Topic
NS5B
About: NS5B is a research topic. Over the lifetime, 1314 publications have been published within this topic receiving 59534 citations.
Papers published on a yearly basis
Papers
More filters
TL;DR: The crystal structure of the enzyme was exploited to elucidate the involvement of these two structural motifs in enzyme activity by site-directed mutagenesis and resulted in an enzyme that was consistently more active in de novo synthesis as well as by “copy-back” mechanism of a self-primed substrate when compared with the wild type NS5B enzyme.
74 citations
TL;DR: The details of NS5B polymerase/inhibitor binding interactions coupled with the observed induced conformational changes provide new insights into the design of novel NNIs of HCV.
74 citations
TL;DR: In this article, a review of hepatitis C virus resistant variants is presented, which depend on the class of direct-acting antiviral drugs used and also vary between hepatitisC virus genotypes and subtypes.
Abstract: There has been a remarkable transformation in the treatment of chronic hepatitis C in recent years with the development of direct acting antiviral agents targeting virus encoded proteins important for viral replication including NS3/4A, NS5A and NS5B. These agents have shown high sustained viral response (SVR) rates of more than 90% in phase 2 and phase 3 clinical trials; however, this is slightly lower in real-life cohorts. Hepatitis C virus resistant variants are seen in most patients who do not achieve SVR due to selection and outgrowth of resistant hepatitis C virus variants within a given host. These resistance associated mutations depend on the class of direct-acting antiviral drugs used and also vary between hepatitis C virus genotypes and subtypes. The understanding of these mutations has a clear clinical implication in terms of choice and combination of drugs used. In this review, we describe mechanism of action of currently available drugs and summarize clinically relevant resistance data.
74 citations
TL;DR: It is found that ribavirin triphosphates have no inhibitory effect, providing direct experimental proof that the therapeutic effect observed in patients is not related to a direct inhibition of the viral polymerase.
Abstract: Hepatitis C virus (HCV), the major causative agent of chronic and sporadic non-A, non-B hepatitis worldwide, is a distinct member of the Flaviviridae virus family. These viruses have in common a plus-strand RNA genome that is replicated in the cytoplasm of the infected cell via minus-strand RNA intermediates. Owing to the lack of reliable cell culture systems and convenient animal models for HCV, the mechanisms governing RNA replication are not known. As a first step towards the development of appropriate in vitro systems, we expressed the NS5B RNA-dependent RNA polymerase (RdRp) in insect cells, purified the protein to near homogeneity and studied its biochemical properties. It is a primer- and RNA template-dependent RNA polymerase able to copy long heteropolymeric templates without additional viral or cellular cofactors. We determined the optimal reaction parameters, the kinetic constants and the substrate specificity of the enzyme, which turned out to be similar to those described for the 3D polymerase of poliovirus. By analysing a series of nucleosidic and non-nucleosidic compounds for their effect on RdRp activity, we found that ribavirin triphosphates have no inhibitory effect, providing direct experimental proof that the therapeutic effect observed in patients is not related to a direct inhibition of the viral polymerase. Finally, mutation analysis was performed to map the minimal NS5B sequence required for enzymatic activity and to identify the ‘classical’ polymerase motifs important for template and NTP binding and catalysis.
73 citations
TL;DR: Compared the efficacy of a number of shRNAs directed against different target regions of the HCV genome, the possibility that the recombinant lentivirus expressing shRNA against NS3–1 would be a useful tool to inhibit HCV‐1b infection is implied.
Abstract: RNA interference (RNAi) is a phenomenon in which small interfering RNA (siRNA), an RNA duplex 21 to 23 nucleotides (nt) long, or short hairpin RNA (shRNA) resembling siRNA, mediates degradation of the target RNA molecule in a sequence-specific manner. RNAi is now expected to be a useful therapeutic strategy for hepatitis C virus (HCV) infection. In the present study we compared the efficacy of a number of shRNAs directed against different target regions of the HCV genome, such as 5′-untranslated region (5′UTR) (nt 286 to 304), Core (nt 371 to 389), NS3–1 (nt 2052 to 2060), NS3–2 (nt 2104 to 2122), and NS5B (nt 7326 to 7344), all of which except for NS5B are conserved among most, if not all, HCV subtype 1b (HCV-1b) isolates in Japan. We utilized two methods to express shRNAs, one utilizing an expression plasmid (pAVU6+27) and the other utilizing a recombinant lentivirus harboring the pAVU6+27-derived expression cassette. Although 5′UTR has been considered to be the most suitable region for therapeutic siRNA and/or shRNA because of its extremely high degree of sequence conservation, we observed only a faint suppression of an HCV subgenomic replicon by shRNA against 5′UTR. In both plasmid- and lentivirus-mediated expression systems, shRNAs against NS3–1 and NS5B suppressed most efficiently the replication of the HCV replicon without suppressing host cellular gene expression. Synthetic siRNA against NS3–1 also inhibited replication of the HCV replicon in a dose-dependent manner. Taken together, the present results imply the possibility that the recombinant lentivirus expressing shRNA against NS3–1 would be a useful tool to inhibit HCV-1b infection.
73 citations