659 HCV NS5A PROTEIN BEARS TRANSACTIVATION PROPERTIES THAT CORRELATE WITH HCV REPLICATION CAPACITY IN VITRO

659 HCV NS5A PROTEIN BEARS TRANSACTIVATION PROPERTIES THAT CORRELATE WITH HCV REPLICATION CAPACITY IN VITRO

POSTERS histological feature of these subjects. Metabolic cofactors and direct viral activities on intracellular lipid pathways are only a part of the...

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POSTERS histological feature of these subjects. Metabolic cofactors and direct viral activities on intracellular lipid pathways are only a part of the several mechanisms implicated in the pathogenesis of hepatic steatosis. A plethora of studies suggest a direct role of HCV in inducing this pathology. Oxysterols, peroxidation products of cholesterol, are considered reliable markers of oxidative stress (OS) in vivo, and are potentially involved in the evolution of liver damage. These molecules are able to bind and activate Liver X Receptor (LXR), a well-known regulator of cholesterol and lipid metabolism, modulating the expression of several involved proteins (SREBP1c, FAS, ACC1). OS is a common feature of HCV infection, thus we investigated the possible link between OS, steatosis and HCV-related chronic hepatitis in 90 patients. Methods: Estimation of oxysterols was performed through Gas-Chromatography/Mass-Spectrometry technique in the culture media of HUH7 HCV-JFH1 infected cells, constitutively expressing the entire genome of HCV, and in plasma samples collected from 90 patients. HBV- and NAFLD-related hepatitis has been considered as controls. At the same time we performed comparative RealTime PCR for ACC1, SREBP1c and FAS mRNAs quantitation in HUH7 HCV-JFH1 infected cells. Results: Plasma concentration of oxysterols (7b-hydroxycholesterol and 7-ketocholesterol) resulted statistically higher in HCV patients than in the control groups. In order to clarify the mechanism by which the virus interferes with oxysterols metabolism we analyzed their levels in culture medium of infected cells. As well as in patients, HCV enhances oxysterols’ levels in cell model. Using Real Time PCR, we observed that the expression of HCV viral proteins up-regulates, at least of two fold, the expression levels of LXRdependent ACC1, FAS, SREBP1c genes, involved in lipid metabolism. LXR activation in infected cells by chromatin immunoprecipitation assays is under investigation. Conclusions: These data suggest a key role for HCV in modulating the expression of LXR-depending genes involved in lipid metabolism through two different mechanisms: directly by viral proteins and indirectly by oxysterols, playing in this way an important role in inducing metabolic syndrome and hepatic steatosis. 659 HCV NS5A PROTEIN BEARS TRANSACTIVATION PROPERTIES THAT CORRELATE WITH HCV REPLICATION CAPACITY IN VITRO M.A. Maqbool, H. Lerat, J.M. Pawlotsky. Department of Virology, INSERM U955 (EQ.18) Institut Mondor de Recherche Biom´edicale, Hˆ opital Henri Mondor, Universit´e Paris 12, Creteil, France E-mail: [email protected] Background and Aims: Hepatitis C virus (HCV) non-structural protein 5A (NS5A) regulates HCV replication through its interaction with the HCV RNA-dependent RNA polymerase and alters infected cell metabolism through complex interactions with host cell proteins. Among these properties, NS5A has been shown to act as a transcriptional activator, potentially after cleavage and transport to the nucleus by means of a nuclear localization signal (NLS). Here, we demonstrate for the first time that NS5A transactivation capacity correlates with HCV replication capacity in vitro. Methods and Results: 21 full-length NS5A quasispecies variants and the corresponding transcriptional activation domains were isolated and amplified from tumoral and adjacent non-tumoral hepatic tissues from an HCV-infected patient. Each of them was cloned into a yeast expression vector to produce a Gal4 fusion protein that regulates the transcription of the lacZ gene within yeast strain Y187. Five full-length NS5A clones with different transcriptional activities were chosen according to their ability to induce lacZ transcription and cloned into a bicistronic HCV sub-genomic replicon expressing luciferase. In this model, there was a strong correlation between NS5A variant ability to activate transcription and the replication capacity of the

corresponding sub-genomic replicon. Acid-rich domain 2 (AR2) of NS5A played an important role in the level of NS5A transactivation. Swapping the AR2 domains of two NS5A variants with strong and weak transactivation potentials, respectively, similarly altered the replication capacity of the corresponding replicons in Huh7 cells. Mutation of the NLS of NS5A decreased the replication capacity of the replicon, suggesting that translocation of NS5A to the nucleus is at least in part required for the effect of NS5A on HCV replication. Conclusions: These results demonstrate that HCV NS5A protein transactivation properties and HCV replication capacity correlate in vitro. This effect appears to be mediated by NS5A acid-rich domain 2. The role of NS5A nuclear localization is in keeping with the involvement of NS5A transactivation properties. 660 MTOR ACTIVITY THAT INDUCED BY INSULIN HAS ANTI-HEPATITIS C VIRUS ACTIVITY T. Muraoka, T. Ichikawa, N. Taura, H. Miyaaki, S. Takeshita, M. Akiyama, S. Miuma, E. Ozawa, T. Matsuzaki, M. Otani, F. Takeshima, K. Nakao. Gastroenterology and Hepatology, Nagasaki University Hospital, Nagasaki, Japan E-mail: [email protected] Background and Aims: The mammalian target of rapamycin (mTOR) is one of the influential molecules for the interferon (IFN) induced anti-hepatitis C virus (HCV) signal. The IFN induced mTOR activity, independent of PI3K and Akt, is the critical factor for its anti-HCV activity and Jak independent mTOR activity involved STAT-1 phosphorylation and nuclear location, and then PKR is expressed in hepatocytes (T.Ichikawa, et al. Journal of Gastroenterology, 2009; 44:856–863). Insulin (INS) is a major cytokine for metabolism and has the PI3-K-Akt-mTOR signal pathway in hepatocytes. An alternation of mTOR activity was reported in chronically HCV infected patients with over nutrition and insulin resistance. Therefore, this experiment investigated whether INS raises the HCV-activity via mTOR activity. Methods: This study used a genome-length HCV RNA (strain O of genotype 1b) replicon reporter system (OR6), which is an effective screening tool. OR6 cells were incubated in IFN or INS with or without pretreatment with rapamycin (Rapa) or LY294002, and then the cells were lysed and used for Western blot or ELISA or Rennila luciferase activity assay. Result: The INS induced anti-HCV mTOR activity, through independent STAT-1 tyrosine phosphorylation, in a dose and time dependent manner. INS induced mTOR activation was PI3-K-mTOR dependent in OR6. Interestingly, INS induced anti-HCV activity was not associated with the induction of the IFN induced anti-viral protein, PKR. The combination with IFN and INS has an additive anti-HCV effect. INS induced anti-HCV activity was not associated with the IFN induced anti-virus protein. Conclusion: The INS induced mTOR activity was identified to be an anti-HCV signal independent STAT pathway in this study. The mTOR activity may be associated with the HCV life cycle. Future studies should therefore attempt to identify further agents that activate mTOR for anti-HCV activity. Acknowledgements: We thank M. Ikeda and N. Kato (department of molecular biology, Okayama University graduate school of medicine and dentistry) for OR6 cells.

Journal of Hepatology 2010 vol. 52 | S183–S317

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