We address the structural and biochemical mechanisms of activation, regulation and inhibition of RIG-I like helicases in the antiviral interferon response. RIG-I and the related MDA5 protein detect viral RNA and initiate a signal transduction cascade to stimulate innate immunity. The molecular basis for virus versus self RNA differentiation by RIG-I and MDA5 is not understood, but of central importance to understand intrinsic antiviral functions of our cells. We use a combination of X-ray crystallography, small angle X-ray scattering and biochemical techniques to understand key principles of how RIG-I recognizes viral RNA patterns, how ATP binding and hydrolysis by RIG-I is used in the process of pattern recognition and finally, how viral protein inhibitors interfere with pattern recognition and activation. Based on existing, X-ray diffracting crystals we aim at deriving in the first aim a structure of the helicase domain of RIG-I. This structure will guide the analysis of the mechanism of activation of RIG-I by viral patterns. We will aim at deriving molecular determinants for the recognition of 5'triphosphate RNA as well as double stranded RNA by the ATPase and regulatory domains of RIG-I. We will address how regulatory domains as well as ATPase domain of RIG-I are mechanistically linked and test the hypothesis that RIG-I integrates several patterns into an active "signal on" conformation. We will then aim at deriving a detailed molecular and mechanistic picture of this "signal on" conformation of RIG-I using a multidisciplinary and collaborative approach. This apprach will include the interlink between ATP dependent pattern recognition and posttranslational modification of RIG-I and we will address whether these posttranslational modifications manifest the "signal on" conformation. Finally, we will aim at revealing how viruses counteract MDA5 signalling by using a proteinaceous inhibitor against MDA5. All in all, the expected outcome will advance our understanding of the specific and proofread pattern recognition of viral RNA by RIG-I like helicases at the molecular mechanistic and atomic level.

Public Health Relevance

Our approach will contribute to the overall objectives with detailed insights into the molecular and structural mechanism of activation and inhibition of RIG-I and MDA5. The structural and biochemical results will help to interpret and rationalize in vivo as well as single molecule data within atomic models and guide mutagenesis to test further hypotheses. Finally, atomic models may help to guide rational drug design against virulence.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Program--Cooperative Agreements (U19)
Project #
5U19AI083025-05
Application #
8509577
Study Section
Special Emphasis Panel (ZAI1-BDP-I)
Project Start
Project End
Budget Start
2013-05-01
Budget End
2014-04-30
Support Year
5
Fiscal Year
2013
Total Cost
$263,985
Indirect Cost
$47,817
Name
University of Southern California
Department
Type
DUNS #
072933393
City
Los Angeles
State
CA
Country
United States
Zip Code
90089
Rajsbaum, Ricardo; Garcia-Sastre, Adolfo; Versteeg, Gijs A (2014) TRIMmunity: the roles of the TRIM E3-ubiquitin ligase family in innate antiviral immunity. J Mol Biol 426:1265-84
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Deimling, Tobias; Cui, Sheng; Lammens, Katja et al. (2014) Crystal and solution structure of the human RIG-I SF2 domain. Acta Crystallogr F Struct Biol Commun 70:1027-31
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Rajsbaum, Ricardo; Versteeg, Gijs A; Schmid, Sonja et al. (2014) Unanchored K48-linked polyubiquitin synthesized by the E3-ubiquitin ligase TRIM6 stimulates the interferon-IKK? kinase-mediated antiviral response. Immunity 40:880-95
Ayllon, Juan; Domingues, Patricia; Rajsbaum, Ricardo et al. (2014) A single amino acid substitution in the novel H7N9 influenza A virus NS1 protein increases CPSF30 binding and virulence. J Virol 88:12146-51
Roth, Susanne; Rottach, Andrea; Lotz-Havla, Amelie S et al. (2014) Rad50-CARD9 interactions link cytosolic DNA sensing to IL-1? production. Nat Immunol 15:538-45
Pulloor, Niyas Kudukkil; Nair, Sajith; McCaffrey, Kathleen et al. (2014) Human genome-wide RNAi screen identifies an essential role for inositol pyrophosphates in Type-I interferon response. PLoS Pathog 10:e1003981

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