Measurement of paracentral stereopsis using a new method with the binocular open perimeter imovifa® in normal adults.

PURPOSE: No method to quantitatively evaluate stereopsis within the 15º visual field has been clinically established. We developed a program to measure paracentral stereopsis and evaluated its feasibility in visually normal participants. STUDY DESIGN: Experimental investigation METHODS: Ten visually normal volunteers with stereopsis of 60 arcseconds or better were included. The Stereo Eccentricity Analysis (SEA) program for stereopsis measurement across the visual field was integrated into the binocular visual field analyzer imovifa®. Subjects with established binocular stereopsis detected a stereoscopic circular target presented with crossed disparity on random dots at the fovea, 3°, 5°, 10°, and 15° on the 45°, 135°, 225°, and 315° meridians. The subjects performed two tasks for measurement in the periphery: a detection task by pressing the response button when the circular target was perceived and a localization task by tilting a joystick to indicate in which quadrant the circular target was perceived. The duration of the target presentation was 500 ms. RESULTS: The stereo thresholds at 0º and 3° did not significantly differ. The thresholds at 10º and 15º were significantly higher than at 0° (P < 0.01). While no inter-individual threshold difference was observed at the fovea, the difference was large at 15°. The stereo thresholds for the detection and localization tasks also did not differ significantly. CONCLUSION: With the SEA program, paracentral stereopsis can be measured and the stereo threshold increases with eccentricity. The SEA program appears to be a feasible clinical method to evaluate paracentral stereopsis.

Cholesterol-binding motifs in STING that control endoplasmic reticulum retention mediate anti-tumoral activity of cholesterol-lowering compounds.

The cGAS-STING pathway plays a crucial role in anti-tumoral responses by activating inflammation and reprogramming the tumour microenvironment. Upon activation, STING traffics from the endoplasmic reticulum (ER) to Golgi, allowing signalling complex assembly and induction of interferon and inflammatory cytokines. Here we report that cGAMP stimulation leads to a transient decline in ER cholesterol levels, mediated by Sterol O-Acyltransferase 1-dependent cholesterol esterification. This facilitates ER membrane curvature and STING trafficking to Golgi. Notably, we identify two cholesterol-binding motifs in STING and confirm their contribution to ER-retention of STING. Consequently, depletion of intracellular cholesterol levels enhances STING pathway activation upon cGAMP stimulation. In a preclinical tumour model, intratumorally administered cholesterol depletion therapy potentiated STING-dependent anti-tumoral responses, which, in combination with anti-PD-1 antibodies, promoted tumour remission. Collectively, we demonstrate that ER cholesterol sets a threshold for STING signalling through cholesterol-binding motifs in STING and we propose that this could be exploited for cancer immunotherapy.

SAM68 directs STING signaling to apoptosis in macrophages.

DNA is a danger signal sensed by cGAS to engage signaling through STING to activate innate immune functions. The best-studied downstream responses to STING activation include expression of type I interferon and inflammatory genes, but STING also activates other pathways, including apoptosis. Here, we report that STING-dependent induction of apoptosis in macrophages occurs through the intrinsic mitochondrial pathway and is mediated via IRF3 but acts independently of gene transcription. By intersecting four mass spectrometry datasets, we identify SAM68 as crucial for the induction of apoptosis downstream of STING activation. SAM68 is essential for the full activation of apoptosis. Still, it is not required for STING-mediated activation of IFN expression or activation of NF-κB. Mechanistic studies reveal that protein trafficking is required and involves SAM68 recruitment to STING upon activation, with the two proteins associating at the Golgi or a post-Golgi compartment. Collectively, our work identifies SAM68 as a STING-interacting protein enabling induction of apoptosis through this DNA-activated innate immune pathway.

The HIF transcription network exerts innate antiviral activity in neurons and limits brain inflammation.

