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1.
EuropePMC;
Preprint in English | EuropePMC | ID: ppcovidwho-327539

ABSTRACT

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.

2.
EuropePMC; 2020.
Preprint in English | EuropePMC | ID: ppcovidwho-306122

ABSTRACT

Effective control of COVID-19 requires antivirals directed against SARS-CoV-2 virus. Here we assess ten available HCV protease inhibitor drugs as potential SARS-CoV-2 antivirals. There is a striking structural similarity of the substrate binding clefts of SARS-CoV-2 Mpro and HCV NS3/4A proteases, and virtual docking experiments show that all ten HCV drugs can potentially bind into the Mpro binding cleft. Seven of these HCV drugs inhibit SARS-CoV-2 Mpro protease activity, while four dock well into the PLpro substrate binding cleft and inhibit PLpro protease activity. These same seven HCV drugs inhibit SARS-CoV-2 virus replication in Vero and/or human cells, demonstrating that HCV drugs that inhibit Mpro, or both Mpro and PLpro, suppress virus replication. Two HCV drugs, simeprevir and grazoprevir synergize with the viral polymerase inhibitor remdesivir to inhibit virus replication, thereby increasing remdesivir inhibitory activity as much as 10-fold.Funding: This research was supported by grants from the National Institutes of Health (R01-GM120574 to GTM) and RPI Center for Computational Innovations (to KB and GTM). This research was also partly funded by CRIP (Center for Research for Influenza Pathogenesis), a NIAID supported Center of Excellence for Influenza Research and Surveillance (CEIRS, contract #,HHSN272201400008C), by DARPA grant HR0011-19-2-0020, by supplements to NIAID grant U19AI142733 U19AI135972 and DoD grant W81XWH-20-1-0270, and by the generous support of the JPB Foundation, the Open Philanthropy Project (research grant 2020-215611 (5384)), and anonymous donors to AG-S.Conflict of Interest: A provisional patent application related to these, studies has been filed. GTM is a founder of Nexomics Biosciences, Inc. This, relationship has no conflict of interest with respect to this study. GTM and RMK are inventors in patents owned jointly by Rutgers University and the University of Texas at Austin concerning the use of specific compounds as antivirals against influenza virus. These patents have no conflict of interest for this study. AG-S is inventor in patents and patent application owned by the Icahn School of Medicine concerning the use of specific antiviral compounds. This inventorship has no conflict of interest with respect to this study.

4.
2021.
Preprint in English | Other preprints | ID: ppcovidwho-295806

ABSTRACT

Germicidal potential of specific wavelengths within the electromagnetic spectrum is an area of growing interest. While ultra-violet (UV) based technologies have shown satisfactory virucidal potential, the photo-toxicity in humans coupled with UV associated polymer degradation limit its use in occupied spaces. Alternatively, longer wavelengths with less irradiation energy such as visible light (405 nm) have largely been explored in the context of bactericidal and fungicidal applications. Such studies indicated that 405 nm mediated inactivation is caused by the absorbance of porphyrins within the organism creating reactive oxygen species which result in free radical damage to its DNA and disruption of cellular functions. The virucidal potential of visible-light based technologies has been largely unexplored and speculated to be ineffective given the lack of porphyrins in viruses. The current study demonstrated increased susceptibility of lipid-enveloped respiratory pathogens of importance such as SARS-CoV-2 (causative agent of COVID-19) as well as the influenza A virus to 405nm, visible light in the absence of exogenous photosensitizers indicating a potential porphyrin-independent alternative mechanism of visible light mediated viral inactivation. These results were obtained using less than expected irradiance levels which are generally safe for humans and commercially achievable. Our results support further exploration of the use of visible light technology for the application of continuous decontamination in occupied areas within hospitals and/or infectious disease laboratories, specifically for the inactivation of respiratory pathogens such as SARS-CoV-2 and Influenza A.

