Researches on Mechanism of Interferon-beta Expression and Bovine Parvovirus VP1 Protein Infection in the Host Cells
Objectives To explore systematically the interferon-beta(IFN-β)expression level effects and underlying mechanism following bovine parvovirus(BPV)VP1 protein infecting the host cells.Methods The protein sequence of BPV Haden strain(GenBank No:DQ335247)was designed.DNAs were extracted from the viruses and amplified with PCR.The eukaryotic expression vectors of BPV VP1 and VP2 were constructed.Expressions of VP1 and VP2 and cell viability were detected using qRT-PCR in HEK-293T cells.Western blotting was used to verify the protein expression levels and determine copy numbers of viruses.HEK-293T cells were transfected with BPV VP1 recombinant plasmid.IFN-β transcription level,expression levels of Mx1,OAS1,ISG15,ISG56,TBK1 and IRF3 mRNAs were tested,respectively by applying qRT PCR.After transfection of HEK-293T cells with pCMV Myc BPV-VP1 protein,expression levels of TBK1,IRF3(5D),MDA5,and MAVS were evaluated respectively with both Western blotting and qRT PCR.Results VP1 and VP2 fragments about 2 022 bp and 1 611 bp were obtained by performing double enzyme digestion on the recombinant plasmid,which were consistent in the expectated size.The mRNA expression levels of VP1 and VP2 were increased by 5.5×104 and 7.4×105 folds as compared to the control group(P<0.001).Copy numbers of viruses in BPV VP1 treatment group were increased by 5.8×104 as compared to the control group(P<0.001).BPV VP1 significantly inhibited IFN-β production in HEK-293T cells induced by vesicular stomatitis virus(VSV).BPV VP1 protein significantly downregulated the transcription levels of Mx,OAS,ISG15,and ISG56 with the decrement of 30%,90%,84%,and 20%,respectively(P<0.01).TBK1 and IRF3(5D)activated IFN-β production in HEK-293T cells,while BPV VP1 reduced their transcription levels of TBK1 and IRF3 by 81%and 89%,respectively.These decreased IFN-β production by 97%and 90%.All of TBK1,IRF3(5D),MDA5 and MAVS were distributed and expressed in HEK-293T cells,with sizes of 84 kDa,55 kDa,118 kDa and 58 kDa,respectively.After transfection of pCMV Myc BPV-VP1 protein,the expression levels of these four factors were significantly reduced(P<0.001).Conclusions The genes of BPV VP1 and VP2 were highly expressed in HEK-293T cells,and BPV VP1 protein significantly promoted the in vitro proliferation of BPV.BPV VP1 protein reduced vesicular stomatitis virus(VSV)-mediated the transcription levels of Mx,OAS,ISG15 and ISG56 by inhibiting ISGs transcription levels,besides downregulating IFN-β production induced by BK1 and IRF3(5D).Such IFN-β production was significantly depressed.Namely,BPV VP1 protein can reduce the antiviral effect of IFN-I through IRF3 or IRF3 pathway.PCMV-Myc-BPV-VP1 can reduce the expression levels of TBK1,IRF3(5D),MDA5 and MAVS in the RLRs pathway.
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