Sayuri Nitta

Hepatitis C virus NS4B protein targets STING and abrogates RIG‐I–mediated type I interferon‐dependent innate immunity

Hepatitis C virus (HCV) infection blocks cellular interferon (IFN)‐mediated antiviral signaling through cleavage of Cardif by HCV‐NS3/4A serine protease. Like NS3/4A, NS4B protein strongly blocks IFN‐β production signaling mediated by retinoic acid–inducible gene I (RIG‐I); however, the underlying molecular mechanisms are not well understood. Recently, the stimulator of interferon genes (STING) was identified as an activator of RIG‐I signaling. STING possesses a structural homology domain with flaviviral NS4B, which suggests a direct protein‐protein interaction. In the present study, we investigated the molecular mechanisms by which NS4B targets RIG‐I–induced and STING‐mediated IFN‐β production signaling. IFN‐β promoter reporter assay showed that IFN‐β promoter activation induced by RIG‐I or Cardif was significantly suppressed by both NS4B and NS3/4A, whereas STING‐induced IFN‐β activation was suppressed by NS4B but not by NS3/4A, suggesting that NS4B had a distinct point of interaction. Immunostaining showed that STING colocalized with NS4B in the endoplasmic reticulum. Immunoprecipitation and bimolecular fluorescence complementation (BiFC) assays demonstrated that NS4B specifically bound STING. Intriguingly, NS4B expression blocked the protein interaction between STING and Cardif, which is required for robust IFN‐β activation. NS4B truncation assays showed that its N terminus, containing the STING homology domain, was necessary for the suppression of IFN‐β promoter activation. NS4B suppressed residual IFN‐β activation by an NS3/4A‐cleaved Cardif (Cardif1‐508), suggesting that NS3/4A and NS4B may cooperate in the blockade of IFN‐β production. Conclusion: NS4B suppresses RIG‐I–mediated IFN‐β production signaling through a direct protein interaction with STING. Disruption of that interaction may restore cellular antiviral responses and may constitute a novel therapeutic strategy for the eradication of HCV. (HEPATOLOGY 2013)

Association of gene expression involving innate immunity and genetic variation in interleukin 28B with antiviral response

Innate immunity plays an important role in host antiviral response to hepatitis C viral (HCV) infection. Recently, single nucleotide polymorphisms (SNPs) of IL28B and host response to peginterferon α (PEG‐IFNα) and ribavirin (RBV) were shown to be strongly associated. We aimed to determine the gene expression involving innate immunity in IL28B genotypes and elucidate its relation to response to antiviral treatment. We genotyped IL28B SNPs (rs8099917 and rs12979860) in 88 chronic hepatitis C patients treated with PEG‐IFNα‐2b/RBV and quantified expressions of viral sensors (RIG‐I, MDA5, and LGP2), adaptor molecule (IPS‐1), related ubiquitin E3‐ligase (RNF125), modulators (ISG15 and USP18), and IL28 (IFNλ). Both IL28B SNPs were 100% identical; 54 patients possessed rs8099917 TT/rs12979860 CC (IL28B major patients) and 34 possessed rs8099917 TG/rs12979860 CT (IL28B minor patients). Hepatic expressions of viral sensors and modulators in IL28B minor patients were significantly up‐regulated compared with that in IL28B major patients (≈3.3‐fold, P < 0.001). However, expression of IPS‐1 was significantly lower in IL28B minor patients (1.2‐fold, P = 0.028). Expressions of viral sensors and modulators were significantly higher in nonvirological responders (NVR) than that in others despite stratification by IL28B genotype (≈2.6‐fold, P < 0.001). Multivariate and ROC analyses indicated that higher RIG‐I and ISG15 expressions and RIG‐I/IPS‐1 expression ratio were independent factors for NVR. IPS‐1 down‐regulation in IL28B minor patients was confirmed by western blotting, and the extent of IPS‐1 protein cleavage was associated with the variable treatment response. Conclusion: Gene expression involving innate immunity is strongly associated with IL28B genotype and response to PEG‐IFNα/RBV. Both IL28B minor allele and higher RIG‐I and ISG15 expressions and RIG‐I/IPS‐1 ratio are independent factors for NVR. (Hepatology 2012)

Analysis of Interferon Signaling by Infectious Hepatitis C Virus Clones with Substitutions of Core Amino Acids 70 and 91

