Generation And Analysis Of USP13 Deficient Mice

Diseases caused by viral infection have long posed great threat to human life and social production.According to genetic material composition,Double stranded DNA(dsDNA)generated by DNA virus and retrovirus during infection and replication constitutes an important type of pathogen-associated molecular patterns(PAMPs),which can be recognized by the pattern recognition receptors(PPRs)encoded by the host cells such as cGAS,IFI16,DDX41.These PPRs either directly recruit downstream adaptor molecule MITA(also known as STING),or catalyze the synthesis of 2'-3'cGAMP which binds to and triggers oligomerization and activation of MITA.MITA further recruits downstream protein kinase TBK1 and transcription factor IRF3 and promotes phosphorylation of IRF3.Previous research has shown that the activity of MITA is under strict control of ubiquitination.For example,the E3 uniquitin ligase RNF5 catalyzes K48-linked ubiquitination of MITA and promotes proteasome-dependent degradation,whereas AMFR catalyzes K27-linked ubiquitination and promotes the recruitment of TBK1 to MITA.However,the deubiquitination regulation of MITA has yet to be demonstrated.We screened the deubiquitinating enzymes(DUBs)that interacted with MITA.This effort led to the identification of USP13.Our preliminary data showed that knockdown of USP13 promotes type I interferons expression and IRF3 activation induced by DNA virus infection,indicating that USP13 suppresses host immune responses against DNA virus.To further investigate the mechanism of USP13 in regulating host antiviral immune responses in vivo,we generated USP13-deficient mice through CRISPR/Cas9 gene editing technology and analyzed the influence of knocking out USP 13 on mouse development and immune cells differentiation.Results indicated that a single cytosine was inserted into the first exon of Usp13 gene,which led to an early translational termination of USP13.The Usp13-deficient mice bred normally with the Mendelian inheritance ratio and showed no development defect.The composition of immune cells in major immune organs was comparable between the Usp13-deficient mice and their wildtype counterparts.the interaction between MITA and TBK1 in USP13-deficient cells after HSV-1 infection was significantly enhanced,along with a higher expression level of cytokines such as type ? interferons.All these data indicated that USP13 restricts the antiviral immune signaling in host cells.