[Show abstract][Hide abstract] ABSTRACT: Background
Hepatitis C virus (HCV) is a major global health problem, causing chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. Development of well-tolerated regimens with high cure rates and fewer side effects is still much needed. Recently, natural antimicrobial peptides (AMPs) are attracting more attention as biological compounds and can be a good template to develop therapeutic agents, including antiviral agents against a variety of viruses. Various AMPs have been characterized from the venom of different venomous animals including scorpions.
The possible antiviral activities of crude venoms obtained from five Egyptian scorpion species (Leiurus quinquestriatus, Androctonus amoreuxi, A. australis, A. bicolor and Scorpio maurus palmatus) were evaluated by a cell culture method using Huh7.5 cells and the J6/JFH1-P47 strain of HCV. Time-of-addition experiments and inactivation of enzymatic activities of the venoms were carried out to determine the characteristics of the anti-HCV activities.
S. maurus palmatus and A. australis venoms showed anti-HCV activities, with 50% inhibitory concentrations (IC50) being 6.3 ± 1.6 and 88.3 ± 5.8 μg/ml, respectively. S. maurus palmatus venom (30 μg/ml) impaired HCV infectivity in culture medium, but not inside the cells, through virocidal effect. The anti-HCV activity of this venom was not inhibited by a metalloprotease inhibitor or heating at 60°C. The antiviral activity was directed preferentially against HCV.
S. maurus palmatus venom is considered as a good natural source for characterization and development of novel anti-HCV agents targeting the entry step. To our knowledge, this is the first report describing antiviral activities of Egyptian scorpion venoms against HCV, and may open a new approach towards discovering antiviral compounds derived from scorpion venoms.
[Show abstract][Hide abstract] ABSTRACT: Hepatitis B virus (HBV) is a widespread human pathogen that often causes chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. Detailed mechanisms underlying the HBV pathogenesis remain poorly understood. The HBV X protein (HBx) is a multifunctional regulator that modulates viral replication and host cell functions, such as cell cycle progression, apoptosis and protein degradation through interaction with a variety of host factors. Recently, the nonstructural protein 5A (NS5A) of hepatitis C virus has been reported to interact with methyltransferase SET and MYND domain-containing 3 (SMYD3), which is implicated in chromatin modification and development of cancer. Since HBx shares fundamental regulatory functions on viral replication and pathogenesis with NS5A, we became interested to examine whether HBx possibly interacts with SMYD3. In the present study, we demonstrated by co-immunoprecipitation analysis that HBx interacted with both ectopically and endogenously expressed SMYD3 in Huh-7.5 cells. Deletion mutation analysis revealed that C-terminal region of HBx (amino acids [aa] 131-154) and an internal region of SMYD3 (aa 269-288) were responsible for their interaction. Immunofluorescence and proximity ligation assays showed that HBx and SMYD3 co-localized predominantly in the cytoplasm. Luciferase reporter assay demonstrated that the interaction between HBx and SMYD3 activated the activator protein 1 (AP-1) signaling, but not that of nuclear factor-kappa B (NF-κB). On the other hand, neither overexpression nor knockdown of SMYD3 altered the levels of HBV transcripts and HBV surface antigen (HBsAg) production. In conclusion, we identified a novel HBx-interacting protein, SMYD3, and propose a novel mechanism of AP-1 activation in HBV-infected cells.