5.
J Virol ; 96(2): e0106321, 2022 01 26.
Article in English | MEDLINE | ID: covidwho-1476388

ABSTRACT

COVID-19 affects multiple organs. Clinical data from the Mount Sinai Health System show that substantial numbers of COVID-19 patients without prior heart disease develop cardiac dysfunction. How COVID-19 patients develop cardiac disease is not known. We integrated cell biological and physiological analyses of human cardiomyocytes differentiated from human induced pluripotent stem cells (hiPSCs) infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the presence of interleukins (ILs) with clinical findings related to laboratory values in COVID-19 patients to identify plausible mechanisms of cardiac disease in COVID-19 patients. We infected hiPSC-derived cardiomyocytes from healthy human subjects with SARS-CoV-2 in the absence and presence of IL-6 and IL-1ß. Infection resulted in increased numbers of multinucleated cells. Interleukin treatment and infection resulted in disorganization of myofibrils, extracellular release of troponin I, and reduced and erratic beating. Infection resulted in decreased expression of mRNA encoding key proteins of the cardiomyocyte contractile apparatus. Although interleukins did not increase the extent of infection, they increased the contractile dysfunction associated with viral infection of cardiomyocytes, resulting in cessation of beating. Clinical data from hospitalized patients from the Mount Sinai Health System show that a significant portion of COVID-19 patients without history of heart disease have elevated troponin and interleukin levels. A substantial subset of these patients showed reduced left ventricular function by echocardiography. Our laboratory observations, combined with the clinical data, indicate that direct effects on cardiomyocytes by interleukins and SARS-CoV-2 infection might underlie heart disease in COVID-19 patients. IMPORTANCE SARS-CoV-2 infects multiple organs, including the heart. Analyses of hospitalized patients show that a substantial number without prior indication of heart disease or comorbidities show significant injury to heart tissue, assessed by increased levels of troponin in blood. We studied the cell biological and physiological effects of virus infection of healthy human iPSC-derived cardiomyocytes in culture. Virus infection with interleukins disorganizes myofibrils, increases cell size and the numbers of multinucleated cells, and suppresses the expression of proteins of the contractile apparatus. Viral infection of cardiomyocytes in culture triggers release of troponin similar to elevation in levels of COVID-19 patients with heart disease. Viral infection in the presence of interleukins slows down and desynchronizes the beating of cardiomyocytes in culture. The cell-level physiological changes are similar to decreases in left ventricular ejection seen in imaging of patients' hearts. These observations suggest that direct injury to heart tissue by virus can be one underlying cause of heart disease in COVID-19.


Subject(s)
COVID-19/immunology , Induced Pluripotent Stem Cells , Interleukin-10/immunology , Interleukin-1beta/immunology , Interleukin-6/immunology , Myocytes, Cardiac , Cells, Cultured , Humans , Induced Pluripotent Stem Cells/immunology , Induced Pluripotent Stem Cells/pathology , Induced Pluripotent Stem Cells/virology , Myocytes, Cardiac/immunology , Myocytes, Cardiac/pathology , Myocytes, Cardiac/virology
6.
Sci Rep ; 11(1): 19470, 2021 09 30.
Article in English | MEDLINE | ID: covidwho-1447318

ABSTRACT

The germicidal potential of specific wavelengths within the electromagnetic spectrum is an area of growing interest. While ultra-violet (UV) based technologies have shown satisfactory virucidal potential, the photo-toxicity in humans coupled with UV associated polymer degradation limit their use in occupied spaces. Alternatively, longer wavelengths with less irradiation energy such as visible light (405 nm) have largely been explored in the context of bactericidal and fungicidal applications. Such studies indicated that 405 nm mediated inactivation is caused by the absorbance of porphyrins within the organism creating reactive oxygen species which result in free radical damage to its DNA and disruption of cellular functions. The virucidal potential of visible-light based technologies has been largely unexplored and speculated to be ineffective given the lack of porphyrins in viruses. The current study demonstrated increased susceptibility of lipid-enveloped respiratory pathogens of importance such as SARS-CoV-2 (causative agent of COVID-19) and influenza A virus to 405 nm, visible light in the absence of exogenous photosensitizers thereby indicating a potential alternative porphyrin-independent mechanism of visible light mediated viral inactivation. These results were obtained using less than expected irradiance levels which are considered safe for humans and commercially achievable. Our results support further exploration of the use of visible light technology for the application of continuous decontamination in occupied areas within hospitals and/or infectious disease laboratories, specifically for the inactivation of respiratory pathogens such as SARS-CoV-2 and Influenza A.


