scholarly journals Nanobodies recognizing conserved hidden clefts of all SARS-CoV-2 spike variants

2021 ◽  
Author(s):  
Ryota Maeda ◽  
Junso Fujita ◽  
Yoshinobu Konishi ◽  
Yasuhiro Kazuma ◽  
Hiroyuki Yamazaki ◽  
...  
Keyword(s):  
Infectious Disease ◽  
Viral Infection ◽  
Receptor Binding ◽  
Heavy Chain ◽  
Receptor Binding Domain ◽  
Variable Regions ◽  
Human Antibodies ◽  
Flow Cytometric ◽  
Alpha Beta ◽  
Spike Proteins

We are in the midst of the historic coronavirus infectious disease 2019 (COVID-19) pandemic caused by severe respiratory syndrome coronavirus 2 (SARS-CoV-2). Although countless efforts to control the pandemic have been attempted--most successfully, vaccination--imbalances in accessibility to vaccines, medicines, and diagnostics among countries, regions, and populations have been problematic. Camelid variable regions of heavy chain-only antibodies (VHHs or nanobodies) have unique modalities: they are smaller, more stable, easier to customize, and, importantly, less expensive to produce than conventional antibodies. We present the sequences of nine alpaca nanobodies that detect the spike proteins of four SARS-CoV-2 variants of concern (VOCs)--namely, the alpha, beta, gamma, and delta variants. We show that they can quantify or detect spike variants via ELISA and lateral flow, kinetic, flow cytometric, microscopy, and Western blotting assays. The panel of nanobodies broadly neutralized viral infection by pseudotyped SARS-CoV-2 VOCs. Structural analyses showed that a P86 clone targeted epitopes that were conserved yet unclassified on the receptor-binding domain (RBD) and located inside the N-terminal domain (NTD). Human antibodies have hardly accessed both regions; consequently, the clone buries hidden crevasses of SARS-CoV-2 spike proteins undetected by conventional antibodies and maintains activity against spike proteins carrying escape mutations.

scholarly journals Gonococcal pili. Primary structure and receptor binding domain.

1984 ◽  
Vol 159 (5) ◽  
pp. 1351-1370 ◽  
Author(s):  
G K Schoolnik ◽  
R Fernandez ◽  
J Y Tai ◽  
J Rothbard ◽  
E C Gotschlich
Keyword(s):  
Amino Acids ◽  
Receptor Binding ◽  
Binding Domain ◽  
Structural Basis ◽  
Receptor Binding Domain ◽  
Variable Regions ◽  
Disulfide Linkage ◽  
Polymeric Structure ◽  
Antigenic Diversity ◽  
Single Polypeptide Chain

The complete amino acid sequence of pilin from gonococcal strain MS11 and the sequence of constant and variable regions from strain R10 pilin have been determined in order to elucidate the structural basis for adherence function, antigenic diversity, and polymeric structure. The MS11 pilin sequence consists of 159 amino acids in a single polypeptide chain with two cysteines in disulfide linkage and serine-bonded phosphate residues. TC-2 (31-111), a soluble monomeric pilus peptide prepared by arginine-specific digestion, bound human endocervical, but not buccal or HeLa cells and therefore is postulated to encompass the receptor binding domain. Variable regions of CNBr-3 appear to confer antigenic diversity and comprise segments in which changes in the position of charged residues occur in hydrophilic, beta-turns. Residues 2-21 and 202-221 of gonococcal pilins and lower eucaryotic actins, respectively, exhibit 50% homology. When these residues are arranged at intervals of 100 degrees of arc on "helical wheels," the identical amino acids comprise a hydrophobic face on one side of the helix. This observation, the hydrophobic character of this region and the tendency for TC-1 (residues 1-30) to aggregate in water, suggest that this stretch interacts with other subunits to stabilize polymeric structure.

scholarly journals Unraveling the stability landscape of mutations in the SARS-CoV-2 receptor-binding domain

2020 ◽  
Author(s):  
Mohamed Raef Smaoui ◽  
Hamdi Yahyaoui
Keyword(s):  
Receptor Binding ◽  
Single Point ◽  
Protein Structures ◽  
Point Mutations ◽  
Binding Domain ◽  
Spike Protein ◽  
Receptor Binding Domain ◽  
Point Mutant ◽  
The Stability ◽  
Spike Proteins

