Redox homeostasis maintained by GPX4 facilitates STING activation.

Stimulator-of-interferon genes (STING) is vital for sensing cytosolic DNA and initiating innate immune responses against microbial infection and tumors. Redox homeostasis is the balance of oxidative and reducing reactions present in all living systems. Yet, how the intracellular redox state controls STING activation is unclear. Here, we show that cellular redox homeostasis maintained by glutathione peroxidase 4 (GPX4) is required for STING activation. GPX4 deficiency enhanced cellular lipid peroxidation and thus specifically inhibited the cGAS-STING pathway. Concordantly, GPX4 deficiency inhibited herpes simplex virus-1 (HSV-1)-induced innate antiviral immune responses and promoted HSV-1 replication in vivo. Mechanistically, GPX4 inactivation increased production of lipid peroxidation, which led to STING carbonylation at C88 and inhibited its trafficking from the endoplasmic reticulum (ER) to the Golgi complex. Thus, cellular stress-induced lipid peroxidation specifically attenuates the STING DNA-sensing pathway, suggesting that GPX4 facilitates STING activation by maintaining redox homeostasis of lipids.

The Ca2+ sensor STIM1 regulates the type I interferon response by retaining the signaling adaptor STING at the endoplasmic reticulum.

Stimulator of interferon genes (STING) is an endoplasmic reticulum (ER) signaling adaptor that is essential for the type I interferon response to DNA pathogens. Aberrant activation of STING is linked to the pathology of autoimmune and autoinflammatory diseases. The rate-limiting step for the activation of STING is its translocation from the ER to the ER-Golgi intermediate compartment. Here, we found that deficiency in the Ca2+ sensor stromal interaction molecule 1 (STIM1) caused spontaneous activation of STING and enhanced expression of type I interferons under resting conditions in mice and a patient with combined immunodeficiency. Mechanistically, STIM1 associated with STING to retain it in the ER membrane, and coexpression of full-length STIM1 or a STING-interacting fragment of STIM1 suppressed the function of dominant STING mutants that cause autoinflammatory diseases. Furthermore, deficiency in STIM1 strongly enhanced the expression of type I interferons after viral infection and prevented the lethality of infection with a DNA virus in vivo. This work delineates a STIM1-STING circuit that maintains the resting state of the STING pathway.

Local proliferation maintains a stable pool of tissue-resident memory T cells after antiviral recall responses.

Although tissue-resident memory T cells (TRM cells) are critical in fighting infection, their fate after local pathogen re-encounter is unknown. Here we found that skin TRM cells engaged virus-infected cells, proliferated in situ in response to local antigen encounter and did not migrate out of the epidermis, where they exclusively reside. As a consequence, secondary TRM cells formed from pre-existing TRM cells, as well as from precursors recruited from the circulation. Newly recruited antigen-specific or bystander TRM cells were generated in the skin without displacement of the pre-existing TRM cell pool. Thus, pre-existing skin TRM cell populations are not displaced after subsequent infections, which enables multiple TRM cell specificities to be stably maintained within the tissue.

TMEFF1 is a neuron-specific restriction factor for herpes simplex virus.

The brain is highly sensitive to damage caused by infection and inflammation1,2. Herpes simplex virus 1 (HSV-1) is a neurotropic virus and the cause of herpes simplex encephalitis3. It is unknown whether neuron-specific antiviral factors control virus replication to prevent infection and excessive inflammatory responses, hence protecting the brain. Here we identify TMEFF1 as an HSV-1 restriction factor using genome-wide CRISPR screening. TMEFF1 is expressed specifically in neurons of the central nervous system and is not regulated by type I interferon, the best-known innate antiviral system controlling virus infections. Depletion of TMEFF1 in stem-cell-derived human neurons led to elevated viral replication and neuronal death following HSV-1 infection. TMEFF1 blocked the HSV-1 replication cycle at the level of viral entry through interactions with nectin-1 and non-muscle myosin heavy chains IIA and IIB, which are core proteins in virus-cell binding and virus-cell fusion, respectively4-6. Notably, Tmeff1-/- mice exhibited increased susceptibility to HSV-1 infection in the brain but not in the periphery. Within the brain, elevated viral load was observed specifically in neurons. Our study identifies TMEFF1 as a neuron-specific restriction factor essential for prevention of HSV-1 replication in the central nervous system.

