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1.
Commun Biol ; 4(1): 228, 2021 02 15.
Article in English | MEDLINE | ID: covidwho-1085408

ABSTRACT

SARS-CoV-2 has been mutating since it was first sequenced in early January 2020. Here, we analyze 45,494 complete SARS-CoV-2 geneome sequences in the world to understand their mutations. Among them, 12,754 sequences are from the United States. Our analysis suggests the presence of four substrains and eleven top mutations in the United States. These eleven top mutations belong to 3 disconnected groups. The first and second groups consisting of 5 and 8 concurrent mutations are prevailing, while the other group with three concurrent mutations gradually fades out. Moreover, we reveal that female immune systems are more active than those of males in responding to SARS-CoV-2 infections. One of the top mutations, 27964C > T-(S24L) on ORF8, has an unusually strong gender dependence. Based on the analysis of all mutations on the spike protein, we uncover that two of four SASR-CoV-2 substrains in the United States become potentially more infectious.


Subject(s)
/virology , Mutation/genetics , /genetics , 5' Untranslated Regions/genetics , Amino Acid Sequence , /metabolism , Evolution, Molecular , Female , Humans , Male , Models, Molecular , Nucleocapsid/metabolism , Open Reading Frames/genetics , Polymorphism, Single Nucleotide/genetics , Protein Binding , Protein Domains , Protein Folding , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Thermodynamics , United States
2.
Commun Biol ; 4(1): 225, 2021 02 12.
Article in English | MEDLINE | ID: covidwho-1082956

ABSTRACT

Serodiagnosis of SARS-CoV-2 infection is impeded by immunological cross-reactivity among the human coronaviruses (HCoVs): SARS-CoV-2, SARS-CoV-1, MERS-CoV, OC43, 229E, HKU1, and NL63. Here we report the identification of humoral immune responses to SARS-CoV-2 peptides that may enable discrimination between exposure to SARS-CoV-2 and other HCoVs. We used a high-density peptide microarray and plasma samples collected at two time points from 50 subjects with SARS-CoV-2 infection confirmed by qPCR, samples collected in 2004-2005 from 11 subjects with IgG antibodies to SARS-CoV-1, 11 subjects with IgG antibodies to other seasonal human coronaviruses (HCoV), and 10 healthy human subjects. Through statistical modeling with linear regression and multidimensional scaling we identified specific peptides that were reassembled to identify 29 linear SARS-CoV-2 epitopes that were immunoreactive with plasma from individuals who had asymptomatic, mild or severe SARS-CoV-2 infections. Larger studies will be required to determine whether these peptides may be useful in serodiagnostics.


Subject(s)
/immunology , Peptide Mapping , Peptides/immunology , /physiology , Amino Acid Sequence , Animals , Chiroptera , Epitopes/immunology , Humans , Immunoglobulin G/metabolism , Peptides/chemistry , Proteome/metabolism
3.
Commun Biol ; 4(1): 197, 2021 02 12.
Article in English | MEDLINE | ID: covidwho-1082259

ABSTRACT

In light of the recent accumulated knowledge on SARS-CoV-2 and its mode of human cells invasion, the binding of viral spike glycoprotein to human Angiotensin Converting Enzyme 2 (hACE2) receptor plays a central role in cell entry. We designed a series of peptides mimicking the N-terminal helix of hACE2 protein which contains most of the contacting residues at the binding site, exhibiting a high helical folding propensity in aqueous solution. Our best peptide-mimics are able to block SARS-CoV-2 human pulmonary cell infection with an inhibitory concentration (IC50) in the nanomolar range upon binding to the virus spike protein with high affinity. These first-in-class blocking peptide mimics represent powerful tools that might be used in prophylactic and therapeutic approaches to fight the coronavirus disease 2019 (COVID-19).


