Previously, human hepatitis B virus (HBV) mutant 164, which has a truncation at the C terminus of the HBV core antigen (HBcAg), was speculated to secrete immature genomes. For this study, we further characterized mutant 164 by different approaches. In addition to the 3.5-kb pregenomic RNA (pgRNA), the mutant preferentially encapsidated the 2.2-kb or shorter species of spliced RNA, which can be reverse transcribed into double-stranded DNA before virion secretion. We observed that mutant 164 produced less 2.2-kb spliced RNA than the wild type. Furthermore, it appeared to produce at least two different populations of capsids: one encapsidated a nuclease-sensitive 3.5-kb pgRNA while the other encapsidated a nuclease-resistant 2.2-kb spliced RNA. In contrast, the wild-type core-associated RNA appeared to be resistant to nuclease. When arginines and serines were systematically restored at the truncated C terminus, the core-associated DNA and nucleaseresistant RNA gradually increased in both size and signal intensity. Full protection of encapsidated pgRNA from nuclease was observed for HBcAg 1-171. A full-length positive-strand DNA phenotype requires positive charges at amino acids 172 and 173. Phosphorylation at serine 170 is required for optimal RNA encapsidation and a full-length positive-strand DNA phenotype. RNAs encapsidated in Escherichia coli by capsids of HBcAg 154, 164, and 167, but not HBcAg 183, exhibited nuclease sensitivity; however, capsid instability after nuclease treatment was observed only for HBcAg 164 and 167. A new hypothesis is proposed here to highlight the importance of a balanced charge density for capsid stability and intracapsid anchoring of RNA templates.The hepatitis B virus (HBV) core antigen (HBcAg) is a 22-kDa protein with multiple functions, including interactions with the pregenomic RNA (pgRNA) and the polymerase during encapsidation (10,31,41) and with the viral DNA during reverse transcription and DNA elongation (18), polymerization to form the nucleocapsid or core particles (4, 13), importing of HBV DNA to the nucleus (51), and targeting to the endoplasmic reticulum for envelope formation (5). The C-terminal region of HBcAg contains a protamine-like domain, which is rich in arginine and presumably binds to HBV RNA and DNA during pgRNA encapsidation and DNA replication (13,18,35). This arginine-rich domain has been shown to be dispensable for core particle assembly but not for viral replication (2, 4, 13, 30).

Hepatitis C virus (HCV) polymerase activity is essential for HCV replication. Targeted screening of nucleoside analogs identified R1479 (4-azidocytidine) as a specific inhibitor of HCV replication in the HCV subgenomic replicon system (IC 50 ‫؍‬ 1.28 M) with similar potency compared with 2-C-methylcytidine (IC 50 ‫؍‬ 1.13 M). R1479 showed no effect on cell viability or proliferation of HCV replicon or Huh-7 cells at concentrations up to 2 mM. HCV replicon RNA could be fully cleared from replicon cells after prolonged incubation with R1479. The corresponding 5-triphosphate derivative (R1479-TP) is a potent inhibitor of native HCV replicase isolated from replicon cells and of recombinant HCV polymerase (NS5B)-mediated RNA synthesis activity. R1479-TP inhibited RNA synthesis as a CTP-competitive inhibitor with a K i of 40 nM. On an HCV RNA-derived template substrate (complementary internal ribosome entry site), R1479-TP showed similar potency of NS5B inhibition compared with 3-dCTP. R1479-TP was incorporated into nascent RNA by HCV polymerase and reduced further elongation with similar efficiency compared with 3-dCTP under the reaction conditions. The S282T point mutation in the coding sequence of NS5B confers resistance to inhibition by 2-C-MeATP and other 2-methyl-nucleotides. In contrast, the S282T mutation did not confer cross-resistance to R1479. Hepatitis C virus (HCV)2 infection is a major cause of chronic liver disease, cirrhosis, and hepatocellular carcinoma and is currently the leading cause of liver transplantation (1, 2). Viral genome sequence analysis established six HCV genotype classes (HCV genotypes 1-6), with genotypes 1-3 being the most prevalent in the United States, Europe, and Japan. Current treatment options available to HCV-infected persons are limited, and sustained virological response rates are particularly low for HCV genotype 1-infected patients. Only ϳ50% of individuals infected with HCV genotype 1 with serum viral titers of Ͼ2 ϫ 10 6 copies/ml achieved sustained virological response rates when treated with a combination of pegylated interferon-␣ and ribavirin (3, 4). Response rates are even lower in persons with HIV co-infection or cirrhosis and also decrease with age (1, 5-7). Urgently required improvements in anti-HCV therapy will depend on the development of novel therapeutic approaches, especially in difficult to treat populations.HCV is an enveloped (ϩ)-strand RNA virus that enters host cells via receptor-mediated endocytosis and replicates in the host cell cytoplasm. A membrane-associated replicase complex containing HCV genome-encoded nonstructural proteins and HCV genomic RNA in a tight complex is responsible for the formation of viral RNA for packaging into new virus particles during the HCV replication process. The viral NS5B protein contains the HCV polymerase active site within the replicase complex, an RNA-dependent RNA polymerase. The concept of polymerase inhibition to attain antiviral efficacy has been successfully established in other viral infections (human immunodefi...

