Hepatitis D virus interferes with hepatitis B virus RNA production via interferon-dependent and -independent mechanisms.

BACKGROUND & AIMS: Chronic coinfection with HBV and HDV leads to the most aggressive form of chronic viral hepatitis. Herein, we aimed to elucidate the molecular mechanisms underlying the widely reported observation that HDV interferes with HBV in most coinfected patients. METHODS: Patient liver tissues, primary human hepatocytes, HepaRG cells and human liver chimeric mice were used to analyze the effect of HDV on HBV using virological and RNA-sequencing analyses, as well as RNA synthesis, stability and association assays. RESULTS: Transcriptomic analyses in cell culture and mouse models of coinfection enabled us to define an HDV-induced signature, mainly composed of interferon (IFN)-stimulated genes (ISGs). We also provide evidence that ISGs are upregulated in chronically HDV/HBV-coinfected patients but not in cells that only express HDV antigen (HDAg). Inhibition of the hepatocyte IFN response partially rescued the levels of HBV parameters. We observed less HBV RNA synthesis upon HDV infection or HDV protein expression. Additionally, HDV infection or expression of HDAg alone specifically accelerated the decay of HBV RNA, and HDAg was associated with HBV RNAs. On the contrary, HDAg expression did not affect other viruses such as HCV or SARS-CoV-2. CONCLUSIONS: Our data indicate that HDV interferes with HBV through both IFN-dependent and IFN-independent mechanisms. Specifically, we uncover a new viral interference mechanism in which proteins of a satellite virus affect the RNA production of its helper virus. Exploiting these findings could pave the way to the development of new therapeutic strategies against HBV. IMPACT AND IMPLICATIONS: Although the molecular mechanisms remained unexplored, it has long been known that despite its dependency, HDV decreases HBV viremia in patients. Herein, using in vitro and in vivo models, we showed that HDV interferes with HBV through both IFN-dependent and IFN-independent mechanisms affecting HBV RNA metabolism, and we defined the HDV-induced modulation signature. The mechanisms we uncovered could pave the way for the development of new therapeutic strategies against HBV by mimicking and/or increasing the effect of HDAg on HBV RNA. Additionally, the HDV-induced modulation signature could potentially be correlated with responsiveness to IFN-α treatment, thereby helping to guide management of HBV/HDV-coinfected patients.

Characterization of a Novel Capsid Assembly Modulator for the Treatment of Chronic Hepatitis B Virus Infection.

The standard of care for the treatment of chronic hepatitis B (CHB) is typically lifelong treatment with nucleos(t)ide analogs (NAs), which suppress viral replication and provide long-term clinical benefits. However, infectious virus can still be detected in patients who are virally suppressed on NA therapy, which may contribute to the failure of these agents to cure most CHB patients. Accordingly, new antiviral treatment options are being developed to enhance the suppression of hepatitis B virus (HBV) replication in combination with NAs ("antiviral intensification"). Here, we describe GS-SBA-1, a capsid assembly modulator (CAM) belonging to class CAM-E, that demonstrates potent inhibition of extracellular HBV DNA in vitro (EC50 [50% effective concentration] = 19 nM) in HBV-infected primary human hepatocytes (PHHs) as well as in vivo in an HBV-infected immunodeficient mouse model. GS-SBA-1 has comparable activities across HBV genotypes and nucleos(t)ide-resistant mutants in HBV-infected PHHs. In addition, GS-SBA-1 demonstrated in vitro additivity in combination with tenofovir alafenamide (TAF). The administration of GS-SBA-1 to PHHs at the time of infection prevents covalently closed circular DNA (cccDNA) formation and, hence, decreases HBV RNA and antigen levels (EC50 = 80 to 200 nM). Furthermore, GS-SBA-1 prevents the production of extracellular HBV RNA-containing viral particles in vitro. Collectively, these data demonstrate that GS-SBA-1 is a potent CAM that has the potential to enhance viral suppression in combination with an NA.