No preview · Article · Nov 2015 · Microbiology and Immunology
[Show abstract][Hide abstract] ABSTRACT: We previously reported that hepatitis C virus (HCV) infection induces Bax-triggered, mitochondrion-mediated apoptosis by using the HCV J6/JFH1 strain and Huh-7.5 cells. However, it was still unclear how HCV induced Bax activation. In this study, we showed that the HCV-induced activation and mitochondrial accumulation of Bax were significantly attenuated by treatment with a general antioxidant, N-acetyl cysteine (NAC), or a specific c-Jun N-terminal kinase (JNK) inhibitor, SP600125, with the result suggesting that the reactive oxygen species (ROS)/JNK signaling pathway is upstream of Bax activation in HCV-induced apoptosis. We also demonstrated that HCV infection transcriptionally activated the gene for the pro-apoptotic protein Bim and the protein expression of three major splice variants of Bim (BimEL, BimL and BimS). The HCV-induced increase in the Bim mRNA and protein levels was significantly counteracted by treatment with NAC or SP600125, suggesting that the ROS/JNK signaling pathway is involved in the Bim upregulation. Moreover, HCV infection led to a marked accumulation of Bim on the mitochondria to facilitate its interaction with Bax. On the other hand, downregulation of Bim by siRNA significantly prevented HCV-mediated activation of Bax and caspase 3. Taken together, these observations suggest that the HCV-induced ROS/JNK signaling transcriptionally activates Bim expression, which leads to Bax activation and apoptosis induction.
No preview · Article · Jul 2015 · Journal of General Virology
[Show abstract][Hide abstract] 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, NS5A modulates various cellular pathways through interaction with cellular proteins. It was reported that HCV NS5A as well as other HCV proteins are degraded through the ubiquitin-proteasome pathway, but the physiological roles of ubiquitylation and deubiquitylation in HCV infection are largely unknown. To elucidate the role of deubiquitylation in HCV infection, we sought to identify a deubiquitinase (DUB) that can interact with NS5A protein. Here we identified an ovarian tumor protein (OTU) deubiquitinase 7B (OTUD7B) as a novel NS5A-binding protein. Co-immunoprecipitation analyses showed that NS5A interacted with OTUD7B in Huh-7 cells and in HCV RNA replicon cells. Immunofluorescence staining revealed that HCV NS5A protein colocalizes with OTUD7B in the cytoplasm. Moreover, HCV infection enhanced the nuclear localization of OTUD7B. Using a series of NS5A deletion mutants, we mapped the OTUD7B-binding domain on NS5A. Our results suggest that the domain I of NS5A is important for its interaction. The region from aa 121 to 126 of NS5A is essential for the interaction. Either V121A or V124A mutation in NS5A disrupted the NS5A-OTUD7B interaction. Our in vivo ubiquitylation assay results suggest that HCV NS5A enhanced the OTUD7B DUB activity. Taken together, these results suggest that HCV NS5A protein interacts with OTUD7B, thereby modulating its DUB activity.
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No preview · Article · Jun 2015 · Microbiology and Immunology
[Show abstract][Hide abstract] ABSTRACT: Hepatitis C virus (HCV) infection often causes extrahepatic manifestations, such as type 2 diabetes. We previously reported that HCV infection induces the lysosomal degradation of the transcription factor HNF-1α via an interaction with viral NS5A, thereby suppressing GLUT2 gene expression. However, molecular mechanism of NS5A-induced degradation of HNF-1α is largely unknown. We aimed to identify the determinants necessary for the degradation of HNF-1α induced by NS5A. Co-immunoprecipitation analysis revealed that the POU specific (POUs) domain spanning from aa 91 to 181 of HNF-1α is responsible for the interaction of NS5A. We also found that the region from aa 121 to 126 of NS5A, which is known as the binding motif of HCV replication factor FKBP8, is important for degradation of HNF-1α. NS5A V121A mutation disrupted NS5A-HNF-1α interaction as well as degradation of HNF-1α. Our findings suggest that NS5A Val121 is crucial for viral pathogenesis.
