ABSTRACT
A novel severe acute respiratory syndrome (SARS)-like coronavirus (SARS-CoV-2) is causing the global coronavirus disease 2019 (COVID-19) pandemic. Understanding how SARS-CoV-2 enters human cells is a high priority for deciphering its mystery and curbing its spread. A virus surface spike protein mediates SARS-CoV-2 entry into cells. To fulfill its function, SARS-CoV-2 spike binds to its receptor human ACE2 (hACE2) through its receptor-binding domain (RBD) and is proteolytically activated by human proteases. Here we investigated receptor binding and protease activation of SARS-CoV-2 spike using biochemical and pseudovirus entry assays. Our findings have identified key cell entry mechanisms of SARS-CoV-2. First, SARS-CoV-2 RBD has higher hACE2 binding affinity than SARS-CoV RBD, supporting efficient cell entry. Second, paradoxically, the hACE2 binding affinity of the entire SARS-CoV-2 spike is comparable to or lower than that of SARS-CoV spike, suggesting that SARS-CoV-2 RBD, albeit more potent, is less exposed than SARS-CoV RBD. Third, unlike SARS-CoV, cell entry of SARS-CoV-2 is preactivated by proprotein convertase furin, reducing its dependence on target cell proteases for entry. The high hACE2 binding affinity of the RBD, furin preactivation of the spike, and hidden RBD in the spike potentially allow SARS-CoV-2 to maintain efficient cell entry while evading immune surveillance. These features may contribute to the wide spread of the virus. Successful intervention strategies must target both the potency of SARS-CoV-2 and its evasiveness.
Subject(s)
Severe acute respiratory syndrome-related coronavirus/physiology , Virus Internalization , Angiotensin-Converting Enzyme 2 , Cell Line , Humans , Immune Evasion , Peptidyl-Dipeptidase A/chemistry , Peptidyl-Dipeptidase A/metabolism , Protein Domains , Receptors, Coronavirus , Receptors, Virus/chemistry , Receptors, Virus/metabolism , Severe acute respiratory syndrome-related coronavirus/chemistry , Severe acute respiratory syndrome-related coronavirus/immunology , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism , Virus ActivationABSTRACT
A novel severe acute respiratory syndrome (SARS)-like coronavirus (SARS-CoV-2) recently emerged and is rapidly spreading in humans, causing COVID-191,2. A key to tackling this pandemic is to understand the receptor recognition mechanism of the virus, which regulates its infectivity, pathogenesis and host range. SARS-CoV-2 and SARS-CoV recognize the same receptor-angiotensin-converting enzyme 2 (ACE2)-in humans3,4. Here we determined the crystal structure of the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 (engineered to facilitate crystallization) in complex with ACE2. In comparison with the SARS-CoV RBD, an ACE2-binding ridge in SARS-CoV-2 RBD has a more compact conformation; moreover, several residue changes in the SARS-CoV-2 RBD stabilize two virus-binding hotspots at the RBD-ACE2 interface. These structural features of SARS-CoV-2 RBD increase its ACE2-binding affinity. Additionally, we show that RaTG13, a bat coronavirus that is closely related to SARS-CoV-2, also uses human ACE2 as its receptor. The differences among SARS-CoV-2, SARS-CoV and RaTG13 in ACE2 recognition shed light on the potential animal-to-human transmission of SARS-CoV-2. This study provides guidance for intervention strategies that target receptor recognition by SARS-CoV-2.
Subject(s)
Betacoronavirus/chemistry , Peptidyl-Dipeptidase A/chemistry , Peptidyl-Dipeptidase A/metabolism , Receptors, Virus/chemistry , Receptors, Virus/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Zoonoses/virology , Angiotensin-Converting Enzyme 2 , Animals , Betacoronavirus/drug effects , Betacoronavirus/metabolism , Binding Sites , COVID-19 , China/epidemiology , Chiroptera/virology , Coronavirus/chemistry , Coronavirus/isolation & purification , Coronavirus Infections/drug therapy , Coronavirus Infections/epidemiology , Coronavirus Infections/transmission , Coronavirus Infections/virology , Crystallization , Crystallography, X-Ray , Disease Reservoirs/virology , Eutheria/virology , Humans , Models, Molecular , Pandemics , Pneumonia, Viral/drug therapy , Pneumonia, Viral/epidemiology , Pneumonia, Viral/transmission , Pneumonia, Viral/virology , Protein Binding , Protein Domains , Protein Stability , Severe acute respiratory syndrome-related coronavirus/chemistry , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/genetics , Zoonoses/epidemiology , Zoonoses/transmissionABSTRACT
Coronaviruses recognize a variety of receptors using different domains of their envelope-anchored 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 post-fusion 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.