Pattern recognition receptors (PRRs) induce host defense but can also induce exacerbated inflammatory responses. This raises the question of whether other mechanisms are also involved in early host defense. Using transcriptome analysis of disrupted transcripts in herpes simplex virus (HSV)-infected cells, we find that HSV infection disrupts the hypoxia-inducible factor (HIF) transcription network in neurons and epithelial cells. Importantly, HIF activation leads to control of HSV replication. Mechanistically, HIF activation induces autophagy, which is essential for antiviral activity. HSV-2 infection in vivo leads to hypoxia in CNS neurons, and mice with neuron-specific HIF1/2α deficiency exhibit elevated viral load and augmented PRR signaling and inflammatory gene expression in the CNS after HSV-2 infection. Data from human stem cell-derived neuron and microglia cultures show that HIF also exerts antiviral and inflammation-restricting activity in human CNS cells. Collectively, the HIF transcription factor system senses virus-induced hypoxic stress to induce cell-intrinsic antiviral responses and limit inflammation.

An IKBKE variant conferring functional cGAS/STING pathway deficiency and susceptibility to recurrent HSV-2 meningitis.

The mechanisms underlying susceptibility to recurrent herpes simplex virus type 2 (HSV-2) meningitis remain incompletely understood. In a patient experiencing multiple episodes of HSV-2 meningitis, we identified a monoallelic variant in the IKBKE gene, which encodes the IKKε kinase involved in induction of antiviral IFN genes. Patient cells displayed impaired induction of IFN-ß1 (IFNB1) expression upon infection with HSV-2 or stimulation with double-stranded DNA (dsDNA) and failed to induce phosphorylation of STING, an activation marker of the DNA-sensing cyclic GMP-AMP synthase/stimulator of IFN genes (cGAS/STING) pathway. The patient allele encoded a truncated IKKε protein with loss of kinase activity and also capable of exerting dominant-negative activity. In stem cell-derived microglia, HSV-2-induced expression of IFNB1 was dependent on cGAS, TANK binding kinase 1 (TBK1), and IKBKE, but not TLR3, and supernatants from HSV-2-treated microglia exerted IKBKE-dependent type I IFN-mediated antiviral activity upon neurons. Reintroducing wild-type IKBKE into patient cells rescued IFNB1 induction following treatment with HSV-2 or dsDNA and restored antiviral activity. Collectively, we identify IKKε to be important for protection against HSV-2 meningitis and suggest a nonredundant role for the cGAS/STING pathway in human antiviral immunity.

ER stress induces caspase-2-tBID-GSDME-dependent cell death in neurons lytically infected with herpes simplex virus type 2.

Neurotropic viruses, including herpes simplex virus (HSV) types 1 and 2, have the capacity to infect neurons and can cause severe diseases. This is associated with neuronal cell death, which may contribute to morbidity or even mortality if the infection is not controlled. However, the mechanistic details of HSV-induced neuronal cell death remain enigmatic. Here, we report that lytic HSV-2 infection of human neuron-like SH-SY5Y cells and primary human and murine brain cells leads to cell death mediated by gasdermin E (GSDME). HSV-2-induced GSDME-mediated cell death occurs downstream of replication-induced endoplasmic reticulum stress driven by inositol-requiring kinase 1α (IRE1α), leading to activation of caspase-2, cleavage of the pro-apoptotic protein BH3-interacting domain death agonist (BID), and mitochondria-dependent activation of caspase-3. Finally, necrotic neurons released alarmins, which activated inflammatory responses in human iPSC-derived microglia. In conclusion, lytic HSV infection in neurons activates an ER stress-driven pathway to execute GSDME-mediated cell death and promote inflammation.

A STING antagonist modulating the interaction with STIM1 blocks ER-to-Golgi trafficking and inhibits lupus pathology.

BACKGROUND: Nucleic acids are potent stimulators of type I interferon (IFN-I) and antiviral defense, but may also promote pathological inflammation. A range of diseases are characterized by elevated IFN-I, including systemic lupus erythematosus (lupus). The DNA-activated cGAS-STING pathway is a major IFN-I-inducing pathway, and activation of signaling is dependent on trafficking of STING from the ER to the Golgi. METHODS: Here we used cell culture systems, a mouse lupus model, and material from lupus patients, to explore the mode of action of a STING antagonistic peptide, and its ability to modulate disease processes. FINDINGS: We report that the peptide ISD017 selectively inhibits all known down-stream activities of STING, including IFN-I, inflammatory cytokines, autophagy, and apoptosis. ISD017 blocks the essential trafficking of STING from the ER to Golgi through a mechanism dependent on the STING ER retention factor STIM1. Importantly, ISD017 blocks STING activity in vivo and ameliorates disease development in a mouse model for lupus. Finally, ISD017 treatment blocks pathological cytokine responses in cells from lupus patients with elevated IFN-I levels. INTERPRETATION: These data hold promise for beneficial use of STING-targeting therapy in lupus. FUNDING: The Novo Nordisk Foundation, The European Research Council, The Lundbeck Foundation, European Union under the Horizon 2020 Research, Deutsche Forschungsgemeinschaft, Chulalongkorn University.

