DNA sensors are critical components of innate immunity that enable cells to recognize infection by pathogens with DNA genomes. The interferon-inducible protein X (IFIX), a member of the PYHIN protein family, is a DNA sensor capable of promoting immune signaling after binding to double-stranded DNA (dsDNA) within either the nucleus or cytoplasm. Here, we investigate the impact of IFIX on the cellular proteome upon introduction of foreign DNA to the nucleus or the cytoplasm as well as regulatory hubs that control IFIX subcellular localization. Using quantitative mass spectrometry, we define the effect of CRISPR-mediated IFIX knockout on nuclear and cytoplasmic proteomes in fibroblasts. Proteomes are probed in response to either nuclear viral DNA, during herpes simplex virus 1 (HSV-1) infection, or cytoplasmic viral DNA, following transfection with dsDNA derived from vaccinia virus (VACV 70-mer). We show that IFIX broadly impacts nuclear and cytoplasmic proteomes, inducing alterations in the abundances of immune signaling, DNA damage response, and vesicle-mediated transport proteins. To characterize IFIX properties that regulate its localization during DNA sensing, we perform deletion and mutagenesis assays. We find that IFIX contains a multipartite nuclear localization signal (NLS) and highlight the main contributing motif for its nuclear localization. Using immunoaffinity purification, we identify IFIX acetylation and phosphorylation sites. Mutations to acetyl or charge mimics demonstrate that K138 acetylation, positioned within the NLS, affects nuclear localization. Altogether, our study establishes a mechanism regulating IFIX subcellular localization and contextualizes this localization with the involvement of IFIX in host cell responses to pathogenic DNA. Mammalian cells must be able to detect and respond to invading pathogens to prevent the spread of infection. DNA sensors, such as IFIX, are proteins that bind to pathogen-derived double-stranded DNA and induce antiviral cytokine expression. Here, we characterize the host proteome changes that require IFIX during both viral infection and DNA transfection. We show IFIX mobilizes numerous pathways and proteome alterations within the nucleus and the cytoplasm, pointing to a multifunctional protein with roles in immune signaling, DNA damage response, and transcriptional regulation. We next interrogate the IFIX domains required for nuclear localization, discovering its regulation via a multipartite nuclear localization motif. The acetylation of this motif promotes IFIX cytoplasmic localization, in agreement with its detection of pathogenic DNA in both the nucleus and the cytoplasm. This study established NLS acetylation as a conserved mechanism for regulating the localization of nuclear DNA sensors from the PYHIN family of proteins.
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