A viral E3 ubiquitin ligase produced by herpes simplex virus 1 inhibits the NLRP1 inflammasome.

Guard proteins initiate defense mechanisms upon sensing pathogen-encoded virulence factors. Successful viral pathogens likely inhibit guard protein activity, but these interactions have been largely undefined. Here, we demonstrate that the human pathogen herpes simplex virus 1 (HSV-1) stimulates and inhibits an antiviral pathway initiated by NLRP1, a guard protein that induces inflammasome formation and pyroptotic cell death when activated. Notably, HSV-1 infection of human keratinocytes promotes posttranslational modifications to NLRP1, consistent with MAPK-dependent NLRP1 activation, but does not result in downstream inflammasome formation. We identify infected cell protein 0 (ICP0) as the critical HSV-1 protein that is necessary and sufficient for inhibition of the NLRP1 pathway. Mechanistically, ICP0's cytoplasmic localization and function as an E3 ubiquitin ligase prevents proteasomal degradation of the auto-inhibitory NT-NLRP1 fragment, thereby preventing inflammasome formation. Further, we demonstrate that inhibiting this inflammasome is important for promoting HSV-1 replication. Thus, we have established a mechanism by which HSV-1 overcomes a guard-mediated antiviral defense strategy in humans.

Herpes simplex virus infected cell protein 8 is required for viral inhibition of the cGAS pathway.

DNA virus infection triggers an antiviral type I interferon (IFN) response in cells that suppresses infection of surrounding cells. Consequently, viruses have evolved mechanisms to inhibit the IFN response for efficient replication. The cellular cGAS protein binds to double-stranded DNA and synthesizes the small molecule cGAMP to initiate DNA-dependent type I IFN production. We showed previously that cGAMP production is relatively low during HSV-1 infection compared to plasmid DNA transfection. Therefore, we hypothesized that HSV-1 produces antagonists of the cGAS DNA sensing pathway. In this study, we found that the HSV-1 ICP8 protein is required for viral inhibition of the cGAS pathway by reducing cGAMP levels stimulated by double-stranded DNA transfection. ICP8 alone inhibited the cGAMP response and may inhibit cGAS action by direct interaction with DNA, cGAS, or other infected cell proteins. Our results reveal another cGAS antiviral pathway inhibitor and highlight the importance of countering IFN for efficient viral replication.

Methotrexate suppresses psoriatic skin inflammation by inhibiting muropeptide transporter SLC46A2 activity.

Cytosolic innate immune sensing is critical for protecting barrier tissues. NOD1 and NOD2 are cytosolic sensors of small peptidoglycan fragments (muropeptides) derived from the bacterial cell wall. These muropeptides enter cells, especially epithelial cells, through unclear mechanisms. We previously implicated SLC46 transporters in muropeptide transport in Drosophila immunity. Here, we focused on Slc46a2, which was highly expressed in mammalian epidermal keratinocytes, and showed that it was critical for the delivery of diaminopimelic acid (DAP)-muropeptides and activation of NOD1 in keratinocytes, whereas the related transporter Slc46a3 was critical for delivering the NOD2 ligand MDP to keratinocytes. In a mouse model, Slc46a2 and Nod1 deficiency strongly suppressed psoriatic inflammation, whereas methotrexate, a commonly used psoriasis therapeutic, inhibited Slc46a2-dependent transport of DAP-muropeptides. Collectively, these studies define SLC46A2 as a transporter of NOD1-activating muropeptides, with critical roles in the skin barrier, and identify this transporter as an important target for anti-inflammatory intervention.

An orphan no more: Nur77 senses cytosolic LPS.

Cytosolic LPS activates the NLRP3 inflammasome via a gasdermin D (GSDMD)-dependent mechanism. In this issue of Immunity, Zhu et al.1 provide insight into the events linking these two steps, identifying the orphan nuclear receptor Nur77 as a mediator of NLRP3 activation that senses LPS and GSDMD-dependent accumulation of cytosolic mtDNA.

