Structural insights into the atypical filament assembly of pyrin domain-containing IFI16.

The Cooperative Assembly of IFI16 Filaments on dsDNA Provides Insights into Host Defense Strategy

The innate immune system is the first line of defense against microbial pathogens. The response of innate immunity is initiated by molecules known as pattern recognition receptors (PRRs). Such responses are often triggered by nucleic acids that are delivered to the cytoplasm or nucleus of cells. The ability to recognize foreign nucleic acids in these two locations is an important defense mechanism of the human innate immune system. Several PRRs are located in the cytosol or nucleus and detect foreign DNAs. The pyrin and hematopoietic interferon-inducible nuclear (PYHIN) domain protein is a family of PRRs that includes interferon-inducible protein 16, absent in melanoma 2, PYHIN 1 (or interferon-inducible protein X, as it is also known), myeloid cell nuclear differentiation antigen, and pyrin domain only protein 3. These nuclear and cytosolic sensors play an essential part in host defense of intracellular pathogens. In addition, members of the PYHIN family are critical regulators of immune response, apoptosis, cell growth, differentiation, and transcription. In this review, we summarize important characteristics of these innate immune sensors and their roles in several diseases. A better understanding of the role of DNA sensors in the nucleus and cytoplasm will lead to the development of novel therapeutic approaches to control infections and associated diseases.

Interferon-inducible protein 16 (IFI16) is a member of the HIN-200 protein family, containing two HIN domains and one PYRIN domain. IFI16 acts as a sensor of viral and bacterial DNA and is important for innate immune responses. IFI16 binds DNA and binding has been described to be DNA length-dependent, but a preference for supercoiled DNA has also been demonstrated. Here we report a specific preference of IFI16 for binding to quadruplex DNA compared to other DNA structures. IFI16 binds to quadruplex DNA with significantly higher affinity than to the same sequence in double stranded DNA. By circular dichroism (CD) spectroscopy we also demonstrated the ability of IFI16 to stabilize quadruplex structures with quadruplex-forming oligonucleotides derived from human telomere (HTEL) sequences and the MYC promotor. A novel H/D exchange mass spectrometry approach was developed to assess protein interactions with quadruplex DNA. Quadruplex DNA changed the IFI16 deuteration profile in parts of the PYRIN domain (aa 0–80) and in structurally identical parts of both HIN domains (aa 271–302 and aa 586–617) compared to single stranded or double stranded DNAs, supporting the preferential affinity of IFI16 for structured DNA. Our results reveal the importance of quadruplex DNA structure in IFI16 binding and improve our understanding of how IFI16 senses DNA. IFI16 selectivity for quadruplex structure provides a mechanistic framework for IFI16 in immunity and cellular processes including DNA damage responses and cell proliferation.

Structures of the HIN Domain:DNA Complexes Reveal Ligand Binding and Activation Mechanisms of the AIM2 Inflammasome and IFI16 Receptor

Truncated IFI16 mRNA transcripts can control its viral DNA defense activity.

