Measles virus entry inhibitors: a structural proposal for mechanism of action and the development of resistance

Abstract

Previously, we developed a panel of nonpeptidic compounds specifically preventing fusion of the measles virus (MV) with target cells at IC(50) values of 0.6-3 muM. Mutations in the MV fusion protein (MV F) that render resistance to these blockers were described. The structural basis for both inhibition and resistance was unclear in the earlier work because of the availability of a structural model for only the postfusion conformation of MV F. We have now developed structural models for both pre- and postfusion conformers of the latter protein trimer. The models allow investigation of the large-scale conformational changes occurring in the MV fusion machinery and, in conjunction with antisera binding studies, provide a rationale for how inhibitors may arrest a conformational intermediate by interfering with the formation of interactions between the heptad repeat B (HR-B) linker and DIII domains. The models also show that resistance to inhibition can be explained by a predicted destabilizing effect of the mutations on the HR-B domain within the trimeric prefusion structure. This viewpoint is supported by the temperature-dependent differential fusion activities of MV F variants harboring these mutations.

FIGURE 1

Secondary structure diagrams of MV F protein homology models highlight the domain movements occurring during fusion. Pre-fusion (A) and post-fusion (B) conformations are colored by subunit. Note the 3-helical bundle on the left and the 6-HB on the right. (C) Pre-fusion (green) and post-fusion (cyan) models superimposed by DI and DII domains. Overall backbone RMSD is 50Å. (D) Single subunit of pre-fusion model colored by domains. (E) Post-fusion model of the same subunit as (D), colored by domains, and similarly oriented by DI and DII domains.

FIGURE 2

Potential energy sources in fusion refolding. (A–D) Hydrophobic surfaces of one HR-A and HR-B subunit in pre-fusion and post-fusion models (brown = hydrophobic, green = neutral, blue = hydrophilic). (E–F) Water-filled cavity and channels in pre-fusion and post-fusion models. Protein is shown as cartoon or surface, colored by subunit; water is shown as vdW spheres. (E) Pre-fusion model sustains a cavity with 550 water molecules held inside. Surface representation on the right shows that the cavity is almost completely sealed. (F) Post-fusion model has an axial channel and three radial channels filled with 400 water molecules. Surface representation shows the openings of the channels at the top and the sides.

FIGURE 3

Changes in the Val94 microdomain occurring during fusion. Pre-fusion (A) and post-fusion (B) models are displayed with Val94 microdomain residues as vdW spheres with the protein shown as secondary structure. In the pre-fusion model the microdomain is covered by the fusion peptide and HR-A; structural rearrangements occur in the DIII and HR-C domains to bring the microdomain together. (C) Interaction between Thr270 and Ser144 (shown as sticks) in the pre-fusion model. This interaction appears to anchor HR-A and Fpep to the protein head prior to fusion.

FIGURE 4

Co-immunoprecipitation experiments with AS-48 and two antibodies directed at epitopes in the Val94 microdomain. (A) Ab-359, directed against residues 88–104, and Ab-361, directed against residues 240–259 have opposing effects on the amount of complex precipitated when added prior to AS-48. When introduced after AS-48 addition, both antibodies precipitated amounts of complex similar to antibody-only conditions. Pre-fusion (B) and post-fusion (C) models are shown as surfaces with residues 88–104 and 240–259 colored red and blue, respectively. The location of the Val94 microdomain in both models is highlighted by circles. The epitopes are closer and considerably more exposed post-fusion (337 Ų and 79 Ų increased surface area for residues 88–104 and 240–259, respectively).

