title: 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.
authors.affiliation: Virology Division, Department of Infectious Diseases and Immunity, Faculty of Veterinary Medicine, and Institute of Biomembranes, Utrecht University, 3584 CL Utrecht, The Netherlands.
authors.forename: Berend Jan
authors.initials: BJ
authors.lastname: Bosch
authors.forename: Ruurd
authors.initials: R
authors.lastname: van der Zee
authors.forename: Cornelis A M
authors.initials: CA
authors.lastname: de Haan
authors.forename: Peter J M
authors.initials: PJ
authors.lastname: Rottier
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002942:Circular Dichroism; D017934:Coronavirus / Q000502:physiology* / Q000648:ultrastructure; D017931:DNA Primers; D008561:Membrane Fusion / Q000502:physiology*; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000502:physiology* / Q000648:ultrastructure; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins / Q000737:chemistry* / Q000502:physiology* / Q000648:ultrastructure
chemical_list: D017931:DNA Primers; D008562:Membrane Glycoproteins; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins; C578557:spike glycoprotein, SARS-CoV; C578553:spike protein, mouse hepatitis virus