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Comparative Study
, 81 (9), 4520-32

Polybasic KKR Motif in the Cytoplasmic Tail of Nipah Virus Fusion Protein Modulates Membrane Fusion by Inside-Out Signaling

Affiliations
Comparative Study

Polybasic KKR Motif in the Cytoplasmic Tail of Nipah Virus Fusion Protein Modulates Membrane Fusion by Inside-Out Signaling

Hector C Aguilar et al. J Virol.

Abstract

The cytoplasmic tails of the envelope proteins from multiple viruses are known to contain determinants that affect their fusogenic capacities. Here we report that specific residues in the cytoplasmic tail of the Nipah virus fusion protein (NiV-F) modulate its fusogenic activity. Truncation of the cytoplasmic tail of NiV-F greatly inhibited cell-cell fusion. Deletion and alanine scan analysis identified a tribasic KKR motif in the membrane-adjacent region as important for modulating cell-cell fusion. The K1A mutation increased fusion 5.5-fold, while the K2A and R3A mutations decreased fusion 3- to 5-fold. These results were corroborated in a reverse-pseudotyped viral entry assay, where receptor-pseudotyped reporter virus was used to infect cells expressing wild-type or mutant NiV envelope glycoproteins. Differential monoclonal antibody binding data indicated that hyper- or hypofusogenic mutations in the KKR motif affected the ectodomain conformation of NiV-F, which in turn resulted in faster or slower six-helix bundle formation, respectively. However, we also present evidence that the hypofusogenic phenotypes of the K2A and R3A mutants were effected via distinct mechanisms. Interestingly, the K2A mutant was also markedly excluded from lipid rafts, where approximately 20% of wild-type F and the other mutants can be found. Finally, we found a strong negative correlation between the relative fusogenic capacities of these cytoplasmic-tail mutants and the avidities of NiV-F and NiV-G interactions (P = 0.007, r(2) = 0.82). In toto, our data suggest that inside-out signaling by specific residues in the cytoplasmic tail of NiV-F can modulate its fusogenicity by multiple distinct mechanisms.

