Mutations in the parainfluenza virus 5 fusion protein reveal domains important for fusion triggering and metastability

Abstract

Paramyxovirus membrane glycoproteins F (fusion protein) and HN, H, or G (attachment protein) are critical for virus entry, which occurs through fusion of viral and cellular envelopes. The F protein folds into a homotrimeric, metastable prefusion form that can be triggered by the attachment protein to undergo a series of structural rearrangements, ultimately folding into a stable postfusion form. In paramyxovirus-infected cells, the F protein is activated in the Golgi apparatus by cleavage adjacent to a hydrophobic fusion peptide that inserts into the target membrane, eventually bringing the membranes together by F refolding. However, it is not clear how the attachment protein, known as HN in parainfluenza virus 5 (PIV5), interacts with F and triggers F to initiate fusion. To understand the roles of various F protein domains in fusion triggering and metastability, single point mutations were introduced into the PIV5 F protein. By extensive study of F protein cleavage activation, surface expression, and energetics of fusion triggering, we found a role for an immunoglobulin-like (Ig-like) domain, where multiple hydrophobic residues on the PIV5 F protein may mediate F-HN interactions. Additionally, destabilizing mutations of PIV5 F that resulted in HN trigger-independent mutant F proteins were identified in a region along the border of F trimer subunits. The positions of the potential HN-interacting region and the region important for F stability in the lower part of the PIV5 F prefusion structure provide clues to the receptor-binding initiated, HN-mediated F trigger.

Fig 1

Design of single point mutations in PIV5 F. (A) PIV5 F prefusion trimer showing the positions of mutations (purple) in Ig-like domain II and domain I, designed to disrupt the interaction of PIV5 F with PIV5 HN. One of the PIV5 F protomers is shown as a cartoon representation, while the other two are surface representations. PIV5 F trimer domains are colored as follows: domain I, yellow; domain II, red; domain III, magenta; HRB, blue; fusion peptide, light pink. (B) Enlarged view of panel A, showing the Ig-like domain in cartoon representation. (C) Ribbon representation of the PIV5 F trimer, showing positions of mutations designed to destabilize F (black box). The PIV5 F protomers are colored differently. (D) Enlarged view of the boxed area in panel C, showing the positions of the mutations designed to destabilize F (purple). The previously characterized hyperfusogenic mutation (S443P) is shown in red. (E) Sequence alignment of various paramyxovirus F proteins showing a part of domain II, including the Ig-like domain. Φ, hydrophobic stretch in the PIV5 F sequence. Strongly conserved residues are highlighted in black; moderately conserved residues are highlighted in gray. Numbers indicate PIV5 F residues. MuV, mumps virus; MeV, measles virus; NiV, Nipah virus; HeV, Hendra virus.

Fig 2

Expression of PIV5 F mutants on the surfaces of 293T cells. (A) Detection of proteins by flow cytometry at the surfaces of 293T cells transfected with wt PIV5 F or PIV5 F point mutants. Surface proteins were detected using the prefusion PIV5 F-specific F1a MAb (gray bars) or a PIV5 F R9176 polyclonal antibody (PAb) (white bars). PIV5 F-positive cells were labeled using a fluorescein-conjugated goat anti-mouse secondary antibody or a fluorescein-conjugated goat anti-rabbit secondary antibody. The mean fluorescence intensity (MFI) of 10,000 cells is shown as a percentage of wt protein levels. Results are from three independent experiments. (B) Detection of PIV5 F mutant proteins F-V340A, F-V362A, and F-S364A with the PIV5 F polyclonal antibody R9176 by flow cytometry. Fluorescein-conjugated goat anti-rabbit antibody was used to fluorescently label F protein-positive cells. Flow cytometry was performed 18 h posttransfection upon incubation of transfected cells at 37°C (gray bars) or 33°C (white bars). ns, not significantly different (P > 0.05). Results are from three independent experiments.

