Chronic hepatitis B virus (HBV) infection affects 240 million people worldwide and is a major risk factor for liver failure and hepatocellular carcinoma. Current antiviral therapy inhibits cytoplasmic HBV genomic replication, but is not curative because it does not directly affect nuclear HBV closed circular DNA (cccDNA), the genomic form that templates viral transcription and sustains viral persistence. Novel approaches that directly target cccDNA regulation would therefore be highly desirable. cccDNA is assembled with cellular histone proteins into chromatin, but little is known about the regulation of HBV chromatin by histone posttranslational modifications (PTMs). Here, using a new cccDNA ChIP-Seq approach, we report, to our knowledge, the first genome-wide maps of PTMs in cccDNA-containing chromatin from de novo infected HepG2 cells, primary human hepatocytes, and from HBV-infected liver tissue. We find high levels of PTMs associated with active transcription enriched at specific sites within the HBV genome and, surprisingly, very low levels of PTMs linked to transcriptional repression even at silent HBV promoters. We show that transcription and active PTMs in HBV chromatin are reduced by the activation of an innate immunity pathway, and that this effect can be recapitulated with a small molecule epigenetic modifying agent, opening the possibility that chromatin-based regulation of cccDNA transcription could be a new therapeutic approach to chronic HBV infection.hepatitis B virus | HBV | cccDNA | chromatin | epigenetics H epatitis B virus (HBV) infection is widespread in humans and is a major public health concern. Primary infection outside the newborn period is usually self-limited, but a subset of infected individuals does not eliminate the virus and goes on to a lifelong persistent infection. Worldwide, at least 240 million people are persistently infected, many of whom develop chronic liver injury (chronic hepatitis B or CHB) (1). CHB often progresses to cirrhosis and liver failure, and is also strongly linked to the development of hepatocellular carcinoma (HCC). It is estimated that CHB accounts for more than 80% of HCC cases in areas of high HBV incidence (2).HBV belongs to the family of Hepadnaviridae, a group of small DNA viruses that infect hepatocytes and replicate through the reverse transcription of an RNA intermediate (3). The 3.2-kb HBV genome in viral particles is in a circular, partially doublestranded DNA conformation (relaxed circular DNA or rcDNA), a result of the unusual replication mechanism of HBV. rcDNA is transcriptionally inert and must be converted into covalently closed circular DNA (cccDNA) in the nucleus of infected cells before viral RNAs can be transcribed. cccDNA is the only template for HBV transcription and, because HBV RNA templates genomic reverse transcription, its persistence is required for persistent infection. HBV replication itself is noncytolytic, but it induces an immune response that in the case of CHB leads to persistent liver inflammation. Suppression of HBV re...

Since the advent of genome-wide small interfering RNA screening, large numbers of cellular cofactors important for viral infection have been discovered at a rapid pace, but the viral targets and the mechanism of action for many of these cofactors remain undefined. One such cofactor is cyclophilin A (CyPA), upon which hepatitis C virus (HCV) replication critically depends. Here we report a new genetic selection scheme that identified a major viral determinant of HCV's dependence on CyPA and susceptibility to cyclosporine A. We selected mutant viruses that were able to infect CyPA-knockdown cells which were refractory to infection by wild-type HCV produced in cell culture. Five independent selections revealed related mutations in a single dipeptide motif (D316 and Y317) located in a proline-rich region of NS5A domain II, which has been implicated in CyPA binding. Engineering the mutations into wild-type HCV fully recapitulated the CyPA-independent and CsA-resistant phenotype and four putative proline substrates of CyPA were mapped to the vicinity of the DY motif. Circular dichroism analysis of wild-type and mutant NS5A peptides indicated that the D316E/Y317N mutations (DEYN) induced a conformational change at a major CyPA-binding site. Furthermore, nuclear magnetic resonance experiments suggested that NS5A with DEYN mutations adopts a more extended, functional conformation in the putative CyPA substrate site in domain II. Finally, the importance of this major CsA-sensitivity determinant was confirmed in additional genotypes (GT) other than GT 2a. This study describes a new genetic approach to identifying viral targets of cellular cofactors and identifies a major regulator of HCV's susceptibility to CsA and its derivatives that are currently in clinical trials.

