USP5 Targeting IRF3 Negatively Regulates Antiviral Innate Immune Response And Its Mechanism

Innate immunity is the first line of host cells against virus invasion.Viral nucleic acids are recognized by pattern recognition receptors(PRRs)and activate the transcription factor interferon-regulatory factor 3(IRF3).Activated IRF3 is phosphorylated and dimerized then translocated into the nucleus.The active transcription complex is formed,and the transcription of the type ?interferon(IFN-I)gene is initiated to activate a large amount of antiviral Interferon-stimulated genes(ISGs),and protect the hosts against viral infection.However,the excessive production of IFN-I leads to the occurrence of many autoimmune diseases and autoinflammatory diseases.Therefore,the expression and activation of IFN-I need to be precisely regulated.It has been well established that post-translational modifications,particularly ubiquitination modifications,play key roles in modulating IFN-I production and IFNAR downstream antiviral signaling.Deubiquitinases(DUBs)are a class of proteases related to ubiquitination that reverse the ubiquitination of substrate proteins and regulate cell function.During antiviral innate immunity,there are still few studies on deubiquitinating enzymes compared to E3-mediated ubiquitination,and many DUB molecules involved iin the regulation of antiviral immunity have not been discovered.In this study,we tried to identify several DUBs that can participate in the regulation of antiviral innate immunity,in order to provide new targets for the treatment of infectious diseases.By analyzing the sequencing results of the expression levels of DUBs in A549 cells infected with VSV and human dendritic cells infected with ZIKA virus in the GEO database,we obtained many DUBs related to antiviral innate immunity.A deubiquitination enzyme USP5 was further confirmed to be significantly downregulated in the cells infected with VSV.SeV and WSN infection by RT-qPCR and Western blot.USP5 overexpression,knockdown,and knockout experiments demonstrated that USP5 can significantly promote the replication of VSV,SeV and WSN by inhibiting the production of IFN-I via the assays from RT-qPCR,flow cytometry,and fluorescence microscope.In order to explore the molecular mechanism of the antiviral effect of USP5?RT-qPCR was first used to prove that IFN-p stimulation and viral mimics such as polyI:C and polydArdT inhibit the expression of USP5,and the induced IFN-I was involved in regulating the expression of USP5 by using the Ifnarl-/-and Ifnb-/-macrophages.Meanwhile,the luciferase assay of IFN-p promoter and ISRE promoter has confirmed that USP5 targets IRF3 and thus inhibits IFN-I expression.Proximity ligation assay(PLA)and immunoprecipitation(IP)experiments have further confirmed that USP5 interacts with IRF3 and specifically remove the polyubiquitination modification of K48 binding on IRF3.Then immunofluorescence found that USP5 inhibited the nuclear translocation of IRF3.Finally,we have proved that IFN-I regulates the expression of USP5,and USP5 inhibits the expression of IFN-I to promote the infection of RNA viruses by specifically removing the polyubiquitination modification of k48-linked IRF3 and affecting the nuclear translocation process of IRF3.In summary,our study demonstrated that IFN-I is involved in regulating the expression of USP5,and USP5 can precisely regulate IFN-I and participate in the antiviral immune response.This study has described the role of DUB in antiviral innate immunity,provided potential targets for the treatment of autoimmune diseases and autoinflammatory diseases,and also provided new stratedies for the prevention and treatment of infectious diseases.