We propose to investigate the mechanisms of the anti-viral innate immune response using single molecule fluorescence techniques in vitro and super-resolution imaging of cells. RIG-I (retinoic acid inducible gene-l) and related receptors were recently identified as the initial sensors for viral RNA that signal downstream molecules. RIG-I has a central DExD/H RNA helicase domain. The N-terminus possesses tandem CARDs (caspase activation and recruitment domains) that interact with a mitochondrial antiviral signaling protein (MAVS) and are ubiquitinated by TRIM25 E3 ligase. It also has a C-terminal regulatory domain (RD) that senses 5'triphosphate which is the primary signature of viral RNA. RIG-I also recognizes double stranded (ds) RNA as a viral signature. RIG-I is an RNA-dependent ATPase and its ATPase activity closely correlate with its signaling function. However, the role of its ATPase activity has remained a mystery. Using proteininduced fluorescence enhancement (PIPE) at the single molecule level in collaboration with Project 2. we found that RIG-I translocates rapidly and repeatedly on short dsRNA (20-50 bp). We also showed that its movement is slowed down by CARDs and accelerated by the presence of 5'triphosphate. Combined with previous studies that show a strong correlation between ATPase activity and RIG-I signaling, this data indicates that ATP-powered RNA translocation is essential for RIG-I signaling. In Project 3, we will address many of the outstanding questions such as how RIG-I discriminates between viral RNA and similar-looking host RNA molecules, what the role of RIG-l's ATPase activity is, how RIG-l's function is regulated by nucleic acid composition, what its interactions with other proteins are and how RIG-l's post-transcriptional and posttranslational modifications affect its function. There are three specific aims:
In Aim 1, we will investigate the RNA translocation activities of RIG-I like receptors.
In Aim 2, we will investigate RIG-I loading and oligomerization, and its conformational changes upon viral RNA recognition.
In Aim 3, we will investigate cellular location of RIG-I and its partners such as MAVS and viral RNA using live cell imaging and super-resolution imaging. In addition, RIG-I interaction with MAVS will be studied at the single molecule level in a reconstituted system.

Public Health Relevance

RIG-I like receptors are the primary sensor of viral RNA in the cytosol in all cell types and triggers downsteam signaling cascades leading interferon production as a first line of defense against viral infection. Our studies will provide a fundamental understanding of RIG-I pathway with unprecedented resolution and sensitivity, providing critical insights that may be used for developing new therapeutic approaches.

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 #
8509578
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
$375,976
Indirect Cost
$68,103
Name
University of Southern California
Department
Type
DUNS #
072933393
City
Los Angeles
State
CA
Country
United States
Zip Code
90089
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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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