A family of conserved bacterial virulence factors dampens interferon responses by blocking calcium signaling.

Interferons (IFNs) induce an antimicrobial state, protecting tissues from infection. Many viruses inhibit IFN signaling, but whether bacterial pathogens evade IFN responses remains unclear. Here, we demonstrate that the Shigella OspC family of type-III-secreted effectors blocks IFN signaling independently of its cell death inhibitory activity. Rather, IFN inhibition was mediated by the binding of OspC1 and OspC3 to the Ca2+ sensor calmodulin (CaM), blocking CaM kinase II and downstream JAK/STAT signaling. The growth of Shigella lacking OspC1 and OspC3 was attenuated in epithelial cells and in a murine model of infection. This phenotype was rescued in both models by the depletion of IFN receptors. OspC homologs conserved in additional pathogens not only bound CaM but also inhibited IFN, suggesting a widespread virulence strategy. These findings reveal a conserved but previously undescribed molecular mechanism of IFN inhibition and demonstrate the critical role of Ca2+ and IFN targeting in bacterial pathogenesis.

SARS-CoV-2 Receptor ACE2 Is an Interferon-Stimulated Gene in Human Airway Epithelial Cells and Is Detected in Specific Cell Subsets across Tissues.

There is pressing urgency to understand the pathogenesis of the severe acute respiratory syndrome coronavirus clade 2 (SARS-CoV-2), which causes the disease COVID-19. SARS-CoV-2 spike (S) protein binds angiotensin-converting enzyme 2 (ACE2), and in concert with host proteases, principally transmembrane serine protease 2 (TMPRSS2), promotes cellular entry. The cell subsets targeted by SARS-CoV-2 in host tissues and the factors that regulate ACE2 expression remain unknown. Here, we leverage human, non-human primate, and mouse single-cell RNA-sequencing (scRNA-seq) datasets across health and disease to uncover putative targets of SARS-CoV-2 among tissue-resident cell subsets. We identify ACE2 and TMPRSS2 co-expressing cells within lung type II pneumocytes, ileal absorptive enterocytes, and nasal goblet secretory cells. Strikingly, we discovered that ACE2 is a human interferon-stimulated gene (ISG) in vitro using airway epithelial cells and extend our findings to in vivo viral infections. Our data suggest that SARS-CoV-2 could exploit species-specific interferon-driven upregulation of ACE2, a tissue-protective mediator during lung injury, to enhance infection.

Mutations in the adenosine deaminase ADAR1 that prevent endogenous Z-RNA binding induce Aicardi-Goutières-syndrome-like encephalopathy.

Mutations in the adenosine-to-inosine RNA-editing enzyme ADAR1 p150, including point mutations in the Z-RNA recognition domain Zα, are associated with Aicardi-Goutières syndrome (AGS). Here, we examined the in vivo relevance of ADAR1 binding of Z-RNA. Mutation of W197 in Zα, which abolished Z-RNA binding, reduced RNA editing. Adar1W197A/W197A mice displayed severe growth retardation after birth, broad expression of interferon-stimulated genes (ISGs), and abnormal development of multiple organs. Notably, malformation of the brain was accompanied by white matter vacuolation and gliosis, reminiscent of AGS-associated encephalopathy. Concurrent deletion of the double-stranded RNA sensor MDA5 ameliorated these abnormalities. ADAR1 (W197A) expression increased in a feedback manner downstream of type I interferons, resulting in increased RNA editing at a subset of, but not all, ADAR1 target sites. This increased expression did not ameliorate inflammation in Adar1W197A/W197A mice. Thus, editing of select endogenous RNAs by ADAR1 is essential for preventing inappropriate MDA5-mediated inflammation, with relevance to the pathogenesis of AGS.

Functional landscape of SARS-CoV-2 cellular restriction.

