The Proteomic Analysis Of PAMs Infected With PRRSV And The Effect Of EIF5A On PRRSV Replication

Porcine reproductive and respiratory syndrome（PRRS）caused by porcine reproductive and respiratory syndrome virus（PRRSV）is characterised of miscarriage,stillbirth in the late pregnancy and breathing disorders of all age pigs.As the intracellular parasitic organism,virus lacks its own translation system.It must rely on the host translation mechanism to synthesize its own protein and assemble virus particles.Numerous studies have shown that eukaryotic initiation factors play a key role in virus replication.Proteome analysis was employed to analyze two different pathogenic PRRSV strains infected pig alveolar macrophages（PAMs）.The results showed that both of the highly pathogenic PRRSV strain or classic PRRSV strain could significantly reduce eukaryotic translation initiation factor 5A（eIF5A）protein expression.Knocking down eIF5A by Si RNA can significantly inhibit the replication of PRRSV.Further studies confirmed that mTOR pathway played an important role in regulating PRRSV replication mediated by eIF5A.The results will aid in the understanding of molecular basis of the pathogenesis of PRRSV infection and further studies of the mechanism of PRRSV how to hijack the host’s translation system to promote self-replication.We conducted experiments as follows:1.Proteomic analysis of PRRSV infected PAMsThe samples of PRRSV BJ-4 and HN07-1 strain infected PAMs were detected by LC-MS/MS.A total of 430 significant differentially expressed proteins were identified from PAMs after infected by BJ-4,including 171 up-regulated proteins and 259down-regulated proteins;and 269 significant differentially expressed proteins were identified after HN07-1 infected PAMs including 46 up-regulated proteins and 223down-regulated proteins.Some differentially expressed proteins were identified by Western Blot and Real-time PCR.The results showed that the trend of the differentially expressed protein were consistent with LC-MS/MS results.KEGG Pathway and PPI analysis were performed by bioinformatics software on significantly differentially expressed proteins.The results showed that up regulated proteins were enriched to type I interferon signaling pathways;Down-regulated proteins mainly concentrate on protein translation,cytoskeleton related signaling pathways.2.The effect of eIF5A on PRRSV replicationProteomic analysis showed that the expression of eIF5A protein could be significantly reduced regardless of the highly pathogenic PRRSV strain or the classic PRRSV strain.To further study the effect of PRRSV infection on host cell translation system,PAMs were infected with PRRSV BJ-4 and HN07-1 respectively.Real-time PCR and Western Blot were used to detect the changes in transcription and protein level of eIF4E and eIF5A.The results showed that the protein expression of eIF5A was significantly down-regulated at 24 hpi,but the protein level of eIF4E was not significant changed.There were no significant changes in the transcription level both of eIF4E and eIF5A after PRRSV infection.eIF4E and eIF5A were knockdown by Si RNA in PAMs and CRL-2843-CD163 cells,respectively.The PRRSV replication was significantly inhibited when eIF5A was knockdown by IFA,Real-time PCR and Western Blot analysis.TCID₅₀ assay proved that knockdown eIF5A significantly reduced the PRRSV titer.But eIF4E had no significant effect on PRRSV replication.3.mTOR pathway plays a role in regulating the PRRSV replication of eIF5AeIF5A is the molecule downstream of the mTOR pathway.In order to further analyze the role of mTOR pathway in eIF5A regulating the PRRSV replication.PAMs were infected with PRRSV.The protein expression and phosphorylations of downstream signal molecules were detected by Western Blot.The results showed that p-mTOR and p-4EBP1,p-70S6K were significantly up-regulated after PRRSV infection.It indicated that PRRSV infection activated mTOR signal pathway.Rapamycin was found to inhibit the phosphorylation of 4EBP1 which is the key molecule in the downstream of mTOR signal pathway.And it was also found that Rapamycin can eliminate the inhibition of eIF5A caused by PRRSV infection and promote PRRSV replication.The results showed that mTOR pathway plays a role in regulating the PRRSV replication of eIF5A.4.Development of an immunochromatographic strip for detection of antibodies against porcine reproductive and respiratory syndrome virusThe recombinant PRRSV nonstructural protein 7（Nsp7）prepared by E.Coli.was purified by immobilized-metal affinity chromatography（IMAC）using a poly-histidine tag.The different states of aggregation such as polymer,dimer and monomer of the proteins were further separated by molecular sieve Superdex 200.Indirect ELISA test confirmed that only Nsp7 monomer protein could have specific response to PRRSV positive sera.The PRRSV antibody immunochromatographic test paper was successfully developed using the Nsp7 recombinant protein.The test strip was shown to be of high specificity and sensitivity.It had high coincidence rate with IDEXX ELISA kit and IPMA.It provided basis for rapid clinical diagnosis of PRRSV infection.