Showing papers on "NS5B published in 2011"

Hepatitis C virus stimulates the phosphatidylinositol 4-kinase III alpha-dependent phosphatidylinositol 4-phosphate production that is essential for its replication.

Abstract: Phosphatidylinositol 4-kinase III alpha (PI4KA) is an essential cofactor of hepatitis C virus (HCV) replication. We initiated this study to determine whether HCV directly engages PI4KA to establish its replication. PI4KA kinase activity was found to be absolutely required for HCV replication using a small interfering RNA transcomplementation assay. Moreover, HCV infection or subgenomic HCV replicons produced a dramatic increase in phosphatidylinositol 4-phosphate (PI4P) accumulation throughout the cytoplasm, which partially colocalized with the endoplasmic reticulum. In contrast, the majority of PI4P accumulated at the Golgi bodies in uninfected cells. The increase in PI4P was not observed after infection with UV-inactivated HCV and did not reflect changes in PI4KA protein or RNA abundance. In an analysis of U2OS cell lines with inducible expression of the HCV polyprotein or individual viral proteins, viral polyprotein expression resulted in enhanced cytoplasmic PI4P production. Increased PI4P accumulation following HCV protein expression was precluded by silencing the expression of PI4KA, but not the related PI4KB. Silencing PI4KA also resulted in aberrant agglomeration of viral replicase proteins, including NS5A, NS5B, and NS3. NS5A alone, but not other viral proteins, stimulated PI4P production in vivo and enhanced PI4KA kinase activity in vitro. Lastly, PI4KA coimmunoprecipitated with NS5A from infected Huh-7.5 cells and from dually transfected 293T cells. In sum, these results suggest that HCV NS5A modulation of PI4KA-dependent PI4P production influences replication complex formation.

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).

Contribution of a mutational bias in hepatitis C virus replication to the genetic barrier in the development of drug resistance

Abstract: The development of resistance to direct-acting antivirals (DAAs) targeting the hepatitis C virus (HCV) can compromise therapy. However, mechanisms that determine prevalence and frequency of resistance-conferring mutations remain elusive. Here, we studied the fidelity of the HCV RNA-dependent RNA polymerase NS5B in an attempt to link the efficiency of mismatch formation with genotypic changes observed in vivo. Enzyme kinetic measurements revealed unexpectedly high error rates (approximately 10-3 per site) for G∶U/U∶G mismatches. The strong preference for G∶U/U∶G mismatches over all other mistakes correlates with a mutational bias in favor of transitions over transversions. Deep sequencing of HCV RNA samples isolated from 20 treatment-naive patients revealed an approximately 75-fold difference in frequencies of the two classes of mutations. A stochastic model based on these results suggests that the bias toward transitions can also affect the selection of resistance-conferring mutations. Collectively, the data provide strong evidence to suggest that the nature of the nucleotide change can contribute to the genetic barrier in the development of resistance to DAAs.

Mechanistic Characterization of GS-9190 (Tegobuvir), a Novel Nonnucleoside Inhibitor of Hepatitis C Virus NS5B Polymerase

Abstract: GS-9190 (Tegobuvir) is a novel imidazopyridine inhibitor of hepatitis C virus (HCV) RNA replication in vitro and has demonstrated potent antiviral activity in patients chronically infected with genotype 1 (GT1) HCV. GS-9190 exhibits reduced activity against GT2a (JFH1) subgenomic replicons and GT2a (J6/JFH1) infectious virus, suggesting that the compound's mechanism of action involves a genotype-specific viral component. To further investigate the GS-9190 mechanism of action, we utilized the susceptibility differences between GT1b and GT2a by constructing a series of replicon chimeras where combinations of 1b and 2a nonstructural proteins were encoded within the same replicon. The antiviral activities of GS-9190 against the chimeric replicons were reduced to levels comparable to that of the wild-type GT2a replicon in chimeras expressing GT2a NS5B. GT1b replicons in which the β-hairpin region (amino acids 435 to 455) was replaced by the corresponding sequence of GT2a were markedly less susceptible to GS-9190, indicating the importance of the thumb subdomain of the polymerase in this effect. Resistance selection in GT1b replicon cells identified several mutations in NS5B (C316Y, Y448H, Y452H, and C445F) that contributed to the drug resistance phenotype. Reintroduction of these mutations into wild-type replicons conferred resistance to GS-9190, with the number of NS5B mutations correlating with the degree of resistance. Analysis of GS-9190 cross-resistance against previously reported NS5B drug-selected mutations showed that the resistance pattern of GS-9190 is different from other nonnucleoside inhibitors. Collectively, these data demonstrate that GS-9190 represents a novel class of nonnucleoside polymerase inhibitors that interact with NS5B likely through involvement of the β-hairpin in the thumb subdomain.

