Schematic of bi-molecular H complementation to explore the organization of the physiological complex.

<p>(Left panel) Overview of previously identified functional domains in H, responsible for interaction with F <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002058#ppat.1002058-Paal1" target="_blank">[33]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002058#ppat.1002058-Lee1" target="_blank">[34]</a>, receptor binding <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002058#ppat.1002058-Corey1" target="_blank">[29]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002058#ppat.1002058-Patterson1" target="_blank">[61]</a>, or required for F triggering <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002058#ppat.1002058-Corey2" target="_blank">[53]</a>. For simplicity, an H dimer is shown representing form I as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002058#ppat.1002058-Hashiguchi1" target="_blank">[31]</a>. (Right panel) Co-expression of H variants defective in individual functions in all possible combinations restores F fusion promotion activity through trans-complementation of functionality <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002058#ppat.1002058-Brindley1" target="_blank">[32]</a>. Structural renderings were generated as outlined for <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002058#ppat-1002058-g001" target="_blank">Figure 1</a>.</p

Representation of MeV H head domains complexed with soluble Slam receptor based on the coordinates reported by Hashiguchi and colleagues [<b>31</b>].

<p>Slam moieties (dark green) and covalently linked H dimers (cyan and light purple) in the tetrameric arrangement are highlighted. Receptor binding is proposed to trigger a significant reorganization of the non-covalent dimer-dimer interface (form I versus form II <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002058#ppat.1002058-Hashiguchi1" target="_blank">[31]</a>). In the original X-ray analysis, form II was observed when an additional L482R mutation was introduced into MeV H. This mutation was found to enhance SLAM-dependent fusion and also appeared in a clinical MeV isolate of the D1 genotype <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002058#ppat.1002058-Hashiguchi1" target="_blank">[31]</a>. Structural renderings were prepared as described for <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002058#ppat-1002058-g001" target="_blank">Figure 1</a>. Dotted lines highlight the dimer–dimer intersection. Hypothetical positions of the H stalk domains are marked in the side view representations.</p

Measles virus fusion model.

<p>(Left panel) Model representation of the MeV envelope glycoprotein prefusion hetero-oligomer. The H and F complexes are aligned in a staggered head configuration in which the F head is thought to stand in contact with the H stalk <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002058#ppat.1002058-Paal1" target="_blank">[33]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002058#ppat.1002058-Prussia1" target="_blank">[57]</a>. (Middle and right panels) Hypothetical dissociation model of F triggering. Upon binding to the cellular receptor, H and F dissociate, resulting in triggering of major conformational changes in metastable prefusion F. Refolding into the stable postfusion conformation is considered to occur through a series of intermediate conformations, including a hypothetical pre-hairpin intermediate <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002058#ppat.1002058-Lamb2" target="_blank">[13]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002058#ppat.1002058-Yin1" target="_blank">[56]</a>. Likely, refolding of multiple F complexes is required to open a fusion pore and enable viral entry. For improved clarity, MeV H is represented as a single tetramer, and F as a single trimer in the hetero-oligomeric fusion complex. More than one F trimer may interact, however, with each individual H tetramer. The insert shows an enlarged representation of proposed lipid mixing intermediates. As F refolds, first the outer membranes are thought to fuse, creating a lipid stalk. Membrane merger is then thought to advance through hemifusion to pore formation. For clarity, F complexes have been eliminated from the lipid mixing representations. Structural renderings are based on original crystal structures (form I H head domains as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002058#ppat.1002058-Hashiguchi1" target="_blank">[31]</a>), homology models of MeV F <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002058#ppat.1002058-Lee2" target="_blank">[55]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002058#ppat.1002058-Plemper3" target="_blank">[58]</a> based on coordinates reported for pre- and post-fusion PIV5 and PIV3 F, respectively <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002058#ppat.1002058-Yin1" target="_blank">[56]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002058#ppat.1002058-Yin2" target="_blank">[59]</a>, or hypothetical structural models (F pre-hairpin intermediate). H stalk domains are modeled in an assumed α-helical configuration <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002058#ppat.1002058-Paal1" target="_blank">[33]</a>. High-resolution structural models were aligned at the level of the transmembrane domain (viral envelope) and then morphed into low resolution images using the Sculptor (resolution 12, voxel size 3) package <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002058#ppat.1002058-Birmanns1" target="_blank">[60]</a>.</p

Both regions 9 and 4N modulate NiV membrane fusion.

