Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV

Since 2002, beta coronaviruses (CoV) have caused three zoonotic outbreaks, SARS-CoV in 2002–2003, MERS-CoV in 2012, and the newly emerged SARS-CoV-2 in late 2019. However, little is currently known about the biology of SARS-CoV-2. Here, using SARS-CoV-2 S protein pseudovirus system, we confirm that human angiotensin converting enzyme 2 (hACE2) is the receptor for SARS-CoV-2, find that SARS-CoV-2 enters 293/hACE2 cells mainly through endocytosis, that PIKfyve, TPC2, and cathepsin L are critical for entry, and that SARS-CoV-2 S protein is less stable than SARS-CoV S. Polyclonal anti-SARS S1 antibodies T62 inhibit entry of SARS-CoV S but not SARS-CoV-2 S pseudovirions. Further studies using recovered SARS and COVID-19 patients’ sera show limited cross-neutralization, suggesting that recovery from one infection might not protect against the other. Our results present potential targets for development of drugs and vaccines for SARS-CoV-2. SARS-CoV-2 has spread globally. Here, the authors characterize the entry pathway of SARS-CoV-2, show that the SARS-CoV-2 spike protein is less stable than that of SARS-CoV, and show limited cross-neutralization activities between SARS-CoV and SARS-CoV-2 sera.

[1]  S. Harrison,et al.  Structure of SARS Coronavirus Spike Receptor-Binding Domain Complexed with Receptor , 2005, Science.

[2]  Christian Drosten,et al.  Identification of a novel coronavirus in patients with severe acute respiratory syndrome. , 2003, The New England journal of medicine.

[3]  K. Holmes,et al.  Receptor for mouse hepatitis virus is a member of the carcinoembryonic antigen family of glycoproteins. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[4]  S. Perlman,et al.  Proteolytic processing of Middle East respiratory syndrome coronavirus spikes expands virus tropism , 2016, Proceedings of the National Academy of Sciences.

[5]  Yi Shi,et al.  Molecular basis of binding between novel human coronavirus MERS-CoV and its receptor CD26 , 2013, Nature.

[6]  Y. Sakurai,et al.  Two-pore channels control Ebola virus host cell entry and are drug targets for disease treatment , 2015, Science.

[7]  Xiuyuan Ou,et al.  Identification of the Fusion Peptide-Containing Region in Betacoronavirus Spike Glycoproteins , 2016, Journal of Virology.

[8]  Jonathan H. Epstein,et al.  Bats Are Natural Reservoirs of SARS-Like Coronaviruses , 2005, Science.

[9]  G. Gao,et al.  A Novel Coronavirus from Patients with Pneumonia in China, 2019 , 2020, The New England journal of medicine.

[10]  Haoxing Xu,et al.  Lysosomal Ion Channels as Decoders of Cellular Signals. , 2019, Trends in biochemical sciences.

[11]  A. Walls,et al.  Structures of MERS-CoV spike glycoprotein in complex with sialoside attachment receptors , 2019, Nature Structural & Molecular Biology.

[12]  Pardis C. Sabeti,et al.  Ebola Virus Glycoprotein with Increased Infectivity Dominated the 2013–2016 Epidemic , 2016, Cell.

[13]  Fang Li,et al.  NAADP-dependent Ca2+ signaling regulates Middle East respiratory syndrome-coronavirus pseudovirus translocation through the endolysosomal system , 2018, Cell Calcium.

[14]  A. Osterhaus,et al.  Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia. , 2012, The New England journal of medicine.

[15]  Zhaohui Qian,et al.  Role of the Spike Glycoprotein of Human Middle East Respiratory Syndrome Coronavirus (MERS-CoV) in Virus Entry and Syncytia Formation , 2013, PloS one.

[16]  X. L. Liu,et al.  Isolation and Characterization of Viruses Related to the SARS Coronavirus from Animals in Southern China , 2003, Science.

[17]  M. Marsh.  Faculty Opinions recommendation of Ebola virus. Two-pore channels control Ebola virus host cell entry and are drug targets for disease treatment. , 2019, Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature.

[18]  David H. L. Bishop,et al.  The International Committee on Taxonomy of Viruses , 1995 .

[19]  Xiuyuan Ou,et al.  Identification of H209 as Essential for pH 8-Triggered Receptor-Independent Syncytium Formation by S Protein of Mouse Hepatitis Virus A59 , 2018, Journal of Virology.

[20]  Christian Drosten,et al.  The Spike Protein of the Emerging Betacoronavirus EMC Uses a Novel Coronavirus Receptor for Entry, Can Be Activated by TMPRSS2, and Is Targeted by Neutralizing Antibodies , 2013, Journal of Virology.

[21]  C. Osborne,et al.  Identification of the Receptor-Binding Domain of the Spike Glycoprotein of Human Betacoronavirus HKU1 , 2015, Journal of Virology.

[22]  Edward C. Holmes,et al.  Human Adaptation of Ebola Virus during the West African Outbreak , 2016, Cell.