No preview · Article · May 2015 · Journal of General Virology
[Show abstract][Hide abstract] ABSTRACT: The non-structural protein 5A (NS5A) of hepatitis C virus (HCV) is a multifunctional protein involved in the HCV lifecycle and pathogenesis. The precise molecular mechanisms of NS5A-mediated pathogenesis still remain to be clarified. In this study, we performed cDNA microarray analysis on NS5A-expressing HEK293 cells and the non-expressing control to screen the possible cellular genes dysregulated by NS5A. Subsequent quantitative real time PCR (qRT-PCR) analysis on NS5A-expressing cells and the control confirmed that NS5A upregulated the anterior gradient homolog 3 (AGR3) mRNA expression. The domain III of NS5A was responsible for the activation of AGR3 gene expression. AGR3 mRNA expression levels were upregulated also in Huh7.5 cells harboring a full-genome HCV-1b RNA replicon (FGR) and in those infected with HCV-2a. Moreover, AGR3 promoter activity was activated in NS5A-expressing cells, FGR-harboring cells and HCV-infected cells. Taken together, our present results suggest that HCV NS5A transcriptionally activates the cancer-associated AGR3 gene. This may be a novel mechanism of HCV-mediated pathogenesis, especially hepatocarcinogenesis.
No preview · Article · Apr 2015 · The Kobe journal of medical sciences
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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, whose fruits and leaves are among the food sources and also used as a treatment in traditional medicine. In this study, by using the HCV cell culture system, we demonstrated that a methanol extract, its n-hexane and ethyl acetate fractions from M. citrifolia leaves possessed anti-HCV activities with 50%-inhibitory concentration (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). We also found that pyropheophorbide a possessed 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, with selectivity index 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 the entry and the post-entry steps. The present results suggest that pheophorbide a and its related compounds would be good candidates for seed compounds to develop antivirals against HCV.
No preview · Article · Jan 2014 · Microbiology and Immunology
[Show abstract][Hide abstract] ABSTRACT: Development of complementary and/or alternative drugs for treatment of hepatitis C virus (HCV) infection is still much needed from clinical and economical 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, we examined extracts of G. uralensis roots and their components for anti-HCV activities using the HCV cell culture system. Our results revealed that a methanol extract of G. uralensis roots and its chloroform fraction possessed anti-HCV activities with 50%-inhibitory concentrations (IC50 ) of 20.0 and 8.0 μg/ml, respectively. Through bioactivity-guided purification and structural analysis, we isolated and identified glycycoumarin, glycyrin, glycyrol and liquiritigenin as anti-HCV compounds, with IC50 being 8.8, 7.2, 4.6 and 16.4 μg/ml, respectively. On the other hand, glycyrrhizin, the most common constituent in G. uralensis, and its monoammonium salt showed only marginal levels of anti-HCV activity. We also found that licochalcone A and glabridin, known as the exclusive constituents of G. inflata and G. glabra, respectively, possessed anti-HCV activities, with IC50 being 2.5 and 6.2 μg/ml, respectively. Another chalcone, isoliquiritigenin, also showed anti-HCV activity, with IC50 of 3.7 μg/ml. Time-of-addition analysis revealed that all the Glycyrrhiza-derived anti-HCV compounds tested in this study act at the post-entry step. In conclusion, our 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.
Full-text · Article · Jan 2014 · Microbiology and Immunology
[Show abstract][Hide abstract] ABSTRACT: Hepatitis C virus (HCV) is a major cause of liver disease and a potential cause of substantial morbidity and mortality worldwide. The overall prevalence of HCV infection is 2%, representing 120 million people worldwide. Current standard treatment using pegylated interferon and ribavirin is effective in only 50% of the patients infected with HCV genotype 1, and is associated with significant side effects. Therefore, it is still of importance to develop new drugs for treatment of HCV. Antiviral substances obtained from natural products, including medicinal plants, are potentially good targets to study. In this study, we evaluated Indonesian medicinal plants for their anti-HCV activities.
Ethanol extracts of 21 samples derived from 17 species of medicinal plants explored in the East Java region were tested. Anti-HCV activities were determined by a cell culture method using Huh7.5 cells and HCV strains of 9 different genotypes (1a to 7a, 1b and 2b).
