Mechanisms Of E3Ubiquitin Ligase TRIM4-and TRIM21-mediated Regulation Of Antiviral Innate Immune Signaling

Antiviral immunity is the front line of host defence upon viral infection. The pattern recognition receptors (PRRs) activate downstream signaling in virus infection through pathogen-associated molecular patterns (PAMPs), and induce the production of type I interferons (IFNs) and proinfiammatory cytokines, which then cause the activation of innate immunity response.In the infection and replication of virus, various viral components such as dsRNA can be recongnized by PRRs such as RIG-Ⅰ like receptors (RLRs). RLRs recognize the viral dsRNA, and then recruit a central adapter protein, designated virus-induced signaling adapter (VISA, also known as IPS-1, MAVS and CARDIF), which signals to different pathways. VISA associates with TRAF3, MITA and TBK1to activate IRF3by phosphorylation while VISA contemporarily interacts with TRAF2/TRAF6to activate IKK complex, leading to subsequent activation of another transcription factor, NF-κB. Activated IRF3and NF-κB translocate in nuclear and collaboratively trigger the expression of type ⅠIFN genes.The regulation mechanism of host antiviral response is mediated by the modifications of cellular proteins, the major of which are ubiquitination and deubiquitination. To identify some new proteins which are involved in the regulation of cellular antiviral signaling through ubiquitination and deubiquitination, we chose pGL3-NF-κB as a reporter gene for screening a gene library. We found that transiently transfection of an E3ubiquitin ligase, TRIM4led to the significant activation of the NF-κB, ISRE, and IFNβ reporters, and increased the induction of type I interferons in SeV infection. Consistently, knockdown of endogenous TRIM4remarkably inhibited the production of SeV-induced type I interferons. Endogenous coimmunoprecipitation experiments also indicated that TRIM4associated with RIG-I, and the interaction of them was increased by the induction of viral infection. Further study showed that TRIM4collaboratively triggered the production of IFNs by the associating with RIG-Ⅰ and subsequently catalyzing the K63-linked ubiquitination of RIG-I. There are some new questions from our study, such as whether TRIM25, which was identified as an E3ubiquitin ligase which target RIG-Ⅰ for K63-linked ubiquitination of RIG-Ⅰ, could associate with TRIM4. Moreover, knockout mice of TRIM4should be generated to support our conclusion.Another TRIM family member, TRIM21was identified playing a role as a regulator of IRF3signaling. However, two independent groups received the opposite conclusions. Yang et al demonstrated that TRIM21increases the stability of IRF3to enhance type I interferons induction by interaction with Pinl, a negative regulator of antiviral responses that mediates the ubiquitination and degradation of IRF3during virus infection. In contrast, Higgs et al found that TRIM21interacts with IRF3and promotes the ubiquitination and degradation of IRF3to limit IFNβ induction. Thus, to reveal the function of TRIM21in antiviral pathway, we generated TRIM21deficient mice. In our study, the embryonic fibroblast cells derived from TRIM21deficient mice showed increased production of IFNP and downstream gene RANTES in SeV infection. Our study confirmed that TRIM21in vivo inhibits in virus-induced production of type I IFNs, and the physiological molecular mechanism needs our further study. And comprehensive phenotype analysis of TRIM21deficient mice could furnish more information to us.