[23]  Jie Dong,et al.  Identification of a novel coronavirus causing severe pneumonia in human: a descriptive study , 2020, Chinese medical journal.

[24]  M. Takeda,et al.  Efficient Multiplication of Human Metapneumovirus in Vero Cells Expressing the Transmembrane Serine Protease TMPRSS2 , 2008, Journal of Virology.

[25]  Linqi Zhang,et al.  Structure of MERS-CoV spike receptor-binding domain complexed with human receptor DPP4 , 2013, Cell Research.

[26]  Christian Drosten,et al.  Dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-EMC , 2013, Nature.

[27]  Christian Drosten,et al.  Cleavage and Activation of the Severe Acute Respiratory Syndrome Coronavirus Spike Protein by Human Airway Trypsin-Like Protease , 2011, Journal of Virology.

[28]  J. A. Comer,et al.  A novel coronavirus associated with severe acute respiratory syndrome. , 2003, The New England journal of medicine.

[29]  F. Taguchi,et al.  Localization of neutralizing epitopes and the receptor-binding site within the amino-terminal 330 amino acids of the murine coronavirus spike protein , 1994, Journal of virology.

[30]  Makoto Takeda,et al.  Efficient Activation of the Severe Acute Respiratory Syndrome Coronavirus Spike Protein by the Transmembrane Protease TMPRSS2 , 2010, Journal of Virology.

[31]  Christian Drosten,et al.  Evidence that TMPRSS2 Activates the Severe Acute Respiratory Syndrome Coronavirus Spike Protein for Membrane Fusion and Reduces Viral Control by the Humoral Immune Response , 2011, Journal of Virology.

[32]  M. Habjan,et al.  TMPRSS2 Activates the Human Coronavirus 229E for Cathepsin-Independent Host Cell Entry and Is Expressed in Viral Target Cells in the Respiratory Epithelium , 2013, Journal of Virology.

[33]  Jeremy G. Carlton,et al.  The mammalian phosphatidylinositol 3-phosphate 5-kinase (PIKfyve) regulates endosome-to-TGN retrograde transport , 2006, Journal of Cell Science.

[34]  K. Shirato,et al.  Middle East Respiratory Syndrome Coronavirus Infection Mediated by the Transmembrane Serine Protease TMPRSS2 , 2013, Journal of Virology.

[35]  P. Marik,et al.  A Descriptive Study , 2015 .

[36]  R. Guha,et al.  The phosphatidylinositol-3-phosphate 5-kinase inhibitor apilimod blocks filoviral entry and infection , 2017, PLoS neglected tropical diseases.

[37]  G. Whittaker,et al.  Host cell entry of Middle East respiratory syndrome coronavirus after two-step, furin-mediated activation of the spike protein , 2014, Proceedings of the National Academy of Sciences.

[38]  Lisa E. Gralinski,et al.  SARS-like WIV1-CoV poised for human emergence , 2016, Proceedings of the National Academy of Sciences.

[39]  G. Whittaker,et al.  Activation of the SARS coronavirus spike protein via sequential proteolytic cleavage at two distinct sites , 2009, Proceedings of the National Academy of Sciences.

[40]  G. Herrler,et al.  SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor , 2020, Cell.

[41]  R. Baric,et al.  Human Coronavirus HKU1 Spike Protein Uses O-Acetylated Sialic Acid as an Attachment Receptor Determinant and Employs Hemagglutinin-Esterase Protein as a Receptor-Destroying Enzyme , 2015, Journal of Virology.

[42]  Meitian Wang,et al.  Crystal structure of the receptor binding domain of the spike glycoprotein of human betacoronavirus HKU1 , 2017, Nature Communications.

[43]  Kai Zhao,et al.  A pneumonia outbreak associated with a new coronavirus of probable bat origin , 2020, Nature.

[44]  K. Shokat,et al.  PIKfyve Regulation of Endosome-Linked Pathways , 2009, Traffic.

[45]  P. Vollmar,et al.  Transmission of 2019-nCoV Infection from an Asymptomatic Contact in Germany , 2020, The New England journal of medicine.

[46]  J. Cunningham,et al.  Biochemical Basis for Increased Activity of Ebola Glycoprotein in the 2013-16 Epidemic. , 2017, Cell host & microbe.

[47]  B. Bosch,et al.  Human coronaviruses OC43 and HKU1 bind to 9-O-acetylated sialic acids via a conserved receptor-binding site in spike protein domain A , 2019, Proceedings of the National Academy of Sciences.

[48]  S. Diamond,et al.  Inhibitors of cathepsin L prevent severe acute respiratory syndrome coronavirus entry. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[49]  V. Papavassiliou,et al.  Emerging zoonoses and vector-borne infections affecting humans in Europe , 2007, Epidemiology and Infection.

[50]  Fang Li,et al.  Receptor Recognition Mechanisms of Coronaviruses: a Decade of Structural Studies , 2014, Journal of Virology.

[51]  Y. Hu,et al.  Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China , 2020, The Lancet.