Characterization of distinct molecular interactions responsible for IRF3 and IRF7 phosphorylation and subsequent dimerization.

IRF3 and IRF7 are critical transcription factors in the innate immune response. Their activation is controlled by phosphorylation events, leading to the formation of homodimers that are transcriptionally active. Phosphorylation occurs when IRF3 is recruited to adaptor proteins via a positively charged surface within the regulatory domain of IRF3. This positively charged surface also plays a crucial role in forming the active homodimer by interacting with the phosphorylated sites stabilizing the homodimer. Here, we describe a distinct molecular interaction that is responsible for adaptor docking and hence phosphorylation as well as a separate interaction responsible for the formation of active homodimer. We then demonstrate that IRF7 can be activated by both MAVS and STING in a manner highly similar to that of IRF3 but with one key difference. Regulation of IRF7 appears more tightly controlled; while a single phosphorylation event is sufficient to activate IRF3, at least two phosphorylation events are required for IRF7 activation.

Author Correction: STEEP mediates STING ER exit and activation of signaling.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

STEEP mediates STING ER exit and activation of signaling.

STING is essential for control of infections and for tumor immunosurveillance, but it can also drive pathological inflammation. STING resides on the endoplasmic reticulum (ER) and traffics following stimulation to the ERGIC/Golgi, where signaling occurs. Although STING ER exit is the rate-limiting step in STING signaling, the mechanism that drives this process is not understood. Here we identify STEEP as a positive regulator of STING signaling. STEEP was associated with STING and promoted trafficking from the ER. This was mediated through stimulation of phosphatidylinositol-3-phosphate (PtdIns(3)P) production and ER membrane curvature formation, thus inducing COPII-mediated ER-to-Golgi trafficking of STING. Depletion of STEEP impaired STING-driven gene expression in response to virus infection in brain tissue and in cells from patients with STING-associated diseases. Interestingly, STING gain-of-function mutants from patients interacted strongly with STEEP, leading to increased ER PtdIns(3)P levels and membrane curvature. Thus, STEEP enables STING signaling by promoting ER exit.

Screening for inhibitor of episomal DNA identified dicumarol as a hepatitis B virus inhibitor.

Currently, there is no available therapy to eradicate hepatitis B virus (HBV) in chronically infected individuals. This is due to the difficulty in eliminating viral covalently closed circular (ccc) DNA, which is central to the gene expression and replication of HBV. We developed an assay system for nuclear circular DNA using an integration-deficient lentiviral vector. This vector produced non-integrated circular DNA in nuclei of infected cells. We engineered this vector to encode firefly luciferase to monitor the lentiviral episome DNA. We screened 3,840 chemicals by this assay for luciferase-reducing activity and identified dicumarol, which is known to have anticoagulation activity. We confirmed that dicumarol reduced lentiviral episome DNA. Furthermore, dicumarol inhibited HBV replication in cell culture using NTCP-expressing HepG2 and primary human hepatocytes. Dicumarol reduced intracellular HBV RNA, DNA, supernatant HBV antigens and DNA. We also found that dicumarol reduced the cccDNA level in HBV infected cells, but did not affect HBV adsorption/entry. This is a novel assay system for screening inhibitors targeting nuclear cccDNA and is useful for finding new antiviral substances for HBV.

Rosmarinic acid is a novel inhibitor for Hepatitis B virus replication targeting viral epsilon RNA-polymerase interaction.