The hepatitis C virus (HCV) subgenomic replicon is the primary tool for evaluating the activity of anti-HCV compounds in drug discovery research. Despite the prevalence of HCV genotype 1a (ϳ70% of U.S. HCV patients), few genotype 1a reporter replicon cell lines have been described; this is presumably due to the low replication capacity of such constructs in available Huh-7 cells. In this report, we describe the selection of highly permissive Huh-7 cell lines that support robust replication of genotype 1a subgenomic replicons harboring luciferase reporter genes. These novel cell lines support the replication of multiple genotype 1a replicons (including the H77 and SF9 strains), are significantly more permissive to genotype 1a HCV replication than parental Huh7-Lunet cells, and maintain stable genotype 1a replication levels suitable for antiviral screening. We found that the sensitivity of genotype 1a luciferase replicons to known antivirals was highly consistent between individual genotype 1a clonal cell lines but could vary significantly between genotypes 1a and 1b. Sequencing of the nonstructural region of 12 stable replicon cell clones suggested that the enhanced permissivity is likely due to cellular component(s) in these new cell lines rather than the evolution of novel adaptive mutations in the replicons. These new reagents will enhance drug discovery efforts targeting genotype 1a and facilitate the profiling of compound activity among different HCV genotypes and subtypes.

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 (HCV) is an enveloped positive-strand RNA virus that has only recently been adapted to tissue culture (22,41,46). The full-length genome of isolate JFH-1 was demonstrated to be competent for viral particle production in tissue culture (22,41,46) by using Huh-7-derived cell lines that are permissive to HCV infection and replication (2,20). Several of these HCV cell culture (HCVcc) systems have been described, the most robust of which are based on chimeric J6/JFH-1 viruses or tissue culture-adapted strains of JFH-1 (1,3,4,17,18,26,36,47). However, the quantity of infectious virions these systems can produce is limited, presumably due to currently unidentified constraints on infectious virus particle production in tissue culture (5,16,26,47). After passage of tissue culture-grown J6/JFH-1 virus in animals, the resultant viruses exhibited a higher specific infectivity (23). Similarly, passage of HCV in primary human hepatocytes yielded virus with higher specific infectivity (33). Although Huh-7 cells are currently the most efficient system for culturing of HCV, these data suggest that virus particle production is suboptimal in these cells compared to that in bona fide hepatocytes.Serial passage of HCV in cell culture has yielded virus isolates with increased viral titers (9,10,32,37,44). Interestingly, these ...

Clinical trials of direct-acting antiviral agents in patients chronically infected with hepatitis C virus (HCV) have demonstrated that viral resistance is detected rapidly during monotherapy. In patients, HCV does not exist as a single, genetically homogenous virus but rather as a population of variants termed "quasispecies." Preexisting variants resistant to specific antiviral drugs, overlooked in traditional hit-to-lead discovery efforts, may be responsible for these poor clinical outcomes. To enable real-time studies of resistance emergence in live cells, we established fluorescent protein-labeled HCV replicon cell lines. We validated these cell lines by demonstrating that antiviral susceptibility and the selection of signature resistance mutations for various drug classes are similar to traditional replicon cell lines. By quantifying the kinetics and uniformity of replication within colonies of drug-resistant fluorescent replicon cells, we showed that resistance emerged from a single cell and preexisted in a treatment-naive replicon population. Within this population, we determined the relative frequency of preexisting replicons capable of establishing foci during treatment with distinct antivirals. By measuring relative frequency as a function of dose, we quantitatively ranked distinct antiviral molecules on the basis of their distinct barriers to resistance. These insights into RNA virus quasispecies structure provide guidance for selecting clinical drug concentrations and selecting antiviral drug combinations most likely to suppress resistance.evolution | virology R esistance to antiviral drugs is a significant worldwide health problem. Although we typically discuss drug resistance in language suggesting that resistance "emerges" after administration of an antiviral drug, classical microbial genetics suggests that drug-resistant mutants preexist in viral populations. The Luria and Delbruck experiment (1) provided the first evidence that drug-resistance mutations could preexist. In infected patients, hepatitis C virus (HCV), a positive-strand RNA virus, is composed of a swarm of genetically heterogeneous variants termed "quasispecies." A central question is whether this diversity is so pronounced that drug-resistance mutations preexist in a drugnaïve HCV population. Despite the importance of drug resistance in driving therapeutic outcomes, identification of rare variants within polymorphic virus populations has been fundamentally difficult in vitro and in vivo.Although many new classes of direct-acting anti-HCV drugs are in clinical development, viral resistance to these agents generally is detected within a few days of monotherapy (2, 3). Further supporting the model of preexisting resistance, ultradeep sequencing experiments identified mutations, before drug exposure, that confer resistance to NS3 protease inhibitors (4, 5). Although drugdiscovery campaigns traditionally have prioritized leads and lead classes on the basis of potency and toxicity, quantitative comparisons of the frequency of drug-resista...

