Cryo-EM structure of the vaccinia virus entry fusion complex reveals a multicomponent fusion machinery

Abstract

Membrane fusion is essential for viral entry. Unlike class I-III fusion proteins, vaccinia virus (VACV) uses a multicomponent entry fusion complex (EFC). Using cryo-electron microscopy, we determined the full-length structure of the VACV EFC at near-atomic resolution, revealing a 15-protein asymmetric assembly organized into three layers. The central A16/G9/J5 heterotrimer forms the fusion core, stabilized by conserved PXXCW and Delta motifs, and anchors two A28/H2 adaptor dimers linked to peripheral G3/L5/A21/O3 scaffolds. Structural and evolutionary analyses identify a conserved N-terminal domain in A16 containing a myristoyl-binding pocket and a phenylalanine-rich region that stabilizes the trimer and may regulate lipid engagement. An additional component, F9, binds peripherally to J5, A21, and H2 through Delta-like motifs, reinforcing the prefusion architecture. Together, these results define the VACV EFC as a unique multiprotein fusion machinery and provide a structural framework for understanding the mechanism of poxvirus entry and membrane fusion.

Fig. 1.. Cryo-EM structure of the pre-fusion VACV EFC.

(A) SDS-PAGE analysis of purified VACV EFC eluted from the streptavidin affinity column. Major EFC components are indicated; contaminating host proteins (tubulin/actin) and the viral membrane protein H3 are marked. Molecular weight markers are shown at left. (B) In vitro liposome coflotation assay of purified EFC after neutral or acidic pH treatment. Purified EFC and liposomes were mixed at different pH levels from 7 to 4 for 2 hours, followed by OptiPrep gradient centrifugation and collected into six fractions (top to bottom). The distribution of each EFC component was analyzed by immunoblotting. (C) Cryo-EM density map of the EFC. Left: map colored by local resolution and contoured at 2.5σ. Right: map contoured at 6σ and colored by subunit identity, A16 (red), G9 (green), J5 (yellow), A28 (blue), A28′ (light blue), H2 (magenta), H2′ (light pink), G3 (forest green), G3′ (light green), L5 (orange), L5′ (light orange), A21 (cyan), A21′ (light cyan), O3 (brown), and O3′ (light brown) are highlighted (right). (D) Schematic diagram showing the topology of the 15-protein EFC and its tripartite architecture: the central trimer, two adaptor pairs, and two scaffold assemblies. Single-pass transmembrane (TM) helices are indicated by vertical bars. Colors correspond to those in (C). (E) Atomic model of the 15-protein EFC viewed from two orientations. The model was reconstructed by fitting AlphaFold predicted subunits and manually rebuilding them into the cryo-EM density map, as shown in (C). Approximate dimensions are indicated. (F) Surface representation highlighting the tripartite organization: the central trimer (green), adaptor 1/2 (magenta), and scaffold 1/2 (beige). (G) Cross-sectional cartoon of TM domains along the dashed line in (E), showing the spatial arrangement of the central trimer, adaptors, and scaffolds. Color coding follows (F).

Fig. 2.. Asymmetric organization and hinge flexibility of adaptor and scaffold subcomplexes within the EFC.

[(A), (D), and (E)] Superimposed structures of adaptor and scaffold pairs reveal conserved cores and asymmetric features. (A) Alignment of adaptor 1 (A28/H2) and adaptor 2 (A28′/H2′), with aligned regions shown in gray (RMSD = 0.72 Å). The boxed close-up highlights the secondary structures and conserved contact residues at the A28-H2 and A28′-H2′ interfaces. (B and C) Structures of adaptor 1 and adaptor 2, respectively, with boxed close-ups showing distinct hinge-contact residues. (D) Comparison of scaffold 1 (G3/L5) and scaffold 2 (G3′/L5′) (RMSD = 0.65 Å), showing shared contacts in gray. The inset highlights the secondary structures and contact residues mediating at the G3-L5 and G3′-L5′ interfaces. (E) Alignment of A21 and A21′ within scaffold 1 and scaffold 2 (RMSD = 0.65 Å), with common intersubunit contacts shown in gray. The inset highlights the secondary structures and contact residues mediating at the A21-G3/L5 and A21′-G3′L5′ interfaces. The unaligned regions in each panel are colored as in Fig. 1C. [(B), (C), (F), and (G)] Structural divergence between the two adaptor and scaffold pairs. (F and G) Structures of scaffold 1 (G3/L5/A21/O3) and scaffold 2 (G3′/L5′/A21′/O3′), with insets highlighting secondary structures and differences in hydrogen bonding and ionic interactions at their hinge interfaces. Residues are shown as sticks and labeled. Hydrogen bonds and salt bridges are represented by dashed black and red lines, respectively.

Fig. 3.. Intersubcomplex interfaces connecting adaptor, scaffold, and trimer layers within the EFC ectodomain.

