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Showing papers on "NS5B published in 2009"


Journal ArticleDOI
TL;DR: TMC435350 is a highly specific and potent inhibitor of NS3/4A protease selected from a series of novel macrocyclic inhibitors that has the potential for combination with other anti-HCV agents and the favorable pharmacokinetic profile.
Abstract: The hepatitis C virus (HCV) NS3/4A serine protease has been explored as a target for the inhibition of viral replication in preclinical models and in HCV-infected patients. TMC435350 is a highly specific and potent inhibitor of NS3/4A protease selected from a series of novel macrocyclic inhibitors. In biochemical assays using NS3/4A proteases of genotypes 1a and 1b, inhibition constants of 0.5 and 0.4 nM, respectively, were determined. TMC435350 inhibited HCV replication in a cellular assay (subgenomic 1b replicon) with a half-maximal effective concentration (EC50) of 8 nM and a selectivity index of 5,875. The compound was synergistic with alpha interferon and an NS5B inhibitor in the replicon model and additive with ribavirin. In rats, TMC435350 was extensively distributed to the liver and intestinal tract (tissue/plasma area under the concentration-time curve ratios of >35), and the absolute bioavailability was 44% after a single oral administration. Compound concentrations detected in both plasma and liver at 8 h postdosing were above the EC99 value measured in the replicon. In conclusion, given the selective and potent in vitro anti-HCV activity, the potential for combination with other anti-HCV agents, and the favorable pharmacokinetic profile, TMC435350 has been selected for clinical development.

178 citations


Journal ArticleDOI
TL;DR: A model in which the incorporation of the HCV polymerase into the RC depends on its interaction with a cellular chaperone protein and in which cyclosporine inhibits HCV replication by blocking this critical interaction and the PPIase activity of CyPA is proposed.
Abstract: Replication of hepatitis C virus (HCV) RNA occurs on intracellular membranes, and the replication complex (RC) contains viral RNA, nonstructural proteins, and cellular cofactors. We previously demonstrated that cyclophilin A (CyPA) is an essential cofactor for HCV infection and the intracellular target of cyclosporine's anti-HCV effect. Here we investigate the mechanism by which CyPA facilitates HCV replication. Cyclosporine treatment specifically blocked the incorporation of NS5B into the RC without affecting either the total protein level or the membrane association of the protein. Other nonstructural proteins or viral RNAs in the RC were not affected. NS5B from the cyclosporine-resistant replicon was resistant to this disruption of RC incorporation. We also isolated membrane fractions from both naive and HCV-positive cells and found that CyPA is recruited into membrane fractions in HCV-replicating cells via an interaction with RC-associated NS5B, which is sensitive to cyclosporine treatment. Finally, we introduced point mutations in the prolyl-peptidyl isomerase (PPIase) motif of CyPA and demonstrated a critical role of this motif in HCV replication in cDNA rescue experiments. We propose a model in which the incorporation of the HCV polymerase into the RC depends on its interaction with a cellular chaperone protein and in which cyclosporine inhibits HCV replication by blocking this critical interaction and the PPIase activity of CyPA. Our results provide a mechanism of action for the cyclosporine-mediated inhibition of HCV and identify a critical role of CyPA's PPIase activity in the proper assembly and function of the HCV RC.

152 citations


Journal ArticleDOI
TL;DR: Binding of the HCV‐NS5B protein to GBP‐1 countered the antiviral effect by inhibition of its GTPase activity, which may contribute to resistance to innate, IFN‐mediated antiviral defense and to the clinical persistence of HCV infection.

143 citations


Journal ArticleDOI
TL;DR: The utility of the range of in vitro and in vivo models now available for anti‐HCV drug development and the potential utility of polymerase inhibitors in future combination therapies for HCV treatment are demonstrated.

