J Virol 77(16): 8801-8811

The Coronavirus Spike Protein Is a Class I Virus Fusion Protein: Structural and Functional Characterization of the Fusion Core Complex

Virology Division,^{Immunology Division, Department of Infectious Diseases and Immunity, Faculty of Veterinary Medicine, and Institute of Biomembranes, Utrecht University, 3584 CL Utrecht, The Netherlands}²

^{Corresponding author. Mailing address: Virology Division, Department of Infectious Diseases and Immunology, Yalelaan 1, 3584CL Utrecht, The Netherlands. Phone: 31-30-2532485. Fax: 31-30-2536723. E-mail:}ln.uu.tev@reittor.p.

Received 2003 Jan 10; Accepted 2003 May 22.

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 ∼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.

Abstract

To successfully initiate an infection, viruses need to overcome the cell membrane barrier. Enveloped viruses achieve this by membrane fusion, a process mediated by specialized viral fusion proteins. Most viral fusion proteins are expressed as precursor proteins, which are endoproteolytically cleaved by cellular proteases, giving rise to a metastable complex of a receptor binding subunit and a membrane fusion subunit. Upon receptor binding at the cell membrane or as a result of protonation after endocytosis, the fusion proteins undergo a dramatic conformational transition. A hydrophobic fusion peptide becomes exposed and inserts into the target membrane. The free energy released upon subsequent refolding of the fusion protein to its most stable conformation is believed not only to facilitate the close apposition of viral and cellular membranes but also to effect the actual membrane merger (1, 47, 57). Knowledge about the molecular and biophysical events of this process is required for a thorough understanding of this essential step in the virus life cycle, as well as for the rational design of methods for intervention.

With a positive-stranded RNA genome of 28 to 32 kb, the Coronaviridae are the largest enveloped RNA viruses. Coronaviruses exhibit a broad host range, infecting mammalian and avian species. They are responsible for a variety of acute and chronic diseases of the respiratory, hepatic, gastrointestinal, and neurological systems (59). The spike (S) protein is the sole viral membrane protein responsible for cell entry. It binds to the receptor on the target cell and mediates subsequent virus-cell fusion (6). Spikes can be seen under the electron microscope as clear, 20-nm-long, bulbous surface projections on the virion membrane (14). The spike protein of mouse hepatitis virus strain A59 (MHV-A59) is a 180-kDa heavily N-glycosylated type I membrane protein which occurs in a homodimeric (38, 69) or homotrimeric (16) complex. In most MHV strains, the S protein is cleaved intracellularly into an N-terminal subunit (S1) and a membrane-anchored subunit (S2) of similar sizes which are noncovalently linked and have distinct functions. Binding to the MHV receptor (77) has been mapped to the N-terminal 330 amino acids (aa) of the S1 subunit (65), whereas the membrane fusion function resides in the S2 subunit (81). It has been suggested that the S1 subunit forms the globular head while the S2 subunit constitutes the stalk-like region of the spike (15). Binding of S1 to soluble MHV receptor, or exposure to 37°C and an elevated pH (pH 8.0), induces a conformational change which is accompanied by the separation of S1 and S2 and which might be involved in triggering membrane fusion (22, 28, 63). Cleavage of the S protein into S1 and S2 has been shown to enhance fusogenicity (26, 64), but cleavage is not absolutely required for fusion (2, 27, 62, 64).

The ectodomain of the S2 subunit contains two regions with a 4,3 hydrophobic (heptad) repeat (15), a sequence motif characteristic of coiled coils. These two heptad repeat (HR) regions, designated here HR1 and HR2, are conserved in position and sequence among the members of the three coronavirus antigenic clusters (Fig. (Fig.1).1). A number of studies have shown that the HR1 and HR2 regions are involved in viral fusion. First, a putative internal fusion peptide has been proposed to occur close to (7) or within (41) the HR1 region. Second, viruses with mutations in the membrane-proximal HR2 region exhibited defects in spike oligomerization and in fusion ability (40). Third, it has been suggested that the MHV-4 (JHM) strain can utilize both endosomal and nonendosomal pathways for cell entry but does not require acidification of endosomes for fusion activation (49). However, mutations found in MHVs which do require a low pH for fusion appeared to map to the HR1 region (24).

