NL63 coronavirus (NL63-CoV), a prevalent human respiratory virus, is the only group I coronavirus known to use angiotensin-converting enzyme 2 (ACE2) as its receptor. Incidentally, ACE2 is also used by group II SARS coronavirus (SARS-CoV). We investigated how different groups of coronaviruses recognize the same receptor, whereas homologous group I coronaviruses recognize different receptors. We determined the crystal structure of NL63-CoV spike protein receptorbinding domain (RBD) complexed with human ACE2. NL63-CoV RBD has a novel ␤-sandwich core structure consisting of 2 layers of ␤-sheets, presenting 3 discontinuous receptor-binding motifs (RBMs) to bind ACE2. NL63-CoV and SARS-CoV have no structural homology in RBD cores or RBMs; yet the 2 viruses recognize common ACE2 regions, largely because of a ''virus-binding hotspot'' on ACE2. Among group I coronaviruses, RBD cores are conserved but RBMs are variable, explaining how these viruses recognize different receptors. These results provide a structural basis for understanding viral evolution and virus-receptor interactions.receptor protein ͉ SARS coronavirus ͉ spike protein receptor-binding domain ͉ virus-binding hotspots A fundamental yet unresolved puzzle in virology is how viruses evolve to recognize their receptor proteins (1). Specifically, how do different viruses recognize the same receptor protein, and how do similar viruses recognize different receptor proteins? Do viruses select their receptor proteins by chance, or do they target specific virus-binding hotspots on these receptor proteins? Structural information of virus-receptor interfaces can potentially answer these questions. To date, although a few studies have obtained structural information for a single virus-receptor interface (2-6), no study has provided structural information for the interfaces between different viruses and their common receptor protein.Here we provide such structural information, by showing that nonhomologous receptor-binding proteins of 2 coronaviruses bind to the same ''virus-binding hotspot'' on their common protein receptor.A recently identified human coronavirus, NL63 (NL63-CoV), is associated with common colds, croup, and other respiratory diseases (7,8). Potent neutralizing antibodies against NL63-CoV are detected in sera from nearly all humans older than 8 years, suggesting that NL63-CoV infection is common in childhood (7, 9). NL63-CoV belongs to the coronavirus family, a group of enveloped, positive-stranded RNA viruses that infect many mammalian and avian species. Coronaviruses are classified into 3 serologic and genetic groups: mammalian group I, mammalian group II, and avian group III (10). NL63-CoV is the only group I coronavirus known to use angiotensin-converting enzyme 2 (ACE2) as its receptor (9), whereas the others use aminopeptidase-N (APN) (10-12). Curiously, ACE2 is also the receptor for the severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) (13), a group II coronavirus responsible for SARS (14,15).Coronaviruses enter cells through a larg...

Background:The severe acute respiratory syndrome (SARS) virus has undergone mutations in its receptor-binding domain. Results:We used biochemical, functional, and crystallographic methods to investigate these mutations. Conclusion: These mutations were viral adaptations to either the human or palm civet receptor. Significance: This research elucidates detailed mechanisms of host receptor adaptation by the SARS virus and can help predict and monitor future evolution of the SARS virus in animals.

Coronaviruses have evolved diverse mechanisms to recognize different receptors for their cross-species transmission and hostrange expansion. Mouse hepatitis coronavirus (MHV) uses the N-terminal domain (NTD) of its spike protein as its receptorbinding domain. Here we present the crystal structure of MHV NTD complexed with its receptor murine carcinoembryonic antigenrelated cell adhesion molecule 1a (mCEACAM1a). Unexpectedly, MHV NTD contains a core structure that has the same β-sandwich fold as human galectins (S-lectins) and additional structural motifs that bind to the N-terminal Ig-like domain of mCEACAM1a. Despite its galectin fold, MHV NTD does not bind sugars, but instead binds mCEACAM1a through exclusive protein-protein interactions. Critical contacts at the interface have been confirmed by mutagenesis, providing a structural basis for viral and host specificities of coronavirus/CEACAM1 interactions. Sugar-binding assays reveal that galectin-like NTDs of some coronaviruses such as human coronavirus OC43 and bovine coronavirus bind sugars. Structural analysis and mutagenesis localize the sugar-binding site in coronavirus NTDs to be above the β-sandwich core. We propose that coronavirus NTDs originated from a host galectin and retained sugar-binding functions in some contemporary coronaviruses, but evolved new structural features in MHV for mCEACAM1a binding. Coronaviruses use a variety of cellular receptors and coreceptors, including proteins and sugars. The diverse use of receptors has allowed coronaviruses to infect a wide range of mammalian and avian species and cause respiratory, enteric, systemic, and neurological diseases. How coronaviruses have evolved to do so has been a major puzzle in virology. To solve this puzzle, we have investigated the structural basis for the complex receptorrecognition mechanisms of coronaviruses.

