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

et al. (2007) Hepatitis C virus induces E6AP-dependent degradation of the retinoblastoma protein. PLoS Pathog 3(9): e139. doi:10.1371/journal.ppat.0030139 Hepatitis C Virus Induces E6AP-Dependent Degradation of the Retinoblastoma Protein Tsubasa Munakata 0 1 Yuqiong Liang 0 1 Seungtaek Kim 0 1 David R. McGivern 0 1 Jon Huibregtse 0 1 Akio Nomoto 0 1 Stanley M. Lemon 0 1 0 Editor: John A. T. Young, The Salk Institute for Biological Studies , United States of America 1 1 Center for Hepatitis Research, University of Texas Medical Branch , Galveston, Texas , United States of America, 2 Department of Microbiology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan, 3 Department of Microbiology and Immunology, University of Texas Medical Branch , Galveston, Texas , United States of America, 4 Department of Molecular Genetics and Microbiology, Institute for Cellular and Molecular Biology, University of Texas Austin , Austin, Texas , United States of America, 5 Sealy Center for Cancer Cell Biology, University of Texas Medical Branch , Galveston, Texas , United States of America 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 NS5Bdependent 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. - Among viruses that infect the human liver, hepatitis C virus (HCV) is a leading cause of morbidity and mortality worldwide [1]. Chronic infection with HCV is a major risk factor for the development of cirrhosis as well as hepatocellular carcinoma (HCC) [2,3]. The incidence of this cancer has increased dramatically in recent years in Japan and the United States, reflecting prior increases in the prevalence of HCV infection, and in Japan HCV has replaced hepatitis B virus as the leading infectious cause of liver cancer. The strong association between HCC and HCV infection is particularly notable in that HCV is a positive-strand RNA virus, classified within the genus Hepacivirus of the family Flaviviridae [4]. Its 9.6-kb genome replicates in association with membranes within the cytoplasm of infected cells, and encodes a single polyprotein that is processed by both cellular and viral proteases into ten individual structural and nonstructural viral proteins. Although inflammation associated with chronic hepatitis C is likely to contribute to the development of HCC, there is strong evidence that one or more of the proteins expressed by the virus contribute directly to carcinogenesis. The HCV core protein, a component of the putative viral nucleocapsid, has been shown to modulate the hepatocyte cell cycle [5,6]. Other studies suggest that expression of the nonstructural (NS) proteins, NS3 (a serine proteinase/helicase), NS5A (a replicase-associated phosphoprotein of uncertain function), or NS5B (the viral RNA-dependent RNA polymerase) may also affect control of cellular proliferation [710]. Moreover, transgenic mice expressing a high abundance of the core protein develop steatosis and HCC [11]. Liver cancer also developed in transgenic mice expressing a much lower abundance of the entire viral polyprotein, but not in a companion transgenic lineage expressing a higher abundance of the structural proteins (core, E1, E2, and p7) only [12]. None of these transgenic mouse lineages had demonstrable hepatic inflammation in advance of the development of HCC. Together, these data suggest a direct role for both structural and nonstructural HCV proteins in oncogenesis. Persons infected with hepatitis C virus (HCV) are at increased risk for liver cancer. This is remarkable because HCV is an RNA virus with replication confined to the cytoplasm and no potential for integration of its genome into host cell DNA. While it is likely that chronic inflammation contributes to liver cancer, prior studies with HCV transgenic mice indicate that the viral proteins are intrinsically carcinogenic. In this study, we have examined the interaction of one of these, the RNA-dependent RNA polymerase nonstructural protein 5B, with an important cellular tumor suppressor protein, the retinoblastoma protein (pRb). pRb is a master regulator of the cell cycle, and altered expression of some of the many genes it regulates may lead to cancer. We show that the abundance of pRb is strongly downregulated in cells infected with HCV, and that nonstructural protein 5B targets pRb for destruction via the cells normal protein degradation machinery. The E6-associated protein appears to play a role in this process, which is interesting as it also mediates the degradation of another tumor suppressor, p53, by papillomaviruses. The loss of pRb function in HCV-infected cells likely promotes hepatocellular proliferation as well chromosomal instability, factors important for the development of liver cancer. At least four different pathways that regulate either cell proliferation or cell death, the retinoblastoma (pRb)/E2F, p53, transforming growth factor-b (TGF-b), and b-catenin pathways, are commonly altered in HCCs [2]. Among them, pRb plays a major role in controlling the G1- to S-phase transition and mitotic checkpoints through a repressive effect on E2F transcription factors [13]. pRb functions as a tumor suppressor, and the gene which encodes it (RB) is frequently mutated in various types of tumors, including retinoblastomas, small-cell lung carcinomas, and osteosarcomas [14]. In previously published studies, we d (...truncated)