ARF1 prevents aberrant type I interferon induction by regulating STING activation and recycling.

Type I interferon (IFN) signalling is tightly controlled. Upon recognition of DNA by cyclic GMP-AMP synthase (cGAS), stimulator of interferon genes (STING) translocates along the endoplasmic reticulum (ER)-Golgi axis to induce IFN signalling. Termination is achieved through autophagic degradation or recycling of STING by retrograde Golgi-to-ER transport. Here, we identify the GTPase ADP-ribosylation factor 1 (ARF1) as a crucial negative regulator of cGAS-STING signalling. Heterozygous ARF1 missense mutations cause a previously unrecognized type I interferonopathy associated with enhanced IFN-stimulated gene expression. Disease-associated, GTPase-defective ARF1 increases cGAS-STING dependent type I IFN signalling in cell lines and primary patient cells. Mechanistically, mutated ARF1 perturbs mitochondrial morphology, causing cGAS activation by aberrant mitochondrial DNA release, and leads to accumulation of active STING at the Golgi/ERGIC due to defective retrograde transport. Our data show an unexpected dual role of ARF1 in maintaining cGAS-STING homeostasis, through promotion of mitochondrial integrity and STING recycling.

Regulation of STING activity in DNA sensing by ISG15 modification.

Sensing of human immunodeficiency virus type 1 (HIV-1) DNA is mediated by the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) signaling axis. Signal transduction and regulation of this cascade is achieved by post-translational modifications. Here we show that cGAS-STING-dependent HIV-1 sensing requires interferon-stimulated gene 15 (ISG15). ISG15 deficiency inhibits STING-dependent sensing of HIV-1 and STING agonist-induced antiviral response. Upon external stimuli, STING undergoes ISGylation at residues K224, K236, K289, K347, K338, and K370. Inhibition of STING ISGylation at K289 suppresses STING-mediated type Ⅰ interferon induction by inhibiting its oligomerization. Of note, removal of STING ISGylation alleviates gain-of-function phenotype in STING-associated vasculopathy with onset in infancy (SAVI). Molecular modeling suggests that ISGylation of K289 is an important regulator of oligomerization. Taken together, our data demonstrate that ISGylation at K289 is crucial for STING activation and represents an important regulatory step in DNA sensing of viruses and autoimmune responses.

Modulation of type I interferon responses potently inhibits SARS-CoV-2 replication and inflammation in rhesus macaques.

Type I interferons (IFN-I) are critical mediators of innate control of viral infections but also drive the recruitment of inflammatory cells to sites of infection, a key feature of severe coronavirus disease 2019. Here, IFN-I signaling was modulated in rhesus macaques (RMs) before and during acute SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) infection using a mutated IFN-α2 (IFN-modulator; IFNmod), which has previously been shown to reduce the binding and signaling of endogenous IFN-I. IFNmod treatment in uninfected RMs was observed to induce a modest up-regulation of only antiviral IFN-stimulated genes (ISGs); however, in SARS-CoV-2-infected RMs, IFNmod reduced both antiviral and inflammatory ISGs. IFNmod treatment resulted in a potent reduction in SARS-CoV-2 viral loads both in vitro in Calu-3 cells and in vivo in bronchoalveolar lavage (BAL), upper airways, lung, and hilar lymph nodes of RMs. Furthermore, in SARS-CoV-2-infected RMs, IFNmod treatment potently reduced inflammatory cytokines, chemokines, and CD163+ MRC1- inflammatory macrophages in BAL and expression of Siglec-1 on circulating monocytes. In the lung, IFNmod also reduced pathogenesis and attenuated pathways of inflammasome activation and stress response during acute SARS-CoV-2 infection. Using an intervention targeting both IFN-α and IFN-ß pathways, this study shows that, whereas early IFN-I restrains SARS-CoV-2 replication, uncontrolled IFN-I signaling critically contributes to SARS-CoV-2 inflammation and pathogenesis in the moderate disease model of RMs.

