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.

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.

Impaired STING Activation Due to a Variant in the E3 Ubiquitin Ligase AMFR in a Patient with Severe VZV Infection and Hemophagocytic Lymphohistiocytosis.

Varicella zoster virus (VZV) is a neurotropic alphaherpesvirus exclusively infecting humans, causing two distinct pathologies: varicella (chickenpox) upon primary infection and herpes zoster (shingles) following reactivation. In susceptible individuals, VZV can give rise to more severe clinical manifestations, including disseminated infection, pneumonitis, encephalitis, and vasculopathy with stroke. Here, we describe a 3-year-old boy in whom varicella followed a complicated course with thrombocytopenia, hemorrhagic and necrotic lesions, pneumonitis, and intermittent encephalopathy. Hemophagocytic lymphohistiocytosis (HLH) was strongly suspected and as the condition deteriorated, HLH therapy was initiated. Although the clinical condition improved, longstanding hemophagocytosis followed despite therapy. We found that the patient carries a rare monoallelic variant in autocrine motility factor receptor (AMFR), encoding a ubiquitin ligase involved in innate cytosolic DNA sensing and interferon (IFN) production through the cyclic GMP-AMP synthase-stimulator of IFN genes (cGAS-STING) pathway. Peripheral blood mononuclear cells (PBMCs) from the patient exhibited impaired signaling downstream of STING in response dsDNA and 2'3'-cGAMP, agonists of cGAS and STING, respectively, and fibroblasts from the patient showed impaired type I IFN responses and significantly increased VZV replication. Overexpression of the variant AMFR R594C resulted in decreased K27-linked STING ubiquitination compared to WT AMFR. Moreover, ImageStream technology revealed reduced STING trafficking from ER to Golgi in cells expressing the patient AMFR R594C variant. This was supported by a dose-dependent dominant negative effect of expression of the patient AMFR variant as measured by IFN-ß reporter gene assay. Finally, lentiviral transduction with WT AMFR partially reconstituted 2'3'-cGAMP-induced STING-mediated signaling and ISG expression in patient PBMCs. This work links defective AMFR-STING signaling to severe VZV disease and hyperinflammation and suggests a direct role for cGAS-STING in the control of viral infections in humans. In conclusion, we describe a novel genetic etiology of severe VZV disease in childhood, also representing the first inborn error of immunity related to a defect in the cGAS-STING pathway.

Attenuation of cGAS-STING signaling is mediated by a p62/SQSTM1-dependent autophagy pathway activated by TBK1.

Negative regulation of immune pathways is essential to achieve resolution of immune responses and to avoid excess inflammation. DNA stimulates type I IFN expression through the DNA sensor cGAS, the second messenger cGAMP, and the adaptor molecule STING Here, we report that STING degradation following activation of the pathway occurs through autophagy and is mediated by p62/SQSTM1, which is phosphorylated by TBK1 to direct ubiquitinated STING to autophagosomes. Degradation of STING was impaired in p62-deficient cells, which responded with elevated IFN production to foreign DNA and DNA pathogens. In the absence of p62, STING failed to traffic to autophagy-associated vesicles. Thus, DNA sensing induces the cGAS-STING pathway to activate TBK1, which phosphorylates IRF3 to induce IFN expression, but also phosphorylates p62 to stimulate STING degradation and attenuation of the response.

pol-miR-194a of Japanese flounder (Paralichthys olivaceus) suppresses type I interferon response and facilitates Edwardsiella tarda infection.

MicroRNAs (miRNAs) are a type of small non-coding RNAs that participate in diverse cellular processes including microbial invasion and immune defense. In a previous study, we identified a large amount of Japanese flounder (Paralichthys olivaceus) miRNAs responsive to megalocytivirus infection. In the present study, we examined the function of one of these miRNAs, pol-miR-194a, in association with the infectivity of Edwardsiella tarda, an intracellular bacterial pathogen to many fish species including flounder. We found that pol-miR-194a was induced in expression to a significant extent in the spleen, liver, and gill of Japanese flounder infected by E. tarda. Transfection of flounder cells with pol-miR-194a mimic significantly enhanced the intracellular replication of E. tarda. pol-miR-194a was able to interact specifically with the 3'UTR of IRF7 in a negative manner, resulting in inhibition of IRF7 expression. Consistently, pol-miR-194a significantly blocked the promoter activity of type Ⅰ interferon. Taken together, these results indicate that pol-miR-194a plays an important role in the regulation of flounder immune response as well as microbial infection, and that pol-miR-194a probably serves as a target for E. tarda to manipulate and escape host immune defense.