141 citations


Journal ArticleDOI
01 Sep 2009-RNA
TL;DR: The present data firmly suggest the existence of a higher-order structure that may mediate a protein-independent circularization of the HCV genome and the 5'-3' end bridge may have a role in viral translation modulation and in the switch from protein synthesis to RNA replication.
Abstract: The RNA genome of the hepatitis C virus (HCV) contains multiple conserved structural cis domains that direct protein synthesis, replication, and infectivity. The untranslatable regions (UTRs) play essential roles in the HCV cycle. Uncapped viral RNAs are translated via an internal ribosome entry site (IRES) located at the 59 UTR, which acts as a scaffold for recruiting multiple protein factors. Replication of the viral genome is initiated at the 39 UTR. Bioinformatics methods have identified other structural RNA elements thought to be involved in the HCV cycle. The 5BSL3.2 motif, which is embedded in a cruciform structure at the 39 end of the NS5B coding sequence, contributes to the three-dimensional folding of the entire 39 end of the genome. It is essential in the initiation of replication. This paper reports the identification of a novel, strand-specific, long-range RNA–RNA interaction between the 59 and 39 ends of the genome, which involves 5BSL3.2 and IRES motifs. Mutants harboring substitutions in the apical loop of domain IIId or in the internal loop of 5BSL3.2 disrupt the complex, indicating these regions are essential in initiating the kissing interaction. No complex was formed when the UTRs of the related foot and mouth disease virus were used in binding assays, suggesting this interaction is specific for HCV sequences. The present data firmly suggest the existence of a higher-order structure that may mediate a protein-independent circularization of the HCV genome. The 59–39 end bridge may have a role in viral translation modulation and in the switch from protein synthesis to RNA replication.

124 citations


Journal ArticleDOI
TL;DR: Debio 025 forms an attractive drug candidate for the treatment of HCV infections in combination with standard interferon-based treatment and treatments that directly target the HCV polymerase and/or protease.
Abstract: Debio 025 is a potent inhibitor of hepatitis C virus (HCV) replication (J Paeshuyse et al, Hepatology 43:761-770, 2006) In phase I clinical studies, monotherapy (a Debio 025 dose of 1,200 mg twice a day) resulted in a mean maximal decrease in the viral load of 36 log 10 units (R Flisiak et al, Hepatology 47:817-826, 2008), whereas a reduction of 46 log 10 units was obtained in phase II studies when Debio 025 was combined with interferon (R Flisiak et al, J Hepatol, 48:S62, 2008) We here report on the particular characteristics of the in vitro anti-HCV activities of Debio 025 The combination of Debio 025 with either ribavirin or specifically targeted antiviral therapy for HCV (STAT-C) inhibitors (NS3 protease or NS5B [nucleoside and nonnucleoside] polymerase inhibitors) resulted in additive antiviral activity in short-term antiviral assays Debio 025 has the unique ability to clear hepatoma cells from their HCV replicon when it is used alone or in combination with interferon and STAT-C inhibitors Debio 025, when it was used at concentrations that have been observed in human plasma (01 or 05 μM), was able to delay or prevent the development of resistance to HCV protease inhibitors as well as to nucleoside and nonnucleoside polymerase inhibitors Debio 025 forms an attractive drug candidate for the treatment of HCV infections in combination with standard interferon-based treatment and treatments that directly target the HCV polymerase and/or protease

122 citations


Journal ArticleDOI
TL;DR: CsA is the first anti‐HCV drug shown to act through NS2, and inhibits replication of full‐length HCV Japanese Fulminant Hepatitis genomes about 10‐fold more efficiently than subgenomic replicons.

122 citations


Journal ArticleDOI
TL;DR: A brief review summarizes the current literature concerning resistance and directly acting antiviral agents, and identifies key challenges facing this emerging field.
Abstract: As health care providers, we find ourselves on the verge of a new era in the treatment of chronic hepatitis C virus (HCV) infection. A number of directly acting antiviral agents are now in the latter stages of clinical development. The more promising candidates include direct inhibitors of the HCV nonstructural 3 protease, as well as both nucleoside and non-nucleoside inhibitors of the NS5B RNA-dependent RNA polymerase. Although these agents have demonstrated potent antiviral effect, monotherapy has been complicated by rapid virological breakthrough due to the selection of drug-resistant mutants. As for HIV and HBV, combination therapy will therefore be necessary. This brief review summarizes the current literature concerning resistance and directly acting antiviral agents, and identifies key challenges facing this emerging field.

