HIN Domain Research Articles

Inhibition of AIM2 inflammasome activation by SOX/ORF37 promotes lytic replication of Kaposi's sarcoma-associated herpesvirus

Inflammasomes are one kind of important innate immune defense against viral and bacterial infections. Several inflammasome-forming sensors detect molecular patterns of invading pathogens and then trigger inflammasome activation and/or pyroptosis in infected cells, and viruses employ unique strategies to hijack or subvert inflammasome activation. Infection with herpesviruses induces the activation of diverse inflammasomes, including AIM2 and IFI16 inflammasomes; however, how Kaposi's sarcoma-associated herpesvirus (KSHV) counteracts inflammasome activation largely remains unclear. Here, we reveal that the KSHV ORF37-encoded SOX protein suppresses AIM2 inflammasome activation independent of its viral DNA exonuclease activity and host mRNA turnover. SOX interacts with the AIM2 HIN domain through the C-terminal Motif VII region and disrupts AIM2:dsDNA polymerization and ASC recruitment and oligomerization. The Y443A or F444A mutation of SOX abolishes the inhibition of AIM2 inflammasome without disrupting SOX nuclease activity, and a short SOX peptide is capable of inhibiting AIM2 inflammasome activation; consequently, infection with SOX-null, Y443A, or F444A Bac16 recombinant viruses results in robust inflammasome activation, suppressed lytic replication, and increased pyroptosis in human lymphatic endothelial cells in an AIM2-dependent manner. These results reveal that KSHV SOX suppresses AIM2 inflammasome activation to promote KSHV lytic replication and inhibit pyroptosis, representing a unique mechanism for evasion of inflammasome activation during KSHV lytic cycle.

Discovery of an inhibitor of DNA-driven inflammation that preferentially targets the AIM2 inflammasome

Inflammation driven by DNA sensors is now understood to be important to disease pathogenesis. Here, we describe new inhibitors of DNA sensing, primarily of the inflammasome forming sensor AIM2. Biochemistry and molecular modeling has revealed 4-sulfonic calixarenes as potent inhibitors of AIM2 that likely work by binding competitively to the DNA-binding HIN domain. Although less potent, these AIM2 inhibitors also inhibit DNA sensors cGAS and TLR9 demonstrating a broad utility against DNA-driven inflammatory responses. The 4-sulfonic calixarenes inhibited AIM2-dependent post-stroke Tcell death, highlighting a proof of concept that the 4-sulfonic calixarenes could be effective at combating post-stroke immunosuppression. By extension, we propose a broad utility against DNA-driven inflammation in disease. Finally, we reveal that the drug suramin, by virtue of its structural similarities, is an inhibitor of DNA-dependent inflammation and propose that suramin could be rapidly repurposed to meet an increasing clinical need.

Impact of nsSNPs in human AIM2 and IFI16 gene: a comprehensive in silico analysis

AIM2 and IFI16 are the most studied members of AIM2-like receptors (ALRs) in humans and share a common N-Terminal PYD domain and C-terminal HIN domain. The HIN domain binds to dsDNA in response to the invasion of bacterial and viral DNA, and the PYD domain directs apoptosis-associated speck-like protein via protein–protein interactions. Hence, activation of AIM2 and IFI16 is crucial for protection against pathogenic assaults, and any genetic variation in these inflammasomes can dysregulate the human immune system. In this study, different computational tools were used to identify the most deleterious and disease-causing non-synonymous single nucleotide polymorphisms (nsSNPs) in AIM2 and IFI16 proteins. Molecular dynamic simulation was performed for the top damaging nsSNPs to study single amino acid substitution-induced structural alterations in AIM2 and IFI16. The observed results suggest that the variants G13V, C304R, G266R, and G266D for AIM2, and G13E and C356F are deleterious and affect structural integrity. We hope that the suggested deleterious nsSNPs and structural dynamics of AIM2 and IFI16 variants will guide future research to better understand the function of these variants with large-scale studies and may assist in fresher therapeutics focusing on these polymorphisms. Communicated by Ramaswamy H. Sarma

Cytosolic DNA sensor IFI16 proteins: Potential molecular integrators of interactions among the aging hallmarks.

