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1.
Emerg Microbes Infect ; 11(1): 1488-1499, 2022 Dec.
Article in English | MEDLINE | ID: covidwho-1852834

ABSTRACT

The recent global pandemic was a spillover from the SARS-CoV-2 virus. Viral entry involves the receptor binding domain (RBD) of the viral spike protein interacting with the protease domain (PD) of the cellular receptor, ACE2. We hereby present a comprehensive mutational landscape of the effects of ACE2-PD point mutations on RBD-ACE2 binding using a saturation mutagenesis approach based on microarray-based oligo synthesis and a single-cell screening assay. We observed that changes in glycosylation sites and directly interacting sites of ACE2-PD significantly influenced ACE2-RBD binding. We further engineered an ACE2 decoy receptor with critical point mutations, D30I, L79W, T92N, N322V, and K475F, named C4-1. C4-1 shows a 200-fold increase in neutralization for the SARS-CoV-2 D614G pseudotyped virus compared to wild-type soluble ACE2 and a sevenfold increase in binding affinity to wild-type spike compared to the C-terminal Ig-Fc fused wild-type soluble ACE2. Moreover, C4-1 efficiently neutralized prevalent variants, especially the omicron variant (EC50=16 ng/mL), and rescued monoclonal antibodies, vaccine, and convalescent sera neutralization from viral immune-escaping. We hope to next investigate translating the therapeutic potential of C4-1 for the treatment of SARS-CoV-2.


Subject(s)
COVID-19 , SARS-CoV-2 , Angiotensin-Converting Enzyme 2/genetics , COVID-19/therapy , Humans , Immunization, Passive , Mutagenesis , Protein Binding , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry
2.
Viruses ; 14(4)2022 04 18.
Article in English | MEDLINE | ID: covidwho-1792409

ABSTRACT

In RNA viruses, a small increase in their mutation rates can be sufficient to exceed their threshold of viability. Lethal mutagenesis is a therapeutic strategy based on the use of mutagens, driving viral populations to extinction. Extinction catastrophe can be experimentally induced by promutagenic nucleosides in cell culture models. The loss of HIV infectivity has been observed after passage in 5-hydroxydeoxycytidine or 5,6-dihydro-5-aza-2'-deoxycytidine while producing a two-fold increase in the viral mutation frequency. Among approved nucleoside analogs, experiments with polioviruses and other RNA viruses suggested that ribavirin can be mutagenic, although its mechanism of action is not clear. Favipiravir and molnupiravir exert an antiviral effect through lethal mutagenesis. Both drugs are broad-spectrum antiviral agents active against RNA viruses. Favipiravir incorporates into viral RNA, affecting the G→A and C→U transition rates. Molnupiravir (a prodrug of ß-d-N4-hydroxycytidine) has been recently approved for the treatment of SARS-CoV-2 infection. Its triphosphate derivative can be incorporated into viral RNA and extended by the coronavirus RNA polymerase. Incorrect base pairing and inefficient extension by the polymerase promote mutagenesis by increasing the G→A and C→U transition frequencies. Despite having remarkable antiviral action and resilience to drug resistance, carcinogenic risks and genotoxicity are important concerns limiting their extended use in antiviral therapy.


Subject(s)
COVID-19 , RNA Viruses , Antiviral Agents/pharmacology , Humans , Mutagenesis , Mutagens/pharmacology , Nucleosides/pharmacology , RNA Viruses/genetics , RNA, Viral/genetics , SARS-CoV-2
3.
Int J Mol Sci ; 23(6)2022 Mar 08.
Article in English | MEDLINE | ID: covidwho-1732071