FIGURE 5

Proposed fusion pathways for the entire MV F (A–C, similar to pathway by Yin et al. (6)) and the DIII domains (D). (A) Prior to fusion, MV F exists in a metastable state with water-filled cavity, HR-B in a 3HB, and the fusion peptide and HR-A wrapped around the head of the protein. (B) Activation causes fusion peptides to bury within the cell membrane, formation of the HR-A 3HB, and dissociation of HR-B. (C) Final post-fusion structure with completely collapsed water-filled cavity and fully formed HR-A and HR-B 6HB. (D) Results of morphing simulation of the DIII domain, shown in 6 of the 30 frames. Residue coloring is the same as Figure 3A–B. AS-48 (shown as sticks, colored with pink carbons and outlined in black) is docked into frame 25 and is proposed to inhibit fusion by disrupting interactions between DIII and the HR-B linker. In this frame, the 240–259 epitope (shown as blue surface) is fully exposed for interaction with the Ab-361 antibody.

FIGURE 6

Position of resistance-conferring mutations at Asn462 and Ala367. (A) Pre-fusion model shows that these residues are proximal and part of the key interactions holding HR-B to the head of the protein prior to fusion. (B) Proposed intermediate structure revealing that Asn462 and Ala367 must separate to achieve the conformational intermediate. (C) Post-fusion model showing that Asn462 ends up in the 6HB while Ala367 remains in the DI domain of the MV F head. (D) View of the HR-B/HR-B linker interface with the head of MV F in the pre-fusion model. Residues providing key hydrophobic interactions are shown as sticks. Eight hydrophobic residues provide interaction of the HR-B/HR-B linker to the head. Five of the residues (Leu457, Leu 454, Ile452, Pro451 and Pro450) are from this network. The other three hydrophobic residues (Leu448, Ile446, and Val432, not shown) are located upstream on the HR-B linker.

FIGURE 7

Predicted structures for resistance-conferring mutations. (A–D) Asn462 mutations at the interface of HR-B and DI domains in the pre-fusion model. The protein is shown as secondary structure with its Connolly surface. Selected residues are shown as sticks. In wild-type (A), Asn462 hydrogen bonds the backbone oxygen of Asp458. The Asn462 →Asp mutant (B) introduces a negative charge into this critical hydrophobic environment. The Asn462 → Lys mutant (C) introduces a positive charge. The Asn462 → Ser mutant (D) does not introduce a charge, but as a smaller residue, it increases the solvent exposure of the hydrophobic interactions compared to wild-type. (E–F) Ala367 mutations at the interface of HR-B and DI domains in the pre-fusion model. The protein is shown as secondary structure. Selected residues are shown as sticks. In wild-type (E), Ala367 is part of the network of hydrophobic interactions. The Ala367 → Thr mutant (F) introduces a polar hydroxyl directly into the hydrophobic network.

FIGURE 8

(A) MV F variants N462S and N462K show enhanced fusion activity at 30°C indicating reduced conformational stability. The 462K variant shows increased fusogenicity also at physiological temperature. Quantification of fusion activity of F variants after co-transfection of Vero cells with equal amounts of plasmid DNA encoding MV H and F, and incubation at 30°C or 37°C as indicated. The values represent means of four experiments and are expressed as the percentage of fusion activity observed for unmodified MV F after incubation at 30°C or 37°C, respectively. (B) In the context of infection with recombinant MV, the F-462K variant confers increased lateral spread through the target cell monolayer. Infected cells were subjected to crystal violet-staining 30 hours post-infection to visualize virus-induced syncytia.

FIGURE 9

(A) Predicted binding model for AS-48, in the post-fusion model. This binding pocket is not available in the pre-fusion model (vida Figures 3 and 5). (B) Predicted binding model for AM-4, also in the post fusion model.

FIGURE 10

Predicted geometry of the intersubunit disulfide bond between positions 452 and 460 in the pre-fusion MV F model. The protein is shown as secondary structure and colored by subunit. Cysteines at positions 452 and 460 are shown as blue spheres with orange sulfur atoms (A) Side view of the trimer (B) View looking up from the 3HB of HR-B. (C) Close-up view of disulfide bonds with the cysteines shown as sticks