Abstract The interaction between the receptor-binding domain of the SARS-CoV-2 spike glycoprotein and the ACE2 enzyme is believed to be the entry point of the virus into various cells in the body, including the lungs, heart, liver, and kidneys. The current focus of several therapeutic design efforts explore attempts at affecting the binding interaction between the two proteins to limit the activity of the virus and disease progression. In this work, we analyze the stability of the spike protein under all possible single-point mutations in the receptor-binding domain and computationally explore mutations that can affect the binding with the ACE2 enzyme. We unravel the mutation landscape of the receptor region and assess the toxicity potential of single and multi-point mutations, generating insights for future vaccine efforts on potential mutations that might further stabilize the spike protein and increase its infectivity. We developed a tool, called SpikeMutator, to construct full atomic protein structures of the mutant spike proteins and shared a database of 3,800 single-point mutant structures. We analyzed the recent 65,000 reported spike sequences across the globe and observed the emergence of stable multi-point mutant structures. Using the landscape, we searched through 7.5 million possible 2-point mutation combinations and report that the (R355D K424E) mutation produces one of the strongest spike proteins that therapeutic efforts should investigate for the sake of developing an effective vaccine.

scholarly journals Niclosamide inhibits SARS-CoV2 entry by blocking internalization through pH-dependent CLIC/GEEC endocytic pathway

2020 ◽  
Author(s):  
Chaitra Prabhakara ◽  
Rashmi Godbole ◽  
Parijat Sil ◽  
Sowmya Jahnavi ◽  
Thomas S van Zanten ◽  
...  
Keyword(s):  
Viral Infection ◽  
Receptor Binding ◽  
Endocytic Pathway ◽  
Host Cells ◽  
Entry Inhibitor ◽  
Receptor Binding Domain ◽  
Endosomal Acidification ◽  
Independent Mechanism ◽  
Ph Dependent ◽  
Endosomal Ph

AbstractMany viruses utilize the host endo-lysosomal network to infect cells. Tracing the endocytic itinerary of SARS-CoV2 can provide insights into viral trafficking and aid in designing new therapeutic targets. Here, we demonstrate that the receptor binding domain (RBD) of SARS-CoV2 is internalized via the clathrin and dynamin-independent, pH-dependent CLIC/GEEC (CG) endocytic pathway. Endosomal acidification inhibitors like BafilomycinA1 and NH4Cl, which inhibit the CG pathway, strongly block the uptake of RBD. Using transduction assays with SARS-CoV2 Spike-pseudovirus, we confirmed that these acidification inhibitors also impede viral infection. By contrast, Chloroquine neither affects RBD uptake nor extensively alters the endosomal pH, yet attenuates Spike-pseudovirus entry, indicating a pH-independent mechanism of intervention. We screened a subset of FDA-approved acidification inhibitors and found Niclosamide to be a potential SARS-CoV2 entry inhibitor. Niclosamide, thus, could provide broader applicability in subverting infection of similar category viruses entering host cells via this pH-dependent endocytic pathway.

scholarly journals Decreased neutralization of SARS-CoV-2 global variants by therapeutic anti-spike protein monoclonal antibodies

2021 ◽  
Author(s):  
Takuya Tada ◽  
Belinda M. Dcosta ◽  
Hao Zhou ◽  
Ada Vaill ◽  
Wes Kazmierski ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Receptor Binding ◽  
Binding Domain ◽  
Spike Protein ◽  
Receptor Binding Domain ◽  
Neutralization Activity ◽  
Protein Mutations ◽  
Spike Proteins

AbstractMonoclonal antibodies against the SARS-CoV-2 spike protein, notably, those developed by Regeneron Pharmaceuticals and Eli Lilly and Company have proven to provide protection against severe COVID-19. The emergence of SARS-CoV-2 variants with heavily mutated spike proteins raises the concern that the therapy could become less effective if any of the mutations disrupt epitopes engaged by the antibodies. In this study, we tested monoclonal antibodies REGN10933 and REGN10987 that are used in combination, for their ability to neutralize SARS-CoV-2 variants B.1.1.7, B.1.351, mink cluster 5 and COH.20G/677H. We report that REGN10987 maintains most of its neutralization activity against viruses with B.1.1.7, B.1.351 and mink cluster 5 spike proteins but that REGN10933 has lost activity against B.1.351 and mink cluster 5. The failure of REGN10933 to neutralize B.1.351 is caused by the K417N and E484K mutations in the receptor binding domain; the failure to neutralize the mink cluster 5 spike protein is caused by the Y453F mutation. The REGN10933 and REGN10987 combination was 9.1-fold less potent on B.1.351 and 16.2-fold less potent on mink cluster 5, raising concerns of reduced efficacy in the treatment of patients infected with variant viruses. The results suggest that there is a need to develop additional monoclonal antibodies that are not affected by the current spike protein mutations.