ABSTRACT Substitution of amino acids 70 and 91 in the hepatitis C virus (HCV) core region is a significant predictor of poor responses to peginterferon-plus-ribavirin therapy, while their molecular mechanisms remain unclear. Here we investigated these differences in the response to alpha interferon (IFN) by using HCV cell culture with R70Q, R70H, and L91M substitutions. IFN treatment of cells transfected or infected with the wild type or the mutant HCV clones showed that the R70Q, R70H, and L91M core mutants were significantly more resistant than the wild type. Among HCV-transfected cells, intracellular HCV RNA levels were significantly higher for the core mutants than for the wild type, while HCV RNA in culture supernatant was significantly lower for these mutants than for the wild type. IFN-induced phosphorylation of STAT1 and STAT2 and expression of the interferon-inducible genes were significantly lower for the core mutants than for the wild type, suggesting cellular unresponsiveness to IFN. The expression level of an interferon signal attenuator, SOCS3, was significantly higher for the R70Q, R70H, and L91M mutants than for the wild type. Interleukin 6 (IL-6), which upregulates SOCS3, was significantly higher for the R70Q, R70H, and L91M mutants than for the wild type, suggesting interferon resistance, possibly through IL-6-induced, SOCS3-mediated suppression of interferon signaling. Expression levels of endoplasmic reticulum (ER) stress proteins were significantly higher in cells transfected with a core mutant than in those transfected with the wild type. In conclusion, HCV R70 and L91 core mutants were resistant to interferon in vitro, and the resistance may be induced by IL-6-induced upregulation of SOCS3. Those mechanisms may explain clinical interferon resistance of HCV core mutants.

Effect of interferon-based and -free therapy on early occurrence and recurrence of hepatocellular carcinoma in chronic hepatitis C

BACKGROUND AND AIMS Although treatment for hepatitis C virus has been dramatically improved by the development of direct-acting antiviral agents (DAAs), whether interferon (IFN)-free therapy reduces hepatocarcinogenesis in an equivalent manner to IFN-based therapy remains controversial. The aims of this study were to evaluate the occurrence and recurrence of hepatocellular carcinoma (HCC) in chronic hepatitis C (CHC) patients treated with DAAs and to identify biomarkers of HCC development after antiviral treatment. METHODS A restrospective review of a prospective database of 1,897 CHC patients who were treated with IFN-based (1,145) or IFN-free therapies (752) was carried out. Cumulative HCC occurrence and recurrence rates were compared using propensity score-matched analysis. Predictors of HCC development after viral eradication were identified by multivariate analysis. RESULTS Propensity score-matched analysis showed no significant difference in HCC occurrence (p=0.49) and recurrence rates (p=0.54) between groups treated with IFN-based or IFN-free therapies. In multivariate analysis, higher levels of post-treatment α-fetoprotein (AFP) or Wisteria floribunda agglutinin positive Mac-2 binding protein (WFA+M2BP) were independently associated with HCC occurrence and recurrence after viral eradication. Only post-treatment WFA+M2BP level was significantly associated with HCC occurrence and recurrence among patients without severe fibrosis. The area under the receiver operating characteristic (ROC) curve for WFA+M2BP levels was greater than that for AFP levels in ROC analysis. CONCLUSION The risks of early HCC occurrence and recurrence after viral eradication were similar between IFN-based and IFN-free therapies. Post-treatment levels of WFA+M2BP may be helpful screening biomarkers for assessing the risk of HCC after IFN-free therapy. Patients with high WFA+M2BP levels after antiviral treatment, even without severe fibrosis, must be followed up carefully for HCC development. Lay summary: The risks of early HCC occurrence and recurrence after viral eradication were similar between IFN-based and IFN-free therapies. Post-treatment levels of WFA+M2BP may be helpful screening biomarkers for assessing the risk of HCC after IFN-free therapy.

Wnt5a signaling mediates biliary differentiation of fetal hepatic stem/progenitor cells in mice.

The molecular mechanisms regulating differentiation of fetal hepatic stem/progenitor cells, called hepatoblasts, which play pivotal roles in liver development, remain obscure. Wnt signaling pathways regulate the development and differentiation of stem cells in various organs. Although a β‐catenin–independent noncanonical Wnt pathway is essential for cell adhesion and polarity, the physiological functions of noncanonical Wnt pathways in liver development are unknown. Here we describe a functional role for Wnt5a, a noncanonical Wnt ligand, in the differentiation of mouse hepatoblasts. Wnt5a was expressed in mesenchymal cells and other cells of wild‐type (WT) midgestational fetal liver. We analyzed fetal liver phenotypes in Wnt5a‐deficient mice using a combination of histological and molecular techniques. Expression levels of Sox9 and the number of hepatocyte nuclear factor (HNF)1β+HNF4α− biliary precursor cells were significantly higher in Wnt5a‐deficient liver relative to WT liver. In Wnt5a‐deficient fetal liver, in vivo formation of primitive bile ductal structures was significantly enhanced relative to WT littermates. We also investigated the function of Wnt5a protein and downstream signaling molecules using a three‐dimensional culture system that included primary hepatoblasts or a hepatic progenitor cell line. In vitro differentiation assays showed that Wnt5a retarded the formation of bile duct–like structures in hepatoblasts, leading instead to hepatic maturation of such cells. Whereas Wnt5a signaling increased steady‐state levels of phosphorylated calcium/calmodulin‐dependent protein kinase II (CaMKII) in fetal liver, inhibition of CaMKII activity resulted in the formation of significantly more and larger‐sized bile duct–like structures in vitro compared with those in vehicle‐supplemented controls. Conclusion: Wnt5a‐mediated signaling in fetal hepatic stem/progenitor cells suppresses biliary differentiation. These findings also suggest that activation of CaMKII by Wnt5a signaling suppresses biliary differentiation. (HEPATOLOGY 2013;)

Serum interleukin-6 levels correlate with resistance to treatment of chronic hepatitis C infection with pegylated-interferon-α2b plus ribavirin.