Subject(s)
/chemistry , Peptides/pharmacology , /physiology , Amino Acid Sequence , Cell Line , Circular Dichroism , Humans , Peptides/chemical synthesis , Peptides/chemistry , Peptides/metabolism , Protein Binding/drug effects , Protein Structure, Secondary , Spike Glycoprotein, Coronavirus/metabolism , Virus Replication/drug effects
4.
Nat Commun ; 12(1): 972, 2021 02 09.
Article in English | MEDLINE | ID: covidwho-1075220

ABSTRACT

Among the many questions unanswered for the COVID-19 pandemic are the origin of SARS-CoV-2 and the potential role of intermediate animal host(s) in the early animal-to-human transmission. The discovery of RaTG13 bat coronavirus in China suggested a high probability of a bat origin. Here we report molecular and serological evidence of SARS-CoV-2 related coronaviruses (SC2r-CoVs) actively circulating in bats in Southeast Asia. Whole genome sequences were obtained from five independent bats (Rhinolophus acuminatus) in a Thai cave yielding a single isolate (named RacCS203) which is most related to the RmYN02 isolate found in Rhinolophus malayanus in Yunnan, China. SARS-CoV-2 neutralizing antibodies were also detected in bats of the same colony and in a pangolin at a wildlife checkpoint in Southern Thailand. Antisera raised against the receptor binding domain (RBD) of RmYN02 was able to cross-neutralize SARS-CoV-2 despite the fact that the RBD of RacCS203 or RmYN02 failed to bind ACE2. Although the origin of the virus remains unresolved, our study extended the geographic distribution of genetically diverse SC2r-CoVs from Japan and China to Thailand over a 4800-km range. Cross-border surveillance is urgently needed to find the immediate progenitor virus of SARS-CoV-2.


Subject(s)
Chiroptera/virology , /physiology , Amino Acid Sequence , Animals , Antibodies, Neutralizing/blood , Asia, Southeastern , Chiroptera/blood , Geography , Neutralization Tests , Phylogeny , Protein Domains , Receptors, Cell Surface/chemistry , Receptors, Cell Surface/metabolism
5.
Int J Biol Macromol ; 168: 272-278, 2021 Jan 31.
Article in English | MEDLINE | ID: covidwho-1065145

ABSTRACT

SARS-CoV-2is the causative agent for the ongoing COVID19 pandemic, and this virus belongs to the Coronaviridae family. The nsp14 protein of SARS-CoV-2 houses a 3' to 5' exoribonuclease activity responsible for removing mismatches that arise during genome duplication. A homology model of nsp10-nsp14 complex was used to carry out in silico screening to identify molecules among natural products, or FDA approved drugs that can potentially inhibit the activity of nsp14. This exercise showed that ritonavir might bind to the exoribonuclease active site of the nsp14 protein. A model of the SARS-CoV-2-nsp10-nsp14 complex bound to substrate RNA showed that the ritonavir binding site overlaps with that of the 3' nucleotide of substrate RNA. A comparison of the calculated energies of binding for RNA and ritonavir suggested that the drug may bind to the active site of nsp14 with significant affinity. It is, therefore, possible that ritonavir may prevent association with substrate RNA and thus inhibit the exoribonuclease activity of nsp14. Overall, our computational studies suggest that ritonavir may serve as an effective inhibitor of the nsp14 protein. nsp14 is known to attenuate the inhibitory effect of drugs that function through premature termination of viral genome replication. Hence, ritonavir may potentiate the therapeutic properties of drugs such as remdesivir, favipiravir and ribavirin.


Subject(s)
Antiviral Agents/pharmacology , Exoribonucleases/antagonists & inhibitors , Ritonavir/pharmacology , Viral Nonstructural Proteins/antagonists & inhibitors , Amino Acid Sequence , Antiviral Agents/administration & dosage , Antiviral Agents/chemistry , Catalytic Domain , Computer Simulation , Drug Evaluation, Preclinical , Drug Synergism , Drug Therapy, Combination , Exoribonucleases/chemistry , Exoribonucleases/genetics , Genome, Viral/drug effects , Humans , Molecular Dynamics Simulation , Pandemics , Ritonavir/administration & dosage , Ritonavir/chemistry , /physiology , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/genetics , Virus Replication/drug effects
7.
Sci Signal ; 14(665)2021 01 12.
Article in English | MEDLINE | ID: covidwho-1066811

