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.