Cleavage of the SARS coronavirus spike glycoprotein by airway proteases enhances virus entry into human bronchial epithelial cells in vitro.
BACKGROUND
Entry of enveloped viruses into host cells requires the activation of viral envelope glycoproteins through cleavage by either intracellular or extracellular proteases. In order to gain insight into the molecular basis of protease cleavage and its impact on the efficiency of viral entry, we investigated the susceptibility of a recombinant native full-length S-protein trimer (triSpike) of the severe acute respiratory syndrome coronavirus (SARS-CoV) to cleavage by various airway proteases.


RESULTS
PURIFIED TRISPIKE PROTEINS WERE READILY CLEAVED IN VITRO BY THREE DIFFERENT AIRWAY PROTEASES: trypsin, plasmin and TMPRSS11a. High Performance Liquid Chromatography (HPLC) and amino acid sequencing analyses identified two arginine residues (R667 and R797) as potential protease cleavage site(s). The effect of protease-dependent enhancement of SARS-CoV infection was demonstrated with ACE2 expressing human bronchial epithelial cells 16HBE. Airway proteases regulate the infectivity of SARS-CoV in a fashion dependent on previous receptor binding. The role of arginine residues was further shown with mutant constructs (R667A, R797A or R797AR667A). Mutation of R667 or R797 did not affect the expression of S-protein but resulted in a differential efficacy of pseudotyping into SARS-CoVpp. The R667A SARS-CoVpp mutant exhibited a lack of virus entry enhancement following protease treatment.


CONCLUSIONS
These results suggest that SARS S-protein is susceptible to airway protease cleavage and, furthermore, that protease mediated enhancement of virus entry depends on specific conformation of SARS S-protein upon ACE2 binding. These data have direct implications for the cell entry mechanism of SARS-CoV along the respiratory system and, furthermore expand the possibility of identifying potential therapeutic agents against SARS-CoV.
Kam
Yiu-Wing
YW
HKU-Pasteur Research Center, The University of Hong Kong, Hong Kong SAR, China.
Okumura
Yuushi
Y
Kido
Hiroshi
H
Ng
Lisa F P
LF
Bruzzone
Roberto
R
Altmeyer
Ralf
R
D000595:Amino Acid Sequence; D000085962:Angiotensin-Converting Enzyme 2; D000818:Animals; D001120:Arginine / Q000737:chemistry; D001665:Binding Sites; D001980:Bronchi / Q000378:metabolism*; D002522:Chlorocebus aethiops; D019281:Dimerization; D004847:Epithelial Cells / Q000166:cytology*; D006023:Glycoproteins / Q000737:chemistry; D006801:Humans; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D007703:Peptidyl-Dipeptidase A / Q000235:genetics* / Q000378:metabolism; D045473:Severe acute respiratory syndrome-related coronavirus / Q000378:metabolism*; D064370:Spike Glycoprotein, Coronavirus; D014709:Vero Cells; D014759:Viral Envelope Proteins / Q000378:metabolism*
D006023:Glycoproteins; D008562:Membrane Glycoproteins; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins; C578557:spike glycoprotein, SARS-CoV; C578553:spike protein, mouse hepatitis virus; D001120:Arginine; D007703:Peptidyl-Dipeptidase A; C000705307:ACE2 protein, human; D000085962:Angiotensin-Converting Enzyme 2