Invoving Of Prrsv Nonstructural Transmembrane Proteins In The Viral Replication And Transcription Complex Formation

Porcine reproductive and respiratory syndrome virus(PRRSV),the causative agent of porcine reproductive and respiratory syndrome(PRRS),first occurred in the USA in 1980 s,then spread worldwide and it has contributed great economic losses to the global pig industry.The transmembrane proteins nsp2,nsp3 and nsp5 are very important proteins in PRRSV nonstructural proteins.To investigate the interactions between the transmembrane proteins nsp2,nsp3,and nsp5 and their associations with other non-structural proteins,this study screened and identified non-structural proteins that interact with these three transmembrane proteins.Yeast two-hybrid technology was used to screen proteins that interacted with nsp2,nsp3,and nsp5,and then Biomolecule fluorescence complementation technique was applied to validate these interactions to remove false positives.Through Y2 H and BiFC,it was detected that nsp9,nsp12 and transmembrane proteins nsp3,nsp5 interacted with a number of nsps,indicating that these nsps may serve as the core components of the viral replication and transcription complex(RTC)formation.Finally,some of these multiple interactions were further verified using Pull-down techniques.Hydrophobic nsp2,nsp3 and nsp5 containing predicted membrane-spanning domains are thought to be involved in membrane modification and formation of double membrane vesicles(DMVs),a typical structure induced by arterivirus infection.We found that both nsp2 and nsp5 interacted with nsp3,and nsp3 could also interact with itself,indicating that a scaffold with nsp3 in the center,flanked by nsp2 and nsp5,might serve as an anchor on the DMV membrane to recruit and support the RTC centered with nsp9 and nsp12,and participated in the replication and transcription processes during PRRSV infection.