The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) positive RNA detected in red fox swab samples provide evidence of a natural SARS-CoV-2 infection in this species. Many studies have also demonstrated that the red fox angiotensin converting enzyme 2 (fACE2) could serve as the receptor of SARS-CoV-2 and many SARS-CoV-2 related sarbecoviruses. However, the molecular mechanisms of fACE2 attached by animal-origin coronaviruses remain poorly understood. Here, we found that fACE2 could mediate pseudovirus entry and cell membrane fusion induced by the SARS-CoV-2 and pangolin coronavirus isolated from Guangdong (PCoV-GD) spikes as human ACE2 (hACE2). The binding affinity of fACE2 bound to the receptor-binding domain (RBD) of PCoV-GD was stronger than the SARS-CoV-2 RBD demonstrated by surface plasmon resonance (SPR) assay, while deglycosylation of PCoV-GD RBD N370 by T372A mutation or glycosylation of SARS-CoV-2 RBD N370 by A372T mutation had little effect on fACE2 binding compared with wild-type (WT) RBD. We further solved the crystal structures of fACE2 bound to SARS-CoV-2 RBD and PCoV-GD T372A mutant RBD. Interface analysis and SPR assay revealed that the R417 and H498 of PCoV-GD RBD might account for the binding affinity enhancement with fACE2 compared with K417 and Q498 of SARS-CoV-2 RBD. Moreover, the Y453F mutation in SARS-CoV-2 RBD increased the binding affinity to fACE2 while this mutation in PCoV-GD RBD decreased the binding affinity to fACE2 compared with the corresponding WT RBDs. Furthermore, the RBDs of many prevalent SARS-CoV-2 variants could all bind to fACE2. Our results indicated that the continuous surveillance of SARS-CoV-2 and related sarbecoviruses in fox species was also necessary to better prevent animal-to-human spillover of the coronaviruses.

SARS-CoV-2 has a broad animal host range, but its origin and intermediate hosts are still debated. Arctic ground squirrel is proved to be a susceptible animal host of SARS-CoV-2 and RaTG13, as the Arctic ground squirrel ACE2 (sACE2) could bind to the RBDs of SARS-CoV-2 and RaTG13, and support transduction of the SARS-CoV-2 and RaTG13 pseudovirions. Here, we determined crystal structures of sACE2 bound to the RBDs of RaTG13, PCoV-GD, PCoV-GX, SARS-CoV-2 A372T and SARS-CoV-2 JN.1 variant. SPR assay indicated that RBD residues 493, 498 and 501 might be important for sACE2 recognition. Notably, N322-linked glycans of sACE2 were found to be in contact with S375 of the RaTG13 RBD. Moreover, the recent SARS-CoV-2 KP.2 and KP.3 variant RBDs could also bind to the sACE2. Our results provide further insights into interactions between coronavirus RBD and cell receptor ACE2, which may promote our understanding of SARS-CoV-2 evolution.

Bat coronaviruses ZXC21 and ZC45 were discovered before the COVID-19 outbreak and share approximately 86% genome homology with SARS-CoV-2. Earlier studies indicated that ZXC21 and ZC45 may be involved in the emergence of SARS-CoV-2. However, the cell invasion mechanisms of ZXC21 and ZC45 remain unclear. Here, we determined the crystal structure of the ZXC21 receptor-binding domain (RBD) and found that the core structure shared high similarity with SARS-CoV-2, MERS-CoV, human coronavirus (HCoV)-HKU1, SARS-CoV, and HCoV-OC43 RBDs, whereas the receptor-binding motifs (RBMs) differ. We demonstrated that the ZXC21 RBD had no interaction with the human coronavirus receptors angiotensin-converting enzyme 2 (ACE2), dipeptidylpeptidase 4 (DPP4), aminopeptidase N (APN), or transmembrane serine protease 2 (TMPRSS2) by surface plasmon resonance (SPR). Moreover, the P5S-3B11 Fab can bind to the ZXC21 RBD, indicating that this SARS-CoV-2 core-targeting antibody may retain neutralizing activity toward the ZXC21 coronavirus. Our results revealed the bat coronavirus ZXC21 RBD structure, which may provide further insights into the evolution of SARS-CoV-2 and the other human beta-coronaviruses.

