Conserved molecular signatures in the spike protein provide evidence indicating the origin of SARS-CoV-2 and a Pangolin-CoV (MP789) by recombination(s) between specific lineages of Sarbecoviruses

Both SARS-CoV-2 and SARS coronaviruses (CoVs) are members of the subgenus Sarbecovirus. To understand the origin of SARS-CoV-2, sequences for the spike and nucleocapsid proteins from sarbecoviruses were analyzed to identify molecular markers consisting of conserved inserts or deletions (termed CSIs) that are specific for either a particular clade of Sarbecovirus or are commonly shared by two or more clades of these viruses. Three novel CSIs in the N-terminal domain (NTD) of the spike protein S1-subunit (S1-NTD) are uniquely shared by SARS-CoV-2, Bat-CoV-RaTG13 and most pangolin CoVs (SARS-CoV-2r clade). Three other sarbecoviruses viz. bat-CoVZXC21, -CoVZC45 and -PrC31 (forming CoVZC/PrC31 clade), and a pangolin-CoV_MP789 also contain related CSIs in the same positions. In contrast to the S1-NTD, both SARS and SARS-CoV-2r viruses contain two large CSIs in the S1-C-terminal domain (S1-CTD) that are absent in the CoVZC/PrC31 clade. One of these CSIs, consisting of a 12 aa insert, is also present in the RShSTT clade (Cambodia-CoV strains). Sequence similarity studies show that the S1-NTD of SARS-CoV-2r viruses is most similar to the CoVZC/PrC31 clade, whereas their S1-CTD exhibits highest similarity to the RShSTT- (and the SARS-related) CoVs. Results from the shared presence of CSIs and sequence similarity studies on different CoV lineages support the inference that the SARS-CoV-2r cluster of viruses has originated by a genetic recombination between the S1-NTD of the CoVZC/PrC31 clade of CoVs and the S1-CTD of RShSTT/SARS viruses, respectively. We also present compelling evidence, based on the shared presence of CSIs and sequence similarity studies, that the pangolin-CoV_MP789, whose receptor-binding domain is most similar to the SARS-CoV-2 virus, has resulted from another independent recombination event involving the S1-NTD of the CoVZC/PrC31 CoVs and the S1-CTD of an unidentified SARS-CoV-2r related virus. The SARS-CoV-2 virus involved in this latter recombination event is postulated to be most similar to the SARS-CoV-2. Several other CSIs reported here are specific for other clusters of sarbecoviruses including a clade consisting of bat-SARS-CoVs (BM48-31/BGR/2008 and SARS_BtKY72). Structural mapping studies show that the identified CSIs form distinct loops/patches on the surface of the spike protein. It is hypothesized that these novel loops/patches on the spike protein, through their interactions with other host components, should play important roles in the biology/pathology of SARS-CoV-2 virus. Lastly, the CSIs specific for different clades of sarbecoviruses including SARS-CoV-2r clade provide novel means for the identification of these viruses and other potential applications.

Download Full-text

Conserved Sequence Features in the Spike Protein Provide Evidence Suggesting the Origin of SARS-CoV-2 (COVID-19) Virus by Recombination between SARS virus and Another Sarbecovirus

10.20944/preprints202006.0165.v2 ◽

2020 ◽

Author(s):

Radhey S. Gupta ◽

Bijendra Khadka

Keyword(s):