BACKGROUND Interleukin (IL)-6, a pleiotropic cytokine, is increased in various types of chronic liver disease, including chronic hepatitis C (CHC). It was reported recently that IL-6 is associated with insulin resistance, iron metabolism and interferon resistance, which may affect the outcome of antiviral treatment. In this study, we investigated the association of serum IL-6 levels with outcomes of pegylated interferon (PEG-IFN) plus ribavirin (RBV) combination therapy. METHODS We included 149 CHC patients and measured serum IL-6 levels at baseline and at 4, 8 and 12 weeks, and the end of treatment in 49 patients. We performed univariate and multivariate regression analyses for the association of IL-6 levels and clinical and laboratory parameters and treatment responses. RESULTS Serum IL-6 levels were significantly higher in CHC patients than healthy subjects. Pretreatment IL-6 levels of male patients were inversely correlated with sustained virological response (SVR) in univariate analysis (P=0.012). In male patients with SVR, serum IL-6 levels decreased significantly at 4 weeks of treatment (P=0.029) and remained significantly lower than those of non-SVR patients after 4, 8 and 12 weeks of PEG-IFN plus RBV therapy. CONCLUSIONS Our results suggest that baseline levels of IL-6, as well as their decrease during treatment, are correlated to outcomes of PEG-IFN plus RBV therapy in male patients. Further analyses of IL-6 may provide new strategies for difficult-to-treat CHC patients and prevention of hepatocarcinogenesis.

IL-6-mediated intersubgenotypic variation of interferon sensitivity in hepatitis C virus genotype 2a/2b chimeric clones

Mechanisms of difference in interferon sensitivity between hepatitis C virus (HCV) strains have yet to be clarified. Here, we constructed an infectious genotype2b clone and analyzed differences in interferon-alpha sensitivity between HCV-2b and 2a-JFH1 clones using intergenotypic homologous recombination. The HCV-2b/JFH1 chimeric virus able to infect Huh7.5.1 cells and was significantly more sensitive to IFN than JFH1. IFN-induced expression of MxA and 25-OAS was significantly lower in JFH1 than in 2b/JFH1-infected cells. In JFH1-infected cells, expression of SOCS3 and its inducer, IL-6, was significantly higher than in 2b/JFH1-infected cells. The IFN-resistance of JFH1 cells was negated by siRNA-knock down of SOCS3 expression and by pretreatment with anti-IL6 antibody. In conclusion, intergenotypic differences of IFN sensitivity of HCV may be attributable to the sequences of HCV structural proteins and can be determined by SOCS3 and IL-6 expression levels.

ITPA gene variation and ribavirin-induced anemia in patients with genotype 2 chronic hepatitis C treated with sofosbuvir plus ribavirin

Sofosbuvir (SOF) and ribavirin (RBV) combination therapy produces a sustained response in many patients with genotype 2 chronic hepatitis C. However, RBV‐induced anemia is a troublesome side‐effect that may limit this treatment. Genetic variation leading to inosine triphosphatase (ITPA) deficiency is known to protect against RBV‐induced hemolytic anemia. This study aimed to evaluate the relationships between the efficacy and safety of SOF/RBV treatment and ITPA gene variants.

Impaired induction of interleukin 28B and expression of interferon λ 4 associated with nonresponse to interferon‐based therapy in chronic hepatitis C

Interferon (IFN) λ plays an important role in innate immunity to protect against hepatitis C viral (HCV) infection. Single nucleotide polymorphisms (SNPs) near IL28B (IFNλ3) are strongly associated with treatment response to IFNα therapy in chronic hepatitis C (CHC) patients. Recently, IFNλ4 related to IL28B‐unfavorable allele was discovered. However, the impact of IFNλs on CHC is unknown. We aimed to investigate the mechanism underlying responsiveness to IFN‐based therapy in CHC associated with SNPs near IL28B.

Identification of Novel N-(Morpholine-4-Carbonyloxy) Amidine Compounds as Potent Inhibitors against Hepatitis C Virus Replication

ABSTRACT To identify novel compounds that possess antiviral activity against hepatitis C virus (HCV), we screened a library of small molecules with various amounts of structural diversity using an HCV replicon-expressing cell line and performed additional validations using the HCV-JFH1 infectious-virus cell culture. Of 4,004 chemical compounds, we identified 4 novel compounds that suppressed HCV replication with 50% effective concentrations of ranging from 0.36 to 4.81 μM. N′-(Morpholine-4-carbonyloxy)-2-(naphthalen-1-yl) acetimidamide (MCNA) was the most potent and also produced a small synergistic effect when used in combination with alpha interferon. Structure-activity relationship (SAR) analyses revealed 4 derivative compounds with antiviral activity. Further SAR analyses revealed that the N-(morpholine-4-carbonyloxy) amidine moiety was a key structural element for antiviral activity. Treatment of cells with MCNA activated nuclear factor κB and downstream gene expression. In conclusion, N-(morpholine-4-carbonyloxy) amidine and other related morpholine compounds specifically suppressed HCV replication and may have potential as novel chemotherapeutic agents.