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1.
Cell reports methods ; 1(4), 2021.
Article in English | EuropePMC | ID: covidwho-1801491

ABSTRACT

Summary Multimodal advances in single-cell sequencing have enabled the simultaneous quantification of cell surface protein expression alongside unbiased transcriptional profiling. Here, we present LinQ-View, a toolkit designed for multimodal single-cell data visualization and analysis. LinQ-View integrates transcriptional and cell surface protein expression profiling data to reveal more accurate cell heterogeneity and proposes a quantitative metric for cluster purity assessment. Through comparison with existing multimodal methods on multiple public CITE-seq datasets, we demonstrate that LinQ-View efficiently generates accurate cell clusters, especially in CITE-seq data with routine numbers of surface protein features, by preventing variations in a single surface protein feature from affecting results. Finally, we utilized this method to integrate single-cell transcriptional and protein expression data from SARS-CoV-2-infected patients, revealing antigen-specific B cell subsets after infection. Our results suggest LinQ-View could be helpful for multimodal analysis and purity assessment of CITE-seq datasets that target specific cell populations (e.g., B cells). Graphical Highlights • LinQ-View integrates mRNA and protein expression data to reveal cell heterogeneity• LinQ-View prevents single dominant ADT features from affecting clustering• LinQ-View presents a quantitative purity metric for CITE-seq data• LinQ-View is specialized in handling CITE-seq data with fewer ADT features Motivation Multimodal single-cell sequencing enables multiple aspects for characterizing the dynamics of cell states and developmental processes. Properly integrating information from multiple modalities is a crucial step for interpreting cell heterogeneity. Here, we present LinQ-View, a computational workflow that provides an effective solution for integrating multiple modalities of CITE-seq data for downstream interpretation. LinQ-View balances information from multiple modalities to achieve accurate clustering results and is specialized in handling CITE-seq data with routine numbers of surface protein features. Li et al. present LinQ-View, a computational workflow that provides an effective solution for integrating multiple modalities of CITE-seq data and quantitative assessment of cluster purity. LinQ-View could be helpful for multimodal analysis and purity assessment of CITE-seq datasets that target specific cell populations.

2.
EuropePMC; 2022.
Preprint in English | EuropePMC | ID: ppcovidwho-334317

ABSTRACT

The Papain-like protease (PLpro) is a domain of a multi-functional, non-structural protein 3 of coronaviruses. PLpro cleaves viral polyproteins and posttranslational conjugates with poly-ubiquitin and protective ISG15, composed of two ubiquitin-like (UBL) domains. Across coronaviruses, PLpro showed divergent selectivity for recognition and cleavage of posttranslational conjugates despite sequence conservation. We show that SARS-CoV-2 PLpro binds human ISG15 and K48-linked di-ubiquitin (K48-Ub 2 ) with nanomolar affinity and detect alternate weaker-binding modes. Crystal structures of untethered PLpro complexes with ISG15 and K48-Ub 2 combined with solution NMR and cross-linking mass spectrometry revealed how the two domains of ISG15 or K48-Ub 2 are differently utilized in interactions with PLpro. Analysis of protein interface energetics uncovered differential binding stabilities of the two UBL/Ub domains. We emphasize how substrate recognition can be tuned to cleave specifically ISG15 or K48-Ub 2 modifications while retaining capacity to cleave mono-Ub conjugates. These results highlight alternative druggable surfaces that would inhibit PLpro function.

3.
EuropePMC; 2021.
Preprint in English | EuropePMC | ID: ppcovidwho-315247

ABSTRACT

Single cell RNA sequencing is a powerful tool for characterizing the molecular and cellular heterogeneity of immune cells during their development and activation. Multimodal advances in single cell sequencing have enabled the simultaneous quantification of cell surface protein expression alongside unbiased transcriptional profiling. Here we present LinQ-View, a toolkit designed for multimodal single cell data visualization and analysis that links transcriptional and cell surface protein expression profiling data. Further, we propose a quantitative metric for cluster purity of CITE-seq data, enabling effective determination of clustering algorithms and their parameters, and finally demonstrate the utility of our toolkit through seamless integration with standard single cell analysis workflows on several public datasets. Through comparison to existing multimodal methods, we demonstrate that LinQ-View generates more accurate cell clusters and is highly efficient, even with massive datasets. LinQ-View is specialized in handling CITE-seq data with routine numbers of surface protein features (e.g. less than 50), by preventing variations in a single surface protein feature from affecting results. Finally, we utilized this method to integrate single cell transcriptional and protein expression data from COVID-19-infected patients and influenza-immunized subjects, revealing antigen-specific B cell subsets and previously unknown T cell subsets post-infection and vaccination.

