Role of spike in the pathogenic and antigenic behavior of SARS-CoV-2 BA.1Omicron (preprint)

The recently identified, globally predominant SARS-CoV-2 Omicron variant (BA.1) is highly transmissible, even in fully vaccinated individuals, and causes attenuated disease compared with other major viral variants recognized to date1-7. The Omicron spike (S) protein, with an unusually large number of mutations, is considered the major driver of these phenotypes3,8. We generated chimeric recombinant SARS-CoV-2 encoding the S gene of Omicron in the backbone of an ancestral SARS-CoV-2 isolate and compared this virus with the naturally circulating Omicron variant. The Omicron S-bearing virus robustly escapes vaccine-induced humoral immunity, mainly due to mutations in the receptor-binding motif (RBM), yet unlike naturally occurring Omicron, efficiently replicates in cell lines and primary-like distal lung cells. In K18-hACE2 mice, while Omicron causes mild, non-fatal infection, the Omicron S-carrying virus inflicts severe disease with a mortality rate of 80%. This indicates that while the vaccine escape of Omicron is defined by mutations in S, major determinants of viral pathogenicity reside outside of S.

The oral drug nitazoxanide restricts SARS-CoV-2 infection and attenuates disease pathogenesis in Syrian hamsters (preprint)

A well-tolerated and cost-effective oral drug that blocks SARS-CoV-2 growth and dissemination would be a major advance in the global effort to reduce COVID-19 morbidity and mortality. Here, we show that the oral FDA-approved drug nitazoxanide (NTZ) significantly inhibits SARS-CoV-2 viral replication and infection in different primate and human cell models including stem cell-derived human alveolar epithelial type 2 cells. Furthermore, NTZ synergizes with remdesivir, and it broadly inhibits growth of SARS-CoV-2 variants B.1.351 (beta), P.1 (gamma), and B.1617.2 (delta) and viral syncytia formation driven by their spike proteins. Strikingly, oral NTZ treatment of Syrian hamsters significantly inhibits SARS-CoV-2-driven weight loss, inflammation, and viral dissemination and syncytia formation in the lungs. These studies show that NTZ is a novel host-directed therapeutic that broadly inhibits SARS-CoV-2 dissemination and pathogenesis in human and hamster physiological models, which supports further testing and optimization of NTZ-based therapy for SARS-CoV-2 infection alone and in combination with antiviral drugs.

SARS-CoV-2 ORF8 encoded protein contains a histone mimic, disrupts chromatin regulation, and enhances replication (preprint)

SARS-CoV-2 emerged in China at the end of 2019 and caused the global pandemic of COVID-19, a disease with high morbidity and mortality. While our understanding of this new virus is rapidly increasing, gaps remain in our understanding of how SARS-CoV-2 can effectively suppress host cell antiviral responses. Recent work on other viruses has demonstrated a novel mechanism through which viral proteins can mimic critical regions of human histone proteins. Histone proteins are responsible for governing genome accessibility and their precise regulation is critical for the ability of a cell to control transcription and respond to viral threats. Here, we show that the protein encoded by ORF8 (Orf8) in SARS-CoV-2 functions as a histone mimic of the ARKS motif in histone 3. Orf8 is associated with chromatin, binds to numerous histone-associated proteins, and is itself acetylated within the histone mimic site. Orf8 expression in cells disrupts multiple critical histone post-translational modifications (PTMs) including H3K9ac, H3K9me3, and H3K27me3 and promotes chromatin compaction while Orf8 lacking the histone mimic motif does not. Further, SARS-CoV-2 infection in human cell lines and postmortem patient lung tissue cause these same disruptions to chromatin. However, deletion of the Orf8 gene from SARS-CoV-2 largely blocks its ability to disrupt host-cell chromatin indicating that Orf8 is responsible for these effects. Finally, deletion of the ORF8 gene affects the host-cell transcriptional response to SARS-CoV-2 infection in multiple cell types and decreases the replication of SARS-CoV-2 in human induced pluripotent stem cell-derived lung alveolar type 2 (iAT2) pulmonary cells. These findings demonstrate a novel function for the poorly understood ORF8-encoded protein and a mechanism through which SARS-CoV-2 disrupts host cell epigenetic regulation. Finally, this work provides a molecular basis for the finding that SARS-CoV-2 lacking ORF8 is associated with decreased severity of COVID-19.