Ancestral allele of DNA polymerase gamma modifies antiviral tolerance.

Mitochondria are critical modulators of antiviral tolerance through the release of mitochondrial RNA and DNA (mtDNA and mtRNA) fragments into the cytoplasm after infection, activating virus sensors and type-I interferon (IFN-I) response1-4. The relevance of these mechanisms for mitochondrial diseases remains understudied. Here we investigated mitochondrial recessive ataxia syndrome (MIRAS), which is caused by a common European founder mutation in DNA polymerase gamma (POLG1)5. Patients homozygous for the MIRAS variant p.W748S show exceptionally variable ages of onset and symptoms5, indicating that unknown modifying factors contribute to disease manifestation. We report that the mtDNA replicase POLG1 has a role in antiviral defence mechanisms to double-stranded DNA and positive-strand RNA virus infections (HSV-1, TBEV and SARS-CoV-2), and its p.W748S variant dampens innate immune responses. Our patient and knock-in mouse data show that p.W748S compromises mtDNA replisome stability, causing mtDNA depletion, aggravated by virus infection. Low mtDNA and mtRNA release into the cytoplasm and a slow IFN response in MIRAS offer viruses an early replicative advantage, leading to an augmented pro-inflammatory response, a subacute loss of GABAergic neurons and liver inflammation and necrosis. A population databank of around 300,000 Finnish individuals6 demonstrates enrichment of immunodeficient traits in carriers of the POLG1 p.W748S mutation. Our evidence suggests that POLG1 defects compromise antiviral tolerance, triggering epilepsy and liver disease. The finding has important implications for the mitochondrial disease spectrum, including epilepsy, ataxia and parkinsonism.

Interferon-Independent Activities of Mammalian STING Mediate Antiviral Response and Tumor Immune Evasion.

Type I interferon (IFN) response is commonly recognized as the main signaling activity of STING. Here, we generate the Sting1S365A/S365A mutant mouse that precisely ablates IFN-dependent activities while preserving IFN-independent activities of STING. StingS365A/S365A mice protect against HSV-1 infection, despite lacking the STING-mediated IFN response. This challenges the prevailing view and suggests that STING controls HSV-1 infection through IFN-independent activities. Transcriptomic analysis reveals widespread IFN-independent activities of STING in macrophages and T cells, and STING activities in T cells are predominantly IFN independent. In mouse tumor models, T cells in the tumor experience substantial cell death that is in part mediated by IFN-independent activities of STING. We found that the tumor induces STING-mediated cell death in T cells to evade immune control. Our data demonstrate that mammalian STING possesses widespread IFN-independent activities that are important for restricting HSV-1 infection, tumor immune evasion and likely also adaptive immunity.

Transfer of cGAMP into Bystander Cells via LRRC8 Volume-Regulated Anion Channels Augments STING-Mediated Interferon Responses and Anti-viral Immunity.

The enzyme cyclic GMP-AMP synthase (cGAS) senses cytosolic DNA in infected and malignant cells and catalyzes the formation of 2'3'cGMP-AMP (cGAMP), which in turn triggers interferon (IFN) production via the STING pathway. Here, we examined the contribution of anion channels to cGAMP transfer and anti-viral defense. A candidate screen revealed that inhibition of volume-regulated anion channels (VRACs) increased propagation of the DNA virus HSV-1 but not the RNA virus VSV. Chemical blockade or genetic ablation of LRRC8A/SWELL1, a VRAC subunit, resulted in defective IFN responses to HSV-1. Biochemical and electrophysiological analyses revealed that LRRC8A/LRRC8E-containing VRACs transport cGAMP and cyclic dinucleotides across the plasma membrane. Enhancing VRAC activity by hypotonic cell swelling, cisplatin, GTPγS, or the cytokines TNF or interleukin-1 increased STING-dependent IFN response to extracellular but not intracellular cGAMP. Lrrc8e-/- mice exhibited impaired IFN responses and compromised immunity to HSV-1. Our findings suggest that cell-to-cell transmission of cGAMP via LRRC8/VRAC channels is central to effective anti-viral immunity.

