Cleavage and Activation of the Severe Acute Respiratory Syndrome Coronavirus Spike Protein by Human Airway Trypsin-Like Protease

ABSTRACT The highly pathogenic severe acute respiratory syndrome coronavirus (SARS-CoV) poses a constant threat to human health. The viral spike protein (SARS-S) mediates host cell entry and is a potential target for antiviral intervention. Activation of SARS-S by host cell proteases is essential for SARS-CoV infectivity but remains incompletely understood. Here, we analyzed the role of the type II transmembrane serine proteases (TTSPs) human airway trypsin-like protease (HAT) and transmembrane protease, serine 2 (TMPRSS2), in SARS-S activation. We found that HAT activates SARS-S in the context of surrogate systems and authentic SARS-CoV infection and is coexpressed with the viral receptor angiotensin-converting enzyme 2 (ACE2) in bronchial epithelial cells and pneumocytes. HAT cleaved SARS-S at R667, as determined by mutagenesis and mass spectrometry, and activated SARS-S for cell-cell fusion in cis and trans, while the related pulmonary protease TMPRSS2 cleaved SARS-S at multiple sites and activated SARS-S only in trans. However, TMPRSS2 but not HAT expression rendered SARS-S-driven virus-cell fusion independent of cathepsin activity, indicating that HAT and TMPRSS2 activate SARS-S differentially. Collectively, our results show that HAT cleaves and activates SARS-S and might support viral spread in patients.

[1]  R. Connor,et al.  Vpr is required for efficient replication of human immunodeficiency virus type-1 in mononuclear phagocytes. , 1995, Virology.

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

[3]  S. Pöhlmann,et al.  Cellular entry of the SARS coronavirus , 2004, Trends in Microbiology.

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

[5]  Chawaree Chaipan,et al.  Highly Conserved Regions within the Spike Proteins of Human Coronaviruses 229E and NL63 Determine Recognition of Their Respective Cellular Receptors , 2006, Journal of Virology.

[6]  B. Bosch,et al.  Cathepsin L Functionally Cleaves the Severe Acute Respiratory Syndrome Coronavirus Class I Fusion Protein Upstream of Rather than Adjacent to the Fusion Peptide , 2008, Journal of Virology.

[7]  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.

[8]  K. Pulford,et al.  Detection of anaplastic lymphoma kinase (ALK) and nucleolar protein nucleophosmin (NPM)-ALK proteins in normal and neoplastic cells with the monoclonal antibody ALK1. , 1997, Blood.

[9]  N. Letvin,et al.  Codon usage optimization of HIV type 1 subtype C gag, pol, env, and nef genes: in vitro expression and immune responses in DNA-vaccinated mice. , 2003, AIDS research and human retroviruses.

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

[11]  Young Woo Park,et al.  Type II transmembrane serine proteases in cancer and viral infections. , 2009, Trends in molecular medicine.

[12]  Michael C. Myers,et al.  A Small-Molecule Oxocarbazate Inhibitor of Human Cathepsin L Blocks Severe Acute Respiratory Syndrome and Ebola Pseudotype Virus Infection into Human Embryonic Kidney 293T cells , 2010, Molecular Pharmacology.

[13]  J. Peiris,et al.  Newly discovered coronavirus as the primary cause of severe acute respiratory syndrome , 2003, The Lancet.

[14]  Jincun Zhao,et al.  A Transmembrane Serine Protease Is Linked to the Severe Acute Respiratory Syndrome Coronavirus Receptor and Activates Virus Entry , 2010, Journal of Virology.

[15]  D. Leduc,et al.  The human airway trypsin-like protease modulates the urokinase receptor (uPAR, CD87) structure and functions. , 2007, American journal of physiology. Lung cellular and molecular physiology.

[16]  Chengyu Jiang,et al.  SARS coronavirus entry into host cells through a novel clathrin- and caveolae-independent endocytic pathway , 2008, Cell Research.

[17]  Y. Natkunam,et al.  Jaw1/LRMP, a germinal centre‐associated marker for the immunohistological study of B‐cell lymphomas , 2006, The Journal of pathology.

[18]  K P Lee,et al.  TMPRSS4 promotes invasion, migration and metastasis of human tumor cells by facilitating an epithelial–mesenchymal transition , 2008, Oncogene.

[19]  Shinsei Minoshima,et al.  Insertion of β-satellite repeats identifies a transmembrane protease causing both congenital and childhood onset autosomal recessive deafness , 2001, Nature Genetics.

[20]  G. Navis,et al.  Tissue distribution of ACE2 protein, the functional receptor for SARS coronavirus. A first step in understanding SARS pathogenesis , 2004, The Journal of pathology.

