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1.
Sci Adv ; 8(8): eabi6110, 2022 Feb 25.
Article in English | MEDLINE | ID: covidwho-1714330

ABSTRACT

The spread of SARS-CoV-2 and ongoing COVID-19 pandemic underscores the need for new treatments. Here we report that cannabidiol (CBD) inhibits infection of SARS-CoV-2 in cells and mice. CBD and its metabolite 7-OH-CBD, but not THC or other congeneric cannabinoids tested, potently block SARS-CoV-2 replication in lung epithelial cells. CBD acts after viral entry, inhibiting viral gene expression and reversing many effects of SARS-CoV-2 on host gene transcription. CBD inhibits SARS-CoV-2 replication in part by up-regulating the host IRE1α RNase endoplasmic reticulum (ER) stress response and interferon signaling pathways. In matched groups of human patients from the National COVID Cohort Collaborative, CBD (100 mg/ml oral solution per medical records) had a significant negative association with positive SARS-CoV-2 tests. This study highlights CBD as a potential preventative agent for early-stage SARS-CoV-2 infection and merits future clinical trials. We caution against use of non-medical formulations including edibles, inhalants or topicals as a preventative or treatment therapy at the present time.


Subject(s)
Antiviral Agents/pharmacology , Cannabidiol/pharmacology , Host-Pathogen Interactions/drug effects , Immunity, Innate/drug effects , SARS-CoV-2/drug effects , A549 Cells , Animals , Antiviral Agents/chemistry , COVID-19/drug therapy , COVID-19/virology , Cannabidiol/chemistry , Cannabidiol/metabolism , Chlorocebus aethiops , Endoplasmic Reticulum Stress/drug effects , Endoribonucleases/genetics , Endoribonucleases/metabolism , Epithelial Cells/virology , Female , Gene Expression Regulation, Viral/drug effects , Host-Pathogen Interactions/physiology , Humans , Interferons/metabolism , Mice , /metabolism , SARS-CoV-2/physiology , Vero Cells , Virus Internalization/drug effects , Virus Replication/drug effects
2.
RNA ; 28(2): 227-238, 2022 02.
Article in English | MEDLINE | ID: covidwho-1533393

ABSTRACT

The Bacillus subtilis genome is predicted to encode numerous ribonucleases, including four 3' exoribonucleases that have been characterized to some extent. A strain containing gene knockouts of all four known 3' exoribonucleases is viable, suggesting that one or more additional RNases remain to be discovered. A protein extract from the quadruple RNase mutant strain was fractionated and RNase activity was followed, resulting in the identification of an enzyme activity catalyzed by the YloC protein. YloC is an endoribonuclease and is a member of the highly conserved "YicC family" of proteins that is widespread in bacteria. YloC is a metal-dependent enzyme that catalyzes the cleavage of single-stranded RNA, preferentially at U residues, and exists in an oligomeric form, most likely a hexamer. As such, YloC shares some characteristics with the SARS-CoV Nsp15 endoribonuclease. While the in vivo function of YloC in B. subtilis is yet to be determined, YloC was found to act similarly to YicC in an Escherichia coli in vivo assay that assesses decay of the small RNA, RyhB. Thus, YloC may play a role in small RNA regulation.


Subject(s)
Bacillus subtilis/genetics , Endoribonucleases/genetics , Endoribonucleases/metabolism , Bacillus subtilis/enzymology , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Endoribonucleases/chemistry , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/metabolism , Gene Expression Regulation, Bacterial , Microorganisms, Genetically-Modified , Mutation , RNA Stability , RNA, Bacterial/chemistry , RNA, Bacterial/metabolism , Ribonucleases/genetics , Ribonucleases/metabolism , Substrate Specificity , Viral Nonstructural Proteins/metabolism
3.
J Biol Chem ; 297(4): 101218, 2021 10.
Article in English | MEDLINE | ID: covidwho-1433454