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.

Arginine monomethylation by PRMT7 controls MAVS-mediated antiviral innate immunity.

Accurate control of innate immune responses is required to eliminate invading pathogens and simultaneously avoid autoinflammation and autoimmune diseases. Here, we demonstrate that arginine monomethylation precisely regulates the mitochondrial antiviral-signaling protein (MAVS)-mediated antiviral response. Protein arginine methyltransferase 7 (PRMT7) forms aggregates to catalyze MAVS monomethylation at arginine residue 52 (R52), attenuating its binding to TRIM31 and RIG-I, which leads to the suppression of MAVS aggregation and subsequent activation. Upon virus infection, aggregated PRMT7 is disabled in a timely manner due to automethylation at arginine residue 32 (R32), and SMURF1 is recruited to PRMT7 by MAVS to induce proteasomal degradation of PRMT7, resulting in the relief of PRMT7 suppression of MAVS activation. Therefore, we not only reveal that arginine monomethylation by PRMT7 negatively regulates MAVS-mediated antiviral signaling in vitro and in vivo but also uncover a mechanism by which PRMT7 is tightly controlled to ensure the timely activation of antiviral defense.

An innate antiviral pathway acting before interferons at epithelial surfaces.

Mucosal surfaces are exposed to environmental substances and represent a major portal of entry for microorganisms. The innate immune system is responsible for early defense against infections and it is believed that the interferons (IFNs) constitute the first line of defense against viruses. Here we identify an innate antiviral pathway that works at epithelial surfaces before the IFNs. The pathway is activated independently of known innate sensors of viral infections through a mechanism dependent on viral O-linked glycans, which induce CXCR3 chemokines and stimulate antiviral activity in a manner dependent on neutrophils. This study therefore identifies a previously unknown layer of antiviral defense that exerts its action on epithelial surfaces before the classical IFN response is operative.

E3 ubiquitin ligase RNF128 promotes innate antiviral immunity through K63-linked ubiquitination of TBK1.

TBK1 is essential for interferon-ß (IFN-ß) production and innate antiviral immunity. Here we identified the T cell anergy-related E3 ubiquitin ligase RNF128 as a positive regulator of TBK1 activation. RNF128 directly interacted with TBK1 through its protease-associated (PA) domain and catalyzed the K63-linked polyubiquitination of TBK1, which led to TBK1 activation, IRF3 activation and IFN-ß production. Deficiency of RNF128 expression attenuated IRF3 activation, IFN-ß production and innate antiviral immune responses to RNA and DNA viruses, in vitro and in vivo. Our study identified RNF128 as an E3 ligase for K63-linked ubiquitination and activation of TBK1 and delineated a previously unrecognized function for RNF128.

iRhom2 is essential for innate immunity to DNA viruses by mediating trafficking and stability of the adaptor STING.

STING is a central adaptor in the innate immune response to DNA viruses. However, the manner in which STING activity is regulated remains unclear. We identified iRhom2 ('inactive rhomboid protein 2') as a positive regulator of DNA-virus-triggered induction of type I interferons. iRhom2 deficiency markedly impaired DNA-virus- and intracellular-DNA-induced signaling in cells, and iRhom2-deficient mice were more susceptible to lethal herpes simplex virus type 1 (HSV-1) infection. iRhom2 was constitutively associated with STING and acted in two distinct processes to regulate STING activity. iRhom2 recruited the translocon-associated protein TRAPß to the STING complex to facilitate trafficking of STING from the endoplasmic reticulum to perinuclear microsomes. iRhom2 also recruited the deubiquitination enzyme EIF3S5 to maintain the stability of STING through removal of its K48-linked polyubiquitin chains. These results suggest that iRhom2 is essential for STING activity, as it regulates TRAPß-mediated translocation and EIF3S5-mediated deubiquitination of STING.

