Ube2D3 and Ube2N are essential for RIG-I-mediated MAVS aggregation in antiviral innate immunity

Yuheng Shi et al., Nat Commun.8 :15138 2017 May 4

Speaker: Jia-Bao Chen (陳家寶)                           Time: 15:00~16:00, April. 11, 2018

Commentator: Dr. Chia-Yi Yu (余佳益)       Place: Room 601

Abstract:

Cytosolic RNA sensor RIG-I plays a key role in detecting RNA virus infection, like dengue and influenza, to trigger antiviral immunity. The C-terminal domain of RIG-I functions to bind 5’ triphosphate viral RNA, and then the N-terminal tandem CARD domains of RIG-I will be released for K63-linked ubiquitination, eventually leading to the oligomerization of RIG-I. This RIG-I protein complex subsequently engages with mitochondrial adaptor MAVS to induce downstream signaling to type I IFN-mediated antiviral defenses. However, the mechanism of how RIG-I undergoes ubiquitination and oligomerization after virus infection remains to be further explored. The authors reconstituted a cell-free assay which used purified components to induce prion-like MAVS aggregation¹ for studying mechanism of RIG-I ubiquitination. Ubiquitin modification to a target protein is accomplished through an enzymatic cascade involving three enzymes, ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2) and ubiquitin ligase (E3). First, they demonstrated that E3 ubiquitin ligase-Riplet but not TRIM25 was  essential for RIG-I to induce MAVS aggregation in antiviral signalling in cells. Next, to further identify essential E2 ubiquitin-conjugating enzyme  in the RIG-I-MAVS signaling pathway, they performed the Fraction analysis to identify Ube2D3 and Ube2N critical for RIG-I-mediated antiviral signalling in HEK293T cells. They also examined the involvement of Ube2D3 and Ube2N in mouse primary cells. Different from the result of HEK293T, Ube2N played an essential role in RIG-I-MAVS signaling pathway while  Ube2D3 plays an redundant role in this pathway. By IP-western blot assay, RIG-I, Riplet, and Ube2D3/Ube2N formed a complex to ubiquitize RIG-I. Results from the cell free assay  revealed that Ube2D3 and Ube2N mediated the covalent conjugation of polyubiquitin chains and unanchored polyubiquitin chains respectively for RIG-I ubiquitination. Finally, they identified  three critical residues (K48/96/172R) of RIG-I for polyubiquitin chain conjugation. In conclusion, the authors identified the essential E2 enzymes and two polyubiquitin-mediated mechanisms underlying the activation of RIG-I and MAVS for triggering innate immune signalling in response to viral infection .

References：

1. Hou, F. et al. MAVS forms functional prion-like aggregates to activate and propagate antiviral innate immune response. Cell 146, 448–461 (2011).