ABSTRACT

The SARS-CoV-2 replication-transcription complex is an assembly of nonstructural viral proteins that collectively act to reproduce the viral genome and generate mRNA transcripts. While the structures of the individual proteins involved are known, how they assemble into a functioning superstructure is not. Applying molecular modeling tools, including protein-protein docking, to the available structures of nsp7-nsp16 and the nucleocapsid, we have constructed an atomistic model of how these proteins associate. Our principal finding is that the complex is hexameric, centered on nsp15. The nsp15 hexamer is capped on two faces by trimers of nsp14/nsp16/(nsp10)2, which then recruit six nsp12/nsp7/(nsp8)2 polymerase subunits to the complex. To this, six subunits of nsp13 are arranged around the superstructure, but not evenly distributed. Polymerase subunits that coordinate dimers of nsp13 are capable of binding the nucleocapsid, which positions the 5'-UTR TRS-L RNA over the polymerase active site, a state distinguishing transcription from replication. Analysis of the viral RNA path through the complex indicates the dsRNA that exits the polymerase passes over the nsp14 exonuclease and nsp15 endonuclease sites before being unwound by a convergence of zinc fingers from nsp10 and nsp14. The template strand is then directed away from the complex, while the nascent strand is directed to the sites responsible for mRNA capping. The model presents a cohesive picture of the multiple functions of the coronavirus replication-transcription complex and addresses fundamental questions related to proofreading, template switching, mRNA capping, and the role of the endonuclease.


Subject(s)
Endoribonucleases/metabolism , Models, Molecular , SARS-CoV-2/metabolism , Viral Nonstructural Proteins/metabolism , Binding Sites , COVID-19/pathology , COVID-19/virology , Dimerization , Endoribonucleases/chemistry , Endoribonucleases/genetics , Humans , Molecular Docking Simulation , Protein Structure, Quaternary , RNA, Double-Stranded/chemistry , RNA, Double-Stranded/metabolism , SARS-CoV-2/isolation & purification , Transcription, Genetic , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/genetics , Virus Replication
4.
RNA ; 27(11): 1318-1329, 2021 11.
Article in English | MEDLINE | ID: covidwho-1329126

ABSTRACT

The transcriptional induction of interferon (IFN) genes is a key feature of the mammalian antiviral response that limits viral replication and dissemination. A hallmark of severe COVID-19 disease caused by SARS-CoV-2 is the low presence of IFN proteins in patient serum despite elevated levels of IFN-encoding mRNAs, indicative of post-transcriptional inhibition of IFN protein production. Here, we performed single-molecule RNA visualization to examine the expression and localization of host mRNAs during SARS-CoV-2 infection. Our data show that the biogenesis of type I and type III IFN mRNAs is inhibited at multiple steps during SARS-CoV-2 infection. First, translocation of the interferon regulatory factor 3 (IRF3) transcription factor to the nucleus is limited in response to SARS-CoV-2, indicating that SARS-CoV-2 inhibits RLR-MAVS signaling and thus weakens transcriptional induction of IFN genes. Second, we observed that IFN mRNAs primarily localize to the site of transcription in most SARS-CoV-2 infected cells, suggesting that SARS-CoV-2 either inhibits the release of IFN mRNAs from their sites of transcription and/or triggers decay of IFN mRNAs in the nucleus upon exiting the site of transcription. Lastly, nuclear-cytoplasmic transport of IFN mRNAs is inhibited during SARS-CoV-2 infection, which we propose is a consequence of widespread degradation of host cytoplasmic basal mRNAs in the early stages of SARS-CoV-2 replication by the SARS-CoV-2 Nsp1 protein, as well as the host antiviral endoribonuclease, RNase L. Importantly, IFN mRNAs can escape SARS-CoV-2-mediated degradation if they reach the cytoplasm, making rescue of mRNA export a viable means for promoting the immune response to SARS-CoV-2.


Subject(s)
COVID-19/genetics , Host-Pathogen Interactions/genetics , Interferons/genetics , RNA Stability , SARS-CoV-2/pathogenicity , Viral Nonstructural Proteins/genetics , A549 Cells , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , COVID-19/virology , Cell Line , Endoribonucleases/genetics , Endoribonucleases/metabolism , Humans , In Situ Hybridization, Fluorescence/methods , Interferon Regulatory Factor-3/genetics , Interferon Regulatory Factor-3/metabolism , Interferons/metabolism , RNA, Messenger/metabolism , Single Molecule Imaging
5.
Front Immunol ; 11: 1554, 2020.
Article in English | MEDLINE | ID: covidwho-1194588

ABSTRACT

The RNase T2 family consists of evolutionarily conserved endonucleases that express in many different species, including animals, plants, protozoans, bacteria, and viruses. The main biological roles of these ribonucleases are cleaving or degrading RNA substrates. They preferentially cleave single-stranded RNA molecules between purine and uridine residues to generate two nucleotide fragments with 2'3'-cyclic phosphate adenosine/guanosine terminus and uridine residue, respectively. Accumulating studies have revealed that RNase T2 is critical for the pathophysiology of inflammation and cancer. In this review, we introduce the distribution, structure, and functions of RNase T2, its differential roles in inflammation and cancer, and the perspective for its research and related applications in medicine.


