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1.
Indian J Med Res ; 151(2 & 3): 200-209, 2020.
Article in English | MEDLINE | ID: covidwho-1726321

ABSTRACT

Background & objectives: Since December 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has globally affected 195 countries. In India, suspected cases were screened for SARS-CoV-2 as per the advisory of the Ministry of Health and Family Welfare. The objective of this study was to characterize SARS-CoV-2 sequences from three identified positive cases as on February 29, 2020. Methods: Throat swab/nasal swab specimens for a total of 881 suspected cases were screened by E gene and confirmed by RdRp (1), RdRp (2) and N gene real-time reverse transcription-polymerase chain reactions and next-generation sequencing. Phylogenetic analysis, molecular characterization and prediction of B- and T-cell epitopes for Indian SARS-CoV-2 sequences were undertaken. Results: Three cases with a travel history from Wuhan, China, were confirmed positive for SARS-CoV-2. Almost complete (29,851 nucleotides) genomes of case 1, case 3 and a fragmented genome for case 2 were obtained. The sequences of Indian SARS-CoV-2 though not identical showed high (~99.98%) identity with Wuhan seafood market pneumonia virus (accession number: NC 045512). Phylogenetic analysis showed that the Indian sequences belonged to different clusters. Predicted linear B-cell epitopes were found to be concentrated in the S1 domain of spike protein, and a conformational epitope was identified in the receptor-binding domain. The predicted T-cell epitopes showed broad human leucocyte antigen allele coverage of A and B supertypes predominant in the Indian population. Interpretation & conclusions: The two SARS-CoV-2 sequences obtained from India represent two different introductions into the country. The genetic heterogeneity is as noted globally. The identified B- and T-cell epitopes may be considered suitable for future experiments towards the design of vaccines and diagnostics. Continuous monitoring and analysis of the sequences of new cases from India and the other affected countries would be vital to understand the genetic evolution and rates of substitution of the SARS-CoV-2.


Subject(s)
Betacoronavirus/genetics , Genome, Viral , COVID-19 , Coronavirus Infections , Epitopes, B-Lymphocyte/genetics , Epitopes, T-Lymphocyte/genetics , Humans , India , Models, Molecular , Pandemics , Phylogeny , Pneumonia, Viral , Protein Structure, Tertiary , RNA, Viral/genetics , Reverse Transcriptase Polymerase Chain Reaction , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/genetics
2.
PLoS One ; 17(2): e0262591, 2022.
Article in English | MEDLINE | ID: covidwho-1666759

ABSTRACT

SARS-CoV-2 Nucleocapsid (N) is the most abundant viral protein expressed in host samples and is an important antigen for diagnosis. N is a 45 kDa protein that does not present disulfide bonds. Intending to avoid non-specific binding of SARS-CoV-2 N to antibodies from patients who previously had different coronaviruses, a 35 kDa fragment of N was expressed without a conserved motif in E. coli as inclusion bodies (N122-419-IB). Culture media and IB washing conditions were chosen to obtain N122-419-IB with high yield (370 mg/L bacterial culture) and protein purity (90%). High pressure solubilizes protein aggregates by weakening hydrophobic and ionic interactions and alkaline pH promotes solubilization by electrostatic repulsion. The association of pH 9.0 and 2.4 kbar promoted efficient solubilization of N122-419-IB without loss of native-like tertiary structure that N presents in IB. N122-419 was refolded with a yield of 85% (326 mg/L culture) and 95% purity. The refolding process takes only 2 hours and the protein is ready for use after pH adjustment, avoiding the necessity of dialysis or purification. Antibody binding of COVID-19-positive patients sera to N122-419 was confirmed by Western blotting. ELISA using N122-419 is effective in distinguishing between sera presenting antibodies against SARS-CoV-2 from those who do not. To the best of our knowledge, the proposed condition for IB solubilization is one of the mildest described. It is possible that the refolding process can be extended to a wide range of proteins with high yields and purity, even those that are sensible to very alkaline pH.


