Ligand-induced receptor oligomerization is a ubiquitous mechanism for signal activation in the immune system. Understanding how these receptors oligomerize during ligand recognition and activate downstream signaling pathways is fundamental to understanding their functions and is pre-requisite to therapeutic application of these receptors. In this proposal, we describe our strategies to investigate the oligomerization mechanism, oligomer architecture and signal activation mechanism of a conserved innate immune receptor, retinoic acid inducible gene-1 (RIG-I). RIG-I and its paralog, MDA5, represent a major receptor family that recognizes viral RNAs in the cytoplasm of a broad range of cell types. RIG-I and MDA5 both contain a tandem caspase activation recruitment domain (2CARD) for signal activation and a helicase domain and a C-terminal domain for RNA binding and RNA- dependent ATP hydrolysis. 2CARDs of RIG-I and MDA5 activate their downstream adaptor molecule, MAVS, by promoting its monomer-to-filament transition. MAVS filaments, in turn, recruit further downstream signaling molecules to activate the IFN?/ signaling pathways. We have previously shown that MDA5 cooperatively forms a filament along the length of dsRNA, and that its formation is important for high affinity interaction with dsRNA, oligomerization of 2CARD and dsRNA length dependent regulation of signaling activity. Unlike MDA5, oligomerization of RIG-I has been unclear, and has been thought to strictly require a co-factor, K63-linked polyubiquitin chains. Recently, we found that RIG-I assembles into a filament during ATP hydrolysis, and that the filament can directly activate MAVS in the absence of polyubiquitin chains, suggesting a novel mechanism for RNA recognition and signal activation by RIG-I. We here propose to determine the precise mechanisms by which RIG-I assembles into a filament (Aim 1) and stimulates MAVS filament formation independent of or together with polyubiquitin chains (Aim 2), and how RIG-I filaments form and function in the context of viral infection (Aim 3). This proposal builds upon our previous research on the MDA5 filament (Wu et al, Cell, 2013; Peisley et al, PNAS, 2012 & PNAS, 2011; Rice et al, Nat. Genetics, In press), our discovery of the RIG-I filament (Peisley et al, Mol. Cell, 2013), a very recent crystal structure of RIG-I 2CARD tetramer bound by K63-Ubn (Peisley et al, Nature, Epub) and finally the atomic structures of the MAVS filament as well as the RIG-I 2CARD:MAVS CARD complex (manuscript in preparation). We believe that the proposed research will provide a comprehensive picture of functions of RIG-I and help us dissect commonalities and divergences between RIG-I and MDA5 in viral RNA detection and signal activation mechanisms. Furthermore, detailed mechanistic understanding of the RIG-I filament could potentially offer insights into novel therapeutic strategies to modulat the activity of RIG-I in antiviral and anticancer therapies.

Public Health Relevance

Viral infection poses a major challenge to global health as demonstrated in the recent pandemics of H1N1 influenza virus, SARS and HIV. The goal of the current proposal is to understand the molecular mechanisms of viral recognition and signaling by RIG-I and MAVS, which together constitute one of the principle innate immune pathways. These signaling pathways have been also implicated in pathogenesis of several autoimmune and inflammatory diseases, and were proposed to be a potential therapeutic target in anticancer immunotherapies.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI111784-05
Application #
9543279
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Jiang, Chao
Project Start
2014-09-17
Project End
2019-08-31
Budget Start
2018-09-01
Budget End
2019-08-31
Support Year
5
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Boston Children's Hospital
Department
Type
DUNS #
076593722
City
Boston
State
MA
Country
United States
Zip Code
Ahmad, Sadeem; Mu, Xin; Yang, Fei et al. (2018) Breaching Self-Tolerance to Alu Duplex RNA Underlies MDA5-Mediated Inflammation. Cell 172:797-810.e13
Mu, Xin; Greenwald, Emily; Ahmad, Sadeem et al. (2018) An origin of the immunogenicity of in vitro transcribed RNA. Nucleic Acids Res 46:5239-5249
Ruaud, Lyse; Rice, Gillian I; Cabrol, Christelle et al. (2018) Autosomal-dominant early-onset spastic paraparesis with brain calcification due to IFIH1 gain-of-function. Hum Mutat 39:1076-1080
de Carvalho, Luciana Martins; Ngoumou, Gonza; Park, Ji Woo et al. (2017) Musculoskeletal Disease in MDA5-Related Type I Interferonopathy: A Mendelian Mimic of Jaccoud's Arthropathy. Arthritis Rheumatol 69:2081-2091
Cadena, Cristhian; Hur, Sun (2017) Antiviral Immunity and Circular RNA: No End in Sight. Mol Cell 67:163-164
Wu, Bin; Huoh, Yu-San; Hur, Sun (2016) Measuring Monomer-to-Filament Transition of MAVS as an In Vitro Activity Assay for RIG-I-Like Receptors. Methods Mol Biol 1390:131-42
Sohn, Jungsan; Hur, Sun (2016) Filament assemblies in foreign nucleic acid sensors. Curr Opin Struct Biol 37:134-44
Mu, X; Ahmad, S; Hur, S (2016) Endogenous Retroelements and the Host Innate Immune Sensors. Adv Immunol 132:47-69
Ahmad, Sadeem; Hur, Sun (2015) Helicases in Antiviral Immunity: Dual Properties as Sensors and Effectors. Trends Biochem Sci 40:576-585
Yao, Hui; Dittmann, Meike; Peisley, Alys et al. (2015) ATP-dependent effector-like functions of RIG-I-like receptors. Mol Cell 58:541-548

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