ABSTRACT

Nanobodies provide important advantages over traditional antibodies, including their smaller size and robust biochemical properties such as high thermal stability, high solubility, and the ability to be bioengineered into novel multivalent, multi-specific, and high-affinity molecules, making them a class of emerging powerful therapies against SARS-CoV-2. Recent research efforts on the design, protein engineering, and structure-functional characterization of nanobodies and their binding with SARS-CoV-2 S proteins reflected a growing realization that nanobody combinations can exploit distinct binding epitopes and leverage the intrinsic plasticity of the conformational landscape for the SARS-CoV-2 S protein to produce efficient neutralizing and mutation resistant characteristics. Structural and computational studies have also been instrumental in quantifying the structure, dynamics, and energetics of the SARS-CoV-2 spike protein binding with nanobodies. In this review, a comprehensive analysis of the current structural, biophysical, and computational biology investigations of SARS-CoV-2 S proteins and their complexes with distinct classes of nanobodies targeting different binding sites is presented. The analysis of computational studies is supplemented by an in-depth examination of mutational scanning simulations and identification of binding energy hotspots for distinct nanobody classes. The review is focused on the analysis of mechanisms underlying synergistic binding of multivalent nanobodies that can be superior to single nanobodies and conventional nanobody cocktails in combating escape mutations by effectively leveraging binding avidity and allosteric cooperativity. We discuss how structural insights and protein engineering approaches together with computational biology tools can aid in the rational design of synergistic combinations that exhibit superior binding and neutralization characteristics owing to avidity-mediated mechanisms.


Subject(s)
Binding Sites , Molecular Docking Simulation , Molecular Dynamics Simulation , Single-Domain Antibodies/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Amino Acids , Antibody Affinity , Epitopes/chemistry , Epitopes/metabolism , Humans , Multiprotein Complexes/chemistry , Mutagenesis , Protein Binding , Protein Engineering , Protein Interaction Domains and Motifs , Single-Domain Antibodies/genetics , Single-Domain Antibodies/metabolism , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism
4.
J Virol ; 96(3): e0082621, 2022 02 09.
Article in English | MEDLINE | ID: covidwho-1691430

ABSTRACT

Human adenovirus serotype 26 (Ad26) is used as a gene-based vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and HIV-1. However, its primary receptor portfolio remains controversial, potentially including sialic acid, coxsackie and adenovirus receptor (CAR), integrins, and CD46. We and others have shown that Ad26 can use CD46, but these observations were questioned on the basis of the inability to cocrystallize Ad26 fiber with CD46. Recent work demonstrated that Ad26 binds CD46 with its hexon protein rather than its fiber. We examined the functional consequences of Ad26 for infection in vitro and in vivo. Ectopic expression of human CD46 on Chinese hamster ovary cells increased Ad26 infection significantly. Deletion of the complement control protein domain CCP1 or CCP2 or the serine-threonine-proline (STP) region of CD46 reduced infection. Comparing wild-type and sialic acid-deficient CHO cells, we show that the usage of CD46 is independent of its sialylation status. Ad26 transduction was increased in CD46 transgenic mice after intramuscular (i.m.) injection but not after intranasal (i.n.) administration. Ad26 transduction was 10-fold lower than Ad5 transduction after intratumoral (i.t.) injection of CD46-expressing tumors. Ad26 transduction of liver was 1,000-fold lower than that ofAd5 after intravenous (i.v.) injection. These data demonstrate the use of CD46 by Ad26 in certain situations but also show that the receptor has little consequence by other routes of administration. Finally, i.v. injection of high doses of Ad26 into CD46 mice induced release of liver enzymes into the bloodstream and reduced white blood cell counts but did not induce thrombocytopenia. This suggests that Ad26 virions do not induce direct clotting side effects seen during coronavirus disease 2019 (COVID-19) vaccination with this serotype of adenovirus. IMPORTANCE The human species D Ad26 is being investigated as a low-seroprevalence vector for oncolytic virotherapy and gene-based vaccination against HIV-1 and SARS-CoV-2. However, there is debate in the literature about its tropism and receptor utilization, which directly influence its efficiency for certain applications. This work was aimed at determining which receptor(s) this virus uses for infection and its role in virus biology, vaccine efficacy, and, importantly, vaccine safety.


Subject(s)
Adenovirus Infections, Human/metabolism , Adenovirus Infections, Human/virology , Adenoviruses, Human/classification , Adenoviruses, Human/physiology , Coxsackie and Adenovirus Receptor-Like Membrane Protein/metabolism , Host-Pathogen Interactions , Membrane Cofactor Protein/metabolism , Adenoviruses, Human/ultrastructure , Animals , Biomarkers , Blood Cell Count , CHO Cells , Cell Line , Coxsackie and Adenovirus Receptor-Like Membrane Protein/chemistry , Cricetulus , Disease Models, Animal , Gene Expression , Humans , Membrane Cofactor Protein/chemistry , Membrane Cofactor Protein/genetics , Mice, Transgenic , Models, Biological , Models, Molecular , Mutagenesis , Protein Binding , Protein Conformation , Serogroup , Sialic Acids/metabolism , Sialic Acids/pharmacology , Structure-Activity Relationship
5.
Int J Mol Sci ; 23(4)2022 Feb 14.
Article in English | MEDLINE | ID: covidwho-1686820

