Vertebrates rely on innate immune receptors to sense conserved structures from invading microbes. The overall goal of this project is to understand at the molecular level how innate immune receptors recognize flaviviruses, and how this recognition is translated into an immune response. We focus on innate immune recognition of viral genomic RNA in the cytoplasm by the DExD/H-box RNA helicases RIG-I and MDA5. RIGI recognizes short RNA oligonucleotides bearing 5'-triphosphates, while MDA5 recognizes longer, double stranded RNAs. Our first goal is to measure the binding affinities of RIG-I and MDA5 for RNA substrates of various lengths and secondary structures. We will begin to dissect the mechanism of signal transduction of RIG-I and MDA5 by measuring their rates of ATP hydrolysis and their helicase activities in the presence of their preferred RNA substrates. In the major part of our proposal, we will determine the crystal structures of RIG-I and MDA5 in the presence and absence of their preferred RNA ligands. These crystal structures will reveal the conformational changes caused by RNA binding, which will allow us to understand how binding of RNA is translated into an innate immune signal. To test our structure-based model for signal generation, we will engineer mutations designed to alter the signaling and ligand-binding properties of RIG-I and MDA5. Intracellular signaling in response to viral genomic RNA is modulated by another DExD/H-box helicase, LGP2, which interacts directly with RIG-I and MDA5. To understand how LGP2 regulates RIG-I and MDA5 signaling, we will determine the crystal structure of LGP2 in complex with RIG-I or MDA5. Paramyxoviruses inhibit MDA5 signaling by binding MDA5 directly with their V proteins. In order to understand the molecular basis of this viral immune evasion, we will determine the crystal structure of MDA5 in complex with SV5 V. Our structural approach will provide unique mechanistic insights into the generation and regulation of viral RNA sensing by RIG-I and MDA5. In the long term, this work will guide the rational design of novel vaccine adjuvants and immunomodulatory therapeutics targeting RIG-I or MDA5, thus providing new strategies for the prevention and treatment of viral infections.

Public Health Relevance

Our work will provide a detailed understanding at the molecular level of how the innate immune system recognizes genetic material from certain important viral pathogens, including dengue and West Nile viruses, which afftect approximately 100 million people each year and are rapidly spreading. This project will provide some of the necessary tools to create novel therapeutics against these pathogens, such as less toxic vaccine adjuvants, and anti-inflammatory drugs targeting the innate immune response.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Program Projects (P01)
Project #
5P01GM022778-38
Application #
8450800
Study Section
Special Emphasis Panel (ZRG1-BCMB-K)
Project Start
Project End
Budget Start
2013-04-01
Budget End
2014-03-31
Support Year
38
Fiscal Year
2013
Total Cost
$269,672
Indirect Cost
$106,709
Name
Yale University
Department
Type
DUNS #
043207562
City
New Haven
State
CT
Country
United States
Zip Code
06520
Wang, Jimin; Liu, Zheng; Crabtree, Robert H et al. (2018) On the damage done to the structure of the Thermoplasma acidophilum proteasome by electron radiation. Protein Sci 27:2051-2061
Sherlock, Madeline E; Sadeeshkumar, Harini; Breaker, Ronald R (2018) Variant Bacterial Riboswitches Associated with Nucleotide Hydrolase Genes Sense Nucleoside Diphosphates. Biochemistry :
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Mirihana Arachchilage, Gayan; Sherlock, Madeline E; Weinberg, Zasha et al. (2018) SAM-VI RNAs selectively bind S-adenosylmethionine and exhibit similarities to SAM-III riboswitches. RNA Biol 15:371-378
Wang, Jimin (2018) Determination of chemical identity and occupancy from experimental density maps. Protein Sci 27:411-420
Sherlock, Madeline E; Sudarsan, Narasimhan; Breaker, Ronald R (2018) Riboswitches for the alarmone ppGpp expand the collection of RNA-based signaling systems. Proc Natl Acad Sci U S A 115:6052-6057
Harris, Kimberly A; Breaker, Ronald R (2018) Large Noncoding RNAs in Bacteria. Microbiol Spectr 6:
Yang, Yang; Kang, Dongwei; Nguyen, Laura A et al. (2018) Structural basis for potent and broad inhibition of HIV-1 RT by thiophene[3,2-d]pyrimidine non-nucleoside inhibitors. Elife 7:
Wang, Jimin (2017) On the appearance of carboxylates in electrostatic potential maps. Protein Sci 26:396-402

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