INX-08189, a Phosphoramidate Prodrug of 6-O-Methyl-2′-C-Methyl Guanosine, Is a Potent Inhibitor of Hepatitis C Virus Replication with Excellent Pharmacokinetic and Pharmacodynamic Properties

Abstract: INX-08189 is an aryl-phosphoramidate of 6-O-methyl-2'-C-methyl guanosine. INX-08189 was highly potent in replicon assays, with a 50% effective concentration of 10 ± 6 nM against hepatitis C genotype 1b at 72 h. The inhibitory effect on viral replication was rapid, with a 50% effective concentration (EC50) of 35 ± 8 nM at 24 h. An intracellular 2'-C-methyl guanosine triphosphate (2'-C-MeGTP) concentration of 2.43 ± 0.42 pmol/106 cells was sufficient to achieve 90% inhibition of viral replication. In vitro resistance studies confirmed that the S282T mutation in the NS5b gene conferred an approximately 10-fold reduction in sensitivity to INX-08189. However, the complete inhibition of S282T mutant replicons still could be achieved with an EC90 of 344 ± 170 nM. Drug combination studies of INX-08189 and ribavirin indicated significant synergy in antiviral potency both in wild-type and S282T-expressing replicons. Genotype 1b replicons could be cleared after 14 days of culture when exposed to as little as 20 nM INX-08189. No evidence of mitochondrial toxicity was observed after 14 days of INX-08189 exposure in both HepG2 and CEM human cell lines. In vivo studies of rats and cynomolgus monkeys demonstrated that 2'-C-MeGTP concentrations in liver equivalent to the EC90 could be attained after a single oral dose of INX-08189. Rat liver 2'-C-MeGTP concentrations were proportional to dose, sustained for greater than 24 h, and correlated with plasma concentrations of the nucleoside metabolite 2'-C-methyl guanosine. The characteristics displayed by INX-08189 support its continued development as a clinical candidate for the treatment of chronic HCV infection.

Differential In Vitro Effects of Intravenous versus Oral Formulations of Silibinin on the HCV Life Cycle and Inflammation

Abstract: Silymarin prevents liver disease in many experimental rodent models, and is the most popular botanical medicine consumed by patients with hepatitis C. Silibinin is a major component of silymarin, consisting of the flavonolignans silybin A and silybin B, which are insoluble in aqueous solution. A chemically modified and soluble version of silibinin, SIL, has been shown to potently reduce hepatitis C virus (HCV) RNA levels in vivo when administered intravenously. Silymarin and silibinin inhibit HCV infection in cell culture by targeting multiple steps in the virus lifecycle. We tested the hepatoprotective profiles of SIL and silibinin in assays that measure antiviral and anti-inflammatory functions. Both mixtures inhibited fusion of HCV pseudoparticles (HCVpp) with fluorescent liposomes in a dose-dependent fashion. SIL inhibited 5 clinical genotype 1b isolates of NS5B RNA dependent RNA polymerase (RdRp) activity better than silibinin, with IC50 values of 40–85 µM. The enhanced activity of SIL may have been in part due to inhibition of NS5B binding to RNA templates. However, inhibition of the RdRps by both mixtures plateaued at 43–73%, suggesting that the products are poor overall inhibitors of RdRp. Silibinin did not inhibit HCV replication in subgenomic genotype 1b or 2a replicon cell lines, but it did inhibit JFH-1 infection. In contrast, SIL inhibited 1b but not 2a subgenomic replicons and also inhibited JFH-1 infection. Both mixtures inhibited production of progeny virus particles. Silibinin but not SIL inhibited NF-κB- and IFN-B-dependent transcription in Huh7 cells. However, both mixtures inhibited T cell proliferation to similar degrees. These data underscore the differences and similarities between the intravenous and oral formulations of silibinin, which could influence the clinical effects of this mixture on patients with chronic liver diseases.

