Abstract
The spike (S) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) mediates viral entry into target cells. Cleavage and activation of SARS S by a host cell protease is essential for infectious viral entry and the responsible enzymes are potential targets for antiviral intervention. The type II transmembrane serine protease TMPRSS2 cleaves and activates SARS S in cell culture and potentially also in the infected host. Here, we investigated which determinants in SARS S control cleavage and activation by TMPRSS2. We found that SARS S residue R667, a previously identified trypsin cleavage site, is also required for S protein cleavage by TMPRSS2. The cleavage fragments produced by trypsin and TMPRSS2 differed in their decoration with N-glycans, suggesting that these proteases cleave different SARS S glycoforms. Although R667 was required for SARS S cleavage by TMPRSS2, this residue was dispensable for TMPRSS2-mediated S protein activation. Conversely, residue R797, previously reported to be required for SARS S activation by trypsin, was dispensable for S protein cleavage but required for S protein activation by TMPRSS2. Collectively, these results show that different residues in SARS S control cleavage and activation by TMPRSS2, suggesting that these processes are more complex than initially appreciated.
Highlights
The subfamily Coronavirinae within the family Coronaviridae comprises viruses that cause respiratory, neurological and intestinal symptoms in mammals and birds
In order to identify SARS S residues required for S protein cleavage and activation by TMPRSS2, we first investigated which cleavage fragments are produced upon cotransfection of 293T cells with a constant amount of SARS S plasmid and decreasing amounts of TMPRSS2 plasmid, since expression of low amounts of protease in the transfected cells may best reflect TMPRSS2 expression in the lung
Removal of N-glycans by PNGase F revealed that the latter was the case: The untreated SARS S fragments produced upon cleavage by trypsin and TMPRSS2 migrated with different speed in the gel while migration was identical upon PNGase F treatment (Fig 1B)
Summary
The subfamily Coronavirinae within the family Coronaviridae comprises viruses that cause respiratory, neurological and intestinal symptoms in mammals and birds. The severe acute respiratory syndrome coronavirus (SARS-CoV), a member of the genus Betacoronavirus, emerged in 2002 in Southern China. Mainly in Asia, wrecked massive economic havoc and was associated with almost 800 deaths [1,2]. Another betacoronavirus, the Middle East respiratory syndrome coronavirus (MERS-CoV), emerged in Saudi Arabia in 2012 and causes a SARS-like disease, in patients with underlying chronic illness [3,4], indicating that emerging coronaviruses can constitute a severe threat to human health.
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