The adaptor molecule MAVS forms prion-like aggregates to govern the RIG-I-like receptor (RLR) signaling cascade. Lys63 (K63)-linked polyubiquitination is critical for MAVS aggregation, yet the underlying mechanism and the corresponding E3 ligases and deubiquitinating enzymes (DUBs) remain elusive. Here, we found that the K63-linked polyubiquitin chains loaded on MAVS can be directly recognized by RIG-I to initiate RIG-I-mediated MAVS aggregation with the prerequisite of the CARDRIG-I-CARDMAVS interaction. Interestingly, many K63-linked polyubiquitin chains attach to MAVS via an unanchored linkage. We identified Ube2N as a major ubiquitin-conjugating enzyme for MAVS and revealed that Ube2N cooperates with the E3 ligase Riplet and TRIM31 to promote the unanchored K63-linked polyubiquitination of MAVS. In addition, we identified USP10 as a direct DUB that removes unanchored K63-linked polyubiquitin chains from MAVS. Consistently, USP10 attenuates RIG-I-mediated MAVS aggregation and the production of type I interferon. Mice with a deficiency in USP10 show more potent resistance to RNA virus infection. Our work proposes a previously unknown mechanism for the activation of the RLR signaling cascade triggered by MAVS-attached unanchored K63-linked polyubiquitin chains and establishes the DUB USP10 and the E2:E3 pair Ube2N-Riplet/TRIM31 as a specific regulatory system for the unanchored K63-linked ubiquitination and aggregation of MAVS upon viral infection.
This is a preview of subscription content, access via your institution
Subscribe to this journal
Receive 12 digital issues and online access to articles
$119.00 per year
only $9.92 per issue
Rent or buy this article
Prices vary by article type
Prices may be subject to local taxes which are calculated during checkout
The authors declare that all data supporting the findings of this study are available within the paper and its supplementary information files or are available from the corresponding author upon reasonable request.
Akira S, Uematsu S, Takeuchi O. Pathogen recognition and innate immunity. Cell 2006;124:783–801.
Kawai T, Akira S. Innate immune recognition of viral infection. Nat Immunol. 2006;7:131–7.
Takeuchi O, Akira S. Pattern recognition receptors and inflammation. Cell 2010;140:805–20.
Rehwinkel J, Gack MU. RIG-I-like receptors: their regulation and roles in RNA sensing. Nat Rev Immunol. 2020;20:537–51.
Yoneyama M, Fujita T. RNA recognition and signal transduction by RIG-I-like receptors. Immunol Rev. 2009;227:54–65.
Luo D, Ding SC, Vela A, Kohlway A, Lindenbach BD, Pyle AM. Structural insights into RNA recognition by RIG-I. Cell 2011;147:409–22.
Kowalinski E, Lunardi T, McCarthy AA, Louber J, Brunel J, Grigorov B, et al. Structural basis for the activation of innate immune pattern-recognition receptor RIG-I by viral RNA. Cell 2011;147:423–35.
Jiang F, Ramanathan A, Miller MT, Tang GQ, Gale M Jr., Patel SS, et al. Structural basis of RNA recognition and activation by innate immune receptor RIG-I. Nature 2011;479:423–7.
Peisley A, Wu B, Xu H, Chen ZJ, Hur S. Structural basis for ubiquitin-mediated antiviral signal activation by RIG-I. Nature 2014;509:110–4.
Wu B, Peisley A, Tetrault D, Li Z, Egelman EH, Magor KE, et al. Molecular imprinting as a signal-activation mechanism of the viral RNA sensor RIG-I. Mol Cell. 2014;55:511–23.
Hou F, Sun L, Zheng H, Skaug B, Jiang QX, Chen ZJ. MAVS forms functional prion-like aggregates to activate and propagate antiviral innate immune response. Cell 2011;146:448–61.
Zeng W, Sun L, Jiang X, Chen X, Hou F, Adhikari A, et al. Reconstitution of the RIG-I pathway reveals a signaling role of unanchored polyubiquitin chains in innate immunity. Cell 2010;141:315–30.
Jiang X, Kinch LN, Brautigam CA, Chen X, Du F, Grishin NV, et al. Ubiquitin-induced oligomerization of the RNA sensors RIG-I and MDA5 activates antiviral innate immune response. Immunity 2012;36:959–73.
Okamoto M, Kouwaki T, Fukushima Y, Oshiumi H. Regulation of RIG-I Activation by K63-Linked Polyubiquitination. Front Immunol. 2017;8:1942.
Shi Y, Yuan B, Zhu W, Zhang R, Li L, Hao X, et al. Ube2D3 and Ube2N are essential for RIG-I-mediated MAVS aggregation in antiviral innate immunity. Nat Commun. 2017;8:15138.
Cadena C, Ahmad S, Xavier A, Willemsen J, Park S, Park JW, et al. Ubiquitin-Dependent and -Independent Roles of E3 Ligase RIPLET in Innate Immunity. Cell 2019;177:1187–200 e16.
Song B, Chen Y, Liu X, Yuan F, Tan EYJ, Lei Y, et al. Ordered assembly of the cytosolic RNA-sensing MDA5-MAVS signaling complex via binding to unanchored K63-linked poly-ubiquitin chains. Immunity 2021;54:2218–30 e5.
Liu B, Zhang M, Chu H, Zhang H, Wu H, Song G, et al. The ubiquitin E3 ligase TRIM31 promotes aggregation and activation of the signaling adaptor MAVS through Lys63-linked polyubiquitination. Nat Immunol. 2017;18:214–24.
