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Hepatitis C virus (HCV) is a positive-sense, single-stranded RNA virus that causes chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. The release of infectious HCV particles from infected hepatocytes is a crucial step in viral dissemination and disease progression. While the exact mechanisms of HCV particle release remain poorly understood, emerging evidence suggests that HCV utilizes intracellular membrane trafficking and secretory pathways. These pathways include the Golgi secretory pathway and the endosomal trafficking pathways, such as the recycling endosome pathway and the endosomal sorting complex required for transport (ESCRT)-dependent multivesicular bodies (MVBs) pathway. This review provides an overview of recent advances in understanding the release of infectious HCV particles, with a particular focus on the involvement of the host cell factors that participate in HCV particle release. By summarizing the current knowledge in this area, this review aims to contribute to a better understanding of endosomal pathways involved in the extracellular release of HCV particles and the development of novel antiviral strategies. [ABSTRACT FROM AUTHOR]

Lysosome incorporate and degrade proteins in a process known as autophagy. There are three types of autophagy; macroautophagy, microautophagy, and chaperone-mediated autophagy (CMA). Although autophagy is considered a nonselective degradation process, CMA is known as a selective degradation pathway. All proteins internalized in the lysosome via CMA contain a pentapeptide KFERQ-motif, also known as a CMA-targeting motif, which is necessary for selectivity. CMA directly delivers a substrate protein into the lysosome lumen using the cytosolic chaperone HSC70 and the lysosomal receptor LAMP-2A for degradation. Hepatitis C virus (HCV) NS5A protein interacts with hepatocyte-nuclear factor 1α (HNF-1α) together with HSC70 and promotes the lysosomal degradation of HNF-1α via CMA, resulting in HCV-induced pathogenesis. HCV NS5A promotes recruitment of HSC70 to the substrate protein HNF-1α. HCV NS5A plays a crucial role in HCV-induced CMA. Further investigations of HCV NS5A-interacting proteins containing CMA-targeting motifs may help to elucidate HCV-induced pathogenesis. [ABSTRACT FROM AUTHOR]

ABSTRACT Hepatitis C virus (HCV) non-structural protein 5A (NS5A) is a multifunctional protein that is involved in the HCV life cycle and pathogenesis. In this study, a host protein(s) interacting with NS5A by tandem affinity purification were searched for with the aim of elucidating the role of NS5A. An NS5A-interacting protein, SET and MYND domain-containing 3 (SMYD3), a lysine methyltransferase reportedly involved in the development of cancer, was identified. The interaction between NS5A and SMYD3 was confirmed in ectopically expressing, HCV RNA replicon-harboring and HCV-infected cells. The other HCV proteins did not bind to SMYD3. SMYD3 bound to NS5A of HCV genotypes 1b and 2a. Deletion mutational analysis revealed that domains II and III of NS5A (amino acids [aa] 250 to 447) and the MYND and N-SET domains of SMYD3 (aa 1 to 87) are involved in the full extent of NS5A-SMYD3 interaction. NS5A co-localized with SMYD3 exclusively in the cytoplasm, thereby inhibiting nuclear localization of SMYD3. Moreover, NS5A formed a complex with SMYD3 and heat shock protein 90 (HSP90), which is a positive regulator of SMYD3. The intensity of binding between SMYD3 and HSP90 was enhanced by NS5A. Luciferase reporter assay demonstrated that NS5A significantly induces activator protein 1 (AP-1) activity, this being potentiated by co-expression of SMYD3 with NS5A. Taken together, the present results suggest that NS5A interacts with SMYD3 and induces AP-1 activation, possibly by facilitating binding between HSP90 and SMYD3. This may be a novel mechanism of AP-1 activation in HCV-infected cells. [ABSTRACT FROM AUTHOR]