A deficient interferon (IFN) response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has been implicated as a determinant of severe coronavirus disease 2019 (COVID-19). To identify the molecular effectors that govern IFN control of SARS-CoV-2 infection, we conducted a large-scale gain-of-function analysis that evaluated the impact of human IFN-stimulated genes (ISGs) on viral replication. A limited subset of ISGs were found to control viral infection, including endosomal factors inhibiting viral entry, RNA binding proteins suppressing viral RNA synthesis, and a highly enriched cluster of endoplasmic reticulum (ER)/Golgi-resident ISGs inhibiting viral assembly/egress. These included broad-acting antiviral ISGs and eight ISGs that specifically inhibited SARS-CoV-2 and SARS-CoV-1 replication. Among the broad-acting ISGs was BST2/tetherin, which impeded viral release and is antagonized by SARS-CoV-2 Orf7a protein. Overall, these data illuminate a set of ISGs that underlie innate immune control of SARS-CoV-2/SARS-CoV-1 infection, which will facilitate the understanding of host determinants that impact disease severity and offer potential therapeutic strategies for COVID-19.

Innate immunity: the bacterial connection.

Pathogens have fueled the diversification of intracellular defense strategies that collectively define cell-autonomous innate immunity. In bacteria, innate immunity is manifested by a broad arsenal of defense systems that provide protection against bacterial viruses, called phages. The complexity of the bacterial immune repertoire has only been realized recently and is now suggesting that innate immunity has commonalities across the tree of life: many components of eukaryotic innate immunity are found in bacteria where they protect against phages, including the cGAS-STING pathway, gasdermins, and viperins. Here, I summarize recent findings on the conservation of innate immune pathways between prokaryotes and eukaryotes and hypothesize that bacterial defense mechanisms can catalyze the discovery of novel molecular players of eukaryotic innate immunity.

USP16 is an ISG15 cross-reactive deubiquitinase that targets pro-ISG15 and ISGylated proteins involved in metabolism.

Interferon-induced ubiquitin (Ub)-like modifier ISG15 covalently modifies host and viral proteins to restrict viral infections. Its function is counteracted by the canonical deISGylase USP18 or Ub-specific protease 18. Notwithstanding indications for the existence of other ISG15 cross-reactive proteases, these remain to be identified. Here, we identify deubiquitinase USP16 as an ISG15 cross-reactive protease by means of ISG15 activity-based profiling. Recombinant USP16 cleaved pro-ISG15 and ISG15 isopeptide-linked model substrates in vitro, as well as ISGylated substrates from cell lysates. Moreover, interferon-induced stimulation of ISGylation was increased by depletion of USP16. The USP16-dependent ISG15 interactome indicated that the deISGylating function of USP16 may regulate metabolic pathways. Targeted enzymes include malate dehydrogenase, cytoplasmic superoxide dismutase 1, fructose-bisphosphate aldolase A, and cytoplasmic glutamic-oxaloacetic transaminase 1. USP16 may thus contribute to the regulation of a subset of metabolism-related proteins during type-I interferon responses.

Mechanisms of immune regulation by the placenta: Role of type I interferon and interferon-stimulated genes signaling during pregnancy.

Pregnancy is a unique condition where the maternal immune system is continuously adapting in response to the stages of fetal development and signals from the environment. The placenta is a key mediator of the fetal/maternal interaction by providing signals that regulate the function of the maternal immune system as well as provides protective mechanisms to prevent the exposure of the fetus to dangerous signals. Bacterial and/or viral infection during pregnancy induce a unique immunological response by the placenta, and type I interferon is one of the crucial signaling pathways in the trophoblast cells. Basal expression of type I interferon-ß and downstream ISGs harbors physiological functions to maintain the homeostasis of pregnancy, more importantly, provides the placenta with the adequate awareness to respond to infections. The disruption of type I interferon signaling in the placenta will lead to pregnancy complications and can compromise fetal development. In this review, we focus the important role of placenta-derived type I interferon and its downstream ISGs in the regulation of maternal immune homeostasis and protection against viral infection. These studies are helping us to better understand placental immunological functions and provide a new perspective for developing better approaches to protect mother and fetus during infections.

Mitochondrial DNA: cellular genotoxic stress sentinel.

High copy number, damage prone, and lean on repair mechanisms are unique features of mitochondrial DNA (mtDNA) that are hard to reconcile with its essentiality for oxidative phosphorylation, the primary function ascribed to this maternally inherited component of our genome. We propose that mtDNA is also a genotoxic stress sentinel, as well as a direct second messenger of this type of cellular stress. Here, we discuss existing evidence for this sentinel/effector role through the ability of mtDNA to escape the confines of the mitochondrial matrix and activate nuclear DNA damage/repair responses via interferon-stimulated gene products and other downstream effectors. However, this arrangement may come at a cost, leading to cancer chemoresistance and contributing to inflammation, disease pathology, and aging.

