The Deubiquitinase USP52-mediated Regulation Of Type Ⅰ Interferon Signaling Pathway And Antiviral Immunity

Objective:At present,virus infection accounts for more than 3/4 of infectious diseases in the world,which is a serious threat to human life and health.The purpose of this study is to find that a deubiquitin enzyme(DUBs)affects the antiviral activity of interferon by regulating the ubiquitization of key proteins,which provides a new strategy and target for clinical application of interferon antiviral therapy.method:We collected blood samples from patients with clinically confirmed virus infection,extracted peripheral blood mononuclear cell(PBMC),and analyzed the difference of USP52 between patients with virus infection and normal people by RT-qPCR technique.The CRISPR-Cas9 plasmid that can knock down USP52 was constructed,and the USP52 in cells was knocked down.The antiviral activity of USP52 against VSV virus was analyzed by RT-qPCR,Western Blot and inverted fluorescence microscope.The antiviral ability of USP52 knockdown was evaluated by MEF cells of IFNAR1 gene knockout mice(KO-IFNAR1),USP52 was knocked down in cells,the cells were infected with model virus SeV,and the mRNA level of interferon(IFNβ)was observed by RT-qPCR technique.USP52 was overexpressed or knocked down in cells,and the protein level of interferon stimulating gene(IISGs)and the effect of mRNA level were analyzed by WesternBlot and RT-qPCR.Immunoprecipitation of endogenous USP52 or STAT2,the interaction between USP52 and STAT2 was verified by Western Blot,and the effect of USP52 on the stability of STAT2 protein was analyzed by protein synthesis inhibitor(Cycloheximide,CHX).Using MG132(proteasome inhibitor)and MA(lysosome inhibitor),the regulation of USP52 on STAT2 protein degradation pathway was analyzed by Western Blot method,and the regulation effect of USP52 on STAT2 protein ubiquitin level and ubiquitin type were analyzed by immunoprecipitation experiment.Results:The expression of USP52 in PBMC of patients with clinically confirmed viral infection was lower than that of normal subjects.After knocking down USP52 in cells,the mRNA and protein levels of VSV virus increased.After the knockout of IFN-I pathway,the antiviral ability of USP52 decreased and that of USP52,IFN-I decreased,which suggested that USP52 could achieve antiviral effect through IFN-I pathway.Overexpression of USP52 does not affect the production of IFN,but can enhance the mRNA and protein levels of ISGs downstream of IFN,suggesting that USP52 is related to the downstream signal pathway of IFN-I.The protein level of knocking down USP52,STAT2 was significantly decreased,and the relationship between USP52 and STAT2 was proved by immunoprecipitation experiment.Through CHX experiment,it was found that USP52 inhibited the degradation of STAT2.Through immunoprecipitation assay,it was found that USP52 stabilized STAT2 protein level by down-regulating the ubiquitin modification of K48 of STAT2.Conclusions:A deubiquitinating enzyme USP52 in the deubiquitinating enzyme family was identified.It was found that USP52 stabilized STAT2 protein level by inhibiting K48 polyubiquitination of STAT2 protein and proteasome degradation,which stabilized interferon pathway,promoted the expression of ISGs downstream of IFN-α,and positively regulated the antiviral ability of cells.This study provides a new strategy and a new target for the clinical application of antiviral interferon,At the same time,it can also provide a new idea for the development of drugs to prevent viral infection in the future.