Reduced replication but increased interferon resistance of SARS-CoV-2 Omicron BA.1.

The IFN system constitutes a powerful antiviral defense machinery. Consequently, effective IFN responses protect against severe COVID-19 and exogenous IFNs inhibit SARS-CoV-2 in vitro. However, emerging SARS-CoV-2 variants of concern (VOCs) may have evolved reduced IFN sensitivity. Here, we determined differences in replication and IFN susceptibility of an early SARS-CoV-2 isolate (NL-02-2020) and the Alpha, Beta, Gamma, Delta, and Omicron VOCs in Calu-3 cells, iPSC-derived alveolar type-II cells (iAT2) and air-liquid interface (ALI) cultures of primary human airway epithelial cells. Our data show that Alpha, Beta, and Gamma replicated to similar levels as NL-02-2020. In comparison, Delta consistently yielded higher viral RNA levels, whereas Omicron was attenuated. All viruses were inhibited by type-I, -II, and -III IFNs, albeit to varying extend. Overall, Alpha was slightly less sensitive to IFNs than NL-02-2020, whereas Beta, Gamma, and Delta remained fully sensitive. Strikingly, Omicron BA.1 was least restricted by exogenous IFNs in all cell models. Our results suggest that enhanced innate immune evasion rather than higher replication capacity contributed to the effective spread of Omicron BA.1.

Near-Native Visualization of SARS-CoV-2 Induced Membrane Remodeling and Virion Morphogenesis.

Infection with the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative agent of the COVID-19 pandemic, leads to profound remodeling of cellular membranes, promoting viral replication and virion assembly. A full understanding of this drastic remodeling and the process of virion morphogenesis remains lacking. In this study, we applied room temperature transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM) tomography to visualize the SARS-CoV-2 replication factory in Vero cells, and present our results in comparison with published cryo-EM studies. We obtained cryo-EM-like clarity of the ultrastructure by employing high-pressure freezing, freeze substitution (HPF-FS) and embedding, allowing room temperature visualization of double-membrane vesicles (DMVs) in a near-native state. In addition, our data illustrate the consecutive stages of virion morphogenesis and reveal that SARS-CoV-2 ribonucleoprotein assembly and membrane curvature occur simultaneously. Finally, we show the tethering of virions to the plasma membrane in 3D, and that accumulations of virus particles lacking spike protein in large vesicles are most likely not a result of defective virion assembly at their membrane. In conclusion, this study puts forward a room-temperature EM technique providing near-native ultrastructural information about SARS-CoV-2 replication, adding to our understanding of the interaction of this pandemic virus with its host cell.

Modulation of type I interferon responses potently inhibits SARS-CoV-2 replication and inflammation in rhesus macaques.

Type-I interferons (IFN-I) are critical mediators of innate control of viral infections, but also drive recruitment of inflammatory cells to sites of infection, a key feature of severe COVID-19. Here, and for the first time, IFN-I signaling was modulated in rhesus macaques (RMs) prior to and during acute SARS-CoV-2 infection using a mutated IFNα2 (IFN-modulator; IFNmod), which has previously been shown to reduce the binding and signaling of endogenous IFN-I. In SARS-CoV-2-infected RMs, IFNmod reduced both antiviral and inflammatory ISGs. Notably, IFNmod treatment resulted in a potent reduction in (i) SARS-CoV-2 viral load in Bronchoalveolar lavage (BAL), upper airways, lung, and hilar lymph nodes; (ii) inflammatory cytokines, chemokines, and CD163+MRC1-inflammatory macrophages in BAL; and (iii) expression of Siglec-1, which enhances SARS-CoV-2 infection and predicts disease severity, on circulating monocytes. In the lung, IFNmod also reduced pathogenesis and attenuated pathways of inflammasome activation and stress response during acute SARS-CoV-2 infection. This study, using an intervention targeting both IFN-α and IFN-ß pathways, shows that excessive inflammation driven by type 1 IFN critically contributes to SARS-CoV-2 pathogenesis in RMs, and demonstrates the potential of IFNmod to limit viral replication, SARS-CoV-2 induced inflammation, and COVID-19 severity.