ABSTRACT

SARS-CoV-2 infection elicits a polyclonal neutralizing antibody (nAb) response that primarily targets the spike protein, but it is still unclear which nAbs are immunodominant and what distinguishes them from subdominant nAbs. This information would however be crucial to predict the evolutionary trajectory of the virus and design future vaccines. To shed light on this issue, we gathered 83 structures of nAbs in complex with spike protein domains. We analyzed in silico the ability of these nAbs to bind the full spike protein trimer in open and closed conformations, and predicted the change in binding affinity of the most frequently observed spike protein variants in the circulating strains. This led us to define four nAb classes with distinct variant escape fractions. By comparing these fractions with those measured from plasma of infected patients, we showed that the class of nAbs that most contributes to the immune response is able to bind the spike protein in its closed conformation. Although this class of nAbs only partially inhibits the spike protein binding to the host's angiotensin converting enzyme 2 (ACE2), it has been suggested to lock the closed pre-fusion spike protein conformation and therefore prevent its transition to an open state. Furthermore, comparison of our predictions with mRNA-1273 vaccinated patient plasma measurements suggests that spike proteins contained in vaccines elicit a different nAb class than the one elicited by natural SARS-CoV-2 infection and suggests the design of highly stable closed-form spike proteins as next-generation vaccine immunogens.


Subject(s)
Antibodies, Neutralizing/immunology , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/immunology , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Monoclonal/immunology , Antigen-Antibody Reactions , COVID-19/pathology , COVID-19/virology , Epitopes/immunology , Humans , Mutagenesis , Protein Binding , Protein Conformation , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism
7.
Nature ; 601(7894): 496, 2022 01.
Article in English | MEDLINE | ID: covidwho-1641925

Subject(s)
Antiviral Agents/therapeutic use , COVID-19/drug therapy , COVID-19/virology , Drug Development/trends , Drug Resistance, Viral , Research Personnel , SARS-CoV-2/drug effects , Adenosine Monophosphate/administration & dosage , Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/pharmacology , Adenosine Monophosphate/therapeutic use , Administration, Oral , Alanine/administration & dosage , Alanine/analogs & derivatives , Alanine/pharmacology , Alanine/therapeutic use , Antiviral Agents/administration & dosage , Antiviral Agents/pharmacology , Antiviral Agents/supply & distribution , COVID-19/mortality , COVID-19/prevention & control , COVID-19 Vaccines/supply & distribution , Cytidine/administration & dosage , Cytidine/analogs & derivatives , Cytidine/pharmacology , Cytidine/therapeutic use , Drug Approval , Drug Combinations , Drug Resistance, Viral/drug effects , Drug Resistance, Viral/genetics , Drug Therapy, Combination , Hospitalization/statistics & numerical data , Humans , Hydroxylamines/administration & dosage , Hydroxylamines/pharmacology , Hydroxylamines/therapeutic use , Lactams/administration & dosage , Lactams/pharmacology , Lactams/therapeutic use , Leucine/administration & dosage , Leucine/pharmacology , Leucine/therapeutic use , Medication Adherence , Molecular Targeted Therapy , Mutagenesis , Nitriles/administration & dosage , Nitriles/pharmacology , Nitriles/therapeutic use , Proline/administration & dosage , Proline/pharmacology , Proline/therapeutic use , Public-Private Sector Partnerships/economics , Ritonavir/administration & dosage , Ritonavir/pharmacology , Ritonavir/therapeutic use , SARS-CoV-2/enzymology , SARS-CoV-2/genetics
8.
STAR Protoc ; 3(1): 101101, 2022 03 18.
Article in English | MEDLINE | ID: covidwho-1626432

ABSTRACT

The generation of high-affinity nanobodies for diverse biomedical applications typically requires immunization or affinity maturation. Here, we report a simple protocol using complementarity-determining region (CDR)-swapping mutagenesis to isolate high-affinity nanobodies from common framework libraries. This approach involves shuffling the CDRs of low-affinity variants during the sorting of yeast-displayed libraries to directly isolate high-affinity nanobodies without the need for lead isolation and optimization. We expect this approach, which we demonstrate for SARS-CoV-2 neutralizing nanobodies, will simplify the generation of high-affinity nanobodies. For complete details on the use and execution of this profile, please refer to Zupancic et al. (2021).