Subject(s)
Antibodies, Viral/blood , Antigens, Viral/chemistry , COVID-19/blood , COVID-19/diagnosis , Coronavirus Nucleocapsid Proteins/chemistry , Immunoglobulin G/blood , Inclusion Bodies/chemistry , Protein Refolding , SARS-CoV-2/immunology , Antibodies, Viral/immunology , Antigens, Viral/immunology , COVID-19/virology , Coronavirus Nucleocapsid Proteins/immunology , Enzyme-Linked Immunosorbent Assay/methods , Escherichia coli/genetics , Escherichia coli/metabolism , Humans , Hydrogen-Ion Concentration , Hydrostatic Pressure , Immunoglobulin G/immunology , Phosphoproteins/chemistry , Phosphoproteins/immunology , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/immunology , Solubility
3.
Int Immunol ; 33(10): 541-545, 2021 09 25.
Article in English | MEDLINE | ID: covidwho-1575598

ABSTRACT

The spatial organization of chromatin is known to be highly dynamic in response to environmental stress. However, it remains unknown how chromatin dynamics contributes to or modulates the pathogenesis of immune and infectious diseases. Influenza virus is a single-stranded RNA virus, and transcription and replication of the virus genome occur in the nucleus. Since viral infection is generally associated with virus-driven hijack of the host cellular machineries, influenza virus may utilize and/or affect the nuclear system. In this review article, we focus on recent studies showing that the three-dimensional structure of chromatin changes with influenza virus infection, which affects the pathology of infection. Also, we discuss studies showing the roles of epigenetics in influenza virus infection. Understanding how this affects immune responses may lead to novel strategies to combat immune and infectious diseases.


Subject(s)
Chromatin/metabolism , Histone-Lysine N-Methyltransferase/metabolism , Homeodomain Proteins/metabolism , Influenza A virus/immunology , Influenza, Human/pathology , Cell Cycle Proteins/metabolism , Chromosomal Proteins, Non-Histone/metabolism , Histone Code/physiology , Histone-Lysine N-Methyltransferase/genetics , Host-Pathogen Interactions/immunology , Humans , Neoplasms/pathology , Protein Structure, Tertiary , Severity of Illness Index , Virus Replication/physiology
4.
Sci Rep ; 11(1): 23561, 2021 12 07.
Article in English | MEDLINE | ID: covidwho-1559302

ABSTRACT

N-glycosylation plays an important role in the structure and function of membrane and secreted proteins. The spike protein on the surface of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes COVID-19, is heavily glycosylated and the major target for developing vaccines, therapeutic drugs and diagnostic tests. The first major SARS-CoV-2 variant carries a D614G substitution in the spike (S-D614G) that has been associated with altered conformation, enhanced ACE2 binding, and increased infectivity and transmission. In this report, we used mass spectrometry techniques to characterize and compare the N-glycosylation of the wild type (S-614D) or variant (S-614G) SARS-CoV-2 spike glycoproteins prepared under identical conditions. The data showed that half of the N-glycosylation sequons changed their distribution of glycans in the S-614G variant. The S-614G variant showed a decrease in the relative abundance of complex-type glycans (up to 45%) and an increase in oligomannose glycans (up to 33%) on all altered sequons. These changes led to a reduction in the overall complexity of the total N-glycosylation profile. All the glycosylation sites with altered patterns were in the spike head while the glycosylation of three sites in the stalk remained unchanged between S-614G and S-614D proteins.


Subject(s)
Glycopeptides/analysis , Mass Spectrometry/methods , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/metabolism , COVID-19/pathology , COVID-19/virology , Chromatography, High Pressure Liquid , Glycosylation , Humans , Mutation , Protein Binding , Protein Structure, Tertiary , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/chemistry
5.
J Autoimmun ; 126: 102779, 2022 01.
Article in English | MEDLINE | ID: covidwho-1561067

ABSTRACT

Severe Acute Respiratory Coronavirus (SARS-CoV-2) has been emerging in the form of different variants since its first emergence in early December 2019. A new Variant of Concern (VOC) named the Omicron variant (B.1.1.529) was reported recently. This variant has a large number of mutations in the S protein. To date, there exists a limited information on the Omicron variant. Here we present the analyses of mutation distribution, the evolutionary relationship of Omicron with previous variants, and probable structural impact of mutations on antibody binding. Our analyses show the presence of 46 high prevalence mutations specific to Omicron. Twenty-three of these are localized within the spike (S) protein and the rest localized to the other 3 structural proteins of the virus, the envelope (E), membrane (M), and nucleocapsid (N). Phylogenetic analysis showed that the Omicron is closely related to the Gamma (P.1) variant. The structural analyses showed that several mutations are localized to the region of the S protein that is the major target of antibodies, suggesting that the mutations in the Omicron variant may affect the binding affinities of antibodies to the S protein.