100 citations


Journal ArticleDOI
TL;DR: Deep insights are provided into the initiation of HCV RNA synthesis and the crystal structure of JFH1 NS5B, which displays a very closed conformation that is expected to facilitate de novo initiation.
Abstract: The hepatitis C virus (HCV) isolate JFH1 represents the only cloned wild-type sequence capable of efficient replication in cell culture, as well as in chimpanzees. Previous reports have pointed to the viral polymerase NS5B as a major determinant for efficient replication of this isolate. To understand the underlying mechanisms, we expressed and purified NS5B of JFH1 and of the closely related isolate J6, which replicates below the limit of detection in cell culture. The JFH1 enzyme exhibited a 5- to 10-fold-higher specific activity in vitro, consistent with the polymerase activity itself contributing to efficient replication of JFH1. The higher in vitro activity of the JFH1 enzyme was not due to increased RNA binding, elongation rate, or processivity of the polymerase but to higher initiation efficiency. By using homopolymeric and heteropolymeric templates, we found that purified JFH1 NS5B was significantly more efficient in de novo initiation of RNA synthesis than the J6 counterpart, particularly at low GTP concentrations, probably representing an important prerequisite for the rapid replication kinetics of JFH1. Furthermore, we solved the crystal structure of JFH1 NS5B, which displays a very closed conformation that is expected to facilitate de novo initiation. Structural analysis shows that this closed conformation is stabilized by a sprinkle of substitutions that together promote extra hydrophobic interactions between the subdomains “thumb” and “fingers.” These analyses provide deeper insights into the initiation of HCV RNA synthesis and might help to establish more efficient cell culture models for HCV using alternative isolates.

86 citations


Journal ArticleDOI
TL;DR: The structure-activity relationships within this series led to the discovery of these novel nucleoside analogues with the most potent compound, showing more than a 50-fold increase in antiviral potency as compared to 4'-azidocytidine.
Abstract: The discovery of 4′-azidocytidine (3) (R1479) (J. Biol. Chem. 2006, 281, 3793; Bioorg. Med. Chem. Lett. 2007, 17, 2570) as a potent inhibitor of RNA synthesis by NS5B (EC50 = 1.28 μM), the RNA polymerase encoded by hepatitis C virus (HCV), has led to the synthesis and biological evaluation of several monofluoro and difluoro derivatives of 4′-azidocytidine. The most potent compounds in this series were 4′-azido-2′-deoxy-2′,2′-difluorocytidine and 4′-azido-2′-deoxy-2′-fluoroarabinocytidine with antiviral EC50 of 66 nM and 24 nM in the HCV replicon system, respectively. The structure−activity relationships within this series were discussed, which led to the discovery of these novel nucleoside analogues with the most potent compound, showing more than a 50-fold increase in antiviral potency as compared to 4′-azidocytidine (3).

86 citations


Journal ArticleDOI
TL;DR: The magnitude of reduction of NNI binding affinity for the NS5B proteins with various resistance mutations in the palm and thumb binding sites correlated well with resistance -fold shifts in NS5 B polymerase activity and replicon replication assays.

Journal ArticleDOI
TL;DR: It is demonstrated that an efficient HCV-production system could be obtained by introducing adaptive mutations into the J6/JFH1 genome and evidence suggesting that the mutation N534H in the E2 glycoprotein of the mutant viruses conferred an advantage at the entry level is provided.
Abstract: Robust production of infectious hepatitis C virus (HCV) in cell culture was realized by using the JFH1 strain and the homologous chimeric J6/JFH1 strain in Huh-7.5 cells, a highly HCV-permissive subclone of Huh-7 cells. In this study, we aimed to establish a more efficient HCV-production system and to gain some insight into the adaptation mechanisms of efficient HCV production. By serial passaging of J6/JFH1-infected Huh-7.5 cells, we obtained culture-adapted J6/JFH1 variants, designated P-27, P-38 and P-47. Sequence analyses revealed that the adaptive mutant viruses P-27, P-38 and P-47 possessed eight mutations [four in E2, two in NS2, one in NS5A and one in NS5B), 10 mutations [two additional mutations in the 5'-untranslated region (5'-UTR) and core] and 11 mutations (three additional mutations in 5'-UTR, core and NS5B), respectively. We introduced amino acid substitutions into the wild-type J6/JFH1 clone, generated recombinant viruses with adaptive mutations and analysed their infectivity and ability to produce infectious viruses. The viruses with the adaptive mutations exhibited higher expression of HCV proteins than did the wild type in Huh-7.5 cells. Moreover, we provide evidence suggesting that the mutation N534H in the E2 glycoprotein of the mutant viruses conferred an advantage at the entry level. We thus demonstrate that an efficient HCV-production system could be obtained by introducing adaptive mutations into the J6/JFH1 genome. The J6/JFH1-derived mutant viruses presented here would be a good tool for producing HCV particles with enhanced infectivity and for studying the molecular mechanism of HCV entry.