ABSTRACT

The spike protein of SARS-CoV-2 binds the angiotensin-converting enzyme 2 (ACE2) on the host cell surface and subsequently enters host cells through receptor-mediated endocytosis. Additional cell receptors may be directly or indirectly involved, including integrins. The cytoplasmic tails of ACE2 and integrins contain several predicted short linear motifs (SLiMs) that may facilitate internalization of the virus as well as its subsequent propagation through processes such as autophagy. Here, we measured the binding affinity of predicted interactions between SLiMs in the cytoplasmic tails of ACE2 and integrin ß3 with proteins that mediate endocytic trafficking and autophagy. We validated that a class I PDZ-binding motif mediated binding of ACE2 to the scaffolding proteins SNX27, NHERF3, and SHANK, and that a binding site for the clathrin adaptor AP2 µ2 in ACE2 overlaps with a phospho-dependent binding site for the SH2 domains of Src family tyrosine kinases. Furthermore, we validated that an LC3-interacting region (LIR) in integrin ß3 bound to the ATG8 domains of the autophagy receptors MAP1LC3 and GABARAP in a manner enhanced by LIR-adjacent phosphorylation. Our results provide molecular links between cell receptors and mediators of endocytosis and autophagy that may facilitate viral entry and propagation.


Subject(s)
/physiology , Integrin beta3/physiology , Receptors, Virus/physiology , /pathogenicity , Virus Internalization , Amino Acid Sequence , /genetics , Autophagy/physiology , Endocytosis/physiology , Host Microbial Interactions/genetics , Host Microbial Interactions/physiology , Humans , Integrin beta3/chemistry , Integrin beta3/genetics , Models, Molecular , Pandemics , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/physiology , Phosphorylation , Protein Binding , Protein Interaction Domains and Motifs , Protein Sorting Signals/genetics , Protein Sorting Signals/physiology , Receptors, Virus/chemistry , Receptors, Virus/genetics , /genetics
8.
Genes (Basel) ; 12(2)2021 01 29.
Article in English | MEDLINE | ID: covidwho-1055035

ABSTRACT

SARS-CoV-2 is a recently emerged, novel human coronavirus responsible for the currently ongoing COVID-19 pandemic. Recombination is a well-known evolutionary strategy of coronaviruses, which may frequently result in significant genetic alterations, such as deletions throughout the genome. In this study we identified a co-infection with two genetically different SARS-CoV-2 viruses within a single patient sample via amplicon-based next generation sequencing in Hungary. The recessive strain contained an 84 base pair deletion in the receptor binding domain of the spike protein gene and was found to be gradually displaced by a dominant non-deleterious variant over-time. We have identified the region of the receptor-binding domain (RBD) that is affected by the mutation, created homology models of the RBDΔ84 mutant, and based on the available experimental data and calculations, we propose that the mutation has a deteriorating effect on the binding of RBD to the angiotensin-converting enzyme 2 (ACE2) receptor, which results in the negative selection of this variant. Extending the sequencing capacity toward the discovery of emerging recombinant or deleterious strains may facilitate the early recognition of novel strains with altered phenotypic attributes and understanding of key elements of spike protein evolution. Such studies may greatly contribute to future therapeutic research and general understanding of genomic processes of the virus.


Subject(s)
/metabolism , /metabolism , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Amino Acid Sequence , Animals , Base Sequence , Binding Sites , /virology , Cell Line , Chlorocebus aethiops , Computer Simulation , Humans , Pandemics , Protein Binding , Protein Domains , Sequence Deletion , Vero Cells
9.
Sci Rep ; 11(1): 2636, 2021 01 29.
Article in English | MEDLINE | ID: covidwho-1054050

