Viral RNA recognition by LGP2 and MDA5, and activation of signaling through step-by-step conformational changes.

Ivana Duic,Hisashi Tadakuma,Kunio Takeyasu,Shige H Yoshimura,Takashi Fujita,Katashi Deguchi,Yuki Suzuki,Hiroki Kato,Ryo Yamaue,Yoshie Harada

doi:10.1093/nar/gkaa935

Abstract

Cytoplasmic RIG-I-like receptor (RLR) proteins in mammalian cells recognize viral RNA and initiate an antiviral response that results in IFN-β induction. Melanoma differentiation-associated protein 5 (MDA5) forms fibers along viral dsRNA and propagates an antiviral response via a signaling domain, the tandem CARD. The most enigmatic RLR, laboratory of genetics and physiology (LGP2), lacks the signaling domain but functions in viral sensing through cooperation with MDA5. However, it remains unclear how LGP2 coordinates fiber formation and subsequent MDA5 activation. We utilized biochemical and biophysical approaches to observe fiber formation and the conformation of MDA5. LGP2 facilitated MDA5 fiber assembly. LGP2 was incorporated into the fibers with an average inter-molecular distance of 32 nm, suggesting the formation of hetero-oligomers with MDA5. Furthermore, limited protease digestion revealed that LGP2 induces significant conformational changes on MDA5, promoting exposure of its CARDs. Although the fibers were efficiently dissociated by ATP hydrolysis, MDA5 maintained its active conformation to participate in downstream signaling. Our study demonstrated the coordinated actions of LGP2 and MDA5, where LGP2 acts as an MDA5 nucleator and requisite partner in the conversion of MDA5 to an active conformation. We revealed a mechanistic basis for LGP2-mediated regulation of MDA5 antiviral innate immune responses.

Highlights

retinoic acid inducible gene I (RIG-I)-like receptors (RLRs) are mammalian cytosolic pattern-recognition receptors (PRRs) activated by viralRNA species [1,2]
No significant induction of the IFN-␤ promoter was observed upon EMCV infection in cells expressing melanoma differentiation-associated gene 5 (MDA5) alone (Figure 1C), whereas clear IFN-␤ induction was observed in cells expressing laboratory of genetics and physiology 2 (LGP2) and MDA5
To clarify the mechanism of fiber formation by MDA5 and LGP2, we investigated the recruitment of LGP2 within the MDA5-dsRNA fibers

Summary

Introduction

RIG-I-like receptors (RLRs) are mammalian cytosolic pattern-recognition receptors (PRRs) activated by viral. RNA species [1,2]. The members of the RLR family are: retinoic acid inducible gene I (RIG-I), melanoma differentiation-associated gene 5 (MDA5), and laboratory of genetics and physiology 2 (LGP2). All of the family members have a highly homologous structure, in particular the central DExD/H box RNA helicase domain. The helicase and the C terminal domain (CTD) are responsible for RNA recognition. RIG-I and MDA5 have N terminal caspase activation and recruitment domain (CARD), which is essential for signal transduction.

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Results

Conclusion