Subject(s)
Antibodies, Viral/immunology , COVID-19/virology , SARS-CoV-2/genetics , Binding Sites , COVID-19/immunology , Humans , Mutation , Phylogeny , Protein Structure, Tertiary , Spike Glycoprotein, Coronavirus/genetics
6.
PLoS One ; 16(12): e0260360, 2021.
Article in English | MEDLINE | ID: covidwho-1546953

ABSTRACT

Recent emergence of SARS-CoV-2 and associated COVID-19 pandemic have posed a great challenge for the scientific community. In this study, we performed bioinformatic analyses on SARS-CoV-2 protein sequences, trying to unravel potential molecular similarities between this newly emerged pathogen with non-coronavirus ssRNA viruses. Comparing the proteins of SARS-CoV-2 with non-coronavirus positive and negative strand ssRNA viruses revealed multiple sequence similarities between SARS-CoV-2 and non-coronaviruses, including similarities between RNA-dependent RNA-polymerases and helicases (two highly-conserved proteins). We also observed similarities between SARS-CoV-2 surface (i.e. spike) protein with paramyxovirus fusion proteins. This similarity was restricted to a segment of spike protein S2 subunit which is involved in cell fusion. We next analyzed spike proteins from SARS-CoV-2 "variants of concern" (VOCs) and "variants of interests" (VOIs) and found that some of these variants show considerably higher spike-fusion similarity with paramyxoviruses. The 'spike-fusion' similarity was also observed for some pathogenic coronaviruses other than SARS-CoV-2. Epitope analysis using experimentally verified data deposited in Immune Epitope Database (IEDB) revealed that several B cell epitopes as well as T cell and MHC binding epitopes map within the spike-fusion similarity region. These data indicate that there might be a degree of convergent evolution between SARS-CoV-2 and paramyxovirus surface proteins which could be of pathogenic and immunological importance.


Subject(s)
SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Viral Fusion Proteins/genetics , Epitopes/genetics , Humans , Paramyxoviridae/genetics , Phylogeny , Protein Structure, Tertiary , Spike Glycoprotein, Coronavirus/chemistry
7.
Emerg Microbes Infect ; 11(1): 147-157, 2022 Dec.
Article in English | MEDLINE | ID: covidwho-1537457

ABSTRACT

The repeated emergence of highly pathogenic human coronaviruses as well as their evolving variants highlight the need to develop potent and broad-spectrum antiviral therapeutics and vaccines. By screening monoclonal antibodies (mAbs) isolated from COVID-19-convalescent patients, we found one mAb, 2-36, with cross-neutralizing activity against SARS-CoV. We solved the cryo-EM structure of 2-36 in complex with SARS-CoV-2 or SARS-CoV spike, revealing a highly conserved epitope in the receptor-binding domain (RBD). Antibody 2-36 neutralized not only all current circulating SARS-CoV-2 variants and SARS-COV, but also a panel of bat and pangolin sarbecoviruses that can use human angiotensin-converting enzyme 2 (ACE2) as a receptor. We selected 2-36-escape viruses in vitro and confirmed that K378 T in SARS-CoV-2 RBD led to viral resistance. Taken together, 2-36 represents a strategic reserve drug candidate for the prevention and treatment of possible diseases caused by pre-emergent SARS-related coronaviruses. Its epitope defines a promising target for the development of a pan-sarbecovirus vaccine.


Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , SARS-CoV-2/immunology , Animals , Broadly Neutralizing Antibodies/immunology , COVID-19 , Chlorocebus aethiops , Cryoelectron Microscopy , Epitopes/immunology , HEK293 Cells , Humans , Neutralization Tests , Protein Interaction Domains and Motifs , Protein Structure, Tertiary , Vero Cells
8.
J Med Virol ; 93(12): 6551-6556, 2021 Dec.
Article in English | MEDLINE | ID: covidwho-1530181

ABSTRACT

Lineage B.1.617+, also known as G/452R.V3 and now denoted by WHO with the Greek letters δ and κ, is a recently described SARS-CoV-2 variant under investigation first identified in October 2020 in India. As of May 2021, three sublineages labeled as B.1.617.1 (κ), B.1.617.2 (δ), and B.1.617.3 have been already identified, and their potential impact on the current pandemic is being studied. This variant has 13 amino acid changes, three in its spike protein, which are currently of particular concern: E484Q, L452R, and P681R. Here, we report a major effect of the mutations characterizing this lineage, represented by a marked alteration of the surface electrostatic potential (EP) of the receptor-binding domain (RBD) of the spike protein. Enhanced RBD-EP is particularly noticeable in the B.1.617.2 (δ) sublineage, which shows multiple replacements of neutral or negatively charged amino acids with positively charged amino acids. We here hypothesize that this EP change can favor the interaction between the B.1.617+ RBD and the negatively charged ACE2, thus conferring a potential increase in the virus transmission.


Subject(s)
COVID-19/virology , SARS-CoV-2/pathogenicity , COVID-19/transmission , Humans , Mutation , Protein Structure, Tertiary , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Static Electricity
9.
Eur J Med Chem ; 227: 113966, 2022 Jan 05.
Article in English | MEDLINE | ID: covidwho-1487705

ABSTRACT

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is unprecedented in human history. As a major structural protein, nucleocapsid protein (NPro) is critical to the replication of SARS-CoV-2. In this work, 17 NPro-targeting phenanthridine derivatives were rationally designed and synthesized, based on the crystal structure of NPro. Most of these compounds can interact with SARS-CoV-2 NPro tightly and inhibit the replication of SARS-CoV-2 in vitro. Compounds 12 and 16 exhibited the most potent anti-viral activities with 50% effective concentration values of 3.69 and 2.18 µM, respectively. Furthermore, site-directed mutagenesis of NPro and Surface Plasmon Resonance (SPR) assays revealed that 12 and 16 target N-terminal domain (NTD) of NPro by binding to Tyr109. This work found two potent anti-SARS-CoV-2 bioactive compounds and also indicated that SARS-CoV-2 NPro-NTD can be a target for new anti-virus agents.


Subject(s)
Antiviral Agents/chemistry , Coronavirus Nucleocapsid Proteins/antagonists & inhibitors , Phenanthridines/chemistry , SARS-CoV-2/metabolism , Animals , Antiviral Agents/metabolism , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , Binding Sites , COVID-19/drug therapy , COVID-19/virology , Cell Survival/drug effects , Chlorocebus aethiops , Coronavirus Nucleocapsid Proteins/metabolism , Drug Design , Humans , Kinetics , Molecular Docking Simulation , Phenanthridines/metabolism , Phenanthridines/pharmacology , Phenanthridines/therapeutic use , Phosphoproteins/antagonists & inhibitors , Phosphoproteins/metabolism , Protein Binding , Protein Structure, Tertiary , SARS-CoV-2/isolation & purification , SARS-CoV-2/physiology , Vero Cells
10.
Biochem Pharmacol ; 193: 114800, 2021 11.
Article in English | MEDLINE | ID: covidwho-1471892

ABSTRACT

Remdesivir (GS-5734, Veklury®) has remained the only antiviral drug formally approved by the US FDA for the treatment of Covid-19 (SARS-CoV-2 infection). Its key structural features are the fact that it is a C-nucleoside (adenosine) analogue, contains a 1'-cyano function, and could be considered as a ProTide based on the presence of a phosphoramidate group. Its antiviral spectrum and activity in animal models have been well established and so has been its molecular mode of action as a delayed chain terminator of the viral RdRp (RNA-dependent RNA polymerase). Its clinical efficacy has been evaluated, but needs to be optimized with regard to timing, dosage and duration of treatment, and route of administration. Safety, toxicity and pharmacokinetics need to be further addressed, and so are its potential combinations with other drugs such as corticosteroids (i.e. dexamethasone) and ribavirin.


