A novel receptor-induced activation site in the Nipah virus attachment glycoprotein (G) involved in triggering the fusion glycoprotein (F)

Abstract

Cellular entry of paramyxoviruses requires the coordinated action of both the attachment (G/H/HN) and fusion (F) glycoproteins, but how receptor binding activates G to trigger F-mediated fusion during viral entry is not known. Here, we identify a receptor (ephrinB2)-induced allosteric activation site in Nipah virus (NiV) G involved in triggering F-mediated fusion. We first generated a conformational monoclonal antibody (monoclonal antibody 45 (Mab45)) whose binding to NiV-G was enhanced upon NiV-G-ephrinB2 binding. However, Mab45 also inhibited viral entry, and its receptor binding-enhanced (RBE) epitope was temperature-dependent, suggesting that the Mab45 RBE epitope on G may be involved in triggering F. The Mab45 RBE epitope was mapped to the base of the globular domain (beta6S4/beta1H1). Alanine scan mutants within this region that did not exhibit this RBE epitope were also non-fusogenic despite their ability to bind ephrinB2, oligomerize, and associate with F at wild-type (WT) levels. Although circular dichroism revealed conformational changes in the soluble ectodomain of WT NiV-G upon ephrinB2 addition, no such changes were detected with soluble RBE epitope mutants or short-stalk G mutants. Additionally, WT G, but not a RBE epitope mutant, could dissociate from F upon ephrinB2 engagement. Finally, using a biotinylated HR2 peptide to detect pre-hairpin intermediate formation, a cardinal feature of F-triggering, we showed that ephrinB2 binding to WT G, but not the RBE-epitope mutants, could trigger F. In sum, we implicate the coordinated interaction between the base of NiV-G globular head domain and the stalk domain in mediating receptor-induced F triggering during viral entry.

FIGURE 1.

Enhanced binding of Mab45 to NiV-G (G) induced by ephrinB2 (B2) receptor binding. A, polyclonal (806) and monoclonal (Mab26 and Mab45) anti-NiV-G antibodies binding to full-length NiV-G expressed in CHOpgsA745 (CHO) cells in the presence or absence of soluble B2 protein at room temperature (∼20 °C) as detected by flow cytometry. % normalized binding indicates MFI values normalized to the MFI values obtained in the absence of soluble B2 (set at 100% for each antibody examined). Average ± S.E. are shown, n = 3. B, similar experiment to A, except performed at 4 °C. C, binding of Mab45 to CHO cells expressing G or mutant E533Q at 37 °C when increasing concentrations of soluble ephrinB1 (B1), ephrinB2 (B2), or ephrinB3 (B3) were used. Logarithm of the molar concentration of the soluble ephrinBs used was plotted against % normalized binding, calculated as in A. Average ± S.E. are shown, n = 4. D, the inhibitory properties of 806, Mab26, or Mab45 antibodies were measured by mixing the indicated concentrations with NiV/vesicular stomatitis virus renilla luciferase pseudotyped particles immediately before infection of Vero cells. Data are shown as % inhibition normalized to luciferase activity in the absence of any antibodies. Average ± S.E. are shown; n = 3.

FIGURE 2.

Role of the β6S4/β1H1 region of G in membrane fusion and Mab45 binding enhancement. A, schematic of NiV-G showing the various domains. Deletion sites 1, 2, 3, 4, 9, 11, and 14 are depicted, and their sequences are shown in supplemental Table 1. B, binding of anti-HA, Mab26, and Mab45 to CHO cells expressing WT or deletion mutant G constructs Δ4 and Δ9, as measured by flow cytometry. Average ± S.E. are shown, n = 3. Both Mab45 and Mab26 are conformational antibodies, as they do not Western blot. C, sequence of region 4 of G showing the location of the triple Ala mutations 4-1, 4-2, 4-3, 4-4, and 4-5 constructed in the context of full-length G. D, binding of the full-length WT and the indicated mutant G proteins to Mab26 and Mab45 by flow cytometry, normalized to their anti-HA antibody binding levels. Average ± S.E. are shown, n = 3. E, levels of binding of region 4 mutants to polyclonal anti-NiV-G antiserum 806 or to soluble ephrinB2 (B2) in CHO cells and levels of syncytia formation in 293T cells normalized to anti-HA antibody binding to correct for any discrepancies in cell surface expression levels. Average ± S.E. values are shown, n = 3. F, levels of binding of WT and region 4 mutant proteins to Mab45 at various concentrations of B2, normalized to Mab45 binding in the absence of B2 (set at 100%) as in Fig. 1C. Data were plotted using Prism, and average ± S.E. values are shown, n = 3.