Subject(s)
COVID-19 , Single-Domain Antibodies , Complementarity Determining Regions/genetics , Humans , Mutagenesis , Peptide Library , SARS-CoV-2 , Single-Domain Antibodies/genetics
9.
Proc Natl Acad Sci U S A ; 119(4)2022 01 25.
Article in English | MEDLINE | ID: covidwho-1621333

ABSTRACT

The emergence of new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a major concern given their potential impact on the transmissibility and pathogenicity of the virus as well as the efficacy of therapeutic interventions. Here, we predict the mutability of all positions in SARS-CoV-2 protein domains to forecast the appearance of unseen variants. Using sequence data from other coronaviruses, preexisting to SARS-CoV-2, we build statistical models that not only capture amino acid conservation but also more complex patterns resulting from epistasis. We show that these models are notably superior to conservation profiles in estimating the already observable SARS-CoV-2 variability. In the receptor binding domain of the spike protein, we observe that the predicted mutability correlates well with experimental measures of protein stability and that both are reliable mutability predictors (receiver operating characteristic areas under the curve ∼0.8). Most interestingly, we observe an increasing agreement between our model and the observed variability as more data become available over time, proving the anticipatory capacity of our model. When combined with data concerning the immune response, our approach identifies positions where current variants of concern are highly overrepresented. These results could assist studies on viral evolution and future viral outbreaks and, in particular, guide the exploration and anticipation of potentially harmful future SARS-CoV-2 variants.


Subject(s)
COVID-19/virology , Epistasis, Genetic , Epitopes , Mutation , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Viral Proteins/chemistry , Algorithms , Area Under Curve , Computational Biology/methods , DNA Mutational Analysis , Databases, Protein , Deep Learning , Epitopes/chemistry , Genome, Viral , Humans , Models, Statistical , Mutagenesis , Probability , Protein Domains , ROC Curve
10.
Environ Mol Mutagen ; 63(1): 37-63, 2022 01.
Article in English | MEDLINE | ID: covidwho-1620131

ABSTRACT

This review considers antiviral nucleoside analog drugs, including ribavirin, favipiravir, and molnupiravir, which induce genome error catastrophe in SARS-CoV or SARS-CoV-2 via lethal mutagenesis as a mode of action. In vitro data indicate that molnupiravir may be 100 times more potent as an antiviral agent than ribavirin or favipiravir. Molnupiravir has recently demonstrated efficacy in a phase 3 clinical trial. Because of its anticipated global use, its relative potency, and the reported in vitro "host" cell mutagenicity of its active principle, ß-d-N4-hydroxycytidine, we have reviewed the development of molnupiravir and its genotoxicity safety evaluation, as well as the genotoxicity profiles of three congeners, that is, ribavirin, favipiravir, and 5-(2-chloroethyl)-2'-deoxyuridine. We consider the potential genetic risks of molnupiravir on the basis of all available information and focus on the need for additional human genotoxicity data and follow-up in patients treated with molnupiravir and similar drugs. Such human data are especially relevant for antiviral NAs that have the potential of permanently modifying the genomes of treated patients and/or causing human teratogenicity or embryotoxicity. We conclude that the results of preclinical genotoxicity studies and phase 1 human clinical safety, tolerability, and pharmacokinetics are critical components of drug safety assessments and sentinels of unanticipated adverse health effects. We provide our rationale for performing more thorough genotoxicity testing prior to and within phase 1 clinical trials, including human PIG-A and error corrected next generation sequencing (duplex sequencing) studies in DNA and mitochondrial DNA of patients treated with antiviral NAs that induce genome error catastrophe via lethal mutagenesis.


