Mechanisms And Regulations Of MITA-Mediated Cellular Antiviral Signaling

The mechanisms of viral infection and host immune response have long been recognized as a hot research field in life sciences. Pattern-recognition receptors (PRRs) encoded by the host genome recognize invading viruses, representing the first step for antiviral response. The PRRs initiates a series of signaling, which leads to production of a number of cytokines such as typeⅠinterferons (IFNs) and interleukin-1β(IL-1β). The secreted typeⅠIFNs bind to the receptors in autocrine or paracrine manner and initiate signaling that activates transcription of thousands of genes. The produced proteins collaborate to inhibit viral replication or induce apoptosis of infected cells. On the other hand, typeⅠIFNs activate innate immune cells to induce inflammatory response and/or adaptive immune system, resulting in clearance of invading virus and infected cells. Thus, typeⅠIFNs play a vital role in host antiviral response.The mechanisms of virus-triggered induction of typeⅠIFNs have been extensively investigated during the past decade. Viral infection and replication generate pathogen associated molecular patterns (PAMPs) such as 5'triphosphate panhandle RNA and double-stranded RNA, which are recognized by the pattern-recognition receptors (PRRs). Among the PRRs, the cytoplasmic RIG-Ⅰ-like receptors (RLRs), RIG-I and MDA5, have been demonstrated to bind to viral RNAs. Upon detection of viral RNA, RIG-I or MDA5 is associated with the mitochondrial adaptor protein VISA. VISA is associated with several downstream proteins constitutively or in a viral infection dependent manner, including TRAF2, TRAF3 and TRAF6. On one hand, VISA interacts with TRAF2 and/or TRAF6 to activate IKK complex, which phosphorylates IκB. The phosphorylated IκB is ubiquitinated and degraded through proteasome, leading to the release and activation of NF-κB. On the other hand, VISA recruits TRAF3 and TBKl to phosphorylate and activate IRF3. The activated transcription factors IRF3 and NF-κB enter into the nucleus and collaboratively activate transcription of typeⅠIFN genes.Although breakthrough advances on the virus-triggered typeⅠIFN signaling pathways have been made during the past decade, there are a lot of key questions remaining to be elucidated. For example, it has been suggested that VISA interacts with TRAF2/TRAF6 via its conserved TRAF-interacting motifs to activate NF-κB, whereas how VISA is associated with TBK1 to activate IRF3 is not known yet. Also, what are the unknown proteins involved in virus-triggered typeⅠIFN signaling is another research interest. To identify proteins involved in typeⅠIFNs production, we performed expression cloning experiments, leading to the identification and characterization of MITA (mediator of IRF3 activation). Overexpression of MITA activated IRF3, whereas knockdown of MITA inhibited virus-triggered activation of IRF3, expression of typeⅠIFNs, and cellular antiviral response. MITA contained four putative transmenbrane domains at its N-terminus and was found to localize to the outer membrane of mitochondria and the third transmembrane is critical for its mitochondrial localization, VISA-MITA association and MITA oligomerization. MITA was found to be associated with VISA and IRF3 constitutively and recruited the kinase TBK1 to the VISA-associated complex. The serine 358 of MITA was phosphorylated by TBK1, which is required for MITA-mediated activation of IRF3.During the process to characterize MITA, we found that MITA was basally phosphorylated without viral infection. However, the kinase(s) remained to be identified. In addition, we also observed that MITA was ubiquitinated, whereas the mechanism of this process was unknown. To address these questions, we performed yeast two-hybrid assays with full-length MITA as bait, leading to the identification of an E3 ubiquitin ligase RNF5 as a MITA-interacting protein. RNF5 interacted with MITA through its C-terminus in a viral-infection-dependent manner. Overexpression of RNF5 inhibited virus-triggered IRF3 activation, IFN-βexpression and cellular antiviral response, whereas knockdown of RNF5 had opposite effects. RNF5 targeted MITA at Lys150 for ubiquitination and degradation after viral infection. Both MITA and RNF5 were located at the mitochondria and endoplasmic reticulum (ER) and viral infection caused their redistribution to the ER and mitochondria, respectively. We further found that virus-induced ubiquitination and degradation of MITA by RNF5 occurred at the mitochondria. We also found that RNF5 targeted Lys 362 and Lys 461 of VISA for ubiquitination at the early phase of viral infection, thereby negatively regulating virus-induced type I IFN signaling and preventing excessive immune responses.These studies further our understandings of the mechanisms and regulations of virus-induced type I IFN signaling and contribute to the elucidation of the complicated molecular mechanisms of cellular antiviral response. Our results also indicate that the interplay between mitochondria and ER plays a critical role in host defense against invading viruses as well as avoiding harmful excessive immune responses.