Recombination Event ◽

Sequence Similarity ◽

Protein Sequences ◽

Spike Protein ◽

Amino Acid Sequence Similarity ◽

Receptor Binding Domain ◽

Conserved Sequence ◽

Terminal Domain ◽

Surface Loops ◽

Sequence Characteristics

Both SARS-CoV-2 (COVID-19) and SARS coronaviruses (CoVs) are members of the subgenus Sarbecovirus. To understand the origin of SARS-CoV-2, protein sequences from sarbecoviruses were analyzed to identify highly-specific molecular markers consisting of conserved inserts or deletions (termed CSIs) in the spike (S) and nucleocapsid (N) proteins that are specific for either particular clusters/lineages of these viruses or are commonly shared by specific lineages. Three novel CSIs in the N-terminal domain of the spike protein S1-subunit (S1-NTD) are uniquely shared by the SARS-CoV-2, BatCoV-RaTG13 and most pangolin CoVs, distinguishing this cluster of viruses (SARS-CoV-2r) from all others. In the same positions, where these CSIs are found, related CSIs are also present in two other sarbecoviruses (viz. CoVZXC21 and CoVZC45 forming CoVZC cluster), which form an out group of the SARS-CoV-2r cluster. These three CSIs are not found in the SARS-CoVs. However, both SARS and SARS-CoV-2r CoVs contain two large CSIs in the C-terminal domain of S1 (S1-CTD), which binds the human ACE-2 receptor, that are absent in the CoVZC cluster of CoVs. These results indicate that while the S1-NTD of the SARS-CoV-2r viruses possesses the sequence characteristics of the CoVZC cluster of CoVs, their S1-CTD resembles the SARS viruses. Thus, the spike protein of SARS-CoV-2r viruses has likely originated from a recombination event between the S1-NTD of the CoVZC viruses and the S1-CTD of SARS viruses. This inference is also supported by the amino acid sequence similarity of the S1-NTD and S1-CTD from SARS-CoV-2 compared to the CoVZC and SARS CoVs. We also present evidence that one of the pangolin-CoV_MP789, whose receptor-binding domain is most similar to the SARS-CoV-2, is also derived by a recent recombination between the S1-NTD of the CoVZC CoVs and the S1-CTD of a SARS-CoV-2 related virus. Several other identified CSIs are specific for others clusters of sarbecoviruses including a clade consisting of bat SARS-CoVs (BM48-31/BGR/2008 and SARS_BtKY72). Structural mappings studies show that the identified CSIs are located within surface-exposed loops and form distinct patches on the surface of the spike protein. These surface loops/patches are predicted to interact with other host components and play important role in the biology/pathology of SARS-CoV-2 virus. Lastly, the CSIs specific for the SARS-CoV-2r clade provide novel means for development of new diagnostic and therapeutic targets for these viruses.

Download Full-text

Can the SARS-CoV-2 Spike Protein Bind Integrins Independent of the RGD Sequence?

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2021.765300 ◽

2021 ◽

Vol 11 ◽

Author(s):

Christopher A. Beaudoin ◽

Samir W. Hamaia ◽

Christopher L.-H. Huang ◽

Tom L. Blundell ◽

Antony P. Jackson

Keyword(s):

Receptor Binding ◽

Conformational Changes ◽

Sequence Similarity ◽

Binding Domain ◽

Spike Protein ◽

Receptor Binding Domain ◽

Binding Motifs ◽

Rgd Motif ◽

Rgd Sequence ◽

Protein Receptor

The RGD motif in the Severe Acute Syndrome Coronavirus 2 (SARS-CoV-2) spike protein has been predicted to bind RGD-recognizing integrins. Recent studies have shown that the spike protein does, indeed, interact with αVβ3 and α5β1 integrins, both of which bind to RGD-containing ligands. However, computational studies have suggested that binding between the spike RGD motif and integrins is not favourable, even when unfolding occurs after conformational changes induced by binding to the canonical host entry receptor, angiotensin-converting enzyme 2 (ACE2). Furthermore, non-RGD-binding integrins, such as αx, have been suggested to interact with the SARS-CoV-2 spike protein. Other viral pathogens, such as rotaviruses, have been recorded to bind integrins in an RGD-independent manner to initiate host cell entry. Thus, in order to consider the potential for the SARS-CoV-2 spike protein to bind integrins independent of the RGD sequence, we investigate several factors related to the involvement of integrins in SARS-CoV-2 infection. First, we review changes in integrin expression during SARS-CoV-2 infection to identify which integrins might be of interest. Then, all known non-RGD integrin-binding motifs are collected and mapped to the spike protein receptor-binding domain and analyzed for their 3D availability. Several integrin-binding motifs are shown to exhibit high sequence similarity with solvent accessible regions of the spike receptor-binding domain. Comparisons of these motifs with other betacoronavirus spike proteins, such as SARS-CoV and RaTG13, reveal that some have recently evolved while others are more conserved throughout phylogenetically similar betacoronaviruses. Interestingly, all of the potential integrin-binding motifs, including the RGD sequence, are conserved in one of the known pangolin coronavirus strains. Of note, the most recently recorded mutations in the spike protein receptor-binding domain were found outside of the putative integrin-binding sequences, although several mutations formed inside and close to one motif, in particular, may potentially enhance binding. These data suggest that the SARS-CoV-2 spike protein may interact with integrins independent of the RGD sequence and may help further explain how SARS-CoV-2 and other viruses can evolve to bind to integrins.