Evidence for separation of HCV subtype 1a into two distinct clades.

Abstract: The nucleotide sequence diversity present among Hepatitis C Virus (HCV) isolates allows rapid adjustment to exterior forces including host immunity and drug therapy. This viral response reflects a combination of a high rate of replication together with an error-prone RNA dependent RNA polymerase providing for the selection and proliferation of the viruses with the highest fitness. We examined HCV subtype 1a whole genome sequences to identify positions contributing to genotypic and phenotypic diversity. Phylogenetic tree reconstructions showed two distinct clades existing within the 1a subtype with each clade having a star-like tree topology and lacking definite correlation between time or place of isolation and phylogeny. Identification of significant phylogenetically-informative sites at the nucleotide level revealed positions not only contributing to clade differentiation, but which are located at or proximal to codons associated with resistance to: protease inhibitors (NS3 Q41) or polymerase inhibitors (NS5B S368). Synonymous/nonsynonymous substitution mutation analyses revealed that the majority of nucleotide mutations yielded synonymous amino acids, indicating the presence of purifying selection pressure across the polyprotein with pockets of positive selection also being detected. Despite evidence for divergence at several loci, certain 1a characteristics were preserved including the length of the alternative reading frame/F protein (ARF/F) gene, and a subtype 1a-specific phosphorylation site in NS5A (S349). Our analysis suggests that there may be strain-specific differences in the development of antiviral resistance to viruses infecting patients that are dependent on the genetic variation separating these two clades.

A Comprehensive Structure-Function Comparison of Hepatitis C Virus Strain JFH1 and J6 Polymerases Reveals a Key Residue Stimulating Replication in Cell Culture across Genotypes

Abstract: The hepatitis C virus (HCV) genotype 2a isolate JFH1 represents the only cloned HCV wild-type sequence capable of efficient replication in cell culture as well as in vivo. Previous reports have pointed to NS5B, the viral RNA-dependent RNA polymerase (RdRp), as a major determinant for efficient replication of this isolate. To understand the contribution of the JFH1 NS5B gene at the molecular level, we aimed at conferring JFH1 properties to NS5B from the closely related J6 isolate. We created intragenotypic chimeras in the NS5B regions of JFH1 and J6 and compared replication efficiency in cell culture and RdRp activity of the purified proteins in vitro, revealing more than three independent mechanisms conferring the role of JFH1 NS5B in efficient RNA replication. Most critical was residue I405 in the thumb domain of the polymerase, which strongly stimulated replication in cell culture by enhancing overall de novo RNA synthesis. A structural comparison of JFH1 and J6 at high resolution indicated a clear correlation of a closed-thumb conformation of the RdRp and the efficiency of the enzyme at de novo RNA synthesis, in accordance with the proposal that I405 enhances de novo initiation. In addition, we identified several residues enhancing replication independent of RdRp activity in vitro. The functional properties of JFH1 NS5B could be restored by a few single-nucleotide substitutions to the J6 isolate. Finally, we were able to enhance the replication efficiency of a genotype 1b isolate with the I405 mutation, indicating that this mechanism of action is conserved across genotypes.

