The Study Of Interactions Between PRRSV And Interferon Stimulated Genes Viperin,DCP1a

ISGs?Interferon stimulated genes?belong to a large kind of antiviral effectors,which are induced by interferon.ISGs exert as the ultimate effectors to control the viral infection.Viperin?Virus inhibitory protein,endoplasmic reticulum associated,interferon inducible?and DCP1a?m RNA-decapping enzyme 1a?are two classic ISGs.Viperin belongs to the member of radical SAM?S-adenosylmethionine?superfamily enzymes.DCP1a is one of important factors in P bodies?Processing bodies?.Previous studies showed that Viperin and DCP1a had the ability to inhibit many kinds of viruses,but the antiviral mechanisms were different among diverse viruses.PRRSV?Porcine reproductive and respiratory syndrome virus?has continuously damaged the global swine industry since it was discovered in 1987 in the United States.Previous studies had reported that p Viperin?porcine Viperin?suppressed PRRSV proliferation.However,the mechanism of p Viperin inhibiting PRRSV replication remained mysterious.Besides,the effect of p DCP1a?porcine DCP1a?on PRRSV replication was still unknown.Our study demonstrated that p Viperin inhibited PRRSV replication via degrading the viral nsp1?and p DCP1a slightly inhibited PRRSV replication.Further studies showed that PRRSV nsp4 cleaved p DCP1a to impair its antiviral activity.Herein,our work containing two parts are as follows:1. The mechanism of porcine Viperin inhibiting PRRSV replicationTo investigate the effect of p Viperin on PRRSV replication,it was found that overexpression of p Viperin inhibited PRRSV replication in MARC-145 cells and knockdown of p Viperin promoted PRRSV proliferation in porcine alveolar macrophages.Our study comfirmed that p Viperin had the ability to restrict PRRSV replication.Previous studies reported that Viperin inhibited TBEV?Tick-borne encephalitis virus?and ZIKV?Zika virus?via degrading the viral NS3 proteins.To analyse whether p Viperin degrade PRRSV proteins,cells were cotransfected with the eukaryotic expression plasmids of all PRRSV encoding proteins and p Viperin respectively.Unexpectedly,we found that p Viperin could exclusively degrade the viral nsp1??nonstructural protein 1??.Proteasome pathways,autophagy pathways and apoptosis pathways are main three protein degradation pathways.To further investigate the potential pathway involved in this degradation,cells were treated with the inhibitors of proteasome?MG132?,apoptosis?Z-VAD-FMK?,and autophagy?3-MA?,respectively.It was found that the treatment of either MG132 or 3-MA could partly restore the expression level of nsp1?.Meanwhile,Co-IP?coimmunoprecipitation?experiments demonstrated that p Viperin interacted with nsp1?and the two proteins colocalized in the cytoplasm,indicating that this interaction mediated the degradation.To analyse the mechanism of proteasome pathway,cells were transfected with p Viperin,nsp1?and wild type Ub?ubiquitin?or its mutants?K6-Ub,K11-Ub,K27-Ub,K29-Ub,K33-Ub,K48-Ub,K63-Ub?respectively.It was found that p Viperin promoted the ubiquitination of nsp1?by adding wild type Ub or K29-Ub,indicating that p Viperin catalyzed the K29-linked ubiquitination of nsp1?.Together,the three nsp1?lysine mutant plasmids were constructed?nsp1?-K117A,nsp1?-K150A,nsp1?-K169A?and then were cotransfected with p Viperin to cells respectively.We found that the expression level of nsp1?-K150A was partly restored compared with the wild type nsp1?.At the same time,the Co-IP assay showed that p Viperin hardly catalyzed the K29-linked ubiquitination of nsp1?-K150A compaired with nsp1?.These results indicated that p Viperin-mediated proteasome pathway degraded nsp1?by targeting the K150 site of nsp1?for K29-linked polyubiquitination.To further investigated the mechanism of p Viperin-mediated apoptosis pathway,it was found that overexpression of p Viperin could induce apoptosis through western blot and firefly luciferase reporter assays.Three D?Aspartic acid?residues in nsp1?were subsequently replaced with A?nsp1?-D6A,nsp1?-D149A,nsp1?-D172A?.And then cells were cotransfected with the three nsp1?mutants and p Viperin plasmids respectively.It was found that the abundance of nsp1?-D149A mutant was partly restored compared with the wild type nsp1?.Consistently,cells transfected with nsp1?