4.
EuropePMC; 2020.
Preprint in English | EuropePMC | ID: ppcovidwho-314309

ABSTRACT

The COVID-19 disease caused by the SARS-CoV-2 Coronavirus has become a pandemic health crisis. An attractive target for antiviral inhibitors is the main protease 3CL M pro due to its essential role in processing the polyproteins translated from viral RNA. Here we report the room temperature X-ray structure of unliganded SARS-CoV-2 3CL M pro , revealing the resting structure of the active site and the conformation of the catalytic site cavity. Comparison with previously reported low-temperature ligand-free and inhibitor-bound structures suggest that the room temperature structure may provide more relevant information at physiological temperatures for aiding in molecular docking studies.

5.
EuropePMC; 2021.
Preprint in English | EuropePMC | ID: ppcovidwho-309780

ABSTRACT

Dissecting the evolution of memory B cells (MBCs) against SARS-CoV-2 is critical for understanding antibody recall upon secondary exposure. Here, we utilized single-cell sequencing to profile SARS-CoV-2-reactive B cell subsets in 42 COVID-19 patients. We isolated thousands of B cells in multiple distinct subsets specific to the SARS-CoV-2 spike, endemic coronavirus (HCoV) spikes, nucleoprotein (NP), and open reading frame 8 (ORF8). Spike-specific cells were enriched in the memory compartment of acutely infected and convalescent patients 1.5–5 months post-infection. With severe acute infection, we identified substantial populations of endemic HCoV-reactive antibody-secreting cells with highly mutated variable genes, indicative of preexisting immunity. Finally, MBCs exhibited maturation to NP and ORF8 over time relative to spike, especially in older patients. Monoclonal antibodies against these targets were non-neutralizing and non-protective in vivo. These findings reveal considerable antibody adaptation to non-neutralizing antigens during infection, emphasizing the importance of vaccination for inducing neutralizing spike-specific MBCs.Trial Registration Number: This clinical trial was registered at ClinicalTrials.gov with identifier NCT04340050, and clinical information for patients included in the study is detailed in Table S1–S3.Funding: This project was funded in part by the National Institute of Allergy and Infectious Disease (NIAID);National Institutes of Health (NIH) grant numbers U19AI082724 (P.C.W.), U19AI109946 (P.C.W.), U19AI057266 (P.C.W.), the NIAID Centers of Excellence for Influenza Research and Surveillance (CEIRS) grant numbers HHSN272201400005C(P.C.W.). N.W.A. was supported by the Multi-disciplinary Training program in Cancer Research (MTCR) - NIH T32 CA009594. A.J. and R.P.J were supported by federal funds from the NIAID, NIH, and Department of Health and Human Services under Contract HHSN272201700060C. F.K and F.A. were funded by the NIAID CEIRS contractHHSN272201400008C, Collaborative Influenza Vaccine Innovation Centers (CIVIC) contract 75N93019C00051 and the generous support of the JPB foundation, the Open Philanthropy Project (#2020-215611) and other philanthropic donations. Y.K. and P.H.were funded by the Research Program on Emerging and Re-emerging Infectious Disease grant (JP19fk0108113) and the Japan Program for Infectious Diseases Research and Infrastructure (JP20fk0108272) from the Japan Agency for Medical Research and Development (AMED), NIAID CEIRS contract HHSN272201400008C, and CIVIC contract 75N93019C00051. D.F., C.N, Y.D., and P.D.H, were supported by NIAID contracts HHSN272201700060C and 75N93019C00062. M.S.D. and E.S.W. were supported by NIH grants R01 AI157155 and F30 AI152327, respectively.Conflict of Interest: Several antibodies generated from this work are being used by Now Diagnostics in Springdale, AR for the development of a diagnostic test. M.S.D. is a consultant for Inbios, Vir Biotechnology, NGM Biopharmaceuticals, and Carnival Corporation, and on the Scientific Advisory Boards of Moderna and Immunome. The Diamond laboratory has received funding support in sponsored research agreements from Moderna, Vir Biotechnology, and Emergent BioSolutions.Ethical Approval: All studies were performed with the approval of the University of Chicago institutional review board IRB20-0523 and University of Chicago, University of Wisconsin-Madison, and Washington University in St. Louis institutional biosafety committees. Informed consent was obtained after the research applications and possible consequences of the studies were disclosed to study subjects.