ABSTRACT Hepatitis C virus (HCV) NS5A protein plays crucial roles in viral RNA replication, virus assembly, and viral pathogenesis. Although NS5A has no known enzymatic activity, it modulates various cellular pathways through interaction with cellular proteins. HCV NS5A (and other HCV proteins) are reportedly degraded through the ubiquitin-proteasome pathway; however, the physiological roles of ubiquitylation and deubiquitylation in HCV infection are largely unknown. To elucidate the role of deubiquitylation in HCV infection, an attempt was made to identify a deubiquitinase (DUB) that can interact with NS5A protein. An ovarian tumor protein (OTU), deubiquitinase 7B (OTUD7B), was identified as a novel NS5A-binding protein. Co-immunoprecipitation analyses showed that NS5A interacts with OTUD7B in both Huh-7 and HCV RNA replicon cells. Immunofluorescence staining revealed that HCV NS5A protein colocalizes with OTUD7B in the cytoplasm. Moreover, HCV infection was found to enhance the nuclear localization of OTUD7B. The OTUD7B-binding domain on NS5A was mapped using a series of NS5A deletion mutants. The present findings suggest that the domain I of NS5A is important and the region from amino acid 121 to 126 of NS5A essential for the interaction. Either V121A or V124A mutation in NS5A disrupts the NS5A-OTUD7B interaction. The results of this in vivo ubiquitylation assay suggest that HCV NS5A enhances OTUD7B DUB activity. Taken together, these results suggest that HCV NS5A protein interacts with OTUD7B, thereby modulating its DUB activity. [ABSTRACT FROM AUTHOR]

Effective therapeutic vaccines against virus infection must induce sufficient levels of cell-mediated immune responses against the target viral epitopes and also must avoid concomitant risk factors, such as potential carcinogenic properties. The nonstructural protein 3 (NS3) of hepatitis C virus (HCV) carries a variety of CD4+ and CD8+ T cell epitopes, and induces strong HCV-specific T cell responses, which are correlated with viral clearance and resolution of acute HCV infection. On the other hand, NS3 possesses serine protease and nucleoside triphosphatase (NTPase)/RNA helicase activities, which not only play important roles in viral life cycle but also concomitantly interfere with host defense mechanisms by deregulating normal cellular functions. In this study, we constructed a series of DNA vaccines that express NS3 of HCV. To avoid the potential harm of NS3, we introduced mutations to the catalytic triad of the serine protease (H57A, D81A and S139A) and the NTPase/RNA helicase domain (K210N, F444A, R461Q and W501A) to eliminate the enzymatic activities. Immunization of BALB/c mice with each of the DNA vaccine candidates (pNS3[S139A/K210N], pNS3[S139A/F444A], pNS3[S139A/R461Q] and pNS3[S139A/W501A]) that expresses an NS3 mutant lacking both serine protease and NTPase/helicase activities induced T cell immune responses to the degree comparable to that induced by the wild type NS3 and the NS3/4A complex, as demonstrated by interferon-γ production and cytotoxic T lymphocytes activities against NS3. The present study has demonstrated that plasmids expressing NS3 mutants, NS3(S139A/K210N), NS3(S139A/F444A), NS3(S139A/R461Q) and NS3(S139A/W501A), which lack both serine protease and NTPase/RNA helicase activities, would be good candidates for safe and efficient therapeutic DNA vaccines against HCV infection. [ABSTRACT FROM AUTHOR]

The development of complementary and/or alternative drugs for treatment of hepatitis C virus (HCV) infection is still needed. Antiviral compounds in medicinal plants are potentially good targets to study. Morinda citrifolia is a common plant distributed widely in Indo-Pacific region; its fruits and leaves are food sources and are also used as a treatment in traditional medicine. In this study, using a HCV cell culture system, it was demonstrated that a methanol extract, its n-hexane, and ethyl acetate fractions from M. citrifolia leaves possess anti-HCV activities with 50%-inhibitory concentrations (IC50) of 20.6, 6.1, and 6.6 μg/mL, respectively. Bioactivity-guided purification and structural analysis led to isolation and identification of pheophorbide a, the major catabolite of chlorophyll a, as an anti-HCV compound present in the extracts (IC50 = 0.3 μg/mL). It was also found that pyropheophorbide a possesses anti-HCV activity (IC50 = 0.2 μg/mL). The 50%-cytotoxic concentrations (CC50) of pheophorbide a and pyropheophorbide a were 10.0 and 7.2 μg/mL, respectively, their selectivity indexes being 33 and 36, respectively. On the other hand, chlorophyll a, sodium copper chlorophyllin, and pheophytin a barely, or only marginally, exhibited anti-HCV activities. Time-of-addition analysis revealed that pheophorbide a and pyropheophorbide a act at both entry and the post-entry steps. The present results suggest that pheophorbide a and its related compounds would be good candidates for seed compounds for developing antivirals against HCV. [ABSTRACT FROM AUTHOR]

