Yoshi-Hiro Ide

Hepatitis C Virus Infection Promotes Hepatic Gluconeogenesis through an NS5A-Mediated, FoxO1-Dependent Pathway

ABSTRACT Chronic hepatitis C virus (HCV) infection is often associated with type 2 diabetes. However, the precise mechanism underlying this association is still unclear. Here, using Huh-7.5 cells either harboring HCV-1b RNA replicons or infected with HCV-2a, we showed that HCV transcriptionally upregulated the genes for phosphoenolpyruvate carboxykinase (PEPCK) and glucose 6-phosphatase (G6Pase), the rate-limiting enzymes for hepatic gluconeogenesis. In this way, HCV enhanced the cellular production of glucose 6-phosphate (G6P) and glucose. PEPCK and G6Pase gene expressions are controlled by the transcription factor forkhead box O1 (FoxO1). We observed that although neither the mRNA levels nor the protein levels of FoxO1 expression were affected by HCV, the level of phosphorylation of FoxO1 at Ser319 was markedly diminished in HCV-infected cells compared to the control cells, resulting in an increased nuclear accumulation of FoxO1, which is essential for sustaining its transcriptional activity. It was unlikely that the decreased level of FoxO1 phosphorylation was mediated through Akt inactivation, as we observed an increased phosphorylation of Akt at Ser473 in HCV-infected cells compared to control cells. By using specific inhibitors of c-Jun N-terminal kinase (JNK) and reactive oxygen species (ROS), we demonstrated that HCV infection induced JNK activation via increased mitochondrial ROS production, resulting in decreased FoxO1 phosphorylation, FoxO1 nuclear accumulation, and, eventually, increased glucose production. We also found that HCV NS5A mediated increased ROS production and JNK activation, which is directly linked with the FoxO1-dependent increased gluconeogenesis. Taken together, these observations suggest that HCV promotes hepatic gluconeogenesis through an NS5A-mediated, FoxO1-dependent pathway.

17β-estradiol inhibits the production of infectious particles of hepatitis C virus.

Persistent infection with hepatitis C virus causes serious liver diseases, such as chronic hepatitis, hepatic cirrhosis and hepatocellular carcinoma. The male gender is one of the critical factors in progression of hepatic fibrosis due to chronic HCV infection; thus female hormones may play a role in delaying the progression of hepatic fibrosis. It has also been reported that women are more likely than men to clear HCV in the acute phase of infection. These observations lead the present authors to the question: do female hormones inhibit HCV infection? In this study using HCV J6/JFH1 and Huh‐7.5 cells, the possible inhibitory effect(s) of female hormones such as 17β‐estradiol (the most potent physiological estrogen) and progesterone on HCV RNA replication, HCV protein synthesis and production of HCV infectious particles (virions) were analyzed. It was found that E2, but not P4, significantly inhibited production of the HCV virion without inhibiting HCV RNA replication or HCV protein synthesis. E2–mediated inhibition of HCV virion production was abolished by a nuclear estrogen receptor (ER) antagonist ICI182780. Moreover, treatment with the ERα‐selective agonist 4, 4′, 4″‐ (4‐propyl‐[1H]‐pyrazole‐1, 3, 5‐triyl)trisphenol (PPT), but not with the ERβ‐selective agonist 2, 3‐bis (4‐hydroxyphenyl)‐propionitrile (DPN) or the G protein‐coupled receptor 30 (GPR30)‐selective agonist 1‐(4‐[6‐bromobenzo 1, 3 dioxol‐5‐yl]‐3a, 4, 5, 9b‐tetrahydro‐3H‐cyclopenta [c] quinolin‐8‐yl)‐ethanone (G‐1), significantly inhibited HCV virion production. Taken together, the present results suggest that the most potent physiological estrogen, E2, inhibits the production of HCV infectious particles in an ERα–dependent manner.

Polymorphisms of Hepatitis C Virus Non-Structural Protein 5A and Core Protein and Clinical Outcome of Pegylated-Interferon/Ribavirin Combination Therapy

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.

Sequence heterogeneity of NS5A and core proteins of hepatitis C virus and virological responses to pegylated‐interferon/ribavirin combination therapy

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 (Gln70) and non‐SVR (P= 0.02). Notably, Gln70 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.

Sequence Heterogeneity in NS5A of Hepatitis C Virus Genotypes 2a and 2b and Clinical Outcome of Pegylated-Interferon/Ribavirin Therapy

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.

Single-point mutations of the M protein of a measles virus variant obtained from a patient with subacute sclerosing panencephalitis critically affect solubility and subcellular localization of the M protein and cell-free virus production

Subacute sclerosing panencephalitis (SSPE) is caused by variants of wild-type measles virus (MV). Such MV variants lack almost completely the ability to produce cell-free progeny virus. We recently isolated an MV variant that has only three amino acid mutations (L165P,L250P and Y282H) in the M protein compared with MV field isolates of the same genotype. In the present study, we analyzed the significance of these mutations with regard to the characteristics of the M protein and progeny virus production. We found that each of the three mutations rendered the M protein insoluble in 0.5% Triton X-100 and altered its subcellular localization, either when ectopically expressed alone using a plasmid-based expression system or when expressed in the context of viral replication. Moreover, each of the three mutations markedly, but not completely, impaired the ability of MV to produce cell-free progeny virus, with the degree of impairment being the same as for all three mutations together. These results suggest the possibility that the changes in the solubility and subcellular localization of the M protein determine the ability to produce cell-free progeny virus, at least to some extent, and play a role in the pathogenicity of variants causing SSPE.

Prediction of Response to Pegylated Interferon/Ribavirin Combination Therapy for Chronic Hepatitis C Genotypes 2a and 2b and High Viral Load

Objective: We investigated the impact of host genetics represented by the single nucleotide polymorphism (SNP) of the IL28B gene and viral genetic variations within the nonstructural protein 5A (NS5A) [including the interferon (IFN)/ribavirin (RBV) resistance-determining region (IRRDR) and the IFN sensitivity-determining region (ISDR)] on the outcome of pegylated IFN and RBV (PEG-IFN/RBV) treatment. Methods: Sixty-six patients infected with hepatitis C virus (HCV)-2a or HCV-2b who received PEG-IFN/RBV for 24 weeks were examined. Results: In HCV-2a, the major genotype of IL28B SNP showed a tendency toward association with sustained virological response (SVR) and rapid virological response (RVR), and four or more mutations in IRRDR (IRRDR[2a] ≥4) and one or more mutations in ISDR plus its carboxyl-flanking region (ISDR/+C[2a] ≥1) were significantly associated with SVR and RVR. In HCV-2b, one or more mutations in the N-terminal part of IRRDR (IRRDR/N[2b] ≥1) were significantly associated with RVR. Multivariate analysis identified the major genotype of IL28B SNP and IRRDR[2a] ≥4 as independent predictive factors of SVR in HCV-2a, with IRRDR[2a] ≥4 being more powerful than the IL28B SNP. Also, IRRDR[2a] ≥4 in HCV-2a and IRRDR/N[2b] ≥1 in HCV-2b were significant determiners of RVR. Conclusion: The NS5A sequence heterogeneity and IL28B SNP are useful factors to predict the sensitivity to PEG-IFN/RBV therapy in HCV-2a and HCV-2b infections.