The Regulatory Mechanism And Function Of Deubiquitinase USP4 Against EV71 Infection Through RLR Signaling Pathway

Objective: To study the regulatory mechanism and function of deubiquitinase USP4 against EV71 infection through RLR signaling pathway and further understand the relationship between EV71 infection and its escape immune defense,which could provide a novel theoretical basis for the treatment of EV71 infection in clinical.Methods: The differential expression of deubiquitinase genes was screened by PCR microarray.The left hind leg muscle tissue from EV71 infected-AG129 mouse was collected,and the viral load and expression of USP4 were detected by RT-PCR.The levels of IRF3,NF-?B p65 protein and their phosphorylation were analysed in EV71 infected-rhabdomyosarcoma(RD)cells by Western blot.RLR signaling pathway in RD cells was activated by poly(I:C).When USP4 was knockdowned or overexpressed by si RNA or plasmid transfetion,the activity of NF-?B luciferase reporter genes was detected.The interaction between USP4 and TRAF6 was analyzed by immunoprecipitation.Study on the function of TRAF6 by deubiquitinating enzyme USP4 after co-transfection of USP4,TRAF6 and Ub.USP4 expression plasmid was transfected into RD cells,RT-PCR and Western blot were used to detect the replication of EV71,and cells apoptosis was detected by flow cytometry.In addition,the plasmids of non-structural protein 2A and 3C in EV71 was constructed and the mechanism of USP4 degradation was investigated.MG132,Q-VD and STS reagents were added to further investigate the mechanism of 2A protease cleavage of USP4.Results: The expression of USP4 reduced in the process of EV71 infection,while after EV71 infected mouse,the transcription level of USP4 was attenuated.The phosphorylation of IRF3 and NF-?B p65 were suppressed in EV71-infected cells.USP4 regulates RLR-induced NF-?B signaling positively and has no significant change to IFN signaling pathway.TRAF6 is a key protein in the RLR-induced NF-?B signal pathway which plays essential roles in the activation of NF-?B.We also found that USP4 could interact with TRAF6 for removing K48 ubiquitin chain to reduce the degradation of TRAF6 and activate the expression of downstream cytokines to exert antiviral function.Meanwhile,the multiplication of EV71 can be inhibited by USP4 overexpression.In addition,non structural protein 2A in EV71 may cleave the USP4 protein and reduce its function.STS induces cell apoptosis without Cutting of USP4.2A protease still cuts USP4 after MG132 and Q-VD inhibition.Conclusion: USP4 plays a positive regulatory role in RLR antiviral signaling pathway,2A proteinases cleaves USP4 protein to promote TRAF6 degradation through proteasome pathway,so as to suppress immune response of host cells,maintain virus proliferation itself,and escape host immune defense.