Current therapeutics for hepatitis B virus (HBV) patients such as nucleoside analogs (NAs) are effective; however, new antiviral drugs against HBV are still desired. Since the interaction between the epsilon (ε) sequence of HBV pregenomic RNA and viral polymerase (Pol) is a key step in the HBV replication cycle, we aimed to identify small compounds for its inhibition, and established a pull-down assay system for the detection of ε-RNA-binding-Pol. Screening showed that 5 out of 3,965 compounds inhibited ε-Pol binding, and we identified rosmarinic acid, which exhibited specificity, as a potential antiviral agent. In order to examine the anti-HBV effects of rosmarinic acid, HBV-infected primary human hepatocytes from a humanized mouse liver were treated with rosmarinic acid. The rosmarinic acid treatment decreased HBV components including the amounts of extracellular HBV DNA with negligible cytotoxicity. We also investigated the combined effects of rosmarinic acid and the NA, lamivudine. rosmarinic acid slightly enhanced the anti-HBV activity of lamivudine, suggesting that the HBV replication step targeted by rosmarinic acid is distinct from that of NA. We analyzed an additional 25 rosmarinic acid derivatives, and found that 5 also inhibited ε-Pol. Structural comparisons between these derivatives implied that the "two phenolic hydroxyl groups at both ends" and the "caffeic acid-like structure" of rosmarinic acid are critical for the inhibition of ε-Pol binding. Collectively, our results demonstrate that rosmarinic acid inhibits HBV replication in HBV-infected cells by specifically targeting ε-Pol binding.

RIG-I-Like Receptor and Toll-Like Receptor Signaling Pathways Cause Aberrant Production of Inflammatory Cytokines/Chemokines in a Severe Fever with Thrombocytopenia Syndrome Virus Infection Mouse Model.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease caused by a tick-borne phlebovirus of the family Bunyaviridae, SFTS virus (SFTSV). Wild-type and type I interferon (IFN-I) receptor 1-deficient (IFNAR1-/-) mice have been established as nonlethal and lethal models of SFTSV infection, respectively. However, the mechanisms of IFN-I production in vivo and the factors causing the lethal disease are not well understood. Using bone marrow-chimeric mice, we found that IFN-I signaling in hematopoietic cells was essential for survival of lethal SFTSV infection. The disruption of IFN-I signaling in hematopoietic cells allowed an increase in viral loads in serum and produced an excess of multiple inflammatory cytokines and chemokines. The production of IFN-I and inflammatory cytokines was abolished by deletion of the signaling molecules IPS-1 and MyD88, essential for retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) and Toll-like receptor (TLR) signaling, respectively. However, IPS-1-/- MyD88-/- mice exhibited resistance to lethal SFTS with a moderate viral load in serum. Taken together, these results indicate that adequate activation of RLR and TLR signaling pathways under low to moderate levels of viremia contributed to survival through the IFN-I-dependent antiviral response during SFTSV infection, whereas overactivation of these signaling pathways under high levels of viremia resulted in abnormal induction of multiple inflammatory cytokines and chemokines, causing the lethal disease.IMPORTANCE SFTSV causes a severe infectious disease in humans, with a high fatality rate of 12 to 30%. To know the pathogenesis of the virus, we need to clarify the innate immune response as a front line of defense against viral infection. Here, we report that a lethal animal model showed abnormal induction of multiple inflammatory cytokines and chemokines by an uncontrolled innate immune response, which triggered the lethal SFTS. Our findings suggest a new strategy to target inflammatory humoral factors to treat patients with severe SFTS. Furthermore, this study may help the investigation of other tick-borne viruses.

Structure-dependent antiviral activity of catechol derivatives in pyroligneous acid against the encephalomycarditis virus.

The pyrolysis product, wood vinegar (WV), from Japanese larch exhibited strong antiviral activity against the encephalomycarditis virus (EMCV). Catechol, 3-methyl-, 4-methyl-, 4-ethyl-, and 3-methoxycatechol, and 2-methyl-1,4-benzenediol were identified as the major antiviral compounds. The viral inhibition ability of these compounds was affected by the structure and position of the substituent group attached to the aromatic skeleton. The IC50 of catechol was 0.67 mg mL-1 and those of its derivatives were <0.40 mg mL-1. Methyl and ethyl substitution in the para position relative to a hydroxyl group obviously increased the antiviral activities. The mode of antiviral action was investigated by adding catechol derivatives at different times of the viral life cycle. It was found that direct inactivations of EMCV by these compounds were the major pathway for the antiviral activity. The effect of catechol derivatives on the host immune system was studied by quantification of Il6 and Ifnb1 expression levels. Increased Il6 expression levels indicate NF-κB activation by reactive oxygen species from auto-oxidations of catechol derivatives, which is also a possible antiviral route. The present research provides indices for production of potent antiviral agents form lignocellulose biomass.