Cellular changes that are linked to aging in humans include genomic instability, telomere attrition, epigenetic alterations, mitochondrial dysfunction, cellular senescence, and altered intercellular communications. The extent of the changes in these aging hallmarks and their interactions with each other are part of the human aging. However, the molecular mechanisms through which the aging hallmarks interact with each other remain unclear. Studies have indicated a potential role for the type I interferon (IFN) and p53-inducible IFI16 proteins in interactions with the aging hallmarks. The IFI16 proteins are members of the PYHIN protein family. Proteins in the family share a DNA-binding domain (the HIN domain) and a protein-protein interaction pyrin domain (PYD). IFI16 proteins are needed for cytosolic DNA-induced activation of the cGAS-STING pathway for type I IFN (IFN-β) expression. The pathway plays an important role in aging-related inflammation (inflammaging). Further, increased levels of the IFI16 proteins potentiate the cell growth inhibitory functions of the p53 and pRb tumor suppressors proteins. Moreover, IFI16 proteins are needed for most aging hallmarks. Therefore, here we discuss how an improved understanding of the role of the IFI16 proteins in integration of the aging hallmarks has potential to improve the human health and lifespan.

Functions and roles of IFIX, a member of the human HIN-200 family, in human diseases.

Pyrin and hematopoietic expression, interferon-inducible nature, and nuclear localization (HIN) domain family member 1 (PYHIN1), also known as IFIX, belongs to the family of pyrin proteins. This family includes structurally and functionally related mouse (e.g., p202, p203, and p204 proteins) and human (e.g., the interferon-inducible protein 16, absent in melanoma 2 protein, myeloid cell nuclear differentiation antigen, and pyrin and HIN domain family 1 or IFIX) proteins. The IFIX protein belongs to the HIN-200 family of interferon-inducible proteins that have a 200-amino acid signature motif at their C-termini. The increased expression of pyrin proteins in most cell types inhibits cell cycle control and modulates cell survival. Consistent with this role for pyrin proteins, IFIX is a potential antiviral DNA sensor that is essential for immune responses, the detection of viral DNA in the nucleus and cytoplasm, and the binding of foreign DNA via its HIN domain in a sequence non-specific manner. By promoting the ubiquitination and subsequent degradation of MDM2, IFIX acts as a tumor suppressor, thereby leading to p53/TP53 stabilization, HDAC1 regulation via the ubiquitin-proteasome pathway, and tumor-cell-specific silencing of the maspin gene. These data demonstrate that the potential molecular mechanism(s) underlying the action of the IFIX protein might be associated with the development of human diseases, such as viral infections, malignant tumors, and autoimmune diseases. This review summarizes the current insights into IFIX functions and how its regulation affects the outcomes of various human diseases.

Targeting NLRP3 Inflammasome in Translational Treatment of Nervous System Diseases: An Update

Neuroinflammatory response is the immune response mechanism of the innate immune system of the central nervous system. Both primary and secondary injury can activate neuroinflammatory response. Among them, the nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome plays a key role in the inflammatory response of the central system. Inflammasome is a type of pattern recognition receptor, a cytoplasmic polyprotein complex composed of members of the Nod-like receptor (NLR) family and members of the pyrin and HIN domain (PYHIN) family, which can be affected by a variety of pathogen-related molecular patterns or damage-related molecular patterns are activated. As one of the research hotspots in the field of medical research in recent years, there are increasing researches on immune function abnormalities in the onset of neurological diseases such as depression, AD, ischemic brain injury and cerebral infarction, the NLRP3 inflammasome causes the activated caspase-1 to cleave pre-interleukin-1β and pre-interleukin-18 into mature interleukin-1β and interleukin-18, in turn, a large number of inflammatory factors are produced, which participate in the occurrence and development of the above-mentioned diseases. Targeted inhibition of the activation of inflammasomes can reduce the inflammatory response, promote the survival of nerve cells, and achieve neuroprotective effects. This article reviews NLRP3 inflammasome’s role in neurological diseases and related regulatory mechanisms, which providing references for future research in this field.