[21]  Christian Drosten,et al.  Differential Downregulation of ACE2 by the Spike Proteins of Severe Acute Respiratory Syndrome Coronavirus and Human Coronavirus NL63 , 2009, Journal of Virology.

[22]  Mark Chappell,et al.  A crucial role of angiotensin converting enzyme 2 (ACE2) in SARS coronavirus–induced lung injury , 2005, Nature Medicine.

[23]  A. Lesner,et al.  Substrate specificity and inhibitory study of human airway trypsin-like protease. , 2010, Bioorganic & medicinal chemistry.

[24]  G. Fey,et al.  Susceptibility to SARS coronavirus S protein-driven infection correlates with expression of angiotensin converting enzyme 2 and infection can be blocked by soluble receptor , 2004, Biochemical and Biophysical Research Communications.

[25]  Gregory P Cosgrove,et al.  SARS-CoV replicates in primary human alveolar type II cell cultures but not in type I-like cells , 2007, Virology.

[26]  N. Seidah,et al.  Implication of proprotein convertases in the processing and spread of severe acute respiratory syndrome coronavirus , 2004, Biochemical and Biophysical Research Communications.

[27]  H. Klenk,et al.  Proteolytic Activation of Influenza Viruses by Serine Proteases TMPRSS2 and HAT from Human Airway Epithelium , 2006, Journal of Virology.

[28]  S. Pöhlmann,et al.  Novel insights into proteolytic cleavage of influenza virus hemagglutinin , 2010, Reviews in medical virology.

[29]  Shibo Jiang,et al.  Identification of Immunodominant Sites on the Spike Protein of Severe Acute Respiratory Syndrome (SARS) Coronavirus: Implication for Developing SARS Diagnostics and Vaccines , 2004, The Journal of Immunology.

[30]  R. Doms,et al.  DC-SIGN and DC-SIGNR bind ebola glycoproteins and enhance infection of macrophages and endothelial cells. , 2003, Virology.

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

[32]  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.

[33]  T. Sano,et al.  Localization of human airway trypsin-like protease in the airway: an immunohistochemical study , 2001, Histochemistry and Cell Biology.

[34]  K. Schughart,et al.  TMPRSS2 and TMPRSS4 Facilitate Trypsin-Independent Spread of Influenza Virus in Caco-2 Cells , 2010, Journal of Virology.

[35]  John L. Sullivan,et al.  Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus , 2003, Nature.

[36]  Chawaree Chaipan,et al.  Different host cell proteases activate the SARS-coronavirus spike-protein for cell–cell and virus–cell fusion , 2011, Virology.

[37]  D. A. Stein,et al.  Inhibition of Influenza Virus Infection in Human Airway Cell Cultures by an Antisense Peptide-Conjugated Morpholino Oligomer Targeting the Hemagglutinin-Activating Protease TMPRSS2 , 2010, Journal of Virology.

[38]  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.

[39]  A. Lo,et al.  Exploring the pathogenesis of severe acute respiratory syndrome (SARS): the tissue distribution of the coronavirus (SARS‐CoV) and its putative receptor, angiotensin‐converting enzyme 2 (ACE2) , 2004, The Journal of pathology.

[40]  G. Simmons,et al.  Characterization of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) spike glycoprotein-mediated viral entry , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[41]  Kwok-Hung Chan,et al.  Severe acute respiratory syndrome coronavirus-like virus in Chinese horseshoe bats. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[42]  A. Danchin,et al.  The Severe Acute Respiratory Syndrome , 2003 .

[43]  Semi Kim,et al.  Proteolytic Activation of the 1918 Influenza Virus Hemagglutinin , 2009, Journal of Virology.

[44]  Arthur S Slutsky,et al.  Angiotensin-converting enzyme 2 protects from severe acute lung failure , 2005, Nature.

[45]  O. Jahn,et al.  Technical innovations for the automated identification of gel-separated proteins by MALDI-TOF mass spectrometry , 2006, Analytical and bioanalytical chemistry.

[46]  G. Tse,et al.  Tissue and cellular tropism of the coronavirus associated with severe acute respiratory syndrome: an in‐situ hybridization study of fatal cases , 2004, The Journal of pathology.

[47]  K. Überla,et al.  S Protein of Severe Acute Respiratory Syndrome-Associated Coronavirus Mediates Entry into Hepatoma Cell Lines and Is Targeted by Neutralizing Antibodies in Infected Patients , 2004, Journal of Virology.

[48]  R. Rappuoli,et al.  SARS — beginning to understand a new virus , 2003, Nature Reviews Microbiology.