The Anti-PRRSV By The Porcine Innate Immune Signaling Adaptor Proteins And The Antagonism By PRRSV Protein

Porcine Reproductive and respiratory syndrome(PRRS)is the viral infectious disease caused by porcine reproductive and respiratory syndrome virus(PRRSV),which has caused serious damage to the global swine industry.The main clinical manifestations of the virus infection are reproductive disorders such as abortion,fetal death or weak birth of sows and severe respiratory diseases of pigs of various ages.At present,because of the high genomic diversity,antigenic heterogeneity and antibody-dependent enhancement(ADE),vaccination strategies are very limited for controlling PRRSV infections.Innate immunity is a crucial factor in the host’s resistance to the invasion of pathogenic microorganisms and is the primary defense against infections.It is typically initiated by various pathogen-associated molecular patterns(PAMPs)recognized by the body’s pattern recognition receptors(PRRs),activating the natural immune signaling pathways and anti-infection effects.The PRRs including Tolllike receptors(TLRs),RIG-I-like receptors(RLRs),C-type lectin family(CLRs),NOD-like receptors(NLRS),and Cytoplasmic DNA receptors(CDRs).When activated,these receptors transmit signals to the signaling adapter proteins,which in turn activate the expression of transcription factors IRF3/IRF7 and NF-κB through a series of signal cascades,thereby inducing the production of antiviral type I interferon(IFN-I)and pro-inflammatory cytokines to resist the infection of pathogens.Viral DNA or RNA is the essential PAMP of viruses.As an RNA virus,PRRSV is primarily sensed by innate immune RNA receptors,and the DNA sensors in PRRSV infection have not been investigated and its role remains unclear.Here,the porcine innate immune signaling pathways were systematicly investigated for anti-PRRSV function,the role of DNA receptor cGAS-STING pathway in PRRSV infection was studied,and the interactions between signal adaptor protein STING and PRRSV proteins and the effects of interactive regions on cellular signaling and PRRSV replication were also studied.1.Screening of porcine innate immune signaling adaptors revealed several anti-PRRSV signaling pathwaysPRRSV shows extensive and strong inhibitory effect on innate immunity,and the innate immunity plays an important role in the recognition and defense of PRRSV.The current strategies for controlling PRRSV are limited and complete understanding of anti-PRRSV innate immunity is needed.In this study,we expressed the nine porcine signaling adaptors(pmCherry-Cl-MyD88,TRIF,MAVS,RIPK2,ASC,CARD9,BCL10,MALT1 and STING)which represent all currently known innate immune receptor signaling pathways.The analysis of PRRSV N gene transcription and protein expression both suggested the multiple ectopic adaptors exhibited varying degrees of anti-PRRSV activities.Among these,TRIF and MAVS were most effective,MyD88,RIPK2,CARD9/BCL10/MALT1(CBM)and STING were intermediate in inhibition,and ASC was barely effective in inhibition of PRRSV.In order to better evaluate the antiviral effects of innate immune adaptor proteins,the GFP signal of recombinant PRRSV(rPRRSV)from reverse genetics was measured by flow cytometry and similar anti-PRRSV activities by different signaling adaptors were observed.These results demonstrated that the seven innate adaptors signaling are all likely involved in the recognition and defense during PRRSV infection.Based on the screening data and considering the importance of viral nucleic acid in innate immune response,endogenous TRIF,MAVS and STING were selected for further examination of anti-PRRSV activity.Several agonists were used to trigger the endogenous innate signaling,including the poly(I:C)-HMW for TLR3-TRIF,poly(I:C)-LMW for RIGI/MDA5-MAVS,poly(dA:dT)for cGAS-STING and 2’3’-cGAMP for STING.These results suggested that endogenous MAYS and STING other than TRIF in Marc-145 cells can inhibit PRRSV replication.On the other hand,the siRNA knockdown experiment showed that TRIF,MAVS and STING are all involved in