Structural mechanism of DNA recognition by the p204 HIN domain.

The interferon gamma-inducible protein 16 (IFI16) and its murine homologous protein p204 function in non-sequence specific dsDNA sensing; however, the exact dsDNA recognition mechanisms of IFI16/p204, which harbour two HIN domains, remain unclear. In the present study, we determined crystal structures of p204 HINa and HINb domains, which are highly similar to those of other PYHIN family proteins. Moreover, we obtained the crystal structure of p204 HINab domain in complex with dsDNA and provided insights into the dsDNA binding mode. p204 HINab binds dsDNA mainly through α2 helix of HINa and HINb, and the linker between them, revealing a similar HIN:DNA binding mode. Both HINa and HINb are vital for HINab recognition of dsDNA, as confirmed by fluorescence polarization assays. Furthermore, a HINa dimerization interface was observed in structures of p204 HINa and HINab:dsDNA complex, which is involved in binding dsDNA. The linker between HINa and HINb reveals dynamic flexibility in solution and changes its direction at ∼90° angle in comparison with crystal structure of HINab:dsDNA complex. These structural information provide insights into the mechanism of DNA recognition by different HIN domains, and shed light on the unique roles of two HIN domains in activating the IFI16/p204 signaling pathway.

Role of NLRP3 Inflammasome Activation in Obesity-Mediated Metabolic Disorders.

NLRP3 inflammasome is one of the multimeric protein complexes of the nucleotide-binding domain, leucine-rich repeat (NLR)-containing pyrin and HIN domain family (PYHIN). When activated, NLRP3 inflammasome triggers the release of pro-inflammatory interleukins (IL)-1β and IL-18, an essential step in innate immune response; however, defective checkpoints in inflammasome activation may lead to autoimmune, autoinflammatory, and metabolic disorders. Among the consequences of NLRP3 inflammasome activation is systemic chronic low-grade inflammation, a cardinal feature of obesity and insulin resistance. Understanding the mechanisms involved in the regulation of NLRP3 inflammasome in adipose tissue may help in the development of specific inhibitors for the treatment and prevention of obesity-mediated metabolic diseases. In this narrative review, the current understanding of NLRP3 inflammasome activation and regulation is highlighted, including its putative roles in adipose tissue dysfunction and insulin resistance. Specific inhibitors of NLRP3 inflammasome activation which can potentially be used to treat metabolic disorders are also discussed.

HUWE1 mediates inflammasome activation and promotes host defense against bacterial infection.

The mechanism by which inflammasome activation is modulated remains unclear. In this study, we identified an AIM2-interacting protein, the E3 ubiquitin ligase HUWE1, which was also found to interact with NLRP3 and NLRC4 through the HIN domain of AIM2 and the NACHT domains of NLRP3 and NLRC4. The BH3 domain of HUWE1 was important for its interaction with NLRP3, AIM2, and NLRC4. Caspase-1 maturation, IL-1β release, and pyroptosis were reduced in Huwe1-deficient bone marrow-derived macrophages (BMDMs) compared with WT BMDMs in response to stimuli to induce NLRP3, NLRC4, and AIM2 inflammasome activation. Furthermore, the activation of NLRP3, NLRC4, and AIM2 inflammasomes in both mouse and human cells was remarkably reduced by treatment with the HUWE1 inhibitor BI8622. HUWE1 mediated the K27-linked polyubiquitination of AIM2, NLRP3, and NLRC4, which led to inflammasome assembly, ASC speck formation, and sustained caspase-1 activation. Huwe1-deficient mice had an increased bacterial burden and decreased caspase-1 activation and IL-1β production upon Salmonella, Francisella, or Acinetobacter baumannii infection. Our study provides insights into the mechanisms of inflammasome activation as well as a potential therapeutic target against bacterial infection.

Nuclear PYHIN proteins target the host transcription factor Sp1 thereby restricting HIV-1 in human macrophages and CD4+ T cells.

