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NS5B
About: NS5B is a research topic. Over the lifetime, 1314 publications have been published within this topic receiving 59534 citations.
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TL;DR: Valopicitabine and interferon appear to have synergistic antiviral effects both in vitro and in vivo, and the compound is generally well tolerated, with gastrointestinal effects being the most commonly observed treatment-related adverse events.
Abstract: Chronic hepatitis C is caused by infection with the hepatitis C virus (HCV), a member of the Flaviviridae family of viruses. Currently available treatment for HCV, including the standard combination therapy with interferon and ribavirin, is often unsuccessful at eradicating infection. In addition, the therapies now used to treat chronic hepatitis C are associated with substantial side effects. Therefore, new therapeutic strategies such as the use of antiviral drugs targeted to HCV-specific viral enzymes are being explored. One such option is the RNA-directed RNA polymerase (NS5B) inhibitor valopicitabine (NM-283), an orally bioavailable prodrug of the novel ribonucleoside analogue NM-107. This compound has shown in vitro activity against HCV-related bovine viral diarrhea virus (BVDV) polymerase. In patients with HCV-1 infection, valopicitabine produced reductions in HCV RNA viral load when administered either as monotherapy or in combination with pegylated interferon. When used together, valopicitabine and interferon appear to have synergistic antiviral effects both in vitro and in vivo. The compound is generally well tolerated, with gastrointestinal effects being the most commonly observed treatment-related adverse events.
16 citations
TL;DR: The adjuvant activity of NT(gp96) was more efficient in the induction of immune responses when fused to the C-terminal end of the HCV DNA polytope, and this immunomodulatory effect was dependent on the position of the fusion.
Abstract: Induction of a strong hepatitis C virus (HCV)-specific immune response plays a key role in control and clearance of the virus. A polytope (PT) DNA vaccine containing B- and T-cell epitopes could be a promising vaccination strategy against HCV, but its efficacy needs to be improved. The N-terminal domain of heat shock protein gp96 (NT(gp96)) has been shown to be a potent adjuvant for enhancing immunity. We constructed a PT DNA vaccine encoding four HCV immunodominant cytotoxic T lymphocyte epitopes (two HLA-A2- and two H2-Dd-specific motifs) from the Core, E2, NS3 and NS5B antigens in addition to a T-helper CD4+ epitope from NS3 and a B-cell epitope from E2. The NT(gp96) was fused to the C- or N-terminal end of the PT DNA (PT-NT(gp96) or NT(gp96)-PT), and their potency was compared. Cellular and humoral immune responses against the expressed peptides were evaluated in CB6F1 mice. Our results showed that immunization of mice with PT DNA vaccine fused to NT(gp96) induced significantly stronger T-cell and antibody responses than PT DNA alone. Furthermore, the adjuvant activity of NT(gp96) was more efficient in the induction of immune responses when fused to the C-terminal end of the HCV DNA polytope. In conclusion, the NT(gp96) improved the efficacy of the DNA vaccine, and this immunomodulatory effect was dependent on the position of the fusion.
16 citations
TL;DR: The truncated GBV‐B NS5B (NS5BΔCT19) was purified to homogeneity and shown to possess an RNA‐dependent RNA polymerase (RdRp) activity in both gel‐based and scintillation proximity assays, and provides a good surrogate assay system for HCV polymerase studies.
Abstract: Phylogenetic analysis and polyprotein organization comparison have shown that GB virus-B (GBV-B) is closely related to hepatitis C virus (HCV). In this study, the coding region for GBV-B non-structural protein 5B (NS5B) was isolated by reverse transcription-polymerase chain reaction (RT-PCR) from pooled serum of GBV-B-infected tamarins. Expression of soluble GBV-B NS5B protein in Escherichia coli was achieved by removal of a 19-amino acid hydrophobic domain at the C-terminus of the protein. The truncated GBV-B NS5B (NS5BDeltaCT19) was purified to homogeneity and shown to possess an RNA-dependent RNA polymerase (RdRp) activity in both gel-based and scintillation proximity assays. NS5BDeltaCT19 required the divalent cation Mn2+ for enzymatic activity, at an optimal concentration of 15 mM. Interestingly, Mg2+, at concentrations up to 20 mM, did not support the GBV-B NS5B activity. This differs from HCV NS5B where both Mn2+ and Mg2+ can support RdRp activity. Zn2+ was found to inhibit the activity of GBV-B NS5B, with a 50% inhibitory concentration (IC50) of 5-10 microM. Higher concentrations of monovalent salts (NaCl or KCl > 100 mM) and glycerol (> 3%) were also inhibitory. NS5BDeltaCT19 was able to bind to RNA homopolymers, but utilized most efficiently poly(C), the one with the lowest binding affinity for RNA synthesis. Mutational analysis of GBV-B NS5B demonstrated the importance of several conserved sequence motifs for enzymatic activity. Based on sequence homology ( approximately 37% identity and 52% similarity) between GBV-B and HCV NS5B proteins, the active GBV-B RdRp provides a good surrogate assay system for HCV polymerase studies.
16 citations
TL;DR: Structural analysis and molecular dynamics simulations, binding free energy calculations and free energy decomposition analysis reveal that the location of PF-00868554 and the binding mode changed due to mutation of the residue at the 423 site of NS5B polymerase from methionine to threonine, isoleucine or valine, which resulted in the loss of binding ability.
Abstract: NS5B, a hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) that plays a key role in viral replication, is an important target in the discovery of antiviral agents. PF-00868554 is a potent non-nucleoside inhibitor (NNI) that binds to the Thumb II allosteric pocket of NS5B polymerase and has shown significant promise in phase II clinical trials. Unfortunately, several PF-00868554 resistant mutants have been identified. M423 variants were the most common NS5B mutations that occurred after PF-00868554 monotherapy. In this study, we used molecular dynamics (MD) simulations, binding free energy calculations and free energy decomposition to explore the drug resistance mechanism of HCV to PF-00868554 resulting from three representative mutations (M423T/V/I) in NS5B polymerase. Free energy decomposition analysis reveals that the loss of binding affinity mainly comes from the reduction of both van der Waals (ΔEvdw) and electrostatic interaction contributions in the gas phase (ΔEele). Further structural analysis indicates that the location of PF-00868554 and the binding mode changed due to mutation of the residue at the 423 site of NS5B polymerase from methionine to threonine, isoleucine or valine, which further resulted in the loss of binding ability of PF-00868554 to NS5B polymerase. The obtained computational results will have important value for the rational design of novel non-nucleoside inhibitors targeting HCV NS5B polymerase.
16 citations
TL;DR: Several unknown HLA class I-restricted HCV variants which in part impair binding to predicted HCV CD8+ T cell epitopes with remarkable differences between HCV subtypes 1a and 1b quasispecies are identified.
16 citations