Specific inhibitors of hepatitis C virus (HCV) replication that target the NS3/4A protease (e.g., VX-950) or the NS5B polymerase (e.g., R1479/R1626, PSI-6130/R7128, NM107/NM283, and HCV-796) have advanced into clinical development. Treatment of patients with VX-950 or HCV-796 rapidly selected for drug-resistant variants after a 14-day monotherapy treatment period. However, no viral resistance was identified after monotherapy with R1626 (prodrug of R1479) or NM283 (prodrug of NM107) after 14 days of monotherapy. Based upon the rapid selection of resistance to the protease and nonnucleoside inhibitors during clinical trials and the lack of selection of resistance to the nucleoside inhibitors, we used the replicon system to determine whether nucleoside inhibitors demonstrate a higher genetic barrier to resistance than protease and nonnucleoside inhibitors. Treatment of replicon cells with nucleoside inhibitors at 10 and 15 times the 50% effective concentration resulted in clearance of the replicon, while treatment with a nonnucleoside or protease inhibitor selected resistant colonies. In combination, the presence of a nucleoside inhibitor reduced the frequency of colonies resistant to the other classes of inhibitors. These results indicate that the HCV replicon presents a higher barrier to the selection of resistance to nucleoside inhibitors than to nonnucleoside or protease inhibitors. Furthermore, the combination of a nonnucleoside or protease inhibitor with a nucleoside polymerase inhibitor could have a clear clinical benefit through the delay of resistance emergence.Hepatitis C virus (HCV) is a positive-strand RNA virus that is a member of the Hepacivirus genus within the Flaviridae family. There are an estimated 170 million individuals chronically infected with HCV worldwide, which amounts to almost 3% of the global population (1). In the United States, an estimated 20,000 new HCV infections occurred in 2005, adding to the approximately 4 million individuals previously infected with HCV (2, 38). Liver cirrhosis, as a result of HCV infection, is currently the leading reason justifying liver transplantation; however, reinfection occurs immediately posttransplantation and can result in graft loss (39).The current treatment of pegylated alpha interferon in combination with ribavirin results in a sustained viral response in approximately 50% of HCV patients infected with genotype (GT) 1 virus, the most prevalent GT worldwide. Therefore, a specific HCV antiviral therapy is highly desirable. Viral proteases and viral polymerases have been validated as clinically effective targets for a number of different viruses, including human immunodeficiency virus, hepatitis B virus, and herpesviruses (6,7,14,15). Two potential drug targets encoded by HCV are the NS3/4A serine protease and the NS5B RNA-dependent RNA polymerase (5). Several anti-HCV compounds that inhibit the activity of either the NS3/4A protease or the NS5B RNA-dependent RNA polymerase have resulted in decreased viral loads when administered to HCV-infected pat...

The HCV polymerase is an attractive target for the development of new and specific anti-HCV drugs. Herein, the characterization of the inhibitory effect of 2'-C-Methyl-Cytidine shows that it is a potent inhibitor of both genotype 1b and 1a HCV replicon replication, both of laboratory-optimized as well as of NS5B clinical isolates-chimera replicons. The corresponding 5'-triphosphate derivative is a potent inhibitor of native HCV replicase isolated from replicon cells and of the recombinant genotype 1b and 1a HCV polymerase-mediated RNA synthesis. Resistance to 2'-C-Methyl-Cytidine was mapped to amino acid substitution S282T in the NS5B coding region. Cross-resistance was observed to 2'-C-Methyl-Adenosine but not to interferon alpha-2a, to non-nucleoside HCV polymerase inhibitors or to R1479, a new and potent nucleoside inhibitor of NS5B polymerase. In vitro studies mapped resistance to R1479 to amino acid substitutions S96T and S96T/N142T of the NS5B polymerase. These mutations did not confer resistance to 2-C-Methyl-Cytidine, thus confirming the lack of cross-resistance between these two HCV inhibitors. These data will allow the optimization of new polymerase inhibitors and their use in combination therapy.

The higher frequency of known NNI resistance mutations or polymorphisms known to affect their antiviral potency when compared with the lack of detection of resistance mutations to the nucleoside analogues suggests a potential for primary reduced responsiveness as well as faster development of clinically significant resistance.