Several host cell nuclear factors are known to restrict herpes simplex virus 1 (HSV-1) replication, but their mechanisms of action remain to be defined. Interferon-inducible protein 16 (IFI16) and the nuclear domain 10-associated proteins, such as promyelocytic leukemia (PML) protein, localize to input viral genomes, but they are also capable of restricting progeny viral transcription. In this study, we used structured illumination microscopy to show that after HSV DNA replication, IFI16 forms nuclear filamentous structures on DNA within a subset of nuclear replication compartments in HSV-1 ICP0-null mutant virus-infected human cells. The ability to form filaments in different cell types correlates with the efficiency of restriction, and the kinetics of filament formation and epigenetic changes are similar. Thus, both are consistent with the filamentous structures being involved in epigenetic silencing of viral progeny DNA. IFI16 filaments recruit other restriction factors, including PML, Sp100, and ATRX, to aid in the restriction. Although the filaments are only in a subset of the replication compartments, IFI16 reduces the levels of elongation-competent RNA polymerase II (Pol II) in all replication compartments. Therefore, we propose that IFI16 filaments with associated restriction factors that form in replication compartments constitute a "restrictosome" structure that signals in cis and trans to silence the progeny viral DNA throughout the infected cell nucleus. The IFI16 filamentous structure may constitute the first known nuclear supramolecular organizing center for signaling in the cell nucleus.IMPORTANCE Mammalian cells exhibit numerous strategies to recognize and contain viral infections. The best-characterized antiviral responses are those that are induced within the cytosol by receptors that activate interferon responses or shut down translation. Antiviral responses also occur in the nucleus, yet these intranuclear innate immune responses are poorly defined at the receptor-proximal level. In this study, we explored the ability of cells to restrict infection by assembling viral DNA into transcriptionally silent heterochromatin within the nucleus. We found that the IFI16 restriction factor forms filaments on DNA within infected cells. These filaments recruit antiviral restriction factors to prevent viral replication in various cell types. Mechanistically, IFI16 filaments inhibit the recruitment of RNA polymerase II to viral genes. We propose that IFI16 filaments with associated restriction factors constitute a "restrictosome" structure that can signal to other parts of the nucleus where foreign DNA is located that it should be silenced.

The zebrafish NLRP3 inflammasome has functional roles in ASC-dependent interleukin-1β maturation and gasdermin E–mediated pyroptosis

Local acidosis has been demonstrated in ischemic tissues and at inflammatory sites. Acidic extracellular pH triggers NLRP3 inflammasome activation and interleukin-1β secretion in human macrophages. Acidic pH represents a novel danger signal alerting the innate immunity. Local acidosis may promote inflammation at ischemic and inflammatory sites. Local extracellular acidification has been demonstrated at sites of ischemia and inflammation. IL-1β is one of the key proinflammatory cytokines, and thus, its synthesis and secretion are tightly regulated. The NLRP3 (nucleotide-binding domain leucine-rich repeat containing family, pyrin domain containing 3) inflammasome complex, assembled in response to microbial components or endogenous danger signals, triggers caspase-1-mediated maturation and secretion of IL-1β. In this study, we explored whether acidic environment is sensed by immune cells as an inflammasome-activating danger signal. Human macrophages were exposed to custom cell culture media at pH 7.5-6.0. Acidic medium triggered pH-dependent secretion of IL-1β and activation of caspase-1 via a mechanism involving potassium efflux from the cells. Acidic extracellular pH caused rapid intracellular acidification, and the IL-1β-inducing effect of acidic medium could be mimicked by acidifying the cytosol with bafilomycin A1, a proton pump inhibitor. Knocking down the mRNA expression of NLRP3 receptor abolished IL-1β secretion at acidic pH. Remarkably, alkaline extracellular pH strongly inhibited the IL-1β response to several known NLRP3 activators, demonstrating bipartite regulatory potential of pH on the activity of this inflammasome. The data suggest that acidic environment represents a novel endogenous danger signal alerting the innate immunity. Low pH may thus contribute to inflammation in acidosis-associated pathologies such as atherosclerosis and post-ischemic inflammatory responses.

Animal cells use pattern-recognition receptors (PRRs) to detect specific pathogens. Pathogen detection mounts an appropriate immune response, including interferon and cytokine induction. The intracellular PRR-signaling pathways that detect DNA viruses have been characterized, particularly in myeloid cells. In these pathways, cGMP-AMP synthase (cGAS) and the pyrin and HIN domain family member (PYHIN) protein interferon-γ–inducible protein 16 (IFI16) detect DNA and signal via stimulator of interferon genes protein (STING). However, although airway epithelial cells are frontline sentinels in detecting pathogens, information on how they respond to DNA viruses is limited, and the roles of PYHIN proteins in these cells are unknown. Here, we examined expression and activities of cGAS, STING, and PYHINs in human lung epithelial cells. A549 epithelial cells, commonly used for RNA-sensing studies, failed to respond to DNA because they lacked STING expression, and ectopic STING expression restored a cGAS-dependent DNA response in these cells. In contrast, NuLi-1 immortalized human bronchial epithelial cells did express STING, which was activated after DNA stimulation and mediated DNA-dependent gene induction. PYHIN1, which like IFI16 has been proposed to be a viral DNA sensor, was the only PYHIN protein expressed in both airway epithelial cell types. However, rather than having a role in DNA sensing, PYHIN1 induced proinflammatory cytokines in response to interleukin-1 (IL-1) or tumor necrosis factor α (TNFα) stimulation. Of note, PYHIN1, via its HIN domain, directly induced IL-6 and TNFα transcription, revealing that PYHIN proteins play a role in proinflammatory gene induction in airway epithelial cells.