ABSTRACT Development of complementary and/or alternative drugs for treatment of hepatitis C virus (HCV) infection is still much needed from clinical and economic points of view. Antiviral substances obtained from medicinal plants are potentially good targets to study. Glycyrrhiza uralensis and G. glabra have been commonly used in both traditional and modern medicine. In this study, extracts of G. uralensis roots and their components were examined for anti-HCV activity using an HCV cell culture system. It was found that a methanol extract of G. uralensis roots and its chloroform fraction possess anti-HCV activity with 50%-inhibitory concentrations (IC50) of 20.0 and 8.0 μg/mL, respectively. Through bioactivity-guided purification and structural analysis, glycycoumarin, glycyrin, glycyrol and liquiritigenin were isolated and identified as anti-HCV compounds, their IC50 being 8.8, 7.2, 4.6 and 16.4 μg/mL, respectively. However, glycyrrhizin, the major constituent of G. uralensis, and its monoammonium salt, showed only marginal anti-HCV activity. It was also found that licochalcone A and glabridin, known to be exclusive constituents of G. inflata and G. glabra, respectively, did have anti-HCV activity, their IC50 being 2.5 and 6.2 μg/mL, respectively. Another chalcone, isoliquiritigenin, also showed anti-HCV activity, with an IC50 of 3.7 μg/mL. Time-of-addition analysis revealed that all Glycyrrhiza-derived anti-HCV compounds tested in this study act at the post-entry step. In conclusion, the present results suggest that glycycoumarin, glycyrin, glycyrol and liquiritigenin isolated from G. uralensis, as well as isoliquiritigenin, licochalcone A and glabridin, would be good candidates for seed compounds to develop antivirals against HCV. [ABSTRACT FROM AUTHOR]

Objective: Hepatitis C virus (HCV genome) polymorphisms are thought to influence the outcome of pegylated-interferon/ribavirin (PEG-IFN/RBV) therapy. This study aimed to examine non-structural protein 5A (NS5A) polymorphisms, e.g. IFN/RBV resistance-determining region (IRRDR) and IFN sensitivity-determining region (ISDR), and core protein polymorphism as predictive therapeutic markers. Methods: Pretreatment sequences of NS5A and core regions were analyzed in 68 HCV-1b-infected patients treated with PEG-IFN/RBV. Results: Of 24 patients infected withHCV having an IRRDR with 6 or more mutations (IRRDR≥6), 18 (75%) patients achieved sustained virological response (SVR), whereas only 11 (25%) of 44 patients infected with HCV having IRRDR≤5 did. IRRDR≥6 was significantly associated with SVR (p < 0.0001). On the other hand, ISDR≥2 was significantly associated with relapse (either before [breakthrough] or after end-of-treatment response [ETR[-]relapse]) (p < 0.05) and a point mutation of the core protein from Arg to Gln at position 70 (Gln70) was significantly associated with null-response (p < 0.05). Multivariate analysis identified IRRDR≥6 as the only viral genetic factor that independently predicted SVR. Conclusion: NS5A (IRRDR and ISDR) and core protein polymorphisms are associated with the outcome of PEG-IFN/RBV therapy for chronic hepatitis C. In particular, IRRDR≥6 is a useful marker for prediction of SVR. Copyright © 2011 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Both host and viral factors have been implicated in influencing the response to pegylated-interferon/ribavirin (PEG-IFN/RBV) therapy for hepatitis C virus (HCV) infection. Among the viral factors, sequence heterogeneity within NS5A and core regions has been proposed. This study aimed to clarify the relationship between virological responses to PEG-IFN/RBV therapy and sequence heterogeneity within NS5A, including the IFN/RBV resistance-determining region (IRRDR), the interferon sensitivity-determining region (ISDR) and the core region. Pretreatment sequences of NS5A and the core regions were analyzed in 57 HCV-1b-infected patients who were to be treated with PEG-IFN/RBV. Of 40 patients infected with HCV having an IRRDR with four or more mutations (IRRDR ≥ 4), 28 (70%) patients achieved a sustained virological response (SVR). On the other hand, only 4 (24%) of 17 patients infected with HCV having an IRRDR with three or fewer mutations (IRRDR ≤ 3) achieved a SVR ( P= 0.001). Similarly, 22 (71%) of 31 patients infected with HCV and having an ISDR with one or more mutations (ISDR ≥ 1) achieved a SVR while 10 (38%) of 26 patients infected with HCV and having an ISDR without any mutations (ISDR = 0) achieved a SVR ( P= 0.014). As for the core region, there was significant correlation between a single mutation at position 70 (Gln) and non-SVR ( P= 0.02). Notably, Gln was more prominently associated with the null response ( P= 0.0007). In conclusion, sequence heterogeneity within the IRRDR and ISDR, and a single point mutation at position 70 of the core region of HCV-1b are likely to be correlated with virological responses to PEG-IFN/RBV therapy. [ABSTRACT FROM AUTHOR]