Download Full-text

N-terminal domain (NTD) of SARS-CoV-2 spike-protein structurally resembles MERS-CoV NTD sialoside-binding pocket

10.21203/rs.3.rs-37300/v1 ◽

2020 ◽

Cited By ~ 1

Author(s):

Mayanka Awasthi ◽

Sahil Gulati ◽

Debi P. Sarkar ◽

Swasti Tiwari ◽

Suneel Kateriya ◽

...

Keyword(s):

Sequence Similarity ◽

Secondary Infection ◽

Respiratory Illness ◽

Cell Receptor ◽

Binding Pocket ◽

Therapeutic Interventions ◽

Spike Protein ◽

High Sequence Similarity ◽

Terminal Domain ◽

Novel Coronavirus

Abstract COVID-19 novel coronavirus disease caused by SARS-CoV-2 causes severe lethal respiratory illness in humans and has recently developed into a worldwide pandemic. The lack of effective treatment strategy and vaccines against SARS-CoV-2 poses a threat to human health. Extremely high infection rate and multi-organ secondary infection within a short time period makes this virus more deadly and challenging for therapeutic interventions. Despite of high sequence similarity and utilization of common host-cell receptor, human angiotensin-converting enzyme-2 (ACE2) for virus entry, SARS-CoV-2 is much infectious than SARS-CoV. Structure-based sequence comparison of the N-terminal domain (NTD) of spike protein of MERS-CoV, SARS-CoV and SARS-CoV-2 illustrate three short stretches of amino acid motifs in SARS-CoV-2 , which appears to be the reminiscent of MERS-CoV sialoside binding pockets. These key differences with SARS-CoV and similarity with MERS-CoV, suggest an evolutionary adaptation of SARS-CoV-2 spike protein reciprocal interaction with host surface sialosides.

Download Full-text

Structure-activity relationships of B.1.617 and other SARS-CoV-2 spike variants

10.1101/2021.09.12.459978 ◽

2021 ◽

Author(s):

Tzu-Jing Yang ◽

Pei-Yu Yu ◽

Yuan-Chih Chang ◽

Ning-En Chang ◽

Yu-Xi Tsai ◽

...

Keyword(s):

Receptor Binding ◽

Host Immunity ◽

Binding Domain ◽

Spike Protein ◽

Receptor Binding Domain ◽

Structure Activity Relationships ◽

Terminal Domain ◽

Structure Activity ◽

Functional States ◽

Binding Interfaces

The surge of COVID-19 infection cases is spurred by emerging SARS-CoV-2 variants such as B.1.617. Here we report 38 cryo-EM structures, corresponding to the spike protein of the Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2) and Kappa (B.1.617.1) variants in different functional states with and without its receptor, ACE2. Mutations on the N-terminal domain not only alter the conformation of the highly antigenic supersite of the Delta variant, but also remodel the glycan shield by deleting or adding N-glycans of the Delta and Gamma variants, respectively. Substantially enhanced ACE2 binding was observed for all variants, whose mutations on the receptor binding domain modulate the electrostatics of the binding interfaces. Despite their abilities to escape host immunity, all variants can be potently neutralized by three unique antibodies.