Human airway lineages derived from pluripotent stem cells reveal the epithelial responses to SARS-CoV-2 infection (preprint)

There is an urgent need to understand how SARS-CoV-2 infects the airway epithelium and in a subset of individuals leads to severe illness or death. Induced pluripotent stem cells (iPSCs) provide a near limitless supply of human cells that can be differentiated into cell types of interest, including airway epithelium, for disease modeling. We present a human iPSC-derived airway epithelial platform, composed of the major airway epithelial cell types, that is permissive to SARS-CoV-2 infection. Subsets of iPSC-airway cells express the SARS-CoV-2 entry factors ACE2 and TMPRSS2. Multiciliated cells are the primary initial target of SARS-CoV-2 infection. Upon infection with SARS-CoV-2, iPSC-airway cells generate robust interferon and inflammatory responses and treatment with remdesivir or camostat methylate causes a decrease in viral propagation and entry, respectively. In conclusion, iPSC-derived airway cells provide a physiologically relevant in vitro model system to interrogate the pathogenesis of, and develop treatment strategies for, COVID-19 pneumonia.

SARS-CoV-2 desensitizes host cells to interferon through inhibition of the JAK-STAT pathway (preprint)

SARS-CoV-2 can infect multiple organs, including lung, intestine, kidney, heart, liver, and brain. The molecular details of how the virus navigates through diverse cellular environments and establishes replication are poorly defined. Here, we performed global proteomic analysis of the virus-host interface in a newly established panel of phenotypically diverse, SARS-CoV-2-infectable human cell lines representing different body organs. This revealed universal inhibition of interferon signaling across cell types following SARS-CoV-2 infection. We performed systematic analyses of the JAK-STAT pathway in a broad range of cellular systems, including immortalized cell lines and primary-like cardiomyocytes, and found that several pathway components were targeted by SARS-CoV-2 leading to cellular desensitization to interferon. These findings indicate that the suppression of interferon signaling is a mechanism widely used by SARS-CoV-2 in diverse tissues to evade antiviral innate immunity, and that targeting the viral mediators of immune evasion may help block virus replication in patients with COVID-19.

In vitro assessment of the virucidal activity of four mouthwashes containing Cetylpyridinium Chloride, ethanol, zinc and a mix of enzyme and proteins against a human coronavirus (preprint)

Background: saliva is established to contain high counts SARS-CoV-2 virus and contact with saliva droplets, contaminated surfaces or airborne particles are sources of viral transmission. The generation of infective aerosols during clinical procedures is of particular concern. Therefore, a fuller understanding of the potential of mouthwash to reduce viral counts and modulate the risk of transmission in medical professional and public context is an important research topic. Method: we determined the virucidal activity of four anti-bacterial mouthwashes against a surrogate for SARS-CoV-2, Human CoV-SARS 229E, using a standard ASTM suspension test, with dilution and contact times applicable to recommended mouthwash use. Results: the mouthwash formulated with 0.07% Cetylpyridinium Chloride exhibited virucidal effects providing a [≥]3.0 log reduction HCoV-229E viral count. Mouthwashes containing 15.7% ethanol, 0.2% zinc sulphate heptahydrate and a mix of enzymes and proteins did not demonstrate substantive virucidal activity in this test. Conclusion: mouthwash containing 0.07% Cetylpyridinium Chloride warrants further laboratory and clinical assessment to determine their potential benefit in reducing the risk of SARS-CoV-2.

