PMC

N-glycans protect NiV from neutralizing antibodies. Cell-cell fusion mediated by WT NiV-F and the indicated mutants was differentially inhibited by a polyclonal anti-NiV-F-specific antiserum. The fusion assay was performed in 293T cells as described above, except that serial dilutions of the anti-NiV-F antiserum were incubated with the transfected cells during the overnight incubation. The amount of fusion seen in the absence of anti-NiV-F antiserum was normalized to 100%. The inhibition curves were regressed, and the IC₅₀s were calculated using GraphPad PRISM. Data are shown as normalized averages ± standard deviations from two separate experiments.

Fusogenicity is inversely correlated with the avidity of F/G interactions. (A) Reciprocal coimmunoprecipitation of NiV-F and NiV-G. Cell lysates of 293T cells transfected with WT NiV-G and NiV-F or the indicated N-glycan mutants were immunoprecipitated using rabbit anti-NiV-G-specific antisera or rabbit anti-F-specific antisera. The immunoprecipitates containing directly immunoprecipitated and coimmunoprecipitated proteins were split in half and analyzed by Western blotting. Left panels (a and b) were blotted with mouse anti-AU1 to detect NiV-F, and right panels (c and d) were blotted with mouse antihemagglutinin to detect NiV-G. (B) Relative avidities of NiV-G interactions for the WT or mutant NiV-F proteins. The amounts of co-IP NiV-F and NiV-G in panel A (a and c, respectively) were quantified by densitometry as described in the text using a VersaDoc Imaging System (Bio-Rad). The avidity of F/G interactions is represented by the ratio of the amount of NiV-F protein coimmunoprecipitated with anti-NiV-G antisera (a) to the amount of NiV-F directly immunoprecipitated from total cell lysates (b) in panel A or the ratio of the amount of NiV-G protein coimmunoprecipitated with anti-NiV-F antisera (c) to the amount of NiV-G protein directly immunoprecipitated from total cell lysates (d). (C) The ratiometric values representing F/G interaction avidities from panel B were plotted against the fusion/CSE ratios (fusion index) from Table . Pearson correlation analysis was performed using GraphPad PRISM.

Multiple N-glycans in combination on NiV-F inhibit cell-cell fusion and viral entry. (A) Relative levels of fusion versus CSE for WT NiV-F and multiple N-glycan mutants in 293T cells. Data are presented as values normalized to that of WT NiV-F (set at 100%) and are shown as averages ± standard errors from three separate experiments. (B and C) Relative entry levels of NiV-F/G-pseudotyped VSV-Renilla-luciferase reporter viruses (VSV-rluc). NiV-G and the indicated WT or mutant NiV-F proteins were pseudotyped onto luciferase reporter viruses as described in Materials and Methods. RLU were quantified 24 h postinfection and plotted against the number of viral genomes per ml. Relative amounts of genome copies in the viral preparations were analyzed by reverse transcription-PCR as described in Materials and Methods. Data shown are averages ± standard deviations from three independent experiments. Sometimes the error bars are not visible because they are too small to be seen in a logarithmic scale. (D) Western blot analysis of WT or mutant NiV-F proteins contained in viral preparations used in panels B and C and blotted with a mouse anti-AU1 monoclonal antibody, as described in Materials and Methods, showing F₀ and F₁ subunits. One representative experiment out of three is shown.

The role of N-glycans on NiV-F in syncytium formation. (A) NiV-F (or the indicated N-glycan mutants) and NiV-G plasmids were transfected at a 1:1 ratio with Lipofectamine 2000 (0.3 μg NiV-F and -G/12-well plate). Cells were DAPI stained 18 h posttransfection, and representative images of syncytia formed are shown as DAPI images overlaid onto bright-field images. DAPI-stained nuclei appear blue, and syncytia appear as large clusters of blue nuclei surrounded by relatively clear cytoplasm. Syncytia are particularly evident in the F3 panel. (B) Relative levels of fusion and CSE obtained for WT NiV-F or single N-glycan mutant proteins in 293T cells. CSE was determined by flow cytometry as in Fig. . Both fusion and CSE levels were separately normalized to levels seen for WT NiV-F protein, which were set at 100%. Data shown are averages ± standard errors from three independent experiments. (C) Titration of transfected NiV-F/G plasmids with the degree of CSE observed. The indicated amounts of NiV-F/NiV-G expression plasmids were cotransfected at a 1:1 ratio, and the total DNA amount transfected was kept constant with pcDNA3.1. Data for 293T cells are shown as averages ± standard deviations. One representative experiment out of three is shown. (D) Titration of transfected NiV-F/G plasmids with the degree of cell-cell fusion observed. The indicated amounts of NiV-F/NiV-G expression plasmids were cotransfected at a 1:1 ratio, and the total DNA amount transfected was kept constant with pcDNA3.1. Fusion was quantified in 293T and Vero cells as described in Materials and Methods. Data for 293T cells are shown as averages ± standard deviations from three independent experiments. One representative experiment out of three is shown. (E) The measure of cell-cell fusion from panel D was plotted against CSE from panel C. Pearson correlation analysis was performed using GraphPad PRISM.