Hou J, Han L, Zhao Z, Liu H, Zhang L, Ma C, et al. USP18 positively regulates innate antiviral immunity by promoting K63-linked polyubiquitination of MAVS. Nat Commun. 2021;12:2970.
Yoneyama M, Kikuchi M, Natsukawa T, Shinobu N, Imaizumi T, Miyagishi M, et al. The RNA helicase RIG-I has an essential function in double-stranded RNA-induced innate antiviral responses. Nat Immunol. 2004;5:730–7.
Seth RB, Sun L, Ea CK, Chen ZJ. Identification and characterization of MAVS, a mitochondrial antiviral signaling protein that activates NF-kappaB and IRF 3. Cell 2005;122:669–82.
Xu LG, Wang YY, Han KJ, Li LY, Zhai Z, Shu HB. VISA is an adapter protein required for virus-triggered IFN-beta signaling. Mol Cell. 2005;19:727–40.
He L, Bardiaux B, Ahmed M, Spehr J, Konig R, Lunsdorf H, et al. Structure determination of helical filaments by solid-state NMR spectroscopy. Proc Natl Acad Sci USA. 2016;113:E272–81.
Xu H, He X, Zheng H, Huang LJ, Hou F, Yu Z, et al. Structural basis for the prion-like MAVS filaments in antiviral innate immunity. Elife 2014;3:e01489.
Cai X, Xu H, Chen ZJ. Prion-Like Polymerization in Immunity and Inflammation. Cold Spring Harb Perspect Biol. 2017;9:a023580.
Frias-Staheli N, Giannakopoulos NV, Kikkert M, Taylor SL, Bridgen A, Paragas J, et al. Ovarian tumor domain-containing viral proteases evade ubiquitin- and ISG15-dependent innate immune responses. Cell Host Microbe. 2007;2:404–16.
Xia ZP, Sun L, Chen X, Pineda G, Jiang X, Adhikari A, et al. Direct activation of protein kinases by unanchored polyubiquitin chains. Nature 2009;461:114–9.
Reyes-Turcu FE, Horton JR, Mullally JE, Heroux A, Cheng X, Wilkinson KD. The ubiquitin binding domain ZnF UBP recognizes the C-terminal diglycine motif of unanchored ubiquitin. Cell 2006;124:1197–208.
Gack MU, Shin YC, Joo CH, Urano T, Liang C, Sun L, et al. TRIM25 RING-finger E3 ubiquitin ligase is essential for RIG-I-mediated antiviral activity. Nature 2007;446:916–20.
Li S, Zheng H, Mao AP, Zhong B, Li Y, Liu Y, et al. Regulation of virus-triggered signaling by OTUB1- and OTUB2-mediated deubiquitination of TRAF3 and TRAF6. J Biol Chem. 2010;285:4291–7.
Peng Y, Guo J, Sun T, Fu Y, Zheng H, Dong C, et al. USP39 Serves as a Deubiquitinase to Stabilize STAT1 and Sustains Type I IFN-Induced Antiviral Immunity. J Immunol. 2020;205:3167–78.
Xu S, Han L, Wei Y, Zhang B, Wang Q, Liu J, et al. MicroRNA-200c-targeted contactin 1 facilitates the replication of influenza A virus by accelerating the degradation of MAVS. PLoS Pathog. 2022;18:e1010299.
Meyer C, Garzia A, Morozov P, Molina H, Tuschl T. The G3BP1-Family-USP10 Deubiquitinase Complex Rescues Ubiquitinated 40S Subunits of Ribosomes Stalled in Translation from Lysosomal Degradation. Mol Cell. 2020;77:1193–205 e5.
Higuchi M, Kawamura H, Matsuki H, Hara T, Takahashi M, Saito S, et al. USP10 Is an Essential Deubiquitinase for Hematopoiesis and Inhibits Apoptosis of Long-Term Hematopoietic Stem Cells. Stem Cell Rep. 2016;7:1116–29.
Blount JR, Johnson SL, Todi SV. Unanchored Ubiquitin Chains, Revisited. Front Cell Dev Biol. 2020;8:582361.
Hodge CD, Spyracopoulos L, Glover JN. Ubc13: the Lys63 ubiquitin chain building machine. Oncotarget 2016;7:64471–504.
Yuan J, Luo K, Zhang L, Cheville JC, Lou Z. USP10 regulates p53 localization and stability by deubiquitinating p53. Cell 2010;140:384–96.
Niu J, Shi Y, Xue J, Miao R, Huang S, Wang T, et al. USP10 inhibits genotoxic NF-kappaB activation by MCPIP1-facilitated deubiquitination of NEMO. EMBO J. 2013;32:3206–19.
We thank Dr FaJian Hou (Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences) for the DDX58−/−, UBE2N−/− and MAVS−/− HEK293T cells. This work was supported by grants from the National Natural Science Foundation of China (31730026, 81930039, 32000633), National Key Research and Development Program (2021YFC2300603), Natural Science Foundation of Shandong Province (ZR2020QH136), China Postdoctoral Science Foundation (2020M682187), and Postdoctoral Innovation Project of Shandong Province (202002012).
The authors declare no competing interests.
About this article
Cite this article
Liu, F., Zhuang, W., Song, B. et al. MAVS-loaded unanchored Lys63-linked polyubiquitin chains activate the RIG-I-MAVS signaling cascade. Cell Mol Immunol 20, 1186–1202 (2023). https://doi.org/10.1038/s41423-023-01065-2