Ethacridine inhibits SARS-CoV-2 by inactivating viral particles in cellular models (preprint)

More than a million people have now died from COVID-19, because of infection with the SARS-CoV-2 coronavirus. Currently, the FDA has approved remdesivir, an inhibitor of SARS-CoV-2 replication, to treat COVID-19, though very recent data from WHO showed little if any COVID19 protective effect. Here we report that ethacridine, a safe and potent antiseptic use in humans, effectively inhibits SARS-CoV-2, at very low concentrations (EC50 ~ 0.08 M). Ethacridine was identified through a high-throughput screening of an FDA-approved drug library in living cells using a fluorescent assay. Interestingly, the main mode of action of ethacridine is to inactivate virus particles, preventing binding to the host cells. Thus, our work has identified a potent drug with a distinct mode of action against SARS-CoV-2.

SARS-CoV-2 replication triggers an MDA-5-dependent interferon production which is unable to efficiently control replication (preprint)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the third highly pathogenic coronavirus to spill over to humans in less than 20 years, after SARS-CoV-1 in 2002-2003 and Middle East respiratory syndrome (MERS)-CoV in 2012. SARS-CoV-2 is the etiologic agent of coronavirus disease 19 (COVID-19), which ranges from mild respiratory symptoms to severe lung injury and death in the most severe cases. The COVID-19 pandemic is currently a major health issue worldwide. Immune dysregulation characterized by altered innate cytokine responses is thought to contribute to the pathology of COVID-19 patients, which is a testimony of the fundamental role of the innate immune response against SARS-CoV-2. Here, we further characterized the host cell antiviral response against SARS-CoV-2 by using primary human airway epithelia and immortalized model cell lines. We mainly focused on the type I and III interferon (IFN) responses, which lead to the establishment of an antiviral state through the expression of IFN-stimulated genes (ISGs). Our results demonstrate that both primary airway epithelial cells and model cell lines elicit a robust immune response characterized by a strong induction of type I and III IFN through the detection of viral pathogen molecular patterns (PAMPs) by melanoma differentiation associated gene (MDA)-5. However, despite the high levels of type I and III IFNs produced in response to SARS-CoV-2 infection, the IFN response was unable to control viral replication, whereas IFN pre-treatment strongly inhibited viral replication and de novo production of infectious virions. Taken together, these results highlight the complex and ambiguous interplay between viral replication and the timing of IFN responses.

Cyclooxgenase-2 is induced by SARS-CoV-2 infection but does not affect viral entry or replication (preprint)

Identifying drugs that regulate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and its symptoms has been a pressing area of investigation during the coronavirus disease 2019 (COVID-19) pandemic. Nonsteroidal anti-inflammatory drugs (NSAIDs), which are frequently used for the relief of pain and inflammation, could modulate both SARS-CoV-2 infection and the host response to the virus. NSAIDs inhibit the enzymes cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2), which mediate the production of prostaglandins (PGs). PGE2, one of the most abundant PGs, has diverse biological roles in homeostasis and inflammatory responses. Previous studies have shown that NSAID treatment or inhibition of PGE2 receptor signaling leads to upregulation of angiotensin-converting enzyme 2 (ACE2), the cell entry receptor for SARS-CoV-2, thus raising concerns that NSAIDs could increase susceptibility to infection. COX/PGE2 signaling has also been shown to regulate the replication of many viruses, but it is not yet known whether it plays a role in SARS-CoV-2 replication. The purpose of this study was to dissect the effect of NSAIDs on COVID-19 in terms of SARS-CoV-2 entry and replication. We found that SARS-CoV-2 infection induced COX-2 upregulation in diverse human cell culture and mouse systems. However, suppression of COX-2/PGE2 signaling by two commonly used NSAIDs, ibuprofen and meloxicam, had no effect on ACE2 expression, viral entry, or viral replication. Our findings suggest that COX-2 signaling driven by SARS-CoV-2 may instead play a role in regulating the lung inflammation and injury observed in COVID-19 patients.

