Mechanism Of Antagonizing RNAi In Tembusu Virus

Tembusu virus(TMUV)is a member of the Flavivirus family,and is a single-stranded positive-stranded RNA virus.Since 2010,Tembusu virus disease has been prevalent in China,which mainly causes a decrease in egg production and neurological symptoms in ducklings.And it has become an important infectious disease that seriously threatens the healthy and sustainable development of China's poultry industry.RNA interference(RNAi)plays an important function in the host's fight against viral infection.However,many viruses can evade host RNAi by encoding Viral suppressor of RNAi(VSR)during the long-term game.Current studies on flaviviruses have shown that their encoded non-structural proteins function as VSR to block RNAi,confirming the immunosuppressive effect of flaviviruses.In order to elucidate the mechanism of TMUV antagonistic RNAi,in the study,we established shRNA and siRNA-mediated EGFP-RNAi system on HEK293 cells,and explored the mechanism of TMUV antagonistic RNAi immune response with the prepared TMUV-NS2 A polyclonal antibody,then screened the VSR encoded by TMUV,and established dsRNAmediated EGFP-RNAi system on Drosophila S2 cells and validated it.1.Establishment of EGFP-RNAi system in HEK293 cellsIn the study,we firstly constructed anti-EGFP shRNA-mediated RNAi system,and overexpressed pEGFP-C1 eukaryotic expression plasmid with anti-EGFP shRNA in HEK293 cells.The results showed that anti-EGFP shRNA could significantly inhibit EGFP expression level.The pEGFP-C1 eukaryotic expression plasmid was cotransfected with the synthesized siRNA in HEK293 cells,and the results showed that anti-EGFP siRNA could significantly inhibit the EGFP expression level.2.Inhibition of shRNA and siRNA-mediated RNAi by TMUVIn order to investigate whether TMUV can antagonize RNAi,we constructed p ET-32aNS2 A prokaryotic expression vector,and transferred it into E.coli BL21(DE3)to express the recombinant protein and purified it,which was used as antigen to immunize Kunming mice,then the corresponding polyclonal antibody was prepared and characterized.The results showed that TMUV-NS2 A mice polyclonal antibody could be used for the detection of TMUV replication by Western blot with IFA.Subsequently,HEK293 cells were transfected with pEGFP-C1 eukaryotic expression plasmid and anti-EGFP shRNA were infected with TMUV.The results showed that TMUV significantly inhibited the shRNA-mediated RNAi response.Infection of HEK293 cells transfected with pEGFP-C1 eukaryotic expression plasmid and antiEGFP siRNA with TMUV showed that TMUV significantly inhibited siRNA-mediated RNAi responses.The above results showed that infection with TMUV upregulated the expression level of EGFP in shRNA and siRNA-mediated RNAi,indicating that TMUV hindered the RNAi process in HEK293 cells.3.Study on the role of TMUV-NS2 A in blocking RNAiIt has been shown that the non-structural proteins of flaviviruses evade host RNAi as VSR.In the study,seven non-structural proteins of TMUV(NS1,NS2 A,NS2B,NS3,NS4 A,NS4B and NS5)were used to investigate the molecular mechanism of inhibition of siRNA-mediated RNAi.The pEGFP-C1 eukaryotic expression plasmid,anti-EGFP siRNA and NS eukaryotic expression plasmid were cotransfected with HEK293 cells.The results showed that TMUVNS2 A protein had a significant inhibitory effect on RNAi response mediated by siRNA in HEK293 cells.To further verify the interaction between TMUV-NS2 A and RNAi,in the study,the pEGFP-C1 eukaryotic expression plasmid,anti-EGFP shRNA and NS2 A eukaryotic expression plasmid were cotransfected with HEK293 cells.The results showed that the TMUV-NS2 A protein in HEK293 cells had a significant inhibitory effect,indicating that NS2 A protein is a VSR encoded by TMUV that can inhibit the function of both siRNA and shRNA,further suggesting that the efficacy of NS2 A protein to exert VSR may occur during and after the production of mature siRNA.4.Establishment and validation of EGFP-RNAi system in S2 cellsIt has been shown that Drosophila has been a well-studied model organism for innate immunity.It is the RNAi pathway mediated by dsRNA that exerts primary antiviral immune efficacy.In the study,EGFP eukaryotic expression plasmid was constructed and EGFP-dsRNA was transcribed in vitro and overexpressed in Drosophila S2 cells.The results showed that EGFP-dsRNA significantly inhibited the expression level of EGFP.FB2 is a classical VSR.To further verify the above results,the protein expression level of EGFP in FB2 overexpressed cells was examined.The results showed that FB2 protein significantly up-regulated the EGFP protein expression level.In summary,the study investigated the mechanism of TMUV antagonism against shRNA and siRNA-mediated RNAi immune response,and demonstrated that TMUV NS2 A protein is a VSR.It provides more theoretical basis for understanding flavivirus and RNAi antiviral natural immunity.