Subject(s)
Disinfection/methods , Influenza A Virus, H1N1 Subtype/radiation effects , SARS-CoV-2/radiation effects , Disinfection/instrumentation , Dose-Response Relationship, Radiation , Encephalomyocarditis virus/radiation effects , Light , Time Factors , Virus Inactivation/radiation effects
7.
J Virol ; 95(17): e0040221, 2021 08 10.
Article in English | MEDLINE | ID: covidwho-1350001

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the viral pathogen responsible for the current coronavirus disease 2019 (COVID-19) pandemic. As of 19 May 2021, John Hopkins University's COVID-19 tracking platform reported 3.3 million deaths associated with SARS-CoV-2 infection. Currently, the World Health Organization has granted emergency use listing (EUL) to six COVID-19 vaccine candidates. However, much of the pathogenesis observed during SARS-CoV-2 infection remains elusive. To gain insight into the contribution of individual accessory open reading frame (ORF) proteins in SARS-CoV-2 pathogenesis, we used our recently described reverse-genetics system approach to successfully engineer recombinant SARS-CoV-2 (rSARS-CoV-2) constructs; we removed individual viral ORF3a, -6, -7a, -7b, and -8 proteins from them, and we characterized the resulting recombinant viruses in vitro and in vivo. Our results indicate differences in plaque morphology, with ORF-deficient (ΔORF) viruses producing smaller plaques than those of the wild type (rSARS-CoV-2/WT). However, growth kinetics of ΔORF viruses were like those of rSARS-CoV-2/WT. Interestingly, infection of K18 human angiotensin-converting enzyme 2 (hACE2) transgenic mice with the ΔORF rSARS-CoV-2s identified ORF3a and ORF6 as the major contributors of viral pathogenesis, while ΔORF7a, ΔORF7b, and ΔORF8 rSARS-CoV-2s induced pathology comparable to that of rSARS-CoV-2/WT. This study demonstrates the robustness of our reverse-genetics system to generate rSARS-CoV-2 constructs and the major role for ORF3a and ORF6 in viral pathogenesis, providing important information for the generation of attenuated forms of SARS-CoV-2 for their implementation as live attenuated vaccines for the treatment of SARS-CoV-2 infection and associated COVID-19. IMPORTANCE Despite great efforts put forward worldwide to combat the current coronavirus disease 2019 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to be a human health and socioeconomic threat. Insights into the pathogenesis of SARS-CoV-2 and the contribution of viral proteins to disease outcome remain elusive. Our study aims (i) to determine the contribution of SARS-CoV-2 accessory open reading frame (ORF) proteins to viral pathogenesis and disease outcome and (ii) to develop a synergistic platform combining our robust reverse-genetics system to generate recombinant SARS-CoV-2 constructs with a validated rodent model of infection and disease. We demonstrate that SARS-CoV-2 ORF3a and ORF6 contribute to lung pathology and ultimately disease outcome in K18 hACE2 transgenic mice, while ORF7a, ORF7b, and ORF8 have little impact on disease outcome. Moreover, our combinatory platform serves as a foundation for generating attenuated forms of the virus to develop live attenuated vaccines for the treatment of SARS-CoV-2.