Members of the family of pyrin and HIN domain containing (PYHIN) proteins play an emerging role in innate immunity. While absent in melanoma 2 (AIM2) acts a cytosolic sensor of non-self DNA and plays a key role in inflammasome assembly, the γ-interferon-inducible protein 16 (IFI16) restricts retroviral gene expression by sequestering the transcription factor Sp1. Here, we show that the remaining two human PYHIN proteins, i.e. myeloid cell nuclear differentiation antigen (MNDA) and pyrin and HIN domain family member 1 (PYHIN1 or IFIX) share this antiretroviral function of IFI16. On average, knock-down of each of these three nuclear PYHIN proteins increased infectious HIV-1 yield from human macrophages by more than an order of magnitude. Similarly, knock-down of IFI16 strongly increased virus transcription and production in primary CD4+ T cells. The N-terminal pyrin domain (PYD) plus linker region containing a nuclear localization signal (NLS) were generally required and sufficient for Sp1 sequestration and anti-HIV-1 activity of IFI16, MNDA and PYHIN1. Replacement of the linker region of AIM2 by the NLS-containing linker of IFI16 resulted in a predominantly nuclear localization and conferred direct antiviral activity to AIM2 while attenuating its ability to form inflammasomes. The reverse change caused nuclear-to-cytoplasmic relocalization of IFI16 and impaired its antiretroviral activity but did not result in inflammasome assembly. We further show that the Zn-finger domain of Sp1 is critical for the interaction with IFI16 supporting that pyrin domains compete with DNA for Sp1 binding. Finally, we found that human PYHIN proteins also inhibit Hepatitis B virus and simian vacuolating virus 40 as well as the LINE-1 retrotransposon. Altogether, our data show that IFI16, PYHIN1 and MNDA restrict HIV-1 and other viral pathogens by interfering with Sp1-dependent gene expression and support an important role of nuclear PYHIN proteins in innate antiviral immunity.

PYHIN1 regulates pro-inflammatory cytokine induction rather than innate immune DNA sensing in airway epithelial cells

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.

Interferon-Responsive Genes Are Targeted during the Establishment of Human Cytomegalovirus Latency.

Human cytomegalovirus (HCMV) latency is an active process which remodels the latently infected cell to optimize latent carriage and reactivation. This is achieved, in part, through the expression of viral genes, including the G-protein-coupled receptor US28. Here, we use an unbiased proteomic screen to assess changes in host proteins induced by US28, revealing that interferon-inducible genes are downregulated by US28. We validate that major histocompatibility complex (MHC) class II and two pyrin and HIN domain (PYHIN) proteins, myeloid cell nuclear differentiation antigen (MNDA) and IFI16, are downregulated during experimental latency in primary human CD14+ monocytes. We find that IFI16 is targeted rapidly during the establishment of latency in a US28-dependent manner but only in undifferentiated myeloid cells, a natural site of latent carriage. Finally, by overexpressing IFI16, we show that IFI16 can activate the viral major immediate early promoter and immediate early gene expression during latency via NF-κB, a function which explains why downregulation of IFI16 during latency is advantageous for the virus.IMPORTANCE Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus which infects 50 to 100% of humans worldwide. HCMV causes a lifelong subclinical infection in immunocompetent individuals but is a serious cause of mortality and morbidity in the immunocompromised and neonates. In particular, reactivation of HCMV in the transplant setting is a major cause of transplant failure and related disease. Therefore, a molecular understanding of HCMV latency and reactivation could provide insights into potential ways to target the latent viral reservoir in at-risk patient populations.

P200 family protein IFI204 negatively regulates type I interferon responses by targeting IRF7 in nucleus.