FIGURE 3.

EphrinB2 binding to NiV-G induces secondary structure changes in NiV-G that are correlated with fusogenicity. A, changes in the secondary structure of soluble G (blue) at 1.2 μm were monitored using CD spectroscopy upon the addition of B2 (red) at 2.5 μm. A theoretical spectrum is shown using a black dashed line, and the experimental G:B2 scan is shown in green. The difference between the latter two lines reflects differences in secondary structure. B and C, same experiment as in A, except replacing WT G for soluble 4-5 and GΔstalk proteins, respectively. The GΔStalk protein lacked amino acids 71–154 of the stalk domain. D, the difference between experimental and theoretical data (Δ [θ]) were generated by averaging the CD signals between 223 and 208 nm and subtracting the experimental signal (green line) coming from the indicated G and B2 mixture from the theoretical signal (black dashed line). Data are the average ± S.E., n = 3. E, soluble WT G, 4-5, or GΔStalk mutant proteins coating a maxisorb plate bound soluble B2 similarly in an ELISA enzyme-linked immunosorbent assay format assay. A sigmoidal dose response curve is shown (data are the average ± S.D., performed in triplicate).

FIGURE 4.

Lack of G activation correlates with lack of F dissociation from G. A, co-immunoprecipitation of NiV-F using anti-NiV-G antibodies (top) from F/G/vesicular stomatitis virus pseudotyped virions allowed to mix with CHO cells (negative control) for 2 h at 4 °C then triggered at 37 °C for 0, 15, or 60 min, and lysed in 1% Triton X-100. Amounts of NiV-F in the viral/cell lysate (middle) and amounts of immunoprecipitated G under each condition (bottom) are shown. Co-IP, co-immunoprecipitation. B, same experiment as in A but using CHOB2 instead of CHO cells. C, same experiment as in B except using the 4-5 mutant G protein instead of the WT G protein. One representative experiment of three is shown. D, quantification of the effects of ephrinB2 binding on F/G interactions. The bands in A–C were quantified by densitometry using a VersaDoc Imaging System (Bio-Rad). For each time point and experimental condition, the normalized co-immunoprecipitation (Co-IP) F ratio was calculated as the ratio of co-immunoprecipitated F₀ or F₁ signals (from A–C, top panels) over the total F₀ or F₁ signals in the cell lysates (A–C, middle panels) (to normalize for F expression levels) divided by the total amount of immunoprecipitated G (A–C, bottom panels), as shown in the formulas below. For each experimental condition, the normalized co-immunoprecipitation F ratio at various time points was normalized to the ratio obtained at time 0, which was set at 1 unit. Note that F₁ dissociated from WT G more than F₀ in the presence of B2. Data are the average ± S.E. are shown, n = 3. Normalized co-immunoprecipitation (Co-IP) F₀ ratio = (Co-IP F₀/(F₀ in cell lysates))/(G IP); normalized co-IP F₁ ratio = (Co-IP F₁/(F₁ in cell lysates))/(G IP).

FIGURE 5.

Lack of G activation results in inability to trigger F. A, CHO cells expressing F and G were mixed with either CHO cells (negative control) or CHOB2 cells for 2 h at 4 °C. Cell mixtures were brought to 37 °C or kept at 4 °C for 45 more min to allow for triggering of F or not in the presence of 500 nm biotinylated HR2 peptide. B, the MFI values are summarized for 4 or 37 °C and in the absence (-B2) or presence (+B2) of B2 for the F/G (WT), F/4-4, and F/4-5 protein pairs. Data are the average ± S.E. are shown, n = 3. The p value for the MFI of the (+B2) versus (-B2) conditions at 37 °C for WT F/G was 0.02.