CRISPA: A Non-viral, Transient Cas9 Delivery System Based on Reengineered Anthrax Toxin.

Translating the CRISPR/Cas9 genome editing technology into clinics is still hampered by rather unspecific, unsafe and/or inconvenient approaches for the delivery of its main components - the Cas9 endonuclease and a guide RNA - into cells. Here, we describe the development of a novel transient and non-viral Cas9 delivery strategy based on the translocation machinery of the Bacillus anthracis anthrax toxin, PA (protective antigen). We show that Cas9 variants fused to the N-terminus of the lethal factor or to a hexahistidine tag are shuttled through channels formed by PA into the cytosol of human cells. As proof-of-principle, we applied our new approach, denoted as CRISPA, to knock out lipolysis-stimulated lipoprotein receptor (LSR) in the human colon cancer cell line HCT116 and green-fluorescent protein (GFP) in human embryonic kidney 293T cells stably expressing GFP. Notably, we confirmed that the transporter PA can be adapted to recognize specific host cell-surface receptor proteins and may be optimized for cell type-selective delivery of Cas9. Altogether, CRISPA provides a novel, transient and non-viral way to deliver Cas9 into specific cells. Thus, this system is an additional step towards safe translation of the CRISPR/Cas9 technology into clinics.

Luciferase reporter assays to monitor interferon signaling modulation by SARS-CoV-2 proteins.

We present a protocol for analyzing the impact of SARS-CoV-2 proteins in interferon signaling using luciferase reporter assays. Here, the induction of defined promoters can be quantitatively assessed with high sensitivity and broad linear range. The results are similar to those obtained using qPCR to measure endogenous mRNA induction. The assay requires stringent normalization and confirmation of the results in more physiological settings. The protocol is adaptable for other viruses and other innate immune stimuli. For complete details on the use and execution of this protocol, please refer to Hayn et al. (2021).

Implications of Innate Immunity in Post-Acute Sequelae of Non-Persistent Viral Infections.

Non-persistent viruses classically cause transient, acute infections triggering immune responses aimed at the elimination of the pathogen. Successful viruses evolved strategies to manipulate and evade these anti-viral defenses. Symptoms during the acute phase are often linked to dysregulated immune responses that disappear once the patient recovers. In some patients, however, symptoms persist or new symptoms emerge beyond the acute phase. Conditions resulting from previous transient infection are termed post-acute sequelae (PAS) and were reported for a wide range of non-persistent viruses such as rota-, influenza- or polioviruses. Here we provide an overview of non-persistent viral pathogens reported to be associated with diverse PAS, among them chronic fatigue, auto-immune disorders, or neurological complications and highlight known mechanistic details. Recently, the emergence of post-acute sequelae of COVID-19 (PASC) or long COVID highlighted the impact of PAS. Notably, PAS of non-persistent infections often resemble symptoms of persistent viral infections, defined by chronic inflammation. Inflammation maintained after the acute phase may be a key driver of PAS of non-persistent viruses. Therefore, we explore current insights into aberrant activation of innate immune signaling pathways in the post-acute phase of non-persistent viruses. Finally, conclusions are drawn and future perspectives for treatment and prevention of PAS are discussed.

Manipulation of autophagy by SARS-CoV-2 proteins.

