Date Published: November 17, 2009
Publisher: Public Library of Science
Author(s): Yiu-Wing Kam, Yuushi Okumura, Hiroshi Kido, Lisa F. P. Ng, Roberto Bruzzone, Ralf Altmeyer, Georges Snounou. http://doi.org/10.1371/journal.pone.0007870
Abstract: 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.
Partial Text: Proteolytic cleavage of the viral envelope glycoprotein into a receptor binding and a fusogenic transmembrane subunit is important to regulate virus entry and infectivity . Previous studies showed that viral glycoprotein activation is mediated by secreted proteases recognizing either monobasic or multibasic cleavage sites . Cleavage of viral glycoprotein has been demonstrated in retrovirus, ortho and paramyxoviruses to regulate virus entry and fusion , , . The extracellular processing of the envelope glycoprotein has a major impact on the infectivity of virulent or avirulent strains of influenza viruses, Sendai virus and Newcastle disease virus , , . A typical example is influenza A virus, where virus-cell fusion activity is induced by post-translational proteolytic cleavage of the envelope glycoprotein that is mediated by trypsin-like protease in the bronchial epithelium and airway secretion . Several proteases such as tryptase clara, mini-plasmin, ectopic anionic trypsin, mast-cell tryptase and tryptase TC30, which have been isolated from airway epithelial, can selectivity cleave the consensus cleavage motif of human influenza A virus envelope glycoprotein , , ,  and determine the virus tropism and infectivity. Recent advance of human genome studies identified a large number of transmembrane serine protease (TMPRSS). Various TMPRSS members with known airway localization have been identified from the respiratory tract. TMPRSS11a, one of the newly identified members of type II transmembrane serine proteases, is expressed in upper respiratory tract (pharynx and trachea) (unpublished data). However, less is known about TMPRSS that activate pneumotropic virus under natural infection.
The spike glycoproteins of many enveloped viruses, including various strain of human influenza viruses and Sendai virus are cleaved by host-derived proteases into two non-covalently linked subunits , . Proteolytic modification of spike glycoproteins is the major determinant of virus tropism and pathogenicity as shown in pneumotropic viruses whose infectivity is determined by airway proteases , , . A monobasic cleavage site has been identified in various viral glycoproteins and is recognized by proteases secreted by epithelial cells . Our data demonstrate that SARS spike glycoprotein has two monobasic cleavage sites that are susceptible to airway protease cleavage. We show here that specific airway proteases secreted along the respiratory tract, such as trypsin, plasmin and TMPRSS11a, recognize and cleave the same two monobasic motifs on the SARS spike glycoprotein. Furthermore, we have taken advantage of the flexibility of pseudotyped particles to investigate the molecular mechanisms underlying initial steps of virus entry following cell attachment. We have observed that protease cleavage of spike glycoprotein greatly enhances entry but only after receptor-binding interaction at the cell surface. These observations suggest that proteolytic processing of bound spike results in a conformational change that favors entry of the virus. This is the first report which demonstrates the functional impact of disrupting cleavage at position 667, which had not been addressed before. More importantly, it clarifies the mechanism of virus entry enhancement by several airway proteases. Our data are in agreement with the recent study showing that SARS-CoV S-mediated virus entry is dependent on the sequential proteolytic cleavage of monobasic sites .