GS-9451 is a selective hepatitis C virus (HCV) NS3 protease inhibitor in development for the treatment of genotype 1 (GT1) HCV infection. Key preclinical properties of GS-9451, including in vitro antiviral activity, selectivity, cross-resistance, and combination activity, as well as pharmacokinetic properties, were determined. In multiple GT1a and GT1b replicon cell lines, GS-9451 had mean 50% effective concentrations (EC 50 s) of 13 and 5.4 nM, respectively, with minimal cytotoxicity; similar potency was observed in chimeric replicons encoding the NS3 protease gene of GT1 clinical isolates. GS-9451 was less active in GT2a replicon cells (EC 50 ‫؍‬ 316 nM). Additive to synergistic in vitro antiviral activity was observed when GS-9451 was combined with other agents, including alpha interferon, ribavirin, and the polymerase inhibitors GS-6620 and tegobuvir (GS-9190), as well as the NS5A inhibitor ledipasvir (GS-5885). GS-9451 retained wild-type activity against multiple classes of NS5B and NS5A inhibitor resistance mutations. GS-9451 was stable in hepatic microsomes and hepatocytes from human and three other tested species. Systemic clearance was low in dogs and monkeys but high in rats. GS-9451 showed good oral bioavailability in all three species tested. In rats, GS-9451 levels were ϳ40-fold higher in liver than plasma after intravenous dosing, and elimination of GS-9451 was primarily through biliary excretion. Together, these results are consistent with the antiviral activity observed in a recent phase 1b study. The results of in vitro cross-resistance and combination antiviral assays support the ongoing development of GS-9451 in combination with other agents for the treatment of chronic HCV infection.T he NS3 serine protease of hepatitis C virus (HCV), which liberates essential nonstructural proteins from the HCV polyprotein, is required for viral replication (1) and may promote infection by blunting host innate immunity (2). Inhibitors of the HCV NS3/4A serine protease can induce rapid and substantial reductions in viral load. The NS3 protease inhibitors telaprevir (Incivek) and boceprevir (Victrelis) are separately indicated for use in triple therapy combinations with pegylated alpha interferon (PEG-IFN) and ribavirin (RBV) for treating chronic genotype 1 (GT1) HCV infection. When added to PEG-IFN and RBV, telaprevir and boceprevir independently have increased rates of sustained virologic response (SVR) in GT1 HCV-infected patients (3-13). However, the standard of care still has many limitations, including a complex treatment regimen, significant drug-drug interaction potential, and adverse effects that can limit tolerability.There is continued need for novel NS3 protease inhibitors that are well tolerated, have minimal potential for drug-drug interactions, and provide more favorable treatment regimens to improve compliance. GS-9451 is a novel acyclic HCV protease inhibitor being developed for the treatment of GT1 HCV infection. GS-9451 inhibits NS3 protease by binding the active site of the enzyme in a reve...

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A novel series of HCV replication inhibitors based on a pyrido[3,2-d]pyrimidine core were optimized for pharmacokinetics (PK) in rats. Several associations between physicochemical properties and PK were identified and exploited to guide the design of compounds. In addition, a simple new metric that may aid in the prediction of bioavailability for compounds with higher polar surface area is described (3*HBD-cLogP).