The Mechanism Of Membrane Proteins PCSK9 And BST2 Inhibiting PRRSV Replication

Porcine Reproductive and Respiratory Syndrome(PRRS)have seriously impacted the development of swine industry since the outbreak in North America,1987,especially the outbreak of Highly Pathogenic PRRS(Highly Pathogenic PRRS,HP-PRRS)in China,2006.HP-PRRSV is characterized by high morbidity and mortality,with morbidity up to 50%-100%and mortality up to 20%-100%(TONG et al.,2007).In order to explore the difference in pathogenicity between HP-PRRSV Hu N4 and its attenuated vaccine strain Hu N4-F112,we analyzed the infection efficiency of Hu N4 and Hu N4-F112on PAM by flow cytometry,and found that the infection efficiency of Hu N4 on PAM was significantly higher than that of Hu N4-F112.The PAM cells infected with Hu N4 or Hu N4-F112 were successfully separated by flow cytometry.The cell membranes of the blank PAM cells,Hu N4 or Hu N4-F112infected PAMs were extracted,respectively,and the membrane proteins of differential expression were screened by shotgun mass spectrum technology.A series of differentially expressed membrane proteins were identified.In this study,two membrane proteins,Proprotein convertase subtilisin kexin 9(PCSK9)and bone marrow stromal cell antigen 2(BST2)were studied deeply in its influence on PRRSV replication.PCSK9,also known as Neural apoptosis-regulated convertase 1(NARC-1),belongs to the family of Proprotein Convertases(PC),and plays an important role in cholesterol transportion.However,there are relatively few studies on the antiviral process of PCSK9.Previous studies have shown that PCSK9can promote the degradation of HCV receptor.Thus,it can inhibite HCV replication.In our previous study,it was found that PCSK9 could significantly inhibit the replication of Hu N4 and Hu N4-F112,and the inhibition of Hu N4 was significantly stronger than that of Hu N4-F112.In this study,the mechanism of PCSK9's antiviral role was further studied.Western blot,RT-q PCR,and TCID₅₀showed that the C-terminal domain of PCSK9 had antiviral effect,and PCSK9 could interact with PRRSV receptor CD163,and degraded the PRRSV receptor CD163 mainly through lysosomal pathway by K48/K63ubiquitination modification.The expression of endogenous PCSK9 was further detected by PCSK9antibodies.As the results showed that the expression of endogenous PCSK9 was up-regulated in host cells at the early stage of PRRSV infection,but PRRSV could inhibit the expression of endogenous PCSK9 when infected with enough dose of PRRSV,and PRRSV can antagonize the antiviral effect of PCSK9.Further research showed that PRRSV Nsp11 could inhibit the expression of PCSK9,and nsp11mutants with inactivated endoribonuclease activity lost the function of antagonizing PCSK9's antiviral activity,whereas those nsp11 mutants that lost their deubiquitinating activity did not,and PRRSV Nsp11 could antagonize the antiviral effect of PCSK9 through its endonuclease activity.BST2,also known as tetherin,CD317 and HM1.24,is a type II transmembrane protein induced by interferon,which can hijake envolope virions to the host cell membrane by its special topological structure,thereby it could inhibit the release process of the virus.In this study,it was found that the overexpression of BST2 could inhibit the replication of PRRSV while knockdown of BST2 could promote the replication of PRRSV by overexpression and si RNA interference experiments.Further research showed that BST2 could inhibit PRRSV replication by different ways.It can inhibit the expression of E protein via m RNA level,and inhibit the expression of Nsp12 through ubiquitin-protease pathway.Low dose of PRRSV can up-regulate the expression of BST2 while high dose of PRRSV can inhibit the expression of BST2.Further studies showed that PRRSV Nsp11 could inhibit the expression of BST2 through its endonuclease activity,and PRRSV Nsp5 could interact or co-located with BST2,and Nsp5 could inhibit the expression of BST2 in cytoplasm and cell membrane,which plays an important role in antagonizing the antiviral role of BST2.In addition,porcine PCSK9 monoclonal antibody was successfully prepared,which provided tool support for exploring the relationship between the abundance of membrane proteins and PRRSV infection in future studies via screening PAM cells with different abundance of membrane proteins and being infected with highly and attenuted PRRSV strains.In summary,this study identified the impact of membrane proteins PCSK9 and BST2 on PRRSV replication,and analyzed the antiviral mechanism of PCSK9 and BST2.Meanwhile,PRRSV could antagonize the antiviral activities of PCSK9 and BST2,which provided a reference for understanding the replication mechanism of PRRSV.