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

Abstract

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.

Hepatitis C Virus NS5B Protein Delays S Phase Progression in Human Hepatocyte-derived Cells by Relocalizing Cyclin-dependent Kinase 2-interacting Protein (CINP)

“…8A). The down-regulation of pRb by NS5B has been consistently reported by Lemon and co-workers (19,39,40), and we confirmed this result in HepG2 Tet-On NS5B stable cells (Fig. 8B).…”

“…NS5B induces the redistribution of the RNA helicase p68 from the nucleus to the cytoplasm where it assists in HCV negativestrand RNA synthesis (34). A striking cytoplasmic relocalization of pRb in JFH-1-infected cells and in cells with NS5B present has also been demonstrated (19,39). Together with our results, these studies indicate that the NS5B-induced relocalization of nuclear proteins may be a common strategy used by HCV to aid in persistence and pathogenesis.…”

“…Recently, other studies on a possible mechanism of cell cycle regulation by NS5B have been reported. Studies performed by Lemon and co-workers (19,39) demonstrated that HCV NS5B interacts with and down-regulates Rb through a proteasome-dependent pathway. Rb degradation results in the release of E2F and the transcription of a series of genes responsible for DNA replication, promoting entry into S phase and cell division.…”

Roles of the two distinct proteasome pathways in hepatitis C virus infection

“…The abundance of the retinoblastoma tumor-suppressor protein (pRb) is negatively regulated in HCV RNA replicon cells [61] and HCVcc-infected cells [19] . The HCV RNAdependent RNA polymerase NS5B protein forms a complex with pRb, targeting it for degradation, resulting in a reduction of pRb, the activation of the E2F-responsive promoter, and the promotion of cell proliferation [61] .…”

“…The HCV life cycle is tightly regulated by both viral and cellular proteins [5] and evidence is accumulating to show that the stability of HCV proteins is regulated through both the ubiquitin-dependent and ubiquitin-independent proteasome pathways [14][15][16][17][18] . Moreover, HCV infection has been shown to trigger the degradation of host factors [19] . It is well known that many viruses manipulate the ubiquitin-proteasome pathway to promote their propagation by redirecting the cellular ubiquitin machinery to enable replication, egress and evasion of the host immune system [20] .…”