Four of the 21 samples tested showed antiviral activities against HCV: Toona sureni leaves (TSL) with 50% inhibitory concentrations (IC50) of 13.9 and 2.0 mug/ml against the HCV J6/JFH1-P47 and -P1 strains, respectively, Melicope latifolia leaves (MLL) with IC50 of 3.5 and 2.1 mug/ml, respectively, Melanolepis multiglandulosa stem (MMS) with IC50 of 17.1 and 6.2 mug/ml, respectively, and Ficus fistulosa leaves (FFL) with IC50 of 15.0 and 5.7 mug/ml, respectively. Time-of-addition experiments revealed that TSL and MLL inhibited both at the entry and post-entry steps while MMS and FFL principally at the entry step. TSL and MLL inhibited all of 11 HCV strains of all the genotypes tested to the same extent. On the other hand, FFL showed significantly weaker inhibitory activities against the HCV genotype 1a strain, and MMS against the HCV strains of genotypes 2b and 7a to a lesser extent, compared to the other HCV genotypes.
Ethanol extracts of TSL, MLL, MMS and FFL showed antiviral activities against all the HCV genotypes tested with the exception that some genotype(s) showed significant resistance to FFL and to MMS to a lesser extent. These plant extracts may be good candidates for the development of anti-HCV drugs.
[Show abstract][Hide abstract] ABSTRACT: Background: Hepatitis C virus (HCV) is a major cause of liver disease and a potential cause of substantial morbidity and mortality worldwide. The overall prevalence of HCV infection is 2%, representing 120 million people worldwide. Current standard treatment using pegylated interferon and ribavirin is effective in only 50% of the patients infected with HCV genotype 1, and is associated with significant side effects. Therefore, it is still of importance to develop new drugs for treatment of HCV. Antiviral substances obtained from natural products, including medicinal plants, are potentially good targets to study. In this study, we evaluated Indonesian medicinal plants for their anti-HCV activities. Methods: Ethanol extracts of 21 samples derived from 17 species of medicinal plants explored in the East Java region were tested. Anti-HCV activities were determined by a cell culture method using Huh7.5 cells and HCV strains of 9 different genotypes (1a to 7a, 1b and 2b). Results: Four of the 21 samples tested showed antiviral activities against HCV: Toona sureni leaves (TSL) with 50% inhibitory concentrations (IC 50) of 13.9 and 2.0 μg/ml against the HCV J6/JFH1-P47 and -P1 strains, respectively, Melicope latifolia leaves (MLL) with IC 50 of 3.5 and 2.1 μg/ml, respectively, Melanolepis multiglandulosa stem (MMS) with IC 50 of 17.1 and 6.2 μg/ml, respectively, and Ficus fistulosa leaves (FFL) with IC 50 of 15.0 and 5.7 μg/ml, respectively. Time-of-addition experiments revealed that TSL and MLL inhibited both at the entry and post-entry steps while MMS and FFL principally at the entry step. TSL and MLL inhibited all of 11 HCV strains of all the genotypes tested to the same extent. On the other hand, FFL showed significantly weaker inhibitory activities against the HCV genotype 1a strain, and MMS against the HCV strains of genotypes 2b and 7a to a lesser extent, compared to the other HCV genotypes. Conclusions: Ethanol extracts of TSL, MLL, MMS and FFL showed antiviral activities against all the HCV genotypes tested with the exception that some genotype(s) showed significant resistance to FFL and to MMS to a lesser extent. These plant extracts may be good candidates for the development of anti-HCV drugs.
[Show abstract][Hide abstract] ABSTRACT: Unlabelled:
Hepatocellular carcinoma (HCC) is one of the common sequelae of hepatitis C virus (HCV) infection. It remains controversial, however, whether HCV itself plays a direct role in the development of HCC. Although HCV core, NS3, and NS5A proteins were reported to display tumorigenic activities in cell culture and experimental animal systems, their clinical impact on HCC development in humans is still unclear. In this study we investigated sequence polymorphisms in the core protein, NS3, and NS5A of HCV genotype 1b (HCV-1b) in 49 patients who later developed HCC during a follow-up of an average of 6.5 years and in 100 patients who did not develop HCC after a 15-year follow-up. Sequence analysis revealed that Gln at position 70 of the core protein (core-Gln(70) ), Tyr at position 1082 plus Gln at 1112 of NS3 (NS3-Tyr(1082) /Gln(1112) ), and six or more mutations in the interferon/ribavirin resistance-determining region of NS5A (NS5A-IRRDR≥6) were significantly associated with development of HCC. Multivariate analysis identified core-Gln(70) , NS3-Tyr(1082) /Gln(1112) , and α-fetoprotein (AFP) levels (>20 ng/L) as independent factors associated with HCC. Kaplan-Meier analysis revealed a higher cumulative incidence of HCC for patients infected with HCV isolates with core-Gln(70) , NS3-Tyr(1082) /Gln(1112) or both than for those with non-(Gln(70) plus NS3-Tyr(1082) /Gln(1112) ). In most cases, neither the residues at position 70 of the core protein nor positions 1082 and 1112 of the NS3 protein changed during the observation period.
