Inflammasomes are multiprotein complexes that recognize pathogens and pathogen-or danger-associated molecular patterns. They induce the maturation and secretion of powerful proinflammatory interleukin-1B (IL-1␤), IL-18, and IL-33 cytokines, which in turn activate expression of other immune genes and lymphocyte recruitment to the site of primary infection, thereby controlling invading pathogens. Inflammasomes are comprised of cytoplasmic sensor molecules, such as NLRP3 and AIM2 or nuclear sensor IFI16, the adaptor protein ASC (apoptosis-associated speck-like protein containing CARD), and the effector protein procaspase-1. Herpes simplex virus 1 (HSV-1), a ubiquitous virus that infects humans and establishes life-long latency, has evolved numerous mechanisms to evade host detection and immune responses. Here, we show that early during in vitro infection of human foreskin fibroblasts (2 to 4 h), HSV-1 induced the activation of the IFI16 and NLRP3 inflammasomes and maturation of IL-1␤. Independent of viral gene expression, IFI16 recognized the HSV-1 genome in infected cell nuclei, relocalized, and colocalized with ASC in the cytoplasm. However, HSV-1 specifically targeted IFI16 for rapid proteasomic degradation at later times postinfection, which was dependent on the expression of ICP0, an immediate early protein of HSV-1. In contrast, NLRP3, AIM2, and ASC levels were not decreased. Also, caspase-1 was "trapped" in actin clusters at later time points that likely blocked the NLRP3/IFI16 inflammasome activity. In addition, the secretion of mature IL-1␤ was inhibited. These results suggest that though the host cell responds to HSV-1 infection by IFI16 and NLRP3 inflammasomes early during infection, HSV-1 has evolved mechanisms to shut down these responses to evade the proinflammatory consequences.

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The IL-1β and type I interferon-β (IFN-β) molecules are important inflammatory cytokines elicited by the eukaryotic host as innate immune responses against invading pathogens and danger signals. Recently, a predominantly nuclear gamma-interferon-inducible protein 16 (IFI16) involved in transcriptional regulation has emerged as an innate DNA sensor which induced IL-1β and IFN-β production through inflammasome and STING activation, respectively. Herpesvirus (KSHV, EBV, and HSV-1) episomal dsDNA genome recognition by IFI16 leads to IFI16-ASC-procaspase-1 inflammasome association, cytoplasmic translocation and IL-1β production. Independent of ASC, HSV-1 genome recognition results in IFI16 interaction with STING in the cytoplasm to induce interferon-β production. However, the mechanisms of IFI16-inflammasome formation, cytoplasmic redistribution and STING activation are not known. Our studies here demonstrate that recognition of herpesvirus genomes in the nucleus by IFI16 leads into its interaction with histone acetyltransferase p300 and IFI16 acetylation resulting in IFI16-ASC interaction, inflammasome assembly, increased interaction with Ran-GTPase, cytoplasmic redistribution, caspase-1 activation, IL-1β production, and interaction with STING which results in IRF-3 phosphorylation, nuclear pIRF-3 localization and interferon-β production. ASC and STING knockdowns did not affect IFI16 acetylation indicating that this modification is upstream of inflammasome-assembly and STING-activation. Vaccinia virus replicating in the cytoplasm did not induce nuclear IFI16 acetylation and cytoplasmic translocation. IFI16 physically associates with KSHV and HSV-1 genomes as revealed by proximity ligation microscopy and chromatin-immunoprecipitation studies which is not hampered by the inhibition of acetylation, thus suggesting that acetylation of IFI16 is not required for its innate sensing of nuclear viral genomes. Collectively, these studies identify the increased nuclear acetylation of IFI16 as a dynamic essential post-genome recognition event in the nucleus that is common to the IFI16-mediated innate responses of inflammasome induction and IFN-β production during herpesvirus (KSHV, EBV, HSV-1) infections.

The innate immune system pattern recognition receptors (PRR) are the first line of host defenses recognizing the various pathogen- or danger-associated molecular patterns and eliciting defenses by regulating the production of pro-inflammatory cytokines such as IL-1β, IL-18 or interferon β (IFN-β). NOD-like receptors (NLRs) and AIM2-like receptors (ALRs) are cytoplasmic inflammasome sensors of foreign molecules, including DNA. IFI16, a sequence-independent nuclear innate sensor ALR, recognizes episomal dsDNA genomes of herpes viruses such as KSHV, EBV, and HSV-1 in the infected cell nuclei, forms an inflammasome complex with ASC and procaspase1, and relocates into the cytoplasm leading into Caspase-1 and IL-1β generation. IFI16 also induces IFN-β during HSV-1 infection via the cytoplasmic STING-TBK1-IRF3 pathway. Thus far, whether IFI16 recognizes foreign DNA directly or utilizes other host protein(s) is unknown. Here, we demonstrate that BRCA1, a DNA damage repair sensor and transcription regulator, is in complex with IFI16 in the host cell nucleus, and their association increases in the presence of nuclear viral genomes during de novo KSHV, EBV and HSV-1 infection, and in latent KSHV or EBV infection, but not by DNA damage responses (DDR) induced by bleomycin and vaccinia virus cytoplasmic dsDNA. BRCA1 is a constituent of the triggered IFI16-inflammasome and is translocated into the cytoplasm after genome recognition along with the IFI16-inflammasome. The absence of BRCA1 abrogated IFI16-viral genome association, inflammasome assembly, IFI16 cytoplasmic localization, and Caspase-1 and IL-1β production. The absence of BRCA1 also abolished the cytoplasmic IFI16-STING interaction, downstream IRF3 phosphorylation, nuclear translocation of pIRF3 and IFN-β production during de novo KSHV and HSV-1 infection. These findings highlight that BRCA1 plays a hitherto unidentified innate immunomodulatory role by facilitating nuclear foreign DNA sensing by IFI16, subsequent assembly and cytoplasmic distribution of IFI16-inflammasomes leading into IL-1β formation and the induction of IFN-β via cytoplasmic signaling through IFI16-STING, TBK1 and IRF3.