Role Of N Protein Phosphorylation Site Mutation In PRRSV Pathogenicity And Molecular Mechanisms Of Ginsenoside Rg1 In Anti-PRRSV

Porcine reproductive and respiratory syndrome virus(PRRSV)can cause clinical symptoms such as fever,dyspnea,and reproductive disorders in sows,and brought huge losses to pig industry in China.Nucleocapsid(N)protein of PRRSV participates in viral genome replication and virion assembly.Our previous study had found that there are phosphorylation modifications at Ser 105 and 120 of N protein.In order to clarify the function of it,the present study analyzed the effects of phosphorylation sites mutation of N on PRRSV replication and pathogenicity,and screening for proteins that interacted with N protein in porcine alveolar macrophages(PAMs).It provides a basis for the function of N and the role that N played in the molecular mechanism of PRRSV pathogenesis.In this study,we first studied the effect of mutations in the phosphorylation site of N protein on the replication and subgenomic transcription of PRRSV in PAM cells.N protein A105 and 120 Ser were mutated to Ala,and the results showed that the viral replication rate and final titer of the mutant virus A105-120 was significantly lower than the parental virus XH-GD in Marc-145(PA105<0.01,PA120<0.01,PA105-120<0.01).Although the replication rate of A105-120 was lower than XH-GD between 12-36 hpi,but there was no significant difference in final titer between the two viruses in PAMs.RT-PCR was used to analyze the subgenomic transcription of mutant viruses and parental viruses in PAMs.The transcription levels of gRNA,sgRNA2 and 3 of mutant viruses were significantly reduced(P<0.05;P<0.01;P<0.05),but the expression of short-chain sgmRNA4 and 5 increasing over time(P<0.01;P<0.05).It indicates that the mutation of N protein phosphorylation site can affect the replication of PRRSV in PAMs.Virus replication rate in host cells is a key factor affecting its pathogenicity.Therefore,whether the N protein phosphorylation site affected the pathogenicity of PRRSV piglets was evaluated in the present study.The results showed that compared with the parental strain XH-GD,the mutant strain A105-120 infected piglets showed milder clinical symptoms and lung damage.All 6 piglets in the XH-GD infection group developed hyperthermia(body temperature>40.5℃),with a mortality rate of 50%,while only two animals in the A105-120 infection group developed hyperthermia,with a mortality rate of 16.67%.The weight of piglets in the A105-120 challenge group increased steadily,which was significantly higher than that in the XH-GD infection group(P<0.001)between 9-18 dpi(days post infection).The replication of A105-120 in piglets is significantly lower than that of XH-GD,which is mainly reflected in the serum viral load(P<0.001)between 3-7 dpi and the tissue viral load in the lung or lymph nodes at 7 and 14 dpi(P<0.001).This shows that N protein phosphorylation is related to the PRRSV pathogenicity.Virus-mediated host cell life process is another key mechanism of its pathogenicity.In order to explore potential molecular mechanisms of virus pathogenic differences related to N protein phosphorylation site mutations,Label-Free unlabeled quantitative proteomics analysis was used to identify the differential expressed proteins in lung lavage fluid in infected piglets.A total of 1941 proteins were identified for differential protein expression analysis.GO,KEGG and PPI analysis of 2-fold differential proteins revealed that XH-GD and A105-120 infections can cause a large number of differential proteins that enriched in innate immunity,immune regulation,endoplasmic reticulum protein processing and various metabolic processes.However,the differential proteins between the two group are mainly concentrated in innate immunity and immune regulation,involving biological processes such as membrane signal transduction,fibrin production and complement activation.Further,transcriptomics was used to identify the differential expressed genes in the alveolar macrophages of piglets infected with phosphorylation site mutant viruses,and the differences in potential signal transduction mechanisms were analyzed.A total of 15,746 genes were identified in this study.Compared with A105-120,XH-GD infection upregulated genes were mainly enriched in NF-κB,MAPKs and TNF signal transduction involved in inflammatory and immune response,etc;down-regulated genes are mainly concentrated incell tight junctions and ECM receptor interactions,etc.In vitro infection of PAMs by mutant viruses,P65 phosphorylation and IκB degradation analysis showed that the NF-κB activation induced by A105-120 infection was weaker than XH-GD.And the expression of the inflammatory factors,IL-1β(P<0.01),IL-6(P<0.05),IL-8(P<0.05)and