Nipah Virus Attachment Glycoprotein Stalk C-Terminal Region Links Receptor Binding to Fusion Triggering

Cell culture.

PK13 and 293T cells were cultured in Dulbecco's modified Eagle medium (DMEM), and Vero cells were cultured in minimal essential medium, both supplemented with 10% fetal bovine serum (FBS), 50 IU of penicillin ml^–1, 50 μg of streptomycin ml^–1, and 2 mM glutamine.

Expression plasmids, antisera, and antibodies.

Expression plasmids for codon-optimized wild-type (WT) NiV-G and -F, untagged or tagged at the C terminus with the HA and AU1 tags, respectively, were previously described (32). The hyperfusogenic NiV-F N-glycan mutant F_F3F5 and cytoplasmic tail mutant F_K1A proteins were also previously described (32, 33). The NiV-G headless mutant proteins were constructed by inserting two stop codons (TAA TAA) at the corresponding sites. The NiV-F hyperfusogenic (F_{K1A F3F5}) and hypofusogenic (F_P221A) mutant proteins and NiV-G full-length point mutant proteins were constructed by site-directed mutagenesis (Agilent Technologies Inc.). All mutations were verified by DNA sequencing (Eurofins).

The production of rabbit polyclonal anti-G antisera (806 and Ab167) and conformational monoclonal antibodies Mab45 and Mab213 has been described previously (28, 33, 34). The soluble recombinant mouse ephrinB2 human Fc chimera (B2), containing ectodomain residues 27 to 227 of mouse ephrinB2, was purchased from R&D Systems. The rabbit anti-HA monoclonal antibody (Bethyl Laboratories Inc.) and mouse anti-AU1 monoclonal antibody (Covance) were used at 1:2,000 and 1:500 dilutions in immunoblot assays, respectively. Fluorescently labeled secondary antibody Alexa Fluor 647 goat anti-rabbit IgG (Life Technologies, NY) was used at a 1:200 dilution for flow cytometry. Alexa Fluor 647 goat anti-mouse IgG antibody and Alexa Fluor 488 goat ant-rabbit IgG antibodies were used at a 1:2,000 dilution for immunoblot assays.

Quantification of NiV-F and -G cell surface expression (CSE) and antibody and ephrinB2 binding by flow cytometry.

Two micrograms of WT or mutant NiV-G expression plasmid was transfected into 293T or PK13 cells (per well of a six-well plate, 70% confluence) to achieve a linear correlation between the CSE levels and the amount of DNA transfected (32). Cells were collected at 20 to 24 h posttransfection. To measure CSE and ephrinB2 (B2) binding levels, 293T cells expressing WT or mutant NiV-G protein were incubated with anti-NiV-G polyclonal antisera or soluble B2 (see above) at 4°C for 1 h, respectively. The cells were washed twice with phosphate-buffered saline (PBS) supplemented with 1% FBS and then incubated with a fluorescently labeled secondary antibody for 30 min at 4°C. To measure the binding levels of conformation-dependent antibodies, PK13 cells expressing WT or mutant NiV-G were incubated with soluble B2 for 15 min at 4°C and then subjected to a procedure identical to that described above. Cells were fixed with 0.5% paraformaldehyde, and the mean fluorescence intensity was measured by flow cytometry (Guava easyCyte 8HT; EMD Millipore). Cells transfected with the vector alone (pcDNA3.1) served as a negative control.

Quantification of syncytia and EphB3 inhibition.

To achieve a linear correlation between the amount of DNA transfected and the levels of syncytia, WT NiV-F and WT or mutant NiV-G expression plasmids were transfected into 293T or Vero cells (six-well plates, 70% confluence) at a 1:1 ratio (2 μg total). At 12 to 28 h posttransfection, syncytium formation was quantified by counting the nuclei inside syncytia per 200× field. A syncytium is defined as four or more nuclei within the same cell membrane. EphB3 is a natural ligand of ephrinB2 or ephrinB3 (35). For the EphB3 inhibition experiment, mouse EphB3-human Fc (R&D Systems) was added to the cells at 6 to 8 h posttransfection to achieve a final concentration of 40 nM, whereas PBS was added as a negative control (0 nM EphB3).