Subject(s)
Disease Susceptibility , Endoribonucleases/genetics , Endoribonucleases/metabolism , Inflammation/etiology , Inflammation/metabolism , Neoplasms/etiology , Neoplasms/metabolism , Animals , Biomarkers , Cellular Microenvironment/immunology , Disease Susceptibility/immunology , Endoribonucleases/chemistry , Humans , Immune System/immunology , Immune System/metabolism , Immunomodulation , Inflammation/pathology , Neoplasms/pathology , Structure-Activity Relationship
6.
J Chem Inf Model ; 60(12): 5853-5865, 2020 12 28.
Article in English | MEDLINE | ID: covidwho-1065772

ABSTRACT

Tremendous effort has been given to the development of diagnostic tests, preventive vaccines, and therapeutic medicines for coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Much of this development has been based on the reference genome collected on January 5, 2020. Based on the genotyping of 15 140 genome samples collected up to June 1, 2020, we report that SARS-CoV-2 has undergone 8309 single mutations which can be clustered into six subtypes. We introduce mutation ratio and mutation h-index to characterize the protein conservativeness and unveil that SARS-CoV-2 envelope protein, main protease, and endoribonuclease protein are relatively conservative, while SARS-CoV-2 nucleocapsid protein, spike protein, and papain-like protease are relatively nonconservative. In particular, we have identified mutations on 40% of nucleotides in the nucleocapsid gene in the population level, signaling potential impacts on the ongoing development of COVID-19 diagnosis, vaccines, and antibody and small-molecular drugs.


Subject(s)
COVID-19 , SARS-CoV-2/classification , SARS-CoV-2/metabolism , Antibodies, Viral/metabolism , COVID-19/diagnosis , COVID-19/epidemiology , COVID-19/prevention & control , COVID-19/therapy , Coronavirus 3C Proteases/chemistry , Coronavirus 3C Proteases/genetics , Coronavirus Envelope Proteins/chemistry , Coronavirus Envelope Proteins/genetics , Coronavirus Nucleocapsid Proteins/chemistry , Coronavirus Nucleocapsid Proteins/genetics , Coronavirus Papain-Like Proteases/chemistry , Coronavirus Papain-Like Proteases/genetics , Endoribonucleases/chemistry , Endoribonucleases/genetics , Genome, Viral , Genotype , Geography , Humans , Mutant Proteins/chemistry , Mutant Proteins/genetics , Mutation , Phosphoproteins/chemistry , Phosphoproteins/genetics , Protein Conformation , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Vaccines/metabolism , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/genetics
7.
Ann Allergy Asthma Immunol ; 125(5): 503-504, 2020 11.
Article in English | MEDLINE | ID: covidwho-628802

Subject(s)
Adrenal Cortex Hormones/therapeutic use , Anti-Asthmatic Agents/therapeutic use , Asthma/drug therapy , Coronavirus Infections/prevention & control , Cytokine Release Syndrome/prevention & control , Dexamethasone/therapeutic use , Pandemics/prevention & control , Pneumonia, Viral/prevention & control , Administration, Inhalation , Angiotensin-Converting Enzyme 2 , Asthma/immunology , Asthma/pathology , Asthma/virology , Betacoronavirus/drug effects , Betacoronavirus/immunology , Betacoronavirus/pathogenicity , COVID-19 , Coronavirus Infections/immunology , Coronavirus Infections/pathology , Coronavirus Infections/virology , Cytokine Release Syndrome/immunology , Cytokine Release Syndrome/pathology , Cytokine Release Syndrome/virology , Endoribonucleases/antagonists & inhibitors , Endoribonucleases/genetics , Endoribonucleases/immunology , Gene Expression Regulation/drug effects , Host-Pathogen Interactions/drug effects , Host-Pathogen Interactions/genetics , Host-Pathogen Interactions/immunology , Humans , Interleukin-6/antagonists & inhibitors , Interleukin-6/genetics , Interleukin-6/immunology , Interleukin-8/antagonists & inhibitors , Interleukin-8/genetics , Interleukin-8/immunology , Peptidyl-Dipeptidase A/genetics , Peptidyl-Dipeptidase A/immunology , Pneumonia, Viral/immunology , Pneumonia, Viral/pathology , Pneumonia, Viral/virology , Receptors, Virus/antagonists & inhibitors , Receptors, Virus/genetics , Receptors, Virus/immunology , SARS-CoV-2 , Serine Endopeptidases/genetics , Serine Endopeptidases/immunology , Severity of Illness Index , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/immunology , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/immunology
8.
Emerg Microbes Infect ; 9(1): 1418-1428, 2020 Dec.
Article in English | MEDLINE | ID: covidwho-595042