scholarly journals Structural Analysis of Receptor Binding Domain Mutations in SARS-CoV-2 Variants of Concern that Modulate ACE2 and Antibody Binding

2021 ◽  
Author(s):  
Dhiraj Mannar ◽  
James W Saville ◽  
Xing Zhu ◽  
Shanti S. Srivastava ◽  
Alison M. Berezuk ◽  
...  
Keyword(s):  
Structural Analysis ◽  
South African ◽  
Receptor Binding ◽  
Antibody Binding ◽  
Binding Domain ◽  
Spike Protein ◽  
Receptor Binding Domain ◽  
Biochemical Assays ◽  
The Uk ◽  
Spike Proteins

SummaryThe recently emerged SARS-CoV-2 South African (B. 1.351) and Brazil/Japan (P.1) variants of concern (VoCs) include a key mutation (N501Y) found in the UK variant that enhances affinity of the spike protein for its receptor, ACE2. Additional mutations are found in these variants at residues 417 and 484 that appear to promote antibody evasion. In contrast, the Californian VoCs (B.1.427/429) lack the N501Y mutation, yet exhibit antibody evasion. We engineered spike proteins to express these RBD VoC mutations either in isolation, or in different combinations, and analyzed the effects using biochemical assays and cryo-EM structural analyses. Overall, our findings suggest that the emergence of new SARS-CoV-2 variant spikes can be rationalized as the result of mutations that confer either increased ACE2 affinity, increased antibody evasion, or both, providing a framework to dissect the molecular factors that drive VoC evolution.

scholarly journals SARS-CoV-2 Prion-Like Domains in Spike Proteins Enables Higher Affinity to ACE2

2020 ◽  
Author(s):  
George Tetz ◽  
Victor Tetz
Keyword(s):  
Receptor Binding ◽  
Cell Receptor ◽  
Binding Domain ◽  
Spike Protein ◽  
Striking Difference ◽  
Receptor Binding Domain ◽  
Viral Receptor ◽  
Functional Roles ◽  
Binding Domains ◽  
Spike Proteins

Currently, the world is struggling with the coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Prion-like domains are critical for virulence and the development of therapeutic targets; however, the prion-like domains in the SARS-CoV-2 proteome have not been analyzed. In this in silico study, using the PLAAC algorithm, we identified the presence of prion-like domains in SARS-CoV-2 spike protein. Compared with other viruses, a striking difference was observed in the distribution of prion-like domains in the spike, since SARS-CoV-2 was the only coronavirus with a prion-like domain found in the receptor-binding domain of the S1 region of the spike protein. The presence and unique distribution of prion-like domains in the SARS-CoV-2 receptor-binding domains of spike proteins is particularly interesting, since although SARS-CoV-2 and SARS-CoV S share the same host cell receptor, angiotensin-converting enzyme 2 (ACE2), SARS-CoV-2 demonstrates a 10- to 20-fold higher affinity for ACE2. Finally, we identified prion-like domains in the α1 helix of the ACE2 receptor that interacts with the viral receptor-binding domain of SARS-CoV-2. Taken together, the present findings indicate that the identified PrDs in the SARS-CoV-2 receptor-binding domain (RBD) and ACE2 region that interacts with RBD have important functional roles in viral adhesion and entry.

scholarly journals Role of Variable Regions A and B in Receptor Binding Domain of Amphotropic Murine Leukemia Virus Envelope Protein

1998 ◽  
Vol 72 (11) ◽  
pp. 9101-9108 ◽  
Author(s):  
Jin-Young Han ◽  
Yi Zhao ◽  
W. French Anderson ◽  
Paula M. Cannon
Keyword(s):  
Receptor Binding ◽  
Envelope Protein ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Binding Domain ◽  
Receptor Binding Domain ◽  
Variable Regions ◽  
Amino Terminal ◽  
Amphotropic Murine Leukemia Virus ◽  
Murine Leukemia