Subject(s)
Angiotensin-Converting Enzyme 2/immunology , Open Reading Frames/immunology , SARS-CoV-2 , Viral Proteins , A549 Cells , Angiotensin-Converting Enzyme 2/genetics , Animals , COVID-19 Vaccines/genetics , COVID-19 Vaccines/immunology , Chlorocebus aethiops , HEK293 Cells , Humans , Mice , Mice, Transgenic , SARS-CoV-2/genetics , SARS-CoV-2/immunology , Vaccines, Attenuated/genetics , Vaccines, Attenuated/immunology , Vero Cells , Viral Proteins/genetics , Viral Proteins/immunology
8.
J Interferon Cytokine Res ; 41(6): 205-219, 2021 06.
Article in English | MEDLINE | ID: covidwho-1280059

ABSTRACT

The ongoing coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), represents a public health crisis of unprecedented proportions. After the emergence of SARS-CoV-1 in 2002, and Middle East respiratory syndrome coronavirus (MERS-CoV) in 2012, this is the third outbreak of a highly pathogenic zoonotic coronavirus (CoV) that the world has witnessed in the last 2 decades. Infection with highly pathogenic human CoVs often results in a severe respiratory disease characterized by a delayed and blunted interferon (IFN) response, accompanied by an excessive production of proinflammatory cytokines. This indicates that CoVs developed effective mechanisms to overcome the host innate immune response and promote viral replication and pathogenesis. In this review, we describe the key innate immune signaling pathways that are activated during infection with SARS-CoV-2 and other well studied pathogenic CoVs. In addition, we summarize the main strategies that these viruses employ to modulate the host immune responses through the antagonism of IFN induction and effector pathways.


Subject(s)
COVID-19/immunology , COVID-19/pathology , Immune Evasion/immunology , Immunity, Innate/immunology , SARS-CoV-2/immunology , Animals , Cricetinae , Cytokines/immunology , Genome, Viral/genetics , Humans , Interferons/immunology , Mice , SARS-CoV-2/genetics , Signal Transduction/immunology
9.
Hum Pathol ; 114: 110-119, 2021 08.
Article in English | MEDLINE | ID: covidwho-1213257

ABSTRACT

Coronavirus disease 2019 (COVID-19) is an ongoing pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Although viral infection is known to trigger inflammatory processes contributing to tissue injury and organ failure, it is unclear whether direct viral damage is needed to sustain cellular injury. An understanding of pathogenic mechanisms has been handicapped by the absence of optimized methods to visualize the presence and distribution of SARS-CoV-2 in damaged tissues. We first developed a positive control cell line (Vero E6) to validate SARS-CoV-2 detection assays. We then evaluated multiple organs (lungs, kidneys, heart, liver, brain, intestines, lymph nodes, and spleen) from fourteen COVID-19 autopsy cases using immunohistochemistry (IHC) for the spike and the nucleoprotein proteins, and RNA in situ hybridization (RNA ISH) for the spike protein mRNA. Tissue detection assays were compared with quantitative polymerase chain reaction (qPCR)-based detection. SARS-CoV-2 was histologically detected in the Vero E6 positive cell line control, 1 of 14 (7%) lungs, and none (0%) of the other 59 organs. There was perfect concordance between the IHC and RNA ISH results. qPCR confirmed high viral load in the SARS-CoV-2 ISH-positive lung tissue, and absent or low viral load in all ISH-negative tissues. In patients who die of COVID-19-related organ failure, SARS-CoV-2 is largely not detectable using tissue-based assays. Even in lungs showing widespread injury, SARS-CoV-2 viral RNA or proteins were detected in only a small minority of cases. This observation supports the concept that viral infection is primarily a trigger for multiple-organ pathogenic proinflammatory responses. Direct viral tissue damage is a transient phenomenon that is generally not sustained throughout disease progression.


Subject(s)
COVID-19/pathology , Liver/virology , Lung/virology , SARS-CoV-2/pathogenicity , Animals , Autopsy/methods , COVID-19/virology , Chlorocebus aethiops , Disease Progression , Humans , Immunohistochemistry/methods , Liver/chemistry , Liver/pathology , Lung/pathology , RNA, Viral/metabolism , Vero Cells/virology , Viral Load/methods
10.
Cell Rep ; 35(7): 109133, 2021 05 18.
Article in English | MEDLINE | ID: covidwho-1201632

