Fig 1.
Stabilization and characterization of the soluble hMPV F protein ectodomain as a postfusion trimer.
A) Diagrams of the different constructs of hMPV F used in this study. (1) Scheme of the F protein (grey rectangle, NL/1/00 strain) denoting the last amino acid of its ectodomain (489), the fusion peptide (red), the preceding cleavage site (arrow) and the C-terminal 6xHis-tag (black box). (2) Same scheme but with the Foldon sequence (blue rectangle) and TEV cleavage site (arrow) added, and the change G294E denoted by a red circle. (3) The basic residues of hRSV F cleavage site II are shown in boldface and the amino acids deleted from the fusion peptide are indicated by dashes in the amino acid sequence. (4) Scheme of the protein after TEV cleavage. B) Gel-filtration traces of the four proteins depicted in panel A, labelled and color-coded as in panel A. Inset shows a Coomassie-blue-stained SDS-PAGE, run under reducing conditions, of the major peak of each chromatogram. C) Electron microscopy of negative-stained proteins 1–4. Some cone-shaped molecules are indicated in panel 2 by black arrowheads. Scale bar: 50 nm. D) Proteins 2 (green) and 3 (blue) were heated stepwise at the indicated temperatures, as described in Materials and Methods, before being tested for binding in ELISA to the mAbs indicated in each panel. Results are shown as percent of binding with proteins heated for 10 minutes at 30°C.
Table 1.
Crystallographic data collection and refinement statistics.
Fig 2.
Structure of hMPV F in the postfusion conformation.
Left: One protomer of the postfusion hMPV F trimer is shown as a ribbon colored as a rainbow from the N-terminus of F2 (blue) to the C-terminus of F1 (red). Right: The postfusion hMPV F trimer with one protomer shown as a ribbon and two protomers shown as molecular surfaces colored white and grey. The six-helix bundle (6HB) and fusion peptides are labeled.
Fig 3.
Comparison of paramyxovirus postfusion F structures.
Superposition of one protomer of the postfusion hMPV F structure (white) with the corresponding structures of A) hRSV F, B) hPIV3 F, and C) NDV F. Two neutralizing antigenic sites (II and IV of hRSV F) are magnified.
Fig 4.
Binding of mAb 101F to hMPV and hRSV F proteins.
A) Binding constants of mAb 101F for postfusion F proteins from hMPV NL/1/00 (A1 sublineage) and NL/1/99 (B1 sublineage) strains and from hRSV (Long strain) determined by surface plasmon resonance. B) Biacore binding sensorgrams used to determine the data in panel A.
Fig 5.
mAb 101F ELISA binding and neutralization.
A, B and C) ELISA binding of mAb 101F, the hMPV-specific mAb MF14, and the hRSV-specific mAb 47F to the indicated postfusion proteins. D, E and F) Neutralization of the noted viruses with the three mAbs.
Fig 6.
Structural basis of 101F cross-reactivity.
A) Left: Superposition of the linear 101F epitope derived from the hMPV (white) and hRSV (blue) postfusion F proteins. Side-chains are shown as sticks, with oxygen atoms colored red and nitrogen atoms colored blue. hMPV F residues are labeled and numbered. Right: Model of mAb 101F bound to antigenic site IV derived from the hMPV and hRSV postfusion F proteins. 101F heavy chain is colored red and light chain is yellow. Bottom: Sequence alignment of the antigenic site IV domain from hMPV and hRSV F. Identical residues have white text with black backgrounds, whereas residues that are similar are in bold and have a black border. Open circles denote residues with >10 Å2 buried surface area and filled rectangles denote residues whose side-chains form hydrogen bonds with 101F in the hRSV F peptide-bound crystal structure (PDBID: 3O45). B) Negatively stained electron micrographs of 101F Fab bound to postfusion hRSV F and hMPV F. The top two panels are 2D averages whereas the other panels are examples of individual negatively stained F–Fab complexes. C) Models of a single 101F Fab in complex with postfusion F trimers of hRSV and hMPV. Molecular surfaces of the trimers are shown, and residues within 5.5 Å of 101F atoms are darker.
Fig 7.
Binding and neutralization of sera from mice immunized with postfusion F proteins.
Soluble hMPV F postfusion trimers derived from NL/1/00 (A1 sublineage) or NL/1/99 (B1 sublineage) viruses and the equivalent hRSV F postfusion trimer were used to immunize BALB/c mice. Sera were collected 4 weeks after immunization and tested in (A) ELISA and (B) neutralization assays. ELISA titers refer to serum dilution that yielded 50% of the maximal (saturating) value and neutralization titers refer to dilution that inhibited 50% fluorescence intensity, 48 hours after infection. Mean ELISA and neutralization titers for each group are shown by horizontal bars. Horizontal red lines in each panel indicate detection limits.