6.
IUCrJ ; 7(Pt 6)2020 Sep 21.
Article in English | MEDLINE | ID: covidwho-1546124

ABSTRACT

The emergence of the novel coronavirus SARS-CoV-2 has resulted in a worldwide pandemic not seen in generations. Creating treatments and vaccines to battle COVID-19, the disease caused by the virus, is of paramount importance in order to stop its spread and save lives. The viral main protease, 3CL Mpro, is indispensable for the replication of SARS-CoV-2 and is therefore an important target for the design of specific protease inhibitors. Detailed knowledge of the structure and function of 3CL Mpro is crucial to guide structure-aided and computational drug-design efforts. Here, the oxidation and reactivity of the cysteine residues of the protease are reported using room-temperature X-ray crystallography, revealing that the catalytic Cys145 can be trapped in the peroxysulfenic acid oxidation state at physiological pH, while the other surface cysteines remain reduced. Only Cys145 and Cys156 react with the alkylating agent N-ethylmaleimide. It is suggested that the zwitterionic Cys145-His45 catalytic dyad is the reactive species that initiates catalysis, rather than Cys145-to-His41 proton transfer via the general acid-base mechanism upon substrate binding. The structures also provide insight into the design of improved 3CL Mpro inhibitors.

7.
IUCrJ ; 7(Pt 5): 814-824, 2020 Sep 01.
Article in English | MEDLINE | ID: covidwho-1546123

ABSTRACT

Among 15 nonstructural proteins (Nsps), the newly emerging Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) encodes a large, multidomain Nsp3. One of its units is the ADP-ribose phosphatase domain (ADRP; also known as the macrodomain, MacroD), which is believed to interfere with the host immune response. Such a function appears to be linked to the ability of the protein to remove ADP-ribose from ADP-ribosylated proteins and RNA, yet the precise role and molecular targets of the enzyme remain unknown. Here, five high-resolution (1.07-2.01 Å) crystal structures corresponding to the apo form of the protein and its complexes with 2-(N-morpholino)ethanesulfonic acid (MES), AMP and ADP-ribose have been determined. The protein is shown to undergo conformational changes to adapt to the ligand in the manner previously observed in close homologues from other viruses. A conserved water molecule is also identified that may participate in hydrolysis. This work builds foundations for future structure-based research on ADRP, including the search for potential antiviral therapeutics.

8.
EuropePMC; 2021.
Preprint in English | EuropePMC | ID: ppcovidwho-291462

ABSTRACT

Direct-acting antivirals for the treatment of COVID-19, which is caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), are needed to complement vaccination efforts. The papain-like protease (PLpro) of SARS-CoV-2 is essential for viral proliferation. In addition, PLpro dysregulates the host immune response by cleaving ubiquitin and interferon-stimulated gene 15 protein (ISG15) from host proteins. As a result, PLpro is a promising target for inhibition by small-molecule therapeutics. Here we have designed a series of covalent inhibitors by introducing a peptidomimetic linker and reactive electrophilic “warheads” onto analogs of the noncovalent PLpro inhibitor GRL0617. We show that the most promising PLpro inhibitor is potent and selective, with activity in cell-based antiviral assays rivaling that of the RNA-dependent RNA polymerase inhibitor remdesivir. An X-ray crystal structure of the most promising lead compound bound covalently to PLpro establishes the molecular basis for protease inhibition and selectivity against structurally similar human deubiquitinases. These findings present an opportunity for further development of potent and selective covalent PLpro inhibitors.