e Porcine epidemic diarrhea coronavirus (PEDV) has significantly damaged America's pork industry. Here we investigate the receptor usage and cell entry of PEDV. PEDV recognizes protein receptor aminopeptidase N from pig and human and sugar coreceptor N-acetylneuraminic acid. Moreover, PEDV infects cells from pig, human, monkey, and bat. These results support the idea of bats as an evolutionary origin for PEDV, implicate PEDV as a potential threat to other species, and suggest antiviral strategies to control its spread. P orcine epidemic diarrhea coronavirus (PEDV) causes largescale outbreaks of diarrhea in pigs and an 80 to 100% fatality rate in suckling piglets (1-3). Since 2013, PEDV has swept throughout the United States, wiped out more than 10% of America's pig population in less than a year, and significantly damaged the U.S. pork industry (4-6). No vaccine or antiviral drug is currently available to keep the spread of PEDV in check. PEDV belongs to the ␣ genus of the coronavirus family (7,8), which also includes porcine transmissible gastroenteritis coronavirus (TGEV), bat coronavirus 512/2005 (BtCoV/512/2005), and human NL63 coronavirus (HCoV-NL63). Although both PEDV and TGEV infect pigs, PEDV is genetically more closely related to BtCoV/512/ 2005 than to TGEV, leading to the hypothesis that PEDV originated from bats (9).Receptor binding and cell entry are essential steps in viral infection cycles, critical determinants of viral host range and tropism, and important targets for antiviral interventions. An envelope-anchored spike protein mediates coronavirus entry into cells. The spike ectodomain consists of a receptor-binding subunit, S1, and a membrane fusion subunit, S2. S1 contains two domains, an N-terminal domain (S1-NTD) and a C-terminal domain (S1-CTD), both of which can potentially function as receptor-binding domains (RBDs) (Fig. 1A) (10, 11). The ability of coronavirus RBDs to recognize receptor orthologs from different species is one of the most important determinants of coronavirus host range and tropism (8,(12)(13)(14). HCoV-NL63 S1-CTD recognizes human angiotensin-converting enzyme 2 (ACE2), whereas TGEV S1-CTD recognizes porcine aminopeptidase N (APN), and its S1-NTD recognizes two sugar coreceptors, N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) (15-18). Usage of sugar coreceptors is linked to the enteric tropism of coronaviruses (18,19). It has been shown that PEDV uses porcine APN as its receptor (20). However, it is not known whether PEDV recognizes APN from other species or whether it uses sugar coreceptors. Addressing these questions will be critical for understanding the host range, tropism, and evolutionary origin of PEDV, for evaluating its potential risk to other species, particularly humans, and for developing effective vaccines and antiviral drugs to curb the spread of PEDV in pigs and to other species.To characterize the receptor usage of PEDV, here we identified the two S1 domains of PEDV based on the sequence similarity between PEDV and TGEV S1 subun...

Background: Coronavirus spike protein N-terminal domains (NTDs) bind sugar or protein receptors. Results: We determined crystal structure of bovine coronavirus NTD and located its sugar-binding site using mutagenesis. Conclusion: Bovine coronavirus NTD shares structural folds and sugar-binding sites with human galectins and has subtle yet functionally important differences from protein-binding NTD of mouse coronavirus. Significance: This study explores origin and evolution of coronavirus NTDs.