anti-PRRSV activity.The variation in the effectiveness of endogenous TRIF in inhibiting PRRSV replication may be attributed to the impaired TLR3 signaling in the utilized Marc-145 cells.Nevertheless,the above results further collaborated that both MAVS and STING signaling exert anti-PRRSV function.2.The innate immune DNA sensing cGAS-STING signaling pathway mediated antiPRRSV functionIt has been reported that cGAS-STING signaling pathway plays an important role in DNA virus infection.As an RNA virus,PRRSV is mainly sensed by innate immune RNA receptors,whereas the role of innate immune DNA sensors in the PRRSV infection has not been elucidated.Here,we investigated the roles of DNA sensing cGAS-STING pathway in both PRRSV infected Marc-145 cells and porcine macrophages.To explore the role of STING in PRRSV infection,we first prepared the porcine STING-GFP stable expressing Marc-145 cells after G418 selection.The dsRed signal of rPRRSV from reverse genetics was measured by flow cytometry and the anti-PRRSV activity of stable expressed GFP-STING in cells was observed.Further,Marc-145 cells were stimulated with poly(dA:dT)and 2’3’-cGAMP,respectively,and the results showed that both agonists inhibited the PRRSV replication.To further verify the antiviral effect of STING,we made the homologous STING knockout Marc-145 cell clones by CRISPR-Cas9 method.Four homologous STING knockout cell clones were obtained and two cell clones(2-7-1 and 1-4-2)were selected for subsequent experiments.The results showed that STING exerts antiviral activity against PRRSV in Marc145 cells.To investigate the anti-PRRSV role of STING in porcine macrophages,we developed CD163-PAMs(3D4/21),which stably express GFP tagged porcine CD163 and successfully support PRRSV replication.In CD 163-3D4/21 cells,the replication of PRRSV was inhibited by STING overexpression;Secondly,the CD163-3D4/21 cells were stimulated with poly(dA:dT)and 2’3’-cGAMP,respectively,and the results showed that both agonists suppressed the PRRSV infection significantly.Thirdly,the CD163-3D4/21 cells were treated with STING siRNA,and the results showed that PRRSV replication increased in STING knockdown cells.Taken together,the results demonstrated clearly that STING exerts antiPRRSV function in both Marc-145 cells and porcine macrophages,and cGAS-STING pathway possesses an important function against PRRSV infection.3.PRRSV Nsp5 counteracted the cGAS-STING-IFN-P Study on antiviral signalingIn this study,8 structural proteins and 14 non-structural proteins of PRRSV were cloned and constructed in recombinant expression plasmids.The Co-IP screening of these viral proteins interacting with STING first revealed that the non-structural proteins Nsp3,Nsp5 and structural protein E of PRRSV interact with STING.Among the three STING interacting viral proteins,Nsp5 could inhibit the activity of ISRE and IFN-β promoters mediated by the cGASSTING pathway.In addition,Nsp5 could inhibit the phosphorylation of TBK1 and IRF3 mediated by cGAS-STING signal pathway in a dose-dependent manner.For the underlying mechanism,Nsp5 could interfere the STING to recruit TBK1 and IKKε through its interaction with STING,and thus inhibit the cGAS-STING pathway.In order to dissect the interacting regions between STING and Nsp5,different truncation mutants of both STING and Nsp5 were constructed.The Co-IP using the different truncation mutants showed that the 1-153 aa domain of STING was identified as the key structural domain for its interaction with Nsp5,and the 36-47 aa and the 58-67 aa region of Nsp5 were necessary for the interaction with STING.Next,we attempted to clarify the effect of Nsp5 on the cGAS-STING pathway in the context of PRRSV infection.Based on the rPRRSV XJ175/EGFP strain,the mutant viruses with Nsp5 deletion residues 36-47 aa,58-67 aa,or 36-47 aa/58-67 aa were prepared.However,none of the Nsp5 deletion rPRRSV strains could be successfully rescued despite the successful rescue of control rPRRSV.In conclusion,Nsp5 mediated the counteraction by PRRSV on the antiviral effect of cGAS-STING pathway.