Analysis Of Differentially Expressed Membrane Proteins Of PAMs Infected With Highly Pathogenic And Attenuated PRRSVs

Porcine reproductive and respiratory syndrome(PRRS)is one of the most important infectious diseases which threaten swine industry.In 2006,a highly pathogenic PRRS(HP-PRRS)emerged in China,which caused much higher morbidity and mortality in comparison with classical PRRS.PRRSV has a strict cellular tropism and its target cell is pulmonary alveolar macrophage(PAM).The efficiency of PRRSV in infecting PAM is proportional to its virulence,and the virus infection efficiency was increased with the strengthen of virulence.The results of previous laboratory experiments showed that the HP-PRRSV strain HuN4 infection PAM efficiency is significantly higher than the attenuated strain HuN4-F112,and this experiment is devoted to deeper analysis of the differentially expressed membrane proteins in PRRSV-infected PAM cells,exploring the effect of differentially expressed membrane proteins on PRRSV infection and replication as well as the mechanism of differential infection efficiency between HP-and attenuated PRRSV,and to explore the pathogenesis of HP-PRRSV with high morbidity and high mortality.In this study,pulmonary alveolar macrophages(PAMs)were in vitro infected with 1MOI EGFP labelled HP-PRRS virus HuN4(rHuN4-EGFP)and its attenuated strain HuN4-F112(rHuN4-F112-EGFP),respectively.The infected PAMs were isolated through FITC channel using flow cytometry at 24 hours after infection,and then the membrane proteins of the infected as well as noninfected PAMs were extracted for further shotgun proteomics(liquid chromatography-tandem mass spectrometry,LS-MS/MS)analysis to identify the differentially expressed membrane proteins of PAMs infected with PRRSV.82 differentially expressed membrane proteins were identified,and 72 were from rHuN4-EGFP infected PAMs and 12 were from rHuN4-F112-EGFP infected PAMs.These proteins mostly participated in metabolic process,cellular process and biological regulation which have characteristics of combination and catalytic activities.Using over expression method,we confirmed that PCSK9 can inhibit replication of PRRSV,while Clusterin and Apoliprotein C-II can promote PRRSV replication.Further,overexpressed the differentially expressed membrane protein PCSK9 gene on MARC-145 cells followed by PRRSV infection.IFA,RT-qPCR,and multi-step growth curves all showed that PCSK9 could obviously inhibit the replication of PRRSV,and PCSK9 significantly inhibited the HP-PRRSV HuN4 virulent strain compared with the attenuated strain HuN4-F112.PCSK9 can not only inhibit the replication of North American PRRSV,but also inhibit the European type PRRSV strain Lelysted.PCSK9 is likely to be a broad spectrum inhibitor of PRRSV replication.The effect of PCSK9 on virus was detected by RT-qPCR.It was found that PCSK9 had no effect on the attachment and entry stage.The inhibitory effect of PCSK9 on PRRSV occurred at the post-entry process.In addition,we found that the endogenous mRNA level of PCSK9 was associated with PRRSV infection dose and infection time.In the early stage of virus infection,PCSK9 mRNA expression can be stimulated,and the PCSK9 mRNA levels begin to decline with virus increasing and time going.The maturation of PCSK9 protein and the antiviral action of PCSK9 are related to ubiquitin proteasome degradation pathway.In addition,PRRSV also degrades the PCSK9 protein through the ubiquitin proteasome pathway and impairs the effect of PCSK9.At the same time,the results of co-immunoprecipitation experiment showed that PCSK9 interacted with cholesterol metabolism-related LDLR receptors,and the LDLR also inhibited PRRSV replication.In addition,PCSK9 could also co-locate and interact with the PRRSV receptor CD163,and preliminary results showed that PCSK9 could degrade PRRSV receptor CD163 through ubiquitin proteasome pathway,thus inhibiting PRRSV replication.Taken together,this study successfully identified some differentially expressed membrane proteins which had an impact on the replication of PRRSV by Shotgun Mass Spectrometry,and the PCSK9 inhibitory effect for PRRSV replication were verified,which provided a foundation for further understanding of PRRSV replication property and its pathogenic mechanism.