Structure and mutagenesis of the parainfluenza virus 5 hemagglutinin-neuraminidase stalk domain reveals a four-helix bundle and the role of the stalk in fusion promotion

Abstract

Paramyxovirus entry into cells requires the fusion protein (F) and a receptor binding protein (hemagglutinin-neuraminidase [HN], H, or G). The multifunctional HN protein of some paramyxoviruses, besides functioning as the receptor (sialic acid) binding protein (hemagglutinin activity) and the receptor-destroying protein (neuraminidase activity), enhances F activity, presumably by lowering the activation energy required for F to mediate fusion of viral and cellular membranes. Before or upon receptor binding by the HN globular head, F is believed to interact with the HN stalk. Unfortunately, until recently none of the receptor binding protein crystal structures have shown electron density for the stalk domain. Parainfluenza virus 5 (PIV5) HN exists as a noncovalent dimer-of-dimers on the surface of cells, linked by a single disulfide bond in the stalk. Here we present the crystal structure of the PIV5-HN stalk domain at a resolution of 2.65 Å, revealing a four-helix bundle (4HB) with an upper (N-terminal) straight region and a lower (C-terminal) supercoiled part. The hydrophobic core residues are a mix of an 11-mer repeat and a 3- to 4-heptad repeat. To functionally characterize the role of the HN stalk in F interactions and fusion, we designed mutants along the PIV5-HN stalk that are N-glycosylated to physically disrupt F-HN interactions. By extensive study of receptor binding, neuraminidase activity, oligomerization, and fusion-promoting functions of the mutant proteins, we found a correlation between the position of the N-glycosylation mutants on the stalk structure and their neuraminidase activities as well as their abilities to promote fusion.

Fig. 1.

Structure of PIV5-HN stalk domain. (A) Crystal structure of the parainfluenza virus 5 HN stalk domain. Residues in “a,” “d,” and “h” positions of the hydrophobic core of the 4-helix bundle (yellow) are numbered. (B) Top view of the PIV5-HN stalk domain showing the side chain packing of “a,” “d,” and “h” residues (yellow). (C) Portion of the PIV5-HN stalk structure showing a distortion of the helix near serine 79 following which the 4HB shows a slight superhelical twist. The proline at position 84 is also highlighted. (D) Representative electron density (2Fo-Fc map) showing the 4HB structure of the PIV5-HN stalk.

Fig. 2.

Comparison of PIV5-HN and NDV-HN stalk structures. (A) PIV5-HN stalk 4HB in comparison with NDV-HN stalk 4HB. Structural alignment with backbone atoms of PIV5-HN and NDV-HN stalk crystal structures shows a root mean square deviation (RMSD) value of 1.119 over 394 atoms. (B) Sequence alignment showing heptad repeat of the PIV5-HN and NDV-HN stalks with the “a” and “d” positions (red) followed by the 11-mer repeat with the “a,” “d,” and “h” positions (black). Residue 79 is an “a/d” residue. The hydrophobic residues in the heptad repeat are highlighted in olive while those in the 11-mer repeat region are highlighted in yellow.

Fig. 3.

Design of mutants. (A) Schematic diagram of the PIV5 HN protein and the position of sites introduced for N-linked glycosylation (arrowheads) along the stalk region. CT, cytoplasmic tail, TM, transmembrane domain. (B) Positions of Cα atom of each residue on the PIV5-HN stalk structure that has an additional glycosylation added through mutagenesis (red). The mutants are named according to the residue that harbors the extra carbohydrate moiety. (C) Point mutations introduced in the HN stalk to create the N-linked glycosylation motif Asp-X-Ser/Thr-X, where X is any residue but proline. The point mutations are indicated in bold within the motif.

Fig. 4.

Expression of HN N-glycosylation mutants. (A) Immunoprecipitation of wt HN or N-glycosylation mutants expressed in HeLa-CD4-LTR-βGal cells. The cells were labeled with Tran³⁵S-label for 30 min and then incubated in medium (chased) for 90 min. Cells were then lysed in immunoprecipitation buffer, and proteins were immunoprecipitated using a mixture of HN monoclonal antibodies. Polypeptides were analyzed by SDS-PAGE. Numbers at left are molecular masses in kilodaltons. (B) Detection of proteins at the cell surface of 293T cells transfected with wt HN or HN N-glycosylation mutants using flow cytometry. Surface proteins were detected using a mixture of HN monoclonal antibodies or anti-HN polyclonal antibody R471. The mean fluorescence intensity (MFI) is shown as a percentage of wt protein levels. Results are from four independent experiments.

Fig. 5.

