Hepatitis C Virus Nonstructural Protein 5B Delays S-phase Progression By Hijacking The Cyclin-dependent Kinase 2-interacting Protein (CINP)

Hepatitis C virus always keep low level replication after infection, However, it is easily develops into a chronic infection, liver cirrhosis and ultimately hepatocellular carcinoma, the molecular mechanisms have thus far remained unclear. Cell cycle dysregulation is a critical event in virus infection-associated tumorigenesis. HCV non-structural protein 5B (NS5B) is an RNA-dependent RNA polymerase (RdRp) and plays an essential role in HCV RNA replication. Recently, there is increasing experimental evidence supporting the possibility that the direct interaction of NS5B with host factors results in a series of cellular activities, such as cell cycle progression, host immune responses and so on. There have also been investigations suggesting that NS5B is involved in cell cycle regulation. However, there is still controversy around this phenomenon and confusion about the details of the mechanism.To determine the effect of NS5B on cell cycle progression in hepatocyte-derived cells, a HepG2 Tet-on NS5B stable cell line was established. The success establishment was confirmed by western blotting with both anti tag antibody and anti endogenous NS5B antibody. Then, this cell line was employed to study the effect of NS5B on cell cycle. The result showed that the induction of NS5B lead to obviously S-phase accumulation.Our previous study indicated that the cellular protein CINP which interact with NS5B is closely associated with cell cycle, suggesting NS5B might affect cell cycle through interacting with CINP. Firstly, GST pull-down and co-IP assay was performed to confirmed the association of NS5B with CINP, Immunofluorescence assay also observed their partially colocalization in the perinuclear region. The further protein truncation assay was performed to map the amino acid sequences required for their binding with each other. And results demonstrated that the C-terminal end of CINP and two NS5B sequences (aa 84-95 and 455-464) are required for their binding. CINP was identified as CDK2-interacting protein from a HeLa cDNA expression library. Previously report showed that CINP is associated with chromosome and involve in the initiation of DNA replication. To determine whether CINP has the function to regulate cell cycle progression, HeLa cells were synchronized with thymidine (TdR) in the G1/S transition phase and then transfection with CINP or related empty vectors. Following release, cells transfected with empty vectors were able to resume a normal cell cycle progression. However, cells transfected with CINP were still in G1/S phase. Knockdown CINP with specific siRNA resulted in an obvious S-phase accumulation, which indicates the importance of CINP in host cell cycle regulation, further results came from the NS5B deletion mutant (96-454,?NS5B) that failed to bind CINP has no effect on cell cycle and cell proliferation suggested that the CINP/NS5B association is required for cell cycle delay.CINP has been found to localize to the nucleus, whereas NS5B primarily localizes to the perinuclear region. Our above data showed a perinuclear colocalization of NS5B with CINP, which suggests that NS5B caused the relocalization of CINP from the nucleus to the cytoplasm. To test this hypothesis, GFP-CINP was co-transfected with empty vector, myc-NS5B or myc-?NS5B into Huh7 cells, and cells were analyzed by confocal microscopy after immunological staining with an anti-myc antibody. Strong merged signals were observed in the perinuclear regions in GFP-CINP and full-length NS5B co-transfected cells. However, most CINP remained localized to the nucleus in empty vector- and?NS5B-transfected cells. The HCV subgenomic replicon and JFH-1-infected cells exhibit the similar results. The relocalization of CINP by NS5B was also confirmed by the next extraction of a subcellular fractionation assay.The DNA damage response (DDR) is usually employed by viruses or virus-encoded proteins to modulate the cell cycle to promote their own replication and oncogenic effects. After DNA damage, three cell cycle checkpoints are activated:1) The G1/S checkpoint prevents cells from entering S-phase; 2) The intra-S-phase checkpoint inhibits DNA replication; and 3) The G2/M checkpoint prevents damaged DNA from undergoing mitosis. Recent study identified CINP as a checkpoint protein to maintain genomic stability, thus shedding light on the function of CINP in genomic maintenance and cell cycle regulation. Because CINP translocation by NS5B reduced the amount of CINP available for normal cellular regulation in the nucleus, there might be an alteration of signaling pathways that involve CINP. Directly knocking down CINP by specific siRNA resulted in a significant alteration of DNA damage response and cell cycle checkpoint proteins, such as an increase in p21, a decrease in pRb and pChkl. Similar results were also observed in cells with NS5B present and could be partially restored by the ectopic overexpression of CINP. And after knocking down Chkl with specific siRNA could also lead to S-phase accumulation, which suggested that the DNA damage response might be exploited by NS5B to modify cell cycle progression.Taken together, our data demonstrate that NS5B delays S-phase progression through interacting and relocalizing CINP from the nucleus to the cytoplasm, which provides new insights into understanding persistence and tumorigenesis after HCV infection.