Subject(s)
Adenosine Monophosphate/analogs & derivatives , Alanine/analogs & derivatives , Antiviral Agents/administration & dosage , COVID-19/drug therapy , SARS-CoV-2/drug effects , Adenosine Monophosphate/administration & dosage , Adenosine Monophosphate/chemistry , Adenosine Monophosphate/metabolism , Alanine/administration & dosage , Alanine/chemistry , Alanine/metabolism , Animals , Antiviral Agents/chemistry , Antiviral Agents/metabolism , COVID-19/metabolism , Drug Therapy, Combination , Humans , Protein Structure, Tertiary , SARS-CoV-2/chemistry , SARS-CoV-2/metabolism
11.
Brief Bioinform ; 23(1)2022 01 17.
Article in English | MEDLINE | ID: covidwho-1434365

ABSTRACT

MOTIVATION: The Estimation of Model Accuracy problem is a cornerstone problem in the field of Bioinformatics. As of CASP14, there are 79 global QA methods, and a minority of 39 residue-level QA methods with very few of them working on protein complexes. Here, we introduce ZoomQA, a novel, single-model method for assessing the accuracy of a tertiary protein structure/complex prediction at residue level, which have many applications such as drug discovery. ZoomQA differs from others by considering the change in chemical and physical features of a fragment structure (a portion of a protein within a radius $r$ of the target amino acid) as the radius of contact increases. Fourteen physical and chemical properties of amino acids are used to build a comprehensive representation of every residue within a protein and grade their placement within the protein as a whole. Moreover, we have shown the potential of ZoomQA to identify problematic regions of the SARS-CoV-2 protein complex. RESULTS: We benchmark ZoomQA on CASP14, and it outperforms other state-of-the-art local QA methods and rivals state of the art QA methods in global prediction metrics. Our experiment shows the efficacy of these new features and shows that our method is able to match the performance of other state-of-the-art methods without the use of homology searching against databases or PSSM matrices. AVAILABILITY: http://zoomQA.renzhitech.com.


Subject(s)
COVID-19 , Caspases/chemistry , Machine Learning , Models, Molecular , SARS-CoV-2/chemistry , Viral Proteins/chemistry , Humans , Protein Structure, Quaternary , Protein Structure, Tertiary , Sequence Analysis, Protein
12.
Sci Rep ; 11(1): 18851, 2021 09 22.
Article in English | MEDLINE | ID: covidwho-1434149

ABSTRACT

In this pandemic SARS-CoV-2 crisis, any attempt to contain and eliminate the virus will also stop its spread and consequently decrease the risk of severe illness and death. While ozone treatment has been suggested as an effective disinfection process, no precise mechanism of action has been previously reported. This study aimed to further investigate the effect of ozone treatment on SARS-CoV-2. Therefore, virus collected from nasopharyngeal and oropharyngeal swab and sputum samples from symptomatic patients was exposed to ozone for different exposure times. The virus morphology and structure were monitored and analyzed through Atomic Force Microscopy (AFM), Transmission Electron Microscopy (TEM), Atomic Absorption Spectroscopy (AAS), and ATR-FTIR. The obtained results showed that ozone treatment not only unsettles the virus morphology but also alters the virus proteins' structure and conformation through amino acid disturbance and Zn ion release from the virus non-structural proteins. These results could provide a clearer pathway for virus elimination and therapeutics preparation.


Subject(s)
COVID-19/drug therapy , Ozone/pharmacology , SARS-CoV-2/chemistry , SARS-CoV-2/drug effects , Coronavirus Papain-Like Proteases/chemistry , Coronavirus Papain-Like Proteases/metabolism , Coronavirus RNA-Dependent RNA Polymerase/chemistry , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Humans , Microscopy, Electron, Transmission , Protein Structure, Secondary/drug effects , Protein Structure, Tertiary/drug effects , SARS-CoV-2/ultrastructure , Time Factors , Viral Envelope/chemistry , Viral Envelope/drug effects , Viral Regulatory and Accessory Proteins/chemistry , Viral Regulatory and Accessory Proteins/metabolism , Zinc/chemistry , Zinc/metabolism
13.
J Biol Chem ; 297(4): 101208, 2021 10.
Article in English | MEDLINE | ID: covidwho-1415532