Effector-triggered immunity in mammalian antiviral defense.

Effector-triggered immunity (ETI) is a common defense strategy used by mammalian host cells that is engaged upon detection of the enzymatic activities of pathogen-encoded proteins or the effects of their expression on cellular homeostasis. However, in contrast to the effector-triggered responses engaged upon bacterial infection, much less is understood about the activation and consequences of these responses following viral infection. Several recent studies have identified novel mechanisms by which viruses engage ETI, highlighting the importance of these immune responses in antiviral defense. We summarize recent advances in understanding how mammalian cells sense virus-encoded effector proteins, the downstream signaling pathways that are triggered by these sensing events, and how viruses manipulate these pathways to become more successful pathogens.

Depletion of the apical endosome in response to viruses and bacterial toxins provides cell-autonomous host defense at mucosal surfaces.

Polarized epithelial cells form an essential barrier against infection at mucosal surfaces. Many pathogens breach this barrier to cause disease, often by co-opting cellular endocytosis mechanisms to enter the cell through the lumenal (apical) cell surface. We recently discovered that the loss of the cell polarity gene PARD6B selectively diminishes apical endosome function. Here, we find that in response to the entry of certain viruses and bacterial toxins into the epithelial cells via the apical membrane, PARD6B and aPKC, two components of the PARD6B-aPKC-Cdc42 apical polarity complex, undergo rapid proteasome-dependent degradation. The perturbation of apical membrane glycosphingolipids by toxin- or virus-binding initiates degradation of PARD6B. The loss of PARD6B causes the depletion of apical endosome function and renders the cell resistant to further infection from the lumenal cell surface, thus enabling a form of cell-autonomous host defense.

Virus-mediated inactivation of anti-apoptotic Bcl-2 family members promotes Gasdermin-E-dependent pyroptosis in barrier epithelial cells.

Two sets of innate immune proteins detect pathogens. Pattern recognition receptors (PRRs) bind microbial products, whereas guard proteins detect virulence factor activities by the surveillance of homeostatic processes within cells. While PRRs are well known for their roles in many types of infections, the role of guard proteins in most infectious contexts remains less understood. Here, we demonstrated that inhibition of protein synthesis during viral infection is sensed as a virulence strategy and initiates pyroptosis in human keratinocytes. We identified the BCL-2 family members MCL-1 and BCL-xL as sensors of translation shutdown. Virus- or chemical-induced translation inhibition resulted in MCL-1 depletion and inactivation of BCL-xL, leading to mitochondrial damage, caspase-3-dependent cleavage of gasdermin E, and release of interleukin-1α (IL-1α). Blocking this pathway enhanced virus replication in an organoid model of human skin. Thus, MCL-1 and BCL-xL can act as guard proteins within barrier epithelia and contribute to antiviral defense.

Modular Architecture of the STING C-Terminal Tail Allows Interferon and NF-κB Signaling Adaptation.

Stimulator of interferon genes (STING) is a key regulator of type I interferon and pro-inflammatory responses during infection, cellular stress, and cancer. Here, we reveal a mechanism for how STING balances activation of IRF3- and NF-κB-dependent transcription and discover that acquisition of discrete signaling modules in the vertebrate STING C-terminal tail (CTT) shapes downstream immunity. As a defining example, we identify a motif appended to the CTT of zebrafish STING that inverts the typical vertebrate signaling response and results in dramatic NF-κB activation and weak IRF3-interferon signaling. We determine a co-crystal structure that explains how this CTT sequence recruits TRAF6 as a new binding partner and demonstrate that the minimal motif is sufficient to reprogram human STING and immune activation in macrophage cells. Together, our results define the STING CTT as a linear signaling hub that can acquire modular motifs to readily adapt downstream immunity.

Human Skin Explants Recapitulate Key Features of HSV-1 Infections.

The development of novel antiviral compounds is hindered by the lack of model systems that recapitulate the pathophysiology of human infections. Tajpara et al. developed an ex vivo human abdominal skin model of HSV-1 infection to examine host-pathogen interactions and test the efficacy of antiviral compounds. This approach provides a platform for future development and testing of antiviral drugs.

