Hepatitis C Virus Induces E6AP-Dependent Degradation of the Retinoblastoma Protein

Tsubasa Munakata is with the University of Texas Medical Branch and University of Tokyo, Yuqiong Liang is with the University of Texas Medical Branch, Seungtaek Kim is with the University of Texas Medical Branch, David R McGivern is with the University of Texas Medical Branch, Jon Huibregtse is with UT Austin, Akio Nomoto is with University of Tokyo, Stanley M Lemon is with the University of Texas Medical Branch.Hepatitis C virus (HCV) is a positive-strand RNA virus that frequently causes persistent infections and is uniquely associated with the development of hepatocellular carcinoma. While the mechanism(s) by which the virus promotes cancer are poorly defined, previous studies indicate that the HCV RNA-dependent RNA polymerase, nonstructural protein 5B (NS5B), forms a complex with the retinoblastoma tumor suppressor protein (pRb), targeting it for degradation, activating E2F-responsive promoters, and stimulating cellular proliferation. Here, we describe the mechanism underlying pRb regulation by HCV and its relevance to HCV infection. We show that the abundance of pRb is strongly downregulated, and its normal nuclear localization altered to include a major cytoplasmic component, following infection of cultured hepatoma cells with either genotype 1a or 2a HCV. We further demonstrate that this is due to NS5B-dependent ubiquitination of pRb and its subsequent degradation via the proteasome. The NS5B-dependent ubiquitination of pRb requires the ubiquitin ligase activity of E6-associated protein (E6AP), as pRb abundance was restored by siRNA knockdown of E6AP or overexpression of a dominant-negative E6AP mutant in cells containing HCV RNA replicons. E6AP also forms a complex with pRb in an NS5B-dependent manner. These findings suggest a novel mechanism for the regulation of pRb in which the HCV NS5B protein traps pRb in the cytoplasm, and subsequently recruits E6AP to this complex in a process that leads to the ubiquitination of pRb. The disruption of pRb/E2F regulatory pathways in cells infected with HCV is likely to promote hepatocellular proliferation and chromosomal instability, factors important for the development of liver cancer.This work was supported by grants from the National Institute of Allergy and Infectious Diseases, U19-AI40035 (to SML); the Program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation of Japan (to TM and AN); the Special Coordination Funds for Promoting Science and Technology from The Ministry of Education, Culture, Sports, Science, and Technology of Japan (to AN); and by a grant-in-aid for Scientific Research in Priority Areas from The Ministry of Education, Culture, Sports, Science, and Technology of Japan (to AN).Cellular and Molecular Biolog