Hepatitis C Virus NS5B Protein Research Articles

Targeting non-structural proteins of Hepatitis C virus for predicting repurposed drugs using QSAR and machine learning approaches

Hepatitis C virus (HCV) infection causes viral hepatitis leading to hepatocellular carcinoma. Despite the clinical use of direct-acting antivirals (DAAs) still there is treatment failure in 5–10% cases. Therefore, it is crucial to develop new antivirals against HCV. In this endeavor, we developed the “Anti-HCV” platform using machine learning and quantitative structure–activity relationship (QSAR) approaches to predict repurposed drugs targeting HCV non-structural (NS) proteins. We retrieved experimentally validated small molecules from the ChEMBL database with bioactivity (IC50/EC50) against HCV NS3 (454), NS3/4A (495), NS5A (494) and NS5B (1671) proteins. These unique compounds were divided into training/testing and independent validation datasets. Relevant molecular descriptors and fingerprints were selected using a recursive feature elimination algorithm. Different machine learning techniques viz. support vector machine, k-nearest neighbour, artificial neural network, and random forest were used to develop the predictive models. We achieved Pearson’s correlation coefficients from 0.80 to 0.92 during 10-fold cross validation and similar performance on independent datasets using the best developed models. The robustness and reliability of developed predictive models were also supported by applicability domain, chemical diversity and decoy datasets analyses. The “Anti-HCV” predictive models were used to identify potential repurposing drugs. Representative candidates were further validated by molecular docking which displayed high binding affinities. Hence, this study identified promising repurposed drugs viz. naftifine, butalbital (NS3), vinorelbine, epicriptine (NS3/4A), pipecuronium, trimethaphan (NS5A), olodaterol and vemurafenib (NS5B) etc. targeting HCV NS proteins. These potential repurposed drugs may prove useful in antiviral drug development against HCV.

Difluoromethylornithine (DFMO), an Inhibitor of Polyamine Biosynthesis, and Antioxidant N-Acetylcysteine Potentiate Immune Response in Mice to the Recombinant Hepatitis C Virus NS5B Protein

Hepatitis C virus (HCV) is one of the main triggers of chronic liver disease. Despite tremendous progress in the HCV field, there is still no vaccine against this virus. Potential vaccines can be based on its recombinant proteins. To increase the humoral and, especially, cellular immune response to them, more effective adjuvants are needed. Here, we evaluated a panel of compounds as potential adjuvants using the HCV NS5B protein as an immunogen. These compounds included inhibitors of polyamine biosynthesis and urea cycle, the mTOR pathway, antioxidants, and cellular receptors. A pronounced stimulation of cell proliferation and interferon-γ (IFN-γ) secretion in response to concanavalin A was shown for antioxidant N-acetylcysteine (NAC), polyamine biosynthesis inhibitor 2-difluoromethylornithine (DFMO), and TLR9 agonist CpG ODN 1826 (CpG). Their usage during the immunization of mice with the recombinant NS5B protein significantly increased antibody titers, enhanced lymphocyte proliferation and IFN-γ production. NAC and CpG decreased relative Treg numbers; CpG increased the number of myeloid-derived suppressor cells (MDSCs), whereas neither NAC nor DFMO affected MDSC counts. NAC and DFMO suppressed NO and interleukin 10 (IL-10) production by splenocytes, while DFMO increased the levels of IL-12. This is the first evidence of immunomodulatory activity of NAC and DFMO during prophylactic immunization against infectious diseases.

A Multiancestry Sex-Stratified Genome-Wide Association Study of Spontaneous Clearance of Hepatitis C Virus.