As part of innate immune defenses, macroautophagy/autophagy targets viruses and viral components for lysosomal degradation and exposes pathogen-associated molecular patterns to facilitate recognition. However, viruses evolved sophisticated strategies to antagonize autophagy and even exploit it to promote their replication. In our recent study, we systematically analyzed the impact of individual SARS-CoV-2 proteins on autophagy. We showed that E, M, ORF3a, and ORF7a cause an accumulation of autophagosomes, whereas Nsp15 prevents the efficient formation of autophagosomes. Consequently, autophagic degradation of SQSTM1/p62 is decreased in the presence of E, ORF3a, ORF7a, and Nsp15. Notably, M does not alter SQSTM1 protein levels and colocalizes with accumulations of LC3B-positive membranes not resembling vesicles. Infection with SARS-CoV-2 prevents SQSTM1 degradation and increases lipidation of LC3B, indicating overall that the infection causes a reduction of autophagic flux. Our mechanistic analyses showed that the accessory proteins ORF3a and ORF7a both block autophagic degradation but use different strategies. While ORF3a prevents the fusion between autophagosomes and lysosomes, ORF7a reduces the acidity of lysosomes. In summary, we found that Nsp15, E, M, ORF3a, and ORF7a of SARS-CoV-2 manipulate cellular autophagy, and we determined the molecular mechanisms of ORF3a and ORF7a.

Systematic functional analysis of SARS-CoV-2 proteins uncovers viral innate immune antagonists and remaining vulnerabilities.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) evades most innate immune responses but may still be vulnerable to some. Here, we systematically analyze the impact of SARS-CoV-2 proteins on interferon (IFN) responses and autophagy. We show that SARS-CoV-2 proteins synergize to counteract anti-viral immune responses. For example, Nsp14 targets the type I IFN receptor for lysosomal degradation, ORF3a prevents fusion of autophagosomes and lysosomes, and ORF7a interferes with autophagosome acidification. Most activities are evolutionarily conserved. However, SARS-CoV-2 Nsp15 antagonizes IFN signaling less efficiently than the orthologs of closely related RaTG13-CoV and SARS-CoV-1. Overall, SARS-CoV-2 proteins counteract autophagy and type I IFN more efficiently than type II or III IFN signaling, and infection experiments confirm potent inhibition by IFN-γ and -λ1. Our results define the repertoire and selected mechanisms of SARS-CoV-2 innate immune antagonists but also reveal vulnerability to type II and III IFN that may help to develop safe and effective anti-viral approaches.

Alpha-1 antitrypsin inhibits TMPRSS2 protease activity and SARS-CoV-2 infection.

SARS-CoV-2 is a respiratory pathogen and primarily infects the airway epithelium. As our knowledge about innate immune factors of the respiratory tract against SARS-CoV-2 is limited, we generated and screened a peptide/protein library derived from bronchoalveolar lavage for inhibitors of SARS-CoV-2 spike-driven entry. Analysis of antiviral fractions revealed the presence of α1-antitrypsin (α1AT), a highly abundant circulating serine protease inhibitor. Here, we report that α1AT inhibits SARS-CoV-2 entry at physiological concentrations and suppresses viral replication in cell lines and primary cells including human airway epithelial cultures. We further demonstrate that α1AT binds and inactivates the serine protease TMPRSS2, which enzymatically primes the SARS-CoV-2 spike protein for membrane fusion. Thus, the acute phase protein α1AT is an inhibitor of TMPRSS2 and SARS-CoV-2 entry, and may play an important role in the innate immune defense against the novel coronavirus. Our findings suggest that repurposing of α1AT-containing drugs has prospects for the therapy of COVID-19.

Structural basis for translational shutdown and immune evasion by the Nsp1 protein of SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the current coronavirus disease 2019 (COVID-19) pandemic. A major virulence factor of SARS-CoVs is the nonstructural protein 1 (Nsp1), which suppresses host gene expression by ribosome association. Here, we show that Nsp1 from SARS-CoV-2 binds to the 40S ribosomal subunit, resulting in shutdown of messenger RNA (mRNA) translation both in vitro and in cells. Structural analysis by cryo-electron microscopy of in vitro-reconstituted Nsp1-40S and various native Nsp1-40S and -80S complexes revealed that the Nsp1 C terminus binds to and obstructs the mRNA entry tunnel. Thereby, Nsp1 effectively blocks retinoic acid-inducible gene I-dependent innate immune responses that would otherwise facilitate clearance of the infection. Thus, the structural characterization of the inhibitory mechanism of Nsp1 may aid structure-based drug design against SARS-CoV-2.