Role of sequence and structure of the Hendra fusion protein fusion peptide in membrane fusion

Abstract

Viral fusion proteins are intriguing molecular machines that undergo drastic conformational changes to facilitate virus-cell membrane fusion. During fusion a hydrophobic region of the protein, termed the fusion peptide (FP), is inserted into the target host cell membrane, with subsequent conformational changes culminating in membrane merger. Class I fusion proteins contain FPs between 20 and 30 amino acids in length that are highly conserved within viral families but not between. To examine the sequence dependence of the Hendra virus (HeV) fusion (F) protein FP, the first eight amino acids were mutated first as double, then single, alanine mutants. Mutation of highly conserved glycine residues resulted in inefficient F protein expression and processing, whereas substitution of valine residues resulted in hypofusogenic F proteins despite wild-type surface expression levels. Synthetic peptides corresponding to a portion of the HeV F FP were shown to adopt an α-helical secondary structure in dodecylphosphocholine micelles and small unilamellar vesicles using circular dichroism spectroscopy. Interestingly, peptides containing point mutations that promote lower levels of cell-cell fusion within the context of the whole F protein were less α-helical and induced less membrane disorder in model membranes. These data represent the first extensive structure-function relationship of any paramyxovirus FP and demonstrate that the HeV F FP and potentially other paramyxovirus FPs likely require an α-helical structure for efficient membrane disordering and fusion.

FIGURE 1.

Amino acid composition of fusion peptides from class I fusion proteins. A, shown is a diagram of a paramyxovirus F protein in the uncleaved (F₀) and cleaved, fusogenically active F₁+F₂ forms. HR, heptad repeat; CTD, C-terminal domain (C-tail); * denotes site of proteolytic cleavage. B, shown is alignment of paramyxovirus fusion peptides with completely conserved amino acids shaded and compared with fusion peptides from influenza HA and HIV Env. References for sequences are as follows: Hendra (5), PIV5 (59), measles (60), NDV (61), Mumps (62), Sendai (63), HPIV1 (human parainfluenza virus type 1 (64)), CDV (canine distemper virus (65)), influenza (66), HIV (22). C, percent alanine and glycine composition of the fusion peptides is shown in B as compared with the whole protein.

FIGURE 2.

Cell-surface expression of Hendra F fusion peptide mutants. A, Alignment of Hendra F FP mutants is shown. CTD, C-terminal domain. B, cell-surface expression of FP mutants is shown. After transfection with empty pCAGGS vector or either wild-type or mutant pCAGGS-Hendra F, cells were starved and labeled with Tran³⁵S-label containing Cys/Met, and surface proteins were biotinylated. After cell lysis, Hendra F was immunoprecipitated, and surface proteins were pulled down with Streptavidin beads. Protein was analyzed via 15% SDS-PAGE, and bands were visualized using the Typhoon imaging system. C, exogenous TPCK-trypsin cleavage of surface-expressed wild-type or mutant F proteins in the presence or absence of dominant negative Rab5 (DN-Rab5) is shown. Human metapneumovirus F (HMPV F) was used as a positive control for TPCK-trypsin cleavage and activity. Both B and C are representative gels from one of three independent experiments.

FIGURE 3.

Fusogenicity of wild-type and mutant Hendra F proteins. A, syncytia assays demonstrating cell-cell fusion promotion of wild-type or mutant F proteins in the presence of the Hendra attachment (G) protein are shown. Vero cells transfected with either empty pCAGGS and wild-type pCAGGS-Hendra G or wild-type or mutant pCAGGS-Hendra F and wild-type pCAGGS-Hendra G were monitored for syncytia formation as described under “Experimental Procedures.” 36–48 h after transfection, 10 images from different fields were taken using a Nikon Coolpix995 camera mounted on a Nikon TS100 inverted phase-contrast microscope. Representative images are shown from one of three independent experiments. B, quantitative luciferase reporter gene assays show cell-cell fusion levels (black bars) alongside quantitation of F₁ CSE levels from Fig. 2B. Band densitometry was performed using ImageQuant 5.2 (GE Healthcare) software. Vero cells were transfected with a T7 luciferase construct and either empty pCAGGS and wild-type pCAGGS-Hendra G or wild-type or mutant pCAGGS-Hendra F and wild-type pCAGGS-Hendra G at a 1:3 F:G ratio as described under “Experimental Procedures.” 18–24 h post-transfection Vero cells were overlaid with BSR cells stably expressing the T7 polymerase, cells were incubated for 3 h at 37 °C and lysed, and luciferase activity was measured. All data are presented as the means ± S.E. for at least three independent experiments.