An Antiviral Branch of the IL-1 Signaling Pathway Restricts Immune-Evasive Virus Replication.

Virulent pathogens often cause the release of host-derived damage-associated molecular patterns (DAMPs) from infected cells. During encounters with immune-evasive viruses that block inflammatory gene expression, preformed DAMPs provide backup inflammatory signals that ensure protective immunity. Whether DAMPs exhibit additional backup defense activities is unknown. Herein, we report that viral infection of barrier epithelia (keratinocytes) elicits the release of preformed interleukin-1 (IL-1) family cytokines, including the DAMP IL-1α. Mechanistic studies revealed that IL-1 acts on skin fibroblasts to induce an interferon (IFN)-like state that restricts viral replication. We identified a branch in the IL-1 signaling pathway that induces IFN-stimulated gene expression in infected cells and found that IL-1 signaling is necessary to restrict viral replication in human skin explants. These activities are most important to control immune-evasive virus replication in fibroblasts and other barrier cell types. These findings highlight IL-1 as an important backup antiviral system to ensure barrier defense.

Mechanisms of Host IFI16, PML, and Daxx Protein Restriction of Herpes Simplex Virus 1 Replication.

The initial events after DNA virus infection involve a race between epigenetic silencing of the incoming viral DNA by host cell factors and expression of viral genes. Several host gene products, including the nuclear domain 10 (ND10) components PML (promyelocytic leukemia) and Daxx (death domain-associated protein 6), as well as IFI16 (interferon-inducible protein 16), have been shown to restrict herpes simplex virus 1 (HSV-1) replication. Whether IFI16 and ND10 components work together or separately to restrict HSV-1 replication is not known. To determine the combinatorial effects of IFI16 and ND10 proteins on viral infection, we depleted Daxx or PML in primary human foreskin fibroblasts (HFFs) in the presence or absence of IFI16. Daxx or IFI16 depletion resulted in higher ICP0 mutant viral yields, and the effects were additive. Surprisingly, small interfering RNA (siRNA) depletion of PML in the HFF cells led to decreased ICP0-null virus replication, while short hairpin RNA (shRNA) depletion led to increased ICP0-null virus replication, arguing that different PML isoforms or PML-related proteins may have restrictive or proviral functions. In normal human cells, viral DNA replication increases expression of all classes of HSV-1 genes. We observed that IFI16 repressed transcription from both parental and progeny DNA genomes. Taken together, our results show that the mechanisms of action of IFI16 and ND10 proteins are independent, at least in part, and that IFI16 exerts restrictive effects on both input and replicated viral genomes. These results raise the potential for distinct mechanisms of action of IFI16 on parental and progeny viral DNA molecules.IMPORTANCE Many human DNA viruses transcribe their genomes and replicate in the nucleus of a host cell, where they exploit the host cell nuclear machinery for their own replication. Host factors attempt to restrict viral replication by blocking such events, and viruses have evolved mechanisms to neutralize the host restriction factors. In this study, we provide information about the mechanisms of action of three host cell factors that restrict replication of herpes simplex virus (HSV). We found that these factors function independently and that one acts to restrict viral transcription from parental and progeny viral DNA genomes. These results provide new information about how cells counter DNA virus replication in the nucleus and provide possible approaches to enhance the ability of human cells to resist HSV infection.

Apoptosis and Necroptosis as Host Defense Strategies to Prevent Viral Infection.

Antiviral transcriptional responses and regulated cell death are crucial components of the host response to virus infection. However, in contrast to the signaling pathways that promote antiviral transcription, those that initiate cell death following virus infection are less understood. Several recent studies have identified pattern recognition receptors (PRRs) of the mammalian innate immune system that activate cell death pathways. These same receptors also have established roles in the induction of antiviral gene expression. In this review we discuss the mechanisms by which PRRs can serve dual roles as initiators of inflammatory gene expression and as inducers of apoptosis and necroptosis following virus infection.