ABSTRACT

Effective control of COVID-19 requires antivirals directed against SARS-CoV-2. We assessed 10 hepatitis C virus (HCV) protease-inhibitor drugs as potential SARS-CoV-2 antivirals. There is a striking structural similarity of the substrate binding clefts of SARS-CoV-2 main protease (Mpro) and HCV NS3/4A protease. Virtual docking experiments show that these HCV drugs can potentially bind into the Mpro substrate-binding cleft. We show that seven HCV drugs inhibit both SARS-CoV-2 Mpro protease activity and SARS-CoV-2 virus replication in Vero and/or human cells. However, their Mpro inhibiting activities did not correlate with their antiviral activities. This conundrum is resolved by demonstrating that four HCV protease inhibitor drugs, simeprevir, vaniprevir, paritaprevir, and grazoprevir inhibit the SARS CoV-2 papain-like protease (PLpro). HCV drugs that inhibit PLpro synergize with the viral polymerase inhibitor remdesivir to inhibit virus replication, increasing remdesivir's antiviral activity as much as 10-fold, while those that only inhibit Mpro do not synergize with remdesivir.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/drug therapy , Coronavirus Papain-Like Proteases/antagonists & inhibitors , SARS-CoV-2/drug effects , SARS-CoV-2/enzymology , Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/pharmacology , Alanine/analogs & derivatives , Alanine/pharmacology , COVID-19/virology , Cell Culture Techniques , Cell Line , Coronavirus Papain-Like Proteases/metabolism , Drug Repositioning/methods , Drug Synergism , Hepacivirus/drug effects , Hepatitis C/drug therapy , Humans , Molecular Docking Simulation , Molecular Dynamics Simulation , Protease Inhibitors/pharmacology , Virus Replication/drug effects
11.
Mol Cell ; 81(12): 2656-2668.e8, 2021 06 17.
Article in English | MEDLINE | ID: covidwho-1179919

ABSTRACT

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.


Subject(s)
Antigens, CD/genetics , Host-Pathogen Interactions/genetics , Interferon Regulatory Factors/genetics , Interferon Type I/genetics , SARS-CoV-2/genetics , Viral Proteins/genetics , Animals , Antigens, CD/chemistry , Antigens, CD/immunology , Binding Sites , Cell Line, Tumor , Chlorocebus aethiops , Endoplasmic Reticulum/genetics , Endoplasmic Reticulum/immunology , Endoplasmic Reticulum/virology , GPI-Linked Proteins/chemistry , GPI-Linked Proteins/genetics , GPI-Linked Proteins/immunology , Gene Expression Regulation , Golgi Apparatus/genetics , Golgi Apparatus/immunology , Golgi Apparatus/virology , HEK293 Cells , Host-Pathogen Interactions/immunology , Humans , Immunity, Innate , Interferon Regulatory Factors/classification , Interferon Regulatory Factors/immunology , Interferon Type I/immunology , Molecular Docking Simulation , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , SARS-CoV-2/immunology , Signal Transduction , Vero Cells , Viral Proteins/chemistry , Viral Proteins/immunology , Virus Internalization , Virus Release/genetics , Virus Release/immunology , Virus Replication/genetics , Virus Replication/immunology
12.
Front Pharmacol ; 12: 633680, 2021.
Article in English | MEDLINE | ID: covidwho-1175552

ABSTRACT

SARS-CoV-2 infection is required for COVID-19, but many signs and symptoms of COVID-19 differ from common acute viral diseases. SARS-CoV-2 infection is necessary but not sufficient for development of clinical COVID-19 disease. Currently, there are no approved pre- or post-exposure prophylactic COVID-19 medical countermeasures. Clinical data suggest that famotidine may mitigate COVID-19 disease, but both mechanism of action and rationale for dose selection remain obscure. We have investigated several plausible hypotheses for famotidine activity including antiviral and host-mediated mechanisms of action. We propose that the principal mechanism of action of famotidine for relieving COVID-19 symptoms involves on-target histamine receptor H2 activity, and that development of clinical COVID-19 involves dysfunctional mast cell activation and histamine release. Based on these findings and associated hypothesis, new COVID-19 multi-drug treatment strategies based on repurposing well-characterized drugs are being developed and clinically tested, and many of these drugs are available worldwide in inexpensive generic oral forms suitable for both outpatient and inpatient treatment of COVID-19 disease.