Hepatitis C virus nucleotide inhibitors PSI-352938 and PSI-353661 exhibit a novel mechanism of resistance requiring multiple mutations within replicon RNA

Abstract: PSI-352938, a cyclic phosphate nucleotide, and PSI-353661, a phosphoramidate nucleotide, are prodrugs of β-d-2′-deoxy-2′-α-fluoro-2′-β-C-methylguanosine-5′-monophosphate. Both compounds are metabolized to the same active 5′-triphosphate, PSI-352666, which serves as an alternative substrate inhibitor of the NS5B RNA-dependent RNA polymerase during HCV replication. PSI-352938 and PSI-353661 retained full activity against replicons containing the S282T substitution, which confers resistance to certain 2′-substituted nucleoside/nucleotide analogs. PSI-352666 was also similarly active against both wild-type and S282T NS5B polymerases. In order to identify mutations that confer resistance to these compounds, in vitro selection studies were performed using HCV replicon cells. While no resistant genotype 1a or 1b replicons could be selected, cells containing genotype 2a JFH-1 replicons cultured in the presence of PSI-352938 or PSI-353661 developed resistance to both compounds. Sequencing of the NS5B region identified a number of amino acid changes, including S15G, R222Q, C223Y/H, L320I, and V321I. Phenotypic evaluation of these mutations indicated that single amino acid changes were not sufficient to significantly reduce the activity of PSI-352938 and PSI-353661. Instead, a combination of three amino acid changes, S15G/C223H/V321I, was required to confer a high level of resistance. No cross-resistance exists between the 2′-F-2′-C-methylguanosine prodrugs and other classes of HCV inhibitors, including 2′-modified nucleoside/-tide analogs such as PSI-6130, PSI-7977, INX-08189, and IDX-184. Finally, we determined that in genotype 1b replicons, the C223Y/H mutation failed to support replication, and although the A15G/C223H/V321I triple mutation did confer resistance to PSI-352938 and PSI-353661, this mutant replicated at only about 10% efficiency compared to the wild type.

Targeting the ns5a protein of hcv: an emerging option

Abstract: Hepatitis C virus (HCV) infects more than 3% of the world’s population, leading to an increased risk of cirrhosis and hepatocellular carcinoma. The current standard of care, a combination of pegylated interferon alfa and ribavirin, is poorly tolerated and often ineffective against the most prevalent genotype of the virus, genotype 1. The very recent approval of boceprevir and telaprevir, two HCV protease inhibitors, promises to significantly improve treatment options and outcomes. In addition to the viral protease NS3 and the viral polymerase NS5B, direct-acting antivirals are now in development against NS5A. A multifunctional phosphoprotein, NS5A is essential to HCV genome replication, but has no known enzymatic function. Here we report how the design of small-molecule inhibitors against NS5A has evolved from promising monomers to highly potent dimeric compounds effective against many HCV genotypes. We also highlight recent clinical data and how the inhibitors may bind to NS5A, itself capable of forming dimers.

Peptidyl-Prolyl Isomerase Pin1 Is a Cellular Factor Required for Hepatitis C Virus Propagation

Abstract: The life cycle of hepatitis C virus (HCV) is highly dependent on cellular factors. Using small interfering RNA (siRNA) library screening, we identified peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1) as a host factor involved in HCV propagation. Here we demonstrated that silencing of Pin1 expression resulted in decreases in HCV replication in both HCV replicon cells and cell culture-grown HCV (HCVcc)-infected cells, whereas overexpression of Pin1 increased HCV replication. Pin1 interacted with both the NS5A and NS5B proteins. However, Pin1 expression was increased only by the NS5B protein. Both the protein binding and isomerase activities of Pin1 were required for HCV replication. Juglone, a natural inhibitor of Pin1, inhibited HCV propagation by inhibiting the interplay between the Pin1 and HCV NS5A/NS5B proteins. These data indicate that Pin1 modulates HCV propagation and may contribute to HCV-induced liver pathogenesis.

