Mechanism Of PRRSV Infection-Induced Stress Granule Formation

Stress granules(SGs)are dynamic non-membrane-bound organelles that are quickly assembled in eukaryotic cells due to translation arrest induced by exposure of cells to various stresses,such as heat shock,starvation,oxidation,ultraviolet irradiation,hypoxia,and viral infection.SGs are composed of messenger ribonucleoproteins(mRNPs)with the primary function of storing aborted mRNPs under various environmental stresses to protect them from degradation.SGs are very complex and dynamic in composition,involving many crucial proteins in various signaling pathways,suggesting their potential effects on the occurrence and development of many diseases,such as tumors,neurodegenerative diseases,and viral infections.G3BP1(RAS GTPase-activating protein SH3 domain-binding protein 1),one of the nuclear proteins of SGs,has been proved to be a novel antiviral protein that can inhibit the proliferation of various viruses.Porcine reproductive and respiratory syndrome(PRRS)is one of the most economically important diseases affecting the development of the global swine industry.In our study,we attempted to explore the molecular mechanism of SG formation induced by porcine reproductive and respiratory syndrome virus(PRRSV)and demonstrate that PRRSV induces the assembly of canonical SGs in a PERK-dependent manner.Moreover,PRRSV N protein is shown to evade the antiviral effect of G3BP1 by upregulating its phosphorylation level.This dissertation consists of four parts: 1.PRRSV infection induces the formation of canonical stress granules(SGs)To detect whether PRRSV infection induces SG formation,the localizations of endogenously expressed SG marker proteins(G3BP1 and TIA1)in PRRSV-infected cells were examined by indirect immunofluorescence assays(IFA)at different time points.The results showed that PRRSV infection induced the foci aggregation of TIA1 and G3BP1,leading to the efficient co-localization of G3BP1 with TIA1,suggesting that PRRSV induces SG assembly.Moreover,eukaryotic initiation factor 3?(eIF3?)was chosen as the marker protein of canonical SGs,and the co-localization of eIF3? with G3BP1 was observed at later stage in infection,indicating that PRRSV-induced cytoplasmic foci are canonical SGs.Finally,UV-inactivated PRRSV was identified to be unable to induce SG formation,implying that PRRSV replication is required for induction of SGs.2.PRRSV induces SG assembly in a PERK-dependent mannerTo further investigate the potential signaling pathways involved in the formation of PRRSV-induced SGs,we tested the phosphorylation level of eIF2? in PRRSV-infected cells through Western blot assays and found that PRRSV infection induced eIF2? phosphorylation,suggesting that PRRSV induces SG assembly via eIF2? phosphorylation.Furthermore,there are four eIF2? kinases responsible for the phosphorylation of eIF2?,including PKR,PERK,HRI and GCN2.In order to determine the eIF2? kinase involved in PRRSV-induced SG formation,cells were treated separately with inhibitors of PKR(C16 and 2-AP)or PERK(PERK-I: 516535 PERK Inhibitor I,GSK2606414).The results of indirect immunofluorescence assays indicated that neither C16 nor 2-AP inhibited the formation of PRRSV-induced SGs,while PERK-I significantly reduced the number of PRRSV-induced SGs.Moreover,considering that no specific inhibitors are available for GCN2 and HRI,their roles in PRRSV-induced SG assembly were tested by knockdown using specific siRNAs,and neither of them was found to influence the formation of PRRSV-induced SGs.These results suggested that the formation of SGs induced by PRRSV is dependent on PERK,rather than PKR,GCN2 or HRI.Finally,the results of Western blot assays showed that PERK-I could down-regulate PRRSV-induced eIF2? phosphorylation,further confirming the role of PERK in PRRSV-induced stress response.3.SGs and G3BP1 slightly inhibit PRRSV proliferationPrevious studies have shown that simultaneous knockdown of TIA1,TIAR and G3BP1 can significantly inhibit the production of SGs induced by sodium arsenite.Here,the effect of SGs on PRRSV proliferation was tested by simultaneous silencing of TIA1,TIAR and G3BP1 using specific siRNAs.The results indicated that inhibition of SG formation only slightly promoted PRRSV proliferation,but significantly promoted the expression level of PRRSV-induced inflammatory factors.G3BP1,one of the SG components,is a novel antiviral factor that antagonizes the proliferation of many viruses.However,in this study,both overexpression and stable expression of G3BP1 were shown to slightly inhibit PRRSV proliferation.Therefore,SGs and G3BP1 exhibit a slight inhibitory effect on PRRSV infection,suggesting that PRRSV may antagonize the antiviral effects of SGs and G3BP1.4.PRRSV N protein evades the antiviral effect of G3BP1 by increasing its phosphorylation levelPrevious phosphoproteomics of PRRSV-infected cells revealed that PRRSV increases the phosphorylation level of G3BP1,which was further confirmed by Western blot assays in this study.To detect the biological function of phosphorylation modification of G3BP1,non-phosphorylatable G3BP1 mutant(S149A/S231A)and phosphorylatable G3BP1 mutant(S149E/S231E)were constructed by mutating a pair of serine(S)codons at positions 149 and 231 to alanine(A)and glutamate(E),respectively.Mutant S149A/S231 A showed slightly higher anti-PRRSV ability than the wild-type G3BP1,while mutant S149E/S231 E exhibited no significant inhibitory effect on PRRSV multiplication,suggesting that PRRSV may evade the antiviral immunity of G3BP1 by upregulating the phosphorylation level of G3BP1,but the protein(s)of PRRSV involved in the immune escape is still unclear.Furthermore,previous quantitative interactome of PRRSV N protein revealed the interaction between N protein and G3BP1.Here,the interaction between G3BP1 and N protein was further tested under PRRSV infection or overexpression of N protein by Co-IP experiment,and PRRSV N protein was found to up-regulate the phosphorylation level of G3BP1.Overall,PRRSV escapes the antiviral effect of G3BP1 by inducing its phosphorylation through N protein.In conclusion,this dissertation explored the mechanism by which PRRSV infection induces SG formation.First,we revealed the aggregation of the SG marker proteins(TIA1,G3BP1 and eIF3?)to foci in the cytoplasm of PRRSV infected cells,and their co-localization with each other,indicating that PRRSV induces the formation of canonical SGs.Additionally,we proved that PRRSV induces SG formation by increasing the phosphorylation level of eIF2? through PERK.However,SGs and their component G3BP1 could only slightly inhibit the proliferation of PRRSV.Finally,we demonstrate that PRRSV antagonizes the antiviral effect of G3BP1 by up-regulating the phosphorylation level of G3BP1 through N protein.