Quantification of viral genome copies and viral entry.

NiV-F and NiV-G were pseudotyped onto a vesicular stomatitis virus (VSV) reporter expressing the Renilla Luc reporter gene (NiV/VSV-rLuc) as previously described (32, 33). Briefly, 293T cells were transfected with the respective WT and mutant NiV-G and NiV-F expression plasmids and subsequently infected with recombinant VSV-ΔG-rLuc. NiV/VSV-rLuc pseudotyped virions were collected at ∼30 h postinfection and purified over a 20% sucrose cushion. Virions were resuspended in NaCl-Tris-EDTA (NTE) buffer supplemented with 5% sucrose and stored at −80°C. Viral RNA was extracted with the QIAamp viral RNA minikit (Qiagen), and the isolated RNA was reverse transcribed with the SuperScriptIII first-strand synthesis system for reverse transcription-PCR (Invitrogen). The VSV genome copy number was quantified by using a quantitative TaqMan PCR protocol as previously described (32).

To quantify viral entry, 293T cells were seeded into a 96-well plate and infected with 10-fold serial dilutions of NiV/VSV-rLuc pseudotyped virions in infection buffer (PBS plus 1% FBS) at 37°C. Complete growth medium (DMEM) was added to the cells at 2 h postinfection. 293T cells were lysed at 20 to 24 h postinfection, and luciferase activity was measured in relative light units (RLU) with a Renilla Luciferase Flash Assay kit (Pierce) and an Infinite M1000 microplate reader (Tecan Group Ltd.). RLU were plotted against numbers of genome copies per milliliter and regressed with GraphPad Prism 5 (GraphPad Software Inc.).

Detection of glycoprotein expression and viral incorporation by Western blotting.

293T cells transfected with WT or mutant NiV-G expression plasmids were lysed in 1× radioimmunoprecipitation assay (RIPA) buffer (EMD Millipore) supplemented with protease inhibitors (cOmplete, Mini; Roche) at 24 to 48 h posttransfection. Cell lysates or 10⁹ VSV/NiV-rLuc pseudotyped virions (genome copies) were subjected to SDS-PAGE and immunoblotting. NiV-G proteins were detected with anti-G polyclonal antisera (described above). Fluorescently labeled secondary antibodies (described above) were used at a dilution of 1:2,000 and detected by a Chemidoc gel imager system (Bio-Rad).

Coimmunoprecipitation.

293T cells transfected with WT NiV-F and WT or mutant NiV-G (HA-tagged) expression plasmids were lysed in RIPA buffer at 24 to 36 h posttransfection. Cell lysates were incubated with rabbit anti-HA μMAC Microbeads (Miltenyi Biotec) for 1 h at 4°C with rotation and then purified and eluted over μMAC columns (Miltenyi Biotec). Total cell lysates and purified elutions were subjected to SDS-PAGE and immunoblotting as described above. Protein band intensities were measured by densitometry with a Chemidoc imaging system (Bio-Rad).

PNGase F treatment.

293T cells transfected with headless NiV-G expression plasmids were lysed in RIPA buffer at 20 to 24 h posttransfection. Cell lysates were denatured for 10 min at 60°C and treated with or without peptide-N-glycosidase F (PNGase F; New England BioLabs Inc.) for 1 h at 37°C according to the manufacturer's protocol. The PNGase F-treated cell lysates were subjected to 15% SDS-PAGE and immunoblotting with anti-AU1 monoclonal antibody against F and polyclonal antiserum Ab167 against G.

Raman spectroscopy.