Zebrafish as a Potential Model Organism for Drug Test Against Hepatitis C Virus

Abstract: Screening and evaluating anti- hepatitis C virus (HCV) drugs in vivo is difficult worldwide, mainly because of the lack of suitable small animal models. We investigate whether zebrafish could be a model organism for HCV replication. To achieve NS5B-dependent replication an HCV sub-replicon was designed and created with two vectors, one with HCV ns5b and fluorescent rfp genes, and the other containing HCV's 5′UTR, core, 3′UTR and fluorescent gfp genes. The vectors containing sub-replicons were co-injected into zebrafish zygotes. The sub-replicon amplified in liver showing a significant expression of HCV core RNA and protein. The sub-replicon amplification caused no abnormality in development and growth of zebrafish larvae, but induced gene expression change similar to that in human hepatocytes. As the amplified core fluorescence in live zebrafish was detectable microscopically, it rendered us an advantage to select those with replicating sub-replicon for drug experiments. Ribavirin and oxymatrine, two known anti-HCV drugs, inhibited sub-replicon amplification in this model showing reduced levels of HCV core RNA and protein. Technically, this method had a good reproducibility and is easy to operate. Thus, zebrafish might be a model organism to host HCV, and this zebrafish/HCV (sub-replicon) system could be an animal model for anti-HCV drug screening and evaluation.

Cyclosporin A associated helicase-like protein facilitates the association of hepatitis C virus RNA polymerase with its cellular cyclophilin B.

Abstract: Background Cyclosporin A (CsA) is well known as an immunosuppressive drug useful for allogeneic transplantation. It has been reported that CsA inhibits hepatitis C virus (HCV) genome replication, which indicates that cellular targets of CsA regulate the viral replication. However, the regulation mechanisms of HCV replication governed by CsA target proteins have not been fully understood. Principal Findings Here we show a chemical biology approach that elucidates a novel mechanism of HCV replication. We developed a phage display screening to investigate compound-peptide interaction and identified a novel cellular target molecule of CsA. This protein, named CsA associated helicase-like protein (CAHL), possessed RNA-dependent ATPase activity that was negated by treatment with CsA. The downregulation of CAHL in the cells resulted in a decrease of HCV genome replication. CAHL formed a complex with HCV-derived RNA polymerase NS5B and host-derived cyclophilin B (CyPB), known as a cellular cofactor for HCV replication, to regulate NS5B-CyPB interaction. Conclusions We found a cellular factor, CAHL, as CsA associated helicase-like protein, which would form trimer complex with CyPB and NS5B of HCV. The strategy using a chemical compound and identifying its target molecule by our phage display analysis is useful to reveal a novel mechanism underlying cellular and viral physiology.

A cell-based assay for RNA synthesis by the HCV polymerase reveals new insights on mechanism of polymerase inhibitors and modulation by NS5A.

Abstract: RNA synthesis by the genotype 1b hepatitis C virus (HCV) polymerase (NS5B) transiently expressed in Human embryonic kidney 293T cells or liver hepatocytes was found to robustly stimulate RIG-I-dependent luciferase production from the interferon β promoter in the absence of exogenously provided ligand. This cell-based assay, henceforth named the 5BR assay, could be used to examine HCV polymerase activity in the absence of other HCV proteins. Mutations that decreased de novo initiated RNA synthesis in biochemical assays decreased activation of RIG-I signaling. In addition, NS5B that lacks the C-terminal transmembrane helix but remains competent for RNA synthesis could activate RIG-I signaling. The addition of cyclosporine A to the cells reduced luciferase levels without affecting agonist-induced RIG-I signaling. Furthermore, non-nucleoside inhibitor benzothiadiazines (BTDs) that bind within the template channel of the 1b NS5B were found to inhibit the readout from the 5BR assay. Mutation M414T in NS5B that rendered the HCV replicon resistant to BTD was also resistant to BTDs in the 5BR assay. Co-expression of the HCV NS5A protein along with NS5B and RIG-I was found to inhibit the readout from the 5BR assay. The inhibition by NS5A was decreased with the removal of the transmembrane helix in NS5B. Lastly, NS5B from all six major HCV genotypes showed robust activation of RIG-I in the 5BR assay. In summary, the 5BR assay could be used to validate inhibitors of the HCV polymerase as well as to elucidate requirements for HCV-dependent RNA synthesis.

Very high prevalence of hepatitis C virus genotype 6 variants in southern Vietnam: large-scale survey based on sequence determination.

