NS5B

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

Regulation of De Novo-Initiated RNA Synthesis in Hepatitis C Virus RNA-Dependent RNA Polymerase by Intermolecular Interactions

Abstract: The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) has been proposed to change conformations in association with RNA synthesis and to interact with cellular proteins. In vitro, the RdRp can initiate de novo from the ends of single-stranded RNA or extend a primed RNA template. The interactions between the 1 loop and thumb domain in NS5B are required for de novo initiation, although it is unclear whether these interactions are within an NS5B monomer or are part of a higher-order NS5B oligomeric complex. This work seeks to address how polymerase conformation and/or oligomerization affects de novo initiation. We have shown that an increasing enzyme concentration increases de novo initiation by the genotype 1b and 2a RdRps while primer extension reactions are not affected or inhibited under similar conditions. Initiation-defective mutants of the HCV polymerase can increase de novo initiation by the wild-type (WT) polymerase. GTP was also found to stimulate de novo initiation. Our results support a model in which the de novo initiation-competent conformation of the RdRp is stimulated by oligomeric contacts between individual subunits. Using electron microscopy and single-molecule reconstruction, we attempted to visualize the lowresolution conformations of a dimer of a de novo initiation-competent HCV RdRp. Polymerases undergo a series of conformational changes at different stages of nucleic acid synthesis (14). Of the templatedependent polymerases, the RNA-dependent RNA polymerases (RdRps) are the least understood in terms of their mechanism of action. RdRps are of increasing interest since cellular RdRps play important roles in the defense against nonself RNAs (44). In addition, virus-encoded RdRps are important targets for the development of antivirals. A better understanding of RNA-dependent RNA polymerases is thus important for both basic and applied science. Several model systems for biochemical study of viral RNAdependent RNA synthesis exist (4, 19, 20, 25, 37, 42). Wellcharacterized RdRps include those from the hepatitis C virus (HCV) and poliovirus (5, 17). In the host, the RdRps are complexed with other viral and/or cellular proteins that are usually associated with membranous intracellular structures. The replicases are usually difficult to study biochemically, but the catalytic RdRp subunits of several viruses can be purified for functional and structural analyses (53). These recombinant proteins can reproduce some of the activities of the replicases, including the ability to initiate RNA synthesis by a de novo mechanism (22, 47–49). Furthermore, recombinant RdRps can affect the activities of other replicase subunits in vitro, suggesting that the recombinant RdRp is useful for an in-depth understanding of RNA synthesis by HCV (45, 60). RdRps form a right-hand-like structure with thumb, finger, and palm subdomains. The metal-coordinating residues important for nucleotide binding are positioned within the palm subdomain (26). An interesting feature of viral RdRps is that they tend to exist in a closed conformation, even in the absence of template, in contrast to DNA-dependent RNA polymerases, which transition from open to closed complexes upon template

Cyclophilin inhibitors for the treatment of HCV infection.

Abstract: Cyclophilins (Cyps) constitute one of the three families of peptidyl prolyl isomerase enzymes. CypA is the prototypical member of the Cyp family and is the predominant Cyp expressed in human cells. Recent studies indicate that CypA has an essential role in supporting HCV-specific RNA replication and protein expression. CypA interacts with several virally expressed proteins, including the non-structural (NS) proteins NS2, NS5A and NS5B, and may regulate diverse activities ranging from polypeptide processing to viral assembly. The introduction of non-immunosuppressive Cyp inhibitors into clinical trials confirms that Cyp inhibition is a valid strategy for developing novel therapeutics for the treatment of chronic HCV infection. This review describes the cyclophilin protein family and the potential roles played by cyclophilins in supporting HCV RNA replication and protein expression, as well as the initial clinical results obtained with a novel series of non-immunosuppressive cyclophilin inhibitors that established the clinical proof of concept for this emerging class of therapeutic agents.

Molecular Epidemiology and Interferon Susceptibility of the Natural Recombinant Hepatitis C Virus Strain RF1_2k/1b

Abstract: BACKGROUND Hepatitis C virus (HCV) genotype is an important determinant of virological response to antiviral therapies. Currently, there are no data available on the molecular epidemiology and interferon susceptibility of the natural intergenotypic recombinant RF1_2k/1b (RF1) strain. METHODS Genotyping and RF1-PCR screening were performed on samples from 604 HCV RNA-positive individuals from 7 countries. uPA/SCID mice carrying human hepatocytes (chimeric mice) were infected with the RF1_2k/1b strain, and the susceptibility of the strain to interferon and ribavirin was compared with the susceptibilities of 2 different strains of genotype B, used as references. RESULTS Six new RF1 cases were identified in this study; 5 (2%) of 281 in Russia and 1 (1%) of 90 in Uzbekistan. Phylogenetic analyses based on Core/E1 and NS5b indicated that all RF1 representatives share a common evolutionary ancestor. Infection with RF1 was established in chimeric mice. Reduction of RF1 viral load was observed in response to 3 injections of 3 microg/kg pegylated-interferon alpha-2a alone or in combination with 50 mg/kg of ribavirin (0.5 or 1.4 log-copies/mL). CONCLUSIONS All identified RF1-type strains appear to be introduced from a single source, suggesting that intergenotypic recombination in HCV is sporadic and not associated with cocirculation of different genotypes in a population. The RF1 strain in this study was responsive to interferon in vivo.

High-Affinity Aptamers to Subtype 3a Hepatitis C Virus Polymerase Display Genotypic Specificity

Abstract: Research into antiviral agents directed at hepatitis C virus (HCV) proteins is commonly based and tested on a single genotype, namely, genotype 1. This is despite the high level of variability of the RNA virus and the frequency of infection with genotypes other than genotype 1. The systematic evolution of ligands by exponential enrichment (SELEX) is a novel in vitro approach used in this study that allows rapid screening of vast nucleic acid libraries to isolate sequences (termed aptamers) that bind to target proteins with high affinity. The SELEX approach was used in the present study to isolate DNA aptamers to the RNA-dependent RNA polymerase (RdRp) (nonstructural protein 5B [NS5B]) of HCV subtype 3a, with the aim of inhibiting polymerase activity. Ten rounds of selection were performed using a Biacore 2000 as the partitioning system. Two aptamers, r10/43 and r10/47, were chosen for further studies on the basis of their abilities to bind the HCV RdRp and inhibit polymerase activity. The affinities (equilibrium dissociation constants) of these aptamers for the HCV subtype 3a polymerase were estimated to be 1.3 +/- 0.3 nM (r10/43) and 23.5 +/- 6.7 nM (r10/47). The inhibition constants of r10/43 and r10/47 were estimated to be 1.4 +/- 2.4 nM and 6.0 +/- 2.3 nM, respectively. Inhibition of HCV 3a polymerase was specific for r10/47, while r10/43 also demonstrated some inhibitory effect on norovirus and phi6 polymerase activity. Neither r10/43 nor r10/47 was able to inhibit the RdRp activity of HCV genotype 1a and 1b polymerases. This study is the first description of an inhibitor specific to the HCV subtype 3a polymerase.