-D149A and p Viperin were treated with MG132,it was found that the amount of nsp1?-D149A was almost completely restored.To further comfirmed the above data,the double sites mutant plasmid?nsp1?-D149A/K150A?was constructed and then was cotransfected with p Viperin to cells.The results indicated that p Viperin could hardly degrade nsp1?-D149A/K150A.These data indicated that p Viperin targeted D149 sites of nsp1?for apoptosis pathway degradation.Recent studies have demonstrated that the SAM enzyme activity of human Viperin was necessary for inhibiting flavivirus replication.To investigate whether the SAM enzyme activity of p Viperin participated in degrading nsp1?,cells were constransfected with p Viperin-M?Viperin mutant lacking SAM enzyme activity?and nsp1?.It was found that the abilities to degrade nsp1?between p Viperin-M and p Viperin were similar.Moreover,we found that both p Viperin and p Viperin-M showed a similar antiviral effect on PRRSV replication,demonstrating that the SAM enzyme activity of p Viperin did not take a part in inhibiting PRRSV infection.In conclusion,this study demonstrated that p Viperin degraded viral nsp1?via proteasome pathway and apoptosis pathway to restrict PRRSV replication,revealing a novel streategy employed by p Viperin to inhibit PRRSV infection.2. Porcine DCP1a inhibiting PRRSV infection and PRRSV antagonizing the antiviral activity of porcine DCP1aPrevious studies demonstrated that h DCP1a?human DCP1a?was an ISG.However,whether p DCP1a belongs to ISG remains unknown.It was found that the expression of p DCP1a was upregulated under the treatment of IFN-?in PAMs,indicating that p DCP1a was also an ISG.To investigate the effect of p DCP1a on PRRSV replication,it was found that overexpression of p DCP1a slightly suppressed PRRSV infection and knockdown of p DCP1a promoted virus replication,which suggested that PRRSV may employ some strategies to impair the antiviral activity of p DCP1a.To further analyze the effect of PRRSV infection on p DCP1a expression,it was found that PRRSV infection had no effect on the m RNA level and the location of p DCP1a,but cleaved p DCP1a to downregulate the protein level of p DCP1a.To investigate the mechanism of the cleavage mediated by PRRSV,cells were transfected with all PRRSV encoded protein plasmids with p DCP1a.It was found that PRRSV nsp4,the main proteinase,was responsible for p DCP1a cleavage,and this cleavage was dependent on its protease activity.To confirm the cleavage site of p DCP1a,we constructed a series of E?Glutamic acid?mutants of p DCP1a?p DCP1a-E230A,p DCP1a-E238A,p DCP1a-E240A,p DCP1a-E241A,p DCP1a-E243A?based on the cleaved product.Cells were transfected with these p DCP1a mutants with PRRSV nsp4,it was found that nsp4 could not cleave p DCP1a-E238A mutant,which suggested that the cleavage site is at E238 of p DCP1a.To investigate the significance of PRRSV nsp4 cleaving p DCP1a,cells were transfected with p DCP1a?p DCP1a-E238A or the two deletion mutants(p DCP1a₁–₂₃₈,p DCP1a₂₃₉–₅₈₀)respectively and then were infected with PRRSV.It was found that the mutant p DCP1a-E238A showed a higher anti-PRRSV activity,and the antiviral activities of two deletion mutants were dramatically impaired compared with wild type p DCP1a.At the same time,the similar results were also obtained from the p DCP1a knockout cell line.To further analyze conservation of the cleavage site among diverse species,the amino acid sequences of h DCP1a?p DCP1a and m DCP1a?monkey DCP1a?were chosed for alignment.It was shown that the corresponding amino acid at residue 238 of m DCP1a and h DCP1a was D,which suggested that PRRSV nsp4 probably could not cleave h DCP1a and m DCP1a.To verify this hypothesis,we detected the protein level of m DCP1a in MARC-145 cells infected with PRRSV.It was indicated that PRRSV infection did not degrade m DCP1a.Meanwhile,cells were transfected with m DCP1a and nsp4 plasmids,which further confirm that PRRSV nsp4 could not cleave m DCP1a.Moreover,it was found that m DCP1a showed a higher inhibitory activity against PRRSV than p DCP1a.In summary,these data demonstrated that PRRSV nsp4 cleaved p DCP1a to impair its antiviral activity,revealing a new mechanism employed by PRRSV to dampen the host defense.