Functional analyses of the HN N-glycosylation mutants. (A) Receptor binding activity of the HN N-glycosylation mutants. HN wt or N-glycosylation mutants were transfected into 293T cells, and hemadsorption was measured by determining the amount of chicken red blood cell binding. Cells were washed in PBS and then lysed in H₂O. The hemoglobin absorbance at 540 nm was expressed as a percentage of wt protein hemadsorption. The receptor binding level was also normalized to the surface expression level (numbers beneath histogram bars). Abs, absorption wavelength; S.E., surface expression. Results are from four independent experiments. (B) Neuraminidase (NA) activities of wt and mutant HN proteins were determined in transfected HeLa-CD4-LTR-βGal cells. NA activity of these proteins was calculated from the readout of cleaved MU-NANA fluorogenic product at an emission wavelength of 450 nm. This was expressed as a percentage of wt HN NA activity. Neuraminidase activity, normalized to surface expression level, is also shown as numbers beneath histogram bars. Em, emission wavelength. (C) Luciferase reporter assay of cell-cell fusion. Vero cells transfected with F, HN, and luciferase under the control of a T7 promoter were overlaid with BSR-T7 cells 15 h posttransfection. After 7 h, the cells were lysed and the extent of cell-cell fusion was obtained as relative luciferase units (RLUs). Fusion activity of the mutants was expressed as a percentage of wt HN fusion. Luc, pT7 luciferase transfected alone. (D) Representative micrographs of syncytia, showing cell-cell fusion in BHK-21 cells transfected with F and wt HN or HN N-glycosylation mutants at 18 h posttransfection.

Fig. 6.

Oligomeric form of wt HN and HN N-glycosylation mutants. Sucrose density gradient ultracentrifugation was used to analyze oligomerization patterns of wt HN and the mutants N58, N66, N67, N68, and N90 expressed in transfected HeLa-CD4-LTR-βGal cells. Eighteen hours posttransfection, Tran³⁵S-labeled proteins were extracted using Triton X-100 and the lysates were subjected to ultracentrifugation on a 7.5% to 22.5% (wt/vol) sucrose density gradient in a SW41 rotor at 37,000 rpm for 19 h. Fractions were collected from the top of the gradient, alternate fractions were immunoprecipitated using an HN monoclonal antibody mixture, and polypeptides were analyzed by SDS-PAGE under nonreducing conditions. Fractions are labeled numerically from top to bottom of the gradient. M, monomer; D, dimer; T, tetramer. HN-S-S-HN, disulfide-linked HN dimer. The figure shows representative gel images of wt-HN (A), HN-N58 (B), HN-N66 (C), HN-N67 (D), HN-N68 (E), and HN-N90 (F).

Fig. 7.

Fusion activity of HN N-glycosylation mutants in mixed oligomers. (A) Representative micrographs of syncytia, demonstrating cell-cell fusion in BHK-21 cells transfected with F and titrated mixtures of wt HN and mutant HN constructs. One microgram of F DNA was cotransfected with a 1-μg mixture of wt and mutant HN DNAs at different ratios. Mutants are arranged from left to right according to their position in the PIV5-HN stalk, with the mutant toward the bottom of the stalk on the left and the mutant toward the top of the stalk on the right. (B) Quantitative luciferase reporter assay of cell-cell fusion. Fusion activity of HN N-glycosylation mutants was characterized by forming different combinations of tetramers, which would include wt and mutant monomers in various ratios. These different DNA wt/mutant HN ratios, F, and luciferase under the control of the T7 promoter were expressed in Vero cells, which were overlaid with BSR-T7 cells 15 h posttransfection. Seven hours postoverlay, the cells were lysed and luciferase activity (expressed in RLU) was determined. Fusion-inducing capabilities of the HN mutants in mixed oligomers are shown, with wt fusion considered 100%.

Fig. 8.

Expression of proteins in mixed oligomer experiments. Immunoprecipitations of mixed oligomer proteins of HN wt and the N91 mutant were carried out from transfected HeLa cell lysates. Transfected cells were pulsed with Tran³⁵S-label for 30 min and then chased with complete medium for 90 min. The cells were lysed, and immune complexes were detected using an HN-specific monoclonal antibody mix. The proteins were resolved on an 8% SDS-PAGE gel. The arrowheads indicate the shift in size between the wt and the N91 mutant, the latter carrying an N-glycosyl carbohydrate chain. Numbers above the gel indicate the μg of DNA of wt and N91 transfected. Numbers at left indicate molecular masses in kilodaltons.

Fig. 9.

Mapping of mutants implicated in F interaction and affecting NA activity. (Left) PIV5-HN mutations (pink) and corresponding residues of NDV-HN mutations (lilac) believed to block F interaction (category I mutants) are mapped onto the PIV5-HN stalk structure. Residues that affect only fusion in PIV5-HN and NDV-HN are shown in red. (Right) Mutations in PIV5-HN (olive) and residues corresponding to NDV-HN mutations (orange) implicated in influencing functions of the head in addition to fusion (category II mutants) are mapped on the PIV5-HN stalk structure. Residues that affect fusion and NA activity in both PIV5-HN and NDV-HN are shown in yellow.