Smc5/6 silences episomal transcription by a three-step function.

In addition to its role in chromosome maintenance, the six-membered Smc5/6 complex functions as a restriction factor that binds to and transcriptionally silences viral and other episomal DNA. However, the underlying mechanism is unknown. Here, we show that transcriptional silencing by the human Smc5/6 complex is a three-step process. The first step is entrapment of the episomal DNA by a mechanism dependent on Smc5/6 ATPase activity and a function of its Nse4a subunit for which the Nse4b paralog cannot substitute. The second step results in Smc5/6 recruitment to promyelocytic leukemia nuclear bodies by SLF2 (the human ortholog of Nse6). The third step promotes silencing through a mechanism requiring Nse2 but not its SUMO ligase activity. By contrast, the related cohesin and condensin complexes fail to bind to or silence episomal DNA, indicating a property unique to Smc5/6.

Therapeutic shutdown of HBV transcripts promotes reappearance of the SMC5/6 complex and silencing of the viral genome in vivo.

OBJECTIVE: Therapeutic strategies silencing and reducing the hepatitis B virus (HBV) reservoir, the covalently closed circular DNA (cccDNA), have the potential to cure chronic HBV infection. We aimed to investigate the impact of small interferring RNA (siRNA) targeting all HBV transcripts or pegylated interferon-α (peg-IFNα) on the viral regulatory HBx protein and the structural maintenance of chromosome 5/6 complex (SMC5/6), a host factor suppressing cccDNA transcription. In particular, we assessed whether interventions lowering HBV transcripts can achieve and maintain silencing of cccDNA transcription in vivo. DESIGN: HBV-infected human liver chimeric mice were treated with siRNA or peg-IFNα. Virological and host changes were analysed at the end of treatment and during the rebound phase by qualitative PCR, ELISA, immunoblotting and chromatin immunoprecipitation. RNA in situ hybridisation was combined with immunofluorescence to detect SMC6 and HBV RNAs at single cell level. The entry inhibitor myrcludex-B was used during the rebound phase to avoid new infection events. RESULTS: Both siRNA and peg-IFNα strongly reduced all HBV markers, including HBx levels, thus enabling the reappearance of SMC5/6 in hepatocytes that achieved HBV-RNA negativisation and SMC5/6 association with the cccDNA. Only IFN reduced cccDNA loads and enhanced IFN-stimulated genes. However, the antiviral effects did not persist off treatment and SMC5/6 was again degraded. Remarkably, the blockade of viral entry that started at the end of treatment hindered renewed degradation of SMC5/6. CONCLUSION: These results reveal that therapeutics abrogating all HBV transcripts including HBx promote epigenetic suppression of the HBV minichromosome, whereas strategies protecting the human hepatocytes from reinfection are needed to maintain cccDNA silencing.

Hepatitis B Virus X Protein Function Requires Zinc Binding.