Glutamylation of the DNA sensor cGAS regulates its binding and synthase activity in antiviral immunity.

Cyclic GMP-AMP synthase (cGAS) senses cytosolic DNA during viral infection and catalyzes synthesis of the dinucleotide cGAMP, which activates the adaptor STING to initiate antiviral responses. Here we found that deficiency in the carboxypeptidase CCP5 or CCP6 led to susceptibility to DNA viruses. CCP5 and CCP6 were required for activation of the transcription factor IRF3 and interferons. Polyglutamylation of cGAS by the enzyme TTLL6 impeded its DNA-binding ability, whereas TTLL4-mediated monoglutamylation of cGAS blocked its synthase activity. Conversely, CCP6 removed the polyglutamylation of cGAS, whereas CCP5 hydrolyzed the monoglutamylation of cGAS, which together led to the activation of cGAS. Therefore, glutamylation and deglutamylation of cGAS tightly modulate immune responses to infection with DNA viruses.

S6K-STING interaction regulates cytosolic DNA-mediated activation of the transcription factor IRF3.

Cytosolic DNA-mediated activation of the transcription factor IRF3 is a key event in host antiviral responses. Here we found that infection with DNA viruses induced interaction of the metabolic checkpoint kinase mTOR downstream effector and kinase S6K1 and the signaling adaptor STING in a manner dependent on the DNA sensor cGAS. We further demonstrated that the kinase domain, but not the kinase function, of S6K1 was required for the S6K1-STING interaction and that the TBK1 critically promoted this process. The formation of a tripartite S6K1-STING-TBK1 complex was necessary for the activation of IRF3, and disruption of this signaling axis impaired the early-phase expression of IRF3 target genes and the induction of T cell responses and mucosal antiviral immunity. Thus, our results have uncovered a fundamental regulatory mechanism for the activation of IRF3 in the cytosolic DNA pathway.

The kinase CK1ɛ controls the antiviral immune response by phosphorylating the signaling adaptor TRAF3.

The signaling adaptor TRAF3 is a highly versatile regulator of both innate immunity and adaptive immunity, but how its phosphorylation is regulated is still unknown. Here we report that deficiency in or inhibition of the conserved serine-threonine kinase CK1ɛ suppressed the production of type I interferon in response to viral infection. CK1ɛ interacted with and phosphorylated TRAF3 at Ser349, which thereby promoted the Lys63 (K63)-linked ubiquitination of TRAF3 and subsequent recruitment of the kinase TBK1 to TRAF3. Consequently, CK1ɛ-deficient mice were more susceptible to viral infection. Our findings establish CK1ɛ as a regulator of antiviral innate immune responses and indicate a novel mechanism of immunoregulation that involves CK1ɛ-mediated phosphorylation of TRAF3.

The roles of nuclear orphan receptor NR2F6 in anti-viral innate immunity.

Proper transcription regulation by key transcription factors, such as IRF3, is critical for anti-viral defense. Dynamics of enhancer activity play important roles in many biological processes, and epigenomic analysis is used to determine the involved enhancers and transcription factors. To determine new transcription factors in anti-DNA-virus response, we have performed H3K27ac ChIP-Seq and identified three transcription factors, NR2F6, MEF2D and MAFF, in promoting HSV-1 replication. NR2F6 promotes HSV-1 replication and gene expression in vitro and in vivo, but not dependent on cGAS/STING pathway. NR2F6 binds to the promoter of MAP3K5 and activates AP-1/c-Jun pathway, which is critical for DNA virus replication. On the other hand, NR2F6 is transcriptionally repressed by c-Jun and forms a negative feedback loop. Meanwhile, cGAS/STING innate immunity signaling represses NR2F6 through STAT3. Taken together, we have identified new transcription factors and revealed the underlying mechanisms involved in the network between DNA viruses and host cells.

Progression of herpesvirus infection remodels mitochondrial organization and metabolism.

