Structural basis for coronavirus-mediated membrane fusion. Crystal structure of mouse hepatitis virus spike protein fusion core

Abstract

The surface transmembrane glycoprotein is responsible for mediating virion attachment to cell and subsequent virus-cell membrane fusion. However, the molecular mechanisms for the viral entry of coronaviruses remain poorly understood. The crystal structure of the fusion core of mouse hepatitis virus S protein, which represents the first fusion core structure of any coronavirus, reveals a central hydrophobic coiled coil trimer surrounded by three helices in an oblique, antiparallel manner. This structure shares significant similarity with both the low pH-induced conformation of influenza hemagglutinin and fusion core of HIV gp41, indicating that the structure represents a fusion-active state formed after several conformational changes. Our results also indicate that the mechanisms for the viral fusion of coronaviruses are similar to those of influenza virus and HIV. The coiled coil structure has unique features, which are different from other viral fusion cores. Highly conserved heptad repeat 1 (HR1) and HR2 regions in coronavirus spike proteins indicate a similar three-dimensional structure among these fusion cores and common mechanisms for the viral fusion. We have proposed the binding regions of HR1 and HR2 of other coronaviruses and a structure model of their fusion core based on our mouse hepatitis virus fusion core structure and sequence alignment. Drug discovery strategies aimed at inhibiting viral entry by blocking hairpin formation may be applied to the inhibition of a number of emerging infectious diseases, including severe acute respiratory syndrome.

Fig. 1

Structure determination of the MHV spike protein fusion core trimer.A, schematic representation of coronavirus MHV A59 spike protein and the MHV 2-Helix and nMHV 2-Helix constructs. S1 and S2 are formed after proteolytic cleavage (vertical arrow) and noncovalently linked. The enveloped protein has an N-terminal signal sequence (SS) and a TM domain adjacent to the C terminus. S2 contains two HR regions (hatched bars), termed HR1 and HR2 as indicated. FP (hatched bars) is a putative fusion peptide followed by HR1 region. For the MHV 2-Helix, two HR regions were linked to a single polypeptide with an 8-residue linker (GGSGGSGG). For the nMHV 2-Helix, HR2 and a shortened HR1 were linked with a 22-amino acid linker (LVPRGSGGSGGSGGLEVLFQGP). B, sequence alignment of coronavirus spike protein HR1 and HR2 regions. Letters above the sequence indicate the predicted hydrophobic HR a and d residues, which are highly conserved. C, helical wheel representation of HR1 and HR2. Three HR1 helices and one HR2 helix are represented as helical wheel projections. The view is from the top of the structure. The three central HR1 helices form a central hydrophobic core with the interaction of residues in the a and d positions. The three HR2 helices pack against these hydrophobic surface grooves through interactions with residues in the a and d positions in HR2 and e and g positions in HR1. These residues, mediating the interactions between HR1 and HR2, are always hydrophobic and conserved (see B).

Fig. 2

Overall views of the fusion core structure and superposition of nMHV (new construct for MHV fusion core) and MHV fusion core.A, top view of the MHV fusion core structure showing the 3-fold axis of the trimer. B, side view of the MHV fusion core structure showing the six-helix bundle. C, side view showing the superposition of nMHV fusion core (colored in blue) and MHV fusion core (colored in yellow). The columns at both sides of the map represent two HR1 and HR2 regions of nMHV and MHV fusion cores. The number at the end of these columns represents the end residues in the two structures.

Fig. 4

O-X-O motifs in HR2 regions of MHV and the comparison with those of other fusion proteins.A. Left and center, surface map showing the hydrophobic grooves on the surface of three central HR1 helices. Three HR2 helices pack against the hydrophobic groove in an antiparallel manner. The helical regions in HR2 extended regions could be observed clearly. The helical region of HR2 just packs against the deep groove, and the extended region packs against the shallow groove. Right, detailed structure of O-X-O motifs in MHV HR2 region. One HR2 helix is divided into five parts based on its secondary structure. The helical regions (parts 2 and 4) HR2 are colored in red, and extended regions (parts 1, 3, and 5) are colored in blue. The essential residues of the three extended regions and O-X-O motifs in these regions are shown; residues colored in green represent the hydrophobic residues in O-X-O motifs. The three panels on the left show the enlarged images of parts 1, 3, and 5. The hydrophobic residues in these motifs are all packed against the hydrophobic grooves on the surface of three HR1 helices. B, detailed structures of O-X-O motifs in other fusion proteins including SV5F, HRSV F, MMLV Env-TM, and Ebola GP2. They all contain similar motifs in HR2 regions. The regions in which O-X-O motifs are located form extended regions but not α-helices, in a way similar to the MHV 2-Helix.

Fig. 3

Viral fusion proteins and models for membrane fusion.A, comparison of MHV fusion core with other viral fusion protein structures. The proteins under comparison include SV5F, Ebola GP2, HIV gp41, MMLV Env-TM, and low pH-induced influenza virus HA, tBHA2. Top and side views are shown for the six fusion core structures. B, model for coronavirus-mediated membrane fusion. The first state is the native conformation of coronavirus spike protein on the surface of viral membrane. It has been reported that the spike protein is trimeric in this conformation and about 200 Å in length (6), but the exact structure of the full-length protein remains unknown. The second state is the prehairpin state of the S2 subunit. After several conformational changes, the fusion peptide inserts into the cellular membrane with the aid of other regions of S protein and possibly including the receptor. Although the internal fusion peptide is not exposed at the N-terminal of S2, it could insert into part of the target membrane by means of some hydrophobic residues. This insertion would be stable enough to drive the membrane motion with the conformational changes of HR1 region, which is adjacent to the fusion peptide. The third state is conformational change and juxtaposition of the target and viral membranes. With the help of other regions of S protein, the HR1 and HR2 regions move together and facilitate juxtaposition of the cellular and viral membrane. The last state is the postfusion conformation. The coiled coil will reorient with its long axis parallel to the membrane surface. The fused cellular and viral membranes make it possible for subsequent viral infections.