SILAC-Based Quantitative Proteomic Analysis Of Secretome Of Marc-145 Cells Infected With PRRSV

Porcine reproductive and respiratory syndrome(PRRS),caused by porcine reproductive and respiratory syndrome virus(PRRSV),is characterized by severe reproductive failure in sows and respiratory disease in young and growing pigs.PRRS has been causing significant economic losses to the swine industry worldwide since its first outbreak in USA and Canada in 1987.It is also an importrant infectious disease overwhelming the swine industry in China.Previous research has demonstrated that PRRSV induces the expression of inflammatory cytokines and release of proteins involved in the inflammatory response.For example,PRRSV infection of Marc145 cells or porcine alveolar macrophages(PAMs)triggers the release of HMGB1 from the nucleus to the extracellular milieu,which enhances the efficiency of virus-induced inflammatory responses,suggesting that secretory proteins might play an important role in PRRSV pathogenesis.However,the current research of host response to PRRSV infection relies on the concerted release of proteins with various biological activities,and no comprehensive analysis of the secretory proteins has been conducted to date regarding the host response upon PRRSV infection.In this study,we applied proteomic technique for global profiling of PRRSV-infected Marc-145 cells secretome and performed intensive bioinformatic analysis for secretome data.Subsequently,we verified that PRDX3 plays a role of pro-inflammatory cytokine and enhances the efficiency of PRRSV-induced inflammatory responses.The major studies include:1.Secretome analysis of PRRSV-infected Marc-145 cellsStable isotope labeling with amino acids in cell culture(SILAC)combining with tandem mass spectrometry(LC-MS/MS)was used to quantitatively identify the secretory proteins differentially expressed in Marc-145 cells infected with PRRSV compared against mock control Marc-145 cells.In total,we identified 204 secretory proteins showing significant differences in PRRSV-infected Marc-145 cells(163 up-regulated,41 down-regulated).Revealed by the intensive bioinformatic analysis of secretome data,we found that PRRSV infection strongly activate the nonclassical protein secretion,especially vesicle-mediate release of exosomal proteins,including different DAMPs(danger-associated molecular pattern molecules)and a majority of secreted proteins involved in protein binding and transport,regulation of response to stimulus,metabolic process and immune response.According to the functional proteins analysis,we speculated that proteins functioning in binding,transport and immune response were exploited by PRRSV to participate in virus replication and immune evasion.Immunoblotting and qRT-PCR were used to detect four differentially expressed proteins in the dada,and the result was consistent with the result of mass spectrometry.2.PRDX3 plays a role of pro-inflammatory cytokinePrxs(peroxiredoxins)are important endogenous antioxidants.Previous studies reported that PRDX1 secretion from cancer or virus-infected cells could bind to the cell surface alarm signal receptor toll-like receptor 4(TLR4)and induce the activation of NF-?B and release of IL-6 and TNF-?.In our study,we found that PRRSV infection induced the release of PRDX3,then investigated the potential role of PRDX3 in pro-inflammatory responses.To study the function of extracellular PRDX3,we applied prokaryotic expression and purification.Through dual-luciferase assay,immunoblotting,IFA assay and qRT-PCR,we found that the extracellular addition of purified recombinant PRDX3 protein to Marc-145 cells and PK-15 cells promotes the activation of NF-?B promoter,phosphorylation levels of p65 and nuclear translocation and IL-6,IL-8,TNF-? mRNA expression levels,suggesting that it plays a role of pro-inflammatory cytokine.To further explore whether TLR4 is also involved in PRDX3-induced inflammatory cytokines expression.PK-15 cells were transfected with TLR4-specific siRNAs before treated with purified PRDX3 protein.Cells were collected for detecting cytokines expression by real-time PCR or dual-luciferase assay.The results showed that knockdown of TLR4 inhibited PRDX3-induced IL-6,IL-8,TNF-? mRNA expression levels and the activation of NF-?B promoter.Besides,addition of PRDX3 protein could not induce the activation of NF-?B promoter in TLR4 deficient HEK-293 T cells,but PRDX3 restored this ability in TLR4 overexpressed HEK-293 T cells.suggesting that it plays an important role in enhancing expression of inflammatory cytokines.3.PRDX3 is involved in PRRSV-induced NF-?B activation and expression of inflammatory cytokinesIndirect immunofluorescence assay found that PRRSV infection did not alter the mitochondrial location of PRDX3 in Marc-145 cells.In addition,we found that PRRSV infection up-regulated the expression of PRDX3 in the supernatant and slightly down-regulated the intracellular expression of PRDX3.Previous studies have reported that PRRSV infection would cause mitochondria injury.In our secretome data,PRRSV robustly activated vesicle-mediate release of proteins,so we deduced that the proteins from the injured mitochondria may be packaged into exosomes and released to the extracellular space.We detected whether PRDX3 effects PRRSV replication subsequently.Through immunoblotting and qRT-PCR,we found the extracellular addition of purified recombinant PRDX3 protein to Marc-145 cells does not significantly affect PRRSV proliferation under our experimental conditions.To examine the effects of PRDX3 on PRRSV-induced NF-?B inflammatory responses,we added purified recombinant PRDX3 protein to Marc-145 cell supernatant after PRRSV incubation.The rusults showed that addition of recombinant PRDX3 enhanced PRRSV-induced phosphorylation levels of p65.To further explore the effects of PRDX3 on PRRSV-induced inflammatory cytokines expression.PRRSV-infected Marc-145 cells were transfected with PRDX3-specific siRNAs or treated with purified recombinant PRDX3 protein.Cells were collected for detecting cytokine message expression by real-time PCR.The results showed that addition of recombinant PRDX3 enhanced PRRSV-induced IL-6,IL-8,TNF-? mRNA expression levels,and knockdown of PRDX3 promoted these effects,indicating that the peroxidase activity of intracellular PRDX3 affected the inflammatory response mediated by ROS.The above results suggested that extracellular and intracellular PRDX3 plays an important role in PRRSV-induced inflammatory cytokines expression.