9.
Methods Enzymol ; 661: 407-431, 2021.
Article in English | MEDLINE | ID: covidwho-1439812

ABSTRACT

We present a Chemistry and Structure Screen Integrated Efficiently (CASSIE) approach (named for Greek prophet Cassandra) to design inhibitors for cancer biology and pathogenesis. CASSIE provides an effective path to target master keys to control the repair-replication interface for cancer cells and SARS CoV-2 pathogenesis as exemplified here by specific targeting of Poly(ADP-ribose) glycohydrolase (PARG) and ADP-ribose glycohydrolase ARH3 macrodomains plus SARS CoV-2 nonstructural protein 3 (Nsp3) Macrodomain 1 (Mac1) and Nsp15 nuclease. As opposed to the classical massive effort employing libraries with large numbers of compounds against single proteins, we make inhibitor design for multiple targets efficient. Our compact, chemically diverse, 5000 compound Goldilocks (GL) library has an intermediate number of compounds sized between fragments and drugs with predicted favorable ADME (absorption, distribution, metabolism, and excretion) and toxicological profiles. Amalgamating our core GL library with an approved drug (AD) library, we employ a combined GLAD library virtual screen, enabling an effective and efficient design cycle of ranked computer docking, top hit biophysical and cell validations, and defined bound structures using human proteins or their avatars. As new drug design is increasingly pathway directed as well as molecular and mechanism based, our CASSIE approach facilitates testing multiple related targets by efficiently turning a set of interacting drug discovery problems into a tractable medicinal chemistry engineering problem of optimizing affinity and ADME properties based upon early co-crystal structures. Optimization efforts are made efficient by a computationally-focused iterative chemistry and structure screen. Thus, we herein describe and apply CASSIE to define prototypic, specific inhibitors for PARG vs distinct inhibitors for the related macrodomains of ARH3 and SARS CoV-2 Nsp3 plus the SARS CoV-2 Nsp15 RNA nuclease.


Subject(s)
COVID-19 , Nucleic Acids , Severe Acute Respiratory Syndrome , DNA Repair , Humans , Molecular Docking Simulation , SARS-CoV-2
10.
Biophys J ; 120(15): 3152-3165, 2021 08 03.
Article in English | MEDLINE | ID: covidwho-1385180

ABSTRACT

The replication transcription complex (RTC) from the virus SARS-CoV-2 is responsible for recognizing and processing RNA for two principal purposes. The RTC copies viral RNA for propagation into new virus and for ribosomal transcription of viral proteins. To accomplish these activities, the RTC mechanism must also conform to a large number of imperatives, including RNA over DNA base recognition, basepairing, distinguishing viral and host RNA, production of mRNA that conforms to host ribosome conventions, interfacing with error checking machinery, and evading host immune responses. In addition, the RTC will discontinuously transcribe specific sections of viral RNA to amplify certain proteins over others. Central to SARS-CoV-2 viability, the RTC is therefore dynamic and sophisticated. We have conducted a systematic structural investigation of three components that make up the RTC: Nsp7, Nsp8, and Nsp12 (also known as RNA-dependent RNA polymerase). We have solved high-resolution crystal structures of the Nsp7/8 complex, providing insight into the interaction between the proteins. We have used small-angle x-ray and neutron solution scattering (SAXS and SANS) on each component individually as pairs and higher-order complexes and with and without RNA. Using size exclusion chromatography and multiangle light scattering-coupled SAXS, we defined which combination of components forms transient or stable complexes. We used contrast-matching to mask specific complex-forming components to test whether components change conformation upon complexation. Altogether, we find that individual Nsp7, Nsp8, and Nsp12 structures vary based on whether other proteins in their complex are present. Combining our crystal structure, atomic coordinates reported elsewhere, SAXS, SANS, and other biophysical techniques, we provide greater insight into the RTC assembly, mechanism, and potential avenues for disruption of the complex and its functions.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , Models, Molecular , RNA, Viral/genetics , Scattering, Small Angle , Viral Nonstructural Proteins , Virus Replication , X-Ray Diffraction
11.
Cell Reports Methods ; : 100056, 2021.
Article in English | ScienceDirect | ID: covidwho-1322060

