Screening Of Cellular Proteins Of Classical Swine Fever Virus P7 And Study On The Interaction Of P7 With MAPRE1 And VDAC1

Classical swine fever(CSF)is a severe infectious disease caused by pigs infected with Classical swine fever virus(CSFV).Studies have shown that CSFV p7 protein can form ion channels and cytoplasmic rings on the cell membrane,which is related to the formation of ion channels during CSFV replication.In addition,the CSFV p7 protein can be degraded by the host proteasome,and the secretion of the inflammatory cytokine IL-1? can be induced by activating caspase1,which further causes the host cell immune response.In order to build the recombinant bait plasmid pGBKT7-p7,the CSFV p7 gene was firstly amplified by RT-PCR,and then the CSFV p7 gene was cloned into the bait vector pGBKT7 of the yeast two-hybrid system to obtain the recombinant bait plasmid pGBKT7-p7,which was identified by PCR.After the gene sequence was determined correctly,the recombinant plasmid pGBKT7-p7 was transformed into yeast cell Y2 HGold,its toxic effect on yeast,self-activation and protein expression were detected.At the same time,pGBKT7 control and auto-activation control were established.The results showed that the bands with a molecular weight of about 35 kDa were matched with the expected p7 protein size.The growth of Y2HGold(pGBKT7-p7)and Y2HGold(pGBKT7)on SD/-Trp plates was basically the same.Y2HGold(pGBKT7-p7)was a white colony on SD/-Trp/X plates,and did not grow on SD/-trp /X/Aba tablets.It was shown that the recombinant bait plasmid pGBKT7-p7 could be expressed in yeast and had no toxic effect and self-activation on yeast.In order to study the interaction between CSFV p7 protein and host cell protein,we hybridized the recombinant bait plasmid pGBKT7-p7 with the porcine peripheral blood mononuclear cell cDNA library constructed in our laboratory,and then screened 7 host cellproteins interacting with CSFV p7 proteins.Potential interacting host cell proteins are proteasome mature protein(POMP),microtubule associated protein EB1(MAPRE1),mitochondrial voltage-dependent anion channel protein 1(VDAC1),and protein inhibitor of activated STAT1(PIAS1).Gamettin binding protein 2(GGNBP2),COP9 signaling complex 2 subunit protein(COSP2),contact protein 1(CNTN1).After protein functional analysis,these seven proteins were found to be involved in processes such as ubiquitin-proteasome system,mitochondrial autophagy,antigen presentation processing,immune response,intracellular substance transport,signal transduction,ion exchange,and virus replication and so on.Studies have shown that MAPRE1 as a microtubule-associated protein plays an important regulatory role in the integration of retroviral genes into the host cell genome,and is closely related to the replication of the virus;VDAC1 acts as a voltage-dependent anion channel protein that canregulate the replication of the virus by interacting with viral proteins or changing their expression levels.To verify the direct interaction between cells protein MAPRE1,VDAC1 and CSFV p7 protein,we transfected HEK293 T cells with the recombinant plasmids p3×flag-MAPRE1 and p3×flag-VDAC1 which carrying the MAREP1 and VDAC1 genes,and then subjected to GST-pulldown treatment.The results showed that the transfected cells with p3×flag-MAPRE1 and p3×flag-VDAC1 could detect protein bands with molecular weight of about 35 kDa after GST-pulldown treatment,which was consistent with the expected protein levels of MAPRE1 and VDAC1.The results show that there is a direct interaction between the cell proteins MAPRE1,VDAC1 and CSFV p7 protein.In order to further verify the interaction between cells MAPRE1,VDAC1 and CSFV p7 protein,we transfected HEK293 T cells with the recombinant plasmids p3×flag-MAPRE1 and p3×flag-VDAC1,and conduct immunoprecipitation.The results showed the transfected cells with p3×flag-MAPRE1 and p3×flag-VDAC1 could detect protein bands with molecular weight of about 35 kDa after co-immunoprecipitation,which was consistent with expected protein levels of MAPRE1 and VDAC1.This indicates that there is an interaction between the cell proteins MAPRE1,VDAC1 and CSFV p7 protein.In summary,this study screened seven potential host cell proteins interacting with theCSFV p7 protein from the porcine peripheral blood mononuclear cell cDNA library,and further validated the interaction between the cell proteins MAPRE1,VDAC1 and CSFV p7 protein.This study lays a foundation for the interaction relationship of CSFV p7 and host cell proteins,providing scientific basis for in-depth study of the mechanism of CSFV replication.