The host structural maintenance of chromosomes 5/6 complex (Smc5/6) suppresses hepatitis B virus (HBV) transcription. HBV counters this restriction by expressing the X protein (HBx), which redirects the cellular DNA damage-binding protein 1 (DDB1)-containing E3 ubiquitin ligase to target Smc5/6 for degradation. However, the details of how HBx modulates the interaction between DDB1 and Smc5/6 remain to be determined. In this study, we performed biophysical analyses of recombinant HBx and functional analysis of HBx mutants in HBV-infected primary human hepatocytes (PHH) to identify key regions and residues that are required for HBx function. We determined that recombinant HBx is soluble and exhibits stoichiometric zinc binding when expressed in the presence of DDB1. Mass spectrometry-based hydrogen-deuterium exchange and cysteine-specific chemical footprinting of the HBx:DDB1 complex identified several HBx cysteine residues (located between amino acids 61 and 137) that are likely involved in zinc binding. These cysteine residues did not form disulfide bonds in HBx expressed in human cells. In line with the biophysical data, functional analysis demonstrated that HBx amino acids 45 to 140 are required for Smc6 degradation and HBV transcription in PHH. Furthermore, site-directed mutagenesis determined that C61, C69, C137, and H139 are necessary for HBx function, although they are likely not essential for DDB1 binding. This CCCH motif is highly conserved in HBV as well as in the X proteins from various mammalian hepadnaviruses. Collectively, our data indicate that the essential HBx cysteine and histidine residues form a zinc-binding motif that is required for HBx function.IMPORTANCE The structural maintenance of chromosomes 5/6 complex (Smc5/6) is a host restriction factor that suppresses HBV transcription. HBV counters this restriction by expressing HBV X protein (HBx), which redirects a host ubiquitin ligase to target Smc5/6 for degradation. Despite this recent advance in understanding HBx function, the key regions and residues of HBx required for Smc5/6 degradation have not been determined. In the present study, we performed biochemical, biophysical, and cell-based analyses of HBx. By doing so, we mapped the minimal functional region of HBx and identified a highly conserved CCCH motif in HBx that is likely responsible for coordinating zinc and is essential for HBx function. We also developed a method to produce soluble recombinant HBx protein that likely adopts a physiologically relevant conformation. Collectively, this study provides new insights into the HBx structure-function relationship and suggests a new approach for structural studies of this enigmatic viral regulatory protein.

Spatiotemporal Analysis of Hepatitis B Virus X Protein in Primary Human Hepatocytes.

The structural maintenance of chromosomes 5/6 complex (Smc5/6) is a host restriction factor that suppresses hepatitis B virus (HBV) transcription. HBV counters this restriction by expressing the X protein (HBx), which redirects the host DNA damage-binding protein 1 (DDB1) E3 ubiquitin ligase to target Smc5/6 for degradation. HBx is an attractive therapeutic target for the treatment of chronic hepatitis B (CHB), but it is challenging to study this important viral protein in the context of natural infection due to the lack of a highly specific and sensitive HBx antibody. In this study, we developed a novel monoclonal antibody that enables detection of HBx protein in HBV-infected primary human hepatocytes (PHH) by Western blotting and immunofluorescence. Confocal imaging studies with this antibody demonstrated that HBx is predominantly located in the nucleus of HBV-infected PHH, where it exhibits a diffuse staining pattern. In contrast, a DDB1-binding-deficient HBx mutant was detected in both the cytoplasm and nucleus, suggesting that the DDB1 interaction plays an important role in the nuclear localization of HBx. Our study also revealed that HBx is expressed early after infection and has a short half-life (∼3 h) in HBV-infected PHH. In addition, we found that treatment with small interfering RNAs (siRNAs) that target DDB1 or HBx mRNA decreased HBx protein levels and led to the reappearance of Smc6 in the nuclei of HBV-infected PHH. Collectively, these studies provide the first spatiotemporal analysis of HBx in a natural infection system and also suggest that HBV transcriptional silencing by Smc5/6 can be restored by therapeutic targeting of HBx.IMPORTANCE Hepatitis B virus X protein (HBx) is a promising drug target since it promotes the degradation of the host structural maintenance of chromosomes 5/6 complex (Smc5/6) that inhibits HBV transcription. To date, it has not been possible to study HBx in physiologically relevant cell culture systems due to the lack of a highly specific and selective HBx antibody. In this study, we developed a novel monoclonal HBx antibody and performed a spatiotemporal analysis of HBx in a natural infection system. This revealed that HBx localizes to the nucleus of infected cells, is expressed shortly after infection, and has a short half-life. In addition, we demonstrated that inhibiting HBx expression or function promotes the reappearance of Smc6 in the nucleus of infected cells. These data provide new insights into HBx and underscore its potential as a novel target for the treatment of chronic HBV infection.

In vitro resistance profile of hepatitis C virus NS5A inhibitor velpatasvir in genotypes 1 to 6.