Spontaneous clearance of acute hepatitis C virus (HCV) infection is more common in women than in men, independent of known risk factors. To identify sex-specific genetic loci, we studied 4423 HCV-infected individuals (2903 male, 1520 female) of European, African, and Hispanic ancestry. We performed autosomal, and X chromosome sex-stratified and combined association analyses in each ancestry group. A male-specific region near the adenosine diphosphate-ribosylation factor-like 5B (ARL5B) gene was identified. Individuals with the C allele of rs76398191 were about 30% more likely to have chronic HCV infection than individuals with the T allele (OR, 0.69; P = 1.98 × 10-07), and this was not seen in females. The ARL5B gene encodes an interferon-stimulated gene that inhibits immune response to double-stranded RNA viruses. We also identified suggestive associations near septin 6 and ribosomal protein L39 genes on the X chromosome. In box sexes, allele G of rs12852885 was associated with a 40% increase in HCV clearance compared with the A allele (OR, 1.4; P = 2.46 × 10-06). Septin 6 facilitates HCV replication via interaction with the HCV NS5b protein, and ribosomal protein L39 acts as an HCV core interactor. These novel gene associations support differential mechanisms of HCV clearance between the sexes and provide biological targets for treatment or vaccine development.

Hepatitis C Virus RNA-Dependent RNA Polymerase Is Regulated by Cysteine S-Glutathionylation.

Hepatitis C virus (HCV) triggers massive production of reactive oxygen species (ROS) and affects expression of genes encoding ROS-scavenging enzymes. Multiple lines of evidence show that levels of ROS production contribute to the development of various virus-associated pathologies. However, investigation of HCV redox biology so far remained in the paradigm of oxidative stress, whereas no attention was given to the identification of redox switches among viral proteins. Here, we report that one of such redox switches is the NS5B protein that exhibits RNA-dependent RNA polymerase (RdRp) activity. Treatment of the recombinant protein with reducing agents significantly increases its enzymatic activity. Moreover, we show that the NS5B protein is subjected to S-glutathionylation that affects cysteine residues 89, 140, 170, 223, 274, 521, and either 279 or 295. Substitution of these cysteines except C89 and C223 with serine residues led to the reduction of the RdRp activity of the recombinant protein in a primer-dependent assay. The recombinant protein with a C279S mutation was almost inactive in vitro and could not be activated with reducing agents. In contrast, cysteine substitutions in the NS5B region in the context of a subgenomic replicon displayed opposite effects: most of the mutations enhanced HCV replication. This difference may be explained by the deleterious effect of oxidation of NS5B cysteine residues in liver cells and by the protective role of S-glutathionylation. Based on these data, redox-sensitive posttranslational modifications of HCV NS5B and other proteins merit a more detailed investigation and analysis of their role(s) in the virus life cycle and associated pathogenesis.

A review on HCV inhibitors: Significance of non-structural polyproteins

Hepatitis C virus (HCV) mortality and morbidity is a world health misery with an approximate 130–150 million chronically HCV tainted and suffering individuals and it initiate critical liver malfunction like cirrhosis, hepatocellular carcinoma or liver HCV cancer. HCV NS5B protein one of the best studied therapeutic target for the identification of new drug candidates to be added to the combination or multiple combination medication recently approved. During the past few years, NS5B has thus been an important object of attractive medicinal chemistry endeavors, which induced to the surfacing of betrothal preclinical drug molecules. In this scenario, the current review set limit to discuss research published on NS5B and few other therapeutic functional inhibitors concentrating on hit investigation, hit to lead optimization, ADME parameters evaluation, and the SAR data which was out for each compound type and similarity taken into consideration. The discussion outlined in this specific review will surly helpful and vital tool for those medicinal chemists investigators working with HCV research programs mainly pointing on NS5B and set broad spectrum identification of creative anti HCV compounds. This mini review also tells each and every individual compound ability related how much they are active against NS5B and few other targets.

Osteopontin Regulates Hepatitis C Virus (HCV) Replication and Assembly by Interacting with HCV Proteins and Lipid Droplets and by Binding to Receptors αVβ3 and CD44

