Molecular Mechanism Of The Host Proteins CH25H And S100A9 Regulating The Replication Of Porcine Reproductive And Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome virus(PRRSV)is a highly immunosuppressive respiratory transmission pathogen,which cause a huge impact on pork production worldwide.The viral genome is highly variable,prone to gene recombination,and has multiple immunosuppressive mechanisms.Mixed or secondary infections are usually observed in clinic.Currently,commercial vaccines cannot provide adequate protection against the increasingly mutated PRRSV infection.Although we have been studying the virus for more than 30 years and have made some progress,the pathogenesis of the virus has not been thoroughly understanded.Cholesterol-25-hydrogenase(CH25H)is an interferon-inducible protein that oxidizes cholesterol to 25-hydroxy-cholesterol(25HC).Studies have shown that CH25H and 25HC have broad-spectrum antiviral activities against many kinds of envelope and non-envelope viral replication.However,the effects and mechanisms of CH25H on PRRSV replication remain unclear.S100A9 is a damage-related molecular pattern(DAMP),which can regulate host immune function.S100A9 activates NF-?B and MAPK pathways through TLRs and RAGE receptors to regulate host injury,inflammation and apoptosis.Our previous proteomics data showed that PRRSV infection could promote the expression of S100A9,but the role of S100A9 in PRRSV replication is not clear.This study is divided into the following three parts:1.Cholesterol 25-hydroxylase is an interferon-inducible factor that protects against PRRSV infection.In this study,we found that PRRSV infection significantly promotes CH25H transcription in Marc-145 and PAM cells,and the effect is most obvious at 6 hours after infection,then gradually decreased,it indicated that CH25H is an early gene induced byPRRSV.The expression of CH25H was significantly up-regulated in IFN-? treated Marc-145 cells,suggesting that CH25H is an interferon-inducible gene(ISG).CH25H was overexpressed in Marc-145 cells by transfection of eukaryotic expression plasmids,and its effect on PRRSV replication was analyzed by western blot,IFA and RT-qPCR.The results showed that CH25H significantly inhibited PRRSV replication in a dose-dependent manner.The mutant CH25H-M,which lost enzyme activity,was obtained by site direct mutation and was transfected into Marc-145 cells,it was found that the mutant CH25H-M could still inhibit PRRSV replication,but its antiviral activity was significantly lower than that of CH25H.In addition,25HC also showed significant anti-PRRSV activity.Then we studied the effect of 25HC on PRRSV replication cycle.We found that 25HC could significantly inhibit the internalization of PRRSV through the analysis of viral adsorption,internalization,replication and release,while 25HC had no toxic effect on PRRSV activity.Moreover,we also studied the effect of 25HC on the replication of different PRRSV strains,and the results showed that 25HC had good inhibitory activity against various PRRSV strains.These results indicate that CH25H,as an ISG,can inhibit the internalization of PRRSV by producing 25HC.25HC may become a good anti-PRRSV drug in the future.2.25-Hydroxycholesterol provides antiviral protection against PRRSV challenge in swine.In this study,the toxicity of 25HC on PAM cells(CC50)and the half inhibitory concentration of PRRSV(IC50)were measured,it showed that 25HC had good antiviral activity and much low cytotoxicity.In addition,the levels of IL-1? and IL-8 in PAM cells were significantly up-regulated by treating with 25HC.In vitro mimicry treatment experiments showed that 25HC treatment pre-and post-infection significantly inhibited the replication of PRRSV in PAM cells.In the pig treatment experiment,25HC was given daily for 10 days,and the control group was given the same amount of ethanol.The results showed that the 25HC treatment group had better mental status and lower body temperature than the control group,and the piglets survival rate increased significantly.The viral loads in lungs,nasal swabs and blood were measured.It was found that 25HC could significantly reduce the viral loads in lungs,nasal swabs and blood.Lung pathological sections and immunohistochemical results showed that the lungs of 25HC treatment group had only slight lesions and contained a small number of positive cells infected with PRRSV.In the control group,the lung lesions were obvious,and the interstitial mass was significantly widened,the alveoli collapsed,and contained a large number of positive cells infected with PRRSV.This study shows that 25HC has good clinical application prospects.3.S100A9 inhibits PRRSV replication by interacting with PRRSV N proteinCalcium binding protein S100A9 is a damage associated molecular pattern(DAMP)discovered in recent years.Generally,S100A9,a regulatory factor,forms a complex with S100A8 to regulate host injury,inflammation and apoptosis through NF-?B and MAPK signaling pathways.However,little research has been done on the relationship between S100A9 and viral replication.This study explored the effect of PRRSV infection on the expression of S100A9 and S100A9 on PRRSV replication,as well as its related mechanisms.The results showed that PRRSV infection could significantly promote the expression of S100A9.Overexpression and knockdown analysis showed that S100A9 had significant anti-PRRSV activity.Adding intracellular calcium chelating agent BAPTA to Marc-145 cells overexpressing S100A9 can reduce the antiviral activity of S100A9,which proves that the antiviral activity of S100A9 depends on the presence of intracellular Ca2+.Using immunoprecipitation and laser confocal technique,we found that S100A9 interacted with N protein of PRRSV and colocated in cytoplasm.Using site-direct mutation technique revealed that the key sites of S100A9 and N protein interaction were located at E78 of S100A9 and S36-R37 of N protein,respectively.Reverse genetic manipulation technique was used to rescue mutant virus rBB(36/37),and we found that the inhibitory activity of S100A9 on the mutant virus was decreased.Moreover,the interaction between S100A9 and N protein antagonized the inhibitory effect of N protein on host IFN-(3 pathway.The inhibition effect of S100A9 on PRRSV replication was further confirmed by lentivirus-mediated overexpression or RNA interference mediated knock-down of S100A9 in PAM cells.In addition,the study also found that S100A9 can inhibit PRRSV replication by regulating intracellular autophagy levels.The study firstly discovered the antiviral function of S100A9 on PRRSV and elaborated its anti-PRRSV mechanism.It should be helpful to understand the pathogenesis mechanism of PRRSV.