Velpatasvir is a pan-genotypic hepatitis C virus (HCV) NS5A inhibitor, which is used with sofosbuvir for treatment of infection with HCV genotypes 1-6. In vitro resistance studies were performed to characterize NS5A changes that might confer reduced velpatasvir susceptibility in vivo. Resistance selection studies using HCV replicon cells for subtypes 1a, 1b, 2a, 2b, 3a, 4a, 5a and 6a identified NS5A resistance-associated substitutions (RASs) at nine positions, most often 28M/S/T, 31F/I/M/P/V and 93D/H/N/S. In subtype 1a, RASs were selected at positions 31 and/or 93, while in subtype 1b, replicons with two or more RASs at positions 31, 54 or 93 were selected. Y93H was selected in subtypes 1a, 1b, 2a, 3a and 4a. In subtype 5a or 6a, L31P or P32L/Q was selected, respectively. Velpatasvir susceptibility of 358 replicons from genotypes 1 to 6 containing one or more NS5A RASs was also evaluated. The majority (63%) of subtypes 1a and 1b single RAS-containing replicons retained susceptibility to velpatasvir (<2.5-fold change in EC50 ). High levels of resistance to velpatasvir were observed for six single mutants in subtype 1a, including M28G, A92K, Y93H/N/R/W and for one mutant, A92K, in subtype 1b. Most single mutants in subtypes 2a, 2b, 3a, 4a and 5a displayed low levels of reduced velpatasvir susceptibility. High-level resistance was observed for C92T and Y93H/N in subtype 2b, Y93H/S in 3a, and L31V and P32A/L/Q/R in 6a, and several double mutants in these subtypes. Overall, velpatasvir maintained activity against most common RASs that are known to confer resistance to first-generation NS5A inhibitors.

Toll-like receptor 7 agonist GS-9620 induces prolonged inhibition of HBV via a type I interferon-dependent mechanism.

BACKGROUND & AIMS: GS-9620, an oral agonist of toll-like receptor 7 (TLR7), is in clinical development for the treatment of chronic hepatitis B (CHB). GS-9620 was previously shown to induce prolonged suppression of serum viral DNA and antigens in the woodchuck and chimpanzee models of CHB. Herein, we investigated the molecular mechanisms that contribute to the antiviral response to GS-9620 using in vitro models of hepatitis B virus (HBV) infection. METHODS: Cryopreserved primary human hepatocytes (PHH) and differentiated HepaRG (dHepaRG) cells were infected with HBV and treated with GS-9620, conditioned media from human peripheral blood mononuclear cells treated with GS-9620 (GS-9620 conditioned media [GS-9620-CM]), or other innate immune stimuli. The antiviral and transcriptional response to these agents was determined. RESULTS: GS-9620 had no antiviral activity in HBV-infected PHH, consistent with low level TLR7 mRNA expression in human hepatocytes. In contrast, GS-9620-CM induced prolonged reduction of HBV DNA, RNA, and antigen levels in PHH and dHepaRG cells via a type I interferon (IFN)-dependent mechanism. GS-9620-CM did not reduce covalently closed circular DNA (cccDNA) levels in either cell type. Transcriptional profiling demonstrated that GS-9620-CM strongly induced various HBV restriction factors - although not APOBEC3A or the Smc5/6 complex - and indicated that established HBV infection does not modulate innate immune sensing or signaling in cryopreserved PHH. GS-9620-CM also induced expression of immunoproteasome subunits and enhanced presentation of an immunodominant viral peptide in HBV-infected PHH. CONCLUSIONS: Type I IFN induced by GS-9620 durably suppressed HBV in human hepatocytes without reducing cccDNA levels. Moreover, HBV antigen presentation was enhanced, suggesting additional components of the TLR7-induced immune response played a role in the antiviral response to GS-9620 in animal models of CHB. LAY SUMMARY: GS-9620 is a drug currently being tested in clinical trials for the treatment of chronic hepatitis B virus (HBV) infection. GS-9620 has previously been shown to suppress HBV in various animal models, but the underlying antiviral mechanisms were not completely understood. In this study, we determined that GS-9620 does not directly activate antiviral pathways in human liver cells, but can induce prolonged suppression of HBV via induction of an antiviral cytokine called interferon. However, interferon did not destroy the HBV genome, suggesting that other parts of the immune response (e.g. activation of immune cells that kill infected cells) also play an important role in the antiviral response to GS-9620.

