Molecular insights into receptor binding of recent emerging SARS-CoV-2 variants.

Multiple SARS-CoV-2 variants of concern (VOCs) have been emerging and some have been linked to an increase in case numbers globally. However, there is yet a lack of understanding of the molecular basis for the interactions between the human ACE2 (hACE2) receptor and these VOCs. Here we examined several VOCs including Alpha, Beta, and Gamma, and demonstrate that five variants receptor-binding domain (RBD) increased binding affinity for hACE2, and four variants pseudoviruses increased entry into susceptible cells. Crystal structures of hACE2-RBD complexes help identify the key residues facilitating changes in hACE2 binding affinity. Additionally, soluble hACE2 protein efficiently prevent most of the variants pseudoviruses. Our findings provide important molecular information and may help the development of novel therapeutic and prophylactic agents targeting these emerging mutants.

Binding and molecular basis of the bat coronavirus RaTG13 virus to ACE2 in humans and other species.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been spreading worldwide, causing a global pandemic. Bat-origin RaTG13 is currently the most phylogenetically related virus. Here we obtained the complex structure of the RaTG13 receptor binding domain (RBD) with human ACE2 (hACE2) and evaluated binding of RaTG13 RBD to 24 additional ACE2 orthologs. By substituting residues in the RaTG13 RBD with their counterparts in the SARS-CoV-2 RBD, we found that residue 501, the major position found in variants of concern (VOCs) 501Y.V1/V2/V3, plays a key role in determining the potential host range of RaTG13. We also found that SARS-CoV-2 could induce strong cross-reactive antibodies to RaTG13 and identified a SARS-CoV-2 monoclonal antibody (mAb), CB6, that could cross-neutralize RaTG13 pseudovirus. These results elucidate the receptor binding and host adaption mechanisms of RaTG13 and emphasize the importance of continuous surveillance of coronaviruses (CoVs) carried by animal reservoirs to prevent another spillover of CoVs.

Molecular basis of cross-species ACE2 interactions with SARS-CoV-2-like viruses of pangolin origin.

Pangolins have been suggested as potential reservoir of zoonotic viruses, including SARS-CoV-2 causing the global COVID-19 outbreak. Here, we study the binding of two SARS-CoV-2-like viruses isolated from pangolins, GX/P2V/2017 and GD/1/2019, to human angiotensin-converting enzyme 2 (hACE2), the receptor of SARS-CoV-2. We find that the spike protein receptor-binding domain (RBD) of pangolin CoVs binds to hACE2 as efficiently as the SARS-CoV-2 RBD in vitro. Furthermore, incorporation of pangolin CoV RBDs allows entry of pseudotyped VSV particles into hACE2-expressing cells. A screen for binding of pangolin CoV RBDs to ACE2 orthologs from various species suggests a broader host range than that of SARS-CoV-2. Additionally, cryo-EM structures of GX/P2V/2017 and GD/1/2019 RBDs in complex with hACE2 show their molecular binding in modes similar to SARS-CoV-2 RBD. Introducing the Q498H substitution found in pangolin CoVs into the SARS-CoV-2 RBD expands its binding capacity to ACE2 homologs of mouse, rat, and European hedgehog. These findings suggest that these two pangolin CoVs may infect humans, highlighting the necessity of further surveillance of pangolin CoVs.

Broad host range of SARS-CoV-2 and the molecular basis for SARS-CoV-2 binding to cat ACE2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the recent pandemic COVID-19, is reported to have originated from bats, with its intermediate host unknown to date. Here, we screened 26 animal counterparts of the human ACE2 (hACE2), the receptor for SARS-CoV-2 and SARS-CoV, and found that the ACE2s from various species, including pets, domestic animals and multiple wild animals, could bind to SARS-CoV-2 receptor binding domain (RBD) and facilitate the transduction of SARS-CoV-2 pseudovirus. Comparing to SARS-CoV-2, SARS-CoV seems to have a slightly wider range in choosing its receptor. We further resolved the cryo-electron microscopy (cryo-EM) structure of the cat ACE2 (cACE2) in complex with the SARS-CoV-2 RBD at a resolution of 3 Å, revealing similar binding mode as hACE2 to the SARS-CoV-2 RBD. These results shed light on pursuing the intermediate host of SARS-CoV-2 and highlight the necessity of monitoring susceptible hosts to prevent further outbreaks.

Structural and Functional Basis of SARS-CoV-2 Entry by Using Human ACE2.

The recent emergence of a novel coronavirus (SARS-CoV-2) in China has caused significant public health concerns. Recently, ACE2 was reported as an entry receptor for SARS-CoV-2. In this study, we present the crystal structure of the C-terminal domain of SARS-CoV-2 (SARS-CoV-2-CTD) spike (S) protein in complex with human ACE2 (hACE2), which reveals a hACE2-binding mode similar overall to that observed for SARS-CoV. However, atomic details at the binding interface demonstrate that key residue substitutions in SARS-CoV-2-CTD slightly strengthen the interaction and lead to higher affinity for receptor binding than SARS-RBD. Additionally, a panel of murine monoclonal antibodies (mAbs) and polyclonal antibodies (pAbs) against SARS-CoV-S1/receptor-binding domain (RBD) were unable to interact with the SARS-CoV-2 S protein, indicating notable differences in antigenicity between SARS-CoV and SARS-CoV-2. These findings shed light on the viral pathogenesis and provide important structural information regarding development of therapeutic countermeasures against the emerging virus.