Mechanism Of PEDV Nonstructural Protein 15 Inhibiting IFN?

Porcine epidemic diarrhea virus is the momentous pathogen of porcine intestinal diarrhea disease.The essential symptoms of porcine epidemic diarrhea were vomiting,watery diarrhea,dehydration and loss weight.As the second most considerable pathogen of pig industry in China,PEDV has caused huge financial losses to the pig industry.Currently studies had shown that rapid and strong natural immune response was not produced after PEDV infected.It suggested that may evade the antiviral immune response of host cells through some strategies.Previous studies was found that PEDV significantly inhibited the production of interferon in Vero cells.Previous studies was found that PEDV infected Vero cells can significantly inhibit the production of interferon.The study found that at least 11 viral proteins of PEDV can inhibit the production of interferon,such as ORF3,E,M,N,Nsp1,NSP3,Nsp5,Nsp8,Nsp14,Nsp15,Nsp16 inhibited IFN activity by inhibiting nuclear translocation of NF-?B,TBK1,IRF3,IRF1,and degrading STAT1,interference with RIG-I/STING ubiquitination and other different signal pathways.In this study,PEDV(named PEDV-BJ19 strain)was successfully isolated from Vero cells.By analyzing the proliferation of PEDV on different cells,Vero cells were determined to be the most susceptible cells of PEDV-BJ19 strain,which not only produced lesions,but also showed obvious fluorescence 24 hours after infected.Nextgeneration sequencing of PEDV-BJ19 was successful and submitted to the Gen Bank database.The results of PEDV-BJ19 genetic evolutionary tree analysis showed that the strain belonged to GI type.The susceptibility Porcine epithelial cells to infected PEDV was poor.In order to explore the reasons for the poor sensitivity of IPEC-J2 cells to PEDV,we analyzed the transcriptomics of IPEC-J2 cells 12 hpi,24hpi and 48 hpi after PEDV infection.The results showed that the number of differentially expressed genes up-regulated and down-regulated was the highest in the 24 hpi group.At the same time,we verified that the transcriptional levels of IRF1,IRF7,IFITM1 and IFITM3 were consistent with the transcriptomic results,which proved that the transcriptomic results were highly reliable.Afterwards we also found that porcine IFITM3 could inhibit the proliferation of PEDV in IPEC-J2 and Vero cells.Meanwhile,porcine IFITM3 also inhibited the proliferation of rVSV-GFP in A549,BHK21 and DF1 cells,indicating that porcine IFITM3 had broad-spectrum antiviral activityIt is well known that the replication of PEDV in Vero affected the production of interferon.Our experiments showed that PEDV infection of Vero cells inhibited IFN ?by the degradation of MAVS which depended on the ubiquitin proteasome system.The supernatant collected from PEDV infected Vero cells also significantly promoted the proliferation of rVSV-GFP compared with the supernatant without infection..PEDV Nsp15 was a potent protein to antagonist of interferon.We explored the mechanism of Nsp15 involved in antagonizing interferon.Previous studies found that PEDV Nsp15 inhibited poly(I:C)that inducted.Co-localization and double luciferase reporting experiment of Nsp15 and RLR upstream protein indicated that Nsp15 inhibited IFN? by inhibiting RLR pathway and antagonized upstream of IRF3 or before IRF3 was activated.Regulatory effect of Nsp15 on MAVS was contrary to the trend at the transcriptional level and protein level by Western blot and QRT PCR experiments.Degradation of MAVS was mainly through the ubiquitin proteasome system by proteasome inhibition and autophagy inhibition experiments.As we all know,Nsp15 was an endonuclease that involved in the formation of transcriptional complex.In order to investigate whether the endonuclease activity of Nsp15 affected the degradation of MAVS,we found that the degradation of MAVS didn't depend on the endonuclease activity of Nsp15.CO-IP experiment also showed that Nsp15 interacted with MAVS protein.In addition,overexpression of PEDV Nsp15 protein also promoted the replication of rVSV-GFP.This study clarified that PEDV and Nsp15 antagonized IFN ? by inhibited MAVS for the first time.It provided train of thought for further exploring the mechanism of escaping innate immune response of porcine epidemic diarrhea virus and the research and development of antiviral small molecule drugs.