The discovery of the RaTG13 coronavirus in Rhinolophus affinis bats in 2013, sharing 96.3% genome homology with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), suggested bats as the origin of SARS-CoV-2. Although both human angiotensin-converting enzyme 2 (hACE2) and Rhinolophus affinis bat ACE2 (bACE2-Ra, seven polymorphic variants named 01-07) are known to serve as entry receptors for these coronaviruses, the binding mechanism of RaTG13 receptor binding domain (RBD) bound to bACE2-Ra remains poorly understood. Here, we found that RaTG13 RBD bound to bACE2-Ra-07 with a weaker affinity (2.42 μM) compared with SARS-CoV-2 RBD (372 nM). Additional glycosylation at residue N370 of RaTG13 had little influence on bACE2-Ra-07 binding by RaTG13 RBD. Crystal structures of the SARS-CoV-2 and RaTG13 N370Q RBD bound to bACE2-Ra-07 were solved. Interface analysis and surface plasmon resonance (SPR) assay indicated that residue substitutions at 493, 498, 501, and 505 may play a more important role in the cross-species recognition of bACE2-Ra-07 by the SARS-CoV-2 RBD. Besides, the N370Q mutation enhanced the binding affinity between the RBD of pangolin coronavirus isolated from Guangxi (PCoV-GX) and the bACE2-Ra-07 receptor by over 10-fold. Furthermore, the recently prevalent SARS-CoV-2 variant RBDs extensively retained the interaction with the bACE2-Ra-07 receptor. Our findings give new lights on the cross-species evolution of SARS-CoV-2 and prompt the urgency to monitor the circulation of coronaviruses in bats to better prevent future spillover.

The global spread of COVID-19 remains a significant threat to human health. The SARS-CoV-2 BQ.1.1 lineage, including BA.5.2, BF.7, BQ.1 and BQ.1.1, caused a new soaring of infection cases due to rapid transmission. However, the receptor binding mechanism and immune evasion capacity of these variants need to be explored further. Our study found that while the BA.5.2, BF.7 and BQ.1.1 variants pseudovirus had similar cell entry efficiency, the BF.7 and BQ.1.1 RBD bound to human ACE2 (hACE2) with a slightly stronger affinity than the BA.5.2 RBD. Structural analysis revealed R346T, K444T, and N460K mutations altered RBD-hACE2 binding interface details and surface electrostatic potential of BQ.1.1 RBD. Serum neutralization tests showed BQ.1.1 variant had stronger immune evasion capacity than BA.5.2 and BF.7 variants. Our findings illustrated the receptor binding mechanism and serological neutralization activity of the BA.5.2, BF.7 and BQ.1.1 variants, which verified the necessity for further antibody therapy optimization and vaccination development.

Langya virus (LayV) was recently detected in patients with acute pneumonic diseases in China. Genome alignment indicated that LayV is a type of zoonotic henipavirus (HNV) that might also infect domestic animals. Previous studies revealed that HNVs mainly use ephrin-B1, ephrin-B2, or ephrin-B3 as cell receptors and the attachment glycoprotein (G) is the host cell receptor-binding protein. However, the LayV receptor remains unknown. Here, we present the 2.77 Å crystal structure of the LayV G C-terminal domain (CTD). We show that the LayV G protein CTD possesses a similar architecture as the Mojiang virus (MojV) G protein but is markedly different from the Nipah virus (NiV), Hendra virus (HeV), and Cedar virus (CedV) G proteins. Surface plasmon resonance (SPR) experiments indicate that LayV G does not bind ephrin-B proteins. Steric hindrance may prevent interactions between LayV G and ephrin-B. Our data might facilitate drug development targeting LayV.

Bacillus thuringiensis Vip3Aa has been widely used in transgenic crops to resist the erosion of insects. The Scavenger Receptor-C (SR-C) and Fibroblast Growth Factor Receptor (FGFR) of Spodoptera frugiperda (Sf-SR-C and Sf-FGFR) have formerly been identified as the cell receptors of Vip3Aa. However, the interaction mechanism of Vip3Aa binding to Sf-SR-C or Sf-FGFR is still unknown. Here, we purified the MAM domain of Sf-SR-C (Sf-MAM) and the Sf-FGFR ectodomain expressed extracellularly by Sf9 cells. We then solved the crystal structure of the Sf-MAM domain. Structure docking analysis of the Sf-MAM and Vip3Aa C-terminal domain (CTD) excluded the possibility of the two proteins binding. A further surface plasmon resonance (SPR) assay also revealed that the Sf-MAM and Sf-FGFR ectodomain could not bind to the Vip3Aa protein. Our results have raised the urgency of determining the authentic cell receptor for Vip3Aa.