FIGURE 4.

Hemolytic ability and CD spectra of synthetic fusion peptides. A, shown are synthesized peptides where the first 16 amino acids of the FP for either wild-type or mutant Hendra F are coupled to a flexible highly charged host peptide. B, hemolytic ability of synthetic fusion peptides is shown. Chicken RBCs were incubated with increasing concentrations of peptide for 45 min at 37 °C. Supernatants were then cleared by centrifugation at 11,000 × g, and absorbance was measured at 520 nm. All values are expressed as a percent of maximum hemolysis determined by RBC lysis with 0.5% Triton X-100. Data are presented as the means ± S.E. for at least three independent experiments. C, CD spectra of all five peptides in the absence of DPC in 5 mm Hepes, 10 mm MES pH 7.4 with peptide concentrations of 100 μm are shown. D, shown are spectra in the presence of 10 mm DPC micelles prepared in the above buffer as described under “Experimental Procedures.” Spectra in both C and D are representative of one of three independent experiments where each spectrum is an average of four scans.

FIGURE 5.

Cell-surface expression of single alanine FP mutants. A, alignment of the four single alanine FP mutants is shown. B, cell-surface biotinylation of the four single alanine mutants performed as described in Fig. 2 and under “Experimental Procedures” is shown. These lanes are from the same gel as Fig. 2, thus allowing for a direct comparison between mutants. This is a representative gel from one of three independent experiments. CTD, C-terminal domain.

FIGURE 6.

Fusogenicity of wild-type and single alanine mutant F proteins. A, shown is a syncytia assay for each of the four mutants as compared with wild type. The experiment was performed as described in Fig. 3A and under “Experimental Procedures.” Images are representative of one of three independent experiments. B and C, shown is a quantitative reporter gene analysis and quantitation of F₁ cell-surface expression for FP mutants as compared with wild-type F performed as described in Fig. 3B and under “Experimental Procedures.” All data are presented as the means ± S.E. for at least three independent experiments.

FIGURE 7.

CD spectra of synthetic fusion peptides in POPG:POPG SUVs. A, shown are CD spectra of wild-type Hendra F fusion peptides in the presence or absence of SUVs composed of differing POPC:POPG ratios. B and C, CD spectra of wild-type and mutant Hendra F fusion peptides in the absence (B) or presence (C) of POPC:POPG 95:5 SUVs are shown. Spectra were obtained using a peptide concentration of 100 μm in solution or with 5 mm SUVs composed of POPC 100% or POPC:POPG 98:2, 95:5, and 92:8. All spectra were recorded at 22 ± 2 °C in a 0.5-mm quartz cuvette. Spectra were obtained from two independent sets of SUVs where each spectrum is an average of five scans.

FIGURE 8.

Peptide-induced membrane disordering obtained using ATR-FTIR. A, lipid order parameters, S_L, obtained for single planar bilayers before and after incubation with 100 μm Hendra WT, M115A, and VM114/115AA peptides are shown. B, ATR dichroic ratios of lipid methylene stretching vibrations and derived acyl chain order parameters in the absence and presence of wild-type or mutant Hendra F fusion peptides are shown. For all cases the bottom layer of the bilayer was composed of 1,2-dimyristoyl-sn-glycero-3-phosphocholine, and the top was formed with POPC:POPG 95:5. Experiments were performed in D₂O buffered with 5 mm HEPES and 10 mm MES with 150 mm NaCl. Data represent two independent bilayer preparations for each peptide.

FIGURE 9.

Dependence of peptide secondary structure on effective peptide concentration. A, CD spectra of wild-type peptide at various DPC concentrations are shown. B, CD spectra of VM114/115AA peptide at various DPC concentrations are shown. Spectra in both A and B are representative of one of three independent experiments where each spectrum is an average of four scans.