We reported that the ephrinB2-containing VSV pseudotyped virions (VSV/B2) were able to induce membrane fusion on receptor-negative cells expressing NiV-G and -F (33). In this study, VSV/B2 virions were used in a cell-free system to initiate the conformational changes in NiV-G and -F on virions as previously described (36). Briefly, pseudotyped VSV containing WT or mutant NiV-G was incubated with VSV/B2 virions for 30 min on ice to allow receptor binding. The mixture was then transferred from the ice bath directly onto a glass microarray slide coated with gold (Thermo Scientific Inc.). The viral samples on the gold-coated microarray slide were immediately subjected to Raman spectral collection by using a confocal micro-Raman spectrometer above 25°C (Renishaw). The spectra of the virions containing only WT or mutant NiV-G were collected for spectral subtraction. The Raman spectroscopic settings and spectral processing were identical to those described in our recent publication (36).

Statistics.

P values were calculated by unpaired Student t test and corrected by using the respective Bonferroni correction factors. Average values ± the standard errors of the means (SEM) from at least three independent experiments are presented.

The C-terminal region of the NiV-G stalk (aa 159 to 167) is important for fusion triggering.

Our previous studies revealed that NiV-G headless mutant protein 167 (aa 1 to 167) can trigger NiV-F-mediated 293T cell-cell fusion to approximately WT (full-length) levels, as measured by counting relative amounts of syncytial nuclei (28). These results indicate that headless mutant G protein 167 contains the necessary and sufficient F-triggering domain(s). To pinpoint the region(s) in G important for F triggering, we made two more G headless truncation versions, mutant proteins 158 (aa 1 to 158) and 164 (aa 1 to 164), by inserting stop codons after their most C-terminal residues (Fig. 1A). Compared to mutant 167, which triggered 293T cell-cell fusion at levels similar to those of the WT, mutant 164 induced fusion at only ∼20% of WT G levels (Fig. 1B and C) and mutant protein 158 did not induce any 293T cell-cell fusion, although both were expressed to at least 50% of the WT NiV-G level at the cell surface (Fig. 1B and C). We used flow cytometry to determine the 293T CSE of WT and headless mutant NiV-G by using anti-G polyclonal antiserum Ab167, raised against headless mutant protein 167 (28). All of the mutant proteins were expressed on the cell surface to various degrees (Fig. 1B), resembling results obtained by Western blot analysis of total cell lysates with Ab167 (Fig. 1D).

Interestingly, we observed a double banding pattern for mutant protein 158 and a triple banding pattern for mutant proteins 164 and 167. We confirmed that the loss of the upper band for mutant protein 158 was due to the removal of an N-glycosylation site from this mutant protein (an N-glycan is normally added to N₁₅₉ in the NiV-G stalk, as previously shown by Biering et al. [18]). Treatment with PNGase F eliminated the upper band from mutant proteins 164 and 167 (Fig. 1D). We do not know what caused the remaining duplet band, but O-glycosylation and/or other posttranslational modifications of these truncated mutant proteins may be the reason (37). We also confirmed by flow cytometry that the fusion-triggering ability of the three NiV-G headless mutant proteins was independent of their ephrinB2 receptor-binding capabilities (Fig. 1B). As expected, all three headless mutant proteins (158, 164, and 167) were unable to bind soluble ephrinB2, since the known ephrinB2-binding site lies within the G head (10). Overall, these results suggest that the NiV-G stalk C-terminal region from aa 158 to aa 167 is important for F triggering and fusion promotion.

Residues 159 to 163 within the NiV-G stalk modulate NiV-induced cell-cell fusion.

To corroborate the importance of the region from aa 158 to aa 167 in modulating fusion and to map potential specific residues within the region from aa 158 to aa 167 that modulate fusion, we performed alanine scanning mutagenesis of this region in the context of the full-length NiV-G glycoprotein (Fig. 2A). Cysteine 162 was left unchanged, since it has previously been shown, together with two other cysteine residues (C146 and C158), to be important for the tetrameric stability of G (30).