ABSTRACT

Summary Multimodal advances in single-cell sequencing have enabled the simultaneous quantification of cell surface protein expression alongside unbiased transcriptional profiling. Here, we present LinQ-View, a toolkit designed for multimodal single-cell data visualization and analysis. LinQ-View integrates transcriptional and cell surface protein expression profiling data to reveal more accurate cell heterogeneity and proposes a quantitative metric for cluster purity assessment. Through comparison with existing multimodal methods on multiple public CITE-seq datasets, we demonstrate that LinQ-View efficiently generates accurate cell clusters, especially in CITE-seq data with routine numbers of surface protein features, by preventing variations in a single surface protein feature from affecting results. Finally, we utilized this method to integrate single-cell transcriptional and protein expression data from SARS-CoV-2-infected patients, revealing antigen-specific B cell subsets after infection. Our results suggest LinQ-View could be helpful for multimodal analysis and purity assessment of CITE-seq datasets that target specific cell populations (e.g., B cells).

12.
Science ; 373(6557): 931-936, 2021 08 20.
Article in English | MEDLINE | ID: covidwho-1319369

ABSTRACT

There is an urgent need for antiviral agents that treat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We screened a library of 1900 clinically safe drugs against OC43, a human beta coronavirus that causes the common cold, and evaluated the top hits against SARS-CoV-2. Twenty drugs significantly inhibited replication of both viruses in cultured human cells. Eight of these drugs inhibited the activity of the SARS-CoV-2 main protease, 3CLpro, with the most potent being masitinib, an orally bioavailable tyrosine kinase inhibitor. X-ray crystallography and biochemistry show that masitinib acts as a competitive inhibitor of 3CLpro. Mice infected with SARS-CoV-2 and then treated with masitinib showed >200-fold reduction in viral titers in the lungs and nose, as well as reduced lung inflammation. Masitinib was also effective in vitro against all tested variants of concern (B.1.1.7, B.1.351, and P.1).


Subject(s)
Antiviral Agents/pharmacology , COVID-19/drug therapy , Coronavirus 3C Proteases/antagonists & inhibitors , Coronavirus OC43, Human/drug effects , Cysteine Proteinase Inhibitors/pharmacology , SARS-CoV-2/drug effects , Thiazoles/pharmacology , A549 Cells , Animals , Antiviral Agents/chemistry , Antiviral Agents/metabolism , Antiviral Agents/therapeutic use , Benzamides , COVID-19/virology , Catalytic Domain , Coronavirus 3C Proteases/chemistry , Coronavirus 3C Proteases/metabolism , Coronavirus OC43, Human/physiology , Cysteine Proteinase Inhibitors/chemistry , Cysteine Proteinase Inhibitors/metabolism , HEK293 Cells , Humans , Inhibitory Concentration 50 , Mice , Mice, Transgenic , Microbial Sensitivity Tests , Piperidines , Pyridines , SARS-CoV-2/enzymology , SARS-CoV-2/physiology , Thiazoles/chemistry , Thiazoles/metabolism , Thiazoles/therapeutic use , Viral Load/drug effects , Virus Replication/drug effects
13.
Biophys J ; 120(15): 3152-3165, 2021 08 03.
Article in English | MEDLINE | ID: covidwho-1316407

ABSTRACT

The replication transcription complex (RTC) from the virus SARS-CoV-2 is responsible for recognizing and processing RNA for two principal purposes. The RTC copies viral RNA for propagation into new virus and for ribosomal transcription of viral proteins. To accomplish these activities, the RTC mechanism must also conform to a large number of imperatives, including RNA over DNA base recognition, basepairing, distinguishing viral and host RNA, production of mRNA that conforms to host ribosome conventions, interfacing with error checking machinery, and evading host immune responses. In addition, the RTC will discontinuously transcribe specific sections of viral RNA to amplify certain proteins over others. Central to SARS-CoV-2 viability, the RTC is therefore dynamic and sophisticated. We have conducted a systematic structural investigation of three components that make up the RTC: Nsp7, Nsp8, and Nsp12 (also known as RNA-dependent RNA polymerase). We have solved high-resolution crystal structures of the Nsp7/8 complex, providing insight into the interaction between the proteins. We have used small-angle x-ray and neutron solution scattering (SAXS and SANS) on each component individually as pairs and higher-order complexes and with and without RNA. Using size exclusion chromatography and multiangle light scattering-coupled SAXS, we defined which combination of components forms transient or stable complexes. We used contrast-matching to mask specific complex-forming components to test whether components change conformation upon complexation. Altogether, we find that individual Nsp7, Nsp8, and Nsp12 structures vary based on whether other proteins in their complex are present. Combining our crystal structure, atomic coordinates reported elsewhere, SAXS, SANS, and other biophysical techniques, we provide greater insight into the RTC assembly, mechanism, and potential avenues for disruption of the complex and its functions.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , Models, Molecular , RNA, Viral/genetics , Scattering, Small Angle , Viral Nonstructural Proteins , Virus Replication , X-Ray Diffraction
14.
Immunity ; 54(6): 1290-1303.e7, 2021 06 08.
Article in English | MEDLINE | ID: covidwho-1237724

