Inhibitory Effects And Mechanisms Of Porcine MSTNpro-antiviral Peptide On The Porcine Reproductive And Respiratory Syndrome Virus In Vitro

Myostatin propeptide is an inhibitor of myostatin mature peptide and can stimulate muscle growth in livestock.EB and CVN are broad-spectrum antiviral bioactive peptides.In previous studies,the fusion expression vectors of porcine myostatin propeptide and EB or CVN antiviral peptide(pcDNA3.1-MSTNpro-EB/CVN)were successfully constructed and their abilities to promote muscle growth and resist PRRSV were demonstrated in our laboratory.It is relatively clear how myostatin propeptide promote muscle growth.But the inhibitory mechanisms of EB and CVN against PRRSV are unclear.Thus,using Marc-145 as an in vitro model,we further studied the molecular mechanisms of EB and CVN antiviral peptides against PRRSV in order to provide basic theory for developing new antiviral gene engineering drugs.In the test,the cells were divided into normal cells and the cells treated by PRRSV,in which non-transfected group,pcDNA3.1 empty vector group,pcDNA3.1-MSTNpro group,pcDNA3.1-MSTNpro-EB group and pcDNA3.1-MSTNpro-CVN group were established respectively.The inhibitory effects of porcine MSTNpro-antiviral peptide against PRRSV were detected by Annexin V-EGFP/PI double staining and its influence on some PRRSV-related receptors and cytokines mRNA expression were detected by qRT-PCR.The results showed that:? Compared with the empty vector group,the mRNA levels of MSTNpro,MSTNpro-EB and MSTNpro-CVN were significantly increased in the corresponding transfection groups.The results showed that the three target genes were highly expressed in Marc-145 cells.?Compared with the empty vector group,the cells survival rates in the MSTNpro-EB group and the MSTNpro-CVN group significantly increased after cells were treated by PRRSV,indicating that EB and CVN antiviral peptides had inhibitory effects on PRRSV.? For the non-transfected cells,compared with normal cells,the expression of CD 163 mRNA significantly decreased and the expression of IL-6 and TNF-a mRNA significantly increased after cells were treated by PRRSV.? For the normal cells,compared with the empty vector group,the expression of CD 163 and CD151 mRNA significantly decreased in the pcDNA3.1-MSTNpro-EB group and the expression of CD 163 mRNA significantly decreased in the pcDNA3.1-MSTNpro-CVN group;For the cells treated by PRRSV,compared with the empty vector group,the expression of CD 163 and CD151 mRNA significantly decreased in the pcDNA3.1-MSTNpro-EB group and the expression of CD 163 mRNA significantly decreased in the pcDNA3.1-MSTNpro-CVN group.? For the normal cells,compared with the empty vector group,the expression of IL-6 mRNA significantly increased and the expression of TNF-a significantly decreased in the pcDNA3.1-MSTNpro-EB group;For the cells treated by PRRSV,compared with the empty vector group,the expression of IL-6 mRNA significantly increased and the expressions of TNF-a and HSPB1 mRNA significantly decreased in the pcDNA3.1-MSTNpro-EB group,and the expression levels of TNF-a and HSPB1 mRNA significantly decreased in the pcDNA3.1-MSTNpro-CVN group.The above results showed that the cell itself tended to improve the ability to resist PRRSV by down-regulating the expression of CD 163 receptor mRNA and up-regulating the expressions of immune response factors IL-6 and TNF-a mRNA.EB peptide tended to resist PRRSV invasion by down-regulating the expressions of CD151 and CD 163 receptors mRNA.CVN protein tended to resist PRRSV invasion by down-regulating the expression of CD 163 receptors mRNA.EB and CVN could down-regulate the mRNA expression levels of TNF-a and HSPB1 that were immune response and stress response factors needed for resisting virus,which might because EB and CVN inhibited virus invasion;meanwhile EB could up-regulate the mRNA expression level of IL-6 to promote a part of the immune response and to increase the resistance of cells to PRRSV.This study will lay a foundation for further illuminating the antiviral mechanisms of EB and CVN at protein level.