independently of HIV reverse transcriptase

Inhibition of hepatitis B virus replication by APOBEC3G

Comment on "Inhibition of hepatitis B virus replication by APOBEC3G

Author reply to Comment on "Inhibition of hepatitis B virus replication by APOBEC3G

Transcriptional regulation of APOBEC3G, a cytidine deaminase that hypermutates human immunodeficiency virus

Distribution of hepatitis B virus DNA sequences in different peripheral blood mononuclear cell subsets in HBs antigen-positive and negative patients

RNA editing enzyme APOBEC1 and some of its homologs can act as DNA mutators

Rapid evolution of primate antiviral enzyme APOBEC3G

A new group of hepadnaviruses naturally infecting orangutans (Pongo pygmaeus)

Complete sequencing of a gibbon hepatitis B virus genome reveals a unique genotype distantly related to the chimpanzee hepatitis B virus

APOBEC3G targets specific virus species