LuoJVirol2006 - J OURNAL OF VIROLOGY, Aug. 2006, p....

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Unformatted text preview: J OURNAL OF VIROLOGY, Aug. 2006, p. 7364–7374 Vol. 80, No. 15 0022-538X/06/$08.00 1 doi:10.1128/JVI.00586-06 Copyright © 2006, American Society for Microbiology. All Rights Reserved. Replication of Hepatitis C Virus (HCV) RNA in Mouse Embryonic Fibroblasts: Protein Kinase R (PKR)-Dependent and PKR- Independent Mechanisms for Controlling HCV RNA Replication and Mediating Interferon Activities Kyung-Soo Chang, 1 Zhaohui Cai, 1 Chen Zhang, 1 Ganes C. Sen, 2 Bryan R. G. Williams, 2,3 and Guangxiang Luo 1 * Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky College of Medicine, 800 Rose Street, Lexington, Kentucky 40536-0298, 1 ; and Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195 2 ; and Monash Institute of Medical Research, Monash University, Melbourne, Australia 3 Received 23 March 2006/Accepted 9 May 2006 Hepatitis C virus (HCV) infection causes chronic hepatitis and is currently treated with alpha interferon (IFN- a )-based therapies. The underlying mechanisms of chronic HCV infection and IFN-based therapies, however, have not been defined. Protein kinase R (PKR) was implicated in the control of HCV replication and mediation of IFN-induced antiviral response. In this report, we demonstrate that a subgenomic RNA replicon of genotype 2a HCV replicated efficiently in mouse embryonic fibroblasts (MEFs), as determined by cell colony formation efficiency and the detection of HCV proteins and both positive- and negative-strand RNAs. Addi- tionally, the subgenomic HCV RNA was found to replicate more efficiently in the PKR knockout (PKR 2 / 2 ) MEF than in the wild-type (PKR 1 / 1 ) MEF. The knockdown expression of PKR by specific small interfering RNAs significantly enhanced the level of HCV RNA replication, suggesting that PKR is involved in the control of HCV RNA replication. The level of ISG56 (p56) was induced by HCV RNA replication, indicating the activation of PKR-independent antiviral pathways. Furthermore, IFN- a / b inhibited HCV RNA replication in PKR 2 / 2 MEFs as efficiently as in PKR 1 / 1 MEFs. These findings demonstrate that PKR-independent antiviral pathways play important roles in controlling HCV replication and mediating IFN-induced antiviral effect. Our findings also provide a foundation for the development of transgenic mouse models of HCV replication and set a stage to further define the roles of cellular genes in the establishment of chronic HCV infection and the mediation of intracellular innate antiviral response by using MEFs derived from diverse gene knockout animals. Hepatitis C virus (HCV) is a member of the Flaviviridae family, with a single-stranded and positive-sense RNA genome of approximately 9.6 kb in length (12, 51). The viral RNA genome contains a single open reading frame flanked by un- translated regions (UTR) at both the 5 9 and 3 9 ends. A large viral polyprotein precursor of about 3,010 amino acids is trans- lated and is subsequently cleaved by both cellular and viral...
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LuoJVirol2006 - J OURNAL OF VIROLOGY, Aug. 2006, p....

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