RESUMO
Cell cycle dysregulation is a critical event in virus infection-associated tumorigenesis. Previous studies have suggested that hepatitis C virus NS5B modulates cell cycle progression in addition to participating in RNA synthesis as an RNA-dependent RNA polymerase. However, the molecular mechanisms have thus far remained unclear. In this study, a HepG2 Tet-On NS5B stable cell line was generated to confirm the effect of NS5B on the cell cycle. To better understand the role of NS5B in cell cycle regulation, yeast two-hybrid assays were performed using a human liver cDNA library. The cyclin-dependent kinase 2-interacting protein (CINP) was identified. The interaction between NS5B and CINP was further demonstrated by in vivo and in vitro assays, and their association was found to be indispensable for S phase delay and cell proliferation suppression. Further experiments indicated that NS5B relocalized CINP from the nucleus to the cytoplasm. Directly knocking down CINP by specific siRNA resulted in a significant alteration in the DNA damage response and expression of cell cycle checkpoint proteins, including an increase in p21 and a decrease in phosphorylated Retinoblastoma and Chk1. Similar results were observed in cells expressing NS5B, and the effects were partially reversed upon ectopic overexpression of CINP. These studies suggest that the DNA damage response might be exploited by NS5B to hinder cell cycle progression. Taken together, our data demonstrate that NS5B delays cells in S phase through interaction with CINP and relocalization of the protein from the nucleus to the cytoplasm. Such effects might contribute to hepatitis C virus persistence and pathogenesis.
Assuntos
Proteínas de Transporte/metabolismo , Núcleo Celular/metabolismo , Hepacivirus/metabolismo , Hepatócitos/metabolismo , Fase S , Proteínas não Estruturais Virais/metabolismo , Transporte Ativo do Núcleo Celular/genética , Proteínas de Transporte/genética , Núcleo Celular/genética , Quinase 1 do Ponto de Checagem , Citoplasma/genética , Citoplasma/metabolismo , Dano ao DNA/genética , Células HeLa , Células Hep G2 , Hepacivirus/genética , Hepatite C/genética , Hepatite C/metabolismo , Humanos , Fosforilação/genética , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteína do Retinoblastoma/genética , Proteína do Retinoblastoma/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Proteínas não Estruturais Virais/genéticaRESUMO
Autophagy is a conserved eukaryotic mechanism that mediates the removal of long-lived cytoplasmic macromolecules and damaged organelles via a lysosomal degradative pathway. Recently, a multitude of studies have reported that viral infections may have complex interconnections with the autophagic process. The findings reported here demonstrate that hepatitis B virus (HBV) can enhance the autophagic process in hepatoma cells without promoting protein degradation by the lysosome. Mutation analysis showed that HBV small surface protein (SHBs) was required for HBV to induce autophagy. The overexpression of SHBs was sufficient to induce autophagy. Furthermore, SHBs could trigger unfolded protein responses (UPR), and the blockage of UPR signaling pathways abrogated the SHB-induced lipidation of LC3-I. Meanwhile, the role of the autophagosome in HBV replication was examined. The inhibition of autophagosome formation by the autophagy inhibitor 3-methyladenine (3-MA) or small interfering RNA duplexes targeting the genes critical for autophagosome formation (Beclin1 and ATG5 genes) markedly inhibited HBV production, and the induction of autophagy by rapamycin or starvation greatly contributed to HBV production. Furthermore, evidence was provided to suggest that the autophagy machinery was required for HBV envelopment but not for the efficiency of HBV release. Finally, SHBs partially colocalized and interacted with autophagy protein LC3. Taken together, these results suggest that the host's autophagy machinery is activated during HBV infection to enhance HBV replication.
Assuntos
Autofagia/fisiologia , Antígenos de Superfície da Hepatite B/metabolismo , Vírus da Hepatite B/fisiologia , Hepatócitos/virologia , Interações Hospedeiro-Patógeno , Linhagem Celular Tumoral , Antígenos de Superfície da Hepatite B/genética , Vírus da Hepatite B/genética , Vírus da Hepatite B/metabolismo , Humanos , Imunoprecipitação , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação , Fagossomos/metabolismoRESUMO
It is obvious that the albumin is a kind of important large biophysical molecular. The albumin is the main component of plasma within cell. It is also the matter basis of life phenomenon. The fluorescence spectroscopy of human serum albumin (HSA) and the interaction of HSA and the rifampicin capsules (lfp) were studied. When the rifampicin capsules (lfp) was added into HSA solution gradually, an interesting new phenomenon emerged in emission spectrum. The combination constant of rifampicin capsules (lfp) is about Ks = 5.149 x 10(4) L x mol(-1), and the dissociation constant is about Kd = 19.42 x 10(-6) mol x L(-1). The quenching process of rifampicin capsules (lfp)-HSA is not dynamic quenching, which resulted from the molecular diffusion and collision. That is the static quenching process resulting from the chemical component between molecules. The energy transfer efficiency between rifampicin capsules (lfp) and HSA is E=0.42. According to these calculation results, the combination position between the binding site of rifampicin capsules (lfp) and the tryptophane of HSA is about R=2.567 nm. The critical distance, when transfer efficiency equals 50%, is about R0 = 2.433 nm.