Identifying and Characterizing Interplay between Hepatitis B Virus X Protein and Smc5/6.

Hepatitis B X protein (HBx) plays an essential role in the hepatitis B virus (HBV) replication cycle, but the function of HBx has been elusive until recently. It was recently shown that transcription from the HBV genome (covalently-closed circular DNA, cccDNA) is inhibited by the structural maintenance of chromosome 5/6 complex (Smc5/6), and that a key function of HBx is to redirect the DNA-damage binding protein 1 (DDB1) E3 ubiquitin ligase to target this complex for degradation. By doing so, HBx alleviates transcriptional repression by Smc5/6 and stimulates HBV gene expression. In this review, we discuss in detail how the interplay between HBx and Smc5/6 was identified and characterized. We also discuss what is known regarding the repression of cccDNA transcription by Smc5/6, the timing of HBx expression, and the potential role of HBx in promoting hepatocellular carcinoma (HCC).

The Smc5/6 Complex Restricts HBV when Localized to ND10 without Inducing an Innate Immune Response and Is Counteracted by the HBV X Protein Shortly after Infection.

The structural maintenance of chromosome 5/6 complex (Smc5/6) is a restriction factor that represses hepatitis B virus (HBV) transcription. HBV counters this restriction by expressing HBV X protein (HBx), which targets Smc5/6 for degradation. However, the mechanism by which Smc5/6 suppresses HBV transcription and how HBx is initially expressed is not known. In this study we characterized viral kinetics and the host response during HBV infection of primary human hepatocytes (PHH) to address these unresolved questions. We determined that Smc5/6 localizes with Nuclear Domain 10 (ND10) in PHH. Co-localization has functional implications since depletion of ND10 structural components alters the nuclear distribution of Smc6 and induces HBV gene expression in the absence of HBx. We also found that HBV infection and replication does not induce a prominent global host transcriptional response in PHH, either shortly after infection when Smc5/6 is present, or at later times post-infection when Smc5/6 has been degraded. Notably, HBV and an HBx-negative virus establish high level infection in PHH without inducing expression of interferon-stimulated genes or production of interferons or other cytokines. Our study also revealed that Smc5/6 is degraded in the majority of infected PHH by the time cccDNA transcription could be detected and that HBx RNA is present in cell culture-derived virus preparations as well as HBV patient plasma. Collectively, these data indicate that Smc5/6 is an intrinsic antiviral restriction factor that suppresses HBV transcription when localized to ND10 without inducing a detectable innate immune response. Our data also suggest that HBx protein may be initially expressed by delivery of extracellular HBx RNA into HBV-infected cells.

Hepatitis B virus X protein identifies the Smc5/6 complex as a host restriction factor.

Chronic hepatitis B virus infection is a leading cause of cirrhosis and liver cancer. Hepatitis B virus encodes the regulatory HBx protein whose primary role is to promote transcription of the viral genome, which persists as an extrachromosomal DNA circle in infected cells. HBx accomplishes this task by an unusual mechanism, enhancing transcription only from extrachromosomal DNA templates. Here we show that HBx achieves this by hijacking the cellular DDB1-containing E3 ubiquitin ligase to target the 'structural maintenance of chromosomes' (Smc) complex Smc5/6 for degradation. Blocking this event inhibits the stimulatory effect of HBx both on extrachromosomal reporter genes and on hepatitis B virus transcription. Conversely, silencing the Smc5/6 complex enhances extrachromosomal reporter gene transcription in the absence of HBx, restores replication of an HBx-deficient hepatitis B virus, and rescues wild-type hepatitis B virus in a DDB1-knockdown background. The Smc5/6 complex associates with extrachromosomal reporters and the hepatitis B virus genome, suggesting a direct mechanism of transcriptional inhibition. These results uncover a novel role for the Smc5/6 complex as a restriction factor selectively blocking extrachromosomal DNA transcription. By destroying this complex, HBx relieves the inhibition to allow productive hepatitis B virus gene expression.

