RIG-I-like receptors (RLRs) are DEAD box helicases and cytoplasmic pathogen recognition receptors that recognize and bind to nonself/viral RNA and trigger innate antiviral immunity. RLRs are essential for detection of RNA virus infection, and include RIG-I, MDA5, and LGP2. LGP2 has been implicated as RIG-I or MDA5 cofactor to regulate their activity. We conducted a yeast 2 hybrid (Y2H) screen (using LGP2 as bait) of a human hepatic cDNA library to identify protein binding partners of LGP2. Identified proteins were validated as LGP2 interactors through overexpression and co-immunoprecipitation assays of LGP2 binding. The validated proteins were then submitted to a cell-based interferon beta-promoter-luciferase assay model of Sendai virus (SenV) infection as well as a cell based NFkB-luciferase assay of the same system. Endogenous expression of the validated proteins was then assayed through the Huh7 human hepatoma cell line system with various stimuli including viral infection with SenV and Interferon-beta treatment. Through these studies we will define the LGP2 interactome and identify proteins that act as co-factors in RLR signaling.

RIG-I-like receptors (RLRs) are DEAD-box helicases and cytoplasmic pathogen recognition receptors that recognize and bind to nonself/viral RNA and trigger innate antiviral immunity. RLRs are essential for the detection of RNA virus infection and include RIG-I, MDA5, and LGP2. LGP2 has been implicated as RIG-I or MDA5 co-factor to regulate their activity. We conducted a yeast 2-hybrid (Y2H) screen (using LGP2 as bait) of a human hepatic cDNA library to identify protein binding partners of LGP2. Identified proteins were validated as LGP2 interactors through overexpression and co-immunoprecipitation assays of LGP2 binding. DDX39A, a DEAD Box helicase protein with known roles in RNA splicing, was identified as a binding partner. DDX39A overexpression was found to inhibit interferon-beta production from RLR-signaling in a cell-based ligand-free model in a dose-dependent manner. Further, in a cell-based interferon beta-promoter-luciferase assay model of Sendai virus (SenV) infection, DDX39A and LGP2 synergistically inhibited promoter activity. Similar results were seen in a cell-based NFkB-luciferase assay of the same system. Endogenous expression of DDX39A was assayed through the Huh7 human hepatoma cell line system with various stimuli including viral infection with SenV and Interferon-beta treatment showing that DDX39A is constitutively expressed in those cells. Structure-function mutants of DDX39A and LGP2 were used to determine the protein domains and functions required for interaction via co-immunoprecipitation assays. Through these studies, we will define the interactions between LGP2 and DDX39A, and reveal the roles these proteins have as co-factors in RLR signaling.