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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.

HR regions appear to be a common motif in many viral fusion proteins (60). There are usually two of them; one N-terminal HR region (HR1) adjacent to the fusion peptide and a C-terminal HR region (HR2) close to the transmembrane anchor. Structural studies of viral fusion proteins reveal that the HR regions form a six-helix bundle structure implicated in viral entry (reviewed in reference 19). The structure consists of a homotrimeric coiled coil of HR1 domains, in the exposed hydrophobic grooves of which the HR2 regions are packed in an antiparallel manner. This conformation brings the N-terminal fusion peptide into close proximity to the transmembrane anchor. Because the fusion peptide inserts into the cell membrane during the fusion event, such a conformation facilitates a close apposition of the cellular and viral membrane (reviewed in reference 19). Recent evidence suggests that the actual six-helix bundle formation is directly coupled to the merging of the membranes (47, 57). The similarities in the structures of the six-helix bundle complexes elucidated for influenza virus hemagglutinin (HA) (4, 11), human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) gp41 (5, 8, 42, 66, 72, 79), Moloney murine leukemia virus type1 gp21 (20), Ebola virus GP2 (43, 71), human T-cell leukemia virus type I gp21 (32), Visna virus TM (44), simian parainfluenza virus 5 (SV5) F1 (1), and human respiratory syncytial virus (HRSV) F1 (83) all point to a common fusion mechanism for these viruses.

Based on structural similarities, two classes of viral fusion proteins have been distinguished (37). Proteins containing HR regions and an N-terminal or N-proximal fusion peptide are classified as class I viral fusion proteins. Class II viral fusion proteins (e.g., the alphavirus E1 and the flavivirus E fusion proteins) lack HR regions and have an internal fusion peptide. Their fusion protein is folded in tight association with a second protein as a heterodimer. Here, fusion activation takes place upon cleavage of the second protein.

The coronavirus fusion protein (S) shares several features with class I virus fusion proteins. It is a type I membrane protein, synthesized in the endoplasmic reticulum, and is transported to the plasma membrane. It contains two HR sequences, one located downstream of the fusion peptide and one in close proximity to the transmembrane region. Despite its similarity to class I fusion proteins, there are several characteristics that make the coronavirus S protein exceptional. One is the absence of an N-terminal or even N-proximal fusion peptide in the membrane-anchored subunit. Another peculiarity is the relatively large size of the HR regions (∼100 and ∼40 aa). Third, cleavage of the S protein is not required for membrane fusion; in fact, it does not occur at all in the group 1 coronaviruses.

In the present study, we have investigated the biochemical and functional characteristics of the HR regions of the MHV-A59 spike protein. We show that peptides corresponding to the HR regions assembled into a thermostable, oligomeric, alpha-helical rod-like complex, with the HR1 and HR2 helices oriented in an antiparallel manner. HR2 was found to be a strong inhibitor of both virus entry into the cell and cell-cell fusion. Our findings show that the coronavirus MHV spike fusion protein belongs to the class I viral fusion proteins.

Acknowledgments

We are grateful to Mayken Grosveld and Alida Noordzij for their technical assistance with the HPLC and to Maurits de Planque, Bianca van Duyl, and Antoinette Killian for their assistance with the CD spectophotometer. We thank Raoul de Groot and Bert Jan Haijema for helpful discussions and Jean Lepault for his assistance with the electron microscope.

These investigations were supported by financial aid from The Netherlands Foundation for Chemical Research (CW) and The Netherlands Organization for Scientific Research (NWO) to B.J.B. and P.J.M.R.

Acknowledgments

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