Subject(s)
Antiviral Agents/adverse effects , COVID-19/drug therapy , Cytidine/analogs & derivatives , DNA Damage/drug effects , Hydroxylamines/adverse effects , Nucleosides/adverse effects , SARS-CoV-2/genetics , Amides/adverse effects , Amides/therapeutic use , Antiviral Agents/therapeutic use , Cytidine/adverse effects , Cytidine/therapeutic use , Deoxyuridine/adverse effects , Deoxyuridine/analogs & derivatives , Deoxyuridine/therapeutic use , Genome, Human/drug effects , Humans , Hydroxylamines/therapeutic use , Mutagenesis/drug effects , Nucleosides/therapeutic use , Pyrazines/adverse effects , Pyrazines/therapeutic use , Ribavirin/adverse effects , Ribavirin/therapeutic use , SARS-CoV-2/drug effects
12.
J Virol ; 96(3): e0082621, 2022 02 09.
Article in English | MEDLINE | ID: covidwho-1522911

ABSTRACT

Human adenovirus serotype 26 (Ad26) is used as a gene-based vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and HIV-1. However, its primary receptor portfolio remains controversial, potentially including sialic acid, coxsackie and adenovirus receptor (CAR), integrins, and CD46. We and others have shown that Ad26 can use CD46, but these observations were questioned on the basis of the inability to cocrystallize Ad26 fiber with CD46. Recent work demonstrated that Ad26 binds CD46 with its hexon protein rather than its fiber. We examined the functional consequences of Ad26 for infection in vitro and in vivo. Ectopic expression of human CD46 on Chinese hamster ovary cells increased Ad26 infection significantly. Deletion of the complement control protein domain CCP1 or CCP2 or the serine-threonine-proline (STP) region of CD46 reduced infection. Comparing wild-type and sialic acid-deficient CHO cells, we show that the usage of CD46 is independent of its sialylation status. Ad26 transduction was increased in CD46 transgenic mice after intramuscular (i.m.) injection but not after intranasal (i.n.) administration. Ad26 transduction was 10-fold lower than Ad5 transduction after intratumoral (i.t.) injection of CD46-expressing tumors. Ad26 transduction of liver was 1,000-fold lower than that ofAd5 after intravenous (i.v.) injection. These data demonstrate the use of CD46 by Ad26 in certain situations but also show that the receptor has little consequence by other routes of administration. Finally, i.v. injection of high doses of Ad26 into CD46 mice induced release of liver enzymes into the bloodstream and reduced white blood cell counts but did not induce thrombocytopenia. This suggests that Ad26 virions do not induce direct clotting side effects seen during coronavirus disease 2019 (COVID-19) vaccination with this serotype of adenovirus. IMPORTANCE The human species D Ad26 is being investigated as a low-seroprevalence vector for oncolytic virotherapy and gene-based vaccination against HIV-1 and SARS-CoV-2. However, there is debate in the literature about its tropism and receptor utilization, which directly influence its efficiency for certain applications. This work was aimed at determining which receptor(s) this virus uses for infection and its role in virus biology, vaccine efficacy, and, importantly, vaccine safety.


Subject(s)
Adenovirus Infections, Human/metabolism , Adenovirus Infections, Human/virology , Adenoviruses, Human/classification , Adenoviruses, Human/physiology , Coxsackie and Adenovirus Receptor-Like Membrane Protein/metabolism , Host-Pathogen Interactions , Membrane Cofactor Protein/metabolism , Adenoviruses, Human/ultrastructure , Animals , Biomarkers , Blood Cell Count , CHO Cells , Cell Line , Coxsackie and Adenovirus Receptor-Like Membrane Protein/chemistry , Cricetulus , Disease Models, Animal , Gene Expression , Humans , Membrane Cofactor Protein/chemistry , Membrane Cofactor Protein/genetics , Mice, Transgenic , Models, Biological , Models, Molecular , Mutagenesis , Protein Binding , Protein Conformation , Serogroup , Sialic Acids/metabolism , Sialic Acids/pharmacology , Structure-Activity Relationship
13.
J Infect Dis ; 224(3): 415-419, 2021 08 02.
Article in English | MEDLINE | ID: covidwho-1526165

ABSTRACT

Mutagenic ribonucleosides can act as broad-based antiviral agents. They are metabolized to the active ribonucleoside triphosphate form and concentrate in genomes of RNA viruses during viral replication. ß-d-N4-hydroxycytidine (NHC, initial metabolite of molnupiravir) is >100-fold more active than ribavirin or favipiravir against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with antiviral activity correlated to the level of mutagenesis in virion RNA. However, NHC also displays host mutational activity in an animal cell culture assay, consistent with RNA and DNA precursors sharing a common intermediate of a ribonucleoside diphosphate. These results indicate highly active mutagenic ribonucleosides may hold risk for the host.


