CINP Regulate HCV Replication Via Accommodating ATM/ATR Pathway

Hepatitis C virus (HCV) infection is a major cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma (HCC) worldwide. With approximately 3% of the world’s population infected (estimates vary from 130 to 170 million individuals), HCV infection is a major global health challenge. Although much research has been focused on the development of anti-HCV agents, no vaccine is available to date and there is no effective therapy for all genotypes of HCV. HCV is a member of the Hepacivirus family and has a positive single-strand RNA genome composed of approximately 9600 nucleotides. The viral genome composed of a 5’-NCR, a long open reading frame encoding a polyprotein precursor of about 3,000 amino acids,and a 3’-NCR. The polyprotein is processed by a host cell signal peptidase and two virally encoded proteases into three structural proteins (Core,E1,E2,) and seven nonstructural proteins (p7,NS2 to NS5B). An alternative reading frame (ARF) was identified in the HCV core coding region and the functions of the F proteins in the lifecycle and pathogenesis of HCV remain to be elucidated.HCV replication proceeds by the synthesis of a complementary negative-strand RNA using the genome as a template and the subsequent synthesis of genomic positive-strand RNA from this negative-strand RNA template. The key enzyme responsible for both of these steps is the NS5B RdRp. Thus, it represents a good target for antiviral investigations. The HCV RdRp is a so called’tail-anchored protein’. Membrane association is mediated by the C-terminal 21 amino-acid residues, which are dispensable for polymerase activity in vitro but indispensable for RNA replication in cells. Recent studies have revealed a complex interaction between HCV RNA replication and cellular lipid metabolism, presumably through the trafficking and association of viral and host proteins with intracellular membranes.The mechanism of HCV RNA replication are still not declared. Recent studies have identified additional host factors involved in HCV RNA replication. Cyclosporin A (CsA) was observed that it could inhibit HCV RNA replication in vitro via cyclophilin B action, which was the target of CsA. It was shown that cyclophilin B interacts with NS5B, thereby stimulating its RNA binding activity.To further investigate the role of NS5B in HCV replication, we first found interaction between NS5B and CDK2 interacting protein (CINP) by yeast two-hybrid syste, and then confirmed their relationship using GST-pull down and co-IP. CINP is identified as CDK2 interacting protein and is a component of CDK2/cyclinE or CDK2/cyclinA complexes. To determine whether CINP could influence HCV replication, we first knocked down CINP expression with specific siRNA in replicon cells. It was shown that HCV RNA replication was notably inhibited in CINP-depleted cells. The expression of NS5A and NS3 were also decreased. Then the immunofluorescence results indicated that positive staining of NS5A was reduced with CINP silencing. Additionally, we also detected the role of CINP in newly established HCVcc system. We have found that the newly synthesized HCV RNA and NS expression were significantly inhibited in siCINP treated cells. And single-cycle assay showed that siCINP could greatly impair HCV replication. To confirm the hypothesis that CINP was a component of HCV replication complexes(RC), and might interact with other NS proteins, we performed coIP assay and revealed that CINP could not bind to other NS proteins. Then confocal microscopy analysis confirmed that CINP was not co-localized with NS5A. What’s more, our experiments had demonstrated that the regions of NS5B responsible for binding CINP was not in pclymerase domain. All these results indicated that CINP may not participate in the formation of HCV RC.Recent reports have identified that CINP is a checkpoint protein of cell cycle and forms a complex with ATR-ATRIP through ATRIP, promotes cell viability in response to replication stress. Ariumi et al recently demonstrated that replication of both genome-length HCV RNA and the subgenomic replicon RNA were notably suppressed in ATM kinase inhibitor treated cells. All theses studies suggested that CINP might regulate HCV replication through ATM/ATR pathway. To confirm this hypothesis, we used HU or aphidicolin which was ATM/ATR inducer to stimulate cells with CINP silencing. We found that silencing of CINP significantly reduced ATM/ATR phosphorylation and Chk1/Chk2 phophorylation after both HU and aphidicolin treated. This result indicated that CINP was essential for both efficient ATM and ATR signaling to multiple effector proteins. We next examined the effect of specific small-molecule inhibitor of the ATM and ATR kinase (KU55933 and CGK733) on HCV RNA replication. As expected, both of these inhibitors could efficiently suppress genome-length HCV RNA replication. Consistent with this finding, the expression levels of NS5A protein was also significantly decreased. The result revealed that both ATM and ATR were crucial for HCV RNA replication, and these small-molecule inhibitors of the ATM and ATR kinase mimiced the function defects of CINP silencing.The above results suggested that CINP participated in the activation of ATM/ATR pathway, which is essential for HCV replication. CINP regulate HCV replication through accommodating ATM/ATR activation and may represent a novel target for the clinical treatment of patients with chronic hepatitis C.