Previously, it has been demonstrated that SARS-CoV infections can be activated by exogenous elastase (12
). For these studies, pancreatic elastase (or elastase-1) was used as a source of protease. In addition, it has been shown previously that introduction of a furin site at position 797 of SARS-CoV S induced cell-cell fusion independent of the addition of exogenous protease (6
). We added a furin cleavage site at different positions in the region 667–797; however, only the addition of a furin cleavage site at position 797 results in cell-cell fusion. Addition of a furin cleavage site at position 773 has no effect on cell-cell fusion (supplemental Fig. 1
). Therefore, we hypothesized that to activate fusion, the cleavage event has to occur in the vicinity of the S2′ region.
To identify potential elastase cleavage sites at the S2′ position within SARS-CoV S, we examined the S2′ region for possible consensus cleavage sites utilizing the MEROPS
peptidase database ID S01–153. In contrast to trypsin, elastase requires an amino acid with a nonpolar aliphatic, or polar uncharged, R group in the P1 position, with a preference for proline in the P2 position. Although it was not an exact match for the optimal MEROPS
cleavage site consensus sequence for elastase-1 (A/−/P/AV↓−/−/−/−), we identified residue threonine 795, within the sequence 792
as a possible P1 residue for elastase-1, with proline 794 in the P2 position. In this sequence Arg797
represents the expected site of trypsin cleavage (6
), and 803
represent the core residues of the S2 fusion peptide (11
To test the importance of Thr795
for elastase-mediated activation of SARS-CoV S fusion, we performed site-directed mutagenesis and modified residue 795 from threonine to aspartic acid, to create the mutant SARS T795D. This protein showed cell surface expression comparable with the wild type S protein (supplemental Fig. 2
) and was used in quantitative cell-cell fusion assays, with elastase-1 as a fusion trigger. The wild type SARS-CoV S protein showed a dose-dependent activation of membrane fusion when elastase was added, whereas elastase-mediated fusion was essentially abrogated for the T795D mutant ().
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 (more ...)
To confirm the importance of residue Thr795 in the context of the fusion event(s) mediating virus entry, we examined the wild type and mutant S protein incorporated into viral pseudoparticles. Both wild type and T795D S proteins were equally incorporated into pseudoparticles (not shown). These wild type and T795D pseudovirions infected cells via the endosomal routes at comparable levels, and this route of infection was efficiently blocked by neutralizing endosomal acidification with NH4Cl (see ). Although the cell surface-bound wild type S pseudovirions could be induced to enter cells after treatment with elastase-1, entry was severely limited in the case of the T795D mutant pseudovirions (A). As a control, we repeated these experiments using trypsin as a fusion trigger, and in this case both wild type and T795D mutant pseudovirions were able to enter cells efficiently, indicating that residue Thr795 is critical for SARS-CoV entry via an alternate protease trigger utilizing elastase.
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 (more ...)
As seen in , elastase-induced infection at the cell surface is less efficient compared with infection induced by trypsin. This relatively low infection-induced efficiency could be due to the nature of the recognition site, or to its position, which is shifted from the trypsin-exposed N terminus of the fusion peptide.
Because the sequence 792LKPTKRSFIEDLLF805 is not an ideal cleavage site consensus sequence for elastase-1, we next mutated threonine 795 to alanine to create a more optimal cleavage site, to create the mutant T795A. As the identified elastase cleavage site is shifted in position from the expected cleavage site based on previous studies using trypsin (which identified Arg797 as the cleavage site), we also generated a series of alanine mutations in the vicinity of S2′ using T795D as a backbone. These mutants comprised K793A/T795D, P794A/T795D, T795D/K796A, and T795D/R797A and were used in fusion and entry assays with the goal of examining the effect of cleavage site positioning relative to the S2 fusion peptide.
