Prefusion structure of human cytomegalovirus glycoprotein B and structural basis for membrane fusion

Abstract

Human cytomegalovirus (HCMV) causes congenital disease with long-term morbidity. HCMV glycoprotein B (gB) transitions irreversibly from a metastable prefusion to a stable postfusion conformation to fuse the viral envelope with a host cell membrane during entry. We stabilized prefusion gB on the virion with a fusion inhibitor and a chemical cross-linker, extracted and purified it, and then determined its structure to 3.6-Å resolution by electron cryomicroscopy. Our results revealed the structural rearrangements that mediate membrane fusion and details of the interactions among the fusion loops, the membrane-proximal region, transmembrane domain, and bound fusion inhibitor that stabilized gB in the prefusion state. The structure rationalizes known gB antigenic sites. By analogy to successful vaccine antigen engineering approaches for other viral pathogens, the high-resolution prefusion gB structure provides a basis to develop stabilized prefusion gB HCMV vaccine antigens.

Fig. 1. Key structural features.

(A) Linear representation of HCMV gB (Towne strain) domains labeled and color-coded as follows: I, blue; II, green; III, yellow; IV, orange; V, red; membrane-proximal region (MPR), cyan; transmembrane (TM), olive; unmodeled N-terminal residues (N-term), residues 436 to 483, and cytoplasmic residues (Cyto), white. The boundary amino acid numbers are indicated at the top. The fusion loops (FL), furin cleavage site, and key α helices are indicated. Domain color scheme is maintained in all panels. (B) Ribbon diagram of prefusion gB with space-filling model of the bound inhibitor WAY-174865 (magenta). In one protomer, the domains are colored, and the fusion loops (FLs) are indicated with an asterisk. (C) Prefusion gB ribbon diagram fit to the cryo-ET density map of the CMV viron surface (EMD-9328) (6). Protomers of prefusion and postfusion gB are compared in (D) and (E). The dimensions of the trimeric ectodomains are indicated on space-filling models of prefusion (F) and postfusion (G) gB. The approximate height of an unbuildable membrane-distal part of the density map of prefusion gB is indicated. Red dashed lines connect equivalent positions of domains III and IV of the prefusion and postfusion gB structures in (D) and (E) and in (F) and (G). In (B), (F), and (G), the N- to C-terminal direction of the domain III coiled coil is indicated by vertical arrows.

Fig. 2. Structural features of gB mapped to the primary amino acid sequence.

Secondary structural elements of the prefusion structure (Towne strain) are color-coded as in Fig. 1A and indicated above the sequence: α helices, cylinders; β strands, thick arrows; loops, solid lines; residues missing from the model, dashed lines. Postfusion secondary structural elements that differ from the prefusion structural elements are indicated above the prefusion elements in black: α helices, waved lines; β strands, double lines; loops, straight lines. Features of the primary amino acid sequence are indicated by the color of the letters or colored outline or solid boxes around the letters: secretion signal sequence, brown bold letters; fusion loops, black bold letters; predicted N-linked glycosylation sites, solid light blue boxes; glycosylation sites observed in the density map, solid light blue boxes with black outlines; residues that contact the fusion inhibitor, solid magenta boxes; furin cleavage site, gray solid boxes; residues that contact antigenic domain 4 FAb SM5-1 in the complex, green outline boxes; escape mutations for antigenic domain 5 mAbs 1G2 (Y280 and N284) and 2C2 (N293 and Y280), blue outline boxes.

Fig. 3. Structural details of prefusion gB.

When multiple protomers are shown, they are labeled with superscripts. (A) Domain V interprotomer interactions. Ribbon diagrams of domains from one protomer are colored as in Fig. 1A. Backbone of domain V of an adjacent protomer is a gray tube in a semitransparent gray space-filling model. Helices α3, α6, and α7 are labeled. Key residues described in the main text are indicated. (B) MPR structure. Representative solvent-exposed hydrophilic residues are labeled with black type, and representative hydrophobic residues that interact with the membrane are labeled with red type. (C) TM domain structure. Residues and dimensions described in the main text are indicated. (D) Interaction between the MPR and the fusion loop of domain I from an adjacent protomer. (E) Interaction of the MPR of one protomer with the hinge between the MPR and TM of an adjacent protomer. (F) Hydrophobic interactions of the inhibitor and gB. Inhibition escape mutation locations are red. (G) Polar interactions between the inhibitor and gB. Hydrogen bonds and dipole interactions described in the text are indicated with dashed lines.

Fig. 4. Model of gB rearrangement.

(A) In prefusion gB (left), the fusion loops (purple star) interacts with the MPR (cyan), embedded in the viral envelope’s (black parallel lines) outer leaflet. To form the hypothesized extended intermediate (middle), fusion loop interactions with the MPR are broken, and domains I (blue) and II (green) rotate almost 180° (left dashed arrow) so that the fusion loop, now at the viral envelope–distal end of gB, interacts with the host cell membrane (red parallel lines), bridging the viral envelope to the host cell membrane. Domain V (red), which had been covered by domain I, is exposed. During the transition from the extended intermediate to postfusion gB (right model), domain V extends toward the cell membrane (middle dashed arrow), packing between domain I from the other two protomers. The MPR and TM (olive) are juxtaposed, facilitating formation of the hemifusion intermediate between the viral envelope and host cell membrane, which resolves to complete fusion. (B) Breaking of the interactions between the fusion loops of one protomer with the MPR/TM hinge of an adjacent protomer during the prefusion to extended intermediate transition. (C) Movement of MPRs away from the threefold axis during the extended intermediate to postfusion transition.

Fig. 5. Antigenic surfaces and potential gH/gL binding site on the prefusion gB ectodomain (residues 86 to 721).

(A) View perpendicular to the threefold axis. (B) Oblique view from below the viral envelope. The model is rotated 70° as indicated around the black vector perpendicular to the threefold axis. Domains are colored as in Fig. 1A; glycans are beige. Surfaces from residues that bind anti-gB complement-independent neutralizing antibodies are white and labeled in orange type for AD1, green type for AD4, and blue type for AD5. Residues in AD1 were identified by peptide mapping; residues in AD4 and AD5 were identified by x-ray crystal structures of FAb-antigen complexes and mAb neutralization escape mutations (references provided in table S2). The most N-terminal residue in buildable density, T86, is black. Approximate gH/gL binding site (fig. S8) based on published cryo-EM image of CMV virion (6) is indicated with black dashed circle. Threefold axis is indicated with a black triangle.