TNF-α(P<0.01),were lower than that of XH-GD.It shows that the mutation can attenuate PRRSV-mediated NF-κB activation and inflammatory factor expression.The interaction between proteins is the basis of host life activities,but the host interaction protein of PRRSV N protein lacks identification.In this study,the yeast twohybrid technique was used to construct the PAM cell cDNA library pGADT-PAM,and the sequence of N protein of PRRSV XH-GD strain was used to construct the bait vector pGBKT7-ORF7.After toxicity test,self-activation test,co-transformation and sequencing,18 candidate interaction proteins were obtained,15 candidates were not reported in PRRSV related studies.Silencing the gene expression of candidate interaction proteins by siRNA found that RPL23,UBR5,TERF2IP/Rap1,PCBP1,ESRRG,SFPQ,FTH1 and AIF1 silencing significantly affect the expression of Nsp9 in infected Marc-145 cells.Construction of eukaryotic expression plasmids pcDNA3.1-PCBP1,pcDNA3.1TERF2IP/Rap1,pcDNA3.1-AIF1,pcDNA3.1-RPL23 and pcDNA3.1-FTH1,after transfection with Marc-145 cells,they colocalized with N protein.And over-expression of PCBP1,TERF2IP/Rapl and FTH1 can promote the replication of PRRSV in Marc-145 cells,and after overexpression of RPL23 and AIF1 can inhibit the replication of PRRSV.In addition,the protein interaction between N protein and TERF2IP/Rapl was verified in the yeast two-hybrid system,suggesting that PRRSV could promote its own replication through the interaction between N protein and TERF2IP/Rapl.Immuno-suppression caused by PRRSV infection poses a huge challenge to vaccine development,and new antiviral strategies are urgently needed.This study investigated whether ginsenoside Rg1 has anti-PRRSV effects in vivo and in vitro,and its antiviral molecular mechanism.The results showed that Rg1 had less cytotoxicity and could inhibit the replication of different lineages of type II PRRSV in both Marc-145 and PAM cells.Rg1 can inhibit virus replication from virus attachment,replication and release.Rg1 can reduce the expression of IL-1β,IL-6,IL-8 and TNF-α in PRRSV-infected Marc-145 cells and PAM cells,and inhibit the PRRSV-mediated p65 phosphorylation and IκB degradation.By detecting the phosphorylation level of mTOR,p70S6K and 4E-BP1,it was found that Rgl attenuates the inhibitory effect of PRRSV on mTOR signaling by activating mTOR.Further,using puromycin to label nascent polypeptides,it was found that Rgl activation of mTOR can attenuate the translational shut-off caused by PRRSV infection in a dosedependent manner.At the same time,the increased level of autophagy marker LC3-Ⅱprotein the upregulated mRNA levels of autophagy-related genes ATG5,ATG7 and Beclinl caused by PRRSV infection were significantly reduced in the Rg1 treatment group,indicating that Rg1 can attenuate autophagy caused by PRRSV infection.In addition,4 weeks old piglets infected with HP-PRRSV JXA1 were treated with Rg1 injection.The clinical symptoms and lung lesion scores of the piglets in the Rg1 treatment group at 7 days were significantly lower than those of the challenge control group(P<0.05),and the course of the disease was extended after Rg1 treated.The survival rate of the piglets in the challenge control group at 11 dpi was 0%,while in the Rg1 treatment group at 14 dpi was 40%.The serum viral load of the Rg1 treatment group was significantly lower than that of the challenge control at 7(P<0.05)and 10 dpi(P<0.01)after challenge,and the viral load of the lung,lymph nodes and thymus tissue was also significantly lower than the challenge control(P<0.01).It shows that ginsenoside Rg1 can be used as a potential natural compound for PRRSV prevention and control.In summary,it was confirmed that the mutation of Ser to Ala at positions 105 and 120 of N protein reduced PRRSV replication and subgenome transcription in PAM cells,and reduced virus pathogenicity to piglets.The potential molecular mechanism of this mutation affecting the pathogenicity of PRRSV was preliminarily analyzed by means of proteomics and transcriptome.Further,the interacted protein of N protein in PAMs was screened,and the effect of candidate interaction proteins on PRRSV replication was analyzed initially.The existence of protein interaction between N protein and TERF2IP/Rapl was verified in yeast two-hybrid system.Finally,the efficacy and molecular mechanism of ginsenoside Rg1 against PRRSV were described in vitro,and the therapeutic effect of Rg1 against PRRSV in vivo was preliminarily evaluated.This study provides a research basis for pathogenic mechanism of PRRSV and the effect of phosphorylation on the function of N protein.It also provides theoretical support and clinical data for the use of traditional Chinese medicine as a new PRRSV antiviral strategy.