ABSTRACT

Dissecting the evolution of memory B cells (MBCs) against SARS-CoV-2 is critical for understanding antibody recall upon secondary exposure. Here, we used single-cell sequencing to profile SARS-CoV-2-reactive B cells in 38 COVID-19 patients. Using oligo-tagged antigen baits, we isolated B cells specific to the SARS-CoV-2 spike, nucleoprotein (NP), open reading frame 8 (ORF8), and endemic human coronavirus (HCoV) spike proteins. SARS-CoV-2 spike-specific cells were enriched in the memory compartment of acutely infected and convalescent patients several months post symptom onset. With severe acute infection, substantial populations of endemic HCoV-reactive antibody-secreting cells were identified and possessed highly mutated variable genes, signifying preexisting immunity. Finally, MBCs exhibited pronounced maturation to NP and ORF8 over time, especially in older patients. Monoclonal antibodies against these targets were non-neutralizing and non-protective in vivo. These findings reveal antibody adaptation to non-neutralizing intracellular antigens during infection, emphasizing the importance of vaccination for inducing neutralizing spike-specific MBCs.


Subject(s)
Antibodies, Viral/immunology , Antibody Formation/immunology , B-Lymphocytes/immunology , COVID-19/immunology , Host-Pathogen Interactions/immunology , Immunodominant Epitopes/immunology , SARS-CoV-2/immunology , Antibodies, Neutralizing/immunology , Antibody Formation/genetics , B-Lymphocytes/metabolism , Computational Biology/methods , Cross Reactions/immunology , Epitope Mapping , Female , Gene Expression Profiling , High-Throughput Nucleotide Sequencing , Host-Pathogen Interactions/genetics , Humans , Immunodominant Epitopes/genetics , Immunologic Memory , Male , Neutralization Tests , Single-Cell Analysis/methods , Spike Glycoprotein, Coronavirus/immunology , Transcriptome
15.
Proc Natl Acad Sci U S A ; 118(21)2021 05 25.
Article in English | MEDLINE | ID: covidwho-1223143

ABSTRACT

The genome of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) coronavirus has a capping modification at the 5'-untranslated region (UTR) to prevent its degradation by host nucleases. These modifications are performed by the Nsp10/14 and Nsp10/16 heterodimers using S-adenosylmethionine as the methyl donor. Nsp10/16 heterodimer is responsible for the methylation at the ribose 2'-O position of the first nucleotide. To investigate the conformational changes of the complex during 2'-O methyltransferase activity, we used a fixed-target serial synchrotron crystallography method at room temperature. We determined crystal structures of Nsp10/16 with substrates and products that revealed the states before and after methylation, occurring within the crystals during the experiments. Here we report the crystal structure of Nsp10/16 in complex with Cap-1 analog (m7GpppAm2'-O). Inhibition of Nsp16 activity may reduce viral proliferation, making this protein an attractive drug target.