293T cells expressing the WT or mutant NiV-G protein were lysed and subjected to SDS-PAGE and Western blot analysis with Ab167. As expected, mutations in positions 159 and 161 (N159A, S161A) resulted in a faster migration pattern of these proteins because of the disruption of the N-glycosylation site (NIS) we previously reported (Fig. 2B) (18). Interestingly, mutation of the isoleucine centering the glycosylation site resulted in additional bands of slightly higher molecular weight, indicating that this mutation might also have an effect on G folding, glycosylation, and/or other posttranslational modifications. All of the alanine mutant proteins were expressed at levels roughly similar to that of WT G, except the N159A and S161A mutant proteins, which showed slightly lower total cell expression levels (Fig. 2B). Next, we determined by flow cytometry with anti-G polyclonal antiserum 806 whether expression levels at the cell surface corresponded to that of the whole-cell lysates (34). These data indicate that the alanine point mutations had no drastic effects on the CSE levels of G (Fig. 2C).

To measure the ability of the NiV-G alanine mutant proteins to promote cell-cell fusion, we quantified the relative amounts of 293T cell-cell fusion induced by WT or mutant G in combination with WT NiV-F. We previously showed that cell-cell fusion and CSE levels are linearly correlated during transfection with the amount of DNA used in this study (2 μg per well of a six-well plate) (32). Table 1 shows the calculated fusion index (fusion/CSE) of each mutant protein, which is defined as the ratio of the cell-cell fusion level to the CSE level (each normalized to WT levels) (18, 32). Thus, the fusion index of WT G and F was set to 1, meaning that fusion indices higher than 1 describe a hyperfusogenic phenotype and fusion indices below 1 describe a hypofusogenic phenotype. Interestingly, the N159A and I160A mutant proteins did not induce any 293T cell-cell fusion (fusion indices of 0), although they were expressed on the cell surface (Fig. 2C; Table 1). In contrast, the S161A mutation induced a higher level of cell-cell fusion, despite the slightly lower CSE level than that of WT G (fusion index of 2.63) (Fig. 2C; Table 1), and the P163A and L166A mutant proteins displayed severe and mild hypofusogenic phenotypes (fusion indices of 0.05 and 0.42), respectively (Fig. 2C; Table 1). Overall, alanine mutations at N159, I160, S161, and P163 had pronounced effects on the fusion promotion ability of NiV-G (P < 0.001 for each).

Generally, the avidity of cell receptor (ephrinB2) and NiV-G interactions affects the fusion promotion capability of the viral attachment protein (2, 3, 7). Thus, we quantified the ability of WT and mutant NiV-G proteins to bind ephrinB2 via flow cytometry. The normalized receptor-binding level of each mutant was roughly similar to its normalized CSE level (Fig. 2C). These data indicate that the hyper- or hypofusogenic phenotypes of the alanine mutant proteins are not due to aberrant receptor-binding abilities, and aa 159 to 163 are indeed important in modulating cell-cell fusion.

The stalk domain is known to be important for tetramerization of the paramyxovirus attachment proteins, including NiV-G (27, 30, 38). Since cysteines at positions 146, 158, and 162 (C146, C158, and C162) have been implicated as important for G tetramerization (30), we investigated whether the hyper- and/or hypofusogenic phenotypes of the 159 to 163 alanine mutant proteins resulted from alterations in the tetrameric stability of G. We expressed WT or mutant NiV-G in 293T cells and subjected the cell lysates to nonreducing SDS-PAGE and immunoblotting with polyclonal antiserum 806. While for the WT and most of the mutant NiV-G proteins, the most prominent structures were tetrameric and dimeric forms of G, the I160A mutant protein showed less tetrameric (5.8-fold decrease) and more prominent dimeric and monomeric species (2.0- and 4.7-fold increases, respectively) than WT G (Fig. 2D). The relatively weaker tetrameric structure of the I160A mutant protein may be attributed to reduced hydrophobic interactions (lack of a hydrophobic isoleucine). This result, in combination with results for NiV-G stalk cysteine mutant proteins (30), suggests that severe weakening of the tetrameric structure has profound effects on the fusion promotion ability of G. Interestingly, the I160A mutant protein monomers and dimers migrated more slowly than those of WT NiV-G, possibly because of altered posttranslational modifications of this mutant protein.

