Screening And Identification Of The Host Cell Proteins Interacted With Porcine Reproductive And Respiratoiry Syndrome Virus NSP12

Porcine reproductive and respiratory syndrome is an infectious disease caused by porcine reproductive and respiratory syndrome virus and charaterized by reproductive disorders in pregnant sows,respiratory diseases in suckling pigs and high mortality.PRRSV often lead to immunosuppression of the infected pigs,which further causes persistant infection due to failure in clearance of the virus.Many nonstructural proteins of PRRSV have shown to participate in antagonizing host celluar innate immunity and regulating viral replication.However,the biological significance of NSP12 remianed unknown.In this study,the NSP12 gene of an highly pathogenic PRRSV strain,HuN4 was used for constructing the bait vector,pGBKT7.By yeast two-hybrid system(Y2H),cellular proteins of the porcine alveloar macrophages interacted with PRRSV NSP12 were screened and verified.In order to estabilsh the research foundation of NSP12,the NSP12 gene was cloned to prokaryotic expression vector pCold-I and successfully expressed target protein.The expressed protein was purified and immunized to BALB/C mice and the serume was collected after 4 times immunization.By Western blot analysis and indirect immuno-fluorescence assay,the prepared polyclonal antibodies showed good reactivities to viral and eukaryotic expressed NSP12.The bait vector pGBKT7-NSP12 was further used to screen host proteins interacted to NSP12 by Y2H method after showing non-toxicity to yeast cells and self-actvaing effect.The results revealed that 8 host proteins could interact to PRRSV NSP12.Based on sequeces BLAST and GO analysis,these proteins were mainly chaperonins such as FTH1,ZNF598 and apotosis related proteins such as LGALS3 and UCP2.In order to verify these protein-protein interactions,immuno-precipitation and cofocal microscopy analysis were used.The result showed that PRRSV NSP12 could do interact with cellular CD63 nd LGALS3,and mainly colocalized in the cytoplasma.LGALS3 overexpression by transfecting eukaryotic expression vector to MARC-145 cells shown to inhibit virus replication.According to the previous study of the function of LGALS3.The above results provide a new theoretical basis for further analysis of PRRSV virus infection mechanism.