Download Full-text

A neutralizing human antibody binds to the N-terminal domain of the Spike protein of SARS-CoV-2

Science ◽

10.1126/science.abc6952 ◽

2020 ◽

Vol 369 (6504) ◽

pp. 650-655 ◽

Cited By ~ 166

Author(s):

Xiangyang Chi ◽

Renhong Yan ◽

Jun Zhang ◽

Guanying Zhang ◽

Yuanyuan Zhang ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Severe Acute Respiratory Syndrome ◽

Receptor Binding ◽

Binding Domain ◽

Spike Protein ◽

Human Antibody ◽

Receptor Binding Domain ◽

S Protein ◽

Terminal Domain ◽

Promising Target

Developing therapeutics against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) could be guided by the distribution of epitopes, not only on the receptor binding domain (RBD) of the Spike (S) protein but also across the full Spike (S) protein. We isolated and characterized monoclonal antibodies (mAbs) from 10 convalescent COVID-19 patients. Three mAbs showed neutralizing activities against authentic SARS-CoV-2. One mAb, named 4A8, exhibits high neutralization potency against both authentic and pseudotyped SARS-CoV-2 but does not bind the RBD. We defined the epitope of 4A8 as the N-terminal domain (NTD) of the S protein by determining with cryo–eletron microscopy its structure in complex with the S protein to an overall resolution of 3.1 angstroms and local resolution of 3.3 angstroms for the 4A8-NTD interface. This points to the NTD as a promising target for therapeutic mAbs against COVID-19.

Download Full-text

A potent neutralizing human antibody reveals the N-terminal domain of the Spike protein of SARS-CoV-2 as a site of vulnerability

10.1101/2020.05.08.083964 ◽

2020 ◽

Cited By ~ 21

Author(s):

Xiangyang Chi ◽

Renhong Yan ◽

Jun Zhang ◽

Guanying Zhang ◽

Yuanyuan Zhang ◽

...

Keyword(s):

Spike Protein ◽

Human Antibody ◽

Receptor Binding Domain ◽

Global Public Health ◽

S Protein ◽

Angiotensin Converting Enzyme 2 ◽

Terminal Domain ◽

Public Health Threat ◽

A Site ◽

Structural Characterizations

AbstractThe pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) presents a global public health threat. Most research on therapeutics against SARS-CoV-2 focused on the receptor binding domain (RBD) of the Spike (S) protein, whereas the vulnerable epitopes and functional mechanism of non-RBD regions are poorly understood. Here we isolated and characterized monoclonal antibodies (mAbs) derived from convalescent COVID-19 patients. An mAb targeting the N-terminal domain (NTD) of the SARS-CoV-2 S protein, named 4A8, exhibits high neutralization potency against both authentic and pseudotyped SARS-CoV-2, although it does not block the interaction between angiotensin-converting enzyme 2 (ACE2) receptor and S protein. The cryo-EM structure of the SARS-CoV-2 S protein in complex with 4A8 has been determined to an overall resolution of 3.1 Angstrom and local resolution of 3.4 Angstrom for the 4A8-NTD interface, revealing detailed interactions between the NTD and 4A8. Our functional and structural characterizations discover a new vulnerable epitope of the S protein and identify promising neutralizing mAbs as potential clinical therapy for COVID-19.

Download Full-text

The ongoing evolution of variants of concern and interest of SARS-CoV-2 in Brazil revealed by convergent indels in the amino (N)-terminal domain of the Spike protein

10.1101/2021.03.19.21253946 ◽

2021 ◽

Author(s):

Paola Cristina Resende ◽

Felipe Gomes Naveca ◽

Roberto Dias Lins ◽

Filipe Zimmer Dezordi ◽

Matheus V. F. Ferraz ◽

...

Keyword(s):

Receptor Binding ◽

Immune Escape ◽

Binding Domain ◽

Spike Protein ◽

Receptor Binding Domain ◽

S Protein ◽

Terminal Domain

Mutations at both the receptor-binding domain (RBD) and the amino (N)-terminal domain (NTD) of the SARS-CoV-2 Spike (S) glycoprotein can alter its antigenicity and promote immune escape. We identified that SARS-CoV-2 lineages circulating in Brazil with mutations of concern in the RBD independently acquired convergent deletions and insertions in the NTD of the S protein, which altered the NTD antigenic-supersite and other predicted epitopes at this region. These findings support that the ongoing widespread transmission of SARS-CoV-2 in Brazil is generating new viral lineages that might be more resistant to neutralization than parental variants of concern.

Download Full-text