ABSTRACT

Emergence of new severe acute respiratory syndrome coronavirus 2 variants has raised concerns related to the effectiveness of vaccines and antibody therapeutics developed against the unmutated wildtype virus. Here, we examined the effect of the 12 most commonly occurring mutations in the receptor-binding domain of the spike protein on its expression, stability, activity, and antibody escape potential. Stability was measured using thermal denaturation, and the activity and antibody escape potential were measured using isothermal titration calorimetry in terms of binding to the human angiotensin-converting enzyme 2 and to neutralizing human antibody CC12.1, respectively. Our results show that mutants differ in their expression levels. Of the eight best-expressed mutants, two (N501Y and K417T/E484K/N501Y) showed stronger affinity to angiotensin-converting enzyme 2 compared with the wildtype, whereas four (Y453F, S477N, T478I, and S494P) had similar affinity and two (K417N and E484K) had weaker affinity than the wildtype. Compared with the wildtype, four mutants (K417N, Y453F, N501Y, and K417T/E484K/N501Y) had weaker affinity for the CC12.1 antibody, whereas two (S477N and S494P) had similar affinity, and two (T478I and E484K) had stronger affinity than the wildtype. Mutants also differ in their thermal stability, with the two least stable mutants showing reduced expression. Taken together, these results indicate that multiple factors contribute toward the natural selection of variants, and all these factors need to be considered to understand the evolution of the virus. In addition, since not all variants can escape a given neutralizing antibody, antibodies to treat new variants can be chosen based on the specific mutations in that variant.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Neutralizing/immunology , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/genetics , Antigen-Antibody Reactions , COVID-19/pathology , COVID-19/virology , HEK293 Cells , Humans , Polymorphism, Single Nucleotide , Protein Binding , Protein Domains/genetics , Protein Stability , Protein Structure, Tertiary , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Transition Temperature
14.
Nat Struct Mol Biol ; 28(9): 755-761, 2021 09.
Article in English | MEDLINE | ID: covidwho-1406396

ABSTRACT

Bradykinin and kallidin are endogenous kinin peptide hormones that belong to the kallikrein-kinin system and are essential to the regulation of blood pressure, inflammation, coagulation and pain control. Des-Arg10-kallidin, the carboxy-terminal des-Arg metabolite of kallidin, and bradykinin selectively activate two G protein-coupled receptors, type 1 and type 2 bradykinin receptors (B1R and B2R), respectively. The hyperactivation of bradykinin receptors, termed 'bradykinin storm', is associated with pulmonary edema in COVID-19 patients, suggesting that bradykinin receptors are important targets for COVID-19 intervention. Here we report two G protein-coupled complex structures of human B1R and B2R bound to des-Arg10-kallidin and bradykinin, respectively. Combined with functional analysis, our structures reveal the mechanism of ligand selectivity and specific activation of the bradykinin receptor. These findings also provide a framework for guiding drug design targeting bradykinin receptors for the treatment of inflammation, cardiovascular disorders and COVID-19.


Subject(s)
Bradykinin/metabolism , COVID-19/pathology , Kallidin/metabolism , Receptors, Bradykinin/metabolism , Cryoelectron Microscopy , Enzyme Activation/physiology , Humans , Protein Structure, Tertiary , Pulmonary Edema/pathology , Pulmonary Edema/virology , SARS-CoV-2
16.
J Biol Chem ; 297(4): 101127, 2021 10.
Article in English | MEDLINE | ID: covidwho-1373108

ABSTRACT

The SARS-CoV-2 spike is the primary target of virus-neutralizing antibodies and critical to the development of effective vaccines against COVID-19. Here, we demonstrate that the prefusion-stabilized two-proline "S2P" spike-widely employed for laboratory work and clinical studies-unfolds when stored at 4 °C, physiological pH, as observed by electron microscopy (EM) and differential scanning calorimetry, but that its trimeric, native-like conformation can be reacquired by low pH treatment. When stored for approximately 1 week, this unfolding does not significantly alter antigenic characteristics; however, longer storage diminishes antibody binding, and month-old spike elicits virtually no neutralization in mice despite inducing high ELISA-binding titers. Cryo-EM structures reveal the folded fraction of spike to decrease with aging; however, its structure remains largely similar, although with varying mobility of the receptor-binding domain. Thus, the SARS-CoV-2 spike is susceptible to unfolding, which affects immunogenicity, highlighting the need to monitor its integrity.


