Fig 1.
Schematic diagram of the locations of mutations.
(A) Domain structure of the HPIV3 F protein and the relative location of the DI-DII linker in the primary sequence. This schematic is based on the secondary structure of the HPIV3 F protein in the postfusion conformation [16]. Structurally important domains are represented as different colors of rectangles, and their corresponding residue ranges are indicated. Individual mutations introduced into the region were listed beneath the wt sequence. Fusion peptide (FP), heptad repeat region (HR), transmembrane domain (TM), cytoplasmic tail domain (CT), and structural domains DI, DII, and DIII are shown. (B) Locations of the DI-DII linker (residues 369–374) in the prefusion HPIV3 F protein structural model. A side view of the ribbon diagram of the structural model of the HPIV3 F protein in the prefusion conformation which was generated based on the crystal structure of the prefusion PIV5 F protein (PDB ID 4GIP) is shown. The three monomers are shown in light pink, aquamarine, and pale green. The mutant residues are shown in space-filling mode in three chains. The figure was generated with PyMOL 0.99.
Fig 2.
Syncytium formation in monolayers coexpressing wt or mutated F and HN proteins.
After 36 h posttransfection, BHK-21 monolayers transfected with vector alone, wt F alone, wt F and wt HN, or mutant F and wt HN were fixed with methanol and stained with Giemsa stain. (A) Photomicrographs from a representative expreriment. Red arrows indicate syncytia. (B) Quantification of syncytia produced by the mutated F proteins. Values were indicated as percentages of syncytium formation detected in cells transfected with wt F and wt HN proteins and are represented as the mean ± standard deviation (SD) from three separate experiments (*P<0.05, **P<0.01, ***P<0.001).
Fig 3.
Content mixing of wt and mutant F proteins.
Quantification of content mixing directed by wt or mutant F proteins with their homologous HN proteins as measured by β-galactosidase activities. Values are expressed as percentages of content mixing detected in cells transfected with wt F and HN proteins. The experiment was performed in triplicate; the data are shown as the mean±SD (*P<0.05, **P<0.01, ***P<0.001).
Fig 4.
Dye transfer directed by the wt or mutated F and HPIV3 HN proteins.
R18-labeled RBCs were bound at 4°C to BHK-21 cells coexpressing the wt or mutanted F and HN proteins. Cells were incubated for 60 min at 37°C to allow membrane fusion, images were acquired by using fluorescent microscopy. (A) Representative images of dye transfer. White arrows indicate the lipid mixing events. (B) Quantification of the events of dye transfer. The extent of dye tranfer is expressed as the average number of R18 lipid dye tranfer events of six microscopic fields. The means and standard errors are from six microscopic fields (**P<0.01, ***P<0.001).
Fig 5.
Kinetics of R18 dye transfer for HPIV3 wt or mutated F and HN proteins.
The effector-target cell complexes was injected into a cuvette containing PBS prewarmed to 37°C, and fluorescence changes were measured as described in Materials and Methods. (A) The kinetics of dye transfer from R18-labeled RBCs to effector cells. A single point that represents data obtained from three different experiments. GraphPad Prism 5.0. was used to perform nonlinear regression on data points. (B) Determination of the initial rates of fusion of the target-effector cell complexes. The rates of hemifusion induced by the mutated F and HN proteins were calculated from maximum slopes of fit curves as shown in Fig 5A, and normalized to the maximum rate of hemifusion induced by wt F and HN proteins. Data are means±SD of three independent experiments (*P<0.05, **P<0.01, ***P<0.001). (C) Determination of the maximum extent of hemifusion. The maximum extent of fluorescence dequenching at 5 min was calculated from kinetics curves analogous to those shown in Fig 5A. The data are expressed as the percent activity relative to the wt F and HN level. The results are the mean±SD (*P<0.05, **P<0.01, ***P<0.001) from three separate experiments.
Fig 6.
Cell surface expression (CSE) of the mutant F proteins.
Cell surface expression was measured by flow cytometry with an anti-F monoclonal antibody followed with an anti-mouse Alexa Fluor 488-conjugated antibody. (A) Representative fluorescent histograms for each mutant were shown. The x axis indicates the fluorescent intensity values shown in log scale and the y axis represents cell counts, respectively. The mean fluorescence intensity (MFI) of each mutant was calculated and listed below. (B) Quantitation of the cell surface expression level. The mean fluorescence intensity of cells expressing the mutated F proteins was represented as a percentage of that of the wt F subtracting background labeling of control cells transfected with the vector alone. The means and standard errors are from triplicate experiments.
Fig 7.
Co-immunoprecipitation analyses of the interactions of the wt or mutated F with HPIV3HN proteins.
The surface proteins of the transfected cells were biotinylated, then the cells were lysed and the lysates were incubated with Dynabeads-cross-linked anti-HN monoclonal antibodies. After forming Dynabeads-HN Ab-HN Ag-F Ag immune complexes, they were solubilized in PBS and then incubated with streptavidin beads at 4°C all night to precipitate the cell surface complexes. Precipitated complexes were separated on a PVDF membrane and probed with anti-F monoclonal antibody (top panels) or a mixture of two anti-HN monoclonal antibodies (bottom panels). All proteins were electrophoresed in the presence of reducing agent. (A) The critical controls of this co-IP assay: Neg.c, empty vector; HN only and F only, these plasmids alone; wild-type F protein (Fwt) and HN. (B) The results of a co-immunopreciptation assay of HPIV3 F proteins carrying mutations in the DI-DII linker. Wild-type F protein (Fwt) and the nine mutants cotranfected with HN are indicated above the gels. (C) The quantitative results for Fig 7B, using densitometry method from three independent experiments. The relative levels of the co-immunoprecipitated F proteins for the mutants are expressed as the percentages of that detected in cells transfected with wt F and HN genes. Error bars indicate the standard deviations (**P<0.01, ***P<0.001).