Hepatitis C virus (HCV) replication and assembly occur at the specialized site of endoplasmic reticulum (ER) membranes and lipid droplets (LDs), respectively. Recently, several host proteins have been shown to be involved in HCV replication and assembly. In the present study, we demonstrated the important relationship among osteopontin (OPN), the ER, and LDs. OPN is a secreted phosphoprotein, and overexpression of OPN in hepatocellular carcinoma (HCC) tissue can lead to invasion and metastasis. OPN expression is also enhanced in HCV-associated HCC. Our recent studies have demonstrated the induction, proteolytic cleavage, and secretion of OPN in response to HCV infection. We also defined the critical role of secreted OPN in human hepatoma cell migration and invasion through binding to receptors integrin αVβ3 and CD44. However, the role of HCV-induced OPN in the HCV life cycle has not been elucidated. In this study, we showed a significant reduction in HCV replication, assembly, and infectivity in HCV-infected cells transfected with small interfering RNA (siRNA) against OPN, αVβ3, and CD44. We also observed the association of endogenous OPN with HCV proteins (NS3, NS5A, NS4A/B, NS5B, and core). Confocal microscopy revealed the colocalization of OPN with HCV NS5A and core in the ER and LDs, indicating a possible role for OPN in HCV replication and assembly. Interestingly, the secreted OPN activated HCV replication, infectivity, and assembly through binding to αVβ3 and CD44. Collectively, these observations provide evidence that HCV-induced OPN is critical for HCV replication and assembly.IMPORTANCE Recently, our studies uncovered the critical role of HCV-induced endogenous and secreted OPN in migration and invasion of hepatocytes. However, the role of OPN in the HCV life cycle has not been elucidated. In this study, we investigated the importance of OPN in HCV replication and assembly. We demonstrated that endogenous OPN associates with HCV NS3, NS5A, NS5B, and core proteins, which are in close proximity to the ER and LDs. Moreover, we showed that the interactions of secreted OPN with cell surface receptors αVβ3 and CD44 are critical for HCV replication and assembly. These observations provide evidence that HCV-induced endogenous and secreted OPN play pivotal roles in HCV replication and assembly in HCV-infected cells. Taken together, our findings clearly demonstrate that targeting OPN may provide opportunities for therapeutic intervention of HCV pathogenesis.

Heat shock proteins HSPB8 and DNAJC5B have HCV antiviral activity.

Hepatitis C is a disease caused by the hepatitis C virus (HCV), and an estimated 3% of the world population is infected with the virus. During replication, HCV interacts with several cellular proteins. Studies have shown that several heat shock proteins (HSPs) have an altered expression profile in the presence of the virus, and some HSPs interact directly with HCV proteins. In the present study, we evaluated the expression levels of heat shock proteins in vitro in the presence and absence of HCV. The differential expression of 84 HSPs and chaperones was observed using a qPCR array, comparing HCV uninfected and infected Huh7.5 cells. To validate qPCR array, the differentially expressed genes were tested by real-time PCR in three different HCV models: subgenomic HCV replicon cells (SGR-JFH-1), JFH-1 infected cells (both genotype 2a) and subgenomic S52 cells (genotype 3). The HSPB8 gene showed increased expression in all three viral models. We silenced HSPB8 expression and observed an increase in viral replication. In contrast, when we increased the expression of HSPB8, a decrease in the HCV replication rate was observed. The same procedure was adopted for DNAJC5B, and HCV showed a similar replication pattern as that observed for HSPB8. These results suggest that HSPB8 may act as an intracellular factor against hepatitis C virus replication and that DNAJC5B has the same function, with more relevant results for genotype 3. We also evaluated the direct interactions between HCV and HSP proteins, and the IP experiments showed that the HCV NS4B protein interacts with HSPB8. These results contribute to a better understanding of the mechanisms involved in HCV replication.

Hepatitis C virus NS4B protein induces epithelial-mesenchymal transition by upregulation of Snail