Viruses target mitochondria to promote their replication, and infection-induced stress during the progression of infection leads to the regulation of antiviral defenses and mitochondrial metabolism which are opposed by counteracting viral factors. The precise structural and functional changes that underlie how mitochondria react to the infection remain largely unclear. Here we show extensive transcriptional remodeling of protein-encoding host genes involved in the respiratory chain, apoptosis, and structural organization of mitochondria as herpes simplex virus type 1 lytic infection proceeds from early to late stages of infection. High-resolution microscopy and interaction analyses unveiled infection-induced emergence of rough, thin, and elongated mitochondria relocalized to the perinuclear area, a significant increase in the number and clustering of endoplasmic reticulum-mitochondria contact sites, and thickening and shortening of mitochondrial cristae. Finally, metabolic analyses demonstrated that reactivation of ATP production is accompanied by increased mitochondrial Ca2+ content and proton leakage as the infection proceeds. Overall, the significant structural and functional changes in the mitochondria triggered by the viral invasion are tightly connected to the progression of the virus infection.

Herpes simplex virus spreads rapidly in human foreskin, partly driven by chemokine-induced redistribution of Nectin-1 on keratinocytes.

HSV infects keratinocytes in the epidermis of skin via nectin-1. We established a human foreskin explant infection model to investigate HSV entry and spread. HSV1 entry could only be achieved by the topical application of virus via high density microarray projections (HD-MAPs) to the epidermis, which penetrated beyond one third of its thickness, simulating in vivo microtrauma. Rapid lateral spread of HSV1 to a mean of 13 keratinocytes wide occurred after 24 hours and free virus particles were observed between keratinocytes, consistent with an intercellular route of spread. Nectin-1 staining was markedly decreased in foci of infection in the epidermis and in the human keratinocyte HaCaT cell line. Nectin-1 was redistributed, at the protein level, in adjacent uninfected cells surrounding infection, inducible by CCL3, IL-8 (or CXCL8), and possibly CXCL10 and IL-6, thus facilitating spread. These findings provide the first insights into HSV1 entry and spread in human inner foreskin in situ.

SIDT2 Transports Extracellular dsRNA into the Cytoplasm for Innate Immune Recognition.

Double-stranded RNA (dsRNA) is a common by-product of viral infections and acts as a potent trigger of antiviral immunity. In the nematode C. elegans, sid-1 encodes a dsRNA transporter that is highly conserved throughout animal evolution, but the physiological role of SID-1 and its orthologs remains unclear. Here, we show that the mammalian SID-1 ortholog, SIDT2, is required to transport internalized extracellular dsRNA from endocytic compartments into the cytoplasm for immune activation. Sidt2-deficient mice exposed to extracellular dsRNA, encephalomyocarditis virus (EMCV), and herpes simplex virus 1 (HSV-1) show impaired production of antiviral cytokines and-in the case of EMCV and HSV-1-reduced survival. Thus, SIDT2 has retained the dsRNA transport activity of its C. elegans ortholog, and this transport is important for antiviral immunity.

The Fc Domain of Immunoglobulin Is Sufficient to Bridge NK Cells with Virally Infected Cells.

Clearance of pathogens or tumor cells by antibodies traditionally requires both Fab and Fc domains of IgG. Here, we show the Fc domain of IgG alone mediates recognition and clearance of herpes simplex virus (HSV1)-infected cells. The human natural killer (NK) cell surface is naturally coated with IgG bound by its Fc domain to the Fcγ receptor CD16a. NK cells utilize the Fc domain of bound IgG to recognize gE, an HSV1-encoded glycoprotein that also binds the Fc domain of IgG but at a site distinct from CD16a. The bridge formed by the Fc domain between the HSV1-infected cell and the NK cell results in NK cell activation and lysis of the HSV1-infected cell in the absence of HSV1-specific antibody in vitro and prevents fatal HSV1 infection in vivo. This mechanism also explains how bacterial IgG-binding proteins regulate NK cell function and may be broadly applicable to Fcγ-receptor-bearing cells.