Host innate immunity is crucial for orchestrating a protective response against dangerous pathogens. Herein, we demonstrate that interferon-inducible protein (IFI204), a DNA sensor, is implicated in protection against pulmonary pathogenic Mannheimia haemolytica (M. haemolytica) infection by driving inflammasome signaling activation. Ifi204−/− mice are more susceptible to pathogenic M. haemolytica infection compared with their wild-type (WT) counterparts, with decreased survival rates, extensive lung architecture destruction, exacerbated inflammatory cells infiltration, and more bacterial colonization. In vivo and in vitro findings elucidate that Ifi204 deficiency leads to a defect in inflammasome signaling activation, and exogenous recombinant IL-18 is sufficient to rescue the susceptibility of Ifi204−/− mice. Inflammasome signaling downstream of IFI204 facilitates early bacterial killing and clearance. Mechanistically, IFI204 promotes gasdermin D (GSDMD)-dependent inflammasome activation, and GSDMD is required for IFI204-mediated host defense. Notably, IFI204 detects pathogenic M. haemolytica-derived genomic DNA for the inflammasome signaling response. Thus, these data highlight the requirement of IFI204 in host defense response to M. haemolytica infection, and reveal that IFI204 may be a potential therapeutic target for pathogen control.

The interferon-inducible protein X (IFIX), a member of the PYHIN family, was recently recognized as an antiviral factor against infection with herpes simplex virus 1 (HSV-1). IFIX binds viral DNA upon infection and promotes expression of antiviral cytokines. How IFIX exerts its host defense functions and whether it is inhibited by the virus remain unknown. Here, we integrated live cell microscopy, proteomics, IFIX domain characterization, and molecular virology to investigate IFIX regulation and antiviral functions during HSV-1 infection. We find that IFIX has a dynamic localization during infection that changes from diffuse nuclear and nucleoli distribution in uninfected cells to discrete nuclear puncta early in infection. This is rapidly followed by a reduction in IFIX protein levels. Indeed, using immunoaffinity purification and mass spectrometry, we define IFIX interactions during HSV-1 infection, finding an association with a proteasome subunit and proteins involved in ubiquitin-proteasome processes. Using synchronized HSV-1 infection, microscopy, and proteasome-inhibition experiments, we demonstrate that IFIX co-localizes with nuclear proteasome puncta shortly after 3 h of infection and that its pyrin domain is rapidly degraded in a proteasome-dependent manner. We further demonstrate that, in contrast to several other host defense factors, IFIX degradation is not dependent on the E3 ubiquitin ligase activity of the viral protein ICP0. However, we show IFIX degradation requires immediate-early viral gene expression, suggesting a viral host suppression mechanism. The IFIX interactome also demonstrated its association with transcriptional regulatory proteins, including the 5FMC complex. We validate this interaction using microscopy and reciprocal isolations and determine it is mediated by the IFIX HIN domain. Finally, we show IFIX suppresses immediate-early and early viral gene expression during infection. Altogether, our study demonstrates that IFIX antiviral functions work in part via viral transcriptional suppression and that HSV-1 has acquired mechanisms to block its functions via proteasome-dependent degradation.