These mutants were first assayed for cell surface expression (supplemental Fig. 2
), which all mutants except for T795D/R797A (see below) expressed at levels of 70% or more of the wild type protein. We then used these mutants in our quantitative cell-cell fusion assay. In the case of the mutants K793A/T795D, P794A/T795D, T795D/K796A, and T795D/R797A it was apparent that alanine at positions 794 and 797 could be used very efficiently by elastase-1 (). In both these cases, the presence of alanine gave higher levels of fusion than was mediated by Thr795
. In addition, maximum fusion was reached with the lowest dose of elastase with the mutant T795D/R797A, even though this mutant shows lower surface expression compared with the wild type protein (27% ± 15) (supplemental Fig. 2
). However, the presence of alanine at position 796 did not allow elastase-induced fusion. The T795A mutant gave a high level of background fusion in the absence of elastase addition (presumably via activation by an endogenous protease), so could not be used in cell-cell fusion assays designed to assess the role of elastase (data not shown).
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 (more ...)
We next assessed the function of our alanine mutants in viral pseudoparticles. Incorporation of the spike protein into pseudotyped virions was monitored, and because cell surface expression and viral particle production were partially impaired in some cases, (e.g.
for the T795D/R797A mutant; see supplemental Fig. 2
), we ensured that equal amounts of viral particles were used in the infectivity assay by adjusting the volume of supernatant. As a result, levels of infectivity obtained by the endosomal pathway for all of the mutants were comparable with the wild type protein.
In this case, we were able to use the T795A mutant for elastase-mediated entry assays. As with cell-cell fusion assays, the presence of alanine at positions 794 and 797 gave very high levels of elastase-mediated entry, and an alanine at position 796 did not allow entry. In this assay, the T795A mutant gave very high levels of activity, which were substantially greater than wild type (A).
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 . Infection was induced by treatment with elastase ( (more ...)
As a control, we repeated these experiments using trypsin as an entry trigger. In this case, there was essentially no difference in entry between wild type and mutant pseudoviruses, with the exception of the T795D/R797A mutant, which was unable to be activated by trypsin (B). As seen in cell-cell fusion assays, infection mediated by elastase was achieved with the lowest dose of elastase for the mutant T795D/R797A, the infection levels obtained were comparable with those obtained with trypsin treatment for the wild type protein.
Next, we analyzed cleavage products by elastase in a cell surface biotinylation assay. A cleavage product corresponding to the cleavage at position S2′ was observed for the wild type protein and the mutants P794A/T795D, T795A, and T795D/R797A (C, lanes 1, 4, 5, and 7). The cleavage was very efficient for the mutant T795D/R797A. Cleavage products were also detected for the mutant T795D, K793A/T795D, and T795D/K796A; however, the bands migrate slower, suggesting that the mutants are cleaved at a different position in the region located between the junction S1/S2 and the S2′ position.
We have previously shown that mutation of Arg797
is sufficient to inhibit trypsin-induced fusion, suggesting that trypsin is not able to induce fusion by using the basic residue Lys796
. Our data suggested that fusion activity could be modulated by repositioning of the elastase cleavage site in the vicinity of the S2 fusion peptide. To determine whether this finding also applied to trypsin-mediated cleavage at S2′, we generated a series of mutants that contained arginine residues at positions 795 and 794 in the context of the K796A/R797N background to inhibit the endogenous cleavage site. To allow us to focus on cleavage at the S2′ position, these mutants were generated in a backbone of an S protein containing a furin consensus sequence at the S1/S2 boundary (Fur667), and cell surface expression was monitored. All mutants were expressed at >80% of wild type levels (supplemental Fig. 3
). As expected for both cell-cell fusion assays and pseudovirions entry assays, both wild type S and the Fur667 mutants could be activated in a dose-dependent manner by treatment with trypsin, whereas the mutant K796A/R797N could not. As with elastase induction, the presence of arginine at position 794 or 795 allows membrane fusion and entry (, A
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′ (more ...)
We then analyzed the cleavage of the SARS-CoV spike protein by trypsin. The wild type protein is cleaved mainly at the junction S1/S2, but a faint band corresponding to the cleavage S2′ could also be detected with longer exposure time. As expected, introduction of the furin cleavage site (Fur667) facilitates the cleavage at the S2′ position. The double mutation K796A/R797N results in the appearance of a cleavage product that migrates slower, probably due to a cleavage at another position in the region 667–797. Introduction of arginine at either position 794 or 795 restores the cleavage at the S2′ position.