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O51 THE RIBOSOMAL PROTEIN RACK1 IS REQUIRED FOR IRES-MEDIATED TRANSLATION OF HEPATITIS C VIRUS INDEPENDENTLY OF miR122
ORAL PRESENTATIONS O50 THE SPECIFIC INFECTIVITY OF HEPATITIS C VIRUS IS REGULATED BY THE CATALYTIC ACTIVITY OF THE LIPID PEROXIDE SCAVENGER GLUTATHIONE PEROXIDASE 4 C. Brault1 , P. Levy1 , M. Michelet1 , A. Salle1 , E.-I. Pecheur1 , E. Vericel2 , A.V. Ivanov3 , H.M. Steffen4 , M. Odenthal5 , F. Zoulim1,6 , B. Bartosch1 . 1 Centre de Recherche en Canc´erologie de Lyon, INSERM, U 1052, Team 15, 2 Universit´e de Lyon, UMR 1060 INSERM CarMeN, IMBL, INSA-Lyon, Lyon, France; 3 Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russian Federation; 4 University Hospital, 5 Institute of Pathology, University of Cologne, Cologne, Germany; 6 Hospices Civils de Lyon, Lyon, France E-mail: [email protected] Background and Aims: Chronic infection with Hepatitis C virus (HCV) is associated with chronic inflammation and metabolic disturbances including steatosis and insulin resistance. All these pathologies are intrinsically linked to oxidative stress, which plays a major role in driving disease progression of chronic hepatitis C towards hepatocellular carcinogenesis. A number of reports have shown that HCV causes oxidative stress and concomitantly triggers potent anti-oxidant responses. However, most data have been observed in non-replicative model systems and several contradictory findings have been reported. We aimed to analyze and confirm the origin and type of reactive oxygen species (ROS) induced by HCV and to investigate how the virus counteracts this stress. Methods: ROS production and scavenging was analyzed in the HCVcc infection model. Key data were validated in liver biopsies of HCV and control patients. Results: HCV infection led to an increase in superoxide anion, but a decrease in peroxide levels. This suggests that HCV increases intracellular oxidative stress, but concomitantly activates peroxide scavengers. Amongst the induced ROS-scavenging enzymes, we identified glutathione peroxidase 4 (GPx4) as a host factor required for efficient HCV infection. In contrast to other peroxidases, GPx4 eliminates lipidperoxides present in membranes or lipoproteins. Knock down of GPx4 resulted in the production of HCV virions with a close to 10 fold reduced specific infectivity and strongly reduced fusogenic activity. Conclusions: HCV creates a predominantly oxidative cellular environment and induces the catalytic activity of GPx4. The lipidperoxide scavenging activity of GPX4 is required to maintain high specific infectivity and fusogenic activity of progeny virions. O51 THE RIBOSOMAL PROTEIN RACK1 IS REQUIRED FOR IRES-MEDIATED TRANSLATION OF HEPATITIS C VIRUS INDEPENDENTLY OF miR122 M.L. Hafirassou1,2 , K. Majzoub3 , S. Marzi4 , E. Crouchet1,2 , J.-L. Imler3 , T.F. Baumert1,2,5 , C. Schuster1,2 . 1 U 1110, Inserm, 2 Universit´e de Strasbourg, 3 UPR 9022, IBMC, 4 UPR 9002, IBMC, CNRS, 5 Pole H´epato-Digestif, Hopitaux Universitaires de Strasbourg, Strasbourg, France E-mail: mohamed-lamine.hafi[email protected] Background and Aims: The development of effective antiviral agents is hindered by the limited number and variability of viral targets leading to resistance. The life cycle of viruses is dependent on cellular molecules rendering these attractive alternative targets, provided that they are dispensable for normal cell function. Using a functional proteomic screen, we recently identified the Receptor for Activated C Kinase 1 (RACK1) as a specific host factor important for internal ribosome entry site (IRES)-mediated translation of hepatitis C virus (HCV). Recent studies report that RACK1 is required for the function of micro-RNAs, including miR122 a miRNA playing an important role in HCV translation.