Mammalian aminopeptidase N (APN) plays multifunctional roles in many physiological processes, including peptide metabolism, cell motility and adhesion, and coronavirus entry. Here we determined crystal structures of porcine APN at 1.85 Å resolution and its complexes with a peptide substrate and a variety of inhibitors. APN is a cell surface-anchored and seahorse-shaped zinc-aminopeptidase that forms head-to-head dimers. Captured in a catalytically active state, these structures of APN illustrate a detailed catalytic mechanism for its aminopeptidase activity. The active site and peptidebinding channel of APN reside in cavities with wide openings, allowing easy access to peptides. The cavities can potentially open up further to bind the exposed N terminus of proteins. The active site anchors the N-terminal neutral residue of peptides/proteins, and the peptide-binding channel binds the remainder of the peptides/ proteins in a sequence-independent fashion. APN also provides an exposed outer surface for coronavirus binding, without its physiological functions being affected. These structural features enable APN to function ubiquitously in peptide metabolism, interact with other proteins to mediate cell motility and adhesion, and serve as a coronavirus receptor. This study elucidates multifunctional roles of APN and can guide therapeutic efforts to treat APN-related diseases.M ammalian aminopeptidase N (APN) plays pivotal roles in many physiological processes, such as pain sensation, blood pressure regulation, tumor angiogenesis and metastasis, immune cell chemotaxis, sperm motility, cell-cell adhesion, and coronavirus entry (1). Accordingly, APN is a major target for treatment of diseases that are related to the above physiological processes. It is puzzling how APN is able to possess such a wide range of physiological functions, some of which are seemingly unrelated to its aminopeptidase activity. This study determines the atomic structures of mammalian APN and its complexes with a variety of APN-targeting ligands, providing structural basis for the multifunctional roles of APN and for the development of novel therapy strategies to treat APN-related diseases.The M1-family of metalloenzymes consists of a large number of zinc-dependent aminopeptidases containing a zinc-binding HEXXH motif. As the most extensively studied member in this family, mammalian APN (also known as CD13 or alanine aminopeptidase) is widely expressed on cell surfaces of tissues, such as intestinal epithelia and the nervous system (1). APN preferentially cleaves neutral amino acids, most notably alanine, off the N terminus of peptides. The general catalytic mechanism of M1-family metalloenzymes is believed to be similar to that of prototypic zincpeptidase thermolysin, which involves catalytic water attacking scissile peptide bonds (2), but detailed catalytic mechanisms of these enzymes remain elusive. To date, crystal structures are available for several members of the M1-family metalloenzymes (3-8). However, these enzymes are monomeric intracellu...

Porcine epidemic diarrhea virus (PEDV) is

Coronaviruses recognize a variety of receptors using different domains of their envelopeanchored spike protein. How these diverse receptor recognition patterns affect viral entry is unknown. Mouse hepatitis coronavirus (MHV) is the only known coronavirus that uses the N-terminal domain (NTD) of its spike to recognize a protein receptor, CEACAM1a. Here we determined the cryo-EM structure of MHV spike complexed with mouse CEACAM1a. The trimeric spike contains three receptor-binding S1 heads sitting on top of a trimeric membrane-fusion S2 stalk. Three receptor molecules bind to the sides of the spike trimer, where three NTDs are located. Receptor binding induces structural changes in the spike, weakening the interactions between S1 and S2. Using protease sensitivity and negative-stain EM analyses, we further showed that after protease treatment of the spike, receptor binding facilitated the dissociation of S1 from S2, allowing S2 to transition from pre-fusion to postfusion conformation. Together these results reveal a new role of receptor binding in MHV entry: in addition to its well-characterized role in viral attachment to host cells, receptor binding also induces the conformational change of the spike and hence the fusion of viral and host membranes. Our study provides new mechanistic insight into coronavirus entry and highlights the diverse entry mechanisms used by different viruses. Author summaryCoronaviruses recognize many receptors using their envelope-anchored spike protein.The role of receptor binding in coronavirus entry into host cells is a fundamental question in virology. Mouse hepatitis coronavirus (MHV) is unique among all coronaviruses in that it uses the N-terminal domain (NTD) of its spike protein to bind a protein receptor CEACAM1a. While extensive research has been performed on the cell entry mechanisms of coronaviruses that use a different domain of their spike protein for receptor binding, the cell entry mechanism for MHV is still elusive. Here we determined the cryo-EM PLOS PATHOGENS PLOS Pathogens | https://doi., et al. (2020) Structure of mouse coronavirus spike protein complexed with receptor reveals mechanism for viral entry. PLoS Pathog 16 (3): e1008392. https://doi.org/10.Data Availability Statement: The cryo-EM map has been deposited in the Electron Microscopy Data Bank (EMD) under accession code EMD-21377. The atomic model has been deposited in the Protein Data Bank (PDB) under accession code 6VSJ.Funding: This work was supported by R01AI089728 (to F. Li) from National Institute of Allergy and Infectious Diseases (https://www.niaid. nih.gov/). The funders had no role in study design, structure of MHV spike protein complexed with CEACAM1a. The structure reveals unique features of receptor binding by MHV spike that facilitate the structural changes of MHV spike and promote cell entry of MHV. We further confirmed the structural results with biochemical and negative-stain EM analyses. These results suggest that receptor binding plays dual roles in MHV entry: it promotes both viral attachm...