The coronavirus spike protein is a class I virus fusion protein: structural and functional characterization of the fusion core complex

Abstract

Coronavirus entry is mediated by the viral spike (S) glycoprotein. The 180-kDa oligomeric S protein of the murine coronavirus mouse hepatitis virus strain A59 is posttranslationally cleaved into an S1 receptor binding unit and an S2 membrane fusion unit. The latter is thought to contain an internal fusion peptide and has two 4,3 hydrophobic (heptad) repeat regions designated HR1 and HR2. HR2 is located close to the membrane anchor, and HR1 is some 170 amino acids (aa) upstream of it. Heptad repeat (HR) regions are found in fusion proteins of many different viruses and form an important characteristic of class I viral fusion proteins. We investigated the role of these regions in coronavirus membrane fusion. Peptides HR1 (96 aa) and HR2 (39 aa), corresponding to the HR1 and HR2 regions, were produced in Escherichia coli. When mixed together, the two peptides were found to assemble into an extremely stable oligomeric complex. Both on their own and within the complex, the peptides were highly alpha helical. Electron microscopic analysis of the complex revealed a rod-like structure approximately 14.5 nm in length. Limited proteolysis in combination with mass spectrometry indicated that HR1 and HR2 occur in the complex in an antiparallel fashion. In the native protein, such a conformation would bring the proposed fusion peptide, located in the N-terminal domain of HR1, and the transmembrane anchor into close proximity. Using biological assays, the HR2 peptide was shown to be a potent inhibitor of virus entry into the cell, as well as of cell-cell fusion. Both biochemical and functional data show that the coronavirus spike protein is a class I viral fusion protein.

Figures

FIG. 1.

(A) Schematic representation of the coronavirus MHV-A59 spike protein structure. The glycoprotein has an N-terminal signal sequence (SS) and a transmembrane domain (TM) close to the C terminus. The protein is proteolytically cleaved (vertical arrow) into an S1 and an S2 subunit, which are noncovalently linked. S2 contains two HR regions (hatched bars), HR1 and HR2, as indicated. (B) Sequence alignment of HR1 and HR2 domains of MHV-A59 with those of HCoV-OC43, HCoV-229E, FIPV strain 79-1146, infectious bronchitis virus strain Beaudette (IBV), and the newly identified HCoV-SARS (strain TOR2). HCoV-229E and FIPV, MHV-A59 and HCoV-OC43, and IBV are representatives of groups 1, 2, and 3, respectively—the three coronavirus subgroups (59). Dark shading marks sequence identity, while lighter shading represents sequence similarity. The alignment shows a remarkable insertion of exactly two HRs (14 aa) in both HR1 and HR2 of HCV-229E and FIPV, a characteristic of all group 1 viruses. The predicted hydrophobic HR a and d residues are indicated above the sequence. The frame shifts in the predicted HRs in HR1 are caused by a stutter (51). The asterisks indicate conserved residues, and the dots represent similar residues. The amino acid sequences of the peptides HR1, HR1a, HR1b, HR1c, and HR2 used in this study are presented in italics below the alignments. N-terminal residues derived from the proteolytic cleavage site of the GST fusion protein are in parentheses. A conserved N-glycosylation sequence in the HR2 region is underlined.

FIG. 2.

Hetero-oligomeric complex formation of HR1 and HR1a with HR2. (A) HR1 and HR2 on their own or as a preincubated equimolar (80 μM) mix were subjected to Tricine SDS-15% PAGE. Before gel loading, samples were either heated at 100°C or left at RT. The positions of HR1, HR2, and the HR1-HR2 complex are indicated on the left, while the positions of molecular mass markers are indicated on the right. (B) Same as panel A but with peptide HR1a instead of HR1.

FIG. 3.

Temperature stability of HR1-HR2 complex. An equimolar mix of HR1 and HR2 (80 μM) was incubated at RT for 1 h. Samples were subsequently heated for 5 min at the indicated temperatures in 1× Tricine sample buffer and analyzed by SDS-PAGE in a 15% Tricine gel, together with HR1 and HR2 alone. The positions of HR1, HR2, and the HR1-HR2 complex are indicated on the left, while the molecular mass markers are indicated on the right.

FIG. 4.

CD spectra (mean residue eliplicity [φ]) of the HR1 peptide (25 μM; open squares), the HR2 peptide (25 μM; solid triangles), and the HR1-HR2 complex (25 μM; solid squares) in water at RT. Note that the HR1 and HR2 spectra virtually coincide.

FIG. 5.

Electron micrographs of HR1-HR2 complex.

FIG. 6.

Proteinase K treatment of HR peptides. The peptides HR2, HR1, HR1a, HR1b, and HR1c were subjected to proteinase K either individually in solution or after mixing of the different HR1 peptides with HR2 at equimolar concentration followed by a 1-h incubation at 37°C. Proteolytic fragments were separated and purified by HPLC and characterized by mass spectrometry. The peptides are represented by bars. The hatched bars indicate the protease-sensitive part(s) of the peptide. Shaded bars represent the HR2 peptide. The N- and C-terminal positions of the peptide and the amino acid numbering are indicated.

FIG. 7.

Inhibition of virus cell entry and cell-cell fusion by HR peptides. (A) Virus cell entry inhibition by HR peptides using a luciferase gene-expressing MHV. LR7 cells were inoculated with virus at an MOI of 5 in the presence of various concentrations of peptide ranging from 0.4 to 50 μM. At 5 h p.i., the cells were lysed, and luciferase activity was measured. CPS, counts per second. The error bars indicate standard deviations. (B) Inhibition of spike-mediated cell-cell fusion by HR peptides. BSR T7/5 effector cells—BHK cells constitutively expressing T7 RNA polymerase (3)—were infected with vaccinia virus for 1 h and subsequently transfected with a plasmid containing the S gene under a T7 promoter. Three hours posttransfection, LR7 target cells transfected with a plasmid carrying the luciferase gene behind a T7 promoter were added to the effector cells. The cells were incubated for another 4 h in the presence or absence of HR peptide. The cells were lysed, and luciferase activity was measured.

FIG. 8.

Schematic representation (approximately to scale) of the viral fusion proteins of six different virus families: MHV-A59 S (Coronaviridae), influenza virus HA (Orthomyxoviridae), HIV-1 gp160 (Retroviridae), SV5 F (Paramyxoviridae), Ebola Gp2 (Filoviridae), and S. exigua multicapsid nucleopolyhedrosis virus (SeMNPV) F (Baculoviridae). Cleavage sites are indicated by triangles; the solid bars represent the (putative) fusion peptides, the vertically hatched bars represent the HR1 domains, and the horizontally hatched bars represent the HR2 domains. Transmembrane domains are indicated by the vertical dashed lines. For each polypeptide, the total length is given at the right.