Furin cleavage of the SARS coronavirus spike glycoprotein enhances cell-cell fusion but does not affect virion entry

Abstract

The fusogenic potential of Class I viral envelope glycoproteins is activated by proteloytic cleavage of the precursor glycoprotein to generate the mature receptor-binding and transmembrane fusion subunits. Although the coronavirus (CoV) S glycoproteins share membership in this class of envelope glycoproteins, cleavage to generate the respective S1 and S2 subunits appears absent in a subset of CoV species, including that responsible for the severe acute respiratory syndrome (SARS). To determine whether proteolytic cleavage of the S glycoprotein might be important for the newly emerged SARS-CoV, we introduced a furin recognition site at single basic residues within the putative S1-S2 junctional region. We show that furin cleavage at the modified R667 position generates discrete S1 and S2 subunits and potentiates membrane fusion activity. This effect on the cell-cell fusion activity by the S glycoprotein is not, however, reflected in the infectivity of pseudotyped lentiviruses bearing the cleaved glycoprotein. The lack of effect of furin cleavage on virion infectivity mirrors that observed in the normally cleaved S glycoprotein of the murine coronavirus and highlights an additional level of complexity in coronavirus entry.

Fig. 1

Sequence alignment to define the putative S1–S2 junctional region of SARS-CoV S glycoprotein. (A) The MAXHOM algorithm (Sander and Schneider, 1991) as accessed in the PredictProtein suite (http://cubic.bioc.columbia.edu/predictprotein) was used for the initial alignment of the full-length amino acid sequences of the following CoV S glycoproteins: SARS-CoV (Urbani; AAP1344), murine hepatitis virus (MHV; AAA87062), bovine CoV (BCoV; AAF25499), human respiratory CoV OC43 (AAT84362), feline infectious peritonitis virus (FIPV; VGIH79), porcine transmissible gastroenteritis virus (TGEV; AAT00645) and the human respiratory CoVs 229E (AAK32191) and NL63 (AAS58177). The regions containing the furin cleavage site (in group 2 viruses; underlined) and the S1 GxCx motif and conserved S2 nonamer (gray) were manually adjusted to yield the alignment shown. A tyrosine (Y) present in all S1 domains is also underlined. Deletions in SARS-CoV and the group 1 viruses (.) are arbitrarily displayed. (B) The two basic residues in the junctional region of the wild-type SARS-CoV S glycoprotein (R667 and K672) are indicated by diamonds. The furin protease recognition sites engineered at these sites are underlined in the HTR, HTVR and SLLR mutants.

Fig. 2

Serine mutation at R667, but not K672, reduces the ability of the S glycoprotein to mediate cell–cell fusion. (A) Wild-type SARS-CoV S glycoprotein and the R667S and K672S mutants were metabolically labeled in COS-7 cells and isolated using the C-terminal S-peptide (Spep) affinity tag (Kim and Raines, 1993). The mock lane represents cells transfected without vTF7-3 infection. Proteins were resolved by SDS polyacrylamide gel electrophoresis in NuPAGE 3–8% Tris–acetate gels (Invitrogen) either as the isolated glycoproteins (above) or following deglycosylation using PNGase F (below). The Endo-H-resistant (S_r) and Endo-H-sensitive (S_s) forms of the S glycoprotein are indicated, as is the fully deglycosylated S polypeptide (S). [¹⁴C]-methylated Rainbow molecular weight markers are indicated. The images are printed dark to highlight the absence of proteolytic cleavage. (B) The ability of the S glycoproteins to promote ACE2-dependent cell–cell fusion was detected using the recombinant vaccinia virus-based β-galactosidase reporter assay (Nussbaum et al., 1994, York et al., 2004). COS-7 cells expressing the wild-type and mutant glycoproteins were co-cultured with COS-7 cells transiently expressing the SARS-CoV cellular receptor ACE2 and infected with the fusion reporter recombinant vaccinia virus vCB21R-lacZ (Nussbaum et al., 1994, York et al., 2004). X-gal was used to detect β-galactosidase activity arising from cell–cell fusion. The number of blue syncytia is shown from two experiments, and error bars represent one standard deviation. Transfection efficiencies were comparable in all cases.

Fig. 3

Proteolytic cleavage of the S glycoprotein at introduced furin sites. Wild-type S glycoprotein and the furin-site-containing mutants HTVR, HTR and SSLR were metabolically labeled in COS-7 cells. The S glycoprotein in cell lysates was affinity isolated (cells; left panels), whereas the S1 subunit in the cell culture supernatant (medium; right panels) was immunoprecipitated using MAb F26G18 (Berry et al., 2004). Polypeptides obtained upon deglycosylation with PNGase F are shown in bottom panels. Molecular weight markers and S glycoforms are as described in Fig. 2A. Both S1 and S2 polypeptide molecular weights are consistent with cleavage in the junctional region.