Journal ArticleDOI
TL;DR: Mutations known to confer resistance to HCV-796 (NS5B C316Y) and boceprevir (NS3 V170A) were present in the combination-resistant replicons, and the replicon bearing both changes exhibited reduced sensitivity to inhibition but had a reduced replicative capacity.
Abstract: HCV-796 is a nonnucleoside inhibitor of the hepatitis C virus (HCV) nonstructural protein 5B (NS5B) polymerase, and boceprevir is an inhibitor of the NS3 serine protease. The emergence of replicon variants resistant to the combination of HCV-796 and boceprevir was evaluated. Combining the inhibitors greatly reduced the frequency with which resistant colonies arose; however, some resistant replicon cells could be isolated by the use of low inhibitor concentrations. These replicons were approximately 1,000-fold less susceptible to HCV-796 and 9-fold less susceptible to boceprevir. They also exhibited resistance to anthranilate nonnucleoside inhibitors of NS5B but were fully sensitive to inhibitors of different mechanisms: a pyranoindole, Hsp90 inhibitors, an NS5B nucleoside inhibitor, and pegylated interferon (Peg-IFN). The replicon was cleared from the combination-resistant cells by extended treatment with Peg-IFN. Mutations known to confer resistance to HCV-796 (NS5B C316Y) and boceprevir (NS3 V170A) were present in the combination-resistant replicons. These changes could be selected together and coexist in the same genome. The replicon bearing both changes exhibited reduced sensitivity to inhibition by HCV-796 and boceprevir but had a reduced replicative capacity.

Journal ArticleDOI
TL;DR: The data shows significant variation in the specificity in humoral immunity in chronic HCV patients, and the major immunogenic HCV antigens were the core, NS4B, NS3 and NS5A proteins which were recognized in 97%, 86%, 68% and 53% of patient sera, respectively.
Abstract: The viral genome of hepatitis C virus constitutes a 9.6-kb single-stranded positive-sense RNA which encodes altogether 11 viral proteins. In order to study the humoral immune responses against different HCV proteins in patients suffering from chronic HCV infection, we produced three structural (core, E1 and E2) and six nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A and NS5B) in Sf 9 insect cells by using the baculovirus expression system. The recombinant HCV core, E1, E2, NS2, NS3, NS4A, NS4B, NS5A and NS5B proteins were purified and used in Western blot analysis to determine antibody responses against individual HCV protein in 68 HCV RNA and antibody positive human sera that were obtained from patients suffering from genotype 1, 2, 3 or 4 infection. These sera were also analysed with INNO-LIA Score test for HCV antibodies against core, NS3, NS4AB and NS5A, and the results were similar to the ones obtained by Western blot method. Based on our Western blot analyses we found that the major immunogenic HCV antigens were the core, NS4B, NS3 and NS5A proteins which were recognized in 97%, 86%, 68% and 53% of patient sera, respectively. There were no major genotype specific differences in antibody responses to individual HCV proteins. A common feature within the studied sera was that all except two sera recognized the core protein in high titers, whereas none of the sera recognized NS2 protein and only three sera (from genotype 3) recognised NS5B. The data shows significant variation in the specificity in humoral immunity in chronic HCV patients.

Journal ArticleDOI
TL;DR: The synthesis and biological evaluation of several derivatives of 4'-azidocytidine by varying the substituents at the ribose 2' and 3'-positions are described.
Abstract: 4'-Azidocytidine 3 (R1479) has been previously discovered as a potent and selective inhibitor of HCV replication targeting the RNA-dependent RNA polymerase of hepatitis C virus, NS5B. Here we describe the synthesis and biological evaluation of several derivatives of 4'-azidocytidine by varying the substituents at the ribose 2' and 3'-positions. The most potent compound in this series is 4'-azidoarabinocytidine with an IC(50) of 0.17 microM in the genotype 1b subgenomic replicon system. The structure-activity relationships within this series of nucleoside analogues are discussed.