BackgroundChronic hepatitis C virus (HCV) infection is an important cause of hepatocellular carcinoma (HCC). Epithelial to mesenchymal transition (EMT) is a key process associated with tumor metastasis and poor prognosis. HCV infection, HCV core and NS5A protein could induce EMT process, but the role of NS4B on EMT remains poorly understood.MethodsWe overexpressed HCV NS4B protein in HepG2 cells or Huh7.5.1 cells infected by HCVcc, the E-cadherin expression, N-cadherin expression and the EMT-associated transcriptional factor Snail were determined. The migration and invasion capabilities of the transfected cells were evaluated using wound-healing assay. Additionally, we used Snail siRNA interference to confirm the relation of HCV NS4B and Snail on EMT promotion.ResultsHCV NS4B increased the expression of EMT related markers and promoted cell migration and invasion. Snail knock-down almost completely eliminated the function of NS4B protein in EMT changes and reversed cell migration capacity to lower level. HCV NS4B protein could reduce the expression of Scribble and Hippo signal pathway were subsequently inactivated, resulting in the activation of PI3K/AKT pathway, which may be the reason for the up-regulation of Snail.ConclusionsThis study demonstrates that HCV NS4B protein induces EMT progression via the upregulation of Snail in HCC, which may be a novel underlying mechanism for HCV-associated HCC development, invasion and metastasis.

Anti-HCV Activity from Semi-purified Methanolic Root Extracts of Valeriana wallichii.

Hepatitis C virus (HCV) is a serious global health problem affecting approximately 130-150 million individuals. Presently available direct-acting anti-HCV drugs have higher barriers to resistance and also improved success rate; however, cost concerns limit their utilization, especially in developing countries like India. Therefore, development of additional agents to combat HCV infection is needed. In the present study, we have evaluated anti-HCV potential of water, chloroform, and methanol extracts from roots of Valeriana wallichii, a traditional Indian medicinal plant. Huh-7.5 cells infected with J6/JFH chimeric HCV strain were treated with water, chloroform, and methanol extracts at different concentrations. Semi-quantitative reverse transcription polymerase chain reaction result demonstrated that methanolic extract showed reduction in HCV replication. The methanolic extract was fractionated by thin layer chromatography, and the purified fractions (F1, F2, F3, and F4) were checked for anti-HCV activity. Significant viral inhibition was noted only in F4 fraction. Further, intrinsic fluorescence assay of purified HCV RNA-dependent RNA polymerase NS5B in the presence of F4 resulted in sharp quenching of intrinsic fluorescence with increasing amount of plant extract. Our results indicated that methanolic extract of V.wallichii and its fraction (F4) inhibited HCV by binding with HCV NS5B protein. The findings would be further investigated to identify the active principle/lead molecule towards development of complementary and alternative therapeutics against HCV. Copyright © 2017 John Wiley & Sons, Ltd.

Low-molecular-weight regulators of biogenic polyamine metabolism affect cytokine production and expression of hepatitis С virus proteins in Huh7.5 human hepatocarcinoma cells

Hepatitis C virus (HCV) induces the expression of the genes of proinflammatory cytokines, the excessive production of which may cause cell death, and contribute to development of liver fibrosis and hepatocarcinoma. The relationship between cytokine production and metabolic disorders in HCV-infected cells remains obscure. The levels of biogenic polyamines, spermine, spermidine, and their precursor putrescine, may be a potential regulator of these processes. The purpose of the present work was to study the effects of the compounds which modulate biogenic polyamines metabolism on cytokine production and HCV proteins expression. Human hepatocarcinoma Huh7.5 cells have been transfected with the plasmids that encode HCV proteins and further incubated with the following low-molecular compounds that affect different stages of polyamine metabolism: (1) difluoromethylornithine (DFMO), the inhibitor of ornithine decarboxylase, the enzyme that catalyzes the biosynthesis of polyamines; (2) N,N'-bis(2,3-butane dienyl)-1,4-diaminobutane (MDL72.527), the inhibitor of proteins involved in polyamine degradation; and (3) synthetic polyamine analog N^(I),N^(II)-diethylnorspermine (DENSpm), an inducer of polyamine degradation enzyme. The intracellular accumulation and secretion of cytokines (IL-6, IL-1β, TNF-α, and TGF-β) was assessed by immunocytochemistry and in the immunoenzyme assay, while the cytokine gene expression was studied using reverse transcription and PCR. The effects of the compounds under analysis on the expression of HCV proteins were analyzed using the indirect immunofluorescence with anti-HCV monoclonal antibodies. It has been demonstrated that, in cells transfected with HCV genes, DFMO reduces the production of three out of four tested cytokines, namely, TNF-α and TGF-β in cells that express HCV core, Е1Е2, NS3, NS5A, and NS5B proteins, and IL-1β in the cells that express HCV core, Е1Е2, and NS3 proteins. MDL72527 and DENSpm decreased cytokine production to a lesser extent. Incubation with DFMO led to a 28-32% decrease in the number of cells expressing NS5B or NS5A, both of which are key components of the HCV replication complex. The results obtained in the work indicate that a further detailed study of the antiviral activity of DFMO is required in order to assess its potential as an anti-hepatitis C therapeutic agent.

