DDX39A interacts with LGP2 to inhibit RLR responses

Abstract

Abstract 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.