Interferon-inducible p200 family protein IFI204 was reported to be involved in DNA sensing, and subsequently induces the production of type I interferons and proinflammatory mediators. However, its function in the regulation of antiviral innate immune signaling pathway remains unclear. Here we reported a novel role of IFI204 that specifically inhibits the IRF7-mediated type I interferons response during viral infection. IFI204 and other p200 family proteins are highly expressed in mouse hepatitis coronavirus-infected bone marrow-derived dendritic cells. The abundant IFI204 could significantly interact with IRF7 in nucleus by its HIN domain and prevent the binding of IRF7 with its corresponding promoter. Moreover, other p200 family proteins that possess HIN domain could also inhibit the IRF7-mediated type I interferons. These results reveal that, besides the positive regulation function in type I interferon response at the early stage of DNA virus infection, the interferon-inducible p200 family proteins such as IFI204 could also negatively regulate the IRF7-mediated type I interferon response after RNA virus infection to avoid unnecessary host damage from hyper-inflammatory responses.

Structural analysis of the HIN1 domain of interferon-inducible protein 204.

Interferon-inducible protein 204 (p204) binds to microbial DNA to elicit inflammatory responses and induce interferon production. p204 also modulates cell proliferation and differentiation by regulating various transcription factors. The C-terminal HIN domains in p204 are believed to be responsible for DNA binding, but the binding mode is not fully understood. The DNA-binding affinity of the p204 HIN1 domain has been characterized and its crystal structure has been determined, providing insight into its interaction with DNA. Surface-charge distribution together with sequence alignment suggests that the p204 HIN domain uses its L12 and L45 loops for DNA binding.

IFI16 Targets the Transcription Factor Sp1 to Suppress HIV-1 Transcription and Latency Reactivation

The interferon γ-inducible protein 16 (IFI16) is known as immune sensor of retroviral DNA intermediates. We show that IFI16 restricts HIV-1 independently ofimmune sensing by binding and inhibiting the host transcription factor Sp1 that drives viral gene expression. This antiretroviral activity and ability to bind Sp1 require the N-terminal pyrin domain and nuclear localization of IFI16, but not the HIN domains involved in DNA binding. Highly prevalent clade C HIV-1 strains are more resistant to IFI16 and less dependent on Sp1 than other HIV-1 subtypes. Furthermore, inhibition of Sp1 by IFI16 or pharmacologically by Mithramycin A suppresses reactivation of latent HIV-1 in CD4+ Tcells. Finally, IFI16 also inhibits retrotransposition of LINE-1, known to engage Sp1, and murine IFI16 homologs restrict Friend retrovirus replication in mice. Thus, IFI16 restricts retroviruses and retrotransposons by interfering with Sp1-dependent gene expression, and evasion from this restriction may facilitate spread of HIV-1 subtype C.

Nature of selection varies on different domains of IFI16-like PYHIN genes in ruminants

BackgroundALRs (AIM2-like Receptors) are germline encoded PRRs that belong to PYHIN gene family of cytokines, which are having signature N-terminal PYD (Pyrin, PAAD or DAPIN) domain and C-terminal HIN-200 (hematopoietic, interferon-inducible nuclear protein with 200 amino acid repeat) domain joined by a linker region. The positively charged HIN-200 domain senses and binds with negatively charged phosphate groups of single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) purely through electrostatic attractions. On the other hand, PYD domain interacts homotypically with a PYD domain of other mediators to pass the signals to effector molecules downwards the pathways for inflammatory responses. There is remarkable inter-specific diversity in the numbers of functional PYHIN genes e.g. one in cow, five in human, thirteen in mice etc., while there is a unique loss of PYHIN genes in the bat genomes which was revealed by Ahn et al. (2016) by studying genomes of ten different bat species belonging to sub-orders yinpterochiroptera and yangochiroptera. The conflicts between host and pathogen interfaces are compared with “Red queen’s arms race” which is also described as binding seeking dynamics and binding avoidance dynamics. As a result of this never-ending rivalry, eukaryotes developed PRRs as antiviral mechanism while viruses developed counter mechanisms to evade host immune defense. The PYHIN receptors are directly engaged with pathogenic molecules, so these should have evolved under the influence of selection pressures. In the current study, we investigated the nature of selection pressure on different domain types of IFI16-like (IFI16-L) PYHIN genes in ruminants.ResultsThree transcript variants of the IFI16-like gene were found in PBMCs of ruminant animals-water buffalo, zebu cattle, goat, and sheep. The IFI16-like gene has one N-terminal PYD domain and one C-terminal HIN-200 domain, separated by an inter-domain linker region. HIN domain and inter-domain region are positively selected while the PYD domain is under the influence of purifying selection.ConclusionHerein, we conclude that the nature of selection pressure varies on different parts (PYD domain, HIN domain, and inter-domain linker region) of IFI16-like PYHIN genes in the ruminants. This data can be useful to predict the molecular determinants of pathogen interactions.