Additionally, lower levels of dimers were detected for the N159A (18%) and S161A (23%) mutant proteins than for WT NiV-G (44%) (Fig. 2D). This suggests that these two mutant G proteins have increased oligomeric stability and that the glycosylation motif NIS is important for this oligomeric stability. However, neither the absence of an N-glycan nor the stability of the tetramer explains why the S161A mutant protein results in a hyperfusogenic phenotype while the N159A mutant protein results in a severely hypofusogenic phenotype, as neither protein is N-glycosylated at this site and both have similar overall distributions of tetrameric, dimeric, and monomeric forms.

Membrane fusion proteins with mutations in aa 159 to 163 within the NiV-G stalk modulate receptor induced conformational changes differently than WT NiV-G.

Since alanine mutations in aa 159 to 163 induced both hyper- and hypofusogenic phenotypes, we used a panel of previously reported conformation-dependent antibodies to investigate whether mutations in this region modulate receptor-induced conformational changes in NiV-G (28, 34). We recently described three receptor-induced conformational steps in NiV-G important for F triggering, detectable by Mab213 (step 1), Mab45 (step 2), and Ab167 (step 3) (28). We showed that ephrinB2 binding to NiV-G decreases the binding of conformational antibody Mab213 (step 1) to NiV-G by approximately 70%. We previously mapped the Mab213-binding epitope to region β3H1/β3H2 (aa 371 to 392) at the membrane-distal portion of the NiV-G head, near the receptor-binding site, and showed that receptor binding does not compete with Mab213 for binding to NiV-G (28, 34). Here, we found that the addition of ephrinB2 to cells expressing the N159A, I160A, S161A, and P163A mutant proteins also resulted in an ∼70% decrease in Mab213 binding (Fig. 3A), indicating that these mutations had no significant effect on the first conformational change at the membrane-distal portion of the NiV-G head.

EphrinB2 binding to NiV-G also induces a roughly 2-fold increase in Mab45 binding to NiV-G (step 2), a step important for F triggering (28, 30, 31, 34). We previously mapped the Mab45 epitope to region β6S4/β1H1 (aa 177 to 194) at the base of the NiV-G head (28). Here, we found no significant enhancement of Mab45 binding to any of the aa 159 to 163 mutant proteins, with the possible exception of a slight, although not statistically significant (P = 0.08), enhancement for the P163A mutant protein. In contrast, ephrinB2 binding to WT NiV-G resulted in a roughly 2-fold increase in Mab45 binding, as expected (Fig. 3A). These results suggest that none of these mutant proteins undergo conformational step 2, which occurs at the base of the NiV-G head, in the same way that WT NiV-G does.

Conformational Ab167 is a polyclonal antiserum raised against the NiV-G stalk (aa 1 to 167) (28). EphrinB2 binding to NiV-G enhances binding of Ab167 to NiV-G by about 1.5- to 2-fold (step 3) (28). As expected, WT NiV-G showed enhanced receptor-induced binding to Ab167 (1.7-fold), in contrast to any of the aa 159 to 163 mutant proteins, with the exception of the P163A mutant protein, in which a very slight enhancement of Ab167 binding was observed (∼20% increase; P < 0.001) (Fig. 3A).

We then asked whether the lack of receptor-induced conformational changes in the mutant G proteins was due to differences from the pre-receptor-binding conformation of the WT NiV-G protein. To account for CSE differences, we calculated the ratio of the levels of normalized conformational antibody binding to CSE for each mutant protein in the absence of the ephrinB2 receptor (Fig. 3B). As somewhat expected, all of the mutant proteins bound Mab213 at levels similar to that of WT G, with the exception of the I160A mutant (Fig. 3B), which showed only 70% of the Mab213 binding of WT NiV-G, possibly because of the aberrant oligomerization observed for I160A (P < 0.001; Fig. 2D). In contrast, all of the mutant proteins had greater Mab45 binding (1.5- to 2-fold) and, with the exception of the P163A mutant protein, greater Ab167 binding (1.2- to 1.7-fold) than WT NiV-G (P < 0.001; Fig. 3B). Interestingly, the overall trends of Mab45 and Ab167 binding to the WT and mutant NiV-G proteins were similar, suggesting that the receptor-induced conformational changes detected by Mab45 and Ab167 are intricately linked. Moreover, the P163A mutant protein bound Ab167 at levels similar to that of WT G (Fig. 3B). Overall, these results suggest that the pre-receptor-binding conformation of G is altered by mutations in aa 159 to 163, affecting the Mab45 and Ab167 but not the Mab213 epitope binding sites. Therefore, premature exposure of the epitopes recognized by Mab45 and Ab167 prior to receptor binding likely prevents receptor-induced conformational changes in these two epitopes, at least for some of these mutant proteins.

