The retinoblastoma tumor-suppressor protein (Rb) plays a critical role in controlling cellular proliferation and apoptosis by regulating E2F transcription factors. Rb is a key target of oncoproteins expressed by DNA tumor viruses, but RNA viruses are not known to regulate Rb function. Here, we show that Rb abundance is negatively regulated in cells containing replicating genomic RNA from hepatitis C virus, a human virus strongly associated with hepatocellular carcinoma. The viral RNA-dependent RNA polymerase NS5B forms a complex with Rb, targeting it for degradation and resulting in reduction of Rb abundance, activation of E2F-responsive promoters, and cell proliferation. NS5B contains a conserved Leu-x-Cys͞Asn-x-Asp motif that is homologous to Rb-binding domains in the oncoproteins of DNA viruses. This domain overlaps the polymerase active site, and mutations within it abrogate Rb binding and reverse the effects of NS5B on E2F promoter activation and cell proliferation. These findings suggest a unique link between an oncogenic RNA virus implicated in the development of liver cancer and a critically important tumor-suppressor protein.cell cycle regulation ͉ chronic hepatitis ͉ E2F transcription factor ͉ hepatocellular carcinoma ͉ viral oncogenesis H epatitis C virus (HCV) infection is a leading cause of morbidity and mortality in many human populations (1). Persons with persistent HCV infection are at increased risk of developing cirrhosis and hepatocellular carcinoma (2). The strong association between hepatocellular carcinoma and HCV infection is particularly notable in that HCV is a positive-strand RNA virus, classified within the genus Hepacivirus of the family Flaviviridae. Its 9.6-kb genome replicates exclusively within the cytoplasm (3) and encodes a single, large polyprotein that is processed by cellular and viral proteases into only 10 individual structural and nonstructural viral proteins. Although inflammation associated with chronic hepatitis C is likely to contribute to the development of hepatocellular carcinoma, there is evidence that one or more of the proteins expressed by the virus contribute directly to carcinogenesis. Transgenic mice expressing a high abundance of the core protein develop hepatocellular carcinoma and steatosis (4). 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 (5). Such data suggest a direct role for both structural and nonstructural proteins of HCV in oncogenesis.Mechanisms that regulate cell-cycle progression are frequently disrupted in hepatocellular carcinomas associated with HCV infection. These regulatory mechanisms include the retinoblastoma protein (Rb) (6), which plays a major role in controlling the G1 to S-phase transition through a repressive effect on E2F transcription factors (7). Rb functions as a tumor suppressor, and the gene which encodes it (RB) is frequently mutated...

Background: Structural changes in chromatin play essential roles in regulating eukaryotic gene expression. Silencing, potent repression of transcription in Saccharomyces cerevisiae, occurs near telomeres and at the silent mating-type loci, as well as at rDNA loci. This type of repression relates to the condensation of chromatin that occurs in the heterochromatin of multicellular organisms. Anti-silencing is a reaction by which silenced loci are de-repressed. Genetic studies revealed that several factors participate in the anti-silencing reaction. However, actions of factors and molecular mechanisms underlying anti-silencing remain unknown.

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.

Adsorbed structures of naphthalene on Cu(111) have been studied using low temperature scanning tunneling microscope (LT-STM) and low energy electron diffraction (LEED). Starting from single molecules, three kinds of long-range ordered superstructures, ( 5 3 × 5 3)R30°, (2 3 × 3)rect-1C 10 H 8 , and ( -4 1 1 -4 ) are observed depending on the molecular concentrations and the substrate temperatures during molecular adsorption.One of the self-assembled ordered phases with a (5 3 × 5 3) R30°periodicity is chiral in adsorptioninduced arrangement though a single naphthalene molecule itself has no inherent chirality. In STM images, isolated single molecules appear as depressions whereas the molecules are seen as protrusions in self-assembled layers. Coverage dependent two-photon photoemission (2PPE) spectra show that the adsorption-induced occupied states is formed at around Cu 3d bands, and this results in the enhanced tunneling of occupied state images in assembled layers.