In-depth profiling and analysis of host and viral microRNAs in Japanese flounder (Paralichthys olivaceus) infected with megalocytivirus reveal involvement of microRNAs in host-virus interaction in teleost fish.

BACKGROUND: MicroRNAs (miRNAs) regulate gene expression by binding to mRNA transcripts in various biological processes. In mammals and birds, miRNAs are known to play vital parts in both host immune defense and viral infection. However, in lower vertebrates such as teleost, systematic investigations on host and viral miRNAs are lacking. RESULTS: In this study, we applied high-throughput sequencing technology to identify and analyze both host and viral miRNAs in Japanese flounder (Paralichthys olivaceus), an economically important teleost fish farmed widely in the world, infected with megalocytivirus at a timescale of 14 days divided into five different time points. The results showed that a total of 381 host miRNAs and 9 viral miRNAs were identified, the latter being all novel miRNAs that have no homologues in the currently available databases. Of the host miRNAs, 251 have been reported previously in flounder and other species, and 130 were discovered for the first time. The expression levels of 121 host miRNAs were significantly altered at 2 d to 14 d post-viral infection (pi), and these miRNAs were therefore classified as differentially expressed host miRNAs. The expression levels of all 9 viral miRNAs increased from 0 d pi to 10 d pi and then dropped from 10 d pi to 14 d pi. For the 121 differentially expressed host miRNAs and the 9 viral miRNAs, 243 and 48 putative target genes, respectively, were predicted in flounder. GO and KEGG enrichment analysis revealed that the putative target genes of both host and viral miRNAs were grouped mainly into the categories of immune response, signal transduction, and apoptotic process. CONCLUSIONS: The results of our study provide the first evidences that indicate existence in teleost fish (i) infection-responsive host and viral miRNAs that exhibit dynamic changes in expression profiles during the course of viral infection, and (ii) potential involvement of miRNAs in host-viral interaction.

Rock bream (Oplegnathus fasciatus) IL-12p40: identification, expression, and effect on bacterial infection.

IL-12p40, also called IL-12ß, is a subunit of the proinflammatory cytokines interleukin (IL)-12 and IL-23. In teleost, IL-12p40 homologues have been identified in several species, however, the biological function of fish IL-12p40 is essentially unknown. In this work, we reported the identification and analysis of an IL-12p40, OfIL-12p40, from rock bream (Oplegnathus fasciatus). OfIL-12p40 is composed of 361 amino acids and possesses a conserved IL-12p40 domain and a WSxWS signature motif characteristic of known IL-12p40. Constitutive expression of OfIL-12p40 occurred in multiple tissues and was highest in kidney. Experimental infection with bacterial pathogen upregulated the expression of OfIL-12p40 in kidney and spleen in a time-dependent manner. Purified recombinant OfIL-12p40 (rOfIL-12p40) stimulated the respiratory burst activity of peripheral blood leukocytes in a dose-dependent manner. rOfIL-12p40 also enhanced the resistance of rock bream against bacterial infection and upregulated the expression of innate immune genes in kidney. Taken together, these results indicate that OfIL-12p40 possesses cytokine-like property and plays a role in immune defense against bacterial infection.

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.

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.

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.

Complete genome sequence and transcription profiles of the rock bream iridovirus RBIV-C1.

The family Iridoviridae consists of 5 genera of double-stranded DNA viruses, including the genus Megalocytivirus, which contains species that are important fish pathogens. In a previous study, we isolated the first rock bream iridovirus from China (RBIV-C1) and identified it as a member of the genus Megalocytivirus. In this report, we determined the complete genomic sequence of RBIV-C1 and examined its in vivo expression profiles. The genome of RBIV-C1 is 112333 bp in length, with a GC content of 55% and a coding density of 92%. RBIV-C1 contains 4584 simple sequence repeats, 89.8% of which are distributed among coding regions. A total of 119 potential open reading frames (ORFs) were identified in RBIV-C1, including the 26 core iridovirus genes; 41 ORFs encode proteins that are predicted to be associated with essential biological functions. RBIV-C1 exhibits the highest degree of sequence conservation and colinear arrangement of genes with orange-spotted grouper iridovirus (OSGIV) and rock bream iridovirus (RBIV). The pairwise nucleotide identities are 99.49% between RBIV-C1 and OSGIV and 98.69% between RBIV-C1 and RBIV. Compared to OSGIV, RBIV-C1 contains 11 insertions, 13 deletions, and 103 single nucleotide mutations. Whole-genome transcription analysis showed that following experimental infection of rock bream with RBIV-C1, all but 1 of the 119 ORFs were expressed at different time points and clustered into 3 hierarchical groups based on their expression patterns. These results provide new insights into the genetic nature and gene expression features of megalocytiviruses.