ABSTRACT

DNA synthesis in vitro has enabled the rapid production of reference standards. These are used as controls, and allow measurement and improvement of the accuracy and quality of diagnostic tests. Current reference standards typically represent target genetic material, and act only as positive controls to assess test sensitivity. However, negative controls are also required to evaluate test specificity. Using a pair of chimeric A/B RNA standards, this allowed incorporation of positive and negative controls into diagnostic testing for the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). The chimeric standards constituted target regions for RT-PCR primer/probe sets that are joined in tandem across two separate synthetic molecules. Accordingly, a target region that is present in standard A provides a positive control, whilst being absent in standard B, thereby providing a negative control. This design enables cross-validation of positive and negative controls between the paired standards in the same reaction, with identical conditions. This enables control and test failures to be distinguished, increasing confidence in the accuracy of results. The chimeric A/B standards were assessed using the US Centres for Disease Control real-time RT-PCR protocol, and showed results congruent with other commercial controls in detecting SARS-CoV-2 in patient samples. This chimeric reference standard design approach offers extensive flexibility, allowing representation of diverse genetic features and distantly related sequences, even from different organisms.


Subject(s)
Chimera , Amino Acid Sequence , /virology , Humans , RNA, Viral/standards , Reference Standards , Reproducibility of Results , /genetics , Sensitivity and Specificity
10.
Langmuir ; 37(5): 1707-1713, 2021 02 09.
Article in English | MEDLINE | ID: covidwho-1053964

ABSTRACT

The outbreak of the coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 has spread globally. SARS-CoV-2 enters human cells by utilizing the receptor-binding domain (RBD) of an envelope homotrimeric spike (S) glycoprotein to interact with the cellular receptor angiotensin-converting enzyme 2 (ACE2). We thoroughly studied the differences between the two RBDs of SARS-CoV and SARS-CoV-2 when they bind with ACE2 through molecular dynamics simulations. The peculiarities of the SARS-CoV-2 RBD are obvious in several aspects such as fluctuation of the binding interface, distribution of binding free energy on residues of the receptor-binding motifs, and the dissociation process. Based on these peculiarities of SARS-CoV-2 revealed by simulations, we proposed a strategy of destroying the RBD of SARS-CoV-2 by employing enzymatic digestion. This unique strategy is promising for developing a skin-friendly, nontoxic, and convenient disinfectant to protect people from infection by SARS-CoV-2.


Subject(s)
/metabolism , Peptide Hydrolases/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Amino Acid Sequence , Molecular Dynamics Simulation , Protein Binding , Protein Domains , Substrate Specificity , Thermodynamics
11.
Nat Commun ; 12(1): 636, 2021 01 27.
Article in English | MEDLINE | ID: covidwho-1049963

ABSTRACT

Nsp15, a uridine specific endoribonuclease conserved across coronaviruses, processes viral RNA to evade detection by host defense systems. Crystal structures of Nsp15 from different coronaviruses have shown a common hexameric assembly, yet how the enzyme recognizes and processes RNA remains poorly understood. Here we report a series of cryo-EM reconstructions of SARS-CoV-2 Nsp15, in both apo and UTP-bound states. The cryo-EM reconstructions, combined with biochemistry, mass spectrometry, and molecular dynamics, expose molecular details of how critical active site residues recognize uridine and facilitate catalysis of the phosphodiester bond. Mass spectrometry revealed the accumulation of cyclic phosphate cleavage products, while analysis of the apo and UTP-bound datasets revealed conformational dynamics not observed by crystal structures that are likely important to facilitate substrate recognition and regulate nuclease activity. Collectively, these findings advance understanding of how Nsp15 processes viral RNA and provide a structural framework for the development of new therapeutics.


Subject(s)
Endoribonucleases/chemistry , Endoribonucleases/ultrastructure , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/ultrastructure , Amino Acid Sequence , Catalytic Domain , Cryoelectron Microscopy , Endoribonucleases/metabolism , Models, Chemical , Models, Molecular , Uridine Triphosphate/metabolism , Viral Nonstructural Proteins/metabolism
12.
Viruses ; 13(2)2021 01 25.
Article in English | MEDLINE | ID: covidwho-1045365