A small-molecule inhibitor of hepatitis C virus infectivity.

One of the most challenging goals of hepatitis C virus (HCV) research is to develop well-tolerated regimens with high cure rates across a variety of patient populations. Such a regimen will likely require a combination of at least two distinct direct-acting antivirals (DAAs). Combining two or more DAAs with different resistance profiles increases the number of mutations required for viral breakthrough. Currently, most DAAs inhibit HCV replication. We recently reported that the combination of two distinct classes of HCV inhibitors, entry inhibitors and replication inhibitors, prolonged reductions in extracellular HCV in persistently infected cells. We therefore sought to identify new inhibitors targeting aspects of the HCV replication cycle other than RNA replication. We report here the discovery of the first small-molecule HCV infectivity inhibitor, GS-563253, also called HCV infectivity inhibitor 1 (HCV II-1). HCV II-1 is a substituted tetrahydroquinoline that selectively inhibits genotype 1 and 2 HCVs with low-nanomolar 50% effective concentrations. It was identified through a high-throughput screen and subsequent chemical optimization. HCV II-1 only permits the production and release of noninfectious HCV particles from cells. Moreover, infectious HCV is rapidly inactivated in its presence. HCV II-1 resistance mutations map to HCV E2. In addition, HCV-II prevents HCV endosomal fusion, suggesting that it either locks the viral envelope in its prefusion state or promotes a viral envelope conformation change incapable of fusion. Importantly, the discovery of HCV II-1 opens up a new class of HCV inhibitors that prolong viral suppression by HCV replication inhibitors in persistently infected cell cultures.

Hepatitis C viral entry inhibitors prolong viral suppression by replication inhibitors in persistently-infected Huh7 cultures.

Efforts to treat HCV patients are focused on developing antiviral combinations that lead to the eradication of infection. Thus, it is important to identify optimal combinations from the various viral inhibitor classes. Based on viral dynamic models, HCV entry inhibitors are predicted to reduce viral load in a monophasic manner reflecting the slow death rate of infected hepatocytes (t1/2 = 2-70 days) and the protection of naïve, un-infected cells from HCV infection. In contrast, replication inhibitors are predicted to reduce viral load in a biphasic manner. The initial rapid reduction phase is due to the inhibition of virus production and elimination of plasma virus (t1/2∼3 hours). The second, slower reduction phase results from the elimination of infected hepatocytes. Here we sought to compare the ability of HCV entry and replication inhibitors as well as combinations thereof to reduce HCV infection in persistently-infected Huh7 cells. Treatment with 5 × EC50 of entry inhibitors anti-CD81 Ab or EI-1 resulted in modest (≤ 1 log10 RNA copies/ml), monophasic declines in viral levels during 3 weeks of treatment. In contrast, treatment with 5 × EC50 of the replication inhibitors BILN-2016 or BMS-790052 reduced extracellular virus levels more potently (~2 log10 RNA copies/ml) over time in a biphasic manner. However, this was followed by a slow rise to steady-state virus levels due to the emergence of resistance mutations. Combining an entry inhibitor with a replication inhibitor did not substantially enhance the rate of virus reduction. However, entry/replication inhibitor and replication/replication inhibitor combinations reduced viral levels further than monotherapies (up to 3 log10 RNA copies/ml) and prolonged this reduction relative to monotherapies. Our results demonstrated that HCV entry inhibitors combined with replication inhibitors can prolong antiviral suppression, likely due to the delay of viral resistance emergence.