Subject(s)
Antiviral Agents/pharmacology , Cytidine/analogs & derivatives , Mutagens/pharmacology , SARS-CoV-2/drug effects , Animals , Antiviral Agents/adverse effects , CHO Cells/drug effects , Cells, Cultured , Cricetulus , Cytidine/adverse effects , Cytidine/pharmacology , Dose-Response Relationship, Drug , Mutagenesis/drug effects , Mutagens/adverse effects , SARS-CoV-2/genetics , Virus Replication/drug effects
14.
Cell Host Microbe ; 29(12): 1788-1801.e6, 2021 12 08.
Article in English | MEDLINE | ID: covidwho-1509671

ABSTRACT

Previous work found that the co-occurring mutations R203K/G204R on the SARS-CoV-2 nucleocapsid (N) protein are increasing in frequency among emerging variants of concern or interest. Through a combination of in silico analyses, this study demonstrates that R203K/G204R are adaptive, while large-scale phylogenetic analyses indicate that R203K/G204R associate with the emergence of the high-transmissibility SARS-CoV-2 lineage B.1.1.7. Competition experiments suggest that the 203K/204R variants possess a replication advantage over the preceding R203/G204 variants, possibly related to ribonucleocapsid (RNP) assembly. Moreover, the 203K/204R virus shows increased infectivity in human lung cells and hamsters. Accordingly, we observe a positive association between increased COVID-19 severity and sample frequency of 203K/204R. Our work suggests that the 203K/204R mutations contribute to the increased transmission and virulence of select SARS-CoV-2 variants. In addition to mutations in the spike protein, mutations in the nucleocapsid protein are important for viral spreading during the pandemic.


Subject(s)
Amino Acid Substitution , COVID-19/pathology , Coronavirus Nucleocapsid Proteins/genetics , Genome, Viral , Mutation , SARS-CoV-2/genetics , Animals , COVID-19/epidemiology , COVID-19/virology , Cell Line , Coronavirus Nucleocapsid Proteins/chemistry , Coronavirus Nucleocapsid Proteins/metabolism , Cricetulus , Epithelial Cells/pathology , Epithelial Cells/virology , Gene Expression , Genetic Fitness , Humans , Models, Molecular , Mutagenesis , Phosphoproteins/chemistry , Phosphoproteins/genetics , Phosphoproteins/metabolism , Phylogeny , Protein Conformation , SARS-CoV-2/classification , SARS-CoV-2/growth & development , SARS-CoV-2/pathogenicity , Selection, Genetic , Severity of Illness Index , Virion/genetics , Virion/growth & development , Virion/pathogenicity , Virulence , Virus Replication
15.
Sci Rep ; 11(1): 21075, 2021 10 26.
Article in English | MEDLINE | ID: covidwho-1493212

ABSTRACT

Bats are potential natural reservoirs for emerging viruses, causing deadly human diseases, such as COVID-19, MERS, SARS, Nipah, Hendra, and Ebola infections. The fundamental mechanisms by which bats are considered "living bioreactors" for emerging viruses are not fully understood. Some studies suggest that tolerance to viruses is linked to suppressing antiviral immune and inflammatory responses due to DNA damage by energy generated to fly. Our study reveals that bats' gut bacteria could also be involved in the host and its microbiota's DNA damage. We performed screening of lactic acid bacteria and bacilli isolated from bats' feces for mutagenic and oxidative activity by lux-biosensors. The pro-mutagenic activity was determined when expression of recA increased with the appearance of double-strand breaks in the cell DNA, while an increase of katG expression in the presence of hydroxyl radicals indicated antioxidant activity. We identified that most of the isolated bacteria have pro-mutagenic and antioxidant properties at the same time. This study reveals new insights into bat gut microbiota's potential involvement in antiviral response and opens new frontiers in preventing emerging diseases originating from bats.


