The Function And Mechanism Of USP21 Negative Regulate Type ? Interferon Signal Pathway Activation By Targeting STING

Antiviral innate immunity that have some characteristics such as universality, diversity,fast response is the first line of host defense against viral infection. Upon viral infection,innate immune response is initiated by the recognition of viral pathogen-associated molecular patterns by germline-encoded pattern recognition receptors to activate the expression of type I interferons. The interferon receptors recognizes secreted type I interferons and further induce expression of a wide antiviral genes through the JAK-STAT signaling pathway. These expression products play a role in suppression of viral replication and induction of apoptosis,then defense against viral infection and replication.STING has been identified as ER-associated membrane protein that is critically involved in type I interferon induction and antiviral innate immunity in response to both RNA and DNA viral infection. Key components in these pathways are frequently regulated by ubiquitination in virus-triggered type I interferon induction and antiviral response. Viral infection can lead to ubiquitination of STING, previous studies have demonstrated that E3 ubiquitin ligase TRIM32/TRIM56 catalyze K63-linked ubiquitination of STING and enhance virus-triggered type I interferon expression. Next, we screened a DUB expression library to determine whether other DUBs could be involved in STING-induced activation of IFNp and virus triggerred ubquitination of STING. In this study, we identified a USP (ubiquitin specific protease) family member, USP21, that interact and deubiquitinate STING, dramatically inhibit STING-mediated induction of IFN?. Overexpression of USP21 lead to inhibit STING-triggered expression of IFN? downstream genes and antivirus ability of cell. while,knockdown of USP21 had opposite effects. In addition, we found that wild-type USP21 but not USP21(C221A), enzymatic inactive mutants, deubiquitinated STING and this mutant could not act as negative regulator to abolish cellular antiviral response. HSV genome assay indicated that knockdown USP21 by USP21-RNAi in L929 cells and knockout MEF inhibited virus replication.These findings suggest that USP21 negitively regulates cellular antiviral immune depends on its deubiquitinating enzyme activity. Mechanically, USP21 interacted with STING, USP21 directly targeted STING for K63-linked deubiquitination,which block the interaction of STING with TBK1 and IRF3. Then USP21 inhibited phosphorylation,dimerization and nuclear location of IRF3.Our study first demonstrates that USP21 plays a key regulatory role in STING dependent cellular antiviral immunity and can target STING for its K63-linked deubiquitination, then inhibits activation,block interaction between TBK1 and STING, further blocks interaction between IRF3 and STING. Researchers have proved that USP21 target RIG-I for its deubiquitination negative regulate RNA virus triggered I interferon signal pathway. To sum up,USP21 play an important role in antiviral immunity, then we set up a system to test deubiquitinase activity, desired to find a compound inhibit USP21 activity,could be a potential antivirus drug.Nanoluc is a new high sensitive reporter, that sensitivity is 150 times higher than relative luciferase report gene Firefly and Renilla,Nanoluc has a protein structure has the advantages of small, light stability compared to Firefly and Renilla, suggests that the report gene Nanoluc could be used as a high throughput reporter for detecting deubiquitinating enzyme activity.Based on the above advantages, we designed the Ub-Nanoluc and Ub-Ub-GS-Nanoluc system for the detection of USPs and OTULIN activity, desired to find small molecule compounds targeted the deubiquitinating enzyme which regulates innate immunity.Nevertheless, a shortage about this study is only cellular level has been proved. Future studies are required to examine whether USP21 is required for host defense against DNA virus at animal level by generating USP21-deficient mice. however, for screening USP21 inhibitors, we established stable reporter gene system at present.The inhibitor screening is still in the initial stage,we need further screen hit inhibitors at the cellular level and animal level.