Cellular growth kinetics distinguish a cyclophilin inhibitor from an HSP90 inhibitor as a selective inhibitor of hepatitis C virus.

During antiviral drug discovery, it is critical to distinguish molecules that selectively interrupt viral replication from those that reduce virus replication by adversely affecting host cell viability. In this report we investigate the selectivity of inhibitors of the host chaperone proteins cyclophilin A (CypA) and heat-shock protein 90 (HSP90) which have each been reported to inhibit replication of hepatitis C virus (HCV). By comparing the toxicity of the HSP90 inhibitor, 17-(Allylamino)-17-demethoxygeldanamycin (17-AAG) to two known cytostatic compounds, colchicine and gemcitabine, we provide evidence that 17-AAG exerts its antiviral effects indirectly through slowing cell growth. In contrast, a cyclophilin inhibitor, cyclosporin A (CsA), exhibited selective antiviral activity without slowing cell proliferation. Furthermore, we observed that 17-AAG had little antiviral effect in a non-dividing cell-culture model of HCV replication, while CsA reduced HCV titer by more than two orders of magnitude in the same model. The assays we describe here are useful for discriminating selective antivirals from compounds that indirectly affect virus replication by reducing host cell viability or slowing cell growth.

An adaptive mutation in NS2 is essential for efficient production of infectious 1b/2a chimeric hepatitis C virus in cell culture.

The development of JFH1 based intergenotypic recombinants which exploit the unique replication characteristics of JFH1 has made it possible to study infectious HCV encoding the structural genes of additional HCV genotypes including genotype 1b. Although, intergenotypic 1b/2a chimeric genomes replicate efficiently in transfected cells they produce very low viral titers, limiting the utility of this system. Here, intergenotypic 1b/2a variants were generated by serially passaging the virus in a novel highly permissive Huh-7 cell clone. The adapted virus was 1000-fold more infectious than the parental unadapted virus and six adapted mutations were identified throughout the genome. Of the mutations identified, L839S in the NS2 gene was the most critical for the adapted phenotype by enhancing the infectivity of assembled viral particles. Overall, the efficient production of infectious 1b/2a virus particles will facilitate the discovery and characterization of inhibitors targeting steps that involve the structural genes of genotype 1b HCV.

Novel mutations in a tissue culture-adapted hepatitis C virus strain improve infectious-virus stability and markedly enhance infection kinetics.

Hepatitis C virus (HCV) establishes persistent infections and leads to chronic liver disease. It only recently became possible to study the entire HCV life cycle due to the ability of a unique cloned patient isolate (JFH-1) to produce infectious particles in tissue culture. However, despite efficient RNA replication, yields of infectious virus particles remain modest. This presents a challenge for large-scale tissue culture efforts, such as inhibitor screening. Starting with a J6/JFH-1 chimeric virus, we used serial passaging to generate a virus with substantially enhanced infectivity and faster infection kinetics compared to the parental stock. The selected virus clone possessed seven novel amino acid mutations. We analyzed the contribution of individual mutations and identified three specific mutations, core K78E, NS2 W879R, and NS4B V1761L, which were necessary and sufficient for the adapted phenotype. These three mutations conferred a 100-fold increase in specific infectivity compared to the parental J6/JFH-1 virus, and media collected from cells infected with the adapted virus yielded infectious titers as high as 1 × 10(8) 50% tissue culture infective doses (TCID(50))/ml. Further analyses indicated that the adapted virus has longer infectious stability at 37°C than the wild type. Given that the adapted phenotype resulted from a combination of mutations in structural and nonstructural proteins, these data suggest that the improved viral titers are likely due to differences in virus particle assembly that result in significantly improved infectious particle stability. This adapted virus will facilitate further studies of the HCV life cycle, virus structure, and high-throughput drug screening.