13.
Mod Pathol ; 34(8): 1456-1467, 2021 08.
Article in English | MEDLINE | ID: covidwho-1164812

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its associated clinical syndrome COVID-19 are causing overwhelming morbidity and mortality around the globe and disproportionately affected New York City between March and May 2020. Here, we report on the first 100 COVID-19-positive autopsies performed at the Mount Sinai Hospital in New York City. Autopsies revealed large pulmonary emboli in six cases. Diffuse alveolar damage was present in over 90% of cases. We also report microthrombi in multiple organ systems including the brain, as well as hemophagocytosis. We additionally provide electron microscopic evidence of the presence of the virus in our samples. Laboratory results of our COVID-19 cohort disclose elevated inflammatory markers, abnormal coagulation values, and elevated cytokines IL-6, IL-8, and TNFα. Our autopsy series of COVID-19-positive patients reveals that this disease, often conceptualized as a primarily respiratory viral illness, has widespread effects in the body including hypercoagulability, a hyperinflammatory state, and endothelial dysfunction. Targeting of these multisystemic pathways could lead to new treatment avenues as well as combination therapies against SARS-CoV-2 infection.


Subject(s)
COVID-19/physiopathology , Lung/physiopathology , Pulmonary Embolism/physiopathology , Adult , Aged , Aged, 80 and over , Autopsy , Blood Coagulation , COVID-19/blood , COVID-19/pathology , COVID-19/virology , Cause of Death , Cytokines/blood , Female , Host-Pathogen Interactions , Humans , Inflammation Mediators/blood , Lung/pathology , Lung/virology , Male , Middle Aged , New York City , Pulmonary Embolism/blood , Pulmonary Embolism/pathology , Pulmonary Embolism/virology , SARS-CoV-2/pathogenicity
14.
Sci Adv ; 7(6)2021 02.
Article in English | MEDLINE | ID: covidwho-1066794

ABSTRACT

The ongoing unprecedented severe acute respiratory syndrome caused by the SARS-CoV-2 outbreak worldwide has highlighted the need for understanding viral-host interactions involved in mechanisms of virulence. Here, we show that the virulence factor Nsp1 protein of SARS-CoV-2 interacts with the host messenger RNA (mRNA) export receptor heterodimer NXF1-NXT1, which is responsible for nuclear export of cellular mRNAs. Nsp1 prevents proper binding of NXF1 to mRNA export adaptors and NXF1 docking at the nuclear pore complex. As a result, a significant number of cellular mRNAs are retained in the nucleus during infection. Increased levels of NXF1 rescues the Nsp1-mediated mRNA export block and inhibits SARS-CoV-2 infection. Thus, antagonizing the Nsp1 inhibitory function on mRNA export may represent a strategy to restoring proper antiviral host gene expression in infected cells.


Subject(s)
COVID-19/metabolism , Gene Expression , Host Microbial Interactions/genetics , RNA, Messenger/metabolism , SARS-CoV-2/metabolism , Viral Nonstructural Proteins/metabolism , Virulence Factors/metabolism , Active Transport, Cell Nucleus/genetics , Animals , COVID-19/virology , Chlorocebus aethiops , HEK293 Cells , Humans , Nuclear Pore/metabolism , Nucleocytoplasmic Transport Proteins/genetics , Nucleocytoplasmic Transport Proteins/metabolism , RNA, Messenger/genetics , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , SARS-CoV-2/chemistry , Transfection , Vero Cells , Viral Nonstructural Proteins/genetics
15.
Science ; 371(6532): 926-931, 2021 02 26.
Article in English | MEDLINE | ID: covidwho-1048642

