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. 2010 Jul 23;285(30):22758-63.
doi: 10.1074/jbc.M110.103275. Epub 2010 May 27.

Elastase-mediated activation of the severe acute respiratory syndrome coronavirus spike protein at discrete sites within the S2 domain

Sandrine Belouzard  1 , Ikenna Madu, Gary R Whittaker
Affiliations

Elastase-mediated activation of the severe acute respiratory syndrome coronavirus spike protein at discrete sites within the S2 domain

Sandrine Belouzard et al. J Biol Chem. .

Abstract

Proteolytic priming is a common method of controlling the activation of membrane fusion mediated by viral glycoproteins. The severe acute respiratory syndrome coronavirus spike protein (SARS-CoV S) can be primed by a variety of host cell proteases, with proteolytic cleavage occurring both as the S1/S2 boundary and adjacent to a fusion peptide in the S2 domain. Here, we studied the priming of SARS-CoV S by elastase and show an important role for residue Thr(795) in the S2 domain. A series of alanine mutants were generated in the vicinity of the S2 cleavage site, with the goal of examining elastase-mediated cleavage within S2. Both proteolytic cleavage and fusion activation were modulated by altering the cleavage site position. We propose a novel mechanism whereby SARS-CoV fusion protein function can be controlled by spatial regulation of the proteolytic priming site, with important implications for viral pathogenesis.

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Figures

FIGURE 1.
FIGURE 1.
Mutation of Thr795 inhibits elastase-induced cell-cell fusion mediated by SARS-CoV S. 293T cells co-expressing the SARS-CoV wild type (WT) or SARS-CoV T795D mutant, along with a plasmid encoding the luciferase under the control of the T7 promoter, were overlaid with Vero E6 cells expressing the T7 polymerase. Fusion was induced by treating the cells with different doses of elastase, and cells were lysed 5 h after fusion induction. Results are presented as relative light units (RLU). Error bars represent the S.E. of three independent experiments.
FIGURE 2.
FIGURE 2.
Mutation of Thr795 inhibits elastase-induced virus entry mediated by SARS-CoV S. Pseudotyped virions containing the SARS-CoV wild type (WT) or SARS-CoV T795D mutant were bound at 4 °C on HEK 293T cells co-expressing ACE2 and DC-SIGN and pretreated with 25 mm NH4Cl to inhibit infection by the endosomal pathway. Fusion of pseudotyped virions was induced at the cell surface by treating the cells for 5 min with elastase (A) or trypsin (B). Results are represented as relative light units (RLU). Error bars represent the S.E. of three independent experiments.
FIGURE 3.
FIGURE 3.
Introduction of alanine residues in the S2′ region modulates SARS-CoV S-mediated cell-cell fusion induced by elastase. Cell-cell fusion assay of cells expressing the wild type (WT) or mutant SARS-CoV spike protein were performed as described in Fig. 2. Results are presented as relative light units (RLU). Error bars represent the S.E. of three independent experiments.
FIGURE 4.
FIGURE 4.
Introduction of alanine residues in the S2′ region modulates elastase-induced virus entry mediated by SARS-CoV S. A and B, pseudotyped virion infections were performed as described for Fig. 2. Infection was induced by treatment with elastase (A) or trypsin (B). Results are presented as relative light units (RLU). Error bars represent the S.E. of three independent experiments. C, cleavage of SARS-CoV by elastase. HEK293T cells expressing the wild type (WT) or mutant SARS-CoV spike protein were treated with 50 μg/ml elastase. Cell surface proteins were biotinylated and precipitated, and cleavage products of the SARS-CoV spike protein were analyzed in Western blot with an antibody recognizing the C9 tag at the C terminus of the protein.
FIGURE 5.
FIGURE 5.
Moving the trypsin cleavage position by introduction of arginine residues in the S2′ region modulates SARS-CoV S-mediated membrane fusion. A and B, mutant SARS-CoV S proteins with potential trypsin cleavage site at different positions in the S2′ region were assayed in cell-cell fusion (A) and in pseudotyped virion infection assays (B), as described in Figs. 2 and 3. Results are presented as relative light units (RLU). Error bars represent the S.E. of three independent experiments. C, cleavage products were analyzed as described in Fig. 4C.
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