Inhibition of AIM2 inflammasome activation by a novel transcript isoform of IFI16.

Mouse p202 is a disease locus for lupus and a dominant-negative inhibitor of AIM2 inflammasome activation. A human homolog of p202 has not been identified so far. Here, we report a novel transcript isoform of human IFI16-designated IFI16-β, which has a domain architecture similar to that of mouse p202. Like p202, IFI16-β contains two HIN domains, but lacks the pyrin domain. IFI16-β is ubiquitously expressed in various human tissues and cells. Its mRNA levels are also elevated in leukocytes of patients with lupus, virus-infected cells, and cells treated with interferon-β or phorbol ester. IFI16-β co-localizes with AIM2 in the cytoplasm, whereas IFI16-α is predominantly found in the nucleus. IFI16-β interacts with AIM2 to impede the formation of a functional AIM2-ASC complex. In addition, IFI16-β sequesters cytoplasmic dsDNA and renders it unavailable for AIM2 sensing. Enforced expression of IFI16-β inhibits the activation of AIM2 inflammasome, whereas knockdown of IFI16-β augments interleukin-1β secretion triggered by dsDNA but not dsRNA Thus, cytoplasm-localized IFI16-β is functionally equivalent to mouse p202 that exerts an inhibitory effect on AIM2 inflammasome.

Therapeutic Targeting of NLRP3 Inflammasomes by Natural Products and Pharmaceuticals: A Novel Mechanistic Approach for Inflammatory Diseases

Inflammasomes are multiprotein complexes having nucleotide-binding domain and leucine-rich repeat consisting members along with pyrin and HIN domain family. An inflammasome mainly consists of cytoplasmic sensor molecule, such as NLRP3, the adaptor apoptosisassociated speck-like protein containing caspase recruitment domain) protein along with effector procaspase-1. The inflammasome regulates caspase-1 activation, resulting in secretion of interleukin- 1β and interleukin-18. The inflammasome activation is linked with infection, stress, or other immunological signals involved in inflammation. The pathophysiological role of NLRP3 inflammasome in immune regulation, inflammatory receptor-ligand interactions, microbial-associated molecular patterns, danger as well as pathogen associated molecular patterns has been demonstrated in last few years. Furthermore, the role of the inflammasome in peripheral and central nervous system involved with cytokine and chemokine inflammatory responses has been demonstrated in preclinical and clinical studies. The understanding of molecular regulation of inflammasome associated pathways is crucial for drug design and delivery. The use of natural product as an alternate therapy is gaining focus because of easy access and cost effectiveness. A number of herbal extracts and its bioactive constituents known as phytochemicals have shown to be effective in inflammatory response mediated by NLRP3 inflammasomes pathways. To understand the interaction of phytochemicals and inflammasome at the molecular level, it is vital to develop effective drugs that can be evaluated further in the clinical settings. Therefore, this review renders an extensive account of all the phytochemicals which are evaluated either in inflammatory experimental animal models or in immortalized human/animal cell lines that modulate NLRP3 inflammasome mediated pathways to mitigate inflammatory responses with the hope that this pathway modulation by phytochemicals may provide a another class of drugs in the armamentarium as well as novel molecular mechanism of natural products targeting NLRP3 inflammasome.

Human Antiviral Protein IFIX Suppresses Viral Gene Expression during Herpes Simplex Virus 1 (HSV-1) Infection and Is Counteracted by Virus-induced Proteasomal Degradation

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.

IFI16 Preferentially Binds to DNA with Quadruplex Structure and Enhances DNA Quadruplex Formation.

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.