The role of HCV proteins on treatment outcomes.

For many years, the standard of treatment for hepatitis C virus (HCV) infection was a combination of pegylated interferon alpha (Peg-IFN-α) and ribavirin for 24–48 weeks. This treatment regimen results in a sustained virologic response (SVR) rate in about 50 % of cases. The failure of IFN-α-based therapy to eliminate HCV is a result of multiple factors including a suboptimal treatment regimen, severity of HCV-related diseases, host factors and viral factors. In recent years, advances in HCV cell culture have contributed to a better understanding of the viral life cycle, which has led to the development of a number of direct-acting antiviral agents (DAAs) that target specific key components of viral replication, such as HCV NS3/4A, HCV NS5A, and HCV NS5B proteins. To date, several new drugs have been approved for the treatment of HCV infection. Application of DAAs with IFN-based or IFN-free regimens has increased the SVR rate up to >90 % and has allowed treatment duration to be shortened to 12–24 weeks. The impact of HCV proteins in response to IFN-based and IFN-free therapies has been described in many reports. This review summarizes and updates knowledge on molecular mechanisms of HCV proteins involved in anti-IFN activity as well as examining amino acid variations and mutations in several regions of HCV proteins associated with the response to IFN-based therapy and pattern of resistance associated amino acid variants (RAV) to antiviral agents.

A Journey around the Medicinal Chemistry of Hepatitis C Virus Inhibitors Targeting NS4B: From Target to Preclinical Drug Candidates.

Hepatitis C virus (HCV) infection is a global health burden with an estimated 130-170 million chronically infected individuals and is the cause of serious liver diseases such as cirrhosis and hepatocellular carcinoma. HCV NS4B protein represents a validated target for the identification of new drugs to be added to the combination regimen recently approved. During the last years, NS4B has thus been the object of impressive medicinal chemistry efforts, which led to the identification of promising preclinical candidates. In this context, the present review aims to discuss research published on NS4B functional inhibitors focusing the attention on hit identification, hit-to-lead optimization, ADME profile evaluation, and the structure-activity relationship data raised for each compound family taken into account. The information delivered in this review will be a useful and valuable tool for those medicinal chemists dealing with research programs focused on NS4B and aimed at the identification of innovative anti-HCV compounds.

HCV induces the expression of Rubicon and UVRAG to temporally regulate the maturation of autophagosomes and viral replication.

Hepatitis C virus (HCV) induces autophagy to enhance its replication. However, how HCV regulates the autophagic pathway remains largely unclear. In this report, we demonstrated that HCV infection could induce the expression of Rubicon and UVRAG, which inhibited and stimulated the maturation of autophagosomes, respectively. The induction of Rubicon by HCV was prompt whereas the induction of UVRAG was delayed, resulting in the accumulation of autophagosomes in the early time points of viral infection. The role of Rubicon in inhibiting the maturation of autophagosomes in HCV-infected cells was confirmed by siRNA knockdown and the over-expression of Rubicon, which enhanced and suppressed the maturation of autophagosomes, respectively. Rubicon played a positive role in HCV replication, as the suppression of its expression reduced HCV replication and its over-expression enhanced HCV replication. In contrast, the over-expression of UVRAG facilitated the maturation of autophagosomes and suppressed HCV replication. The HCV subgenomic RNA replicon, which expressed only the nonstructural proteins, could also induce the expression of Rubicon and the accumulation of autophagosomes. Further analysis indicated that the HCV NS4B protein was sufficient to induce Rubicon and autophagosomes. Our results thus indicated that HCV, by differentially inducing the expression of Rubicon and UVRAG, temporally regulated the autophagic flux to enhance its replication.