Abstract: Hepatitis C virus (HCV) has infected an estimated 130 million people worldwide, and most of them are chronically infected. HCV-infected people serve as a reservoir for transmission and have a high risk of developing cirrhosis and hepatocellular carcinoma. The development of methods for the prevention and treatment of HCV infection is strongly inhibited by viral factors. In fact, HCV has extensive nucleotide sequence diversity. Sequence comparisons of variants from different geographic areas have led to the identification and classification of various genotypes and subtypes. Thus far, sequencing of HCV isolates has identified 6 major genotypes and more than 83 subtypes. Genotypes 1, 2, and 3 are widely distributed throughout the world, whereas genotypes 4 and 5 have mainly been identified in Africa. In contrast, genotype 6 has been found in limited geographic regions, mainly in Southeast Asia (1). Accurate HCV genotyping is important for predicting the response to antiviral therapy since genotypes 1 and 4 are less likely to respond to interferon than genotypes 2 and 3. Since HCV genotypes vary according to the epidemic history in different geographic regions, genotyping is also an essential tool for epidemiological studies and for estimating the infection route (2,3). In addition, epidemiological studies of HCV strains from blood donors, drug addicts, and hospital patients have demonstrated a correlation between subtypes and risk factors (4,5). Asia is an area of high endemicity for HCV infection, which is a substantial risk factor for serious liver diseases. Although a small-scale survey has suggested a relatively restricted distribution of HCV genotype 6 variants in a limited region of Southeast Asia, larger studies are required to explore their distribution in other geographical regions. It is known that HCV genotype 6 viruses have the greatest genetic diversity. Recently, we reported isolating a novel subtype from Vietnamese patients (6), and thus far, this genotype has been found to contain at least 22 (6a–6v) subtypes. To explore the exact genotypic distribution and genetic variation of HCV in Vietnam, we conducted a large-scale survey based on direct sequence analysis, which is a gold standard method. A total of 842 HCV-positive serum samples collected from Vietnamese blood donors and liver disease patients between 2005 and 2011 were analyzed. All serum samples were collected at Cho Ray Hospital, the Hospital for Tropical Diseases, and the Hospital at the University of Medicine and Pharmacy in Ho Chi Minh City, Vietnam. Serum samples were stored at −30° C until analysis. Informed consent for participation in this study was obtained from each individual. This study conforms to ethical guidelines and was approved by the ethics committees of the University of Medicine and Pharmacy, Cho Ray Hospital, the Hospital for Tropical Diseases, Vietnam, and National Institute of Infectious Diseases, Japan. For the detection of HCV RNA, nested reverse transcriptase (RT)-PCR was performed using primers designed to amplify the NS5B gene of HCV. The primer sequences of the HCV used in this study were as follows: 5′-GAG YHT TCA CGG ADG CTA TGA CYA GGT A-3′ (HC3, sense, nt 8623–8650, with Y:C/T, H:A/C/T, D:A/G/T), and 5′-GAC ASG CTG TGA WAW ATG TCB CCC CCG-3′ (HC3R, antisense, nt 9307–9281, with S:G/C, W:A/T, B:G/C/T) for the outer primer pairs (685 bases), and 5′-GAC YTS GAG YTS ATA ACA TC-3′ (HC4, sense, nt 8688–8707, with Y:C/T, S:G/C), and 5′-ADT GGA GTG AGT TTK AGC TT-3′ (HC4R, antisense, nt 9229–9210, with D:A/G/T, K:G/T) for the inner primer pairs (542 bases). Nucleotide position was based on HCV-TV241 (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"EF632069","term_id":"156960158","term_text":"EF632069"}}EF632069). Briefly, total RNA was extracted from 150 μl of serum using an RNA extraction kit (NKRNAPREP kit; Nam Khoa Biotek Co., Ho Chi Minh City, Vietnam). Then, cDNA synthesis was performed using cDNA synthesis kit (NKcDNA synthesis kits; Nam Khoa Biotek Co.). Viral cDNA was synthesized by using random primers with the following cycle conditions: 25° C for 5 min, 42° C for 30 min, and 85° C for 5 min. HCV cDNA was amplified by nested PCR with the following cycle conditions: 40 cycles