Subject(s)
Chiroptera/virology , Gastrointestinal Microbiome , Mutagens , Animals , Antioxidants/metabolism , Antiviral Agents , Bacillus , Bacterial Proteins/genetics , Biosensing Techniques , COVID-19 , DNA , DNA Damage , Disease Reservoirs/virology , Escherichia coli/metabolism , Feces , Immune System , Inflammation , Lactic Acid/metabolism , Mass Spectrometry , Mutagenesis , Oxidative Stress , Rec A Recombinases/metabolism , SARS-CoV-2 , Viruses/isolation & purification , Zoonoses/virology
16.
Mol Cell ; 81(21): 4467-4480.e7, 2021 11 04.
Article in English | MEDLINE | ID: covidwho-1473419

ABSTRACT

Viral RNA-dependent RNA polymerases (RdRps) are a target for broad-spectrum antiviral therapeutic agents. Recently, we demonstrated that incorporation of the T-1106 triphosphate, a pyrazine-carboxamide ribonucleotide, into nascent RNA increases pausing and backtracking by the poliovirus RdRp. Here, by monitoring enterovirus A-71 RdRp dynamics during RNA synthesis using magnetic tweezers, we identify the "backtracked" state as an intermediate used by the RdRp for copy-back RNA synthesis and homologous recombination. Cell-based assays and RNA sequencing (RNA-seq) experiments further demonstrate that the pyrazine-carboxamide ribonucleotide stimulates these processes during infection. These results suggest that pyrazine-carboxamide ribonucleotides do not induce lethal mutagenesis or chain termination but function by promoting template switching and formation of defective viral genomes. We conclude that RdRp-catalyzed intra- and intermolecular template switching can be induced by pyrazine-carboxamide ribonucleotides, defining an additional mechanistic class of antiviral ribonucleotides with potential for broad-spectrum activity.


Subject(s)
Pyrazines/chemistry , RNA Viruses/genetics , RNA, Viral/genetics , RNA-Dependent RNA Polymerase/genetics , Recombination, Genetic , Ribonucleotides/chemistry , Animals , Antiviral Agents , Catalysis , Cells, Cultured , Genetic Techniques , Genome , Genome, Viral , Homologous Recombination , Humans , Kinetics , Mice , Mice, Transgenic , Molecular Dynamics Simulation , Mutagenesis , Nucleotides/genetics , Protein Conformation , RNA/chemistry , RNA-Dependent RNA Polymerase/metabolism , RNA-Seq , Transgenes , Virulence
17.
Hum Mol Genet ; 30(R2): R274-R284, 2021 10 01.
Article in English | MEDLINE | ID: covidwho-1455301

ABSTRACT

The mouse is the pre-eminent model organism for studies of mammalian gene function and has provided an extraordinarily rich range of insights into basic genetic mechanisms and biological systems. Over several decades, the characterization of mouse mutants has illuminated the relationship between gene and phenotype, providing transformational insights into the genetic bases of disease. However, if we are to deliver the promise of genomic and precision medicine, we must develop a comprehensive catalogue of mammalian gene function that uncovers the dark genome and elucidates pleiotropy. Advances in large-scale mouse mutagenesis programmes allied to high-throughput mouse phenomics are now addressing this challenge and systematically revealing novel gene function and multi-morbidities. Alongside the development of these pan-genomic mutational resources, mouse genetics is employing a range of diversity resources to delineate gene-gene and gene-environment interactions and to explore genetic context. Critically, mouse genetics is a powerful tool for assessing the functional impact of human genetic variation and determining the causal relationship between variant and disease. Together these approaches provide unique opportunities to dissect in vivo mechanisms and systems to understand pathophysiology and disease. Moreover, the provision and utility of mouse models of disease has flourished and engages cumulatively at numerous points across the translational spectrum from basic mechanistic studies to pre-clinical studies, target discovery and therapeutic development.


Subject(s)
Genetic Association Studies , Genetic Predisposition to Disease , Genome , Genomics , Alleles , Animals , Disease Models, Animal , Drug Discovery , Gene Expression Regulation , Genetic Association Studies/methods , Genetic Engineering , Genome-Wide Association Study , Genomics/methods , High-Throughput Screening Assays , Humans , Mice , Mutagenesis , Mutation , Phenomics/methods , Phenotype , Precision Medicine , Signal Transduction
18.
Int J Mol Sci ; 22(19)2021 Sep 22.
Article in English | MEDLINE | ID: covidwho-1438627