SARS-CoV-2 induces double-stranded RNA-mediated innate immune responses in respiratory epithelial derived cells and cardiomyocytes (preprint)

Coronaviruses are adept at evading host antiviral pathways induced by viral double-stranded RNA, including interferon (IFN) signaling, oligoadenylate synthetase-ribonuclease L (OAS-RNase L), and protein kinase R (PKR). While dysregulated or inadequate IFN responses have been associated with severe coronavirus infection, the extent to which the recently emerged SARS-CoV-2 activates or antagonizes these pathways is relatively unknown. We found that SARS-CoV-2 infects patient-derived nasal epithelial cells, present at the initial site of infection, induced pluripotent stem cell-derived alveolar type 2 cells (iAT2), the major cell type infected in the lung, and cardiomyocytes (iCM), consistent with cardiovascular consequences of COVID-19 disease. Robust activation of IFN or OAS-RNase L is not observed in these cell types, while PKR activation is evident in iAT2 and iCM. In SARS-CoV-2 infected Calu-3 and A549ACE2 lung-derived cell lines, IFN induction remains relatively weak; however activation of OAS-RNase L and PKR is observed. This is in contrast to MERS-CoV, which effectively inhibits IFN signaling as well as OAS-RNase L and PKR pathways, but similar to mutant MERS-CoV lacking innate immune antagonists. Remarkably, both OAS-RNase L and PKR are activated in MAVS knockout A549ACE2 cells, demonstrating that SARS-CoV-2 can induce these host antiviral pathways despite minimal IFN production. Moreover, increased replication and cytopathic effect in RNASEL knockout A549ACE2 cells implicates OAS-RNase L in restricting SARS-CoV-2. Finally, while SARS-CoV-2 fails to antagonize these host defense pathways, which contrasts with other coronaviruses, the IFN signaling response is generally weak. These host-virus interactions may contribute to the unique pathogenesis of SARS-CoV-2. SignificanceSARS-CoV-2 emergence in late 2019 led to the COVID-19 pandemic that has had devastating effects on human health and the economy. Early innate immune responses are essential for protection against virus invasion. While inadequate innate immune responses are associated with severe COVID-19 diseases, understanding of the interaction of SARS-CoV-2 with host antiviral pathways is minimal. We have characterized the innate immune response to SARS-CoV-2 infections in relevant respiratory tract derived cells and cardiomyocytes and found that SARS-CoV-2 activates two antiviral pathways, oligoadenylate synthetase-ribonuclease L (OAS-RNase L), and protein kinase R (PKR), while inducing minimal levels of interferon. This in contrast to MERS-CoV which inhibits all three pathways. Activation of these pathways may contribute to the distinctive pathogenesis of SARS-CoV-2.

SARS-CoV-2 Infection of Pluripotent Stem Cell-derived Human Lung Alveolar Type 2 Cells Elicits a Rapid Epithelial-Intrinsic Inflammatory Response (preprint)

ABSTRACTThe most severe and fatal infections with SARS-CoV-2 result in the acute respiratory distress syndrome, a clinical phenotype of coronavirus disease 2019 (COVID-19) that is associated with virions targeting the epithelium of the distal lung, particularly the facultative progenitors of this tissue, alveolar epithelial type 2 cells (AT2s). Little is known about the initial responses of human lung alveoli to SARS-CoV-2 infection due in part to inability to access these cells from patients, particularly at early stages of disease. Here we present an in vitro human model that simulates the initial apical infection of the distal lung epithelium with SARS-CoV-2, using AT2s that have been adapted to air-liquid interface culture after their derivation from induced pluripotent stem cells (iAT2s). We find that SARS-CoV-2 induces a rapid global transcriptomic change in infected iAT2s characterized by a shift to an inflammatory phenotype predominated by the secretion of cytokines encoded by NF-kB target genes, delayed epithelial interferon responses, and rapid loss of the mature lung alveolar epithelial program. Over time, infected iAT2s exhibit cellular toxicity that can result in the death of these key alveolar facultative progenitors, as is observed in vivo in COVID-19 lung autopsies. Importantly, drug testing using iAT2s confirmed the efficacy of TMPRSS2 protease inhibition, validating putative mechanisms used for viral entry in human alveolar cells. Our model system reveals the cell-intrinsic responses of a key lung target cell to infection, providing a platform for further drug development and facilitating a deeper understanding of COVID-19 pathogenesis.Competing Interest StatementThe authors have declared no competing interest.View Full Text