Subject(s)
RNA Caps/metabolism , RNA, Messenger/metabolism , RNA, Viral/metabolism , SARS-CoV-2/chemistry , Crystallography , Methylation , Methyltransferases/chemistry , Methyltransferases/metabolism , Multiprotein Complexes/chemistry , Multiprotein Complexes/metabolism , RNA Cap Analogs/chemistry , RNA Cap Analogs/metabolism , RNA Caps/chemistry , RNA, Messenger/chemistry , RNA, Viral/chemistry , S-Adenosylhomocysteine/chemistry , S-Adenosylhomocysteine/metabolism , S-Adenosylmethionine/chemistry , S-Adenosylmethionine/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Synchrotrons , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/metabolism , Viral Regulatory and Accessory Proteins/chemistry , Viral Regulatory and Accessory Proteins/metabolism
16.
MAbs ; 13(1): 1905978, 2021.
Article in English | MEDLINE | ID: covidwho-1177234

ABSTRACT

Monoclonal antibodies (mAbs) are the basis of treatments and diagnostics for pathogens and other biological phenomena. We conducted a structural characterization of mAbs against the N-terminal domain of nucleocapsid protein (NPNTD) from SARS-CoV-2 using small-angle X-ray scattering and transmission electron microscopy. Our solution-based results distinguished the mAbs' flexibility and how this flexibility affects the assembly of multiple mAbs on an antigen. By pairing two mAbs that bind different epitopes on the NPNTD, we show that flexible mAbs form a closed sandwich-like complex. With rigid mAbs, a juxtaposition of the antigen-binding fragments is prevented, enforcing a linear arrangement of the mAb pair, which facilitates further mAb polymerization. In a modified sandwich enzyme-linked immunosorbent assay, we show that rigid mAb-pairings with linear polymerization led to increased NPNTD detection sensitivity. These enhancements can expedite the development of more sensitive and selective antigen-detecting point-of-care lateral flow devices, which are critical for early diagnosis and epidemiological studies of SARS-CoV-2 and other pathogens.


Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Viral/immunology , COVID-19/immunology , Epitopes/immunology , Nucleocapsid Proteins/immunology , SARS-CoV-2/enzymology , Animals , Humans
17.
Commun Biol ; 4(1): 193, 2021 02 09.
Article in English | MEDLINE | ID: covidwho-1075259

ABSTRACT

SARS-CoV-2 Nsp15 is a uridine-specific endoribonuclease with C-terminal catalytic domain belonging to the EndoU family that is highly conserved in coronaviruses. As endoribonuclease activity seems to be responsible for the interference with the innate immune response, Nsp15 emerges as an attractive target for therapeutic intervention. Here we report the first structures with bound nucleotides and show how the enzyme specifically recognizes uridine moiety. In addition to a uridine site we present evidence for a second base binding site that can accommodate any base. The structure with a transition state analog, uridine vanadate, confirms interactions key to catalytic mechanisms. In the presence of manganese ions, the enzyme cleaves unpaired RNAs. This acquired knowledge was instrumental in identifying Tipiracil, an FDA approved drug that is used in the treatment of colorectal cancer, as a potential anti-COVID-19 drug. Using crystallography, biochemical, and whole-cell assays, we demonstrate that Tipiracil inhibits SARS-CoV-2 Nsp15 by interacting with the uridine binding pocket in the enzyme's active site. Our findings provide new insights for the development of uracil scaffold-based drugs.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/drug therapy , COVID-19/virology , Endoribonucleases/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Pyrrolidines/pharmacology , SARS-CoV-2/drug effects , SARS-CoV-2/enzymology , Thymine/pharmacology , Viral Nonstructural Proteins/antagonists & inhibitors , A549 Cells , Antiviral Agents/chemistry , Antiviral Agents/pharmacokinetics , Catalytic Domain , Crystallography, X-Ray , Endoribonucleases/chemistry , Endoribonucleases/metabolism , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacokinetics , Humans , Ligands , Models, Molecular , Protein Conformation , Pyrrolidines/chemistry , Pyrrolidines/pharmacokinetics , Thymine/chemistry , Thymine/pharmacokinetics , Uridine/metabolism , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/metabolism
18.
Nat Commun ; 12(1): 743, 2021 02 02.
Article in English | MEDLINE | ID: covidwho-1061105

ABSTRACT

The pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) continues to expand. Papain-like protease (PLpro) is one of two SARS-CoV-2 proteases potentially targetable with antivirals. PLpro is an attractive target because it plays an essential role in cleavage and maturation of viral polyproteins, assembly of the replicase-transcriptase complex, and disruption of host responses. We report a substantive body of structural, biochemical, and virus replication studies that identify several inhibitors of the SARS-CoV-2 enzyme. We determined the high resolution structure of wild-type PLpro, the active site C111S mutant, and their complexes with inhibitors. This collection of structures details inhibitors recognition and interactions providing fundamental molecular and mechanistic insight into PLpro. All compounds inhibit the peptidase activity of PLpro in vitro, some block SARS-CoV-2 replication in cell culture assays. These findings will accelerate structure-based drug design efforts targeting PLpro to identify high-affinity inhibitors of clinical value.