Figures

FIG. 1.
FIG. 1.
Analysis of NiV-F CT deletion mutants. (A) Schematic of the NiV-F CT deletion mutants. NiV-F CT was divided into four regions (numbered 1, 2, 3, and 4) as described in the text, and the names of the deletion mutants examined are indicated. (B) Western blot analysis of immunoprecipitated surface WT and mutant NiV-F proteins. Briefly, biotinylated cells were lysed, cell surface biotinylated proteins were precipitated with streptavidin agarose beads, and NiV-F was detected in the biotinylated precipitates by Western blotting with either a monoclonal anti-AU1 tag Ab (left part) or a rabbit anti NiV-F2 antipeptide Ab (3) (right part). Percent processing was calculated as the densitometric units of the F1 subunit over those of the sum of the precursor F0 and the F1 subunits (bottom part) (n = 3). (C) The AU1 tag does not affect cleavage and processing of F. Identical cell surface biotinylation experiments were performed with tagged (F) and untagged versions of WT F (FNA) and the −T2 mutant (−T2NA). A rabbit anti NiV-F2 antipeptide Ab (3) was used to detect NiV-F. (D) Relative levels of CSE and fusion obtained for WT NiV-F and the indicated CT deletion mutants. Fusion was determined by counting nuclei in syncytia per field. At least 10 fields were counted per condition. CSE was determined by flow cytometry with polyclonal anti-NiV-F specific antiserum as described previously (3). Both CSE and fusion levels were separately normalized to levels of WT NiV-F protein, set at 100%. Data shown are averages ± standard errors from three independent experiments.
FIG. 2.
FIG. 2.
Analysis of membrane-proximal point mutations in the CTs of NiV-F. (A) Schematic of the NiV-F CT point mutants, showing the sequence of the whole CT, and the positions of the five alanine substitutions in the membrane-proximal region, designated K1A, K2A, R3A, N4A, and T5A. (B) Western blot analysis of immunoprecipitated cell surface biotinylated WT and mutant NiV-F proteins. Surface proteins were analyzed exactly as described in Fig. 1B. Percent processing was also analyzed as described in the legend to Fig. 1B, and the densitometric results are shown graphically. (C) Relative levels of CSE and fusion obtained for WT NiV-F or CT point mutant proteins in 293T cells. Fusion and CSE were determined exactly as for Fig. 1C. Data shown are averages ± standard errors from three independent experiments. (D) Pictures of syncytium formation by the WT NiV-F or the various NiV-F point mutants and WT NiV-G in Vero cells. Representative ×100 fields are shown.
FIG. 3.
FIG. 3.
Reverse-pseudotyped viral entry assay for membrane-proximal CT point mutants. (A) An ephrinB2-pseudotyped VSV-Renilla luciferase reporter virus (B2-VSV-rLuc) was used to infect 293T cells previously transfected with expression plasmids for NiV-F-NiV-G, NiV-G alone, or NiV-F alone. Numbers of RLU are shown on a logarithmic scale. (B) Reverse-pseudotyped viral entry into NiV-F- or NiV-G-transfected 293T cells was inhibited by anti-NiV-F and anti-NiV-G specific antisera 834 and 806, respectively. Data are presented as percent inhibition, where 0% represents infection in the absence of any antiserum. The data were normalized as follows. The number of RLU obtained at each serum dilution was calculated as a percentage of the average number of RLU obtained in the absence of any antiserum. Percent inhibition was then calculated as 100% minus the percent infection at each serum dilution. The percent inhibition values were regressed and graphed with GraphPad PRISM. An average of two experiments is shown, with four independent wells per datum point (serum dilution) ± the standard deviation. (C) Relative entry levels of B2-VSV-rLuc virus into 293T cells expressing the WT NiV-G protein and the WT or mutant NiV-F protein. RLU were quantified 24 h postinfection and graphed against the number of viral genomes per milliliter. A single preparation of B2-VSV-rLuc was used for all of the experiments shown. The number of genome copies in the viral preparation was analyzed by reverse transcription-PCR as described in Materials and Methods. The data shown are averages from three independent experiments ± the standard deviations.
FIG. 4.
FIG. 4.
Specific CT mutants affect the ectodomain conformation as exhibited by differential MAb binding and neutralization. (A) Flow cytometry histograms showing binding of polyclonal anti-NiV-F antiserum 834 or anti-NiV-F MAb 92 or 66 to 293T cells expressing either NiV-F, HeV-F, or neither (pcDNA3 control). Green contours indicate binding of Abs to 293T cells transfected with the pcDNA3.1 backbone only. Overlaid filled purple histograms indicate binding of Abs to NiV-F- or HeV-F-expressing cells, as indicated. (B) MAb binding ratios of pairs of anti-NiV-F Abs. Polyclonal (antiserum 834) or monoclonal (antisera 492 and 66) rabbit Abs were used to stain 293T cells transfected with WT NiV-F or the indicated CT point mutants at a concentration previously determined to be in the linear range of the binding curve. To compare data from