Methods: Using cell culture models of luciferase-based reporter constructs, we investigated whether the contribution of RACK1 to HCV translation was linked to miR122 function. Results: As HEK-293T cells do not express miR122, we generated a HEK-293T cell line that stably over-expressed miR122 (HEK293T-miR122). IRES-mediated translation, but not cap-mediated translation was markedly increased in HEK-293T-miR122 cells compared to HEK-293T cells, indicating that the over-expressed miR122 is effective in promoting IRES-mediated translation in these cells. IRESHCV translation repression by RACK1 silencing occurred at comparable levels in both HEK-293T-miR122 and HEK-293T cells, and was, thus, independent of miR122 expression. Furthermore, silencing the AGO2 protein, which is crucial for miR122 function, did not affect IRESHCV activity, suggesting that RACK1 and miR122 impact on HCV translation by distinct mechanisms. Conclusions: These findings advance our understanding of viral translation and identify RACK1 as a novel and promising target for the development of antivirals addressing resistance. O52 APOLIPOPROTEIN E MEDIATES HCV ESCAPE FROM HOST NEUTRALIZING ANTIBODIES 1,2 D.J. Felmlee1,2 , C. Fauvelle1,2 , M. Le Fevre ´ , L. Heydmann1,2 , M.-S. Hiet3 , R. Bartenschlager3 , I. Fofana1,2 , F. Habersetzer1,2,4 , R. Milne5 , A. Patel6 , K. Vercauteren7 , P. Meuleman7 , M.B. Zeisel1,2 , C. Schuster1,2 , S. Fafi-Kremer1,2,8 , T.F. Baumert1,2,8 . 1 University of Strasbourg, 2 Inserm U 1110, Strasbourg, France; 3 Heidelberg University, Heidelberg, Germany; 4 Pˆ ole H´epato-Digestif, Hˆ opitaux Universitaires de Strasbourg, Strasbourg, France; 5 University of Ottawa Heart Institute, Ottawa, ON, Canada; 6 University of Glasgow Centre for Virus Research, Glasgow, United Kingdom; 7 Ghent University, Ghent University Hospital, Ghent, Belgium; 8 Laboratoire de Virologie, Hˆ opitaux Universitaires de Strasbourg, Strasourg, France E-mail: [email protected] Background and Aims: Vaccine development against hepatitis C virus (HCV) has been hindered by the escape adaptations that enable establishment of chronic infection. A hallmark feature of hepatitis C virus (HCV) is virion association with host lipoproteins to form lipoviral particles, which may contribute to virus-host interactions during viral entry. In this study, we investigate the role of apolipoproteins in viral evasion from B cell responses using acute liver graft infection as a clinical model. Methods: Engineering a variant characterized by immune escape post liver transplantation into the cell culture system (HCVcc), we analyzed escape from heterologous HCV antibodies and used molecular techniques to determine the role of apolipoproteins in immune evasion. Results: We show that HCV utilizes specifically the host factor apolipoprotein E (apoE) to efficiently escape neutralizing antibodies (nAbs) and evade host immune responses. Using functional studies that modulate host apolipoprotein expression, we identify apoE as a key component mediating immune evasion of HCV lipoviral particles. We further identify a residue within the viral envelope glycoprotein E2 that determines apoE-dependency by modulating HCV-apoE interactions, and the extent that apoE antibodies can neutralize infection. Conclusions: Our results identify a functional role of host apoE in viral evasion and persistence, suggesting that this mechanism acts as an adaptive factor shaping HCV evolution during infection. The discovery of apoE-mediated viral evasion provides a novel avenue to improve development of antiviral vaccines and immunopreventive approaches. DJF & CF contributed equally.
Journal of Hepatology 2014 vol. 60 | S1–S22
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Methods: Using cell culture models of luciferase-based reporter constructs, we investigated whether the contribution of RACK1 to HCV translation was linked to miR122 function. Results: As HEK-293T cells do not express miR122, we generated a HEK-293T cell line that stably over-expressed miR122 (HEK293T-miR122). IRES-mediated translation, but not cap-mediated translation was markedly increased in HEK-293T-miR122 cells compared to HEK-293T cells, indicating that the over-expressed miR122 is effective in promoting IRES-mediated translation in these cells. IRESHCV translation repression by RACK1 silencing occurred at comparable levels in both HEK-293T-miR122 and HEK-293T cells, and was, thus, independent of miR122 expression. Furthermore, silencing the AGO2 protein, which is crucial for miR122 function, did not affect IRESHCV activity, suggesting that RACK1 and miR122 impact on HCV translation by distinct mechanisms. Conclusions: These findings advance our understanding of viral translation and identify RACK1 as a novel and promising target for the development of antivirals addressing resistance. O52 APOLIPOPROTEIN E MEDIATES HCV ESCAPE FROM HOST NEUTRALIZING ANTIBODIES 1,2 D.J. Felmlee1,2 , C. Fauvelle1,2 , M. Le Fevre ´ , L. Heydmann1,2 , M.-S. Hiet3 , R. Bartenschlager3 , I. Fofana1,2 , F. Habersetzer1,2,4 , R. Milne5 , A. Patel6 , K. Vercauteren7 , P. Meuleman7 , M.B. Zeisel1,2 , C. Schuster1,2 , S. Fafi-Kremer1,2,8 , T.F. Baumert1,2,8 . 1 University of Strasbourg, 2 Inserm U 1110, Strasbourg, France; 3 Heidelberg University, Heidelberg, Germany; 4 Pˆ ole H´epato-Digestif, Hˆ opitaux Universitaires de Strasbourg, Strasbourg, France; 5 University of Ottawa Heart Institute, Ottawa, ON, Canada; 6 University of Glasgow Centre for Virus Research, Glasgow, United Kingdom; 7 Ghent University, Ghent University Hospital, Ghent, Belgium; 8 Laboratoire de Virologie, Hˆ opitaux Universitaires de Strasbourg, Strasourg, France E-mail: [email protected] Background and Aims: Vaccine development against hepatitis C virus (HCV) has been hindered by the escape adaptations that enable establishment of chronic infection. A hallmark feature of hepatitis C virus (HCV) is virion association with host lipoproteins to form lipoviral particles, which may contribute to virus-host interactions during viral entry. In this study, we investigate the role of apolipoproteins in viral evasion from B cell responses using acute liver graft infection as a clinical model. Methods: Engineering a variant characterized by immune escape post liver transplantation into the cell culture system (HCVcc), we analyzed escape from heterologous HCV antibodies and used molecular techniques to determine the role of apolipoproteins in immune evasion. Results: We show that HCV utilizes specifically the host factor apolipoprotein E (apoE) to efficiently escape neutralizing antibodies (nAbs) and evade host immune responses. Using functional studies that modulate host apolipoprotein expression, we identify apoE as a key component mediating immune evasion of HCV lipoviral particles. We further identify a residue within the viral envelope glycoprotein E2 that determines apoE-dependency by modulating HCV-apoE interactions, and the extent that apoE antibodies can neutralize infection. Conclusions: Our results identify a functional role of host apoE in viral evasion and persistence, suggesting that this mechanism acts as an adaptive factor shaping HCV evolution during infection. The discovery of apoE-mediated viral evasion provides a novel avenue to improve development of antiviral vaccines and immunopreventive approaches. DJF & CF contributed equally.
Journal of Hepatology 2014 vol. 60 | S1–S22
S21