STING is redundant for host defense and pathology of COVID-19-like disease in mice.

Critical COVID-19 is characterized by lack of early type I interferon-mediated host defense and subsequent hyper-inflammation in the lungs. Aberrant activation of macrophages and neutrophils has been reported to lead to excessive activation of innate immunological pathways. It has recently been suggested that the DNA-sensing cGAS-STING pathway drives pathology in the SARS-CoV-2-infected lungs, but mechanistic understanding from in vivo models is needed. Here, we tested whether STING is involved in COVID-19-like disease using the K18-hACE2 mouse model. We report that disease development after SARS-CoV-2 infection is unaltered in STING-deficient K18-hACE2 mice. In agreement with this, STING deficiency did not affect control of viral replication or production of interferons and inflammatory cytokines. This was accompanied by comparable profiles of infiltrating immune cells into the lungs of infected mice. These data do not support a role for STING in COVID-19 pathology and calls for further investigation into the pathogenesis of critical COVID-19.

TNFR2 is a regulatory target of pol-miR-194a and promotes the antibacterial immunity of Japanese flounder Paralichthys olivaceus.

MicroRNAs (miRNAs) are regulatory RNAs that modulate target gene expression after transcription. Pol-miR-194a had been reported to be a miRNA of Japanese flounder (Paralichthys olivaceus) involved in Edwardsiella tarda infection. Here, we identified tumor necrosis factor receptor 2 (TNFR2) as a target gene of pol-miR-194a. Pol-miR-194a markedly repressed the protein expression of flounder TNFR2 (PoTNFR2) via specific interaction with the 3'UTR of PoTNFR2. PoTNFR2 responded to E. tarda infection in a manner that was opposite to that of pol-miR-194a and inhibited E. tarda invasion by activating the NF-κB pathway. Consistently, dysregulation of PoTNFR2 had a significant impact on E. tarda dissemination in flounder tissues. Together, these results add new insights into the regulation mechanism and immune function of fish TNFR2 and pol-miR-194a.

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.

Japanese flounder pol-miR-3p-2 suppresses Edwardsiella tarda infection by regulation of autophagy via p53.

MicroRNAs (miRNAs) are post-transcriptional regulators that play vital roles in diverse physiological processes including immunity. In this study, we investigated the regulatory mechanism and function of a novel Japanese flounder (Paralichthys olivaceus) miRNA, pol-miR-3p-2. pol-miR-3p-2 was responsive in expression to the infection of the bacterial pathogen Edwardsiella tarda. pol-miR-3p-2 negatively regulated the expression of p53 through interaction with the 3'UTR of p53. Overexpression of pol-miR-3p-2 promoted autophagy, resulting in augmented production of LC3-II, while knockdown of p53 increased the level of beclin, a key factor of autophagy. In vivo and in vitro studies showed that E. tarda infection induced autophagy in flounder, and pol-miR-3p-2 inhibited the infectivity of E. tarda. Together these results indicate that pol-miR-3p-2 regulates autophagy through the target gene p53, thus revealing a regulatory link between p53 and autophagy in teleost, and that pol-miR-3p-2 plays an important role in the immune defense against E. tarda.

HSV1 VP1-2 deubiquitinates STING to block type I interferon expression and promote brain infection.

Herpes simplex virus (HSV) is the main cause of viral encephalitis in the Western world, and the type I interferon (IFN) system is important for antiviral control in the brain. Here, we have compared Ifnb induction in mixed murine brain cell cultures by a panel of HSV1 mutants, each devoid of one mechanism to counteract the IFN-stimulating cGAS-STING pathway. We found that a mutant lacking the deubiquitinase (DUB) activity of the VP1-2 protein induced particularly strong expression of Ifnb and IFN-stimulated genes. HSV1 ΔDUB also induced elevated IFN expression in murine and human microglia and exhibited reduced viral replication in the brain. This was associated with increased ubiquitination of STING and elevated phosphorylation of STING, TBK1, and IRF3. VP1-2 associated directly with STING, leading to its deubiquitination. Recruitment of VP1-2 to STING was dependent on K150 of STING, which was ubiquitinated by TRIM32. Thus, the DUB activity of HSV1 VP1-2 is a major viral immune-evasion mechanism in the brain.