Functional IRF3 deficiency in a patient with herpes simplex encephalitis.

Herpes simplex encephalitis (HSE) in children has previously been linked to defects in type I interferon (IFN) production downstream of Toll-like receptor 3. Here, we describe a novel genetic etiology of HSE by identifying a heterozygous loss-of-function mutation in the IFN regulatory factor 3 (IRF3) gene, leading to autosomal dominant (AD) IRF3 deficiency by haploinsufficiency, in an adolescent female patient with HSE. IRF3 is activated by most pattern recognition receptors recognizing viral infections and plays an essential role in induction of type I IFN. The identified IRF3 R285Q amino acid substitution results in impaired IFN responses to HSV-1 infection and particularly impairs signaling through the TLR3-TRIF pathway. In addition, the R285Q mutant of IRF3 fails to become phosphorylated at S386 and undergo dimerization, and thus has impaired ability to activate transcription. Finally, transduction with WT IRF3 rescues the ability of patient fibroblasts to express IFN in response to HSV-1 infection. The identification of IRF3 deficiency in HSE provides the first description of a defect in an IFN-regulating transcription factor conferring increased susceptibility to a viral infection in the CNS in humans.

A novel function of human Pumilio proteins in cytoplasmic sensing of viral infection.

RIG-I-like receptor (RLR) plays a pivotal role in the detection of invading pathogens to initiate type I interferon (IFN) gene transcription. Since aberrant IFN production is harmful, RLR signaling is strictly regulated. However, the regulatory mechanisms are not fully understood. By expression cloning, we identified Pumilio proteins, PUM1 and PUM2, as candidate positive regulators of RIG-I signaling. Overexpression of Pumilio proteins and their knockdown augmented and diminished IFN-ß promoter activity induced by Newcastle disease virus (NDV), respectively. Both proteins showed a specific association with LGP2, but not with RIG-I or MDA5. Furthermore, all of these components were recruited to NDV-induced antiviral stress granules. Interestingly, biochemical analyses revealed that Pumilio increased double-stranded (ds) RNA binding affinity of LGP2; however, Pumilio was absent in the dsRNA-LGP2 complex, suggesting that Pumilio facilitates viral RNA recognition by LGP2 through its chaperon-like function. Collectively, our results demonstrate an unknown function of Pumilio in viral recognition by LGP2.

Virus-induced expression of retinoic acid inducible gene-I and melanoma differentiation-associated gene 5 in the cochlear sensory epithelium.

The inner ear has been regarded as an immunoprivileged site because of isolation by the blood-labyrinthine barrier. Several reports have indicated the existence of immune cells in the inner ear, but there are no reports showing immunocompetence of the cochlear tissue. In this report, we examined the potential involvement of retinoic acid inducible gene-I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5), which are critical for initiating antiviral innate immune responses. We found that RIG-I and MDA5 are expressed in the mouse cochlear sensory epithelium, including Hensen's and Claudius' cells. Ex vivo viral infection using Theiler's murine encephalomyelitis virus revealed that the virus replicates in these cells and that protein levels of RIG-I and MDA5 are up-regulated. Furthermore, the critical antiviral transcription factor, interferon (IFN) regulatory factor-3, is activated in the infected cells as judged by its nuclear translocation and the accumulation of type I IFN transcripts. These results strongly suggest that RIG-I and MDA5 participate in innate antiviral responses in cochlear tissue.

Functional characterization of domains of IPS-1 using an inducible oligomerization system.

