Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Jan 26;113(4):1056-61.
doi: 10.1073/pnas.1523303113. Epub 2015 Dec 28.

Structure and stabilization of the Hendra virus F glycoprotein in its prefusion form

Joyce J W Wong  1 , Reay G Paterson  2 , Robert A Lamb  3 , Theodore S Jardetzky  4
Affiliations

Structure and stabilization of the Hendra virus F glycoprotein in its prefusion form

Joyce J W Wong et al. Proc Natl Acad Sci U S A. .

Abstract

Hendra virus (HeV) is one of the two prototypical members of the Henipavirus genus of paramyxoviruses, which are designated biosafety level 4 (BSL-4) organisms due to the high mortality rate of Nipah virus (NiV) and HeV in humans. Paramyxovirus cell entry is mediated by the fusion protein, F, in response to binding of a host receptor by the attachment protein. During posttranslational processing, the fusion peptide of F is released and, upon receptor-induced triggering, inserts into the host cell membrane. As F undergoes a dramatic refolding from its prefusion to postfusion conformation, the fusion peptide brings the host and viral membranes together, allowing entry of the viral RNA. Here, we present the crystal structure of the prefusion form of the HeV F ectodomain. The structure shows very high similarity to the structure of prefusion parainfluenza virus 5 (PIV5) F, with the main structural differences in the membrane distal apical loops and the fusion peptide cleavage loop. Functional assays of mutants show that the apical loop can tolerate perturbation in length and surface residues without loss of function, except for residues involved in the stability and conservation of the F protein fold. Structure-based disulfide mutants were designed to anchor the fusion peptide to conformationally invariant residues of the F head. Two mutants were identified that inhibit F-mediated fusion by stabilizing F in its prefusion conformation.

