NS5B

About: NS5B is a research topic. Over the lifetime, 1314 publications have been published within this topic receiving 59534 citations.

Antiviral resistance and specifically targeted therapy for HCV (STAT-C).

Abstract: As health care providers, we find ourselves on the verge of a new era in the treatment of chronic hepatitis C virus (HCV) infection. A number of directly acting antiviral agents are now in the latter stages of clinical development. The more promising candidates include direct inhibitors of the HCV nonstructural 3 protease, as well as both nucleoside and non-nucleoside inhibitors of the NS5B RNA-dependent RNA polymerase. Although these agents have demonstrated potent antiviral effect, monotherapy has been complicated by rapid virological breakthrough due to the selection of drug-resistant mutants. As for HIV and HBV, combination therapy will therefore be necessary. This brief review summarizes the current literature concerning resistance and directly acting antiviral agents, and identifies key challenges facing this emerging field.

Mechanism of De Novo Initiation by the Hepatitis C Virus RNA-Dependent RNA Polymerase: Role of Divalent Metals

Abstract: Polymerases are metal-activated enzymes that use divalent metals for nucleotide polymerization. The paradigm for catalysis by DNA-dependent RNA polymerases is that two Mg2+ ions coordinate the nucleotides and catalyze the formation of the phosphodiester bonds (11). These metals are specifically recognized by amino acids in the catalytic pocket of the polymerase. RNA viruses can initiate RNA synthesis by a number of mechanisms. Many use a de novo initiation mechanism in which the first phosphodiester bond is formed between the initiation nucleotide, usually a purine triphosphate, and a second nucleoside triphosphate (NTP). Others, such as poliovirus, use a primer-dependent mechanism (25). Yet others, such as the influenza virus, can use a combination of the two strategies, depending on the RNA to be synthesized (8). Flaviviridae RNA-dependent RNA polymerases (RdRps) can initiate RNA synthesis by a de novo mechanism (12, 13, 14, 21, 23, 33, 40). De novo initiation is likely the mechanism used during flavivirus replication in vivo (3, 13). The hepatitis C virus (HCV) nonstructural protein 5B (NS5B), the RdRp, has a β-loop near the catalytic pocket (4, 18) that has been proposed to discriminate against primer extension and to provide at least part of the structural basis for de novo initiation (9). Despite the presence of the β-loop, the HCV and other flaviviral RdRps are capable of primer extension. In fact, primer extension is so robust that de novo initiation of RNA synthesis was not observed in some early studies (2, 7, 19, 20, 38). These results beg the question of how primer extension and de novo initiation are differentially achieved by the same recombinant flaviviral RdRps. In RNA synthesis assays, the addition of 1 to 2 mM Mn2+ to a reaction mixture containing Mg2+ increased RNA synthesis, while higher concentrations tended to be inhibitory (7, 21). Mn2+ has been reported to affect several polymerase activities, such as decreasing the specificity for the template and in nucleotide incorporation, lowering the Km for template binding, and increasing terminal nucleotide addition (24, 29, 34, 35, 37). These observations and the low physiological concentration of Mn2+ led some researchers to rule out a role for Mn2+ in the normal mechanism of viral RNA synthesis in the infected cell. There is some suggestive evidence that Mn2+ may play a more active role in RNA-dependent RNA synthesis in at least some RdRps. The crystal structures of the RdRp from phage φ6 can, in the absence of NTPs, bind either Mg2+ or Mn2+ at a site approximately 6 A from the catalytic aspartate residues, an unexpected feature in comparison to other polymerases (5). In the presence of nucleotide, the crystal structure of the RdRp from a calicivirus can form a closed and presumably active structure in the presence of two Mn2+ ions (22). Mn2+ also has a higher occupancy rate in the HCV RdRp than Mg2+ (3). Using a template capable of both de novo initiation and primer extension, we found that Mn2+ is strongly preferred for de novo initiation by the HCV RdRp and increases de novo initiation by the RdRp from bovine viral diarrhea virus (BVDV). In contrast, the HCV RdRp preferentially performed primer extension in the presence of Mg2+. Analysis of C-terminally truncated HCV RdRps revealed that the C-terminal portion of the RdRp contributes to Mn2+-dependent de novo initiation and the suppression of primer extension. Spectroscopy examining the intrinsic fluorescence of tyrosine and tryptophan residues in the HCV RdRp confirms that the presence of Mg2+ or Mn2+ resulted in a spectral change in the protein.

Expression of hepatitis C virus core protein inhibits interferon‐induced nuclear import of STATs

Abstract: IFN-alpha combined with ribavirin is used for the treatment of chronic hepatitis C. However, HCV has mechanisms to resist the antiviral actions of IFN-alpha. In order to study the molecular mechanisms of this resistance, the effect of HCV gene expression on IFN-induced nuclear import of STAT transcription factors and the expression of antiviral MxA protein were studied. In transiently transfected hepatoma cells, HCV core and NS5A proteins clearly inhibited the nuclear import of STAT1 and MxA protein expression (core only), whereas other viral proteins had only a marginal effect. To confirm these observations, human osteosarcoma-derived cell lines, which inducibly express HCV core protein, the entire structural region (core-E1-E2-p7), the NS3-4A complex, NS4B, NS5A, or NS5B proteins were also used. IFN-induced nuclear accumulation of STAT1 was almost completely and STAT2 was partially blocked in cell lines expressing high levels of HCV core protein. Subsequently, in these cells, IFN-alpha-induced MxB protein expression was decreased. Tumor necrosis factor-alpha (TNF-alpha)-induced nuclear import of NF-kappaB was only weakly or not at all inhibited, suggesting that the nuclear import machinery in general was not impaired. The results demonstrate a novel mechanism by which HCV gene expression may interfere with IFN-mediated host defence systems.