IFI16 phase separation via multi-phosphorylation drives innate immune signaling.

The interferon inducible protein 16 (IFI16) is a prominent sensor of nuclear pathogenic DNA, initiating innate immune signaling and suppressing viral transcription. However, little is known about mechanisms that initiate IFI16 antiviral functions or its regulation within the host DNA-filled nucleus. Here, we provide in vitro and in vivo evidence to establish that IFI16 undergoes liquid-liquid phase separation (LLPS) nucleated by DNA. IFI16 binding to viral DNA initiates LLPS and induction of cytokines during herpes simplex virus type 1 (HSV-1) infection. Multiple phosphorylation sites within an intrinsically disordered region (IDR) function combinatorially to activate IFI16 LLPS, facilitating filamentation. Regulated by CDK2 and GSK3ß, IDR phosphorylation provides a toggle between active and inactive IFI16 and the decoupling of IFI16-mediated cytokine expression from repression of viral transcription. These findings show how IFI16 switch-like phase transitions are achieved with temporal resolution for immune signaling and, more broadly, the multi-layered regulation of nuclear DNA sensors.

The Nuclear DNA Sensor IFI16 Indiscriminately Binds to and Diminishes Accessibility of the HSV-1 Genome to Suppress Infection.

Human cells identify invading pathogens and activate immune signaling pathways through a wide array of pattern recognition receptors, including DNA sensors. The interferon-inducible protein 16 (IFI16) is a nuclear DNA sensor that recognizes double-stranded DNA from a number of viral sources, including genomes of nuclear-replicating viruses. Among these is the prevalent human pathogen herpes simplex virus 1 (HSV-1). Upon binding to the HSV-1 DNA genome, IFI16 both induces antiviral cytokine expression and suppresses virus gene expression. Here, we used a multiomics approach of DNA sequencing techniques paired with targeted mass spectrometry to obtain an extensive view of the interaction between IFI16 and the HSV-1 genome and how this binding affects the viral DNA structure and protein expression. Through chromatin immunoaffinity purification coupled with next-generation DNA sequencing (ChIP-seq), we found that IFI16 binds to the HSV-1 genome in a sequence-independent manner while simultaneously exhibiting broad enrichment at two loci: UL30, the viral DNA polymerase gene, and US1 to US7. The assay for transposase-accessible chromatin with sequencing (ATAC-seq) revealed that these two regions are among the most accessible stretches of DNA on the genome, thereby facilitating IFI16 binding. Accessibility of the entire HSV-1 genome is elevated upon IFI16 knockout, indicating that expression of IFI16 globally induces chromatinization of viral DNA. Deletion of IFI16 also results in a global increase in the expression of HSV-1 proteins, as measured by parallel reaction monitoring-mass spectrometry of viral proteins representing 80% of the HSV-1 genome. Altogether, we demonstrate that IFI16 interacts with the HSV-1 genome in a sequence-independent manner, coordinating epigenetic silencing of the viral genome and decreasing protein expression and virus replication. IMPORTANCE Mammalian host defense against viral infection includes broad-acting cellular restriction factors, as well as effectors of intrinsic and innate immunity. IFI16 is a critical nuclear host defense factor and intrinsic immune protein involved in binding viral DNA genomes, thereby repressing the replication of nucleus-replicating viruses, including the human herpes simplex virus 1. What has remained unclear is where on the viral genome IFI16 binds and how binding affects both viral DNA structural accessibility and viral protein expression. Our study provides a global view of where and how a nuclear restriction factor of DNA viruses associates with viral genomes to exert antiviral functions during early stages of an acute virus infection. Our study can additionally serve as a systems-level model to evaluate nuclear DNA sensor interactions with viral genomes, as well as the antiviral outcomes of transcriptionally silencing pathogen-derived DNA.

The DNA Sensor IFIX Drives Proteome Alterations To Mobilize Nuclear and Cytoplasmic Antiviral Responses, with Its Acetylation Acting as a Localization Toggle.

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. IMPORTANCE 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.

Interrogating Host Antiviral Environments Driven by Nuclear DNA Sensing: A Multiomic Perspective.