repeat experiments and to control for transfection efficiency and differential expression, a set of binding ratios was calculated by dividing the mean fluorescence intensities obtained for the various Abs (92/66, 92/834, and 66/834). The Ab binding ratios for WT NiV-F is necessarily defined as 1. P values were calculated with a nonpaired Student t test and multiplied by five, which takes into account the Bonferroni correction for the multiple pairwise comparisons (WT versus the five mutants). (C and D) Neutralization of CT mutant proteins by anti-NiV-F Abs. 293T cells expressing the WT NiV-G protein and the WT or mutant NiV-F protein were infected with B2-VSV-rLuc reverse-pseudotyped virus 8 h posttransfection in the presence of increasing amounts of MAb 92 (C) or 66 (D). The amount of viral entry obtained in the absence of anti-NiV-F MAb (artificially represented by the [MAb] = −4.0 datum point) was normalized to 100%, which is equivalent to 0% inhibition. The percent inhibition was then plotted against the logarithm of the Ab concentration. Inhibition curves were regressed, and IC50s were calculated with GraphPad PRISM. The data shown are normalized averages from three separate experiments ± the standard deviations.
FIG. 5.
FIG. 5.
Association of NiV-F and the hyper- and hypofusogenic mutants with lipid raft domains. Lipid raft fractionations were performed as described in Materials and Methods. Caveolin-1 (Cav-1) and transferrin receptor (TFR) were used as markers for raft (top) and nonraft (bottom) domains, respectively. NiV-F and the indicated mutants were detected by Western blotting with the AU1 Ab. The blots were then stripped and reprobed for Cav-1 and TFR to ensure the integrity of each lipid raft fractionation. Percent NiV-F in lipid rafts was calculated as the percentage of the NiV-F signal observed in the peak Cav-1 fractions (lanes 2 and 3 in most cases) over the sum of signals in the peak Cav-1 and peak TFR fractions (lanes 7 and 8 in most cases) for each sample. This controls for any slight variations between tubes. Representative Cav-1 and TFR blots are shown. The experiment was repeated twice with similar results. Band intensities were quantified by densitometry with a VersaDoc Imaging System (Bio-Rad).
FIG. 6.
FIG. 6.
NiV-F CT fusion mutants are differentially resistant to fusion inhibition by NiV-F HR2-Fc and exhibit corresponding rates of fusion kinetics relative to WT NiV-F. (A) The sensitivity of NiV envelope-mediated fusion to inhibition by NiV-HR2-Fc is shown for WT NiV-F and the indicated CT 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. P values were calculated with the Student t test and the Bonferroni correction to account for the multiple pairwise comparisons of significance (F versus K1A, F versus K2A, and F versus R3A). (B) Fusion kinetics of WT or mutant NiV-F protein. NiV-G was expressed with WT NiV-F or the indicated mutants in effector PK13 cells, and the relative rate of fusion was assessed with 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 datum point is an average from three independent experiments.
FIG. 7.
FIG. 7.
Fusogenicity of WT NiV-F and the CT mutants inversely correlates with the avidity of F-G interactions. (A) Western blot analysis of co-IP F0 and F1 (top part a), immunoprecipitated G (bottom part c), and the relative amounts of F0 and F1 present in total cell lysate (middle part b). Cell lysates of 293T cells transfected with WT NiV-G and NiV-F or the indicated CT mutants were immunoprecipitated with rabbit anti-NiV-G specific antisera. The top and middle parts were blotted with mouse anti-AU1 to detect NiV-F, and the bottom part was blotted with mouse anti-HA to detect NiV-G. (B) A coimmunoprecipitation experiment identical to that in panel A was performed with tagged and untagged NiV-F (F and FNA, respectively) but with a rabbit anti-F2 peptide Ab for detection. Parts a, b, and c are as in panel A. (C) Relative avidities of NiV-F-NiV-G interactions for WT NiV-F and the indicated CT mutants. The amounts of co-IP NiV fusion proteins in panel A were quantified by densitometry as described in the text, with a VersaDoc Imaging System (Bio-Rad). The avidity of F-G interactions is represented by the ratio of the amount of NiV-F protein co-IP with anti-NiV-G antiserum to the relative amount of NiV-F expressed in cell lysates (parts a and b, respectively). The data presented are averages ± standard errors from three experiments. (D) Avidity of the F-G interactions from panel C plotted against the fusion/CSE ratios from Table 1. Pearson correlation analysis was performed with GraphPad PRISM. (E) The avidities of F-G interactions for the multiple N-glycan mutants previously reported by Aguilar et al. (3) were overlaid with the datum points from panel C and plotted together against their respective fusogenic indexes. CT mutants and N-glycan mutants are represented by closed and open symbols, respectively. Pearson correlation analysis was performed with GraphPad PRISM.

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