Okayama University Medical School Acta Medica Okayama 0386-300X 69 2 2015 Annexin A1 Negatively Regulates Viral RNA Replication of Hepatitis C Virus 71 78 EN Hiroki Hiramoto Hiromichi Dansako Midori Takeda Shinya Satoh Takaji Wakita Masanori Ikeda Nobuyuki Kato Original Article 10.18926/AMO/53335 Persistent infection with hepatitis C virus (HCV) often causes chronic hepatitis, and then shows a high rate of progression to liver cirrhosis and hepatocellular carcinoma. To clarify the mechanism of the persistent HCV infection is considered to be important for the discovery of new target(s) for the development of anti-HCV strategies. In the present study, we found that the expression level of annexin A1 (ANXA1) in human hepatoma cell line Li23-derived D7 cells was remarkably lower than that in parental Li23 cells, whereas the susceptibility of D7 cells to HCV infection was much higher than that of Li23 cells. Therefore, we hypothesized that ANXA1 negatively regulates persistent HCV infection through the inhibition of viral RNA replication. The results revealed that HCV production was significantly inhibited without a concomitant reduction in the amount of lipid droplets in the D7 cells stably expressing exogenous ANXA1. Further, we demonstrated that ANXA1 negatively regulated the step of viral RNA replication rather than that of viral entry in human hepatocytes. These results suggest that ANXA1 would be a novel target for the development of anti-HCV strategies. No potential conflict of interest relevant to this article was reported.

Blackwell Pub. Acta Medica Okayama 1742-464X 2015 The cyclic GMP-AMP synthetase-STING signaling pathway is required for both the innate immune response against HBV and the suppression of HBV assembly EN Hiromichi Dansako Youki Ueda Nobuaki Okumura Shinya Satoh Masaya Sugiyama Masashi Mizokami Masanori Ikeda Nobuyuki Kato During viral replication, the innate immune response is induced through the recognition of viral replication intermediates by host factor(s). One of these host factors, cyclic GMP-AMP synthetase (cGAS), was recently reported to be involved in the recognition of viral DNA derived from DNA viruses. However, it is uncertain whether cGAS is involved in the recognition of hepatitis B virus (HBV), which is a hepatotropic DNA virus. In the present study, we demonstrated that HBV genome-derived dsDNA induced the innate immune response through cGAS and its adaptor protein, STING, in human hepatoma Li23 cells expressing high levels of cGAS. In addition, we demonstrated that HBV infection induced ISG56 through the cGAS-STING signaling pathway. This signaling pathway also showed an antiviral response towards HBV through the suppression of viral assembly. From these results, we conclude that the cGAS-STING signaling pathway is required for not only the innate immune response against HBV but also the suppression of HBV assembly. The cGAS-STING signaling pathway may thus be a novel target for anti-HBV strategies. No potential conflict of interest relevant to this article was reported.

PUBLIC LIBRARY SCIENCE Acta Medica Okayama 1932-6203 9 3 2014 Genetic Characterization of Hepatitis C Virus in Long-Term RNA Replication Using Li23 Cell Culture Systems e91156 EN Nobuyuki Kato Hiroe Sejima Youki Ueda Kyoko Mori Shinya Satoh Hiromichi Dansako Masanori Ikeda Background@ @ The most distinguishing genetic feature of hepatitis C virus (HCV) is its remarkable diversity and variation. To understand this feature, we previously performed genetic analysis of HCV in the long-term culture of human hepatoma HuH-7-derived HCV RNA-replicating cell lines. On the other hand, we newly established HCV RNA-replicating cell lines using human hepatoma Li23 cells, which were distinct from HuH-7 cells. @ Methodology/Principal Findings@ @ Li23-derived HCV RNA-replicating cells were cultured for 4 years. We performed genetic analysis of HCVs recovered from these cells at 0, 2, and 4 years in culture. Most analysis was performed in two separate parts: one part covered from the 5Œ-terminus to NS2, which is mostly nonessential for RNA replication, and the other part covered from NS3 to NS5B, which is essential for RNA replication. Genetic mutations in both regions accumulated in a time-dependent manner, and the mutation rates in the 5Œ-terminus-NS2 and NS3-NS5B regions were 4.0–9.0~10|3 and 2.7–4.0~10|3 base substitutions/site/year, respectively. These results suggest that the variation in the NS3-NS5B regions is affected by the pressure of RNA replication. Several in-frame deletions (3–105 nucleotides) were detected in the structural regions of HCV RNAs obtained from 2-year or 4-year cultured cells. Phylogenetic tree analyses clearly showed that the genetic diversity of HCV was expanded in a time-dependent manner. The GC content of HCV RNA was significantly increased in a time-dependent manner, as previously observed in HuH-7-derived cell systems. This phenomenon was partially due to the alterations in codon usages for codon optimization in human cells. Furthermore, we demonstrated that these long-term cultured cells were useful as a source for the selection of HCV clones showing resistance to anti-HCV agents. @ Conclusions/Significance@ @ Long-term cultured HCV RNA-replicating cells are useful for the analysis of evolutionary dynamics and variations of HCV and for drug-resistance analysis. No potential conflict of interest relevant to this article was reported.