IntroductionSince the outbreak of SARS-CoV-2, vaccines have demonstrated their effectiveness in resisting virus infection, reducing severity, and lowering the mortality rate in infected individuals. However, due to the rapid and ongoing mutations of SARS-CoV-2, the protective ability of many available vaccines has been challenged. Therefore, there is an urgent need for vaccines capable of eliciting potent broadly neutralizing antibodies against various SARS-CoV-2 variants.MethodsIn this study, we developed a novel subunit vaccine candidate for SARS-CoV-2 by introducing a series of shielding glycans to the Fc-fused receptor-binding domain (RBD) of the prototypic spike protein. This approach aims to mask non-neutralizing epitopes and focus the immune response on crucial neutralizing epitopes.ResultsAll modified sites were confirmed to be highly glycosylated through mass spectrometry analysis. The binding affinity of the glycan-shielded RBD (gsRBD) to the human ACE2 receptor was comparable to that of the wildtype RBD (wtRBD). Immunizing mice with gsRBD when combined with either Freund’s adjuvant or aluminum adjuvant demonstrated that the introduction of the glycan shield did not compromise the antibody-inducing ability of RBD. Importantly, the gsRBD significantly enhanced the generation of neutralizing antibodies against SARS-CoV-2 pseudoviruses compared to the wtRBD. Notably, it exhibited remarkable protective activity against Beta (B.1.351), Delta (B.1.617.2), and Omicron (B.1.1.529), approximately 3-fold, 7- fold, and 17-fold higher than wtRBD, respectively.DiscussionOur data proved this multiple-epitope masking strategy as an effective approach for highly active vaccine production.

The recently emerged SARS-CoV-2 Omicron variant with numerous mutations in the spike protein has rapidly become the dominant strain, thereby raising concerns about the effectiveness of vaccines. Here, we found that the Omicron variant exhibits a reduced sensitivity to serum neutralizing activity induced by a three-dose inactivated vaccine but remains sensitive to entry inhibitors or an ACE2-Ig decoy receptor.

Background:Rapid and efficient strategies are needed to discover neutralizing antibodies (nAbs) from B cells derived from virus-infected patients. Methods:Here, we report a high-throughput single-B-cell cloning method for high-throughput isolation of nAbs targeting diverse epitopes on the SARS-CoV-2-RBD (receptor binding domain) from convalescent COVID-19 patients. This method is simple, fast and highly efficient in generating SARS-CoV-2-neutralizing antibodies from COVID-19 patients' B cells. Results:Using this method, we have developed multiple nAbs against distinct SARS-CoV-2-RBD epitopes. CryoEM and crystallography revealed precisely how they bind RBD. In live virus assay, these nAbs are effective in blocking viral entry to the host cells. Conclusion:This simple and efficient method may be useful in developing human therapeutic antibodies for other diseases and next pandemic.

Salt bridges are important factors in maintaining the stability of proteins, and their contribution to protein folding has received much attention. Although the interaction energies, or stabilizing contributions, of individual salt bridges have been measured in various proteins, a systematic assessment of various types of salt bridges in a relatively uniform environment is still a valuable analysis. Here, we used a collagen heterotrimer as a host-guest platform to construct 48 heterotrimers with the same charge pattern. A variety of salt bridges were formed between the oppositely charged residues Lys, Arg, Asp, and Glu. The melting temperature (Tm) of the heterotrimers was measured with circular dichroism. The atomic structures of 10 salt bridges were shown in three x-ray crystals of heterotrimer. Molecular dynamics simulation based on the crystal structures indicated that strong, intermediate, and weak salt bridges have distinctive N-O distances. A linear regression model was used to predict the stability of heterotrimers with high accuracy (R2 = 0.93). We developed an online database to help readers understand how a salt bridge stabilizes collagen. This work will help us better understand the stabilizing mechanism of salt bridges in collagen folding and provide a new strategy to design collagen heterotrimers.

