Cleavage of the SARS coronavirus spike glycoprotein by airway proteases enhances virus entry into human bronchial epithelial cells in vitro

Abstract

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.

Methodology/principal findings: 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/significance: 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.

Figure 1. Susceptibility of various human airway epithelial cells to SARS-CoV S-mediated infection.

A, VeroE6 (10,000 cells/well), 16HBE, BEAS-2B and A549 (20,000 cells/well) cells were seeded onto 96-well plates 24 h before SARS-CoVpp infection. Pseudotypes (SARS-CoVpp) were collected from culture medium and concentrated as described previously . SARS-CoVpp were incubated with various cell lines and transduction was measured by determination of the luciferase activity expressed as luminescence counts per second (LCPS). VeroE6 cells were used as positive control. All experiments were performed in triplicates and data are presented as means±SE of two or three independent experiments. B, ACE2 expression from various mammalian airway cell lines. Cell lysates were collected and ACE2 RNA molecules were detected by RT-PCR. Amplified ACE2 cDNA products from 16HBE, BEAS-2B and A549 are shown in lanes 2 to 4, respectively. VeroE6 cell line (lane 1) was used as positive control for ACE2 expression. The quantity of total RNA templates was normalized to β-actin expression as shown from the lower panel. Lane M represents the DNA size marker and the size of DNA bands are indicated on the right.

Figure 2. Identification of airway protease cleavage site(s) along the amino acid sequence of SARS-CoV S glycoprotein.

A, Purified triSpike proteins (lane 1: detected by Western immunoblot, lane 2: silver staining) were incubated with 0.2 mU of trypsin (lane 3), plasmin (lane 4) or TMPRSS11a (lane 5) identified and purified from lungs and bronchi. Cleavage products were visualized and prepared as described in Materials and Methods. Amino acid sequences (T1, T2, P1, P2, N1, and N2) corresponding to the cleaved triSpike proteins are shown in the lower panel. B, Three different types of airway proteases (trypsin, plasmin and TMPRSS11a) utilize the same amino acid residues for protein cleavage. A schematic diagram representing the amino acid sequence of SARS-CoV S glycoprotein shows on top of the figure. A red circle indicates the location of potential cleavage site along the Spike glycoprotein. Red dots represent the basic amino acids, potential protease cleavage sites (red letter) and two red arrows indicate the cleavage sites identified. NTD – N-terminal domain, RBD – receptor-binding domain, RBM – receptor-binding motif, FP – fusion peptide, HR-N – N-terminal of heptad-repeat, HR-C – C-terminal of heptad-repeat, IC – Intracellular tail.

Figure 3. Effect of airway proteases treatment on SARS-CoVpp infectivity.

A, SARS-CoVpp was pre-incubated with either trypsin (T) or plasmin (P) (10 µg/ml) at 37°C for 20 min. Luciferase activity (LCPS) was measured from infected 16HBE cells. Asterisk (*) indicates a value of p<0.05 in two-tailed t tests. Experiments were performed in triplicates and values were expressed as means±SE from two independent experiments. SARS-CoVpp entry into susceptible cell lines was enhanced with the presence of airway proteases. B & C, Equal amounts of SARS-CoVpp and empp (normalized to p24 quantity) were pre-incubated with 16HBE cells on ice for 30 min. Cells were washed twice to remove any unbound pp. Cells were incubated with 10 µg/ml of either trypsin (T), TMPRSS11a (N) or 100 µg/ml of plasmin (P) at room temperature for 40 min. Luciferase activity (LCPS) was measured from infected 16HBE cells. Experiments were performed in triplicates and values were expressed as means±SE from two independent experiments. D-F, Next, SARS-CoVpp was pre-incubated with 16HBE cells on ice for 30 min. Cells were washed twice to remove any unbound pp. Cells were incubated with various concentrations of trypsin (T), plasmin (P) or TMPRSS11a (N) at room temperature for 40 min. Luciferase activity (LCPS) was measured from infected 16HBE cells. Asterisk (**) indicates a value of p<0.01 in two-tailed t tests. Experiments were performed in triplicates and values were expressed as means±SE from three independent experiments.

Figure 4. Differential expression of wild-type and mutant SARS S glycoprotein and SARS-CoV pseudotype production from 293T cells.

A, Different putative spike glycoprotein sequences from SARS-CoV isolates were obtained from NCBI. Name of SARS-CoV isolates and GenBank accession numbers are listed. Potential airway protease cleavage residues are highlighted in green. B, Three mutant constructs were made from the wild-type SARS-CoV Spike glycoprotein cDNA cloned in the vector pcDNA3.1 as described in Materials and Methods. C, Cell lysates were collected from 293T cells 48 h post transfection. Pseudotypes were collected from culture medium and concentrated as described previously . Samples (cell lysates or pseduotypes) were denatured, reduced and analyzed by 4–12% Bis-Tris SDS-PAGE gel and Western Blot using M2 monoclonal antibody against the FLAG peptide. Sizes of molecular weight markers are indicated on the right. Wild-type SARS spike (lane 1), R667A (lane 2), R797A (lane 3), R797AR667A (lane 4) and pseudotype without envelope (lane 5). D, Analysis of various types of SARS-CoVpp for viral entry. SARS-CoVpp (wild-type or mutant SARS S glycoprotein) were incubated with 16HBE cells and transduction was measured by determination of the luciferase activity (LCPS). Experiments were performed in duplicates and data are presented as means±SE from two independent experiments.

Figure 5. Role of amino acid residue 667 enhances SARS-CoVpp entry in the presence of airway proteases.

SARS-CoVpp (A) or R667App (B) was pre-incubated with 16HBE cells on ice for 30 min. Cells were washed twice to remove any unbound pp. Cells were incubated with 10 µg/ml of either trypsin (T), TMPRSS11a (N) or 100 µg/ml of plasmin (P) at room temperature for 40 min. Luciferase activity (LCPS) was measured from infected 16HBE cells. Asterisk (*) indicates a value of p<0.05 and (**) indicates a value of p<0.01 in two-tailed t tests. Experiments were performed in triplicates and values were expressed as means±SE from three independent experiments.