Subject(s)
Papain/metabolism , Peptide Hydrolases/metabolism , SARS-CoV-2/drug effects , SARS-CoV-2/enzymology , Antiviral Agents/pharmacology , Humans , Mutation , Polyproteins/metabolism , Substrate Specificity , Virus Replication/drug effects
19.
mBio ; 12(1)2021 01 19.
Article in English | MEDLINE | ID: covidwho-1038406

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently causing a global pandemic. The antigen specificity of the antibody response mounted against this novel virus is not understood in detail. Here, we report that subjects with a more severe SARS-CoV-2 infection exhibit a larger antibody response against the spike and nucleocapsid protein and epitope spreading to subdominant viral antigens, such as open reading frame 8 and nonstructural proteins. Subjects with a greater antibody response mounted a larger memory B cell response against the spike, but not the nucleocapsid protein. Additionally, we revealed that antibodies against the spike are still capable of binding the D614G spike mutant and cross-react with the SARS-CoV-1 receptor binding domain. Together, this study reveals that subjects with a more severe SARS-CoV-2 infection exhibit a greater overall antibody response to the spike and nucleocapsid protein and a larger memory B cell response against the spike.IMPORTANCE With the ongoing pandemic, it is critical to understand how natural immunity against SARS-CoV-2 and COVID-19 develops. We have identified that subjects with more severe COVID-19 disease mount a more robust and neutralizing antibody response against SARS-CoV-2 spike protein. Subjects who mounted a larger response against the spike also mounted antibody responses against other viral antigens, including the nucleocapsid protein and ORF8. Additionally, this study reveals that subjects with more severe disease mount a larger memory B cell response against the spike. These data suggest that subjects with more severe COVID-19 disease are likely better protected from reinfection with SARS-CoV-2.


Subject(s)
COVID-19/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Adult , Antibodies, Neutralizing/immunology , Antibodies, Viral/blood , Antibodies, Viral/immunology , Antigens, Viral/immunology , B-Lymphocytes/immunology , COVID-19/blood , COVID-19/virology , Coronavirus Nucleocapsid Proteins/immunology , Cross Reactions , Epitopes/immunology , Female , Humans , Immunity, Humoral/immunology , Male , Middle Aged , Phosphoproteins/immunology
20.
Nat Commun ; 11(1): 3202, 2020 06 24.
Article in English | MEDLINE | ID: covidwho-981316

ABSTRACT

The COVID-19 disease caused by the SARS-CoV-2 coronavirus has become a pandemic health crisis. An attractive target for antiviral inhibitors is the main protease 3CL Mpro due to its essential role in processing the polyproteins translated from viral RNA. Here we report the room temperature X-ray structure of unliganded SARS-CoV-2 3CL Mpro, revealing the ligand-free structure of the active site and the conformation of the catalytic site cavity at near-physiological temperature. Comparison with previously reported low-temperature ligand-free and inhibitor-bound structures suggest that the room temperature structure may provide more relevant information at physiological temperatures for aiding in molecular docking studies.


Subject(s)
Betacoronavirus/enzymology , Cysteine Endopeptidases/chemistry , Viral Nonstructural Proteins/chemistry , Catalytic Domain , Coronavirus 3C Proteases , Crystallography, X-Ray , Cysteine Endopeptidases/metabolism , Cysteine Proteinase Inhibitors/metabolism , Ligands , Models, Molecular , Molecular Dynamics Simulation , Protein Binding , Protein Conformation , Protein Domains , Protein Structure, Secondary , SARS-CoV-2 , Temperature , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Nonstructural Proteins/metabolism
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