PUBLIC LIBRARY SCIENCE Acta Medica Okayama 1932-6203 8 8 2013 New Preclinical Antimalarial Drugs Potently Inhibit Hepatitis C Virus Genotype 1b RNA Replication e72519 EN Youki Ueda Midori Takeda Kyoko Mori Hiromichi Dansako Takaji Wakita Hye-Sook Kim Akira Sato Yusuke Wataya Masanori Ikeda Nobuyuki Kato BACKGROUND: Persistent hepatitis C virus (HCV) infection causes chronic liver diseases and is a global health problem. Although new triple therapy (pegylated-interferon, ribavirin, and telaprevir/boceprevir) has recently been started and is expected to achieve a sustained virologic response of more than 70% in HCV genotype 1 patients, there are several problems to be resolved, including skin rash/ageusia and advanced anemia. Thus a new type of anti-HCV drug is still needed. @ METHODOLOGY/PRINCIPAL FINDINGS: @ @ Recently developed HCV drug assay systems using HCV-RNA-replicating cells (e.g., HuH-7-derived OR6 and Li23-derived ORL8) were used to evaluate the anti-HCV activity of drug candidates. During the course of the evaluation of anti-HCV candidates, we unexpectedly found that two preclinical antimalarial drugs (N-89 and its derivative N-251) showed potent anti-HCV activities at tens of nanomolar concentrations irrespective of the cell lines and HCV strains of genotype 1b. We confirmed that replication of authentic HCV-RNA was inhibited by these drugs. Interestingly, however, this anti-HCV activity did not work for JFH-1 strain of genotype 2a. We demonstrated that HCV-RNA-replicating cells were cured by treatment with only N-89. A comparative time course assay using N-89 and interferon-ƒΏ demonstrated that N-89-treated ORL8 cells had more rapid anti-HCV kinetics than did interferon-ƒΏ-treated cells. This anti-HCV activity was largely canceled by vitamin E. In combination with interferon-ƒΏ and/or ribavirin, N-89 or N-251 exhibited a synergistic inhibitory effect.@ @ CONCLUSIONS/SIGNIFICANCE:@ @ We found that the preclinical antimalarial drugs N-89 and N-251 exhibited very fast and potent anti-HCV activities using cell-based HCV-RNA-replication assay systems. N-89 and N-251 may be useful as a new type of anti-HCV reagents when used singly or in combination with interferon and/or ribavirin. No potential conflict of interest relevant to this article was reported.

Academic Press Inc Elsevier Science Acta Medica Okayama 0006-291X 430 2 2013 PML tumor suppressor protein is required for HCV production 592 597 EN Misao Kuroki Yasuo Ariumi Makoto Hijikata Masanori Ikeda Hiromichi Dansako Takaji Wakita Kunitada Shimotohno Nobuyuki Kato PML tumor suppressor protein, which forms discrete nuclear structures termed PML-nuclear bodies, has been associated with several cellular functions, including cell proliferation, apoptosis and antiviral defense. Recently, it was reported that the HCV core protein colocalizes with PML in PML-NBs and abrogates the PML function through interaction with PML. However, role(s) of PML in HCV life cycle is unknown. To test whether or not PML affects HCV life cycle, we examined the level of secreted HCV core and the infectivity of HCV in the culture supernatants as well as the level of HCV RNA in HuH-7-derived RSc cells, in which HCV-JFH1 can infect and efficiently replicate, stably expressing short hairpin RNA targeted to PML. In this context, the level of secreted HCV core and the infectivity in the supernatants from PML knockdown cells was remarkably reduced, whereas the level of HCV RNA in the PML knockdown cells was not significantly affected in spite of very effective knockdown of PML. In fact, we showed that PML is unrelated to HCV RNA replication using the subgenomic HCV-JFH1 replicon RNA, JRN/3-5B. Furthermore, the infectivity of HCV-like particle in the culture supernatants was significantly reduced in PML knockdown JRN/3-5B cells expressing core to NS2 coding region of HCV-JFH1 genome using the trans-packaging system. Finally, we also demonstrated that INI1 and DDX5, the PML-related proteins, are involved in HCV production. Taken together, these findings suggest that PML is required for HCV production. No potential conflict of interest relevant to this article was reported.

‰ͺŽRˆγŠw‰ο Acta Medica Okayama 0030-1558 122 1 2010 DNA ‘ΉƒZƒ“ƒT[ATMƒLƒi[ƒ[‚ΖChk2‚Ν‚bŒ^ŠΜ‰ŠƒEƒCƒ‹ƒX‚ΜRNA•‘»‚Ι•K—v‚Ε‚ ‚ι 9 16 EN Yasuo Ariumi Misao Kuroki Hiromichi Dansako Kenichi Abe Masanori Ikeda Takaji Wakita Nobuyuki Kato No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 2004 Differential activation of interferon-inducible genes by hepatitis C virus core protein mediated by the interferon stimulated response element EN Hiromichi Dansako No potential conflict of interest relevant to this article was reported.