Hepatitis C Virus Impairs Natural Killer Cell-Mediated Augmentation of Complement Synthesis

Natural killer (NK) cells and the complement system play critical roles in the first line of defense against pathogens. The synthesis of complement components C4 and C3 is transcriptionally downregulated by hepatitis C virus (HCV) core and NS5A proteins, and this negative regulation is apparent in chronically HCV-infected patients. In this study, we have examined the potential contribution of an NK cell line as a model in regulating complement synthesis. Coculture of NK cells (NK3.3) with human hepatoma cells (Huh7.5) expressing HCV core or NS5A protein led to a significant increase in C4 and C3 complement synthesis via enhanced specific transcription factors. Reestablishment of complement protein expression was found to be mediated by direct interaction between NKG2D on NK cells and the hepatocyte protein major histocompatibility complex class I-related chains A and B (MICA/B) and not to be associated with specific cytokine signaling events. On the other hand, C4 and C3 synthesis remained impaired in a coculture of NK cells and Huh7.5 cells infected with cell culture-grown HCV. The association between these two cell types through NKG2D and MICA/B was examined further, with MICA/B expression in HCV-infected hepatocytes found to remain inhibited during coculture. Further experiments revealed that the HCV NS2 and NS5B proteins are responsible for the HCV-associated decrease in MICA/B. These results suggest that HCV disables a key receptor ligand in infected hepatoma cells, thereby inhibiting the ability of infected cells to respond to stimuli from NK cells to positively regulate complement synthesis. The complement system contributes to the protection of the host from virus infection. However, the involvement of complement in viral hepatitis has not been well documented. Whether NK cells affect complement component expression in HCV-infected hepatocytes remains unknown. Here, we have shown how HCV subverts the ability of NK cells to positively mediate complement protein expression.

Transient replication of a hepatitis C virus genotype 1b replicon chimera encoding NS5A-5B from genotype 3a

Although hepatitis C virus (HCV) is a pathogen of global significance, experimental therapies in current clinical development include highly efficacious all-oral combinations of HCV direct-acting antivirals (DAAs). If approved for use, these new treatment regimens will impact dramatically upon our capacity to eradicate HCV in the majority of virus-infected patients. However, recent data from late-stage clinical evaluations demonstrated that individuals infected with HCV genotype (GT) 3 responded less well to all-oral DAA combinations than patients infected with other HCV GTs. In light of these observations, the present study sought to expand the number of molecular tools available to investigate small molecule-mediated inhibition of HCV GT3 NS5A and NS5B proteins in preclinical tissue-culture systems. Accordingly, a novel subgenomic HCV replicon chimera was created by utilizing a GT1b backbone modified to produce NS5A and NS5B proteins from a consensus sequence generated from HCV GT3a genomic sequences deposited online at the European Hepatitis C Virus database. This approach avoided the need to isolate and amplify HCV genomes from sera derived from HCV-infected patients. The replicon chimera, together with a version engineered to express NS5A encoding a Y93H mutation, demonstrated levels of replication in transient assays robust enough to assess accurate antiviral activities of inhibitors representing different HCV DAA classes. Thus, the replicon chimera represents a new simple molecular tool suitable for drug discovery programmes aimed at investigating, understanding, and improving GT3a activities of HCV DAAs targeting NS5A or NS5B.

Lipid Droplet-Binding Protein TIP47 Regulates Hepatitis C Virus RNA Replication through Interaction with the Viral NS5A Protein