An optimal NiV-G conformation is important for efficient F triggering.

Next, we studied the temporal dynamics of cell-cell fusion induced by the N159A, I160A, S161A, and P163A mutant proteins by measuring cell-cell fusion changes over time (12, 16, 22, and 28 h posttransfection). No cell-cell fusion was observed with the N159A or I160A mutant protein at any time point, in contrast to the P163A mutant protein, which started causing cell-cell fusion at 16 h posttransfection (5% compared to WT G) (Fig. 4A). The cell-cell fusion levels induced by the S161A mutant protein were nearly 2-fold higher than those induced by WT NiV-G at all of the time points studied (Fig. 4A). Note that at 28 h posttransfection, cell-cell fusion had progressed so far for WT G and the S161A mutant protein that cells started lifting off the plate and fusion levels were uncountable. These data suggest that while the N159A and I160A mutant proteins are severely hypofusogenic, the P163A mutant protein is a “slower” fusion mutant.

Interestingly, the S161A mutant protein showed a hyperfusogenic phenotype even though it did not exhibit enhanced Mab45 and Ab167 binding (Fig. 2C and 3A). From these unexpected results, we hypothesized that the S161A mutant protein may have acquired a “prone-to-trigger” conformation, which undergoes the receptor-induced conformational cascade more readily than WT G.

We then tested whether such a prone-to-trigger conformation may occur independently of receptor binding. Thus, we added soluble EphB3 (40 nM), a natural ligand of ephrinB2/B3, to cells transfected with S161A mutant and WT NiV-F (28). Cell-cell fusion was determined at 16 to 24 h posttransfection, and we observed that, at this concentration, EphB3 inhibited S161A mutant protein-induced fusion at levels at least similar to those of WT NiV-G-induced fusion (about 60 to 70% inhibition) (Fig. 4B). We also tested cell-cell fusion of this mutant protein in PK13 cells (receptor deficient) and observed a lack of cell-cell fusion induced by S161A or WT NiV-G (data not shown). Combined, these results indicate that receptor binding is required for S161A mutant G to induce cell-cell fusion.

Next, we asked whether a lower degree of receptor-induced conformational changes may be required for S161A mutant than for WT NiV-G to overcome a presumed energy barrier needed to trigger F (15). Therefore, we tested if S161A mutant G was able to rescue cell-cell fusion induced by our available hypofusogenic F mutant proteins. We observed that S161A mutant G was able to rescue the level of cell-cell fusion of NiV-F hypofusogenic mutant F_P221A, increasing the F_P221A fusion index from 0.15 to 1.0 (WT fusion index) (Fig. 4C). Although we do not fully understand the mechanism of the hypofusogenicity of F_P221A, which is beyond the scope of this study (unpublished data), the fact that the S161A mutant protein can rescue F_P221A suggests that the S161A mutant protein is in a prone-to-trigger F conformation. Such a conformation presumably requires a lower degree of conformational changes to trigger F than WT G does (Fig. 3 and 4C). Notably, the S161A mutant protein rescued F_P221A without increasing the CSE levels of F_P221A (data not shown). Our results also suggest that the S161A conformation may be at least partially downstream of conformational steps 2 and 3, as for S161A receptor-induced conformational steps 2 and 3 were not detected by Mab45 or Ab167, but these antibodies bind S161A at higher-than-WT levels (P < 0.05; Fig. 3).

