NS5B

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

Identification of a Naturally Occurring Recombinant Genotype 2/6 Hepatitis C Virus

Abstract: Hepatitis C viruses (HCVs) display a high level of sequence diversity and are currently classified into six genotypes and an increasing number of subtypes. Most likely, this heterogeneity is caused by genetic drift; evidence for recombination is scarce. To study the molecular heterogeneity of HCV in Vietnam, we analyzed 58 HCV RNA-positive sera from Vietnamese blood donors by sequence analysis of the CORE and NS5B regions. Phylogenetic analyses revealed the presence of genotype 1 (38%), genotype 2 (10.3%), and genotype 6 viruses (51.7%). All samples showed concordant results except for two (D3 and D54). Sample D54 was a mixed infection of genotype 2i and 6h viruses. Whole-genome analysis and bootscan analysis of sample D3, on the other hand, revealed a recombinant virus with genotype 2i and genotype 6p sequences at the 5' and 3' ends, respectively. The crossover point was located between nucleotide positions 3405 to 3464 (numbering according to prototype strain HCV-H, M67463) at the NS2/NS3 junction. The identification of this naturally occurring recombinant virus strengthens the concept that recombination may play a role in HCV epidemiology and evolution. Furthermore, the location of the recombination breakpoint may be relevant for constructing infectious chimeric viruses.

Crystallographic Identification of a Noncompetitive Inhibitor Binding Site on the Hepatitis C Virus NS5B RNA Polymerase Enzyme

Abstract: The virus-encoded nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) is an RNA-dependent RNA polymerase and is absolutely required for replication of the virus. NS5B exhibits significant differences from cellular polymerases and therefore has become an attractive target for anti-HCV therapy. Using a high-throughput screen, we discovered a novel NS5B inhibitor that binds to the enzyme noncompetitively with respect to nucleotide substrates. Here we report the crystal structure of NS5B complexed with this small molecule inhibitor. Unexpectedly, the inhibitor is bound within a narrow cleft on the protein's surface in the "thumb" domain, about 30 A from the enzyme's catalytic center. The interaction between this inhibitor and NS5B occurs without dramatic changes to the structure of the protein, and sequence analysis suggests that the binding site is conserved across known HCV genotypes. Possible mechanisms of inhibition include perturbation of protein dynamics, interference with RNA binding, and disruption of enzyme oligomerization.

Hypervariable region 1 differentially impacts viability of hepatitis C virus strains of genotypes 1 to 6 and impairs virus neutralization.

Abstract: Approximately 180 million people are chronically infected with hepatitis C virus (HCV) with increased risk of developing liver cirrhosis and hepatocellular carcinoma (1). HCV is an enveloped positive-strand RNA virus of the Flaviviridae family. The 9.6-kb genome consists of 5′ and 3′ untranslated regions (5′ and 3′ UTRs) flanking the open reading frame (ORF) encoding a single polyprotein, which is processed into structural proteins (Core and envelope [E] glycoproteins 1 and 2), p7, and nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) (15). Seven HCV genotypes and multiple subtypes exist, differing at the amino acid (aa) level by ∼30% and ∼20%, respectively (15). Genotype-specific differences in response to alpha interferon-based therapy, in the risk of developing liver steatosis, and possibly in viral persistence have been reported (2, 15). HCV immune evasion mechanisms underlying viral persistence are poorly understood, but it has been suggested that these mechanisms rely on rapid virus evolution, mediating escape from humoral and cellular adaptive immunity (9). Studies of virus neutralization were facilitated by development of HCV culture systems producing pseudoparticles (HCVpp) (5) and JFH1-based cell culture infectious viruses (HCVcc) (22, 33). Select sera from chronically infected patients were shown to contain cross-genotype-reactive neutralizing serum antibodies (18, 20, 25, 29), although their neutralization efficacy varied greatly depending on the virus genotype. The failure of these antibodies to control the virus in vivo might be linked to the emergence of escape mutants (32). However, in acutely infected HCV patients, the occurrence of neutralizing antibodies was associated with viral clearance (11, 27). The envelope motif hypervariable region 1 (HVR1) has the highest sequence variability of the HCV genome. HVR1 was classified as the 26 or 27 N-terminal amino acids of E2 and is identifiable by cross-genotypic conserved residues (7). Variation in HVR1 is believed to arise from antibody-driven immune selection, as HVR1 contains at least one neutralization epitope (12) and does not evolve in IgG-deficient patients (21). HVR1 may act as an immunological decoy, diverting the immune system from targeting more-conserved neutralization epitopes (28). However, several studies showed that an acute-phase immune response against HVR1 was associated with viral clearance (11, 13, 36), and although HVR1-deleted genotype 1a virus was attenuated in experimentally infected chimpanzees, it adapted to produce a robust acute infection and establish persistent infection (14). Recently, an in vitro study with a single JFH1-based recombinant Jc1, in which Core-p7 and the N-terminal part of NS2 is encoded by J6CF (35), showed that HVR1 deletion caused viral attenuation with a 10-fold decrease in infectivity. The HVR1-deleted 2a virus was found to have higher density and increased neutralization susceptibility (4). However, the study did not address in vivo relevance of these findings, and the Jc1 virus has not been shown to be infectious in vivo. Also, the focus on a single isolate raised the question of whether reported observations were representative of HCV in general. In independent studies, we compared the viability of HVR1-deleted viruses across HCV genotypes by deleting HVR1 from JFH1-based 2a recombinant J6/JFH1, in which the entire Core-NS2 is encoded by J6CF, as well as from viruses of genotypes 1 to 6 of the recently developed panel of JFH1-based viruses with genotype-specific Core-NS2 (18). This panel of HCV with and without HVR1 allowed us to perform density analysis and patient serum neutralization comparing virus with and without HVR1 across genotypes. In addition, by infecting human liver chimeric mice with 2a virus with and without HVR1, we observed similar rises in HCV RNA titers and in vitro infectivity titers of infected animal samples. These in vivo infections allowed us to verify our in vitro neutralization findings using in vivo-produced viruses with and without HVR1. Taken together, our data showed differential dependency of HVR1 and that HVR1 is probably protecting HCV from neutralization in vivo by shielding cross-genotype conserved neutralization epitopes, thereby substantiating previous reports of involvement of HVR1 in establishment of chronic infections in human patients and in chimpanzees (11, 12).