Fig. 4

Flow cytometric analysis of cell surface S glycoprotein. COS-7 cells transiently expressing the wild-type and HTVR mutant S glycoproteins were stained using either (top) MAb F26G18 (Berry et al., 2004) and a secondary fluorescein isothiocyanate (FITC)-conjugated anti-mouse antibody or (bottom) a recombinant soluble ACE2 receptor, biotinylated goat anti-ACE2 antibody and FITC-conjugated streptavidin. Cells were analyzed using a FACSCalibur flow cytometer (BD Biosciences), and propidium-iodide-staining (dead) cells were excluded. Background staining of mock-transfected cells is shown in the left panel; non-expressing cells are also evident in the transfected cell populations. Expressing cells were defined using a gate of ≥30 (<0.1% of the mock-transfected population).

Fig. 5

Cell–cell fusion is enhanced by proteolytic cleavage. Wild-type S glycoprotein and the furin-site-containing mutants HTVR, HTR and SSLR were expressed in COS-7 cells, and fusogenicity was determined as described in Fig. 2B (Nussbaum et al., 1994, York et al., 2004). The number of blue syncytia relative to that in the wild-type S glycoprotein control is shown from four independent experiments, and error bars represent one standard deviation. Transfection efficiencies within each experiment were comparable.

Fig. 6

Proteolytic processing in furin-deficient FD11 and wild-type CHO cells. Wild-type S glycoprotein and the furin-site-containing mutants HTVR, HTR and SSLR were metabolically labeled in furin-deficient cells and parental CHO cells (Gordon et al., 1995). The S glycoprotein in cell lysates was affinity isolated (top panels), and the S1 subunit in the cell culture medium (bottom panels) was immunoprecipitated using MAb F26G18 (Berry et al., 2004).

Fig. 7

Co-expression of furin cDNA enhances proteolytic cleavage and cell–cell fusion. (A) Wild-type or HTVR S glycoprotein plasmid was co-transfected with a furin-expressing plasmid (+furin cDNA), or a pcDNA3.1⁻ control (−furin cDNA), into furin-deficient FD11 or wild-type CHO cells (Gordon et al., 1995). The S glycoproteins were expressed and metabolically labeled as described in Fig. 3. Isolated glycoproteins from cell lysates (top panel) were also examined following deglycosylation with PNGase F (middle panel); shed S1 subunit was immunoprecipitated from the cell culture medium and examined following deglycosylation (bottom panel). Molecular weight markers and forms of the S glycoprotein are as described in Fig. 3. A 69 kDa molecular weight form of S1 was also detected in the culture medium upon PNGase F-treatment (indicated by *), as was a 140 kDa polypeptide which is consistent with blebbing of the intact S glycoprotein. (B) Cell–cell fusion was assessed using the indicated S-glycoprotein-expressing cells and COS-7 target cells expressing exogenous ACE2 receptor. The number of blue syncytia is shown from two experiments, and error bars represent one standard deviation. Small error bars are not shown.

Fig. 8

Co-expression of furin cDNA results in cleavage and enhanced fusogenicity of the wild-type and K672S glycoproteins. (A) Wild-type or mutant S glycoprotein plasmid (R667S or K672S) was co-transfected into wild-type CHO cells with a furin-expressing plasmid (+furin cDNA) or a pcDNA3.1⁻ control (−furin cDNA). Isolated glycoproteins from cell lysates (cells; top panel) and immunoprecipitated S1 subunit from culture medium (bottom panel) were deglycosylation with PNGase F. Molecular weight markers and forms of the S polypeptide, including the larger S1 fragment (*), are as described in Fig. 7. The intact S polypeptide present in the medium (bottom panel) is shown for internal size reference. (B) Cell–cell fusion was assessed using the S-glycoprotein-expressing CHO cells and COS-7 target cells expressing exogenous ACE2 receptor. The number of blue syncytia is shown from two experiments, and error bars represent one standard deviation. Smaller error bars are not shown.

Fig. 9

Pseudotyped virions bearing HTVR S glycoprotein are not enhanced in their infectivity. (A) Metabolically labeled pseudotyped virions bearing the co-S Δ19 or co-HTVR Δ19 glycoprotein were pelleted through 20% sucrose and solubilized in 1% Triton X-100 lysis buffer prior to immunoprecipitation. HIV proteins were isolated using anti-HIV immunoglobulin from infected individuals (HIVIG; Prince et al., 1991), and SARS-CoV S glycoproteins were immunoprecipitated using MAb F26G18 (Berry et al., 2004). Particles lacking any envelope glycoprotein (null) served as controls. S-glycoprotein-containing samples were heated to 100 °C prior to SDS-PAGE electrophoresis to disrupt S2 oligomers. Molecular weight markers, HIV proteins (Gag p24 and p17) and forms of the S glycoprotein are indicated. (B) Pseudotyped virions bearing the co-S Δ19 or co-HTVR Δ19 glycoprotein, or the VSV G glycoprotein control, were used to infect 293T cells transiently expressing ACE2 (+) or native 293T cells (−). Fresh pseudotyped virion stocks were applied neat to 293T cell microcultures, and infection was determined 2 days later by chemiluminescence of the luciferase reporter (reported in relative light units (RLUs)). No infection by S-glycoprotein-containing virions was detected (ϕ) in the absence of ACE2 expression. Error bars represent one standard deviation.