ISG20L1 acts as a co-activator of DAPK1 in the activation of the p53-dependent cell death pathway.

Our previous studies have revealed that GADD45α is a liable proapoptotic protein, which undergoes MDM2-dependent constitutive ubiquitylation and degradation in resting cancer cells. Under chemotherapeutic agent (such as arsenite, 5-Fu and VP-16) exposure, DAPK1 functions as a novel p53 (also known as TP53) kinase, which induces phosphorylation of p53 at Ser15 and transactivates the p53 target Ets-1, to synergistically repress IKKß-dependent MDM2 stability, and ultimately removes the inhibitory effect of MDM2 on GADD45α, resulting in GADD45α accumulation and cell apoptosis. In the current study, we show that there is a strong induction of ISG20L1 (also known as AEN) expression in several cancer cell lines under exposure of arsenite and other chemotherapeutic agents. Surprisingly, although originally identified as a transcriptional target of p53, ISG20L1 induction was not controlled by p53. Instead, ISG20L1 functioned as upstream activator of p53 by interacting with DAPK1, and plays an essential role in promoting DAPK1-p53 complex formation and the subsequent activation of Ets-1/IKKß/MDM2/GADD45α cascade. Therefore, our findings have revealed novel function of ISG20L1 in mediating cancer cell apoptosis induced by chemotherapeutic agents via modulating activation of the DAPK1- and p53-dependent cell death pathway.

Reverse aging to treat lupus.

Systemic lupus erythematosus (SLE) is a complex autoimmune disease with multifaceted pathogenetic processes, including abnormalities of T-cell subset distribution and function. Accumulation of senescent CD4+ T cells has been found to contribute to the development of the disease. In this issue, Jiang et al. provide compelling evidence that links an expanded pool of CD4+CD57+ senescent T cells in patients with SLE to disease activity favored by interleukin-15. Importantly, treatment of lupus-prone mice with a senolytic drug resulted in decreased autoimmune pathology. The findings of this study suggest possible novel therapeutics to treat patients with SLE.

ISGylation of the SARS-CoV-2 N protein by HERC5 impedes N oligomerization and thereby viral RNA synthesis.

Interferon (IFN)-stimulated gene 15 (ISG15), a ubiquitin-like protein, is covalently conjugated to host immune proteins such as MDA5 and IRF3 in a process called ISGylation, thereby promoting type I IFN induction to limit the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, whether SARS-CoV-2 proteins can be directly targeted for ISGylation remains elusive. In this study, we identified the nucleocapsid (N) protein of SARS-CoV-2 as a major substrate of ISGylation catalyzed by the host E3 ligase HERC5; however, N ISGylation is readily removed through deISGylation by the papain-like protease (PLpro) activity of NSP3. Mass spectrometry analysis identified that the N protein undergoes ISGylation at four lysine residues (K266, K355, K387, and K388), and mutational analysis of these sites in the context of a SARS-CoV-2 replicon (N-4KR) abolished N ISGylation and alleviated ISGylation-mediated inhibition of viral RNA synthesis. Furthermore, our results indicated that HERC5 targets preferentially phosphorylated N protein for ISGylation to regulate its oligomeric assembly. These findings reveal a novel mechanism by which the host ISGylation machinery directly targets SARS-CoV-2 proteins to restrict viral replication and illuminate how an intricate interplay of host (HERC5) and viral (PLpro) enzymes coordinates viral protein ISGylation and thereby regulates virus replication.IMPORTANCEThe role of protein ISGylation in regulating host cellular processes has been studied extensively; however, how ISG15 conjugation influences the activity of viral proteins, particularly coronaviral proteins, is largely unknown. Our study uncovered that the nucleocapsid (N) protein of SARS-CoV-2 is ISGylated by the HERC5 ISGylation machinery and that this modification impedes the functional assembly of N into oligomers ultimately inhibiting viral RNA synthesis. This antiviral restriction mechanism is antagonized by the PLpro deISGylation activity of SARS-CoV-2 NSP3. This study deepens our understanding of SARS-CoV-2 protein regulation by posttranslational modifications and may open new avenues for designing antiviral strategies for COVID-19.

