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.
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.
|Sánchez-Aparicio, Maria T; Feinman, Leighland J; García-Sastre, Adolfo et al. (2018) Paramyxovirus V Proteins Interact with the RIG-I/TRIM25 Regulatory Complex and Inhibit RIG-I Signaling. J Virol 92:|
|Do?anay, Sultan; Lee, Maurice Youzong; Baum, Alina et al. (2017) Single-cell analysis of early antiviral gene expression reveals a determinant of stochastic IFNB1 expression. Integr Biol (Camb) 9:857-867|
|Sánchez-Aparicio, Maria Teresa; Garcin, Dominique; Rice, Charles M et al. (2017) Loss of Sendai virus C protein leads to accumulation of RIG-I immunostimulatory defective interfering RNA. J Gen Virol 98:1282-1293|
|Chen, Chia-Lin; Huang, Jeffrey Y; Wang, Chun-Hsiang et al. (2017) Hepatitis C virus has a genetically determined lymphotropism through co-receptor B7.2. Nat Commun 8:13882|
|Nelson, Emily V; Schmidt, Kristina M; Deflubé, Laure R et al. (2016) Ebola Virus Does Not Induce Stress Granule Formation during Infection and Sequesters Stress Granule Proteins within Viral Inclusions. J Virol 90:7268-7284|
|Pisanelli, Giuseppe; Laurent-Rolle, Maudry; Manicassamy, Balaji et al. (2016) La Piedad Michoacán Mexico Virus V protein antagonizes type I interferon response by binding STAT2 protein and preventing STATs nuclear translocation. Virus Res 213:11-22|
|Weber, Michaela; Sediri, Hanna; Felgenhauer, Ulrike et al. (2015) Influenza virus adaptation PB2-627K modulates nucleocapsid inhibition by the pathogen sensor RIG-I. Cell Host Microbe 17:309-319|
|Wang, Linya; Tian, Yongjun; Ou, Jing-hsiung James (2015) HCV induces the expression of Rubicon and UVRAG to temporally regulate the maturation of autophagosomes and viral replication. PLoS Pathog 11:e1004764|
|Lee, Jiyoung; Tian, Yongjun; Chan, Stephanie Tze et al. (2015) TNF-? Induced by Hepatitis C Virus via TLR7 and TLR8 in Hepatocytes Supports Interferon Signaling via an Autocrine Mechanism. PLoS Pathog 11:e1004937|
|Tian, Y; Kuo, C-F; Sir, D et al. (2015) Autophagy inhibits oxidative stress and tumor suppressors to exert its dual effect on hepatocarcinogenesis. Cell Death Differ 22:1025-34|
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