ABSTRACT

Viral proteases are critical enzymes for the maturation of many human pathogenic viruses and thus are key targets for direct acting antivirals (DAAs). The current viral pandemic caused by SARS-CoV-2 is in dire need of DAAs. The Main protease (Mpro) is the focus of extensive structure-based drug design efforts which are mostly covalent inhibitors targeting the catalytic cysteine. ML188 is a non-covalent inhibitor designed to target SARS-CoV-1 Mpro, and provides an initial scaffold for the creation of effective pan-coronavirus inhibitors. In the current study, we found that ML188 inhibits SARS-CoV-2 Mpro at 2.5 µM, which is more potent than against SAR-CoV-1 Mpro. We determined the crystal structure of ML188 in complex with SARS-CoV-2 Mpro to 2.39 Å resolution. Sharing 96% sequence identity, structural comparison of the two complexes only shows subtle differences. Non-covalent protease inhibitors complement the design of covalent inhibitors against SARS-CoV-2 main protease and are critical initial steps in the design of DAAs to treat CoVID 19.


Subject(s)
Antiviral Agents/chemistry , Protease Inhibitors/chemistry , /enzymology , Amino Acid Sequence , Antiviral Agents/metabolism , Catalytic Domain , /metabolism , Crystallography, X-Ray , Drug Discovery , Inhibitory Concentration 50 , Models, Molecular , Protease Inhibitors/metabolism , Protein Binding , SARS Virus/enzymology
13.
Sci Rep ; 11(1): 2043, 2021 01 21.
Article in English | MEDLINE | ID: covidwho-1042518

ABSTRACT

The recent outbreak of the coronavirus (SARS-CoV2) is an unprecedented threat to human health and society across the globe. In this context, development of suitable interventions is the need of the hour. The viral spike protein (S Protein) and the cognate host cell receptor ACE2 can be considered as effective and appropriate targets for interventions. It is evident from the present computational study, that catechin and curcumin, not only exhibit strong binding affinity to viral S Protein and host receptor ACE2 but also to their complex (receptor-binding domain (RBD) of the spike protein of SARS-CoV2 and ACE2; RBD/ACE2-complex). The binding affinity values of catechin and curcumin for the S protein, ACE2 and RBD/ACE2-complex are - 10.5 and - 7.9 kcal/mol; - 8.9 and - 7.8 kcal/mol; and - 9.1 and - 7.6 kcal/mol, respectively. Curcumin directly binds to the receptor binding domain (RBD) of viral S Protein. Molecular simulation study over a period of 100 ns further substantiates that such interaction within RBD site of S Protein occurs during 40-100 ns out of 100 ns simulation trajectory. Contrary to this, catechin binds with amino acid residues present near the RBD site of S Protein and causes fluctuation in the amino acid residues of the RBD and its near proximity. Both catechin and curcumin bind the interface of 'RBD/ACE2-complex' and intervene in causing fluctuation of the alpha helices and beta-strands of the protein complex. Protein-protein interaction studies in presence of curcumin or catechin also corroborate the above findings suggesting the efficacy of these two polyphenols in hindering the formation of S Protein-ACE2 complex. In conclusion, this computational study for the first time predicts the possibility of above two polyphenols for therapeutic strategy against SARS-CoV2.


Subject(s)
/metabolism , Catechin/metabolism , Curcumin/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Amino Acid Sequence , Binding Sites , /metabolism , Catechin/chemistry , Catechin/pharmacology , Cell Membrane/metabolism , Computational Biology/methods , Curcumin/chemistry , Curcumin/pharmacology , Humans , Molecular Docking Simulation , Protein Binding , Protein Domains , Spike Glycoprotein, Coronavirus/chemistry
14.
PLoS Pathog ; 17(1): e1009233, 2021 01.
Article in English | MEDLINE | ID: covidwho-1040062

ABSTRACT

The spike (S) protein of Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) binds to a host cell receptor which facilitates viral entry. A polybasic motif detected at the cleavage site of the S protein has been shown to broaden the cell tropism and transmissibility of the virus. Here we examine the properties of SARS-CoV-2 variants with mutations at the S protein cleavage site that undergo inefficient proteolytic cleavage. Virus variants with S gene mutations generated smaller plaques and exhibited a more limited range of cell tropism compared to the wild-type strain. These alterations were shown to result from their inability to utilize the entry pathway involving direct fusion mediated by the host type II transmembrane serine protease, TMPRSS2. Notably, viruses with S gene mutations emerged rapidly and became the dominant SARS-CoV-2 variants in TMPRSS2-deficient cells including Vero cells. Our study demonstrated that the S protein polybasic cleavage motif is a critical factor underlying SARS-CoV-2 entry and cell tropism. As such, researchers should be alert to the possibility of de novo S gene mutations emerging in tissue-culture propagated virus strains.