Innate Immune Mechanisms and Herpes Simplex Virus Infection and Disease.

Innate immune responses play a major role in the control of herpes simplex virus (HSV) infections, and a multiplicity of mechanisms have emerged as a result of human evolution to sense and respond to HSV infections. HSV in turn has evolved a number of ways to evade immune detection and to blunt human innate immune responses. In this review, we summarize the major host innate immune mechanisms and the HSV evasion mechanisms that have evolved. We further discuss how disease can result if this equilibrium between virus and host response is disrupted.

Relative Contributions of Herpes Simplex Virus 1 ICP0 and vhs to Loss of Cellular IFI16 Vary in Different Human Cell Types.

UNLABELLED: The herpes simplex virus 1 (HSV-1) ICP0 protein is an E3 ubiquitin ligase that promotes the degradation of several host cell proteins. Most studies have found that ICP0 promotes the loss of IFI16 in infected cells, but one study reported that ICP0 was not necessary or sufficient for loss of IFI16 in a tumor-derived cell line. Therefore, in this study, we examined the requirement for ICP0 in promoting the loss of IFI16 in several normal and tumor-derived cell lines. HSV-1 infection resulted in an observable decrease of IFI16 protein levels in normal human foreskin fibroblasts (HFFs), normal oral keratinocytes (NOKs), and HeLa cells but not in U2OS cells. During infection with an ICP0-null virus, we observed a reduced loss of IFI16 in HFFs and NOKs but not in HeLa cells. Ectopic expression of ICP0 from a transfected plasmid was sufficient to promote the loss of IFI16 in HFFs and NOKs. In the absence of ICP0, we observed a delayed reduction of IFI16 protein that correlated with a reduction in the steady-state levels of IFI16 mRNA. In addition, we show that the ICP0-independent loss of IFI16 in HeLa cells is dependent in part on the activity of the viral virion host shutoff (vhs) tegument protein. Together, these results demonstrate that HSV-1 promotes the loss of IFI16 through at least two mechanisms: (i) by ICP0-dependent degradation of IFI16 and (ii) by vhs-dependent turnover of IFI16 mRNA. In addition, this study highlights a potential intrinsic difference between normal and tumor-derived cells for the activities of IFI16 and HSV-1 ICP0. IMPORTANCE: HSV-1 is a ubiquitous virus that establishes a lifetime persistent infection in humans. The relative success of HSV-1 as a pathogen is, in part, dependent on the expression of viral proteins that counteract host intrinsic defense mechanisms and that modulate immune responses during viral infection. In this study, we examined the relative roles of two viral gene products for the ability to promote loss of the antiviral IFI16 DNA sensor. We demonstrate that the viral immediate early ICP0 protein plays a dominant role in the loss of IFI16 in normal, but not tumor-derived, human cell lines. In contrast, viral vhs-mediated loss of IFI16 by mRNA destabilization is revealed to be dominant in tumor-derived cells in which ICP0 is nonfunctional. Together, these results contribute to our understanding of how HSV-1 modulates IFI16 protein levels and highlight cell-type-dependent differences between normal and tumor-derived cells.

A one-protein signaling pathway in the innate immune system.

Group A Streptococcus protease SpeB directly binds and activates IL-1ß (LaRock et al., this issue).

cGAS-mediated stabilization of IFI16 promotes innate signaling during herpes simplex virus infection.

Interferon γ-inducible protein 16 (IFI16) and cGMP-AMP synthase (cGAS) have both been proposed to detect herpesviral DNA directly in herpes simplex virus (HSV)-infected cells and initiate interferon regulatory factor-3 signaling, but it has been unclear how two DNA sensors could both be required for this response. We therefore investigated their relative roles in human foreskin fibroblasts (HFFs) infected with HSV or transfected with plasmid DNA. siRNA depletion studies showed that both are required for the production of IFN in infected HFFs. We found that cGAS shows low production of cGMP-AMP in infected cells, but instead cGAS is partially nuclear in normal human fibroblasts and keratinocytes, interacts with IFI16 in fibroblasts, and promotes the stability of IFI16. IFI16 is associated with viral DNA and targets to viral genome complexes, consistent with it interacting directly with viral DNA. Our results demonstrate that IFI16 and cGAS cooperate in a novel way to sense nuclear herpesviral DNA and initiate innate signaling.