Abstract: Objective: To construct the point mutation plasmids expressing HCV NS3/4A with different secondary structures at the N-terminus, and to analyze their serine protease activities. Methods: The point mutation plasmid constructs were generated by using the Quick Change site-directed mutagenesis kit with the backbone of M-H05-5 (A1-1), and were named as subgroup A1-2, A2-1, A2-2, B1-1, B1-2, B2-1, and B2-2 respectively. The transient expression of the constructs was investigated by immunofluorescence assay and Western blot analysis. The difference in in cis and in trans NS3 serine protease activity between each subgroup was determined by Western blot analysis. Luciferase reporter assay was used to observe the inhibitory effects of the constructs on RIG-I induced IFN-β promoter activity and on p53-dependent transcriptional activation. Results: The point mutation plasmid constructs were verified for the correct sequence by DNA sequencing. The immunofluorescence assay revealed 4 subcellular localization patterns of NS3, including dot-like staining, diffuse staining, doughnut-like staining, and rod-shape staining. Western blot analysis indicated that the incomplete cleavage of NS3/4A appeared in subgroups A2-1 and B2-1, indicating that the in cis NS3 serine protease activities of subgroup A2-1 and B2-1 were weaker when compared with the other subgroups. By using NS5A/5BΔC as a substrate for NS3/4A serine protease, it was also found that the in trans NS3 serine protease activities of subgroup A2-1 and B2-1 were also weaker compared the other subgroups. Differences in inhibitory effects of HCV NS3 on RIG-I induced IFN-β promoter activity and on p53-dependent transcriptional activation were also observed between subgroup A2-1, B2-1 and the other subgroups. Conclusion: The results suggest that subgroup A2-1 and B2-1 has weaker serine protease activities and weaker inhibitory activities on host cell functions than the other subgroups, which might be explained by the different secondary structure of the 120-aa sequence at N-terminus of NS3. [Copyright &y& Elsevier]

Abstract: The lack of a culture system that efficiently produces progeny virus has hampered hepatitis C virus (HCV) research. Recently, the discovery of a novel HCV isolate JFH1 and its chimeric derivative J6/JFH1 has led to the development of an efficient virus productive culture system. To construct an easy monitoring system for the viral life cycle of HCV, we generated bicistronic luciferase reporter virus genomes based on the JFH1 and J6/JFH1 isolates, respectively. Transfection of the J6/JFH1-based reporter genome to Huh7.5 cells produced significantly greater levels of progeny virus than transfection of the JFH1 genome. Furthermore, the expression of dominant-negative Vps4, a key molecule of the endosomal sorting complex required for transport machinery, inhibited the virus production of JFH1, but not that of J6/JFH1. These results may account for the different abilities to produce progeny virus between JFH1 and J6/JFH1. Using the J6/JFH1/Luc system, we showed that the two polyanions heparin and polyvinyl sulfate decreased the infectivity of J6/JFH1/Luc virus in a dose-dependent manner. We also analyzed the function of microRNA on HCV replication and found that miR-34b could affect the replication of HCV. The reporter virus generated in this study will be useful for investigating the nature of the HCV life cycle and for identification of HCV inhibitors. [Copyright &y& Elsevier]

