Innate Immunity a Chain Reaction

ReseARch highlights

INNAtE ImmuNIty A chain reaction

New research published in Cell adds observed. However, when RIG-I was This involved the synthesis of free to the evidence indicating a crucial purified from infected cells lacking K63-linked polyubiquitin chains that role for protein ubiquitylation in the ubiquitin E3 ligase TRIM25 were shown to bind to the N-terminal regulating immune responses. But or when RIG-I from uninfected caspase-recruitment domains in this example, free polyubiquitin cells was incubated with ATP and (CARDs) of RIG-I but did not require chains in the cytoplasm — rather 5ʹ-triphosphate RNA in the absence ubiquitylation of RIG-I itself. K63- than the direct ubiquitylation of of ubiquitin ligases, IRF3 dimeriza- linked polyubiquitin chains contain- immune factors — are important tion did not occur. So in this cell-free ing more than two ubiquitin moieties, for activating innate antiviral system, as in vivo, ubiquitylation is but not ubiquitin monomers or K48- immunity. Chen and colleagues show required for RIG-I signalling. linked chains, could potently activate that free polyubiquitin chains are Activation of the RIG-I signalling the N-terminal portion of RIG-I an endogenous ligand of the innate pathway by RNA and ATP in vitro in vitro. Importantly, endogenous pattern recognition receptor for viral required TRIM25 and the E2 ligases unanchored K63-linked polyubiquitin RNA known as retinoic acid-inducible UBC5 and UBC13, which are known chains can be isolated from human gene I (RIG-I). to specifically synthesize lysine 63 cells and they potently stimulate RIG-I binds 5ʹ-triphosphate viral (K63)-linked polyubiquitin chains. RIG-I. Binding of K63-linked polyu- RNA and signals through mito- Ubiquitin proteins wih a lysine biquitin chains to full-length RIG-I chondrial antiviral signalling protein substitution at K63 were inactive in required both RNA and the ATPase (MAVS), TANK-binding kinase 1 the cell-free system. Previous studies activity of RIG-I; RNA binding was (TBK1) and the transcription factor have shown that RIG-I can undergo required to precede polyubiquitin interferon regulatory factor 3 (IRF3) K63-linked ubiquitylation at K172 binding for RIG-I activation. to induce the expression of type I and that K172R mutation of RIG-I The authors therefore suggest a interferons (IFNs). Several ubiquitin impairs the induction of type I IFNs. two-step model of RIG-I activation ligases have been shown to regulate But in the cell-free system, RIG-I in which RNA binding activates this pathway, but the precise with intact K172 but mutated lysine the ATPase activity of RIG-I and mechanism was unclear. residues at five other positions could induces a conformational change that The authors set up a cell-free activate IRF3 despite being defec- exposes the N-terminal CARDs for system to investigate the RIG-I tive for ubiquitylation. In addition, polyubiquitin binding. This results in pathway in more detail: when RIG-I deubiquitylation of RIG-I did not additional conformational changes was purified from HEK293 T cells affect IRF3 activation. These results and/or oligomerization, which infected with Sendai virus and mixed indicate that ubiquitylation of RIG-I activates downstream signalling with mitochondria (containing itself is not required for signalling. through MAVS. MAVS) and cytoplasmic extracts Instead, the authors showed that Kirsty Minton

(containing TBK1) from uninfected the amino-terminal portion of RIG-I ORIGINAL RESEARCH PAPER Zeng, W. et al. cells, together with in vitro syn- can activate IRF3 even in the absence Reconstitution of the RIG-I pathway reveals a thesized IRF3, IRF3 activation (as of RNA, dependent on the pres- signaling role of unanchored polyubiquitin chains in innate immunity. Cell 141, 315–330 (2010) indicated by its dimerization) was ence of ubiquitylation components.