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral proteins interact with the eukaryotic translation machinery, and inhibitors of translation have potent antiviral effects. We found that the drug plitidepsin (aplidin), which has limited clinical approval, possesses antiviral activity (90% inhibitory concentration = 0.88 nM) that is more potent than remdesivir against SARS-CoV-2 in vitro by a factor of 27.5, with limited toxicity in cell culture. Through the use of a drug-resistant mutant, we show that the antiviral activity of plitidepsin against SARS-CoV-2 is mediated through inhibition of the known target eEF1A (eukaryotic translation elongation factor 1A). We demonstrate the in vivo efficacy of plitidepsin treatment in two mouse models of SARS-CoV-2 infection with a reduction of viral replication in the lungs by two orders of magnitude using prophylactic treatment. Our results indicate that plitidepsin is a promising therapeutic candidate for COVID-19.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/drug therapy , Depsipeptides/pharmacology , Peptide Elongation Factor 1/antagonists & inhibitors , SARS-CoV-2/drug effects , Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/pharmacology , Adenosine Monophosphate/therapeutic use , Alanine/analogs & derivatives , Alanine/pharmacology , Alanine/therapeutic use , Animals , Antiviral Agents/therapeutic use , COVID-19/prevention & control , COVID-19/virology , Coronavirus Nucleocapsid Proteins/biosynthesis , Coronavirus Nucleocapsid Proteins/genetics , Depsipeptides/administration & dosage , Depsipeptides/therapeutic use , Drug Evaluation, Preclinical , Female , HEK293 Cells , Humans , Lung/virology , Mice, Inbred C57BL , Mutation , Peptides, Cyclic , Phosphoproteins/biosynthesis , Phosphoproteins/genetics , RNA, Viral/biosynthesis , RNA, Viral/genetics , SARS-CoV-2/genetics , SARS-CoV-2/physiology , Virus Replication/drug effects
16.
Cell Rep ; 34(2): 108628, 2021 01 12.
Article in English | MEDLINE | ID: covidwho-1036973

ABSTRACT

Recent studies have profiled the innate immune signatures in patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and suggest that cellular responses to viral challenge may affect disease severity. Yet the molecular events that underlie cellular recognition and response to SARS-CoV-2 infection remain to be elucidated. Here, we find that SARS-CoV-2 replication induces a delayed interferon (IFN) response in lung epithelial cells. By screening 16 putative sensors involved in sensing of RNA virus infection, we found that MDA5 and LGP2 primarily regulate IFN induction in response to SARS-CoV-2 infection. Further analyses revealed that viral intermediates specifically activate the IFN response through MDA5-mediated sensing. Additionally, we find that IRF3, IRF5, and NF-κB/p65 are the key transcription factors regulating the IFN response during SARS-CoV-2 infection. In summary, these findings provide critical insights into the molecular basis of the innate immune recognition and signaling response to SARS-CoV-2.


Subject(s)
Immunity, Innate , Interferon-Induced Helicase, IFIH1/metabolism , SARS-CoV-2/physiology , COVID-19/pathology , COVID-19/virology , Cell Line , Epithelial Cells/cytology , Epithelial Cells/immunology , Epithelial Cells/virology , Humans , Induced Pluripotent Stem Cells/cytology , Interferon Regulatory Factor-3/genetics , Interferon Regulatory Factor-3/metabolism , Interferon Regulatory Factors/genetics , Interferon Regulatory Factors/metabolism , Interferons/genetics , Interferons/metabolism , RNA Helicases/metabolism , RNA Interference , RNA, Double-Stranded/metabolism , RNA, Small Interfering/metabolism , SARS-CoV-2/immunology , SARS-CoV-2/isolation & purification , Signal Transduction , Transcription Factor RelA/metabolism , Virus Replication
17.
SSRN; 2020.
Preprint | SSRN | ID: ppcovidwho-5301