SARS-CoV-2 papain-like protease inhibits ISGylation of the viral nucleocapsid protein to evade host anti-viral immunity.

A severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes mild-to-severe respiratory symptoms, including acute respiratory distress. Despite remarkable efforts to investigate the virological and pathological impacts of SARS-CoV-2, many of the characteristics of SARS-CoV-2 infection still remain unknown. The interferon-inducible ubiquitin-like protein ISG15 is covalently conjugated to several viral proteins to suppress their functions. It was reported that SARS-CoV-2 utilizes its papain-like protease (PLpro) to impede ISG15 conjugation, ISGylation. However, the role of ISGylation in SARS-CoV-2 infection remains unclear. We aimed to elucidate the role of ISGylation in SARS-CoV-2 replication. We observed that the SARS-CoV-2 nucleocapsid protein is a target protein for the HERC5 E3 ligase-mediated ISGylation in cultured cells. Site-directed mutagenesis reveals that the residue K374 within the C-terminal spacer B-N3 (SB/N3) domain is required for nucleocapsid-ISGylation, alongside conserved lysine residue in MERS-CoV (K372) and SARS-CoV (K375). We also observed that the nucleocapsid-ISGylation results in the disruption of nucleocapsid oligomerization, thereby inhibiting viral replication. Knockdown of ISG15 mRNA enhanced SARS-CoV-2 replication in the SARS-CoV-2 reporter replicon cells, while exogenous expression of ISGylation components partially hampered SARS-CoV-2 replication. Taken together, these results suggest that SARS-CoV-2 PLpro inhibits ISGylation of the nucleocapsid protein to promote viral replication by evading ISGylation-mediated disruption of the nucleocapsid oligomerization.IMPORTANCEISG15 is an interferon-inducible ubiquitin-like protein that is covalently conjugated to the viral protein via specific Lys residues and suppresses viral functions and viral propagation in many viruses. However, the role of ISGylation in SARS-CoV-2 infection remains largely unclear. Here, we demonstrated that the SARS-CoV-2 nucleocapsid protein is a target protein for the HERC5 E3 ligase-mediated ISGylation. We also found that the residue K374 within the C-terminal spacer B-N3 (SB/N3) domain is required for nucleocapsid-ISGylation. We obtained evidence suggesting that nucleocapsid-ISGylation results in the disruption of nucleocapsid-oligomerization, thereby suppressing SARS-CoV-2 replication. We discovered that SARS-CoV-2 papain-like protease inhibits ISG15 conjugation of nucleocapsid protein via its de-conjugating enzyme activity. The present study may contribute to gaining new insight into the roles of ISGylation-mediated anti-viral function in SARS-CoV-2 infection and may lead to the development of more potent and selective inhibitors targeted to SARS-CoV-2 nucleocapsid protein.

Extracellular ISG15 Signals Cytokine Secretion through the LFA-1 Integrin Receptor.

ISG15 is a ubiquitin-like protein that functions in innate immunity both as an intracellular protein modifier and as an extracellular signaling molecule that stimulates IFN-γ secretion. The extracellular function, important for resistance to mycobacterial disease, has remained biochemically uncharacterized. We have established an NK-92 cell-based assay for IFN-γ release, identified residues critical for ISG15 signaling, and identified the cell surface receptor as LFA-1 (CD11a/CD18; αLß2 integrin). LFA-1 inhibition blocked IFN-γ secretion, splenocytes from CD11a-/- mice did not respond to ISG15, and ISG15 bound directly to the αI domain of CD11a in vitro. ISG15 also enhanced secretion of IL-10, indicating a broader role for ISG15 in cytokine signaling. ISG15 engagement of LFA-1 led to the activation of SRC family kinases (SFKs) and SFK inhibition blocked cytokine secretion. These findings establish the molecular basis of the extracellular function of ISG15 and the initial outside-in signaling events that drive ISG15-dependent cytokine secretion.

Nuclear translocation of ISG15 regulated by PPP2R2B inhibits cisplatin resistance of bladder cancer.