ABSTRACT For the amphotropic murine leukemia virus (MuLV), a 208-amino-acid amino-terminal fragment of the surface unit (SU) of the envelope glycoprotein is sufficient to bind to its receptor, Pit2. Within this binding domain, two hypervariable regions, VRA and VRB, have been proposed to be important for receptor recognition. In order to specifically locate residues that are important for the interaction with Pit2, we generated a number of site-specific mutations in both VRA and VRB and analyzed the resulting envelope proteins when expressed on retroviral vectors. Concurrently, we substituted portions of the amphotropic SU with homologous regions from the polytropic MuLV envelope protein. The amphotropic SU was unaffected by most of the point mutations we introduced. In addition, the deletion of eight residues in a region of VRA that was previously suggested to be essential for Pit2 utilization only decreased titer on NIH 3T3 cells by 1 order of magnitude. Although the replacement of the amino-terminal two-thirds of VRA with the polytropic sequence abolished receptor binding, smaller nonoverlapping substitutions did not affect the function of the protein. We were not able to identify a single critical receptor contact point within VRA, and we suggest that the amphotropic receptor binding domain probably makes multiple contacts with the receptor and that the loss of some of these contacts can be tolerated.

scholarly journals Neutralization of recombinant RBD-subunit vaccine ZF2001-elicited antisera to SARS-CoV-2 variants including Delta

2021 ◽  
Author(s):  
Xin Zhao ◽  
Anqi Zheng ◽  
Dedong Li ◽  
Rong Zhang ◽  
Huan Sun ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Tandem Repeat ◽  
Dose Regimen ◽  
Subunit Vaccine ◽  
Immune Escape ◽  
Spike Protein ◽  
Receptor Binding Domain ◽  
Protein Receptor ◽  
A Subunit ◽  
Alpha Beta

SARS-CoV-2 variants brought new waves of infection worldwide. In particular, Delta variant (B.1.617.2 lineage) has become predominant in many countries. These variants raised the concern for their potential immune escape to the currently approved vaccines. ZF2001 is a subunit vaccine received emergency use authorization (EUA) in both China and Uzbekistan, with more than 100-million doses administrated with a three-dose regimen. The tandem-repeat dimer of SARS-CoV-2 spike protein receptor binding domain (RBD) was used as the antigen. In this work, we evaluated the neutralization of ZF2001-elicited antisera to SARS-CoV-2 variants including all four variants of concern (Alpha, Beta, Gamma and Delta) and other three variants of interest (Epsilon, Eta and Kappa) by pseudovirus-based assay. We found antisera preserved majority of the neutralizing activity against these variants. E484K/Q substitution is the key mutation to reduce the RBD-elicited sera neutralization. Moreover, ZF2001-elicited sera with a prolonged intervals between the second and third dose enhanced the neutralizing titers and resilience to SARS-CoV-2 variants.

scholarly journals SARS-CoV-2 variants of concern Alpha, Beta, Gamma and Delta have extended ACE2 receptor host-ranges

2021 ◽  
Author(s):  
Nazia Thakur ◽  
Giulia Gallo ◽  
Joseph Newman ◽  
Thomas P Peacock ◽  
Luca Biasetti ◽  
...  
Keyword(s):  
Public Health ◽  
Host Range ◽  
Receptor Binding ◽  
Immune Escape ◽  
Spike Protein ◽  
Receptor Binding Domain ◽  
Public Health Perspective ◽  
Global Prevalence ◽  
Usage Patterns ◽  
Alpha Beta

Following the emergence of SARS-CoV-2 in China in late 2019 a number of variants have emerged, with two of these, Alpha and Delta, subsequently growing to global prevalence. One characteristic of these variants are changes within the Spike protein, in particular the receptor binding domain (RBD). From a public health perspective these changes have important implications for increased transmissibility and immune escape; however, their presence could also modify the intrinsic host-range of the virus. Using viral pseudotyping we examined whether the variants of concern (VOCs) Alpha, Beta, Gamma and Delta have differing host ACE2 receptor usage patterns, focusing on a range of relevant mammalian ACE2 proteins. All four VOCs were able to overcome a previous restriction for mouse ACE2, with demonstrable differences also seen for individual VOCs with rat, ferret or civet ACE2 receptors, changes which we subsequently attribute to N501Y and E484K substitutions within the Spike RBD.

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