NS5B

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

Characterization of an Autonomous Subgenomic Pestivirus RNA Replicon

Abstract: As an initial approach to define the requirements for the replication of bovine viral diarrhea virus (BVDV), a member of the Flaviviridae family with a positive-strand RNA genome, full-length genomic and subgenomic RNAs were originated by in vitro transcription of diverse BVDV cDNA constructs and transfected into eucaryotic host cells. RNA replication was measured either directly by an RNase protection method or by monitoring the synthesis of viral protein. When full-length BVDV cRNA was initially applied, the synthesis of negative-strand RNA intermediates as well as progeny positive-strand RNA was detected posttransfection in the cytoplasm of the host cells. Compared to the negative-strand RNA intermediate, an excess of positive-strand RNA was synthesized. Surprisingly, a subgenomic RNA molecule, DI9c, corresponding to a previously characterized defective interfering particle, was found to support both steps of RNA replication in the absence of a helper virus as well, thus functioning as an autonomous replicon. DI9c comprises the 5' and 3' untranslated regions of the BVDV genome and the coding regions of the autoprotease Npro and the nonstructural proteins NS3, NS4A, NS4B, NS5A, and NS5B. Most interestingly, the NS2 polypeptide was thus determined to be nonessential for RNA replication. As expected, deletion of the genomic 3' end as well as abolition of the catalytic function of the virus-encoded serine protease resulted in DI9c molecules that were unable to replicate. Deletion of the entire Npro gene also destroyed the ability of DI9c molecules to replicate. On the other hand, DI9c derivatives in which the 5' third of the Npro gene was fused to a ubiquitin gene, allowing the proteolytic release of NS3 in trans, turned out to be replication competent. These results suggest that the RNA sequence located at the 5' end of the open reading frame exerts an essential role during BVDV replication. Replication of DI9c and DI9c derivatives was found not to be limited to host cells of bovine origin, indicating that cellular factors functioning as potential parts of the viral replication machinery are well conserved between different mammalian cells. Our data provide an important step toward the ready identification and characterization of viral factors and genomic elements involved in the life cycle of pestiviruses. The implications for other Flaviviridae and, in particular, the BVDV-related human hepatitis C virus are discussed.

DEB025 (Alisporivir) Inhibits Hepatitis C Virus Replication by Preventing a Cyclophilin A Induced Cis-Trans Isomerisation in Domain II of NS5A

Abstract: DEB025/Debio 025 (Alisporivir) is a cyclophilin (Cyp)-binding molecule with potent anti-hepatitis C virus (HCV) activity both in vitro and in vivo. It is currently being evaluated in phase II clinical trials. DEB025 binds to CypA, a peptidyl-prolyl cis-trans isomerase which is a crucial cofactor for HCV replication. Here we report that it was very difficult to select resistant replicons (genotype 1b) to DEB025, requiring an average of 20 weeks (four independent experiments), compared to the typically <2 weeks with protease or polymerase inhibitors. This indicates a high genetic barrier to resistance for DEB025. Mutation D320E in NS5A was the only mutation consistently selected in the replicon genome. This mutation alone conferred a low-level (3.9-fold) resistance. Replacing the NS5A gene (but not the NS5B gene) from the wild type (WT) genome with the corresponding sequence from the DEB025res replicon resulted in transfer of resistance. Cross-resistance with cyclosporine A (CsA) was observed, whereas NS3 protease and NS5B polymerase inhibitors retained WT-activity against DEB025res replicons. Unlike WT, DEB025res replicon replicated efficiently in CypA knock down cells. However, DEB025 disrupted the interaction between CypA and NS5A regardless of whether the NS5A protein was derived from WT or DEB025res replicon. NMR titration experiments with peptides derived from the WT or the DEB025res domain II of NS5A corroborated this observation in a quantitative manner. Interestingly, comparative NMR studies on two 20-mer NS5A peptides that contain D320 or E320 revealed a shift in population between the major and minor conformers. These data suggest that D320E conferred low-level resistance to DEB025 probably by reducing the need for CypA-dependent isomerisation of NS5A. Prolonged DEB025 treatment and multiple genotypic changes may be necessary to generate significant resistance to DEB025, underlying the high barrier to resistance.

Viral RNA Mutations Are Region Specific and Increased by Ribavirin in a Full-Length Hepatitis C Virus Replication System

Abstract: High rates of genetic variation ensure the survival of RNA viruses. Although this variation is thought to result from error-prone replication, RNA viruses must also maintain highly conserved genomic segments. A balance between conserved and variable viral elements is especially important in order for viruses to avoid "error catastrophe." Ribavirin has been shown to induce error catastrophe in other RNA viruses. We therefore used a novel hepatitis C virus (HCV) replication system to determine relative mutation frequencies in variable and conserved regions of the HCV genome, and we further evaluated these frequencies in response to ribavirin. We sequenced the 5' untranslated region (5' UTR) and the core, E2 HVR-1, NS5A, and NS5B regions of replicating HCV RNA isolated from cells transfected with a T7 polymerase-driven full-length HCV cDNA plasmid containing a cis-acting hepatitis delta virus ribozyme to control 3' cleavage. We found quasispecies in the E2 HVR-1 and NS5B regions of untreated replicating viral RNAs but not in conserved 5' UTR, core, or NS5A regions, demonstrating that important cis elements regulate mutation rates within specific viral segments. Neither T7-driven replication nor sequencing artifacts produced these nucleotide substitutions in control experiments. Ribavirin broadly increased error generation, especially in otherwise invariant regions, indicating that it acts as an HCV RNA mutagen in vivo. Similar results were obtained in hepatocyte-derived cell lines. These results demonstrate the potential utility of our system for the study of intrinsic factors regulating genetic variation in HCV. Our results further suggest that ribavirin acts clinically by promoting nonviable HCV RNA mutation rates. Finally, the latter result suggests that our replication model may be useful for identifying agents capable of driving replicating virus into error catastrophe.