Yuki Nishimura-Sakurai

Antiviral effects of the interferon‐induced protein guanylate binding protein 1 and its interaction with the hepatitis C virus NS5B protein

Interferons (IFNs) and the interferon‐stimulated genes (ISGs) play a central role in antiviral responses against hepatitis C virus (HCV) infection. We have reported previously that ISGs, including guanylate binding protein 1 (GBP‐1), interferon alpha inducible protein (IFI)‐6‐16, and IFI‐27, inhibit HCV subgenomic replication. In this study we investigated the effects of these ISGs against HCV in cell culture and their direct molecular interaction with viral proteins. HCV replication and virus production were suppressed significantly by overexpression of GBP‐1, IFI‐6‐16, or IFI‐27. Knockdown of the individual ISGs enhanced HCV RNA replication markedly. A two‐hybrid panel of molecular interaction of the ISGs with HCV proteins showed that GBP‐1 bound HCV‐NS5B directly. A protein truncation assay showed that the guanine binding domain of GBP‐1 and the finger domain of NS5B were involved in the interaction. Binding of NS5B with GBP‐1 inhibited its guanosine triphosphatase GTPase activity, which is essential for its antiviral effect. Taken together, interferon‐induced GBP‐1 showed antiviral activity against HCV replication. Conclusion: Binding of the HCV‐NS5B protein to GBP‐1 countered the antiviral effect by inhibition of its GTPase activity. These mechanisms may contribute to resistance to innate, IFN‐mediated antiviral defense and to the clinical persistence of HCV infection. (HEPATOLOGY 2009.)

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)

ITPA gene variant protects against anemia induced by pegylated interferon-α and ribavirin therapy for Japanese patients with chronic hepatitis C.

Aim:  Host genetic variants leading to inosine triphosphatase (ITPA) deficiency, a condition not thought to be clinically important, protect against hemolytic anemia in chronic hepatitis C patients receiving ribavirin. In this study, we evaluated the clinical significance of ITPA variants in Japanese hepatitis C patients who were treated with pegylated interferon plus ribavirin.

Comparison of HCV-associated gene expression and cell signaling pathways in cells with or without HCV replicon and in replicon-cured cells

BackgroundHepatitis C virus (HCV) replication is affected by several host factors. Here, we screened host genes and molecular pathways that are involved in HCV replication by comprehensive analyses using two genotypes of HCV replicon-expressing cells, their cured cells and naïve Huh7 cells.MethodsHuh7 cell lines that stably expressed HCV genotype 1b or 2a replicon were used. The cured cells were established by treating HCV replicon cells with interferon-alpha. Expression of 54,675 cellular genes was analyzed by GeneChip DNA microarray. The data were analyzed by using the KEGG Pathway database.ResultsHierarchical clustering analysis showed that the gene-expression profiles of each cell group constituted clear clusters of naïve, HCV replicon-expressed, and cured cell lines. The pathway process analysis between the replicon-expressing and the cured cell lines identified significantly altered pathways, including MAPK, steroid biosynthesis and TGF-beta signaling pathways, suggesting that these pathways were affected directly by HCV replication. Comparison of cured and naïve Huh7 cells identified pathways, including steroid biosynthesis and sphingolipid metabolism, suggesting that these pathways were required for efficient HCV replication. Cytoplasmic lipid droplets were obviously increased in replicon-expressing and cured cells as compared to naïve cells. HCV replication was significantly suppressed by peroxisome proliferator-activated receptor (PPAR)-alpha agonists but augmented by PPAR-gamma agonists.ConclusionComprehensive gene expression and pathway analyses show that lipid biosynthesis pathways are crucial to support proficient virus replication. These metabolic pathways could constitute novel antiviral targets against HCV.

Inhibition of Hepatitis C Virus Replication by a Specific Inhibitor of Serine-Arginine-Rich Protein Kinase

ABSTRACT Splicing of messenger RNAs is regulated by site-specific binding of members of the serine-arginine-rich (SR) protein family, and SR protein kinases (SRPK) 1 and 2 regulate overall activity of the SR proteins by phosphorylation of their RS domains. We have reported that specifically designed SRPK inhibitors suppressed effectively several DNA and RNA viruses in vitro and in vivo. Here, we show that an SRPK inhibitor, SRPIN340, suppressed in a dose-dependent fashion expression of a hepatitis C virus (HCV) subgenomic replicon and replication of the HCV-JFH1 clone in vitro. The inhibitory effects were not associated with antiproliferative or nonspecific cytotoxic effects on the host cells. Overexpression of SRPK1 or SRPK2 resulted in augmentation of HCV replication, while small interfering RNA (siRNA) knockdown of the SRPKs suppressed HCV replication significantly. Immunocytochemistry showed that SRPKs and the HCV core and NS5A proteins colocalized to some extent in the perinuclear area. Our results demonstrate that SRPKs are host factors essential for HCV replication and that functional inhibitors of these kinases may constitute a new class of antiviral agents against HCV infection.

Two flavonoids extracts from Glycyrrhizae radix inhibit in vitro hepatitis C virus replication.

Aim:  Traditional herbal medicines have been used for several thousand years in China and other Asian countries. In this study we screened herbal drugs and their purified compounds, using the Feo replicon system, to determine their effects on in vitro HCV replication.

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.

Efficacy of additional radiofrequency ablation after transcatheter arterial chemoembolization for intermediate hepatocellular carcinoma

For intermediate hepatocellular carcinoma (HCC), transcatheter arterial chemoembolization (TACE) therapy is recommended in the guidelines as a monotherapy, although TACE is a non‐curative therapy. The aims of the present study were to evaluate the efficacy of adding radiofrequency ablation (RFA) to TACE in patients with intermediate HCC, and to identify the factors that were associated with favorable survival in these patients.

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.