Inflammasomes are multi-protein signaling platforms that are primed upon sensing pathogen-associated molecular patterns (PAMPs) as well as endogenous damage-associated molecular patterns (Kolliputi et al., 2010, 2012). Once primed, a complex forms between the inflammasome sensor, an adaptor protein with a caspase recruiter domain (ASC), and caspase-1, which leads to the cleavage of pro-inflammatory cytokines Interleukin-1beta and Interleukin-18 (IL-1B, IL-18). A specific inflammasome sensor, nucleoside-triphosphatase domain (NACHT), leucine rich repeat (LRR), and pyrin domain (PYD) domains-containing protein 3 (NLRP3) has garnered the attention of many secondary to its ability to function as a general sensor of cell stress, possessing a large number of endogenous and exogenous activators compared to other inflammasome sensors such as Interferon-inducible protein (AIM2) and NLR family CARD domain-containing protein 4 (NLRC4). This pathway has also been implicated in numerous diseases like gout, alzheimers, obesity, and diabetes (Heneka et al., 2013; Wei et al., 2013). Given the prevalent evidence that this pathway is involved in many medical conditions, current and further studies about inflammasome regulation could potentially allow pharmaceutical researchers to develop drugs for currently incurable diseases. The NLRP3 inflammasome complex can be regulated in three main ways; the degree of which the inflammasome complex is expressed, the amount of NLRP3 activators present and post-transcriptional modulation via microRNAs (miRNA). miRNAs are defined as a class of non-coding oligonucleotides which function in post-transcriptional regulation of gene expression (Bartel, 2009; Tamarapu et al., 2012). A particular miRNA may have multiple targets, and a given mRNA may have several miRNAs that may exhibit regulatory function on itself (Krek et al., 2005; Jalali et al., 2012). These oligonucleotide sequences have become a hot area of research as they are proving to become relevant in the pathogenesis and possible cure to multiple medical conditions (Holohan et al., 2012). In atherosclerosis it has been shown that elevated miR-155 levels are found in pro-inflammatory macrophages as well as in atherosclerotic lesions (Wei et al., 2013). In the September 2012 edition of the Journal of Immunology, Bauernfeind et al. (2012) present exciting findings on inflammasome sensor, NLRP3, by showing its negative regulation via a type of miRNA, miR-223. To investigate the potential role of post-transcriptional regulation on the inflammasome pathway, Bauernfeind et al. (2012) conducted several experiments in different cell lineages. Their initial study involved examining luciferase activity after transfection with a plasmid armed with luciferase on the 3′UTR of human NLRP3, as well as a genome wide miRNA precursor library in 293T cells. Of the miRNA precursor library, miR-223 showed the greatest promise, decreasing the activity of NLRP3. Bauernfeind et al. (2012) showed that miR-223 levels increase during granulopoiesis and are highest in mature neutrophils, while expression is absent in T and B cells. They also noted an inverse relationship with regards to miR-223 and NLRP3 during the maturation process in the monocyte lineage, showing a decrease in miR-223 levels, an increase in NLRP3 transcripts, and caspase activation after culturing with granulocyte-macrophage colony-stimulating factor (GM-CSF). This was also proven by Haneklaus et al. (2012) as they showed low NLRP3 protein levels in monocytes and significantly higher levels in macrophages, indicating that the threshold for inflammasome activation is variable throughout granulopoiesis and different cell lines. The work of Bauernfeind et al. (2012) has shed light on a key mechanism in the inflammatory cascade, a process implicated in numerous diseases. Finding endogenous and exogenous mediators that can control the unchecked inflammation seen in these conditions is an important step to discover pharmaceutical intervention and even possible cures. Hopefully these studies will set the foundation for the exploration of the regulatory effects of miR-223 and other miRNAs in regulating inflammation in numerous medical conditions in the near future.