Subject(s)
/genetics , Serine Endopeptidases/deficiency , Spike Glycoprotein, Coronavirus/genetics , Amino Acid Sequence , Animals , Caco-2 Cells , Cell Line , Chlorocebus aethiops , HEK293 Cells , Humans , Mutation , /growth & development , Sequence Alignment , Serial Passage , Vero Cells , Viral Tropism
15.
Proc Natl Acad Sci U S A ; 118(6)2021 02 09.
Article in English | MEDLINE | ID: covidwho-1039676

ABSTRACT

RNA-dependent RNA polymerases (RdRps) of the Nidovirales (Coronaviridae, Arteriviridae, and 12 other families) are linked to an amino-terminal (N-terminal) domain, called NiRAN, in a nonstructural protein (nsp) that is released from polyprotein 1ab by the viral main protease (Mpro). Previously, self-GMPylation/UMPylation activities were reported for an arterivirus NiRAN-RdRp nsp and suggested to generate a transient state primed for transferring nucleoside monophosphate (NMP) to (currently unknown) viral and/or cellular biopolymers. Here, we show that the coronavirus (human coronavirus [HCoV]-229E and severe acute respiratory syndrome coronavirus 2) nsp12 (NiRAN-RdRp) has Mn2+-dependent NMPylation activity that catalyzes the transfer of a single NMP to the cognate nsp9 by forming a phosphoramidate bond with the primary amine at the nsp9 N terminus (N3825) following Mpro-mediated proteolytic release of nsp9 from N-terminally flanking nsps. Uridine triphosphate was the preferred nucleotide in this reaction, but also adenosine triphosphate, guanosine triphosphate, and cytidine triphosphate were suitable cosubstrates. Mutational studies using recombinant coronavirus nsp9 and nsp12 proteins and genetically engineered HCoV-229E mutants identified residues essential for NiRAN-mediated nsp9 NMPylation and virus replication in cell culture. The data corroborate predictions on NiRAN active-site residues and establish an essential role for the nsp9 N3826 residue in both nsp9 NMPylation in vitro and virus replication. This residue is part of a conserved N-terminal NNE tripeptide sequence and shown to be the only invariant residue in nsp9 and its homologs in viruses of the family Coronaviridae The study provides a solid basis for functional studies of other nidovirus NMPylation activities and suggests a possible target for antiviral drug development.


Subject(s)
Coronavirus 229E, Human/genetics , RNA-Binding Proteins/metabolism , Viral Nonstructural Proteins/metabolism , Virus Replication , Amino Acid Sequence , Amino Acid Substitution , Asparagine/genetics , Cell Line , Conserved Sequence , Coronavirus 229E, Human/physiology , /metabolism , Humans , Manganese/metabolism , Protein Domains , RNA-Binding Proteins/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Transcription, Genetic , Viral Nonstructural Proteins/genetics
16.
Sci Signal ; 14(665)2021 01 12.
Article in English | MEDLINE | ID: covidwho-1029425

ABSTRACT

The first reported receptor for SARS-CoV-2 on host cells was the angiotensin-converting enzyme 2 (ACE2). However, the viral spike protein also has an RGD motif, suggesting that cell surface integrins may be co-receptors. We examined the sequences of ACE2 and integrins with the Eukaryotic Linear Motif (ELM) resource and identified candidate short linear motifs (SLiMs) in their short, unstructured, cytosolic tails with potential roles in endocytosis, membrane dynamics, autophagy, cytoskeleton, and cell signaling. These SLiM candidates are highly conserved in vertebrates and may interact with the µ2 subunit of the endocytosis-associated AP2 adaptor complex, as well as with various protein domains (namely, I-BAR, LC3, PDZ, PTB, and SH2) found in human signaling and regulatory proteins. Several motifs overlap in the tail sequences, suggesting that they may act as molecular switches, such as in response to tyrosine phosphorylation status. Candidate LC3-interacting region (LIR) motifs are present in the tails of integrin ß3 and ACE2, suggesting that these proteins could directly recruit autophagy components. Our findings identify several molecular links and testable hypotheses that could uncover mechanisms of SARS-CoV-2 attachment, entry, and replication against which it may be possible to develop host-directed therapies that dampen viral infection and disease progression. Several of these SLiMs have now been validated to mediate the predicted peptide interactions.