Cellular sensing of viral DNA and viral evasion mechanisms.

Mammalian cells detect foreign DNA introduced as free DNA or as a result of microbial infection, leading to the induction of innate immune responses that block microbial replication and the activation of mechanisms that epigenetically silence the genes encoded by the foreign DNA. A number of DNA sensors localized to a variety of sites within the cell have been identified, and this review focuses on the mechanisms that detect viral DNA and how the resulting responses affect viral infections. Viruses have evolved mechanisms that inhibit these host sensors and signaling pathways, and the study of these antagonistic viral strategies has provided insight into the mechanisms of these host responses. The field of cellular sensing of foreign DNA is in its infancy, but our currently limited knowledge has raised a number of important questions for study.

A promiscuous lipid-binding protein diversifies the subcellular sites of toll-like receptor signal transduction.

The Toll-like receptors (TLRs) of the innate immune system are unusual in that individual family members are located on different organelles, yet most activate a common signaling pathway important for host defense. It remains unclear how this common signaling pathway can be activated from multiple subcellular locations. Here, we report that, in response to natural activators of innate immunity, the sorting adaptor TIRAP regulates TLR signaling from the plasma membrane and endosomes. TLR signaling from both locations triggers the TIRAP-dependent assembly of the myddosome, a protein complex that controls proinflammatory cytokine expression. The actions of TIRAP depend on the promiscuity of its phosphoinositide-binding domain. Different lipid targets of this domain direct TIRAP to different organelles, allowing it to survey multiple compartments for the presence of activated TLRs. These data establish how promiscuity, rather than specificity, can be a beneficial means of diversifying the subcellular sites of innate immune signal transduction.

Nuclear interferon-inducible protein 16 promotes silencing of herpesviral and transfected DNA.

Mammalian cells have evolved mechanisms to silence foreign DNA introduced by viruses or by transfection. Upon herpesviral infection of cells, the viral genome is chromatinized in an attempt by the host cell to restrict expression of the viral genome. HSV ICP0 acts to counter host-intrinsic and innate responses to viral infection. We have found that nuclear interferon (IFN)-inducible protein 16 (IFI16) acts as a restriction factor against ICP0-null herpes simplex virus 1 (HSV-1) to limit viral replication and immediate-early gene expression. IFI16 promoted the addition of heterochromatin marks and the reduction of euchromatin marks on viral chromatin. IFI16 also restricted the expression of plasmid DNAs introduced by transfection but did not restrict SV40 DNA introduced into the cellular nucleus in the form of nucleosomal chromatin by viral infection. These results argue that IFI16 restricts unchromatinized DNA when it enters the cell nucleus by promoting the loading of nucleosomes and the addition of heterochromatin marks. Furthermore, these results indicate that IFI16 provides a broad surveillance role against viral and transfected DNA by promoting restriction of gene expression from the exogenous DNA and inducing innate immune responses.

Identification of regulators of the innate immune response to cytosolic DNA and retroviral infection by an integrative approach.

The innate immune system senses viral DNA that enters mammalian cells, or in aberrant situations self-DNA, and triggers type I interferon production. Here we present an integrative approach that combines quantitative proteomics, genomics and small molecule perturbations to identify genes involved in this pathway. We silenced 809 candidate genes, measured the response to dsDNA and connected resulting hits with the known signaling network. We identified ABCF1 as a critical protein that associates with dsDNA and the DNA-sensing components HMGB2 and IFI204. We also found that CDC37 regulates the stability of the signaling molecule TBK1 and that chemical inhibition of the CDC37-HSP90 interaction and several other pathway regulators potently modulates the innate immune response to DNA and retroviral infection.