Keywords: F-protein atomic structure; Hendra virus; membrane fusion; metastable F-protein stabilization; paramyxovirus F protein.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Crystal structure of prefusion HeV F ectodomain and comparison with prefusion F structures from other paramyxoviruses. (A) HeV F fusion glycoprotein ectodomain in prefusion conformation, orthogonal views. (B) HeV F prefusion monomer. (C) Superposition of prefusion HeV F and PIV5 F (PDB ID code 2B9B), orthogonal views. HeV F is shown in green, and PIV5 F is shown in orange. Crystallographically refined N-linked carbohydrates are circled and indicated by Asn residue number. (D) Superposition of prefusion HeV F and RSV F (PDB ID code 4JHW), orthogonal views. HeV F is shown in green, and RSV F is shown in purple. (E) Rotation between domains of HeV F and RSV F. The axes of rotation are indicated by yellow rods.
Fig. S1.
Fig. S1.
Iterative build omit electron density maps of HeV F and paramyxovirus prefusion F structure comparison. (A) Apical loops, map contoured at 0.8 σ in PyMOL. The map was generated with PHENIX. (B) Fusion peptide region, map contoured at 1.6 σ in PyMOL. The map was generated with PHENIX. (C) Crystal structures of monomeric chains of prefusion HeV F, PIV5 (PDB ID code 2B9B), and RSV F (PDB ID code 4JHW).
Fig. 2.
Fig. 2.
Apical loops of HeV F in comparison to PIV5 F. (A) Apical portion of the HeV F trimer with PIV5 F superposed. HeV F is shown in green, and PIV5 F is shown in orange, with the apical loops lightened to pale green in HeV F and to orange in PIV5 F. The threefold axis of rotational symmetry is indicated. The disulfide bond between the two apical loops is indicated with sticks, and the sulfur atoms are colored olive-yellow. Carbohydrates of HeV F are shown as green sticks, and carbohydrates of PIV5 F are shown as orange sticks. (B) View down the rotational axis of the HeV F and PIV5 F trimers. (C) Sequence alignment of the apical loops of HeV F, PIV5 F, and NiV F generated with ClustalW. Loop structures confirmed by available crystal structures are lightened. Degree of conservation is indicated as follows: *, perfect; :, strong; ., weak.
Fig. 3.
Fig. 3.
Fusion activity of HeV F apical loop mutants. (A) Site-directed mutants of the apical region of HeV F. Residues mutated to Ala are indicated in purple, and Ala insertion mutants are indicated by B. (B) Fusion levels of site-directed mutants measured by luciferase reporter gene assay. R.L.U., relative light units.
Fig. S2.
Fig. S2.
Syncytia formation in HeV F- and G-expressing cells. Representative samples of HeV F apical loop mutants, double-Cys mutants, WT, and empty vector controls are shown at 160× magnification. Cells at 90% confluency were transfected with HeV F and HeV G in pCAGGS, and syncytia were observed 16–20 h later.
Fig. S3.
Fig. S3.
Total and cell surface expression of HeV F mutants. (A) Total immunoprecipitated HeV F fraction. Vero cells were transfected with HeV F and HeV G in pCAGGS, and then grown in Cys−Met− medium with Easy-Tag EXPRESS35S Protein Labeling Mix for radiolabeling. Cells were biotinylated with Sulfo-NHS-Biotin and lysed in radioimmunoprecipitation assay buffer. HeV F was immunoprecipitated from the cell lysate with polyclonal anti-HeV F 527–540 peptide serum and protein A agarose. (B) Cell surface HeV F fraction. The biotinylated cell surface fraction was separated with streptavidin agarose from the total immunoprecipitated HeV F fraction.
Fig. 4.
Fig. 4.
Fusion peptide region of HeV F in comparison to PIV5 F. (A) HeV and PIV5 F fusion peptides and preceding regions superimposed. HeV F is shown in light green, and PIV5 F is shown in light orange, with the fusion peptides darkened in HeV F and PIV5 F. Basic protease cleavage site residues are indicated by spheres, and the N- to C-terminal fusion peptide direction is indicated by arrows. (B) Sequence alignment of the fusion peptide regions of HeV, PIV5, and NiV F generated with ClustalW. The basic residue upstream of the N terminus of the fusion peptide is boxed in red, and the remainder of the fusion peptide is darkened. The degree of conservation is indicated as in Fig. 2.
Fig. S4.
Fig. S4.
Fusion peptide cleavage site of PIV5 and HeV F. (A) Catalytic site of furin in complex with the inhibitor m-guanidinomethyl-phenylacetyl-Arg-Val-Arg-(amidomethyl)-benzamidine (m-guanidinomethyl-Phac-RVR-Amba) (PDB ID code 4OMC). (B) Structural alignment of cathepsin L in complex with a natural peptide substrate, histone H3 (PDB ID code 3K24) and the inhibitor AZ12878478 (PDB ID code 3HHA). (C) Structural alignment of cathepsin L in complex with the natural polypeptide inhibitors chagasin (PDB ID code 2NQD) and MHC class II Ii p41 variant (PDB ID code 1ICF).
Fig. 5.
Fig. 5.
Fusion activity of predicted disulfide bond-forming mutants in HeV F. (A) Disulfide bond double mutants of HeV F HRA. Prefusion conformation of HeV F with residue pairs chosen for Cys mutations (Left) and a model of postfusion HeV F based on postfusion hPIV3 F (PDB ID code 1ZTM) (Right) are shown. Y97, the only ordered aligned residue in the crystal structure, is colored purple. The distance between the last visible residue preceding the fusion peptide and first visible residue of HRB is shown as a yellow dashed line. (B) Fusion levels of disulfide mutants measured by luciferase reporter gene assay. (C) Binding of prefusion-specific mAb 5B3 to HeV F disulfide mutants measured by flow cytometry. PE, phycoerythrin.
Fig. S5.
Fig. S5.
Assay for prefusion conformation of cell surface HeV F by flow cytometry. HeV F-pCAGGS–transfected HEK293T cells were incubated with prefusion conformation-specific mAb 5B3, followed by phycoerythrin (PE)-conjugated 2° Ab. Representative histograms from each sample are shown.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Rota PA, Lo MK. Molecular virology of the henipaviruses. Curr Top Microbiol Immunol. 2012;359:41–58. - PubMed
    1. Field H, et al. The natural history of Hendra and Nipah viruses. Microbes Infect. 2001;3(4):307–314. - PubMed
    1. Marsh GA, et al. Cedar virus: A novel Henipavirus isolated from Australian bats. PLoS Pathog. 2012;8(8):e1002836. - PMC - PubMed
    1. Drexler JF, et al. Bats host major mammalian paramyxoviruses. Nat Commun. 2012;3:796. - PMC - PubMed
    1. Mahalingam S, et al. Hendra virus: An emerging paramyxovirus in Australia. Lancet Infect Dis. 2012;12(10):799–807. - PubMed

Publication types

Associated data

-