Subject(s)
SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/immunology , Animals , Antibodies, Neutralizing/immunology , Antigen-Antibody Reactions , COVID-19/pathology , COVID-19/virology , Calorimetry, Differential Scanning , Cryoelectron Microscopy , Female , Humans , Hydrogen-Ion Concentration , Mice , Mice, Inbred BALB C , Protein Structure, Tertiary , Protein Unfolding , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Time Factors
17.
Brief Bioinform ; 22(2): 1476-1498, 2021 03 22.
Article in English | MEDLINE | ID: covidwho-1352121

ABSTRACT

Coronavirus disease 2019 (COVID-19), an infectious disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been declared a global pandemic by the World Health Organization, and the situation worsens daily, associated with acute increases in case fatality rates. The main protease (Mpro) enzyme produced by SARS-CoV-2 was recently demonstrated to be responsible for not only viral reproduction but also impeding host immune responses. The element selenium (Se) plays a vital role in immune functions, both directly and indirectly. Thus, we hypothesised that Se-containing heterocyclic compounds might curb the activity of SARS-CoV-2 Mpro. We performed a molecular docking analysis and found that several of the selected selenocompounds showed potential binding affinities for SARS-CoV-2 Mpro, especially ethaselen (49), which exhibited a docking score of -6.7 kcal/mol compared with the -6.5 kcal/mol score for GC376 (positive control). Drug-likeness calculations suggested that these compounds are biologically active and possess the characteristics of ideal drug candidates. Based on the binding affinity and drug-likeness results, we selected the 16 most effective selenocompounds as potential anti-COVID-19 drug candidates. We also validated the structural integrity and stability of the drug candidate through molecular dynamics simulation. Using further in vitro and in vivo experiments, we believe that the targeted compound identified in this study (ethaselen) could pave the way for the development of prospective drugs to combat SARS-CoV-2 infections and trigger specific host immune responses.


Subject(s)
Antiviral Agents/pharmacology , Coronavirus 3C Proteases/antagonists & inhibitors , Heterocyclic Compounds/pharmacology , Protease Inhibitors/pharmacology , Selenium/analysis , Antiviral Agents/chemistry , Computational Biology , Computer Simulation , Coronavirus 3C Proteases/chemistry , Heterocyclic Compounds/chemistry , Humans , Ligands , Models, Molecular , Molecular Docking Simulation , Molecular Dynamics Simulation , Protease Inhibitors/chemistry , Protein Structure, Tertiary , Pyrrolidines/chemistry , Pyrrolidines/pharmacology , Reproducibility of Results , Sulfonic Acids
18.
Bioengineered ; 12(1): 2836-2850, 2021 12.
Article in English | MEDLINE | ID: covidwho-1297360

ABSTRACT

Angiotensin I-converting enzyme 2 (ACE2), type II transmembrane serine protease 2 and 4 (TMPRSS2 and TMPRSS4) are important receptors for SARS-CoV-2 infection. In this study, the full-length tree shrewACE2 gene was cloned and sequenced, and its biological information was analyzed. The expression levels of ACE2, TMPRSS2 and TMPRSS4 in various tissues or organs of the tree shrew were detected. The results showed that the full-length ACE2 gene in tree shrews was 2,786 bp, and its CDS was 2,418 bp, encoding 805 amino acids. Phylogenetic analysis based on the CDS of ACE2 revealed that tree shrews were more similar to rabbits (85.93%) and humans (85.47%) but far from mice (82.81%) and rats (82.58%). In silico analysis according to the binding site of SARS-CoV-2 with the ACE2 receptor of different species predicted that tree shrews had potential SARS-CoV-2 infection possibility, which was similar to that of rabbits, cats and dogs but significantly higher than that of mice and rats. In addition, various tissues or organs of tree shrews expressed ACE2, TMPRSS2 and TMPRSS4. Among them, the kidney most highly expressed ACE2, followed by the lung and liver. The esophagus, lung, liver, intestine and kidney had relatively high expression levels of TMPRSS2 and TMPRSS4. In general, we reported for the first time the expression of ACE2, TMPRSS2 and TMPRSS4 in various tissues or organs in tree shrews. Our results revealed that tree shrews could be used as a potential animal model to study the mechanism underlying SARS-CoV-2 infection.