ABSTRACTSince SARS-CoV-2 Omicron variant (B.1.1.529) was reported in November 2021, it has quickly spread to many countries and outcompeted the globally dominant Delta variant in several countries. The Omicron variant contains the largest number of mutations to date, with 32 mutations located at spike (S) glycoprotein, which raised great concern for its enhanced viral fitness and immune escape[1–4]. In this study, we reported the crystal structure of the receptor binding domain (RBD) of Omicron variant S glycoprotein bound to human ACE2 at a resolution of 2.6 Å. Structural comparison, molecular dynamics simulation and binding free energy calculation collectively identified four key mutations (S477N, G496S, Q498R and N501Y) for the enhanced binding of ACE2 by the Omicron RBD compared to the WT RBD. Representative states of the WT and Omicron RBD-ACE2 systems were identified by Markov State Model, which provides a dynamic explanation for the enhanced binding of Omicron RBD. The effects of the mutations in the RBD for antibody recognition were analyzed, especially for the S371L/S373P/S375F substitutions significantly changing the local conformation of the residing loop to deactivate several class IV neutralizing antibodies.

NH–π interactions between polar and aromatic residues are well distributed in proteins whose stabilizing effects have been investigated in globular and fibrous proteins. In order to gain structural insights into side chain NH–π interactions, we solved a crystal structure of a collagen-like peptide containing Gln-Phe pairs. The Gln-Phe NH–π interactions were further characterized by quantum calculations, molecular simulations, and structural bioinformatics. The analyses indicated that the NH–π interactions are robust under various solvent conditions, can be distributed either on the protein surface or in its hydrophobic core and can form at a wide range of distances between residues. This study suggested that NH–π interactions can play a versatile role in protein design, including engineering hydrophobic cores, solvent accessible surfaces, and protein–protein interfaces.

Severe acute respiratory syndrome coronavirus (SARS-CoV), SARS-CoV-2, and human coronavirus (hCoV)-NL63 utilize ACE2 as the functional receptor for cell entry, which leads to zoonotic infection. Horses (Equus caballus) attracted our attention because the spike protein receptor-binding domains (RBDs) of SARS-CoV-2 and SARS-CoV-2-related coronaviruses bind equine ACE2 (eACE2) with high affinity. Here we show that eACE2 binds the RBDs of these three coronaviruses and also SARS-CoV-2 variants but with lower affinities compared with human ACE2 (hACE2). Structural analysis and mutation assays indicated that eACE2-H41 ac-counts for the lower binding affinity of eACE2 to the RBDs of SARS-CoV-2 variants (Alpha, Beta, and Gamma), SARS-CoV, and hCoV-NL63. Pseudovirus infection assays showed that the SARS-CoV-2 Delta strain (B.1.617.2) displayed a significantly increased infection efficiency in eACE2-expressing HeLa cells. Our re-sults reveal the molecular basis of eACE2 binding to the RBDs of SARS-CoV, SARS-CoV-2, and hCoV-NL63, which provides insights into the potential animal transmission of these ACE2-dependent corona -viruses.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, causing COVID-19, is the most challenging pandemic of the modern era. It has resulted in over 5 million deaths worldwide. To quickly explore therapeutics for COVID-19, we utilized a previously-established system, namely CEBIT. We performed a high-throughput screening of FDA-approved drugs to inhibit the interaction between the receptor-binding domain (RBD) of SARS-CoV-2 spike proteinand its obligate receptor ACE2. This interaction is essential for viral entry and therefore represents a promising therapeutic target. Based on the recruitment of interacting molecules into phase-separated condensates as a readout, we identified six positive candidates from a library of 2572 compounds, most of which have been reported to inhibit the entry of SARS-CoV-2 into host cells. Our surface plasmon resonance (SPR) and molecular docking analyses revealed the possible mechanisms via which these compounds interfere with the interaction between RBD and ACE2. Hence, our results indicate that CEBIT is highly versatile for identifying drugs against SARS-CoV-2 entry, and targeting CoV-2 entry by small molecule drugs is a viable therapeutic option to treat COVID-19 in addition to commonly used monoclonal antibodies.