Hepatitis C virus NS2 protein contributes to virus particle assembly via opposing epistatic interactions with the E1-E2 glycoprotein and NS3-NS4A enzyme complexes.

The hepatitis C virus NS2 protein has been recently implicated in virus particle assembly. To further understand the role of NS2 in this process, we conducted a reverse genetic analysis of NS2 in the context of a chimeric genotype 2a infectious cell culture system. Of 32 mutants tested, all were capable of RNA replication and 25 had moderate-to-severe defects in virus assembly. Through forward genetic selection for variants capable of virus spread, we identified second-site mutations in E1, E2, NS2, NS3, and NS4A that suppressed NS2 defects in assembly. Two suppressor mutations, E1 A78T and NS3 Q221L, were further characterized by additional genetic and biochemical experiments. Both mutations were shown to suppress other NS2 defects, often with mutual exclusivity. Thus, several NS2 mutants were enhanced by NS3 Q221L and inhibited by E1 A78T, while others were enhanced by E1 A78T and inhibited by NS3 Q221L. Furthermore, we show that the NS3 Q221L mutation lowers the affinity of native, full-length NS3-NS4A for functional RNA binding. These data reveal a complex network of interactions involving NS2 and other viral structural and nonstructural proteins during virus assembly.

The NS4A protein of hepatitis C virus promotes RNA-coupled ATP hydrolysis by the NS3 helicase.

Nonstructural protein 3 (NS3) is an essential replicative component of the hepatitis C virus (HCV) and a member of the DExH/D-box family of proteins. The C-terminal region of NS3 (NS3hel) exhibits RNA-stimulated NTPase and helicase activity, while the N-terminal serine protease domain of NS3 enhances RNA binding and unwinding by NS3hel. The nonstructural protein 4A (NS4A) binds to the NS3 protease domain and serves as an obligate cofactor for NS3 serine protease activity. Given its role in stimulating protease activity, we sought to determine whether NS4A also influences the activity of NS3hel. Here we show that NS4A enhances the ability of NS3hel to bind RNA in the presence of ATP, thereby acting as a cofactor for helicase activity. This effect is mediated by amino acids in the C-terminal acidic domain of NS4A. When these residues are mutated, one observes drastic reductions in ATP-coupled RNA binding and duplex unwinding by NS3. These same mutations are lethal in HCV replicons, thereby establishing in vitro and in vivo that NS4A plays an important role in the helicase mechanism of NS3 and its function in replication.

Establishing a mechanistic basis for the large kinetic steps of the NS3 helicase.

The NS3 helicase from hepatitis C virus is a prototypical DEx(H/D) RNA helicase. NS3 has been shown to unwind RNA in a discontinuous manner, pausing after long apparent steps of unwinding. We systematically examined the effects of duplex stability and ionic conditions on the periodicity of the NS3 unwinding cycle. The kinetic step size for NS3 unwinding was examined on diverse substrate sequences. The kinetic step size (16 bp/step) was found to be independent of RNA duplex stability and composition, but it exhibited strong dependence on monovalent salt concentration, decreasing to approximately 11 bp/step at low [NaCl]. We addressed this behavior by analyzing the oligomeric state of NS3 at various salt concentrations. Whereas only NS3 oligomers are capable of processive unwinding, we found that monomeric NS3 is an active helicase that unwinds with low processivity. We demonstrate that low salt conditions enhance unwinding by monomeric NS3, which is likely to account for the reduction in apparent step size under low salt conditions. Based on results reported here, as well as available structural and single molecule data, we present an unwinding mechanism that addresses the apparent periodicity of NS3 unwinding, the magnitude of the step size, and that integrates the various stepwise motions observed for NS3. We propose that the large kinetic step size of NS3 unwinding reflects a delayed, periodic release of the separated RNA product strand from a secondary binding site that is located in the NTPase domain (Domain II) of NS3. These findings suggest that the mechanism of product release represents an important and unexplored feature of helicase mechanism.