Subcellular localization dictates IFI16 ligand specificity in a cGAS-independent manner 3601

Abstract Description Cytosolic dsDNA is detected by an array of innate sensors. cGAS is the primary driver of STING-dependent IFN production in response to this ligand, but IFI16 also plays a role in this pathway. In response to dsDNA, IFI16 translocates from the nucleus into the cytosol and forms nucleoprotein filaments. Initially described as a dsDNA sensor, recent reports have suggested that IFI16 also recognizes RNA. Here, we investigated the nucleic acid specificity of IFI16 to better understand its role in the cell-intrinsic IFN response. We stimulated epithelial cells with synthetic nucleic acids: dsDNA, dsRNA, ssDNA, ssRNA and DNA-RNA hybrid. While IFNβ expression was triggered by all double-stranded ligands, STING phosphorylation occurred only with dsDNA. Moreover, cytosolic IFI16 filaments were only observed in response to dsDNA. However, in cells that stably express IFI16 mutants deficient in nuclear localization, cytosolic IFI16 filament assembly occurred with dsRNA or DNA-RNA hybrids. These observations indicate that 1) nuclear localization prevents IFI16 filament assembly on ligands other than dsDNA, and 2) an initial cytosolic sensing event with specificity for dsDNA is required for IFI16 translocation. Surprisingly, in cGAS KO cells, IFI16 retained the ability to translocate into the cytosol following dsDNA transfection. Together, these data indicate that cytosolic translocation of IFI16 is dsDNA-dependent; however, this phenomenon is uncoupled from cGAS-dependent IFN production. Funding Sources NIH K08AR077100 Topic Categories Innate Immune Responses and Host Defense: Molecular Mechanisms (INM)