Effect Of PSMB5 Protein On Proliferation Of Classical Swine Fever Virus And Its Mechanism

Classical swine fever(CSF)is a major infectious disease caused by the infection of pigs with classical swine fever virus(CSFV).It causes significant economic losses to the pig industry in China and seriously affects the development of pig industry.The 20S proteasome subunit beta 5(PSMB5)is a key protein subunit of the cell proteasome.It is located in the catalytic center of the cell proteasome and plays a role in proteolysis,which can affect the proliferation of the virus in cells.At present,there are few studies on the effect of PSMB5 protein on the proliferation of CSFV.The purpose of this study is to study the effect of PSMB5 protein on the proliferation of CSFV and its mechanism,and provide a theoretical basis for the prevention and control of CSF.In order to study the interaction between CSFV and PSMB5 protein,we first investigated the effect of CSFV-infected PK-15 cells on PSMB5 protein expression.CSFV Shimen strain was infected with PK-15 cells,and PSMB5 m RNA and protein levels was detected by q RT-PCR and Western blot.The results showed that m RNA and protein levels of PSMB5 were decreased in CSFV-infected cells compared to uninfected cells,indicating that CSFV infection inhibited the expression of PSMB5 in cells.To further investigate the effect of PSMB5 on CSFV proliferation,we transfected p3×Flag-PSMB5 recombinant plasmid into 3D4/2 cells to overexpress PSMB5 protein,and transfected si RNA-PSMB5targeting PSMB5 gene into 3D4/2 cells to inhibit PSMB5 protein.At the same time,p3×Flag-CMV and si RNA-NC transfected control groups were established,and CSFV Shimen strain was infected 24 h after transfection.CSFV gene copy number was detected by q RT-PCR,and indirect immunofluorescence assay(IFA)determines the TCID₅₀of CSFV.The results showed that compared with the p3×Flag-CMV transfected cell control,the PSMB5 protein level of the p3×Flag-PSMB5 transfected cells was increased,the gene copy number of CSFV was decreased,and the viral titer of CSFV was decreased;Compared with the control of the stained cells,the PSMB5 protein level of the si RNA-PSMB5 transfected cells was decreased,the gene copy number of CSFV was increased,and the viral titer of CSFV was increased.The results showed that overexpression of PSMB5 protein inhibited the proliferation of CSFV,while PSMB5protein gene function interference promoted the proliferation of CSFV.To further investigate whether PSMB5 interacts with CSFV NS3 to affect CSFV proliferation,we co-transfected 3D4/2 cells with p3×Flag-PSMB5 and HA-NS3recombinant plasmids,and HA-labeled antibody and Flag label 48 h after transfection.The antibody was incubated with the corresponding fluorescent secondary antibody,and the fluorescence was observed by a laser confocal microscope.The results showed that specific orange-yellow fluorescence was observed in the cytoplasm of p3×Flag-PSMB5 and HA-NS3 co-transfected.The results showed that PSMB5 and NS3 co-localized in the cytoplasm,and there may be an interaction relationship between the two.Next,we injected0?g,1?g,2?g of p3×Flag-PSMB5 plasmid with 2?g of HA-NS3 plasmid and 3D4/2 cells,respectively,to detect the expression of PSMB5 protein and NS3 protein.The results showed that the expression of NS3 protein decreased with the increase of p3×Flag-PSMB5plasmid transfection dose,indicating that the cellular protein PSMB5 can inhibit the expression of CSFV NS3 protein.To understand whether PSMB5 protein degrades NS3protein via the ubiquitin-proteasome pathway,we transfected HA-NS3 plasmid into 3D4/2cells,treated cells with proteasome inhibitor MG132 24 h after transfection,and established HA-NS3 plasmid transfection.The cell control group stained with DMSO was used to detect whether the NS3 protein was ubiquitinated by immunoprecipitation assay.The results showed that the cells transfected and treated with MG132 were able to detect the expression of ubiquitin protein,while the transfected cells treated with DMSO showed no expression of ubiquitin protein,indicating that CSFV NS3 protein was ubiquitinated in3D4/2 cells.In conclusion,the cellular protein PSMB5 has an inhibitory effect on the expression of NS3 protein,which may be related to the ubiquitination modification and degradation of NS3 protein by the ubiquitin-proteasome pathway.To investigate whether the Janus activated kinase/signal transducers and activators of transcription(JAK/STAT)pathway mediates the regulation of PSMB5 protein expression by CSFV infection,we added Signal transducers and activators of transcription 3(STAT3)inhibitor STATTIC after CSFV infection of PK-15 cells,and established an untreated blank control group and no STATTIC after infection with CSFV.In the cell control group,the protein level of PSMB5 in the cells was measured 72 h after infection.The results showed that the protein level of PSMB5 in the STATTIC treated group was higher than that in the untreated group after infection with CSFV,which was not significantly different from the blank control group,indicating that inhibition of STAT3 by STATTIC reduced the inhibition of PSMB5 by CSFV.Under the stimulation of factors such as interferon-gamma(IFN-?)and viral invasion,some PSMB5 proteins will be replaced by the immunoproteasome protein LMP7,which participate in the process of degrading viral proteins by MHC class I pathway.Enhance the efficiency of MHC class I antigen presentation.To investigate whether PSMB5 and LMP7affect the proliferation of CSFV in cells through MHC-I pathway,we used q RT-PCR and Western blot to detect m RNA and protein levels of LMP7 and MHC class I antigen-presenting pathway transport-related molecules(TAP1,TAP2,Tapasin)in CSFV-infected 3D4/2 cell;TAP1,TAP2 and Tapasin were detected in 3D4/2 cells transfected with p3×Flag-PSMB5,si RNA-PSMB5,p3×Flag-LMP7 and si RNA-LMP7 by Western blot.The results showed that m RNA levels and protein levels of LMP7,TAP1,TAP2 and Tapasin were significantly decreased in CSFV-infected 3D4/2 cells compared to blank cells;compared to p3×Flag-CMV transfected cells.The protein expression levels of TAP1,TAP2 and Tapasin in p3×Flag-PSMB5 transfected cells and p3×Flag-LMP7transfected cells increased significantly;compared with si RNA-NC transfected cells,si RNA-PSMB5 transfected cells and si RNA-LMP7 transfected cell protein expression levels of TAP1,TAP2 and Tapasin stained cells were significantly decreased.The results showed that CSFV infection inhibited the expression of LMP7,TAP1,TAP2 and Tapasin;PSMB5 and LMP7 overexpression promoted the expression of TAP1,TAP2 and Tapasin.Next,we transfected p3×Flag-PSMB5 and p3×Flag-LMP7 into 3D4/2 cells,infected CSFV24 h after transfection,continued incubation for 48 h,and established a cell control of CSFV infection after transfection of p3×Flag-CMV.Protein levels of TAP1,TAP2 and Tapasin were detected by Western blot with a blank control that was not treated.The results showed that compared with the cells infected with CSFV after transfection of p3×Flag-CMV,the proteins level of TAP1,TAP2 and Tapasin in CSFV infected cells then transfected with p3×Flag-LMP7 and transfected with p3×Flag-PSMB5 was significantly increased,with TAP1 and TAP2 protein levels higher than the blank control,indicating that PSMB5 and LMP7 restore protein levels of TAP1,TAP2 and Tapasin in CSFV-infected cells.In summary,this paper studied the interaction between PSMB5 protein and CSFV infection,and provided a scientific basis for further revealing the interaction between CSFV and the mechanism of CSFV infection.