Journal ArticleDOI
TL;DR: Using structure-based design, a 1,5-benzodiazepine NS5B polymerase inhibitor chemotype is optimized into a new sulfone-containing scaffold that yielded potent inhibitor (S)-4c (K(D) = 0.79 nM), which has approximately 20-fold greater affinity for NS 5B than its carbonyl analogue (R)-2c.
Abstract: HCV NS5B polymerase, an essential and virus-specific enzyme, is an important target for drug discovery. Using structure-based design, we optimized a 1,5-benzodiazepine NS5B polymerase inhibitor chemotype into a new sulfone-containing scaffold. The design yielded potent inhibitor (S)-4c (KD = 0.79 nM), which has ∼20-fold greater affinity for NS5B than its carbonyl analogue (R)-2c.

Journal ArticleDOI
TL;DR: It is suggested that NS5A stimulates NS5B during synthesis of the complementary strand of the RNA genome, which is opposite of the replication reaction of the hepatitis C virus.
Abstract: The hepatitis C virus (HCV) NS5B protein contains the RNA dependent RNA polymerase (RdRp) activity that catalyzes the synthesis of the viral genome with other host and viral factors. NS5A is an HCV-encoded protein previously shown to localize to the replisome and be necessary for viral replication. However, its role in replication has not been defined. Using an in vitro biochemical assay, we detected a stimulatory effect of NS5A on the NS5B replication reaction with minimal natural templates. NS5A stimulates replication by NS5B on two templates derived from the 3’ end of the RNA genome (4 fold ± 1.3 fold). A pre-incubation step with the two proteins prior to the replication reaction and substoichiometric levels of NS5A are required for detecting stimulation. With a template derived from the 3’end complementary to the RNA genome (the negative strand) no stimulation was observed. Furthermore, with a synthetic template that allows studying different phases of replication, NS5A stimulates NS5B during elongation. These findings suggest that NS5A stimulates NS5B during synthesis of the complementary (i.e., negative) strand of the RNA genome.

Journal ArticleDOI
TL;DR: Isoquinoline-based non-nucleoside inhibitors of HCV NS5b RNA-dependent RNA-polymerase are described and the synthesis and structure-activity relationships are detailed, along with enzyme and cellular activity.

Journal ArticleDOI
TL;DR: 1H-benzo[de]isoquinoline-1,3(2H)-diones are presented as a new series of selective inhibitors of HCV NS5B polymerase and SAR in this new series reveals inhibitors, such as 20, with low micromolar activity in the HCV replicon and with good activity/toxicity window in cells.
Abstract: The hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase (RdRp) plays a central role in virus replication. NS5B has no functional equivalent in mammalian cells and, as a consequence, is an attractive target for inhibition. Herein, we present 1H-benzo[de]isoquinoline-1,3(2H)-diones as a new series of selective inhibitors of HCV NS5B polymerase. The HTS hit 1 shows submicromolar potency in two different HCV replicons (1b and 2b) and displays no activity on other polymerases (HIV-RT, Polio-pol, GBV-b-pol). These inhibitors act during the pre-elongation phase by binding to NS5B non-nucleoside binding site Thumb Site II as demonstrated by crystal structure of compound 1 with the ΔC55-1b and ΔC21-2b enzymes and by mutagenesis studies. SAR in this new series reveals inhibitors, such as 20, with low micromolar activity in the HCV replicon and with good activity/toxicity window in cells.

Journal ArticleDOI
TL;DR: This study identified ATA as a potent anti- NS5B inhibitor and suggests that its unique mode of action might be exploited for structural refinement and development of novel anti-NS5B agents.
Abstract: Background:Hepatitis C virus (HCV) NS5B is an essential component of the viral replication machinery and an important target for antiviral intervention. Aurintricarboxylic acid (ATA), a broad-spectrum antiviral agent, was evaluated and characterized for its anti-NS5B activity in vitro and in HCV replicon cells.Methods:Recombinant NS5B, HCV replicase and Huh-7 cells harbouring the subgenomic HCV replicon of genotype 1b were employed for biochemical and mechanistic investigations.Results:Analysis of ATA activity in vitro yielded equipotent inhibition of recombinant NS5B and HCV replicase in the submicromolar range (50% inhibition concentration [IC50] approximately 150 nM). Biochemical and mechanistic studies revealed a bimodal mechanism of ATA inhibition with characteristics of pyrophosphate mimics and non-nucleoside inhibitors. Molecular modelling and competition displacement studies were consistent with these parameters, suggesting that ATA might bind to the benzothiadiazine allosteric pocket 3 of NS5B or...