Hyperfusogenicity of the N-glycan mutants is correlated with the sensitivity to heptad repeat region inhibition and the rate of six-helix bundle formation. (A) A novel NiV-heptad-repeat-human-Fc immunoadhesin (NiV-HR2-Fc) inhibited NiV envelope-induced fusion (in 293T cells) but not entry of HIV-1 virions (in TZM-bl HeLa-based luciferase reporter cells) (). A similar HIV-HR2-Fc protein inhibited HIV-1 virus entry but not NiV envelope-induced fusion. Data are presented as percentages of Fc-only control (averages ± standard errors from three separate experiments. (B) The sensitivity of NiV envelope-mediated fusion to inhibition by NiV-HR2-Fc is shown for WT NiV-F and the indicated N-glycan mutants. For each fusion protein, the amount of fusion in the absence of any inhibitor is set at 0% inhibition. One representative experiment out of two is shown. Error bars indicate standard deviations. (C) Fusion kinetics of WT or mutant NiV-F proteins. NiV-G was expressed with the indicated NiV fusion proteins in effector PK13 cells, and the relative rate of fusion was assessed on target 293T cells loaded with CCF2 dye (see Materials and Methods). Relative fusion is the ratio of blue to green fluorescence obtained with NiV-G- and NiV-F-transfected effectors minus the ratio of background blue and green fluorescence obtained with empty-vector (pcDNA3)-transfected cells. Each data point is an average from three independent experiments. Linear regression on data points was performed with GraphPad PRISM.

Comparison of the primary and tertiary structures of the NiV, HeV, MeV, RPV, PPRV, CDV, PDV, SeV, HPIV-1, HPIV-3, SV5, and NDV paramyxovirus F proteins. The 3-D structure of each paramyxovirus F protein was modeled based on the solved crystal structure of the HPIV-3 F protein (see Materials and Methods), and the N-linked glycosylation sites were mapped onto the predicted structure. (A) Linear comparison of the 12 sequences based on Clustal W multiple sequence alignments. The amino acid positions (not to scale) of the potential N-linked glycosylation sites in NiV-F are shown in red in the top sequence representation. Actual glycan attachment sites are indicated by a branched tree symbol at the amino acid position. Positions of the other paramyxovirus F proteins are shown below the NiV-F sequence, with the amino acid sequence position indicated to the left of the glycan symbol and its position in the predicted 3-D structure on the right; circles, triangles, and diamonds represent glycans located in the head, neck, and stalk region, respectively, of the predicted or actual (for HPIV3) 3-D structure. Asterisks denote N-linked glycosylation sites that could not be structurally mapped. (B) Carbon backbone representation of the HPIV-3 F structure (), representing the general overall fold of the paramyxovirus F protein. N-linked glycosylation sites which can be mapped onto this structure using the threading methodology described in Materials and Methods are indicated by circles; see the key in the figure. The heptad repeat regions (HR-A, HR-B, and HR-C) and head, neck, and stalk regions are also indicated.

Analysis of NiV-F N-glycan site utilization. (A) Schematic of NiV-F glycoprotein. NiV-F (F₀) is a type I transmembrane protein that is cleaved into F₁ and F₂ by a host cell protease. Asterisks represent potential N-linked glycosylation sites F1 to F5. The epitope of NiV-F₂ antipeptide antibody is indicated. Codon-optimized NiV-F was AU1 epitope tagged at the C terminus as indicated. (B) Top: analysis of immunoprecipitated WT and mutant NiV-F proteins (F, F1, F2, F3, F4, and F5). Briefly, ³⁵S-radiolabeled cell lysates were cleared out using goat anti-rabbit IgG at a 1:100 dilution, and supernatants were immunoprecipitated using a 1:100 combination of polyclonal anti-NiV-F antisera () and the anti-F₂ peptide antisera described above (A) at a 1:1 ratio. Relative mobilities of the precursor (F₀) and cleaved proteins (F₁ and F₂) showed usage of F2, F3, F4, and F5 glycosylation sites. Bottom: estimation of the relative levels of processing between the various single N-glycan mutant fusion proteins. Percent processing was calculated as the percentage of the sum of F₁ plus F₂ protein subunits divided by the total sum of NiV-F protein precursor and subunits (F₀ + F₁ + F₂), using phosphorimager quantitation of ³⁵S-radiolabeled protein subunits. One out of two representative experiments is shown. (C) Top: Western blot analysis of biotinylated cell surface proteins. Briefly, biotinylated 293T cells were precipitated with streptavidin-agarose beads and detected with a mouse anti-AU1 monoclonal antibody against AU1-tagged NiV-F. Percent processing was calculated as the percentage of F₁ subunits over the total sum of NiV-F protein precursor and the F₁ subunits (F₀ + F₁). One out of two representative experiments is shown. (D) Relative CSE of WT and mutant NiV-F proteins in 293T cells transfected with NiV-G and WT or N-glycan mutant NiV-F expression plasmids at a 1:1 ratio, using an anti-NiV-F-specific antiserum. Negative control (−) corresponds to cells expressing NiV-G only. Data shown are from one representative experiment out of four.