The proinflammatory cytokines interleukin (IL)-1β and IL-18 are products of activation of the inflammasome, an innate sensing system, and important in the pathogenesis of herpes simplex virus type 1 (HSV-1). The release of IL-18 and IL-1β from monocytes/macrophages is critical for protection from HSV-1 based on animal models of encephalitis and genital infection, yet if and how HSV-1 activates inflammasomes in human macrophages is unknown. To investigate this, we utilized both primary human monocyte derived macrophages and human monocytic cell lines (THP-1 cells) with various inflammasome components knocked-out. We found that HSV-1 activates inflammasome signaling in proinflammatory primary human macrophages, but not in resting macrophages. Additionally, HSV-1 inflammasome activation in THP-1 cells is dependent on nucleotide-binding domain and leucine-rich repeat-containing receptor 3 (NLRP3), apoptosis-associated speck-like molecule containing a caspase recruitment domain (ASC), and caspase-1, but not on absent in melanoma 2 (AIM2), or gamma interferon-inducible protein 16 (IFI16). In contrast, HSV-1 activates non-canonical inflammasome signaling in proinflammatory macrophages that results in IL-1β, but not IL-18, release that is independent of NLRP3, ASC, and caspase-1. Ultraviolet irradiation of HSV-1 enhanced inflammasome activation, demonstrating that viral replication suppresses inflammasome activation. These results confirm that HSV-1 is capable of activating the inflammasome in human macrophages through an NLRP3 dependent process and that the virus has evolved an NLRP3 specific mechanism to inhibit inflammasome activation in macrophages.

Death domain superfamily members typically act as adaptors mediating in the assembly of supramolecular complexes with critical apoptosis and inflammation functions. These modular proteins consist of death domains, death effector domains, caspase recruitment domains, and pyrin domains (PYD). Despite the high structural similarity among them, only homotypic interactions participate in complex formation, suggesting that subtle factors differentiate each interaction type. It is thus critical to identify these factors as an essential step toward the understanding of the molecular basis of apoptosis and inflammation. The proteins apoptosis-associated speck-like protein containing a CARD (ASC) and NLRP3 play key roles in the regulation of apoptosis and inflammation through self-association and protein-protein interactions mediated by their PYDs. To better understand the molecular basis of their function, we have characterized ASC and NLRP3 PYD self-association and their intermolecular interaction by solution NMR spectroscopy and analytical ultracentrifugation. We found that ASC self-associates and binds NLRP3 PYD through equivalent protein regions, with higher binding affinity for the latter. These regions are located at opposite sides of the protein allowing multimeric complex formation previously shown in ASC PYD fibril assemblies. We show that NLRP3 PYD coexists in solution as a monomer and highly populated large-order oligomerized species. Despite this, we determined its monomeric three-dimensional solution structure by NMR and characterized its binding to ASC PYD. Using our novel structural data, we propose molecular models of ASC·ASC and ASC·NLRP3 PYD early supramolecular complexes, providing new insights into the molecular mechanisms of inflammasome and apoptosis signaling.

ABSTRACTThe human interferon-inducible protein IFI16 is an important antiviral factor that binds nuclear viral DNA and promotes antiviral responses. Here, we define IFI16 dynamics in space and time and its distinct functions from the DNA sensor cyclic dinucleotide GMP-AMP synthase (cGAS). Live-cell imaging reveals a multiphasic IFI16 redistribution, first to viral entry sites at the nuclear periphery and then to nucleoplasmic puncta upon herpes simplex virus 1 (HSV-1) and human cytomegalovirus (HCMV) infections. Optogenetics and live-cell microscopy establish the IFI16 pyrin domain as required for nuclear periphery localization and oligomerization. Furthermore, using proteomics, we define the signature protein interactions of the IFI16 pyrin and HIN200 domains and demonstrate the necessity of pyrin for IFI16 interactions with antiviral proteins PML and cGAS. We probe signaling pathways engaged by IFI16, cGAS, and PML using clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-mediated knockouts in primary fibroblasts. While IFI16 induces cytokines, only cGAS activates STING/TBK-1/IRF3 and apoptotic responses upon HSV-1 and HCMV infections. cGAS-dependent apoptosis upon DNA stimulation requires both the enzymatic production of cyclic dinucleotides and STING. We show that IFI16, not cGAS or PML, represses HSV-1 gene expression, reducing virus titers. This indicates that regulation of viral gene expression may function as a greater barrier to viral replication than the induction of antiviral cytokines. Altogether, our findings establish coordinated and distinct antiviral functions for IFI16 and cGAS against herpesviruses.