Journal ArticleDOI
TL;DR: Cyclophilins are cellular peptidyl isomerases that have been implicated in regulating hepatitis C virus (HCV) replication and the effects of purified CypB proteins on the enzymatic activity of NS5B were examined.

Journal ArticleDOI
Si Kim1, Jung-Sung Kim1, J.-M. Sun1, Mingoo Kim1, Jongwook Oh1 
TL;DR: The results identify PRK2 inhibitors as potential antiviral drugs that act by suppressing HCV replication via inhibition of viral RNA polymerase phosphorylation.
Abstract: Hepatitis C virus (HCV) infection is a serious threat to human health worldwide. In spite of the continued search for specific and effective anti-HCV therapies, the rapid emergence of drug-resistance variants has been hampering the development of anti-HCV drugs designed to target viral enzymes. Targeting host factors has therefore emerged as an alternative strategy offering the potential to circumvent the ever-present complication of drug resistance. We previously identified protein kinase C-related kinase 2 (PRK2) as a cellular kinase that phosphorylates the HCV RNA-dependent RNA polymerase (RdRp). Here, we report the anti-HCV activity of HA1077, also known as fasudil, and Y27632, which blocks HCV RdRp phosphorylation by suppressing PRK2 activation. Treatment of a Huh7 cell line, stably expressing a genotype 1b HCV subgenomic replicon RNA, with 20 microm each of HA1077 and Y27632 reduced the HCV RNA level by 55% and 30%, respectively. A combination of the inhibitors with 100 IU/mL interferon alpha (IFN-alpha) significantly potentiated the anti-HCV drug activities resulting in approximately a 2-log(10) viral RNA reduction. We also found that IFN-alpha does not activate PRK2 as well as its upstream kinase PDK1 in HCV-replicating cells. Furthermore, treatment of HCV-infected cells with 20 microm each of HA1077 and Y27632 reduced the levels of intracellular viral RNA by 70% and 92%, respectively. Taken together, the results identify PRK2 inhibitors as potential antiviral drugs that act by suppressing HCV replication via inhibition of viral RNA polymerase phosphorylation.

Journal ArticleDOI
TL;DR: A new series of inhibitors for hepatitis C virus NS5B RNA polymerase containing a constrained pentacyclic scaffold containing hexahydroindolo[2,1-a]pyrrolo[3,2-d]benzazepines exposing basic groups is reported.

Journal ArticleDOI
TL;DR: This data indicates that Hepatitis C virus genotype distribution in Indonesia has been reported but the identification of HCV genotype was based on 5′‐UTR or NS5B sequence.
Abstract: Background Hepatitis C virus (HCV) genotype distribution in Indonesia has been reported. However, the identification of HCV genotype was based on 5'-UTR or NS5B sequence. Aims This study was aimed to observe HCV core sequence variation among HCV-associated liver disease patients in Jakarta, and to analyse the HCV genotype diversity based on the core sequence. Methods Sixty-eight chronic hepatitis (CH), 48 liver cirrhosis (LC) and 34 hepatocellular carcinoma (HCC) were included in this study. HCV core variation was analysed by direct sequencing. Results Alignment of HCV core sequences demonstrated that the core sequence was relatively varied among the genotype. Indeed, 237 bases of the core sequence could classify the HCV subtype; however, 236 bases failed to differentiate several subtypes. Based on 237 bases of the core sequences, the HCV strains were classified into genotypes 1 (subtypes 1a, 1b and 1c), 2 (subtypes 2a, 2e and 2f) and 3 (subtypes 3a and 3k). The HCV 1b (47.3%) was the most prevalent, followed by subtypes 1c (18.7%), 3k (10.7%), 2a (10.0%), 1a (6.7%), 2e (5.3%), 2f (0.7%) and 3a (0.7%). HCV 1b was the most common in all patients, and the prevalence increased with the severity of liver disease (36.8% in CH, 54.2% in LC and 58.8% in HCC). These results were similar to a previous report based on NS5B sequence analysis. Conclusion Hepatitis C virus core sequence (237 bases) could identify the HCV subtype and the prevalence of HCV subtype based on core sequence was similar to those based on the NS5B region.