Background/Aims: Persistent infection with hepatitis C virus (HCV) causes extrahepatic diseases, including diabetes. We investigated the possible effect(s) of HCV replication on cellular glucose uptake and expression of the facilitative glucose transporter (GLUT) 2 and 1. Methods: We used Huh-7.5 cells harboring either an HCV subgenomic RNA replicon (SGR) or an HCV full-genomic RNA replicon (FGR), HCV-infected cells, and the respective cells treated with interferon (IFN). We also used liver tissue samples obtained from patients with or without HCV infection. Results: Glucose uptake and surface expression of GLUT2 and GLUT1 were suppressed in SGR, FGR and HCV-infected cells compared to the control cells. Expression levels of GLUT2 mRNA, but not GLUT1 mRNA, were lower in SGR, FGR and HCV-infected cells than in the control. Luciferase reporter assay demonstrated decreased GLUT2 promoter activities in SGR, FGR and HCV-infected cells. IFN treatment restored glucose uptake, GLUT2 surface expression, GLUT2 mRNA expression and GLUT2 promoter activities. Also, GLUT2 expression was reduced in hepatocytes of liver tissues obtained from HCV-infected patients. Conclusions: HCV replication down-regulates cell surface expression of GLUT2 partly at the transcriptional level, and possibly at the intracellular trafficking level as suggested for GLUT1, thereby lowering glucose uptake by hepatocytes. [Copyright &y& Elsevier]

Abstract: The N-terminal domain of NS3 of hepatitis C virus (HCV) possesses serine protease activity, which is essential for virus replication. This portion is also implicated in malignant transformation of hepatocytes. We previously demonstrated that an N-terminal portion of NS3 formed a complex with the tumor suppressor p53 and suppressed actinomycin D-induced apoptosis. We report here that single-point mutations of NS3 at position 106 from Leu to Ala (L106A), and position 43 from Phe to Ala (F43A) to a lesser extent, significantly impaired complex formation with p53. Moreover, the L106A mutation impaired an otherwise more distinct anti-apoptotic activity of NS3. F43A and L106A mutations also inhibited serine protease activity of NS3. These results collectively suggest the possibility that Leu106 and Phe43 are involved in p53 interaction and serine protease activity, and therefore, can be a good target for certain low-molecular-weight compound(s) to inhibit both oncogenic and replicative abilities of HCV. [Copyright &y& Elsevier]

Hepatitis C virus (HCV) induces cytopathic effects in the form of hepatocytes apoptosis thought to be resulted from the interaction between viral proteins and host factors. Using pathway specific PCR array, we identified 9 apoptosis-related genes that are dysregulated during HCV infection, of which the BH3-only pro-apoptotic Bcl-2 family protein, BIK, was consistently up-regulated at the mRNA and protein levels. Depletion of BIK protected host cells from HCV-induced caspase-3/7 activation but not the inhibitory effect of HCV on cell viability. Furthermore, viral RNA replication and release were significantly suppressed in BIK-depleted cells and over-expression of the RNA-dependent RNA polymerase, NS5B, was able to induce BIK expression. Immunofluorescence and co-immunoprecipitation assays showed co-localization and interaction of BIK and NS5B, suggesting that BIK may be interacting with the HCV replication complex through NS5B. These results imply that BIK is essential for HCV replication and that NS5B is able to induce BIK expression. [ABSTRACT FROM AUTHOR]

By using a yeast two-hybrid assay, cyclic AMP-response-element-binding protein-related protein (CREB-RP), also called activating transcription factor 6β (ATF6β), was identified as a cellular protein that interacts with the NS4B protein of hepatitis C virus. An N-terminal half of NS4B and a central portion of CREB-RP/ATF6β containing the basic leucine zipper (bZIP) domain were involved in the interaction. The interaction between NS4B and CREB-RP/ATF6β was demonstrated also in mammalian cells by co-immunoprecipitation and confocal microscopic analyses using specific antibodies. The bZIP domain of ATF6α, which shares high sequence similarity with CREB-RP/ATF6β, was also shown to interact with NS4B in yeast although the interaction was weaker than that between NS4B and CREB-RP/ATF6β. CREB-RP/ATF6β and ATF6α are known as endoplasmic reticulum (ER) stress-induced transcription factors. Collectively, our results imply the possibility that NS4B modulates certain cellular responses upon ER stress through the physical interaction with CREB-RP/ATF6β and ATF6α. [Copyright &y& Elsevier]