Human iPSC-derived alveolar and airway epithelial cells can be cultured at air-liquid interface and express SARS-CoV-2 host factors (preprint)

Development of an anti-SARS-CoV-2 therapeutic is hindered by the lack of physiologically relevant model systems that can recapitulate host-viral interactions in human cell types, specifically the epithelium of the lung. Here, we compare induced pluripotent stem cell (iPSC)-derived alveolar and airway epithelial cells to primary lung epithelial cell controls, focusing on expression levels of genes relevant for COVID-19 disease modeling. iPSC-derived alveolar epithelial type II-like cells (iAT2s) and iPSC-derived airway epithelial lineages express key transcripts associated with lung identity in the majority of cells produced in culture. They express ACE2 and TMPRSS2, transcripts encoding essential host factors required for SARS-CoV-2 infection, in a minor subset of each cell sub-lineage, similar to frequencies observed in primary cells. In order to prepare human culture systems that are amenable to modeling viral infection of both the proximal and distal lung epithelium, we adapt iPSC-derived alveolar and airway epithelial cells to two-dimensional air-liquid interface cultures. These engineered human lung cell systems represent sharable, physiologically relevant platforms for SARS-CoV-2 infection modeling and may therefore expedite the development of an effective pharmacologic intervention for COVID-19.

Reconstructing SARS-CoV-2 response signaling and regulatory networks (preprint)

Several molecular datasets have been recently compiled to characterize the activity of SARS-CoV-2 within human cells. Here we extend computational methods to integrate several different types of sequence, functional and interaction data to reconstruct networks and pathways activated by the virus in host cells. We identify the key proteins in these networks and further intersect them with genes differentially expressed at conditions that are known to impact viral activity. Several of the top ranked genes do not directly interact with virus proteins though some were shown to impact other coronaviruses highlighting the importance of large scale data integration for understanding virus and host activity.

Morphological Cell Profiling of SARS-CoV-2 Infection Identifies Drug Repurposing Candidates for COVID-19 (preprint)

The global spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the associated disease COVID-19, requires therapeutic interventions that can be rapidly translated to clinical care. Unfortunately, traditional drug discovery methods have a >90% failure rate and can take 10-15 years from target identification to clinical use. In contrast, drug repurposing can significantly accelerate translation. We developed a quantitative high-throughput screen to identify efficacious single agents and combination therapies against SARS-CoV-2. Quantitative high-content morphological profiling was coupled with an AI-based machine learning strategy to classify features of cells for infection and stress. This assay detected multiple antiviral mechanisms of action (MOA), including inhibition of viral entry, propagation, and modulation of host cellular responses. From a library of 1,425 FDA-approved compounds and clinical candidates, we identified 16 dose-responsive compounds with antiviral effects. In particular, we discovered that lactoferrin is an effective inhibitor of SARS-CoV-2 infection with an IC50 of 308 nM and that it potentiates the efficacy of both remdesivir and hydroxychloroquine. Lactoferrin also stimulates an antiviral host cell response and retains inhibitory activity in iPSC-derived alveolar epithelial cells, a model for the primary site of infection. Given its safety profile in humans, these data suggest that lactoferrin is a readily translatable therapeutic adjunct for COVID-19. Additionally, several commonly prescribed drugs were found to exacerbate viral infection and warrant clinical investigation. We conclude that morphological profiling for drug repurposing is an effective strategy for the selection and optimization of drugs and drug combinations as viable therapeutic options for COVID-19 pandemic and other emerging infectious diseases.