PSMB10 Interacts With Viral NS3 Protein And Inhibits The Growth Of Classical Swine Fever Virus

Classical swine fever(CSF)is a major infectious disease of pigs caused by the Classical swine fever virus(CSFV),which causes severe economic losses to the pig industry.CSFV nonstructural protein NS3 plays an important role in the process of CSFV proliferation.However,researches about how NS3 interacts with host cell proteins and participate in the process of CSFV proliferation remains few.To study CSFV interaction relationship with cells,our laboratory screened eight cellular proteins which may interact with NS3 protein by yeast two hybrid system.In this study,we selected proteasome subunit beta 10(PSMB10)for further verification.The interaction between host cellular protein PSMB10 and CSFV NS3 protein was confirmed by yeast two hybrid system,co-immunoprecipitation,GST-pulldown and laser confocal.In order to study the interaction between CSFV and PSMB10 protein,3D4/2 cells were infected with CSFV,followed with detected PSMB10 mRNA and protein expression at 24 h and 48 h post-infection.Results revealed that the expression of PSMB10 in the CSFVinfected 3D4/2 cells was decreased at 24 h and 48 h compared with control,which demonstated that CSFV infection inhibis PSMB10 expression.Further study about the effects of PSMB10 in CSFV propagation,we transfected p3×Flag-PSMB10 recombinant plasmid into 3D4/2 cells to overexpression of PSMB10 protein,and transfected shRNA-PSMB10 to suppress expression of PSMB10,followed with CSFV infection at 24 h post-transfection.At 24 h and 48 h post-infection,CSFV genomic copies were detected by qRT-PCR and virus titer of CSFV were detected by IFA.Results show that compared with p3×Flag-CMV transfection cells controls,the expression of PSMB10 in p3×Flag-PSMB10 transfection cells were increased while CSFV titers and quantity of CSFV genomic copies reduced.Compared with shRNA-NC transfection cell controls,the expression of PSMB10 in shRNA-PSMB10 transfection cells were reduced while CSFV titers and quantity of CSFV genomic copies increased.Results indicated that overexpression of PSMB10 protein inhibited the proliferation of CSFV,while suppress expression of PSMB10 promoted the proliferation of CSFV.On the basis of studied the cellular protein PSMB10 and CSFV NS3 protein interaction and effect of PSMB10 protein in CSFV proliferation,in order to further explore molecular mechanism of cellular protein PSMB10 impact of CSFV proliferation,we transfected 0 ?g,0.5 ?g,1 ?g and 2 ?g p3×Flag-PSMB10 with 2 ?g HA-NS3 plasmid into 3D4/2 cells respectively,and then detected expression of PSMB10 protein and NS3 protein.Results showed that with the increase transfection of p3×Flag-PSMB10 plasmid,the expression of NS3 protein decreased,indicating that cellular protein PMSB10 inhibited the expression of CSFV NS3 protein.PSMB10 is one of immune proteasome subunits and participate in the ubiquitin-proteasome pathway,to understand whether PSMB10 protein degradated NS3 protein by the ubiquitin-proteasome pathway,3D4/2 cells were transfected with HA-NS3 and then treated with proteasome inhibitor MG132 after 24 h of transfection,and detected ubiquitin using immune precipitation method.Results show that the transfection and MG132 treatment cells can detected the expression of ubiquitin protein,demonstated that CSFV NS3 protein has been ubiquitined in 3D4/2 cells.In conclusion,cellular protein PSMB10 inhibited the expression of NS3 protein,and its effect may be related to the ubiquitin-proteasome pathway.In order to further explore whether PSMB10 inhibits CSFV proliferation by MHC-I antigen presentation,we used qRT-PCR to detected MHC-I antigen presentation transport related molecules(TAP1,TAP2,Tapasin)mRNA expression level of 3D4/2 cells after infected with CSFV,transfected with p3×Flag-PSMB10 recombinant plasmid or shRNAPSMB10 plasmid respectively.Results showed that the mRNA expression of TAP1,TAP2 and Tapasin of 3D4/2 cells after CSFV infection were significantly decreased compared with the mock cells.Compared with p3×Flag-CMV transfected control cells,the mRNA expression of TAP1,TAP2 and Tapasin of 3D4/2 cells transfected with p3×Flag-PSMB10 increased significantly.Compared with shRNA-NC transfection cells,the mRNA expression of TAP1,TAP2 and Tapasin of 3D4/2 cells transfected with shRNA-PSMB10 were significantly decreased.Results demonstrated that the mRNA expression of TAP1,TAP2 and Tapasin was inhibited by CSFV infection,and PSMB10 had a catalytic effect on the mRNA expression of TAP1,TAP2 and Tapasin.Then we transfected p3×Flag-PSMB10 into 3D4/2 cells,followed by CSFV infected,detected expression of TAP1,TAP2,Tapasin proteins using western blot.Results show that expression of TAP1,TAP2,Tapasin proteins were significantly increased in cells which transfected with p3×Flag-PSMB10 followed by CSFV infected compared to CSFV infected cells.Indicated that PSMB10 protein can restore or even enhance the expression of TAP1 and TAP2,Tapasin proteins in CSFV infected cells.To sum up,this study demonstrates the interaction between PSMB10 and CSFV NS3 protein and CSFV proliferation,which provides a theoretical basis for studying the antiCSFV infection mechanism of host cells.