The innate immune system recognizes viral nucleic acids and stimulates cellular antiviral responses. Intracellular detection of viral RNA is mediated by the Retinoic acid inducible gene (RIG)-I Like Receptor (RLR), leading to production of type I interferon (IFN) and pro-inflammatory cytokines. Once cells are infected with a virus, RIG-I and MDA5 bind to viral RNA and undergo conformational change to transmit a signal through direct interaction with downstream CARD-containing adaptor protein, IFN-ß promoter stimulator-1 (IPS-1, also referred as MAVS/VISA/Cardif). IPS-1 is composed of N-terminal Caspase Activation and Recruitment Domain (CARD), proline-rich domain, intermediate domain, and C-terminal transmembrane (TM) domain. The TM domain of IPS-1 anchors it to the mitochondrial outer membrane. It has been hypothesized that activated RLR triggers the accumulation of IPS-1, which forms oligomer as a scaffold for downstream signal proteins. However, the exact mechanisms of IPS-1-mediated signaling remain controversial. In this study, to reveal the details of IPS-1 signaling, we used an artificial oligomerization system to induce oligomerization of IPS-1 in cells. Artificial oligomerization of IPS-1 activated antiviral signaling without a viral infection. Using this system, we investigated the domain-requirement of IPS-1 for its signaling. We discovered that artificial oligomerization of IPS-1 could overcome the requirement of CARD and the TM domain. Moreover, from deletion- and point-mutant analyses, the C-terminal Tumor necrosis factor Receptor-Associated Factor (TRAF) binding motif of IPS-1 (aa. 453-460) present in the intermediate domain is critical for downstream signal transduction. Our results suggest that IPS-1 oligomerization is essential for the formation of a multiprotein signaling complex and enables downstream activation of transcription factors, Interferon Regulatory Factor 3 (IRF3) and Nuclear Factor-κB (NF-κB), leading to type I IFN and pro-inflammatory cytokine production.

Critical role of an antiviral stress granule containing RIG-I and PKR in viral detection and innate immunity.

Retinoic acid inducible gene I (RIG-I)-like receptors (RLRs) function as cytoplasmic sensors for viral RNA to initiate antiviral responses including type I interferon (IFN) production. It has been unclear how RIG-I encounters and senses viral RNA. To address this issue, we examined intracellular localization of RIG-I in response to viral infection using newly generated anti-RIG-I antibody. Immunohistochemical analysis revealed that RLRs localized in virus-induced granules containing stress granule (SG) markers together with viral RNA and antiviral proteins. Because of similarity in morphology and components, we termed these aggregates antiviral stress granules (avSGs). Influenza A virus (IAV) deficient in non-structural protein 1 (NS1) efficiently generated avSGs as well as IFN, however IAV encoding NS1 produced little. Inhibition of avSGs formation by removal of either the SG component or double-stranded RNA (dsRNA)-dependent protein kinase (PKR) resulted in diminished IFN production and concomitant enhancement of viral replication. Furthermore, we observed that transfection of dsRNA resulted in IFN production in an avSGs-dependent manner. These results strongly suggest that the avSG is the locus for non-self RNA sensing and the orchestration of multiple proteins is critical in the triggering of antiviral responses.

55 Amino acid linker between helicase and carboxyl terminal domains of RIG-I functions as a critical repression domain and determines inter-domain conformation.

In virus-infected cells, viral RNA with non-self structural pattern is recognized by DExD/Hbox RNA helicase, RIG-I. Once RIG-I senses viral RNA, it triggers a signaling cascade, resulting in the activation of genes including type I interferon, which activates antiviral responses. Overexpression of N-terminal caspase activation and recruitment domain (CARD) is sufficient to activate signaling; however basal activity of full-length RIG-I is undetectable. The repressor domain (RD), initially identified as a.a. 735-925, is responsible for diminished basal activity; therefore, it is suggested that RIG-I is under auto-repression in uninfected cells and the repression is reversed upon its encounter with viral RNA. In this report, we further delimited RD to a.a. 747-801, which corresponds to a linker connecting the helicase and the C-terminal domain (CTD). Alanine substitutions of the conserved residues in the linker conferred constitutive activity to full-length RIG-I. We found that the constitutive active mutants do not exhibit ATPase activity, suggesting that ATPase is required for de-repression but not signaling itself. Furthermore, trypsin digestion of recombinant RIG-I revealed that the wild-type, but not linker mutant conforms to the trypsin-resistant structure, containing CARD and helicase domain. The result strongly suggests that the linker is responsible for maintaining RIG-I in a "closed" structure to minimize unwanted production of interferon in uninfected cells. These findings shed light on the structural regulation of RIG-I function.