Subject(s)
/virology , Host Microbial Interactions/physiology , /pathogenicity , Virus Internalization , Amino Acid Sequence , /genetics , Animals , Conserved Sequence , Host Microbial Interactions/genetics , Humans , Integrins/chemistry , Integrins/genetics , Integrins/physiology , Intrinsically Disordered Proteins/chemistry , Intrinsically Disordered Proteins/genetics , Intrinsically Disordered Proteins/physiology , Models, Biological , Models, Molecular , Oligopeptides/chemistry , Oligopeptides/genetics , Oligopeptides/physiology , Protein Interaction Domains and Motifs/genetics , Protein Interaction Domains and Motifs/physiology , Protein Sorting Signals/genetics , Protein Sorting Signals/physiology , Receptors, Virus/chemistry , Receptors, Virus/genetics , Receptors, Virus/physiology , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/physiology
17.
Int J Mol Sci ; 21(24)2020 Dec 15.
Article in English | MEDLINE | ID: covidwho-1024586

ABSTRACT

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease-19 (COVID-19) being associated with severe pneumonia. Like with other viruses, the interaction of SARS-CoV-2 with host cell proteins is necessary for successful replication, and cleavage of cellular targets by the viral protease also may contribute to the pathogenesis, but knowledge about the human proteins that are processed by the main protease (3CLpro) of SARS-CoV-2 is still limited. We tested the prediction potentials of two different in silico methods for the identification of SARS-CoV-2 3CLpro cleavage sites in human proteins. Short stretches of homologous host-pathogen protein sequences (SSHHPS) that are present in SARS-CoV-2 polyprotein and human proteins were identified using BLAST analysis, and the NetCorona 1.0 webserver was used to successfully predict cleavage sites, although this method was primarily developed for SARS-CoV. Human C-terminal-binding protein 1 (CTBP1) was found to be cleaved in vitro by SARS-CoV-2 3CLpro, the existence of the cleavage site was proved experimentally by using a His6-MBP-mEYFP recombinant substrate containing the predicted target sequence. Our results highlight both potentials and limitations of the tested algorithms. The identification of candidate host substrates of 3CLpro may help better develop an understanding of the molecular mechanisms behind the replication and pathogenesis of SARS-CoV-2.


Subject(s)
/virology , /enzymology , Alcohol Oxidoreductases/metabolism , Amino Acid Sequence , /genetics , DNA-Binding Proteins/metabolism , Host-Pathogen Interactions , Humans , Substrate Specificity
18.
Int Immunopharmacol ; 91: 107276, 2021 Feb.
Article in English | MEDLINE | ID: covidwho-1023607

ABSTRACT

SARS-CoV-2 has a high transmission rate and shows frequent mutations, thus making vaccine development an arduous task. However, researchers around the globe are working hard to find a solution e.g. synthetic vaccine. Here, we have performed genome-wide analysis of 566 Indian SARS-CoV-2 genomes to extract the potential conserved regions for identifying peptide based synthetic vaccines, viz. epitopes with high immunogenicity and antigenicity. In this regard, different multiple sequence alignment techniques are used to align the SARS-CoV-2 genomes separately. Subsequently, consensus conserved regions are identified after finding the conserved regions from each aligned result of alignment techniques. Further, the consensus conserved regions are refined considering that their lengths are greater than or equal to 60nt and their corresponding proteins are devoid of any stop codons. Subsequently, their specificity as query coverage are verified using Nucleotide BLAST. Finally, with these consensus conserved regions, T-cell and B-cell epitopes are identified based on their immunogenic and antigenic scores which are then used to rank the conserved regions. As a result, we have ranked 23 consensus conserved regions that are associated with different proteins. This ranking also resulted in 34 MHC-I and 37 MHC-II restricted T-cell epitopes with 16 and 19 unique HLA alleles and 29 B-cell epitopes. After ranking, the consensus conserved region from NSP3 gene is obtained that is highly immunogenic and antigenic. In order to judge the relevance of the identified epitopes, the physico-chemical properties and binding conformation of the MHC-I and MHC-II restricted T-cell epitopes are shown with respect to HLA alleles.


