Molecular Mechanism By Which Zinc Finger Antiviral Protein Limit Replication Of Small Ruminant Viruses

Pete des petits ruminants?PPR?,commonly known as sheep plague,is an acute,highly contagious viral disease caused by small ruminant viruses?peste des petits ruminants virus,PPRV?,which mainly occurs in goats,sheep and camels Wait for ruminants.The main clinical features of the disease are high fever,conjunctivitis,ocular nasal mucositis and erosive stomatitis,and diarrhea.Its pathogen?peste des petits ruminants virus,PPRV?is a member of the Paramyxoviridae,measles virus genus.The genome size of PPRV is 15948 nt,encoding 6 structural proteins and 2 non-structural proteins.PPRV is difficult to stabilize and low-proliferate in sheep-derived cells,which leads to the relatively backward research of PPRV natural immunity and pathogenic mechanism.The host restriction factor is a part of the natural immune system,and is an early line of defense formed by the evolution of the host against infection by pathogenic microorganisms.A variety of restrictive factors have been found,including Zinc finger antiviral protein?ZAP?.ZAP is a host restriction factor encoded and expressed by the ISG ZC3HAV1gene,which has obvious inhibitory effects on various viruses and plays an important role in the natural immune process.At present,there are few anti-virus studies on ovis aries ZAP?oZAP?,especially the relationship with PPRV infection has not been reported.To this end,this paper first cloned the sheep-derived ZAP,and explored the role and molecular mechanism of ZAP in the process of PPRV infection and replication,with a view to providing new clues for further research on the pathogenic mechanism of PPRV and the natural immune mechanism.The research of this paper mainly includes the following three aspects:1.Cloning and sequence analysis of ovis aries ZAP:First,the total nucleic acid of sheep lungs was extracted,and the oZAP gene was successfully amplified using the specific primers designed.Then,bioinformatics analysis showed that the content of serine?Ser?in oZAP protein was the highest.The half-life is 30h,which is an unstable hydrophilic protein;there is no signal peptide and transmembrane structure,there are 114 potential phosphorylation sites and 5 glycosylation sites.In the prediction of secondary structure,it participates in the formation Randomly coiled amino acids are the most;the functional domain prediction found that the protein has Zinc finger domain,WWE domain and polyribose polymerase functional domain.It provides valuable information for further research on the antiviral effect of oZAP protein.2.Research on the relationship between oZAP and PPRV proliferation:First,we constructed the oZAP eukaryotic expression plasmid,and then transfected this plasmid into Vero-SLAM cells to overexpress oZAP.Then,PPRV was used to infect the ZAP overexpressing cell line,and RT-PCR,TCID₅₀,and Western blot were used to detect the proliferation level of PPRV.The results showed that over-expression of oZAP can significantly inhibit the proliferation of PPRV,and showed a relevant dose dependence.To further prove that oZAP can limit the replication of PPRV,we used small RNA interference technology to silence oZAP expression in sheep endometrial cells?EEC?,and then infected the cells with PPRV.Then the same test method was used to detect the proliferation level of PPRV,and it was found that the proliferation level of PPRV was significantly higher than that of the control group.Therefore,we believe that oZAP is a restrictive cytokine that has a significant inhibitory effect on PPRV proliferation.3.Research on the molecular mechanism of oZAP inhibition of PPRV proliferation:Using dual luciferase reporter gene system and RNA electrophoresis mobility analysis?REMSA?experimental technology,we found that oZAP can interact with PPRV structural proteins?N protein,P protein,M protein and F protein?5?UTR interaction of the coding gene;further research we also found that the expression level of N,P,H,V and C was significantly reduced by co-transfection of recombinant plasmids of oZAP and PPRV protein,and showed a dose-dependent The sexual relationship proves that oZAP can inhibit PPRV at the translation level.Through mass spectrometry analysis and confocal microscope observation,we also found that oZAP interacts with PPRV P protein and co-localizes in the cell.In summary,this paper successfully cloned and expressed the oZAP protein.For the first time,experiments have proved that oZAP has a significant inhibitory effect on the proliferation of PPRV,and found that oZAP may limit PPRV by inhibiting various mechanisms such as viral transcription and translation.Proliferation in cells.This study provides important clues for the follow-up study of the pathogenic mechanism and natural immune mechanism of PPRV.