The Study Of OCIAD2 Protein Regulate The Replication Of Hepatitis C Virus

Hepatitis C virus(HCV)poses a serious threat to human health.According to incomplete statistics,about 170 million people worldwide are infected with HCV,and about 400,000 people die from HCV-like diseases every year.HCV infection is the main cause of chronic hepatitis,liver cirrhosis and hepatocellular carcinoma,and there is still no available preventive vaccine.However,the existing therapeutic drugs have the disadvantages of being effective for some patients,high in cost,large in side effects,and prone to drug resistance.In 2013,the US FDA approved the new drug sofosbuvir for the treatment of HCV,improving the cure rate of HCV to more than 90%,but it still cannot reach 100%.Therefore,research on new drugs for the treatment of HCV is still an urgent task.As a kind of non-cellular microorganisms that must be parasitic on living cells,viruses themselves highly rely on host cells to complete their own proliferation.The life cycle of a virus includes five stages:entry,shelling,replication,assembly and release.In each stage,there is a complex interaction between the viral protein and the host protein.Studies have found that the host proteins ISG12a,ISG16 and HMGB1 promote HCV replication,and the host proteins ARF1,PLA1A and PLA2G4C regulate HCV assembly.Therefore,elucidating the interaction of the virus and the host contributes to understanding HCV replication mechanism,and provides a theoretical basis for the development of new drugs for the treatment of HCVIn our laboratory,202 cellular proteins related to NS4B were identified in the previous phase by affinity chromatography and liquid chromatography-tandem mass spectrometry.In-depth study showed that the cellular proteins PREB and Surf4 are new HCV host cofactors that participate in HCV replication and affect the formation of replication complexes.In this study,based on previous studies,we selected eight cellular proteins with high scores,which were respectively:CKAP4,OCIAD2,Prohibitin,MPV17,RAB13,SLC4A2,LRG1 and RNF5.Preliminary RNAi screening experiments showed that silencing OCIAD2 reduced intracellular luciferase expression levels in SGR-2a cells and intracellular HCV RNA levels in HCV-infected JFhl strains.To investigate whether OCIAD2 affected HCV replication in a dose-dependent and genotype-dependent manner,we first treated SGR-2a and SGR-1b cells with siRNA targeting different sites of OCIAD2 and found that silencing OCIAD2 reduced intracellular luciferase expression levels.We then treated SGR-2a and SGR-1b cells with different concentrations of siRNA and found that intracellular luciferase expression levels decreased with increasing concentrations of siRNA.These results suggest that OCIAD2 affects HCV replication in a dose-dependent and genotypedependent manner.In the research on the mechanism of host protein regulating HCV replication,we found that OCIAD2 is a cellular protein related to NS4B.To investigate the interaction between OCIAD2 and NS4B,we first constructed a eukaryotic expression vector of OCIAD2 and NS4B,which was found to be both expressed in Huh7 cells by Western blot analysis,and then co-transfected into Huh7 cells.Immunocoprecipitation and indirect immunofluorescence analysis showed that OCIAD2 interacted with NS4B.To investigate the interaction site of NS4B in OCIAD2,we first constructed a truncated mutant of OCIAD2,designated as OCdl,OCd2,OCd3,and OCd4,respectively.Only OCdl was detected by western blot.Therefore,we constructed replacement mutants to replace OCd2,OCd3 and OCd4,respectively designated as OCd5,OCd6 and OCd7,which were all found to be expressed in Huh7 cells by western blot analysis.OCIAD2 and its mutant and NS4B were then co-transfected into Huh7 cells,and 31-60aa of OCIAD2 was found to be responsible for interaction with NS4B by immunoprecipitation and indirect immunofluorescence analysis.To investigate the interaction site of NS4B with OCIAD2,we constructed a truncated mutant of NS4B,named NS4Bd1,NS4Bd2,and NS4Bd3,which was found to be all expressed in Huh7 cells by Western blot analysis.Then,NS4B and its mutant were co-transfected into Huh7 cells with OCIAD2.Immunocoprecipitation and indirect immunofluorescence analysis showed that 1-133aa of NS4B was responsible for interaction with OCIAD2.In studies investigating the regulation of the HCV replication complex by host proteins,we found that cellular proteins such as PREB and Surf4 were recruited by NS4B into the replication complex.To investigate whether OCIAD2 was also recruited into the replication complex by NS4B,indirect immunofluorescence analysis of SGR2a and SGR-1b cells treated with actinomycin-D and transfected with BrUTP revealed that OCIAD2 was localized to the replication complex.Subsequently,we transfected OCIAD2 and OCd7(which did not interact with NS4B)into SGR-2a and SGR-1b cells,respectively,and indirect immunofluorescence analysis revealed no co-localization of OCd7 and dsRNA.These results suggest that OCIAD2 was recruited by NS4B into the replication complex.In the study of HCV replication mechanism,we found that OCIAD2,PREB and Surf4 are all cellular proteins that regulate HCV replication,and NS5A is involved in the regulation of the formation of replication complexes.To investigate their interaction,immunoprecipitation and indirect immunofluorescence analysis of Huh7 cells cotransfected with OCIAD2 and PREB,Surf4,and NS5A revealed that OCIAD2 interacted with PREB and NS5A,but not with Surf4.These results suggest that interaction of OCIAD2 with any of the NS4B,PREB,and NS5 A proteins may facilitate its efficient entry into the HCV replication complex.In summary,we found that OCIAD2 is a new host cofactor of HCV and participates in HCV replication.OCIAD2 was recruited into replication complex by interacting with NS4B.These results reveal the new function of OCIAD2 and the new mechanism of HCV replication,and provide new ideas and new basis for the research and development of HCV therapeutic drugs.