[(A) to (E)] Structural interfaces linking the central trimer with the surrounding adaptor and scaffold subcomplexes. (A) Interface between the central trimer and adaptor 1. (B) Interface between the central trimer and adaptor 2. (C) Interface between adaptor 1 and scaffold 1. (D) Interface between adaptor 2 and scaffold 2. (E) Interface between the central trimer and scaffold 2. In each panel, the contact interface between two subcomplexes is boxed, with a close-up view showing secondary structures and interacting residues. Residues involved in intersubcomplex contacts are shown as sticks and labeled. Hydrogen bonds and salt bridges are represented by dashed black and red lines, respectively. Proteins are color-coded as in Fig. 1C.

Fig. 4.. The A16/G9/J5 central trimer is stabilized by conserved structural motifs and intersubunit interfaces.

(A) Overall architecture of the A16/G9/J5 heterotrimer shown as a cartoon representation. (B) Close-up view of the contact interface between the N-terminal domains of A16 (cartoon, red) and G9 (surface, green). Interacting residues are highlighted, and non-interacting regions are shown in gray. (C) Trimerization interface formed by conserved PXXCW motifs of A16, G9, and J5. Intermolecular contact residues and disulfide bonds are shown as sticks. Multiple-sequence alignment (MSA) below highlights conserved and contact residues (black and pink shading, respectively), with secondary-structure elements indicated above (helices as columns, β-strands as arrows). (D) Close-up of the Delta motifs in A16, G9, and J5, displayed as cartoons with hydrophobic surfaces overlaid. A hydrophobic core is evident at the parallel motif interface. Conserved and contact residues are marked in the accompanying MSA. (E) Structural rearrangement of the extended loops linking the ectodomain (ECD) and TM domains of A16, G9, and J5. Four cross-sectional views (left) illustrate the intertwined arrangement of the three subunits, summarized schematically at right. (F) Surface representation of the A16/G9/J5 central trimer highlighting residues whose mutations reduce viral infectivity and disrupt EFC assembly (red).

Fig. 5.. Bioinformatic and structural analyses of the central trimer revealed conserved motifs in the N-terminal domains of A16.

(A) Surface representations of A16 (left), G9 (middle), and J5 (right) colored by sequence conservation across 52 orthologs from Poxviridae family, according to the ConSurf scale (bottom right). Highly conserved residues are shown in purple and variable residues in green. Conserved structural motifs, including PXXCW and Delta, as well as the A16 N-terminal domain (N-domain) containing sites A and B, are labeled. (B) Multiple sequence alignment (MSA) of A16 orthologs from 13 representative poxviruses (abbreviations defined in table S3). Three conserved Phenylalanine-rich motifs are underlined. Residues within 5 Å of myristic acid in the AlphaFold model are marked as pink dots, and conserved aromatic residues as purple dots. Secondary structure elements are shown above the alignment (helices as cylinders, β-strands as arrows). (C) Structural superposition of the myristoyl-binding pocket among four A16 structures: the EFC-bound cryo-EM structure (PDB 9UZO, red), the AlphaFold3 model (green), the A16/G9-A56/K2 complex (PDB 9HBK, orange), and the A16/G9 crystal structure (PDB 8GP6, light blue). The position of bound myristic acid is shown. Black arrows indicate two regions showing major deviations. (D) Close-up view of the lipid-binding cavity showing the deviated α3-α4 and β1-β3 elements from superimposed A16 structures [as in (C)]. (E) Structural comparison of site B among the four A16 structures in (C). Black arrows mark the phenylalanine-containing motif. (F) Interaction network between the N-terminal domains of A16 and G9 within the EFC structure (PDB 9UZO). The phenylalanine-containing motif of A16 (red) and the contacting residues of G9 (green) are shown as sticks. Hydrogen bonds and salt bridges are indicated by black and red dashed lines, respectively.

Fig. 6.. Structure of the EFC + F9 complex and conservation of Delta motifs.

(A) Cryo-EM density map of the EFC + F9 complex. Left: consensus map colored by local resolution and contoured at 2.5σ. Right: map contoured at 6σ and colored by subunit identity. F9 is shown in gray; EFC components are colored as in Fig. 1C. (B) Atomic model of the 16-protein EFC + F9 assembly. Subunits are displayed as ribbons and colored as in (A); the viral membrane boundary is indicated. (C) Surface representation of the EFC + F9 complex. Left: overall surface showing the peripheral location of F9 (gray). Right: F9 colored by its interacting partners-J5 (yellow), A21 (cyan), and H2 (magenta), positioning F9 at the membrane-proximal region of the complex. (D) Close-up view of the boxed region in (C) showing F9 contact interface with A16/G9/J5 Delta motifs; disulfide bonds are shown as sticks. (E) Surface hydrophobicity of the oligomerization interface between F9 and the A16/G9/J5 Delta motifs is colored from hydrophilic (teal) to hydrophobic (tan).