HCV isolates with core-Gln(70) and/or NS3-Tyr(1082) /Gln(1112) are more closely associated with HCC development compared to those with non-(Gln(70) plus NS3-Tyr(1082) /Gln(1112) ).
[Show abstract][Hide abstract] ABSTRACT: Hepatitis C virus (HCV) infects B lymphocytes and induces mixed cryoglobulinemia and B cell non-Hodgkin's lymphoma. The molecular mechanism for the pathogenesis of HCV infection-mediated B cell disorders remains obscure. To identify the possible role for HCV nonstructural 5A (NS5A) protein in B cells, we generated the stable B cell lines expressing Myc-His tagged NS5A. Immunoprecipitation study in the presence or absence of pervanadate (PV) implied that NS5A was tyrosine phosphorylated by pervanadate (PV) treatment of the cells. Therefore we examined pull-down assay by using glutathione S-transferase (GST)-fusion proteins of various Src homology 2 (SH2) domains, which associates with phosphotyrosine within a specific amino acid sequence. The results showed that NS5A specifically bound to SH2 domain of Fyn from PV-treated B cells in addition to Src homology 3 (SH3) domain. Substitution of Arg(176) to Lys in the SH2 domain of Fyn abrogated this interaction. Deletion mutational analysis demonstrated that N-terminal region of NS5A was not required for the interaction with the SH2 domain of Fyn. Tyr(334) was identified as a tyrosine phosphorylation site in NS5A. Far-western analysis revealed that SH2 domain of Fyn directly bound to NS5A. Fyn and NS5A were colocalized in the lipid raft. These results suggest that NS5A directly binds to the SH2 domain of Fyn in a tyrosine phosphorylation-dependent manner. Lastly, we showed that the expression of NS5A in B cells increased phosphorylation of activation loop tyrosine in the kinase domain of Fyn. NS5A containing ligand for both SH2 and SH3 domains enhances an aberrant autophosphorylation and kinase activity of Fyn in B cells.
[Show abstract][Hide abstract] ABSTRACT: Hepatitis C virus genotype 4 (HCV-4) is the cause of approximately 20% of the 180 million cases of chronic hepatitis C in the world. HCV-4 infection is common in the Middle East and Africa, with an extraordinary high prevalence in Egypt. Viral genetic polymorphisms, especially within core and NS5A regions, have been implicated to influence the response to pegylated-interferon and ribavirin (PEG-IFN/RBV) combination therapy in HCV-1 infection. However, it has not been confirmed in HCV-4 infection. Here, we investigated the impact of heterogeneity of NS5A and core proteins of HCV-4, mostly the subtype HCV-4a, on clinical outcome of 43 Egyptian patients treated with PEG-IFN/RBV. Sliding window analysis over carboxy-terminus of NS5A protein identified the IFN/RBV resistance-determining region (IRRDR) as the most prominent region associated with sustained virological response (SVR). Indeed, 21 (84%) of 25 patients with SVR, but only 5 (28%) of 18 patients with non-SVR, were infected with HCV having IRRDR with 4 or more mutations (IRRDR≥4) (P=0.0004). Multivariate analysis identified IRRDR≥4 as an independent SVR predictor. The positive predictive value of IRRDR≥4 for SVR was 81% (21/26; P=0.002) while its negative predictive value for non-SVR was 76% (13/17; P=0.02). On the other hand, there was no significant correlation between core protein polymorphisms, either at residues 70 or 91, and treatment outcome. In conclusion, the present results demonstrate for the first time that IRRDR≥4, a viral genetic heterogeneity, would be a useful predictive marker for SVR in HCV-4 infection when treated with PEG-IFN/RBV.