of 94° C for 20 s, 45° C for 20 s, and 72° C for 40 s for the first PCR, and 94° C for 20 s, 50° C for 20 s, and 72° C for 30 s for the seconf PCR. The PCR products were analyzed by electrophoresis on 2% agarose gels stained with ethidium bromide and recovered using the QIAquick gel extraction kit (Qiagen Inc., Chatsworth, Calif., USA). Purified PCR products were subjected to direct sequencing using the ABI PRISM™ Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, Calif., USA). To obtain genotyping results, the nucleotide data obtained by direct sequencing was submitted to the HCV sequence database at Los Alamos National Laboratory in USA (http://hcv.lanl.gov/content/sequence/BASIC_BLAST/basic_blast.html). As a result, 3 different genotypes in the 842 Vietnamese patients tested were found. Genotype 6 variants were most predominant (458 patients; 54.4%) followed by genotype 1 (256; 30.4%) and genotype 2 (128; 15.2%) (Fig. 1). We did not detect any other genotypes in this study. Furthermore, the subtyping results obtained were as follows: 1a (104; 12.4%), 1b (146; 17.3%), 1e (6; 0.7%), 2a (73; 8.7%), 2c (36; 4.3%), 2i (15; 1.8%), 2j (2; 0.2%), 2k (2; 0.2%), 6a (199; 23.6%), 6c (3; 0.4%), 6e (185; 22%), 6f (1; 0.1%), 6h (10; 1.2%), 6k (4; 0.5%), 6l (27; 3.2%), 6n (1; 0.1%), 6o (13; 1.5%), 6p (8; 1%), 6r (1; 0.1%), and 6t (6; 0.7%). There was no significant difference in genotypic distribution between the blood donor group and the liver disease group. Fig. 1 Genotypic distribution of HCV in Ho Chi Minh City, Vietnam. Genotyping of HCV is important to clarify the infection route and pathogenesis of the virus. In particular, examination of the sequence diversity among different isolates of the virus is important because variants may differ in their patterns of serologic reactivity, pathogenicity, virulence, and response to therapy. HCV has genetic variation, which corresponds to its geographic distribution, and it has been proposed that HCV can be classified into at least 6 major genotypes. In this study, we confirmed the genotypic distribution of HCV in the southern part of Vietnam. To our knowledge, this is the largest survey on the genotypic distribution of HCV in Vietnam examined by the gold standard method of direct sequence analysis. Although HCV genotype 6 has been reported in patients from Southeast Asia, its prevalence and clinical characteristics have not been well described in a large patient sample by means of an accurate genotyping method, such as the 5′UTR-core and/or NS5B sequencing test. Surprisingly, our study showed that more than half of the Vietnamese patients who lived in Ho Chi Minh City were infected with genotype 6 variants. The reason why such peculiar HCV variants are circulating in this limited geographical region remains unknown. Accurate HCV genotyping is clinically important for predicting the response to and determining the duration of antiviral therapy, and for the development of effective vaccines and testing kits. This is illustrated by the fact that genotypes 1 and 4 are more resistant to treatment with pegylated alpha interferon and ribavirin than genotypes 2 and 3 (2,7). Moreover, it has been suggested that patients with chronic HCV subtype 1b infection have more serious liver disease than patients infected with other genotypes (8,9). However, only a few studies on the response to antiviral treatment of genotype 6-infected patients have been reported so far (5,10–13). The reason for this is that genotype 6 virus is only seen in very limited regions of Southeast Asia where long-term observation of patients is difficult for economic reasons. However, fortunately, patients infected with HCV genotype 6 respond better to interferon-based therapy than those infected with genotype 1, although patient baseline clinical characteristics and side effect profiles are similar between HCV genotype 6 and other HCV genotypes (5,13). Further studies are required to address this issue. To resolve this issue, we are now investigating the different clinical and treatment outcomes between genotype 6 and other genotypes. In addition, we are conducting a prospective survey of the risk factors for HCV acquisition to explore other potential routes of viral transmission in this population.