ABSTRACT

Recent studies profiling the innate immune signatures in patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) suggest that cellular responses to viral challenge impact disease severity. Yet, the molecular events that underlie cellular recognition and response to SARS-CoV-2 infection remains to be elucidated. Here, we find that SARS-CoV-2 replication induces a delayed interferon (IFN) response in lung epithelial cells. Through a survey of putative sensors involved in detection of RNA virus infection, we found that MDA5 and LGP2 primarily regulate IFN induction in response to SARS-CoV-2 infection. Additionally, we find that IRF-3, -5, and NF-kB/p65 are the key transcription factors regulating the IFN response during SARS-CoV-2 infection. In summary, these findings provide critical insights into the molecular basis of the innate immune recognition and signaling response to SARS-CoV-2. Funding: This work was supported by the following grants to the Sanford Burnham Prebys Medical Discovery Institute: DoD: W81XWH-20-1-0270;DHIPC: U19 AI118610;Fluomics/NOSI: U19 AI135972, as well as generous philanthropic donations from Dinah Ruch and Susan & James Blair. This work was additionally supported by the following grants to Northwestern University Feinberg School of Medicine: a CTSA supplement to NCATS: UL1 TR002389;a CTSA supplement to NUCATS with the generous support of the Dixon family: UL1 TR001422;and a Cancer Center supplement: P30 CA060553. Development and implementation of iPS cell technology for production of airway epithelial cells was supported by Incubation Program from Office of Society Academia Collaboration for Innovation, Kyoto University.

18.
Emerg Microbes Infect ; 10(1): 376-383, 2021 Dec.
Article in English | MEDLINE | ID: covidwho-977353

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been detected in domestic and wild cats. However, little is known about natural viral infections of domestic cats, although their importance for modelling disease spread, informing strategies for managing positive human-animal relationships and disease prevention. Here, we describe the SARS-CoV-2 infection in a household of two human adults and sibling cats (one male and two females) using real-time RT-PCR, an ELISA test, viral sequencing, and virus isolation. On May 5th, 2020, the cat-owners tested positive for SARS-CoV-2. Two days later, the male cat showed mild respiratory symptoms and tested positive. Four days after the male cat, the two female cats became positive, asymptomatically. Also, one human and one cat showed antibodies against SARS-CoV-2. All cats excreted detectable SARS-CoV-2 RNA for a shorter duration than humans and viral sequences analysis confirmed human-to-cat transmission. We could not determine if cat-to-cat transmission also occurred.


Subject(s)
COVID-19/veterinary , COVID-19/virology , Cats/virology , Virus Shedding , Adult , Animals , Chile , Female , Genome, Viral , Humans , Male , Middle Aged , RNA, Viral/analysis , SARS-CoV-2/growth & development , SARS-CoV-2/physiology
19.
Proc Natl Acad Sci U S A ; 117(45): 28344-28354, 2020 11 10.
Article in English | MEDLINE | ID: covidwho-887237

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the ongoing coronavirus disease 2019 (COVID-19) pandemic that is a serious global health problem. Evasion of IFN-mediated antiviral signaling is a common defense strategy that pathogenic viruses use to replicate and propagate in their host. In this study, we show that SARS-CoV-2 is able to efficiently block STAT1 and STAT2 nuclear translocation in order to impair transcriptional induction of IFN-stimulated genes (ISGs). Our results demonstrate that the viral accessory protein Orf6 exerts this anti-IFN activity. We found that SARS-CoV-2 Orf6 localizes at the nuclear pore complex (NPC) and directly interacts with Nup98-Rae1 via its C-terminal domain to impair docking of cargo-receptor (karyopherin/importin) complex and disrupt nuclear import. In addition, we show that a methionine-to-arginine substitution at residue 58 impairs Orf6 binding to the Nup98-Rae1 complex and abolishes its IFN antagonistic function. All together our data unravel a mechanism of viral antagonism in which a virus hijacks the Nup98-Rae1 complex to overcome the antiviral action of IFN.


Subject(s)
COVID-19/metabolism , Interferons/metabolism , Nuclear Pore Complex Proteins/metabolism , Nuclear Pore/metabolism , STAT1 Transcription Factor/metabolism , STAT2 Transcription Factor/metabolism , Viral Proteins/metabolism , Active Transport, Cell Nucleus , Animals , Binding Sites , Chlorocebus aethiops , HEK293 Cells , Humans , Nuclear Matrix-Associated Proteins/chemistry , Nuclear Matrix-Associated Proteins/metabolism , Nucleocytoplasmic Transport Proteins/chemistry , Nucleocytoplasmic Transport Proteins/metabolism , Protein Binding , Signal Transduction , Vero Cells
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