Cisplatin resistance is a major challenge for systemic therapy against advanced bladder cancer (BC). Little information is available on the regulation of cisplatin resistance and the underlying mechanisms require elucidation. Here, we detected that downregulation of the tumor suppressor, PPP2R2B (a serine/threonine protein phosphatase 2 A regulatory subunit), in BC promoted cell proliferation and migration. What's more, low PPP2R2B expression was correlated with cisplatin resistance. In vitro and in vivo experiments verified that PPP2R2B could promote BC sensitivity to cisplatin. In terms of mechanism, we identified a novel function of PPP2R2B as a nucleocytoplasmic transport molecule. PPP2R2B promoted ISG15 entry into the nucleus by mediating binding of IPO5 with ISG15. Nuclear translocation of ISG15 inhibited DNA repair, further increasing ISG15 expression through activation of the STING pathway. Besides, PPP2R2B was down-regulated by SUV39H1-mediated histone 3 lysine 9 trimethylation, which could be restored by the SUV39H1-specific inhibitor, chaetocin. Our data suggest that PPP2R2B expression level is a potential biomarker for chemotherapy response and that chemotherapy in combination with chaetocin may be a feasible treatment strategy for patients with BC.

ISG15: A link between innate immune signaling, DNA replication, and genome stability.

Interferon stimulated gene 15 (ISG15) encodes a ubiquitin-like protein that is highly induced upon activation of interferon signaling and cytoplasmic DNA sensing pathways. As part of the innate immune system ISG15 acts to inhibit viral replication and particle release via the covalent conjugation to both viral and host proteins. Unlike ubiquitin, unconjugated ISG15 also functions as an intracellular and extra-cellular signaling molecule to modulate the immune response. Several recent studies have shown ISG15 to also function in a diverse array of cellular processes and pathways outside of the innate immune response. This review explores the role of ISG15 in maintaining genome stability, particularly during DNA replication, and how this relates to cancer biology. It puts forth the hypothesis that ISG15, along with DNA sensors, function within a DNA replication fork surveillance pathway to help maintain genome stability.

APOBEC3A regulates transcription from interferon-stimulated response elements.

APOBEC3A (A3A) is a cytidine deaminase that inactivates a variety of viruses through introduction of lethal mutations to the viral genome. Additionally, A3A can suppress HIV-1 transcription in a deaminase-independent manner by binding to the long terminal repeat of proviral HIV-1. However, it is unknown whether A3A targets additional host genomic loci for repression. In this study, we found that A3A suppresses gene expression by binding TTTC doublets that are in close proximity to each other. However, one TTTC motif is sufficient for A3A binding. Because TTTC doublets are present in interferon (IFN)-stimulated response elements (ISRE), we hypothesized that A3A may impact IFN-stimulated gene (ISG) expression. After scanning the human genome for TTTC doublet occurrences, we discovered that these motifs are enriched in the proximal promoters of genes associated with antiviral responses and type I IFN (IFN-I) signaling. As a proof of principle, we examined whether A3A can impact ISG15 expression. We found that A3A binding to the ISRE inhibits phosphorylated STAT-1 binding and suppresses ISG15 induction in response to IFN-I treatment. Consistent with these data, our RNA-sequencing analyses indicate that A3A loss results in increased IFN-I­dependent induction of several ISGs. This study revealed that A3A plays an unexpected role in ISG regulation and suggests that A3A contributes to a negative feedback loop during IFN signaling.

Coronaviral PLpro proteases and the immunomodulatory roles of conjugated versus free Interferon Stimulated Gene product-15 (ISG15).

