Molecular Mechanism Of Interferon-stimulated Gene 15 Inhibiting The Replication Of Peste Des Petits Ruminants Virus

Peste des petits ruminants(PPR)is a highly infectious,highly pathogenic,and lethal infectious disease of goats,sheep,and other small ruminants caused by peste des petits ruminants virus(PPRV).Since its emergence in 1942,PPR has been widely prevalent in the world,causing huge economic losses for the world livestock industry.To control and eradicate the occurrence and prevalence of PPR as soon as possible,it is urgent to develop new antiviral drugs while using vaccine immunization prevention.In general,virus infection stimulates the body to produce interferon(IFN)response and stimulates the production of a large number of Interferon-stimulated genes(ISGs)to resist the infection of pathogenic microorganisms.As a kind of important cytokines for resistance to pathogenic microorganisms,there are few kinds of research on the resistance of ISGs to PPRV infection.To screen effective ISGs molecules against PPRV infection,molecular biological methods were used to screen ISGs molecules PPRV infection,their antiviral mechanisms were studied.This study mainly includes the following four aspects.Screening of PPRV cell infection model:Since PPRV is usually cultured on interferon-deficient African green monkey kidney cells(Vero),studies on PPRV infection and restriction of the interferon signaling pathway cannot be carried out.To solve this problem,three goat cell lines that can proliferate PPRV were firstly screened in this study:Caprine endometrial epithelial cells(EECs),goat skin fibroblasts cells(CSFs),and goat fibroblast cells(GFs).By detecting the expression of interferon in infected cells,it was found that these three kinds of cells could activate the interferon signaling pathway.However,it is noteworthy that PPRV infection only stimulated the expression of type I and type III IFNs in EEC cells.The expression of type I and type III IFNs was not stimulated in both CSF and GF cells.Therefore,the EEC cell line was selected as the cell model for this study.Screening of goat ISGs and molecular bioinformatics analysis:By constructing a variety of eukaryotic plasmids that up-regulated the expression of ISGs in transcription,and screening of ISGs molecules with anti-PPRV infection.The results revealed that ISG15 was significantly up-regulated in PPRV-infected cells and could significantly inhibit the proliferation of PPRV.To understand the molecular basis of ISG15 inhibition of PPRV proliferation,bioinformatics analysis was performed in this study.The basic properties,structure,and function of goat ISG15 molecule were interpreted.The role of ISG15 in PPRV proliferation:By constructing ISG15 overexpression cell lines,knockdown cell lines,and si RNA interference assay,the function of ISG15 in inhibiting PPRV proliferation was further demonstrated.Moreover,the inhibitory effect of ISG15 on PPRV proliferation was found to be dose-dependent.Furthermore,we demonstrated that type I IFN could induce significant expression of ISG15 in EEC cells,which in turn up-regulate the expression of the IFN-βpathway and other inflammatory factors in overexpressing ISG15 cell lines.Although this study demonstrated that PPRV infection can cause upregulation of ISGylation in EEC cells,it was also found that the N protein,P protein,and V protein of PPRV could not be ISGylated.Molecular mechanism of ISG15 inhibiting PPRV replication:The experimental results of this study demonstrated that ISG15 could inhibit the PPRV replication process through the free ISG15,but had no significant effect on the processes such as PPRV banding and entry.Further studies showed that free ISG15 could inhibit PPRV replication by competitively binding the first 50 amino acids of the N-terminal of the P protein with the N protein,which prevents the P protein from binding with the N protein to form the N~0-P complex,thereby disrupting the balance between viral transcription and replication.On the basis,the 77-101 amino acids of ISG15 were identified as the main functional domain to inhibit PPRV replication.In conclusion,this study is the first to identify the inhibitory role of ISG15 in PRRV replication and to elucidate its molecular mechanism at the molecular level.The results of this study provide a new theoretical basis for further study on the molecular pathogenesis of PPRV and the interaction between host and microbe.