Swine IFITM Inhibits Japanese Encephalitis Virus Via Iteraction With ABHD16A

Japanese encephalitis B(Epidemic encephalitis B)is a serious zoonoses infectious disease caused by the Japanese encephalitis virus(JEV).The damage of Japanese encephalitis virus in the world is becoming more and more serious.Interferoninduced transmemberane proteins(IFITMs)are small transmembrane proteins induced by interferon(IFNs).Studies have shown that IFITM family proteins have broad spectrum antiviral capabilities.Preliminary laboratory studies preliminarily demonstrated that porcine-derived interferon-induced transmembrane proteins(s IFITMs)inhibit JEV action.To further investigate its mechanism and the role in the development of Japanese encephalitis,related studies were conducted.The results are as follows:1.Real-time fluorescence quantitative PCR was used to detect the replication of JEV when overexpressing IFITMs.The results showed that overexpression of s IFITM1,s IFITM2,and s IFITM3 proteins in PK15 and HEK293 cells significantly inhibited the replication of JEV at 24 h,48 h,and 72 h,of which IFITM1 had the strongest ability to inhibit virus replication.To further verify the effect of s IFITM1 on virus replication,s IFITM1 was knocked out in PK15 cells and ST cells by CRISPR/cas9 technology.The results showed that deletion of s IFITM1 significantly increased JEV virus replication at 24 h.2.In order to explore the anti-JEV mechanism of s IFITMs,s IFITM1 with strong anti-JEV effect was selected as the object,and the interacting proteins were screened and studied.Yeast two-hybrid experiments confirmed the interaction between s IFITM1 and s ABHD16 A.In PK15 and HEK293 cells,the co-localization of s IFITM1 and s ABHD16 A on the membrane structure was found by laser confocal subcellular localization technique.After JEV infected cells,the co-localization of s IFITM1 and s ABHD16 A was transferred with virus infection.3.The cell culture supernatant was detected by overexpressing s ABHD16 A in BHK21,L02 and HEK293 cells at 24 h,48h and 72 h.The results showed that s ABHD16 A significantly inhibited JEV virus replication.The CRISPR/cas9 knockout vector p HMG-sg RNA-ssg RNAabhd16 a was constructed and the sabhd16 a was knocked out in PK15 and ST cells.The infection of the cells with JEV for 24 hours had a significant increase in JEV replication relative to the control group.4.The p HMG-sg RNA-ssg RNAabhd16 a was transfected into HEK293 cells.A monoclonal cell line with stable deletion of s ABHD16A-/-was selected and constructed.DNA sequencing showed that s ABHD16 A was knocked out successfully in HEK293 cells.5.To investigate the effect of s IFITM1 and s ABHD16 A interaction on JEV infection,s IFITM1 and s ABHD16 A expression plasmids were co-transfected into BHK-21 cells.The results showed that the antiviral effect of co-expression of the two proteins was significantly stronger than that of s IFITM1 alone,indicating that s ABHD16 A Synergistic anti-JEV effect.6.In order to study whether the anti-JEV effect is related to the S-palmitoylation of s IFITM1,a S-palmitoylation site mutant expression plasmid ?s IFITM1(s IFITM1-C50A-C51A-C84A)was constructed and transfected into PK15 cells.Compared with transfected s IFITM1,s ABHD16 A and s IFITM1/ s ABHD16 A,the inhibition of JEV was significantly decreased when transfected with s IFITM1,and the synergistic antiJEV activity was significantly decreased when s IFITM1/ s ABHD16 A was cotransfected.This result initially demonstrated that the anti-JEV effect of s IFITM1 is related to its palmitoylation,and the palmitoylation of this site may be related to s ABHD16 A.7.To determine whether s IFITM and s ABHD16 A are involved in the regulation of Japanese encephalitis,fluorescence quantitative PCR was used to determine the expression of inflammation-related factors in cells overexpressing s IFITM1 or s ABHD16 A.The results showed that IL-1?,IL-6,IL-10,TNF? and COX2 levels increased significantly.However,when s IFITM1/s ABHD16 A was co-expressed,the m RNA of these cytokines was significantly reduced to below the control level with the exception of COX2.The m RNA levels of IL-1?,IL-6 and TNF-? increased significantly in JEV-infected cells expressing s ABHD16 A,whereas the expression of inflammatory factors in JEV-infected cells expressing s IFITM1/s ABHD16 A was significantly higher than that in control and other infected cells.Its decrease indicated that s IFITM1 could play an important role in regulating the balance of inflammation.The results above obtained in this study not only provide a series of experimental materials for following research,but also provide theoretical support for the prevention and treatment of Japanese encephalitis.