The Mechanisms Of Virus-Induced Expression Of Type â…  Interferon By TRIM32

Cellular antiviral innate immunity is the first line of host defense against invading viruses. Upon viral infection, innate immune response is initiated by the recognition of viral pathogen-associated molecular patterns (PAMPs) by germline-encoded pattern recognition receptors (PRRs) to activate the expression of type I interferons. The secreted type I interferons are recognized by interferon receptors (IFNRs) and further induce expression of a wide variety of antiviral genes through the JAK-STAT signaling pathway. The transcriptional activation of type I interferons requires several transcription factors, including nuclear factor-kappa B (NF-κB) and interferon regulatory factor (IRF3).MITA has been identified as a mitochondrion-and 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. Virus-triggered type I interferon induction and antiviral response are heavily regulated by ubiquitination of the key components involved in these pathways. Viral infection can lead to ubiquitination of MITA, our previous study has demonstrated that an E3ubiquitin ligase RNF5catalyzes K48-linked ubiquitination of MITA and inhibits virus-triggered type I interferon induction. Next, we further screened our E3ubiquitin ligase expression library to determine whether other E3ligases are involved in virus-induced ubiquitination of MITA. In this study, we identified a TRIM (tripartite motif protein) family member, TRIM32, that specifically ubiquitinated MITA and dramatically enhanced MITA-mediated induction of IFN-β. Overexpression of TRIM32led to activation of NF-κB and IFN-β promoter and potentiated virus-triggered expression of IFNB1and NF-κB downstream genes. while, knockdown of TRIM32had opposite effects. In addition, we found that wild-type TRIM32but not TRIM32-△R or TRIM32(C39S), two enzymatic inactive mutants, ubiquitinated MITA and these mutants could act as dominant negative regulators to abrogate cellular antiviral response. VSV plaque assays indicated that overexpression of TRIM32inhibited virus replication, while overexpression of the enzymatic inactive mutants or knockdown TRIM32by TRIM32-RNAi potentiated virus replication. These findings suggest that TRIM32positively regulates cellular antiviral immune which depends on its E3ubiquitin ligase activity. Mechanically, TRIM32interacted with MITA, and was located at the mitochondria and endoplasmic reticulum. TRIM32directly targeted MITA for K63-linked ubiquitination at K20/150/224/236of MITA, which promoted the interaction of MITA with TBK1. Collectively, TRIM32is a key regulatory protein for innate immunity against both RNA and DNA viruses by targeting MITA for K63-linked ubiquitination.TRIM32has been shown to induce ubiquitination and degradation of multiple substrates and participate in diverse pathological processes including carcinogenesis, muscular dystrophy and nervous system diseases. Our study for the first time demonstrates that TRIM32plays a key regulatory role in cellular antiviral immunity and can target a protein for K63-linked ubiquitination, thereby promoting its activation. Future studies are required to examine whether TRIM32is required for host defense against DNA or RNA viral infection at animal level by generating TRIM32-deficient mice.