Fig 3

Expression and cleavage of PIV5 F mutant proteins. PIV5 wt F or F point mutants expressed in 293T cells were immunoprecipitated. The cells were labeled with [³⁵S]TranS label for 30 min (pulse) in medium lacking methionine and cysteine, followed by incubation in complete medium (chase) for 90 min. Cells were then lysed in RIPA buffer, and proteins were immunoprecipitated using the PIV5 F polyclonal antibody R9176. Polypeptides were analyzed by SDS-PAGE. Numbers indicate molecular masses (kDa). The positions of the uncleaved F protein (F0) and F protein cleavage products F1 and F2 are indicated. (A) PIV5 F wt and F point mutants with mutations located in the Ig-like domain and the adjacent hydrophobic pocket. (B) PIV5 F wt and point mutants with mutations located in domain I near the F-protomer junction.

Fig 4

Fusion mediated by the PIV5 F point mutants in baby hamster kidney cells. Baby hamster kidney (BHK-21F) cells transfected with PIV5 F or its point mutants alone or cotransfected with PIV5 HN or the PIV5 HN 1-117 headless stalk were incubated at 37°C or 42°C. At 18 h posttransfection, cells were fixed, stained, and imaged. (A) Point mutations in PIV5 F located in the Ig-like domain and in an adjacent hydrophobic pocket. (B) Locations of residues A392, V393, L395, and L384 within the Ig-like domain in the PIV5 F prefusion structure. Green, single mutations at these residues disrupt the HN interaction; blue, combined mutations at these residues disrupt the HN interaction. (C) Syncytial assay of point mutations in PIV5 F that are located in domain I near the protomer-protomer junction of the F trimer; cells were transfected or not transfected with PIV5 HN.

Fig 5

Quantitative luciferase reporter assay of cell-cell fusion activities of PIV5 F point mutants. The fusion activities of PIV5 F point mutants were characterized by using a quantitative luciferase reporter assay. wt PIV5 F or its point mutants were coexpressed with PIV5 HN and luciferase under the control of the T7 promoter 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. Results are from three independent experiments. (A and B) Fusion activities of PIV5 F point mutants in the presence of PIV5 HN. The fusion-inducing capabilities of the F point mutants are shown, with those of wt F and HN considered 100%. (A) Mutants located in the Ig-like domain and the adjoining hydrophobic pocket. (B) Mutants located in domain I near the protomer-protomer junction of the F trimer. (C) Fusion activities of PIV5 F point mutants with mutations located in the Ig-like domain hydrophobic pocket. Cells were transfected without HN at 37°C (white bars) and 42°C (gray bars). The fusion of F point mutants is shown; wt F fusion at 37°C was considered 100% fusion. (D) Fusion activation of PIV5 F hyperfusogenic mutants with (gray bars) or without (white bars) PIV5 HN or with the PIV5 HN 1-117 stalk construct (black bars). (E) Fusion activation of PIV5 F hyperfusogenic mutants in the presence (white bars) or absence (gray bars) of an independent nonactivating receptor-binding protein, influenza virus Udorn HA. Fusion is expressed in RLU as a percentage of wt F fusion in the absence of a receptor-binding protein. *, P = 0.01 to 0.05; **, P = 0.001 to 0.01. Results in all panels constitute data from at least three independent experiments.

Fig 6

Model suggesting the role of Ig-like domains of PIV5 F in interactions with the HN stalk. Images are reconstructed from atomic structures by aligning the PIV5 HN stalk structure (PDB ID 3TSI) (right panel) with the PIV5 HN 4-heads-up structure (PDB ID 1Z4X). The distance between the last residue visible in the HN stalk structure and the viral or cellular membrane (gray bar) is estimated to be ∼28.5Å, if the assumption is made that these 20 residues are helical (dotted lines). Cleaved PIV5 F-GCNt (PDB identfier 4GIP) was modeled adjacent to the constructed PIV5 HN in the 4-heads-up conformation described above. The distance between the last residue before GCNt in the PIV5 F-cleaved structure and the viral or cellular membrane (gray bar) is estimated to be ∼10Å, if the assumption is made that these residues are helical (dotted lines). The model predicts that the PIV5 F Ig-like domain corresponds in height to the middle region of the PIV5 HN stalk, which has been implicated to be involved in F triggering (34). The adjoining key shows the color labeling of PIV5 F or measles virus F residues, which are according to function and sequence alignment.