We then asked if previously described hyperfusogenic NiV-F mutant proteins (F_F3F5, F_K1A, and F_{K1A F3F5}) could rescue cell-cell fusion induced by the hypofusogenic N159A, I160A, and P163A mutant NiV-G proteins. F_F3F5 lacks two N-glycosylation sites in the ectodomain of F, while F_K1A is a cytoplasmic tail mutant (32, 33). Interestingly, hyperfusogenic F_{K1A F3F5}, when coexpressed with the N159A mutant protein, reached only 10% of the cell-cell fusion level obtained with WT G in 293T cells (Fig. 4D). Next, as NiV glycoproteins induce cell-cell fusion more readily in Vero cells than in 293T cells, we investigated the fusion promotion of some mutant G proteins in Vero cells (32, 33; data not shown). Interestingly, the N159A mutant protein induced Vero cell-cell fusion at about 40% of the WT G levels in combination with WT NiV-F (Fig. 4E). These results suggest that in highly fusogenic environments, the N159A mutant protein retains limited membrane fusion promotion ability. Interestingly, none of the hyperfusogenic F mutant proteins rescued I160A-induced 293T cell-cell fusion (Fig. 4D). In contrast, all of the hyperfusogenic F mutant proteins rescued P163A mutant protein-induced 293T cell-cell fusion, reaching nearly WT F and G levels when coexpressed with the F_{K1A F3F5} mutant protein (Fig. 4D). These results indicate that an intact oligomeric structure is required for the fusion promotion ability of NiV-G, as shown by all of the results obtained with the I160A mutant protein. Additionally, these results, together with results in Fig. 3, suggest that mutations at positions N159 and P163 resulted in a suboptimal G conformation for F triggering, causing low levels of fusion and/or slow fusion kinetics (see Fig. 7).

Aa 159 to 163 in the NiV-G stalk C-terminal portion modulate viral entry.

To test whether proteins with mutations in aa 159 to 163 trigger NiV-F equivalently on cell or viral surfaces, we produced NiV/VSV pseudotyped virions expressing a Renilla luciferase reporter gene by using our previously established quantitative BSL2 viral entry assay (18, 28). When compared at several logs of viral input (as determined by VSV genome copy numbers), WT NiV/VSV virions showed up to 3 orders of magnitude increased viral entry compared to the negative-control virus that lacks NiV-F (G alone). In general, the cell-cell fusion phenotypes of the alanine mutant proteins correlated with their viral entry phenotypes (Fig. 2C and 5A). For example, the fusion-dead I160A mutant protein was unable to promote viral entry (similarly to the negative-control “G-alone” virions [Fig. 2C and 5A]), and the hypofusogenic P163A mutant protein yielded about 1-log lower levels than WT “G” virions (Fig. 2C and 5A). The hyperfusogenic S161A mutant virions exhibited entry similar to that of WT virions, as expected, since previous studies showed that generally 3- to 6-fold increases in cell-cell fusion levels are required to detect an increase in viral entry levels (Fig. 5A) (18, 32, 33). Not so surprisingly, the 293T fusion-dead N159A mutant protein induced viral entry at greater-than-background G alone levels, as the N159A mutant protein retained limited F-triggering ability (Fig. 4D and E).

We then monitored the levels of F and G incorporation into virions by Western blotting. Point mutations in the NiV-G region from aa 159 to aa 167 mostly did not significantly affect the incorporation levels of NiV-G, except for the N159, I160, and P163 mutant proteins, for which the incorporation levels were relatively lower than those of WT G (Fig. 5B). The low incorporation level of the I160A mutant protein into virions is likely due to its defective oligomerization stability. Interestingly, the levels of incorporation of NiV-F appeared to be somewhat lower for those mutant G proteins with lower G incorporation levels (Fig. 5B). An overexposed version of these images shows that I160A mutant G is incorporated at very low levels.

Overall, the viral entry and cell-cell fusion results were roughly consistent. In addition, the viral entry levels induced by the hypofusogenic and less-incorporated N159A mutant protein suggest that viral entry might be a more efficient process than cell-cell fusion, at least for this mutant protein (Fig. 2C and 5).

NiV-G stalk aa 159 to 163 modulate F triggering and G-F interactions.