Current perspective of HCV NS5B inhibitors: a review.

Abstract: Hepatitis C virus (HCV) infection has emerged as one of the most significant disease to affect humans. Despite its large medical and economical impact, there are no vaccines or efficient therapies without major side effects. The HCV non-structural protein 5B (NS5B) is the RNA-dependent RNA polymerase responsible for the complete copy of the RNA viral genome and is a target of choice for the development of anti-HCV drugs. Although many small molecules have been identified as allosteric inhibitors of NS5B, very few are active in clinical applications. Developments in the field have prompted us to review the research work on HCV NS5B polymerase inhibitors, especially their structure activity relationships and molecular modeling studies. This review will focus on the journey of drug discovery of HCV NS5B inhibitors covering both nucleoside and non-nucleosides.

Compensatory Mutations Restore the Replication Defects Caused by Cytotoxic T Lymphocyte Escape Mutations in Hepatitis C Virus Polymerase

Abstract: While human leukocyte antigen B57 (HLA-B57) is associated with the spontaneous clearance of hepatitis C virus (HCV), the mechanisms behind this control remain unclear. Immunodominant CD8(+) T cell responses against the B57-restricted epitopes comprised of residues 2629 to 2637 of nonstructural protein 5B (NS5B(2629-2637)) (KSKKTPMGF) and E2(541-549) (NTRPPLGNW) were recently shown to be crucial in the control of HCV infection. Here, we investigated whether the selection of deleterious cytotoxic T lymphocyte (CTL) escape mutations in the NS5B KSKKTPMGF epitope might impair viral replication and contribute to the B57-mediated control of HCV. Common CTL escape mutations in this epitope were identified from a cohort of 374 HCV genotype 1a-infected subjects, and their impact on HCV replication assessed using a transient HCV replicon system. We demonstrate that while escape mutations at residue 2633 (position 5) of the epitope had little or no impact on HCV replication in vitro, mutations at residue 2629 (position 1) substantially impaired replication. Notably, the deleterious mutations at position 2629 were tightly linked in vivo to upstream mutations at residue 2626, which functioned to restore the replicative defects imparted by the deleterious escape mutations. These data suggest that the selection of costly escape mutations within the immunodominant NS5B KSKKTPMGF epitope may contribute in part to the control of HCV replication in B57-positive individuals and that persistence of HCV in B57-positive individuals may involve the development of specific secondary compensatory mutations. These findings are reminiscent of the selection of deleterious CTL escape and compensatory mutations by HLA-B57 in HIV-1 infection and, thus, may suggest a common mechanism by which alleles like HLA-B57 mediate protection against these highly variable pathogens.

Genetic diversity of near genome-wide hepatitis C virus sequences during chronic infection: evidence for protein structural conservation over time.

Abstract: Infection with hepatitis C virus (HCV) is one of the leading causes of chronic hepatitis, liver cirrhosis and end-stage liver disease worldwide. The genetics of HCV infection in humans and the disease course of chronic hepatitis C are both remarkably variable. Although the response to interferon treatment is largely dependent on HCV genotypes, whether or not a relationship exists between HCV genome variability and clinical course of hepatitis C disease still remains unknown. To more thoroughly understand HCV genome evolution over time in association with disease course, near genome-wide HCV genomes present in 9 chronically infected participants over 83 total study years were sequenced. Overall, within HCV genomes, the number of synonymous substitutions per synonymous site (dS) significantly exceeded the number of non-synonymous substitutions per site (dN). Although both dS and dN significantly increased with duration of chronic infection, there was a highly significant decrease in dN/dS ratio in HCV genomes over time. These results indicate that purifying selection acted to conserve viral protein structure despite persistence of high level of nucleotide mutagenesis inherent to HCV replication. Based on liver biopsy fibrosis scores, HCV genomes from participants with advanced fibrosis had significantly greater dS values and lower dN/dS ratios compared to participants with mild liver disease. Over time, viral genomes from participants with mild disease had significantly greater annual changes in dN, along with higher dN/dS ratios, compared to participants with advanced fibrosis. Yearly amino acid variations in the HCV p7, NS2, NS3 and NS5B genes were all significantly lower in participants with severe versus mild disease, suggesting possible pathogenic importance of protein structural conservation for these viral gene products.