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 ...

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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.O ne of the most challenging goals of hepatitis C virus (HCV) research is to develop well-tolerated regimens with high cure rates. The standard of care for HCV patients over the past decade has been to treat with pegylated interferon combined with ribavirin. Relatively recently, the HCV NS3-4A protease inhibitors teleprevir and boceprevir were added to this standard of care and have improved the sustained virologic response (1). However, poor tolerability to treatments containing pegylated interferon has motivated researchers to attempt to develop a variety of other interferon-free treatments (2). An interferon-free cure will likely require a combination of at least two antivirals with differing modes of action (3, 4). Combining multiple antivirals with different resistance profiles increases the number of resistance mutations required for viral breakthrough (5). Studies to determine optimal antiviral combinations which take into account the probability of emergence of specific resistance mutations as well as the subsequent viral fitness (3, 6, 7) are under way.We recently demonstrated that the in vitro combination of HCV entry inhibitors with HCV replication inhibitors could prolong the efficacy compared to a single treatment with either inhibitor class. Specifically, we studied the entry inhibitor anti-CD81 anti...

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.