Subject(s)
Angiotensin-Converting Enzyme 2/genetics , COVID-19/etiology , Membrane Proteins/genetics , SARS-CoV-2 , Serine Endopeptidases/genetics , Tupaiidae/genetics , Tupaiidae/metabolism , Amino Acid Sequence , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/metabolism , Animals , Bioengineering , COVID-19/enzymology , COVID-19/genetics , Computational Biology , Disease Models, Animal , Female , Humans , Male , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Models, Molecular , Phylogeny , Protein Structure, Tertiary , Sequence Homology, Amino Acid , Serine Endopeptidases/chemistry , Serine Endopeptidases/metabolism , Structural Homology, Protein , Tissue Distribution , Tupaiidae/virology
19.
Front Immunol ; 12: 612807, 2021.
Article in English | MEDLINE | ID: covidwho-1282384

ABSTRACT

Since being identified as a key receptor for SARS-CoV-2, Angiotensin converting enzyme 2 (ACE2) has been studied as one of the potential targets for the development of preventative and/or treatment options. Tissue expression of ACE2 and the amino acids interacting with the spike protein of SARS-CoV-2 have been mapped. Furthermore, the recombinant soluble extracellular domain of ACE2 is already in phase 2 trials as a treatment for SARS-CoV-2 infection. Most studies have continued to focus on the ACE2 extracellular domain, which is known to play key roles in the renin angiotensin system and in amino acid uptake. However, few also found ACE2 to have an immune-modulatory function and its intracellular tail may be one of the signaling molecules in regulating cellular activation. The implication of its immune-modulatory role in preventing the cytokine-storm, observed in severe COVID-19 disease outcomes requires further investigation. This review focuses on the regulated proteolytic cleavage of ACE2 upon binding to inducer(s), such as the spike protein of SARS-CoV, the potential of cleaved ACE2 intracellular subdomain in regulating cellular function, and the ACE2's immune-modulatory function. This knowledge is critical for targeting ACE2 levels for developing prophylactic treatment or preventative measures in SARS-CoV infections.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , COVID-19/pathology , Cell Membrane/metabolism , SARS-CoV-2/pathogenicity , Angiotensin-Converting Enzyme 2/chemistry , COVID-19/metabolism , COVID-19/virology , Humans , Immunomodulation , Protein Structure, Tertiary , Proteolysis , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism
20.
Emerg Microbes Infect ; 10(1): 1293-1299, 2021 Dec.
Article in English | MEDLINE | ID: covidwho-1268057

ABSTRACT

The SARS-CoV-2 B.1.1.7 lineage is highly infectious and as of April 2021 accounted for 92% of COVID-19 cases in Europe and 59% of COVID-19 cases in the U.S. It is defined by the N501Y mutation in the receptor-binding domain (RBD) of the Spike (S) protein, and a few other mutations. These include two mutations in the N terminal domain (NTD) of the S protein, HV69-70del and Y144del (also known as Y145del due to the presence of tyrosine at both positions). We recently identified several emerging SARS-CoV-2 variants of concerns, characterized by Membrane (M) protein mutations, including I82T and V70L. We now identify a sub-lineage of B.1.1.7 that emerged through sequential acquisitions of M:V70L in November 2020 followed by a novel S:D178H mutation first observed in early February 2021. The percentage of B.1.1.7 isolates in the US that belong to this sub-lineage increased from 0.15% in February 2021 to 1.8% in April 2021. To date, this sub-lineage appears to be U.S.-specific with reported cases in 31 states, including Hawaii. As of April 2021, it constituted 36.8% of all B.1.1.7 isolates in Washington. Phylogenetic analysis and transmission inference with Nextstrain suggest this sub-lineage likely originated in either California or Washington. Structural analysis revealed that the S:D178H mutation is in the NTD of the S protein and close to two other signature mutations of B.1.1.7, HV69-70del and Y144del. It is surface exposed and may alter NTD tertiary configuration or accessibility, and thus has the potential to affect neutralization by NTD directed antibodies.


Subject(s)
Mutation , SARS-CoV-2/classification , Spike Glycoprotein, Coronavirus/genetics , Viral Matrix Proteins/genetics , Whole Genome Sequencing/methods , Binding Sites , Humans , Models, Molecular , Phylogeny , Protein Domains , Protein Structure, Tertiary , SARS-CoV-2/genetics , Sequence Analysis, RNA , Spike Glycoprotein, Coronavirus/chemistry , United States
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