Gale Jr., MichaelPattabhi, Sowmya2015-02-242015-02-242014Pattabhi_washington_0250E_13879.pdfhttp://hdl.handle.net/1773/27377Thesis (Ph.D.)--University of Washington, 2014Retinoic acid inducible gene-I (RIG-I) is a cytoplasmic pathogen recognition receptor essential for recognition of RNA viruses. RIG-I engagement of viral RNA activates downstream signaling through the mitochondrial antiviral-signaling adaptor (MAVS) and results in the production of type I interferons, pro-inflammatory cytokines, and the expression of innate immune genes that suppress virus infection. My work has been focused on understanding the regulation of RIG-I signaling and developing small molecule agonists that target the RIG-I-like receptors as a strategy to develop host-targeted, broad-spectrum antivirals that work to inhibit emerging and re-emerging RNA virus infections. This thesis focuses on the regulation of RIG-I signaling in the first part. To identify cofactors of RIG-I signaling, a proteomic approach coupled with assessment of innate immune signaling function was used to identify the DEAH-box RNA helicase, DHX15 as a RIG-I signaling partner. My work identifies DHX15, as a positive regulator that is essential for innate immune response to control viruses of the family- Paramyxoviridae (Sendai virus), Picornaviridae (Encephalomyocarditis virus) and Rhabdoviridae (Vesicular stomatitis virus) and characterizes DHX15 as a RNA helicase that binds RIG-I and melanoma differentiation associated factor 5 (MDA5) basally through interactions mediated by the DHX15 N-terminus and the RLR CARDs. RNA binding studies identify DHX15 as a pathogen recognition receptor that binds non-self single-stranded RNA (ssRNA) and double-stranded RNA (dsRNA). DHX15 and RIG-I bind dsRNA more efficiently than either protein alone, indicating that DHX15 serves as a PAMP co-receptor to enhance RLR signaling. In the second part, I hypothesize that small molecule agonists that target the RLR pathway and induce cell and tissue-wide innate antiviral immunity can be effectively used in therapeutic approaches to control RNA virus infections. A small molecule diversity library designed based on the ligand binding pocket within RIG-I, was used in a cell-based screening approach to identify compounds that drive IRF3 activation. Hit compounds identified in the screen differentially induce a discrete subset of proinflammatory cytokines, chemokines and antiviral genes. Correspondingly, compound administration before or after infection significantly decreased the viral RNA load in cultured cells that were infected with the family Flaviviridae West Nile virus (WNV), dengue virus (DV) and hepatitis C virus (HCV), the Filoviridae Ebola virus (EBOV, Zaire), Orthomyxoviridae Influenza A virus (IAV, H3N2, Udorn strain) and Paramyxoviridae respiratory syncytial virus (RSV) and concomitantly suppressed infectious virus production. This study thus identifies a novel class of immune-modulatory molecules that activate RLR signaling to promote host antiviral responses to broadly suppress infection by RNA viruses of distinct genera. These compounds target the host and therefore could be broad-spectrum and effectively developed as a first line of defense in controlling RNA viruses.application/pdfen-USCopyright is held by the individual authors.Antiviral small molecules; DHX15; Innate immune response; Regulator; RIG-I agonists; RIG-I signalingImmunologyVirologyglobal healthThesis