Journal ArticleDOI
TL;DR: It is demonstrated that synthesized pyrrolidine nucleosides represent a new template for antiviral or other biological studies and could be considered for novel combination therapy against HCV infection using nucleoside inhibitors and non-nucleoside inhibitor of HCV NS5B.
Abstract: 3'-Deoxy-4'-azaribonucleosides (15a-d) were synthesized starting from the commercially available (4R)-trans-4-hydroxy-L-proline 7. From biological evaluations, 15b and 15d emerged as potent inhibitors of HCV replication on a replicon assay. These findings demonstrate that synthesized pyrrolidine nucleosides represent a new template for antiviral or other biological studies and could be considered for novel combination therapy against HCV infection using nucleoside inhibitors and non-nucleoside inhibitors of HCV NS5B.

Journal ArticleDOI
TL;DR: An increased level of CsA resistance is associated with distinct mutations in the NS5B gene that increase RNA binding in the presence of C'sA, and the intramolecular communications between residues of the thumb and the C‐terminal domains are important for HCV replicase function.

Journal ArticleDOI
TL;DR: BVDV NS4B was found to colocalize with mitochondria suggesting that this organelle might play a role in BVDV genome replication or cytopathogenicity, and results show it is an integral membrane protein associated with the Golgi apparatus and virus-induced membranes, the putative site for BVDv genome replication.
Abstract: Very little is known about BVDV NS4B, a protein of approximately 38 kDa. However, a missense mutation in NS4B has been implicated in changing BVDV from a cytopathic to noncytopathic virus, suggesting that NS4B might play a role in BVDV pathogenesis. Though this is one possible function, it is also likely that NS4B plays a role in BVDV genome replication. For example, BVDV NS4B interacts with NS3 and NS5A, implying that NS4B is part of a complex, which contains BVDV replicase proteins. Other possible BVDV NS4B functions can be inferred by analogy to hepatitis C virus (HCV) NS4B protein. For instance, HCV NS4B remodels host membranes to form the so-called membranous web, the site for HCV genome replication. Finally, HCV NS4B is membrane-associated, implying that HCV NS4B may anchor the virus replication complex to the membranous web structure. Unlike its HCV counterpart, we know little about the subcellular distribution of BVDV NS4B protein. Further, it is not clear whether NS4B is localized to host membrane alterations associated with BVDV infection. We show first that release of infectious BVDV correlates with the kinetics of BVDV genome replication in infected cells. Secondly, we found that NS4B subcellular distribution changes over the course of BVDV infection. Further, BVDV NS4B is an integral membrane protein, which colocalizes mainly with the Golgi compartment when expressed alone or in the context of BVDV infection. Additionally, BVDV induces host membrane rearrangement and these membranes contain BVDV NS4B protein. Finally, NS4B colocalizes with replicase proteins NS5A and NS5B proteins, raising the possibility that NS4B is a component of the BVDV replication complex. Interestingly, NS4B was found to colocalize with mitochondria suggesting that this organelle might play a role in BVDV genome replication or cytopathogenicity. These results show that BVDV NS4B is an integral membrane protein associated with the Golgi apparatus and virus-induced membranes, the putative site for BVDV genome replication. On the basis of NS4B Colocalization with NS5A and NS5B, we conclude that NS4B protein is an integral component of the BVDV replication complex.

Journal ArticleDOI
TL;DR: Novel NS5B polymerase inhibitors are identified by virtual screening and in vitro evaluation of their inhibitory activities, and kinetic analyses suggest that these compounds are noncompetitive inhibitors with respect to the ribonucleotide substrate.

Journal ArticleDOI
TL;DR: Research in the laboratories has identified a novel series of tetracyclic benzothiadiazine inhibitors of HCV polymerase bearing a benzylamino substituent on the B-ring, which has good oral bioavailability and a favorable tissue distribution drug profile, with high liver to plasma ratios.
Abstract: Benzothiadiazine inhibitors of the HCV NS5B RNA-dependent RNA polymerase are an important class of non-nucleoside inhibitors that have received considerable attention in the search for novel HCV therapeutics. Research in our laboratories has identified a novel series of tetracyclic benzothiadiazine inhibitors of HCV polymerase bearing a benzylamino substituent on the B-ring. Compounds in this series exhibit low-nanomolar activities in both genotypes 1a and 1b polymerase inhibition assays and subgenomic replicon assays. Optimization of pharmacokinetic properties in rat led to compound 30, which has good oral bioavailability (F = 56%) and a favorable tissue distribution drug profile, with high liver to plasma ratios. Compound 30 is a potent inhibitor in replicon assays, with EC(50) values of 10 and 6 nM against genotypes 1a and 1b, respectively.