The Molecular Mechanism Of TRIM21 And TRIM25 Affecting GETV Replication

Gatah virus(GETV)is a mosquito-borne virus with many host species and wide transmission range.GETV infection can cause fetal death in sows and morbidity in neonatal piglets at the beginning of pregnancy.Pigs are the main amplified host of GETV in nature and play a key role in the transmission of GETV.Currently,it has been reported that Tripartite motif(TRIM)family molecules,as E3 ubiquitin ligases,are involved in the regulation of the pathogenesis of various diseases.In this study,eukaryotic expression vectors of TRIM21 and TRIM25 genes were used to explore the roles of TRIM21 and TRIM25 in GETV infection.TRIM21 is widely expressed in various cells,especially immune cells,and plays an important role in antiviral and antimicrobial immune regulation of host.In this study,we found that TRIM21 was upregulated after GETV infected PK-15 cells.At the same time,TRIM21 was overexpressed on PK-15 cells and then the cells were infected with GETV.The GETV E2 protein was detected by Western blot,qPCR and TCID50.The results showed that: TRIM21 promoted the replication of GETV on PK-15cells;On the contrary,PK-15 cells interfered with endogenous TRIM21 were infected with GETV showed that interference TRIM21 inhibited the replication of GETV.This relults fully demonstrates that TRIM21 promotes GETV replication in PK-15 cells.GETV is an enveloped virus that cannot be recognized and intercepted by cytoplasmic TRIM21,and cannot exert an antibody-dependent intracellular neutralisation(ADIN).Therefore,we focused on the effect of TRIM21 on the replication of GETV through the regulation of innate immunity.Given the complex regulation of TRIM21 on IRF3 and its promotion of GETV replication,we suspected that TRIM21 promoted GETV replication by promoting the degradation of IRF3 and inhibiting the expression of IFN-?.Next,we overexpressed TRIM21 in PK-15 cells and detected the expression of IFN-? in m RNA level by qPCR,which proved that overexpression of TRIM21 could inhibit the production of IFN-? in PK-15 cells whether infected or not infected with GETV.We detected the activity of IFN-? promoter by double luciferase assay and found that TRIM21 could inhibit the activity of IFN-? promoter,which further verified this conclusion.PK-15 cells that overexpressed TRIM21 were stimulated by GETV or poly I:C.Western blot results showed that the expression levels of IRF3 and p-IRF3 were decreased.In this study,we demonstrated that TRIM21 promoted GETV replication by inhibiting the expression of IRF3 and then down-regulating IFN-?.TRIM 25 is involved in a variety of cellular processes,including the regulation of antiviral innate immune responses.In recent years,other roles of TRIM25 in early innate immunity have emerged,including negative regulation of RIG-I,activation of the antiviral axis of melanoma differentiation-associated protein 5 and mitochondrial antiviral signaling protein TRAF6,and regulation of p53 levels and activity.In this study,we found that overexpression of TRIM25 significantly inhibited the replication of GETV in PK-15 cells by overexpression of TRIM25.On the contrary,knockdown TRIM25 can promote GETV replication.Therefore,TRIM25 can inhibit the replication of GETV in PK-15 cells and is an anti-GETV factor.In the experiment of overexpression of TRIM25,it was found that the overexpression of TRIM25 gradually decreased with the extension of GETV infection time,so it was speculated that the protein encoded by GETV might degrade TRIM25.The capsid protein(Cap protein)of GETV was analyzed as a serine protease,so we focused on the relationship between Cap protein and TRIM25.The eukaryotic expression plasmid of Cap protein was co-transfected with TRIM25-Flag in 293 T cells,it was found that CAP protein could degrade TRIM25 protein in a dose-dependent manner.By immunoprecipitation and laser confocal experiments,we further confirmed the interaction between Cap protein and TRIM25.By truncating the Cap protein,we found that the key region of the interaction between Cap protein and TRIM25 protein to play the peptidase degradation function was between 1-112 amino acids.In addition,we examined the effect of Cap protein and TRIM25 on IFN-? promoter activity,and found that Cap protein antagonized the promoter effect of TRIM25 on IFN-?.In this study,we found that while TRIM25 exerts a strong antiviral effect against GETV invasion and replication,GETV Cap protein antagonizes the antiviral effect of TRIM25 by degrading TRIM25 protein.In summary,the molecular mechanisms of TRIM21 promoting GETV replication and TRIM25 inhibiting GETV replication were analyzed in this study.This study deepens the understanding of the interaction between the virus and the host,and fills in the gap of basic research on GETV,which is of great significance for the treatment and prevention and control of GETV,and also provides an important theoretical basis for the development of drugs against GETV and other mosquito-borne viruses.