Subject(s)
/immunology , Epitopes, B-Lymphocyte/immunology , Epitopes, T-Lymphocyte/immunology , Genome, Viral/immunology , Immunogenicity, Vaccine/immunology , /immunology , Amino Acid Sequence , Genome-Wide Association Study/methods , Humans , Vaccines, Synthetic/immunology
19.
J Chem Inf Model ; 61(1): 423-431, 2021 01 25.
Article in English | MEDLINE | ID: covidwho-1014975

ABSTRACT

Membrane fusion, a key step in the early stages of virus propagation, allows the release of the viral genome in the host cell cytoplasm. The process is initiated by fusion peptides that are small, hydrophobic components of viral membrane-embedded glycoproteins and are typically conserved within virus families. Here, we attempted to identify the correct fusion peptide region in the Spike protein of SARS-CoV-2 by all-atom molecular dynamics simulations of dual membrane systems with varied oligomeric units of putative candidate peptides. Of all of the systems tested, only a trimeric unit of a 40-amino-acid region (residues 816-855 of SARS-CoV-2 Spike) was effective in triggering the initial stages of membrane fusion, within 200 ns of simulation time. Association of this trimeric unit with dual membranes resulted in the migration of lipids from the upper leaflet of the lower bilayer toward the lower leaflet of the upper bilayer to create a structural unit reminiscent of a fusion bridge. We submit that residues 816-855 of Spike represent the bona fide fusion peptide of SARS-CoV-2 and that computational methods represent an effective way to identify fusion peptides in viral glycoproteins.


Subject(s)
/metabolism , Membrane Fusion , Spike Glycoprotein, Coronavirus/metabolism , Virus Internalization , Amino Acid Sequence , Host-Pathogen Interactions , Humans , Molecular Dynamics Simulation , Peptides/chemistry , Peptides/metabolism , Protein Multimerization , Spike Glycoprotein, Coronavirus/chemistry
20.
Int J Mol Sci ; 22(1)2020 Dec 23.
Article in English | MEDLINE | ID: covidwho-1011558

ABSTRACT

Our evolutionary and structural analyses revealed that the severe acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV-2) spike gene is a complex mosaic resulting from several recombination events. Additionally, the fixation of variants has mainly been driven by purifying selection, suggesting the presence of conserved structural features. Our dynamic simulations identified two main long-range covariant dynamic movements of the novel glycoprotein, and showed that, as a result of the evolutionary duality, they are preserved. The first movement involves the receptor binding domain with the N-terminal domain and the C-terminal domain 2 and is maintained across human, bat and pangolin coronaviruses. The second is a complex network of long-range dynamics specific to SARS-CoV-2 involving the novel PRRA and the conserved KR*SF cleavage sites, as well as conserved segments in C-terminal domain 3. These movements, essential for host cell binding, are maintained by hinges conserved across human, bat, and pangolin coronaviruses glycoproteins. The hinges, located around Threonine 333 and Proline 527 within the N-terminal domain and C-terminal domain 2, represent candidate targets for the future development of novel pan-coronavirus inhibitors. In summary, we show that while recombination created a new configuration that increased the covariant dynamic movements of the SARS-CoV-2 glycoprotein, negative selection preserved its inter-domain structure throughout evolution in different hosts and inter-species transmissions.


Subject(s)
Recombination, Genetic , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Amino Acid Sequence , Animals , Chiroptera/virology , Coronavirus/chemistry , Coronavirus/genetics , Evolution, Molecular , Host Specificity , Humans , Molecular Dynamics Simulation , Phylogeny , Protein Binding , Protein Domains , /genetics
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