Nuclear DNA sensors are critical components of the mammalian innate immune system, recognizing the presence of pathogens and initiating immune signaling. These proteins act in the nuclei of infected cells by binding to foreign DNA, such as the viral genomes of nuclear-replicating DNA viruses herpes simplex virus type 1 (HSV-1) and human cytomegalovirus (HCMV). Upon binding to pathogenic DNA, the nuclear DNA sensors were shown to initiate antiviral cytokines, as well as to suppress viral gene expression. These host defense responses involve complex signaling processes that, through protein-protein interactions (PPIs) and post-translational modifications (PTMs), drive extensive remodeling of the cellular transcriptome, proteome, and secretome to generate an antiviral environment. As such, a holistic understanding of these changes is required to understand the mechanisms through which nuclear DNA sensors act. The advent of omics techniques has revolutionized the speed and scale at which biological research is conducted and has been used to make great strides in uncovering the molecular underpinnings of DNA sensing. Here, we review the contribution of proteomics approaches to characterizing nuclear DNA sensors via the discovery of functional PPIs and PTMs, as well as proteome and secretome changes that define a host antiviral environment. We also highlight the value of and future need for integrative multiomic efforts to gain a systems-level understanding of DNA sensors and their influence on epigenetic and transcriptomic alterations during infection.

Workflows and considerations for investigating protein interactions of viral DNA sensors.

DNA sensors are a core component of innate immunity in mammalian cells. In response to pathogen infection, these specialized proteins sense pathogenic DNA from bacteria or viruses and initiate immune signaling cascades. These defense mechanisms rely on the rapid formation and temporal regulation of protein-protein interactions. Similarly, protein interactions underlie virus immune evasion mechanisms, as proteins from diverse viruses associate with and inhibit DNA sensors. Here, we describe experimental protocols for identifying protein interactions of DNA sensors, and discuss considerations for optimal isolation of protein complexes when targeting either endogenous or tagged proteins. Additionally, as viral infections and immune responses are known to induce prominent changes in cellular protein abundances, we provide a workflow for investigating these protein associations in the context of proteome alterations.

Charge-Mediated Pyrin Oligomerization Nucleates Antiviral IFI16 Sensing of Herpesvirus DNA.

The formation of multimerized protein assemblies has emerged as a core component of immune signal amplification, yet the biochemical basis of this phenomenon remains unclear for many mammalian proteins within host defense pathways. The interferon-inducible protein 16 (IFI16) is a viral DNA sensor that oligomerizes upon binding to nuclear viral DNA and induces downstream antiviral responses. Here, we identify the pyrin domain (PYD) residues that mediate IFI16 oligomerization in a charge-dependent manner. Based on structure modeling, these residues are predicted to be surface exposed within distinct α-helices. By generating oligomerization-deficient mutants, we demonstrate that IFI16 homotypic clustering is necessary for its assembly onto parental viral genomes at the nuclear periphery upon herpes simplex virus 1 (HSV-1) infection. Preventing oligomerization severely hampered the capacity of IFI16 to induce antiviral cytokine expression, suppress viral protein levels, and restrict viral progeny production. Restoring oligomerization via residue-specific charge mimics partially rescued IFI16 antiviral roles. We show that pyrin domains from PYHIN proteins are functionally interchangeable, facilitating cooperative assembly with the IFI16 HINs, highlighting an inherent role for pyrin domains in antiviral response. Using immunoaffinity purification and targeted mass spectrometry, we establish that oligomerization promotes IFI16 interactions with proteins involved in transcriptional regulation, including PAF1C, UBTF, and ND10 bodies. We further discover PAF1C as an HSV-1 restriction factor. Altogether, our study uncovers intrinsic properties that govern IFI16 oligomerization, which serves as a signal amplification platform to activate innate immune responses and to recruit transcriptional regulatory proteins that suppress HSV-1 replication.IMPORTANCE The ability of mammalian cells to detect the genomes of nuclear-replicating viruses via cellular DNA sensors is fundamental to innate immunity. Recently, mounting evidence is supporting the universal role of polymerization in these host defense factors as a signal amplification strategy. Yet, what has remained unclear are the intrinsic properties that govern their immune signal transmission. Here, we uncover the biochemical basis for oligomerization of the nuclear DNA sensor, IFI16. Upon infection with herpes simplex virus 1 (HSV-1) in human fibroblasts, we characterize the contribution of IFI16 oligomerization to downstream protein interactions and antiviral functions, including cytokine induction and suppression of HSV-1 replication. Until now, the global characterization of oligomerization-dependent protein interactions for an immune receptor has never been explored. Our integrative quantitative proteomics, molecular CRISPR/Cas9-based assays, mutational analyses, and confocal microscopy shed light on the dynamics of immune signaling cascades activated against pathogens.