ABSTRACT

The Coronavirus disease 2019 (COVID-19), which is caused by the novel SARS-CoV-2 virus, is now causing a tremendous global health concern. Since its first appearance in December 2019, the outbreak has already caused over 5.8 million infections worldwide (till 29 May 2020), with more than 0.35 million deaths. Early virus-mediated immune suppression is believed to be one of the unique characteristics of SARS-CoV-2 infection and contributes at least partially to the viral pathogenesis. In this study, we identified the key viral interferon antagonists of SARS-CoV-2 and compared them with two well-characterized SARS-CoV interferon antagonists, PLpro and orf6. Here we demonstrated that the SARS-CoV-2 nsp13, nsp14, nsp15 and orf6, but not the unique orf8, could potently suppress primary interferon production and interferon signalling. Although SARS-CoV PLpro has been well-characterized for its potent interferon-antagonizing, deubiquitinase and protease activities, SARS-CoV-2 PLpro, despite sharing high amino acid sequence similarity with SARS-CoV, loses both interferon-antagonising and deubiquitinase activities. Among the 27 viral proteins, SARS-CoV-2 orf6 demonstrated the strongest suppression on both primary interferon production and interferon signalling. Orf6-deleted SARS-CoV-2 may be considered for the development of intranasal live-but-attenuated vaccine against COVID-19.


Subject(s)
Betacoronavirus/metabolism , Coronavirus Infections/metabolism , Endoribonucleases/metabolism , Exoribonucleases/metabolism , Interferons/antagonists & inhibitors , Interferons/metabolism , Methyltransferases/metabolism , Pneumonia, Viral/metabolism , RNA Helicases/metabolism , Viral Nonstructural Proteins/metabolism , Viral Proteins/metabolism , Betacoronavirus/genetics , COVID-19 , Cell Line , Coronavirus Infections/genetics , Coronavirus Infections/virology , Endoribonucleases/genetics , Exoribonucleases/genetics , Host-Pathogen Interactions , Humans , Interferons/genetics , Methyltransferases/genetics , Pandemics , Pneumonia, Viral/genetics , Pneumonia, Viral/virology , RNA Helicases/genetics , SARS-CoV-2 , Viral Nonstructural Proteins/genetics , Viral Proteins/genetics
9.
J Allergy Clin Immunol ; 146(2): 330-331, 2020 08.
Article in English | MEDLINE | ID: covidwho-597639
10.
In Vivo ; 34(3 Suppl): 1629-1632, 2020 Jun.
Article in English | MEDLINE | ID: covidwho-534630

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a positive-sense single-stranded RNA virus. It is contagious in humans and is the cause of the coronavirus disease 2019 (COVID-19) pandemic. In the current analysis, we searched for SARS-CoV-2 sequences within the human genome. To compare the SARS-CoV-2 genome to the human genome, we used the blast-like alignment tool (BLAT) of the University of California, Santa Cruz Genome Browser. BLAT can align a user sequence of 25 bases or more to the genome. BLAT search results revealed a 117-base pair SARS-CoV-2 sequence in the human genome with 94.6% identity. The sequence was in chromosome 1p within an intronic region of the netrin G1 (NTNG1) gene. The sequence matched a sequence in the SARS-CoV-2 orf1b (open reading frames) gene. The SARS-CoV-2 human sequence lies within non-structural proteins 14 and 15 (NSP14 and NSP15), and is quite close to the viral spike sequence, separated only by NSP16, a 904-base pair sequence. The mechanism for SARS-CoV-2 infection is the binding of the virus spike protein to the membrane-bound form of angiotensin-converting enzyme 2 and internalization of the complex by the host cell. It is probably no accident that a sequence from the SARS-CoV-2 orf1b gene is found in the human NTNG1 gene, implicated in schizophrenia, and that haloperidol, used to treat schizophrenia, may also be a treatment for COVID-19. We suggest, therefore, that it is important to investigate other haloperidol analogs. Among them are benperidol, bromperidol, bromperidol decanoate, droperidol, seperidol hydrochloride, and trifluperidol. These analogs might be valuable in the treatment of COVID-19 and other coronavirus infections.


