Mechanism Of IRAV And TRIM21 Regulating PEDV Proliferation

Porcine epidemic diarrhea is a highly contagious intestinal infectious disease caused by porcine epidemic diarrhea virus(PEDV).This disease is susceptible to pigs of all ages,and can cause 100 %fatal effects on piglets within three days.In recent years,the outbreak of variant PEDV in China,even the world has caused huge economic losses to the existing pig industry.PEDV,one of the main pathogens causing porcine epidemic diarrhea,is a single-stranded plus stranded RNA(ssRNA)virus.The protein with the highest content in virions is PEDV N protein.Meanwhile,PEDV N protein is also the structural protein most produced in the process of intracellular replication,playing an important role in the process of virus proliferation.A novel antiviral protein-IRAV interacting with PEDV N protein was screened out through co-IP technology in the preliminary experiment of our laboratory.In order to further explore the effect of IRAV on PEDV proliferation and its mechanism,this study determined the effect of IRAV on PEDV through overexpression and interference,and conducted in-depth research from the analysis of transcription factors regulating IRAV after PEDV infection of host cells and the inhibitory effect of IRAV on PEDV proliferation.The results are as follows:Due to the absence of IRAV specific antibodies,monoclonal antibodies against swine IRAV were obtained by hybrid tumor technology in this study: First,the prokaryotic expression vector of porcine IRAV was constructed successfully.The pig IRAV protein with the expected size was successfully induced in escherichia coli.Next,the purified IRAV protein was immunized against female BALB/c mice,and five strains of hybrid tumor cells secreting monoclonal antibodies against IRAV were obtained,named as 2B10,2G12,2H1,5A8 and 2C5,respectively.Finally,Western-blot and indirect immunofluorescence analysis showed that the antibody had a specific reaction with PK-15 overexpressed porcine IRAV protein.In conclusion,monoclonal antibodies against porcine IRAV protein were successfully prepared in this study,laying a foundation for further experiments.After PEDV infected Vero or PK-15 cells,RT-PCR results showed that the expression of IRAV was up-regulated.Amplifing and analyzing the promoter sequence of IRAV,the results showed that PEDV was able to promote IRAV by up-regulating the transcription factor EGR1 expression.co-IP and colocalization experiments confirmed that IRAV can interact with PEDV N protein.When investigating the interaction between them,it was found that IRAV can degrade N protein by gradient.After inhibitor treatment and interference with key molecules in the degradation pathway,IRAV's degradation of PEDV N protein was inhibited.The above proves that IRAV can utilize the ubiquitinated protease MARCH8 ubiquitinated N protein,and then finally degrade PEDV N protein through the autophagy-lysosomal pathway and ubiquitination pathway,and exert its effect of inhibiting PEDV proliferation;This study also found that IRAV can degrade other coronavirus N proteins in a gradient.In order to further explore the molecular mechanism of host cells using IRAV to inhibit PEDV proliferation,TRIM21 was screened molecules by co-IP and mass spectrometry sequencing.This study found that: TRIM21 molecule can also inhibit PEDV proliferation by degrading PEDV N protein.Above all: In this study,the anti-virus mechanism was clarify from two aspects of transcription factors regulating IRAV after PEDV infection in host cells and the inhibitory effect of IRAV and TRIM21 on PEDV proliferation.The mechanism of IRAV and TRIM21 inhibiting PEDV proliferation was elaborated in detail,providing new research ideas for preventing and controlling PEDV.