[Show abstract][Hide abstract] ABSTRACT: Hepatitis C virus (HCV) infection causes not only intrahepatic diseases but also extrahepatic manifestations, including type 2 diabetes. We previously reported that HCV replication suppresses cellular glucose uptake by down-regulation of cell surface expression of glucose transporter 2 (GLUT2) (J Hepatol. 2009, 50: 883-94). GLUT2 mRNA levels were decreased in both HCV RNA replicon cells and HCV J6/JFH1-infected cells. To elucidate molecular mechanisms of HCV-induced suppression of GLUT2 gene expression, we analyzed transcriptional regulation of GLUT2 promoter using a series of GLUT2 promoter-luciferase reporter plasmids. HCV-induced suppression of GLUT2 promoter activity was abrogated when hepatocyte nuclear factor 1α (HNF-1α)-binding motif was deleted from the GLUT2 promoter. HNF-1α mRNA levels were significantly reduced in HCV J6/JFH1-infected cells. Furthermore, HCV infection remarkably decreased the HNF-1α protein levels. We assessed the effects of proteasome inhibitor or lysosomal protease inhibitors on the HCV-induced reduction of HNF-1α protein levels. Treatment of the HCV-infected cells with a lysosomal protease inhibitor, but not with a proteasome inhibitor, restored HNF-1α protein levels, suggesting that HCV infection promotes lysosomal degradation of HNF-1α protein. Overexpression of NS5A protein enhanced lysosomal degradation of HNF-1α protein and suppressed GLUT2 promoter activity. Immunoprecipitation analyses revealed that the domain I of the NS5A protein physically interacts with HNF-1α protein. Taken together, our results suggest that HCV infection suppresses GLUT2 gene expression via down-regulation of HNF-1α expression at transcriptional and posttranslational levels. HCV-induced down-regulation of HNF-1α expression may play a crucial role in glucose metabolic disorders caused by HCV.
Full-text · Article · Sep 2012 · Journal of Virology
[Show abstract][Hide abstract] ABSTRACT: This study explores pretreatment predictive factors for ultimate virological responses to pegylated interferon-α (1.5 μg/kg/week) and ribavirin (600-1000 mg/day) (PEG-IFN/RBV) combination therapy for patients infected with hepatitis C virus (HCV)-1b and a high viral load.
A total of 75 patients underwent PEG-IFN/RBV combination therapy for 48 weeks. HCV amino acid (aa) substitutions in non-structural protein 5a, including those in the IFN/RBV resistance-determining region (IRRDR) and the IFN sensitivity-determining region and the core regions, as well as the genetic variation (rs8099917) near the interleukin 28B (IL28B) gene (genotype TT) were analyzed.
Of the 75 patients, 49 % (37/75) achieved a sustained virological response (SVR), 27 % (20/75) showed relapse, and 24 % (18/75) showed null virological response (NVR). Multivariate logistic regression analysis identified IRRDR with 6 or more mutations (IRRDR ≥6) [odds ratio (OR) 11.906, p < 0.0001] and age <60 years (OR 0.228, p = 0.015) as significant determiners of SVR and IL28B minor (OR 14.618, p = 0.0019) and platelets <15 × 10(4)/mm(3) (OR 0.113, p = 0.0096) as significant determiners of NVR. A combination of IRRDR ≥6 and age <60 years improved SVR predictability (93.3 %), and that of IRRDR ≤5 and age ≥60 years improved non-SVR predictability (84.0 %). Similarly, a combination of IL28B minor and platelets <15 × 10(4)/mm(3) improved NVR predictability (85.7 %), and that of IL28B major and platelets ≥15 × 10(4)/mm(3) improved non-NVR (response) (97.1 %) predictability.