ABSTRACT

The ongoing pandemic coronavirus (CoV) disease 2019 (COVID-19) by severe acute respiratory syndrome CoV-2 (SARS-CoV-2) has already caused substantial morbidity, mortality, and economic devastation. Reverse genetic approaches to generate recombinant viruses are a powerful tool to characterize and understand newly emerging viruses. To contribute to the global efforts for countermeasures to control the spread of SARS-CoV-2, we developed a passage-free SARS-CoV-2 clone based on a bacterial artificial chromosome (BAC). Moreover, using a Lambda-based Red recombination, we successfully generated different reporter and marker viruses, which replicated similar to a clinical isolate in a cell culture. Moreover, we designed a full-length reporter virus encoding an additional artificial open reading frame with wild-type-like replication features. The virus-encoded reporters were successfully applied to ease antiviral testing in cell culture models. Furthermore, we designed a new marker virus encoding 3xFLAG-tagged nucleocapsid that allows the detection of incoming viral particles and, in combination with bio-orthogonal labeling for the visualization of viral RNA synthesis via click chemistry, the spatiotemporal tracking of viral replication on the single-cell level. In summary, by applying BAC-based Red recombination, we developed a powerful, reliable, and convenient platform that will facilitate studies answering numerous questions concerning the biology of SARS-CoV-2.


Subject(s)
COVID-19/virology , Cloning, Molecular/methods , Genome, Viral , SARS-CoV-2/genetics , Animals , Chlorocebus aethiops , HEK293 Cells , Humans , Mutagenesis , Plasmids/genetics , Recombination, Genetic , Vero Cells
20.
Nature ; 592(7853): 277-282, 2021 04.
Article in English | MEDLINE | ID: covidwho-1387425

ABSTRACT

The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical for virus infection through the engagement of the human ACE2 protein1 and is a major antibody target. Here we show that chronic infection with SARS-CoV-2 leads to viral evolution and reduced sensitivity to neutralizing antibodies in an immunosuppressed individual treated with convalescent plasma, by generating whole-genome ultra-deep sequences for 23 time points that span 101 days and using in vitro techniques to characterize the mutations revealed by sequencing. There was little change in the overall structure of the viral population after two courses of remdesivir during the first 57 days. However, after convalescent plasma therapy, we observed large, dynamic shifts in the viral population, with the emergence of a dominant viral strain that contained a substitution (D796H) in the S2 subunit and a deletion (ΔH69/ΔV70) in the S1 N-terminal domain of the spike protein. As passively transferred serum antibodies diminished, viruses with the escape genotype were reduced in frequency, before returning during a final, unsuccessful course of convalescent plasma treatment. In vitro, the spike double mutant bearing both ΔH69/ΔV70 and D796H conferred modestly decreased sensitivity to convalescent plasma, while maintaining infectivity levels that were similar to the wild-type virus.The spike substitution mutant D796H appeared to be the main contributor to the decreased susceptibility to neutralizing antibodies, but this mutation resulted in an infectivity defect. The spike deletion mutant ΔH69/ΔV70 had a twofold higher level of infectivity than wild-type SARS-CoV-2, possibly compensating for the reduced infectivity of the D796H mutation. These data reveal strong selection on SARS-CoV-2 during convalescent plasma therapy, which is associated with the emergence of viral variants that show evidence of reduced susceptibility to neutralizing antibodies in immunosuppressed individuals.


Subject(s)
COVID-19/drug therapy , COVID-19/therapy , COVID-19/virology , Evolution, Molecular , Mutagenesis/drug effects , SARS-CoV-2/drug effects , SARS-CoV-2/genetics , Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/pharmacology , Adenosine Monophosphate/therapeutic use , Aged , Alanine/analogs & derivatives , Alanine/pharmacology , Alanine/therapeutic use , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/immunology , Chronic Disease , Genome, Viral/drug effects , Genome, Viral/genetics , High-Throughput Nucleotide Sequencing , Humans , Immune Evasion/drug effects , Immune Evasion/genetics , Immune Evasion/immunology , Immune Tolerance/drug effects , Immune Tolerance/immunology , Immunization, Passive , Male , Mutant Proteins/chemistry , Mutant Proteins/genetics , Mutant Proteins/immunology , Mutation , Phylogeny , SARS-CoV-2/immunology , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Time Factors , Viral Load/drug effects , Virus Shedding
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