The Relationship Between TRIM22 And JEV Nonstructural Protein 1

Japanese encephalitis(JE)is a zoonosis arbovirus disease caused by Japanese encephalitis virus(JEV).This disease is harmful to humans.It’s one of the the most common arbovirus diseases of human central nervous system.JE has also brought big damage to swine industry.It can cause pigs reproductive disorders and piglets encephalitis.Pigs are the amplifying host and the reservoir host of JEV,and it plays a very important role in the pig-mosquito-human spread chain.At present,there are no effective drugs for JE treatment,so it’s a priority to enhance the JEV anti-virus research and search for the effective prenvention and trentment methords.Many members of TRIM(Tripartite motif-containing)family proteins can regulate the innate immune signaling pathways and have the antiviral function,and they are important regulatory factors in antiviral innate immune response.It is reported that TRIM22 could inhibit the proliferation of HIV and influenza virus and other viruses.Based on these reports,we did a preliminary study on the JEV proliferation modulated by TRIM22 and the mechanism between them.These results provide new methods to the prevention and researching effective drugs of JEV.The main contents are as follows:1.The influence of TRIM22 on the proliferation of JEVWe connected human TRIM22 gene with vector p TRIP-CMV-3Flag-IRES-RFP,and successfully constructed the recombinant plasmid.Then we overexpressed TRIM22 on HEK293T cells,and infected these cells with JEV SX09S-01 by 1.0MOI after 24 h of the overexpression.We collected the supernatant and cells to do the plaque-forming assay on BHK-21,the quantitative Western blot and the quantitative Real-Time PCR experiment.The plaque forming assay statistics showed that the number of plaques formed after JEV infection in cells with transient transfection of TRIM22 recombinant plasmid is significantly reduced by 30%-40% relative to the control group.We analysised the expression of NS3 protein in HEK293 T cells after JEV infection by quantitative Western blot test.The result showed that the expression of NS3 protein in negative control group was significantly higher than that in transfected group.The quantitative Real-Time PCR result also showed that the copy number of viral particles in the positive group is significantly lower than the negative group.All the results of these experiments showed that overexpression of TRIM22 on cells could inhibit JEV significantly.2.The preliminary mechanism of TRIM22 antiviral action to JEVIn order to investigate whether TRIM22 inhibits the proliferation of JEV by degradating proteins durning the replication through ubiquitylation pathway,we co-transfected pc DNA3.0-TRIM22-HA with plasmids that can express C、E、NS1、NS2A、NS2B、NS3、NS4A、NS4B and NS5 proteins of JEV on the HEK293 T cells respectively.Co-immunoprecipitation experiment showed that TRIM22 protein can interact with NS1 protein.Indirect immunofluorescence result also showed that TRIM22 protein with JEV NS1 protein co-localized in the cytoplasm.At the same time,the result of co-transfected of pc DNA3.0-TRIM22-HA and PCR3.1-NS1-Flag showed that the expression of NS1 is on the rise with the increase of TRIM22 protein expression.So the role of the specific interaction between TRIM22 and NS1 in anti-proliferation needs in-depth study.