Ubiquitin-like proteins (Ubls) share some features with ubiquitin (Ub) such as their globular 3D structure and the ability to attach covalently to other proteins. Interferon Stimulated Gene 15 (ISG15) is an abundant Ubl that similar to Ub, marks many hundreds of cellular proteins, altering their fate. In contrast to Ub, , ISG15 requires interferon (IFN) induction to conjugate efficiently to other proteins. Moreover, despite the multitude of E3 ligases for Ub-modified targets, a single E3 ligase termed HERC5 (in humans) is responsible for the bulk of ISG15 conjugation. Targets include both viral and cellular proteins spanning an array of cellular compartments and metabolic pathways. So far, no common structural or biochemical feature has been attributed to these diverse substrates, raising questions about how and why they are selected. Conjugation of ISG15 mitigates some viral and bacterial infections and is linked to a lower viral load pointing to the role of ISG15 in the cellular immune response. In an apparent attempt to evade the immune response, some viruses try to interfere with the ISG15 pathway. For example, deconjugation of ISG15 appears to be an approach taken by coronaviruses to interfere with ISG15 conjugates. Specifically, coronaviruses such as SARS-CoV, MERS-CoV, and SARS-CoV-2, encode papain-like proteases (PL1pro) that bear striking structural and catalytic similarities to the catalytic core domain of eukaryotic deubiquitinating enzymes of the Ubiquitin-Specific Protease (USP) sub-family. The cleavage specificity of these PLpro enzymes is for flexible polypeptides containing a consensus sequence (R/K)LXGG, enabling them to function on two seemingly unrelated categories of substrates: (i) the viral polyprotein 1 (PP1a, PP1ab) and (ii) Ub- or ISG15-conjugates. As a result, PLpro enzymes process the viral polyprotein 1 into an array of functional proteins for viral replication (termed non-structural proteins; NSPs), and it can remove Ub or ISG15 units from conjugates. However, by de-conjugating ISG15, the virus also creates free ISG15, which in turn may affect the immune response in two opposite pathways: free ISG15 negatively regulates IFN signaling in humans by binding non-catalytically to USP18, yet at the same time free ISG15 can be secreted from the cell and induce the IFN pathway of the neighboring cells. A deeper understanding of this protein-modification pathway and the mechanisms of the enzymes that counteract it will bring about effective clinical strategies related to viral and bacterial infections.

SARS-CoV-2 papain-like protease plays multiple roles in regulating cellular proteins in the endoplasmic reticulum.

Nsp3s are the largest nonstructural proteins of coronaviruses. These transmembrane proteins include papain-like proteases (PLpro) that play essential roles in cleaving viral polyproteins into their mature units. The PLpro of SARS-CoV viruses also have deubiquitinating and deISGylating activities. As Nsp3 is an endoplasmic reticulum (ER)-localized protein, we asked if the deubiquitinating activity of SARS-CoV-2 PLpro affects proteins that are substrates for ER-associated degradation (ERAD). Using full-length Nsp3 as well as a truncated transmembrane form we interrogated, by coexpression, three potential ERAD substrates, all of which play roles in regulating lipid biosynthesis. Transmembrane PLpro increases the level of INSIG-1 and decreases its ubiquitination. However, different effects were seen with SREBP-1 and SREBP-2. Transmembrane PLpro cleaves SREBP-1 at three sites, including two noncanonical sites in the N-terminal half of the protein, resulting in a decrease in precursors of the active transcription factor. Conversely, cleavage of SREBP-2 occurs at a single canonical site that disrupts a C-terminal degron, resulting in increased SREBP-2 levels. When this site is mutated and the degron can no longer be interrupted, SREBP-2 is still stabilized by transmembrane PLpro, which correlates with a decrease in SREBP-2 ubiquitination. All of these observations are dependent on PLpro catalytic activity. Our findings demonstrate that, when anchored to the ER membrane, SARS-CoV-2 Nsp3 PLpro can function as a deubiquitinating enzyme to stabilize ERAD substrates. Additionally, SARS-CoV-2 Nsp3 PLpro can cleave ER-resident proteins, including at sites that could escape analyses based on the established consensus sequence.

Mechanism and inhibition of the papain-like protease, PLpro, of SARS-CoV-2.

The SARS-CoV-2 coronavirus encodes an essential papain-like protease domain as part of its non-structural protein (nsp)-3, namely SARS2 PLpro, that cleaves the viral polyprotein, but also removes ubiquitin-like ISG15 protein modifications as well as, with lower activity, Lys48-linked polyubiquitin. Structures of PLpro bound to ubiquitin and ISG15 reveal that the S1 ubiquitin-binding site is responsible for high ISG15 activity, while the S2 binding site provides Lys48 chain specificity and cleavage efficiency. To identify PLpro inhibitors in a repurposing approach, screening of 3,727 unique approved drugs and clinical compounds against SARS2 PLpro identified no compounds that inhibited PLpro consistently or that could be validated in counterscreens. More promisingly, non-covalent small molecule SARS PLpro inhibitors also target SARS2 PLpro, prevent self-processing of nsp3 in cells and display high potency and excellent antiviral activity in a SARS-CoV-2 infection model.

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.