The nonstructural protein NS5A has emerged as a new drug target in antiviral therapies for Hepatitis C Virus (HCV) infection. NS5A is critically involved in viral RNA replication that takes place at newly formed membranes within the endoplasmic reticulum (membranous web) and assists viral assembly in the close vicinity of lipid droplets (LDs). To identify host proteins that interact with NS5A, we performed a yeast two-hybrid screen with the N-terminus of NS5A (amino acids 1–31), a well-studied α-helical domain important for the membrane tethering of NS5A. Our studies identified the LD-associated host protein, Tail-Interacting Protein 47 (TIP47) as a novel NS5A interaction partner. Coimmunoprecipitation experiments in Huh7 hepatoma cells confirmed the interaction of TIP47 with full-length NS5A. shRNA-mediated knockdown of TIP47 caused a more than 10-fold decrease in the propagation of full-length infectious HCV in Huh7.5 hepatoma cells. A similar reduction was observed when TIP47 was knocked down in cells harboring an autonomously replicating HCV RNA (subgenomic replicon), indicating that TIP47 is required for efficient HCV RNA replication. A single point mutation (W9A) in NS5A that disrupts the interaction with TIP47 but preserves proper subcellular localization severely decreased HCV RNA replication. In biochemical membrane flotation assays, TIP47 cofractionated with HCV NS3, NS5A, NS5B proteins, and viral RNA, and together with nonstructural viral proteins was uniquely distributed to lower-density LD-rich membrane fractions in cells actively replicating HCV RNA. Collectively, our data support a model where TIP47—via its interaction with NS5A—serves as a novel cofactor for HCV infection possibly by integrating LD membranes into the membranous web.

Development of Global Consensus Sequence and Analysis of Highly Conserved Domains of HCV NS5B Protein. [10.5812/hepatmon.6142

BackgroundHepatitis C virus (HCV) is a plus stranded RNA virus which encodes 10 different genes. The HCV NS5B gene encodes a polymerase, which is responsible for the replication of the virus and is a potential target for the development of antiviral agents. HCV has a high mutation rate and is classified into six major genotypes.ObjectivesThe aim of this study was to draw a representing consensus sequence of each HCV genotype, align all six consensus sequences to draw a global consensus sequence and also study the highly conserved residues.Materials and Methods236 HCV NS5B sequences, belonging to all six genotypes, reported from all over the world were aligned then a representing phylogenetic tree wasdrawn.ResultsThe active site residues D220, D225, D318 and D319, which bind the divalent cations, are highly conserved among all the HCV genotypes. The other catalytic pocket residues, R158, S367, R386, and T390 and R394, which interact with the triphosphate of NTPs, are also highly conserved while T390 is mutated to valine in the genotype 5. The motif B residues G283, T286, T287 and N291, which take part in sugar selection by RdRp, are also highly conserved except for T286 which is mutated to proline in the genotypes 3 and 6. The residues E18, Y191, C274, Y276 and H502, which take part in primer/template interaction, are also high conserved except for H502 which is mutated to serine in genotype 2. High variation in all the six consensus sequences was observed in a 12 amino acid beta hairpin loop, which interacts with the double stranded RNA. Nine different peptides from the highly conserved regions of HCV NS5B protein were drawn which can be used as a peptide vaccine. The HCV NS5B phylogenetic tree shows the clusters of different genotypes and their evolutionary association.ConclusionsIn spite of a high mutation rate in HCV, the residues which are present in the catalytic pocket, sugar selection and template/primer interaction are highly conserved. These are target sites for the development of antiviral agents or peptide vaccines. The phylogenetic analysis suggests that different HCV genotypes have been evolved from the genotype 1a.

Prevalence of amino acid mutations in hepatitis C virus core and NS5B regions among Venezuelan viral isolates and comparison with worldwide isolates