To determine directly whether the alanine mutant proteins are able to trigger NiV-F in NiV/VSV pseudotyped virions, we took advantage of a confocal micro-Raman spectroscopic technique we recently developed (36). This technique allows the detection of receptor-induced conformational changes in NiV-F embedded in the surfaces of the NiV/VSV pseudotyped virions (36). In our recent study, we showed that ephrinB2-induced conformational changes in NiV-F, observed as a downward shift of a Raman signal at a wave number of 1,409 cm⁻¹, corresponded to the extent of conformational changes in F during F triggering. As shown in Fig. 6A, no downward shift was observed in negative-control virions containing NiV-F only (No G, red line), whereas a significant downward shift was observed in WT NiV/VSV virions that harbor both WT F and G (G). As expected, I160A mutant virions yielded a Raman spectral pattern similar to that of negative-control virions. In addition, S161A and P163A mutant virions yielded levels of F triggering similar to those of WT virions, and an intermediate F triggering phenotype was observed for the N159A mutant protein, corroborating its intermediate viral entry phenotype (Fig. 5A and 6A). Although the Raman spectroscopic analysis was not sensitive enough to show differences in receptor-induced conformational changes in F between the P163A and S161A mutant and WT virions, our overall results still suggest important roles for residues in aa 159 to 163 within the NiV-G stalk in F triggering.

For paramyxoviruses, the cell receptor type appears to determine the nature of the interactions between the attachment and fusion glycoproteins. For MeV and NiV, protein receptor binding appears to induce the dissociation of previously associated H-F or G-F complexes, and such a dissociation is a critical step in membrane fusion modulation (14, 32, 33). Therefore, it is plausible that the hypofusogenic phenotypes of the N159A, I160A, and P163A mutant proteins are partly due to their decreased ability to release the cognate F upon receptor binding. We used HA-tagged WT and mutant NiV-G proteins to determine the avidities of G-F interactions, since HA-tagged WT and mutant NiV-G had fusion phenotypes similar to those of their untagged counterparts (data not shown). We immunoprecipitated NiV-G from 293T cells expressing NiV-F and WT or mutant NiV-G, with μMACS anti-HA Microbeads. The cell lysates, as well as immunoprecipitated proteins, were separated by SDS-PAGE and analyzed by immunoblotting with antibodies specific for F and G. Whereas immunoprecipitated WT and mutant NiV-G proteins were detected at roughly equal levels, the levels of coimmunoprecipitated NiV-F (F₀, F₁) varied among the mutant NiV-G proteins (Fig. 6B, right panel). In general, hypofusogenic the N159A and P163A mutant proteins exhibited somewhat greater G-F avidities than WT G, whereas the hyperfusogenic S161A mutant protein did not. We speculate that the hypofusogenic I160A mutant protein did not show increased G-F avidities likely because of its anomalous tetrameric structure. As expected, no F was pulled down from the F-only-containing cell lysate. These results also indicated that all of the mutant G proteins retain the ability to interact with F.

We measured G-F interaction avidities by calculating the ratios of coimmunoprecipitated F to the corresponding amount of NiV-F in the total cell lysates for WT G and each mutant protein. The ratios were then normalized to that of WT G, which was set as 1. For those mutant NiV-G proteins that retained an intact tetrameric structure, we observed an inverse correlation between the fusion phenotype of our mutant NiV-G proteins and their avidities of G-F interactions, similarly to previous experiments with NiV-F mutant proteins (Fig. 6B; Table 2) (32, 33). The hyperfusogenic S161A mutant protein was able to pull down F at a level similar to that of WT G (G-F interaction avidity of 1.3; P > 0.05), whereas the hypofusogenic N159A and P163A mutant proteins pulled down F at greater levels than WT G (G-F interaction avidities of 5.0 and 2.7, respectively; P < 0.05) (Fig. 6B, right panel; Table 2). Therefore, the hypofusogenic phenotypes of at least the N159A and P163A mutant proteins may be related to their decreased ability to release NiV-F. These data suggest that mutations in aa 159 to 163 within the NiV-G stalk affect the avidities of G-F interactions and likely G-F dissociation upon fusion triggering.