Cathepsin L functionally cleaves the severe acute respiratory syndrome coronavirus class I fusion protein upstream of rather than adjacent to the fusion peptide

Abstract

Unlike other class I viral fusion proteins, spike proteins on severe acute respiratory syndrome coronavirus virions are uncleaved. As we and others have demonstrated, infection by this virus depends on cathepsin proteases present in endosomal compartments of the target cell, suggesting that the spike protein acquires its fusion competence by cleavage during cell entry rather than during virion biogenesis. Here we demonstrate that cathepsin L indeed activates the membrane fusion function of the spike protein. Moreover, cleavage was mapped to the same region where, in coronaviruses carrying furin-activated spikes, the receptor binding subunit of the protein is separated from the membrane-anchored fusion subunit.

FIG. 1.

Schematic diagrams of the fusion proteins of parainfluenza virus 5 (PIV5; formerly known as simian virus 5), human immunodeficiency virus type 1 (HIV-1), influenza virus, Ebola virus, SARS-CoV (strain BJ01), MHV-A59, and HCoV-NL63. Filled bars represent the fusion peptide. Dark and light shaded bars, HR1 and HR2 regions, respectively. Open arrowheads indicate the positions of the furin cleavage sites. The fusion proteins are C-terminally anchored in the viral membrane (long vertical shaded bar).

FIG. 2.

(A) Confocal fluorescence microscopy of Vero cells transfected with plasmids expressing the SARS-CoV spike or GFP gene and either mock treated or treated with cathepsin L protease (CTSL; 2 μg/ml) or trypsin (2 μg/ml). Nuclei (blue) were labeled with ToPro-3 (Molecular Probes). (B) Semiquantitation of syncytium formation. The number of nuclei in SARS-CoV spike protein-expressing cells/syncytia was counted in mock-treated, CTSL (2 μg/ml)-treated, and trypsin (2 μg/ml)-treated Vero cell cultures. pos., positive.

FIG. 3.

Cathepsin L (CTSL) cleavage of the SARS-CoV spike protein. Purified trimeric spike ectodomains of SARS-CoV and HCoV-NL63 were incubated with different concentrations of CTSL or TPCK-treated trypsin for 30 min at room temperature. (A and B) Samples were analyzed by SDS-PAGE and stained with GelCode Blue reagent. (C and D) SARS-CoV samples were also subjected to Western blot analysis using antibodies recognizing the N-terminal part (α-S1) or the C-terminal part (α-S2) of the SARS-CoV spike ectodomain, respectively. The positions of molecular weight (Mw) marker proteins (in thousands) are indicated alongside the gels and blots.

FIG. 4.

Sequence alignment of the SARS-CoV (strain BJ01; GenBank accession no. AY278488) and MHV-A59 (primary accession no. P11224) spike proteins at the S1-S2 junction. Identical residues are asterisked. The filled, shaded, and open arrowheads indicate the cathepsin L, trypsin, and furin cleavage sites, respectively, with their cleavage positions given in parentheses.