Subject(s)
Betacoronavirus/genetics , Chromosomes, Human, Pair 1/genetics , Exoribonucleases/genetics , Genes, Viral , Netrin-1/genetics , Viral Nonstructural Proteins/genetics , Viral Proteins/genetics , Animals , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , Base Sequence , COVID-19 , Coronavirus Infections/drug therapy , DNA, Complementary/genetics , Endoribonucleases/genetics , Haloperidol/analogs & derivatives , Haloperidol/pharmacology , Haloperidol/therapeutic use , Humans , Introns/genetics , Pan troglodytes/genetics , Pandemics , Pneumonia, Viral/drug therapy , Polyproteins , RNA, Viral/genetics , SARS-CoV-2 , Schizophrenia/drug therapy , Schizophrenia/genetics , Sequence Alignment , Sequence Homology, Nucleic Acid , Species Specificity
11.
Protein Sci ; 29(7): 1596-1605, 2020 07.
Article in English | MEDLINE | ID: covidwho-71902

ABSTRACT

Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) is rapidly spreading around the world. There is no existing vaccine or proven drug to prevent infections and stop virus proliferation. Although this virus is similar to human and animal SARS-CoVs and Middle East Respiratory Syndrome coronavirus (MERS-CoVs), the detailed information about SARS-CoV-2 proteins structures and functions is urgently needed to rapidly develop effective vaccines, antibodies, and antivirals. We applied high-throughput protein production and structure determination pipeline at the Center for Structural Genomics of Infectious Diseases to produce SARS-CoV-2 proteins and structures. Here we report two high-resolution crystal structures of endoribonuclease Nsp15/NendoU. We compare these structures with previously reported homologs from SARS and MERS coronaviruses.


Subject(s)
Betacoronavirus/chemistry , Endoribonucleases/chemistry , Middle East Respiratory Syndrome Coronavirus/chemistry , Oligonucleotides/chemistry , SARS Virus/chemistry , Viral Nonstructural Proteins/chemistry , Amino Acid Sequence , Betacoronavirus/genetics , Betacoronavirus/metabolism , Catalytic Domain , Cloning, Molecular , Crystallography, X-Ray , Endoribonucleases/genetics , Endoribonucleases/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Humans , Middle East Respiratory Syndrome Coronavirus/genetics , Middle East Respiratory Syndrome Coronavirus/metabolism , Models, Molecular , Oligonucleotides/metabolism , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , SARS Virus/genetics , SARS Virus/metabolism , SARS-CoV-2 , Sequence Alignment , Sequence Homology, Amino Acid , Substrate Specificity , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism
12.
Proc Natl Acad Sci U S A ; 117(14): 8094-8103, 2020 04 07.
Article in English | MEDLINE | ID: covidwho-11430

ABSTRACT

Coronaviruses (CoVs) are positive-sense RNA viruses that can emerge from endemic reservoirs and infect zoonotically, causing significant morbidity and mortality. CoVs encode an endoribonuclease designated EndoU that facilitates evasion of host pattern recognition receptor MDA5, but the target of EndoU activity was not known. Here, we report that EndoU cleaves the 5'-polyuridines from negative-sense viral RNA, termed PUN RNA, which is the product of polyA-templated RNA synthesis. Using a virus containing an EndoU catalytic-inactive mutation, we detected a higher abundance of PUN RNA in the cytoplasm compared to wild-type-infected cells. Furthermore, we found that transfecting PUN RNA into cells stimulates a robust, MDA5-dependent interferon response, and that removal of the polyuridine extension on the RNA dampens the response. Overall, the results of this study reveal the PUN RNA to be a CoV MDA5-dependent pathogen-associated molecular pattern (PAMP). We also establish a mechanism for EndoU activity to cleave and limit the accumulation of this PAMP. Since EndoU activity is highly conserved in all CoVs, inhibiting this activity may serve as an approach for therapeutic interventions against existing and emerging CoV infections.


Subject(s)
Coronavirus Infections/immunology , Coronavirus Infections/virology , Coronavirus/metabolism , Endoribonucleases/metabolism , Poly U/metabolism , Viral Nonstructural Proteins/metabolism , Animals , Antiviral Agents/pharmacology , Cell Line , Chlorocebus aethiops , Coronavirus/enzymology , Coronavirus/immunology , Endoribonucleases/genetics , Host Microbial Interactions/physiology , Humans , Interferons/pharmacology , Poly U/chemistry , RNA, Viral/genetics , RNA, Viral/metabolism , Uridine/chemistry , Vero Cells , Viral Nonstructural Proteins/genetics , Virus Replication/physiology
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