CHARACTERIZATION OF THE ANTIVIRAL MECHANISMS OF THE NUCLEAR DNA SENSORS IFIX AND IFI16 DURING HERPESVIRUS INFECTION

Abstract

Mammalian cells express a wide array of pattern recognition receptors (PRRs) to identify invading pathogens and activate immune signaling pathways. The nuclear PRRs interferon-inducible protein X (IFIX) and interferon-inducible protein 16 (IFI16) are DNA sensors that bind to double-stranded DNA (dsDNA) within the nucleus and initiate antiviral programs. This viral dsDNA originates from a number of sources, including the genomes of nuclear replicating viruses (e.g., herpesviruses and papillomaviruses) and the dsDNA intermediates formed during retrovirus (e.g., human immunodeficiency virus) replication. Our lab and others have found that, upon binding to the dsDNA genome of herpes simplex virus 1 (HSV-1), IFIX and IFI16 each induce antiviral cytokine expression and suppress virus gene expression. However, many facets of IFIX- and IFI16-mediated antiviral defense have remained uncharacterized. Here, we explore the mechanisms of IFIX and IFI16 cytokine induction and suppression of viral gene expression through an interdisciplinary study, combining classical molecular biology and virology techniques with a multi-omics approach of proteomics and genomics methods. Altogether, we find that IFIX can localize to foreign DNA in both the cytoplasm and the nucleus, and subcellular IFIX localization is regulated by a multipartite nuclear localization signal which can be acetylated to promote cytoplasmic localization. Further, via IFIX-knockout proteome studies, we determine IFIX mediates distinct subcellular proteome changes in the cytoplasm and nucleus in response to pathogenic dsDNA within each compartment. Regarding IFI16, we identify the biochemical properties of the pyrin domain which mediate IFI16 oligomerization and facilitate immune response during HSV-1 infection. We additionally map the interaction between IFI16 and the HSV-1 genome during early timepoints of infection, and we establish that this interaction is DNA sequence-independent but is augmented at particularly accessible genomic loci. We also observed that IFI16 expression epigenetically regulates HSV-1 DNA by globally reducing genome accessibility, resulting in suppression of viral protein expression across the entire genome. Moreover, we find that IFI16 oligomerization promotes interactions with the chromatin modulating proteins UBTF and PAF1C. Thus, we put forward that IFI16 binding to highly accessible genomic loci stimulates IFI16 oligomerization, thereby recruiting regulatory proteins to the viral genome to dampen gene expression.