IRRDR ≥6 and age <60 years were significantly associated with SVR. IL28B minor and platelets <15 × 10(4)/mm(3) were significantly associated with NVR. Certain combinations of these factors improved SVR and NVR predictability and could, therefore, be used to design therapeutic strategies.
No preview · Article · Mar 2012 · Journal of Gastroenterology
[Show abstract][Hide abstract] ABSTRACT: Pegylated-interferon plus ribavirin (PEG-IFN/RBV) therapy is a current standard treatment for chronic hepatitis C. We previously reported that the viral sequence heterogeneity of part of NS5A, referred to as the IFN/RBV resistance-determining region (IRRDR), and a mutation at position 70 of the core protein of hepatitis C virus genotype 1b (HCV-1b) are significantly correlated with the outcome of PEG-IFN/RBV treatment. Here, we aimed to investigate the impact of viral genetic variations within the NS5A and core regions of other genotypes, HCV-2a and HCV-2b, on PEG-IFN/RBV treatment outcome. Pretreatment sequences of NS5A and core regions were analyzed in 112 patients infected with HCV-2a or HCV-2b, who were treated with PEG-IFN/RBV for 24 weeks and followed up for another 24 weeks. The results demonstrated that HCV-2a isolates with 4 or more mutations in IRRDR (IRRDR[2a]≥4) was significantly associated with rapid virological response at week 4 (RVR) and sustained virological response (SVR). Also, another region of NS5A that corresponds to part of the IFN sensitivity-determining region (ISDR) plus its carboxy-flanking region, which we referred to as ISDR/+C[2a], was significantly associated with SVR in patients infected with HCV-2a. Multivariate analysis revealed that IRRDR[2a]≥4 was the only independent predictive factor for SVR. As for HCV-2b infection, an N-terminal half of IRRDR having two or more mutations (IRRDR[2b]/N≥2) was significantly associated with RVR, but not with SVR. No significant correlation was observed between core protein polymorphism and PEG-IFN/RBV treatment outcome in HCV-2a or HCV-2b infection. CONCLUSION: The present results suggest that sequence heterogeneity of NS5A of HCV-2a (IRRDR[2a]≥4 and ISDR/+C[2a]), and that of HCV-2b (IRRDR[2b]/N≥2) to a lesser extent, is involved in determining the viral sensitivity to PEG-IFN/RBV therapy.
[Show abstract][Hide abstract] ABSTRACT: The molecular basis of antibody neutralization against hepatitis C virus (HCV) is poorly understood. The E2 glycoprotein of HCV is critically involved in viral infectivity through specific binding to the principal virus receptor component CD81, and is targeted by anti-HCV neutralizing antibodies. A previous study showed that a mutation at position 534 (N534H) within the sixth N-glycosylation motif of E2 of the J6/JFH1 strain of HCV genotype 2a (HCV-2a) was responsible for more efficient access of E2 to CD81 so that the mutant virus could infect the target cells more efficiently. The purpose of this study was to analyze the sensitivity of the parental J6/JFH1, its cell culture-adapted variant P-47 possessing 10 amino acid mutations and recombinant viruses with the adaptive mutations to neutralization by anti-HCV antibodies in sera of HCV-infected patients. The J6/JFH1 virus was neutralized by antibodies in sera of patients infected with HCV-2a and -1b, with mean 50% neutralization titers being 1:670 and 1:200, respectively (P < 0.00001). On the other hand, the P-47 variant showed 50- to 200-times higher sensitivity to antibody neutralization than the parental J6/JFH1 without genotype specificity. The N534H mutation, and another one at position 416 (T416A) near the first N-glycosylation motif to a lesser extent, were shown to be responsible for the enhanced sensitivity to antibody neutralization. The present results suggest that the residues 534, and 416 to a lesser extent, of the E2 glycoprotein are critically involved in the HCV infectivity and antibody neutralization.
No preview · Article · Feb 2012 · Journal of Medical Virology