<p>CSE (HA) and cell-cell fusion levels of region 4N (<b>A</b>) and 9 (<b>B</b>) mutants. CSE levels were measured in 293T cells as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003770#ppat-1003770-g004" target="_blank">Fig. 4E</a>. 293T cell-cell fusion levels induced by wt NiV-F and wt or mutant NiV-G, normalized to values of wt NiV-F/G. n = 3–8. <b>C) & D)</b> Depictions of regions 4N (C), or 9 (D), from the crystalized NiV-G head structure in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003770#ppat-1003770-g004" target="_blank">Fig. 4G</a>. Blue and red colored residues mark hypo- or hyper-fusogenic mutants, respectively.</p

Receptor binding to the NiV-G head exposes a stalk domain that triggers F.

<p><b>A</b>) Relative levels of Ab167 binding to PK13 cells expressing NiV-G +/− 100 nM soluble ephrinB2-Fc (B2), normalized to the level observed in the absence of B2. <b>B</b>) Relative levels of 293T cell-cell fusion induced by wt NiV-F or hypofusogenic NiV-F mutants and wt NiV-G or mutant 167, 18 h post transfection, normalized to the level induced by wt NiV-G. <b>C</b>) Relative entry levels of NiV/VSV <i>Renilla</i> luciferase reporter virions containing wt NiV-F and wt NiV-G (solid black line), wt NiV-F and mutant 167, wt NiV-G alone, wt NiV-F alone (various black or gray dashed lines), or vector alone (solid gray line). RLU were quantified 18–24 h post infection and plotted against the number of viral genomes/ml. Data shown are averages± S.D. from three independent experiments. <b>D</b>) Representative Western blot analysis of 10⁹ NiV/VSV pseudotyped virions (genome copies) from 3C showing incorporation of NiV-F and wt NiV-G or mutant 167. <b>E</b>) Second derivative transformed Raman spectral features of F glycoproteins triggered by ephrinB2-bound wt NiV-G virions (G), unbound mutant 167 virions (167), unbound control mutant 176 virions (176), or ephrinB2-bound NiV-F-alone virions (No NiV-G). The lower the 1409 cm⁻¹ peak, the greater the extent of NiV-F triggering <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003770#ppat.1003770-Lu1" target="_blank">[33]</a>.</p

Headless NiV-G can trigger cell-cell fusion.

<p><b>A</b>) Schematic representation of full-length wt NiV-G protein (residues 1–602), and NiV-G truncation mutants named after their most C-terminal residue. The sequence of the stalk C-terminus is shown. CT, cytoplasmic tail; TM, transmembrane domain. <b>B</b>) Relative levels of CSE measured using antibodies 806 or Ab167 by flow cytometry, normalized to wt NiV-G or mutant 167, respectively; and 293T cell-cell fusion levels induced by wt NiV-F and wt or mutant NiV-G, normalized to those of wt NiV-G. Average ± S.D. are shown. Five fields per experiment were counted, n = 3. <i>P</i> values calculated between fusion data for mutant 167 and the other mutants, or between any of the mutants and our negative control, were all <0.001, even when multiplied by the Bonferroni factor (n−1) = 7. <b>C</b>) Western blot analysis of wt NiV-G or truncation mutants blotted with 806 or Ab167. To facilitate the observation of the difference in apparent molecular weight between mutant samples, mutants 164 and 187 were run side-by side on a separate gel, shown on the right. <b>D</b>) Representative images of 293T cell-cell fusion induced by wt NiV-F and wt NiV-G or headless NiV-G mutants 17 h post transfection. Arrows point to syncytia.</p