Hepatitis C virus (HCV) is a leading cause of chronic liver disease worldwide and establishes a persistent infection in more than 60% of infected individuals. This high frequency of persistent infection indicates that HCV has evolved efficient strategies to interfere with the adaptive and innate immune response and to occupy and use host cell infrastructure. The present study provides evidence that c-Src, a member of the Src family kinases that participates in many signal transduction pathways, represents an essential host factor exploited for viral replication. c-Src directly interacts with the viral RNA-dependent RNA polymerase (NS5B) via its SH3 domain and with the nonstructural phosphoprotein NS5A via its SH2 domain. Both interactions are required to maintain the protein-protein interaction of NS5A and NS5B, which has been previously demonstrated to be essential for viral replication. Accordingly, HCV genome replication and production of the viral proteins was strongly reduced upon small interfering RNA-mediated knockdown of c-Src or in the presence of the tyrosine kinase inhibitor herbimycin A. This effect could not be rescued by supplementation of the two other ubiquitously expressed Src family kinases Fyn or Yes. Conclusion: Our data suggest that c-Src participates in the formation of an NS5A/NS5B protein complex that is required for efficient replication of HCV. (HEPATOLOGY 2011;53:1127-1136 H epatitis C virus (HCV) is a global health burden and is a major cause for chronic liver disease leading to liver cirrhosis and subsequent complications, such as portal hypertension and hepatocellular carcinoma. 1 For reasons that are not well understood, persistent infection will develop in over 60% of infected individuals. The virus thus must have evolved strategies to subvert the host antiviral defense, to temper the inflammatory response, to prevent the virus-infected cell from undergoing apoptosis, and to use host cell infrastructure without major cytopathogenicity.The 9.6-kb, positive strand RNA of HCV encodes a large open reading frame flanked by highly structured untranslated regions at the 5 0 and 3 0 end. The translation of the open reading frame results in a precursor polyprotein that is co-and posttranslationally processed into three structural proteins and seven nonstructural proteins, termed the hydrophobic peptide p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B, of which the nonstructural proteins NS3 through NS5B are essentially required for autonomous replication. 2 NS3/ 4A is a virus-encoded protease/helicase, whereas NS5A is a phosphoprotein with multiple functions and NS5B is the RNA-dependent RNA polymerase representing Abbreviations: DMSO, dimethyl sulfoxide; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GST, glutathione S-transferase; HCV, hepatitis C virus; Hsp90, heat shock protein 90; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SFK, Src family kinase; siRNA, small interfering RNA.From the