Cellular Requirements for Sensing and Elimination of Incoming HSV-1 DNA and Capsids.

Incoming viruses challenge the cell with diverse foreign molecules, which need to be sensed quickly to initiate immune responses and to remove the viral components. In this study, we investigate the cellular requirements for sensing and degradation of incoming viral DNA and capsids during herpes simplex virus type 1 (HSV-1) infections. Using click chemistry labeling of the viral genome, we found that HSV-1 DNA was released from a subset of capsids into the cytosol early in infection. By next-generation sequencing of cyclic GMP-AMP (cGAMP) synthase (cGAS)-bound DNA from HSV-1-infected cells, we show that HSV-1 DNA was bound by the cytosolic DNA sensor cGAS. Activation of cGAS enzymatic activity by viral DNA did not require proteasomal activity, indicating that viral DNA release into the cytosol is not proteasome-dependent. However, induction of interferon (IFN)-ß expression was blocked by inhibition of the proteasome, suggesting a contribution of the proteasome to IFN-ß induction through the cGAS-stimulator of interferon genes pathway. Viral DNA was cleared from the cytosol within few hours, in a manner dependent on TREX1 and a cGAS-dependent process. Capsid material in the cytoplasm was also degraded rapidly. This was partially blocked by treatment with a proteasome inhibitor. This treatment led to accumulation of DNA-containing viral capsids near the nucleus and reduced nuclear entry of viral DNA. Thus, cells infected with HSV-1 use a panel of mechanisms to eliminate viral DNA and capsids. This represents a barrier for establishment of infection and potentially enables the host to gear the IFN-ß response to a level required for antiviral defense without causing immunopathology.

Intracellular bacteria engage a STING-TBK1-MVB12b pathway to enable paracrine cGAS-STING signalling.

The innate immune system is crucial for eventual control of infections, but may also contribute to pathology. Listeria monocytogenes is an intracellular Gram-positive bacteria and a major cause of food-borne disease. However, important knowledge on the interactions between L. monocytogenes and the immune system is still missing. Here, we report that Listeria DNA is sorted into extracellular vesicles (EVs) in infected cells and delivered to bystander cells to stimulate the cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS)-stimulator of interferon genes (STING) pathway. This was also observed during infections with Francisella tularensis and Legionella pneumophila. We identify the multivesicular body protein MVB12b as a target for TANK-binding kinase 1 phosphorylation, which is essential for the sorting of DNA into EVs and stimulation of bystander cells. EVs from Listeria-infected cells inhibited T-cell proliferation, and primed T cells for apoptosis. Collectively, we describe a pathway for EV-mediated delivery of foreign DNA to bystander cells, and suggest that intracellular bacteria exploit this pathway to impair antibacterial defence.

pol-miR-731, a teleost miRNA upregulated by megalocytivirus, negatively regulates virus-induced type I interferon response, apoptosis, and cell cycle arrest.

Megalocytivirus is a DNA virus that is highly infectious in a wide variety of marine and freshwater fish, including Japanese flounder (Paralichthys olivaceus), a flatfish that is farmed worldwide. However, the infection mechanism of megalocytivirus remains largely unknown. In this study, we investigated the function of a flounder microRNA, pol-miR-731, in virus-host interaction. We found that pol-miR-731 was induced in expression by megalocytivirus and promoted viral replication at the early infection stage. In vivo and in vitro studies revealed that pol-miR-731 (i) specifically suppresses the expression of interferon regulatory factor 7 (IRF7) and cellular tumor antigen p53 in a manner that depended on the integrity of the pol-miR-731 complementary sequences in the 3' untranslated regions of IRF7 and p53, (ii) disrupts megalocytivirus-induced Type I interferon response through IRF7, (iii) inhibits megalocytivirus-induced splenocyte apoptosis and cell cycle arrest through p53. Furthermore, overexpression of IRF7 and p53 abolished both the inhibitory effects of pol-miR-731 on these biological processes and its stimulatory effect on viral replication. These results disclosed a novel evasion mechanism of megalocytivirus mediated by a host miRNA. This study also provides the first evidence that a virus-induced host miRNA can facilitate viral infection by simultaneously suppressing several antiviral pathways.