BackgroundRecent reports show that R70Q and L/C91M amino acid substitutions in the core from different hepatitis C virus (HCV) genotypes have been associated with variable responses to interferon (IFN) and ribavirin (RBV) therapy, as well to an increase of hepatocellular carcinoma (HCC) risk, liver steatosis and insulin resistance (IR). Mutations in NS5B have also been associated to IFN, RBV, nucleoside and non-nucleoside inhibitors drug resistance. The prevalence of these mutations was studied in HCV RNA samples from chronically HCV-infected drug-naïve patients.MethodsAfter amplification of core and NS5B region by nested-PCR, 12 substitutions were analyzed in 266 Venezuelan HCV isolates subtype 1a, 1b, 2a, 2c, 2b, 2j (a subtype frequently found in Venezuela) and 3a (n = 127 and n = 228 for core and NS5B respectively), and compared to isolates from other countries (n = 355 and n = 646 for core and NS5B respectively).ResultsR70Q and L/C91M core substitutions were present exclusively in HCV G1b. Both substitutions were more frequent in American isolates compared to Asian ones (69% versus 26%, p < 0.001 and 75% versus 45%, p < 0.001 respectively). In Venezuelan isolates NS5B D310N substitution was detected mainly in G3a (100%) and G1a (13%), this later with a significantly higher prevalence than in Brazilian isolates (p = 0.03). The NS5B mutations related to IFN/RBV treatment D244N was mainly found in G3a, and Q309R was present in all genotypes, except G2. Resistance to new NS5B inhibitors (C316N) was only detected in 18% of G1b, with a significantly lower prevalence than in Asian isolates, where this polymorphism was surprisingly frequent (p < 0.001).ConclusionsGenotypical, geographical and regional differences were found in the prevalence of substitutions in HCV core and NS5B proteins. The substitutions found in the Venezuelan G2j type were similar to that found in G2a and G2c isolates. Our results suggest a high prevalence of the R70Q and L/C91M mutations of core protein for G1b and D310N substitution of NS5B protein for the G3a. C316N polymorphism related with resistance to new NS5B inhibitors was only found in G1b. Some of these mutations could be associated with a worse prognosis of the disease in HCV infected patients.

Interaction with membranes of the full C-terminal domain of protein NS4B from Hepatitis C virus

Hepatitis C virus (HCV) NS4B protein is a transmembrane highly hydrophobic protein responsible for many key aspects of the viral replication process. The C-terminal part of NS4B is essential for replication and is a potential target for HCV replication inhibitors. In this work we have carried out a study of the binding to and interaction with model biomembranes of a peptide corresponding to the C-terminal domain of NS4B, NS4BCter. We show that NS4BCter partitions into phospholipid membranes, is capable of rupturing membranes even at very low peptide-to-lipid ratios and its membrane-activity is modulated by lipid composition. NS4BCter is located in a shallow position in the membrane but it is able to affect the lipid environment from the membrane surface down to the hydrophobic core. Our results identify the C-terminal region of the HCV NS4B protein as a membrane interacting domain, and therefore directly implicated in the HCV life cycle and possibly in the formation of the membranous web.

No influence of antiretroviral therapy on the mutation rate of the HCV NS5B polymerase in HIV/HCV-coinfected patients

ObjectivesTo assess the impact of antiretroviral treatment (ART), including nucleoside analogues retrotranscriptase inhibitors (NRTIs), on the mutation rate of hepatitis C virus (HCV) NS5B polymerase and on the ratio of substitution at synonymous and nonsynonymous sites (dN/dS) this polymerase in HIV/HCV-coinfected patients. Patients and methodsSixty-one patients on defined ART were included in this study. The NS5B polymerase of HCV was sequenced at baseline and after at least two years of ART. The mutation rate and the dN/dS were calculated at both times. ResultsThe NS5B gene from forty-nine (80.3%) patients including; 19 HCV-1a (38.8%), 13 HCV-1b (26.5%), 8 HCV-3a (16.3%) and 9 HCV-4d (18.4%), could be sequenced. Thirty-two (65.3%) patients received non-nucleoside analogues and 41 (83.7%) received protease inhibitor. The mean estimated substitution rates at baseline and at the end of follow-up were from 1.38 to 3.5×10−3substitution/site/year (s/s/y) and from 1.39 to 3.18×10−3s/s/y, respectively, varying according to HCV genotype. All HCV genotypes at baseline and the end time point had values of dN/dS <1. At the end of follow-up, most of sites experienced negative selection and positive selection occurred only in a few sites. ConclusionThe mutation rate of NS5B in HIV/HCV-coinfected patients is within the range previously reported in studies in HCV-monoinfected patients. Additionally, the use of ART, including NRTIs, in these patients does not affect neither mutation rate nor the dN/dS of the HCV NS5B protein, suggesting that its use would not generate new resistance mutants to the polymerase inhibitors of HCV.