The neutralizing antibody is a potential therapeutic for the ongoing COVID-19 pandemic. As an antiviral agent, numerous mAbs recognize the epitopes that overlap with ACE2-binding sites in the SARS-CoV-2-RBD. Some studies have shown that residual changes on the spike protein can significantly decrease the efficiency of neutralizing antibodies. To address this issue, a therapeutic cocktail could be an effective countermeasure. In the present study, we isolated a fully human neutralizing antibody, JS026, from a convalescent patient. The comparative analysis revealed that JS026 binding to SARS-CoV-2-RBD mainly located between epitopes for class 2 and class 3 mAbs as opposed to that of class 1 (etesevimab) antibodies. A cocktail of etesevimab and JS026 increased neutralizing efficacy against both wild-type SARS-CoV-2 and the recent emergence of Alpha, Beta, Gamma, and Delta variants. JS026 and the cocktail reduced virus titers in the infected lungs of hACE2 transgenic mice and relieved pathological changes. These findings would benefit antibody-based therapeutic countermeasures in the treatment of COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants continue to emerge during the global pandemic and may facilitate escape from current antibody therapies and vaccine protection. Here we showed that the South African variant B.1.351 was the most resistant to current monoclonal antibodies and convalescent plasma from coronavirus disease 2019 (COVID-19)-infected individuals, followed by the Brazilian variant P.1 and the United Kingdom variant B.1.1.7. This resistance hierarchy corresponded with Y144del and 242-244del mutations in the N-terminal domain and K417N/T, E484K, and N501Y mutations in the receptor-binding domain (RBD) of SARS-CoV-2. Crystal structure analysis of the B.1.351 triple mutant (417N-484K-501Y) RBD complexed with the monoclonal antibody P2C-1F11 revealed the molecular basis for antibody neutralization and escape. B.1.351 and P.1 also acquired the ability to use mouse and mink ACE2 receptors for entry. Our results demonstrate major antigenic shifts and potential broadening of the host range for B.1.351 and P.1 variants, which poses serious challenges to current antibody therapies and vaccine protection.

In recognizing the host cellular receptor and mediating fusion of virus and cell membranes, the spike (S) glycoprotein of coronaviruses is the most critical viral protein for cross-species transmission and infection. Here we determined the cryo-EM structures of the spikes from bat (RaTG13) and pangolin (PCoV_GX) coronaviruses, which are closely related to SARS-CoV-2. All three receptor-binding domains (RBDs) of these two spike trimers are in the “down” conformation, indicating they are more prone to adopt the receptor-binding inactive state. However, we found that the PCoV_GX, but not the RaTG13, spike is comparable to the SARS-CoV-2 spike in binding the human ACE2 receptor and supporting pseudovirus cell entry. We further identified critical residues in the RBD underlying different activities of the RaTG13 and PCoV_GX/SARS-CoV-2 spikes. These results collectively indicate that tight RBD–ACE2 binding and efficient RBD conformational sampling are required for the evolution of SARS-CoV-2 to gain highly efficient infection.

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in more than 235 million cases worldwide and 4.8 million deaths (October 2021), with various incidences and mortalities among regions/ethnicities. The coronaviruses SARS-CoV, SARS-CoV-2, and HCoV-NL63 utilize the angiotensin-converting enzyme 2 (ACE2) as the receptor to enter cells. We hypothesized that the genetic variability in ACE2 may contribute to the variable clinical outcomes of COVID-19. To test this hypothesis, we first conducted an in silico investigation of single-nucleotide polymorphisms (SNPs) in the coding region of ACE2. We then applied an integrated approach of genetics, biochemistry, and virology to explore the capacity of select ACE2 variants to bind coronavirus spike proteins and mediate viral entry. We identified the ACE2 D355N variant that restricts the spike protein-ACE2 interaction and consequently limits infection both in vitro and in vivo. In conclusion, ACE2 polymorphisms could modulate susceptibility to SARS-CoV-2, which may lead to variable disease severity. IMPORTANCE There is considerable variation in disease severity among patients infected with SARS-CoV-2, the virus that causes COVID-19. Human genetic variation can affect disease outcome, and the coronaviruses SARS-CoV, SARS-CoV-2, and HCoV-NL63 utilize human ACE2 as the receptor to enter cells. We found that several missense ACE2 single-nucleotide variants (SNVs) that showed significantly altered binding with the spike proteins of SARSCoV, SARS-CoV-2, and NL63-HCoV. We identified an ACE2 SNP, D355N, that restricts the spike protein-ACE2 interaction and consequently has the potential to protect individuals against SARS-CoV-2 infection. Our study highlights that ACE2 polymorphisms could impact human susceptibility to SARS-CoV-2, which may contribute to ethnic and geographical differences in SARS-CoV-2 spread and pathogenicity.