Fig 1.
The NiV-F trimer has a tree-like overall shape with the three copies of the F glycoprotein (colored in blue, green and violet) twined around a central axis (Axis-T). The fusion Peptide (FP) is colored in red. The NiV-F trimer is shown viewed from side, and from the top. Glycosylation moieties and disulfide bonds are shown as sticks. The inset shows a close-up view of the FP, which is located at the N-terminus of the F1 subunit, and docks into a groove formed by the F2 subunit of a neighboring F molecule within the trimer. Furthermore, the C-terminus of F2 and N-terminus of FP fold into a β-hairpin conformation, forming a continuous β-sheet (strands S1-S6) with the F1 subunit, which fixes the position of FP and stabilizes the pre-fusion state. The N-terminal FP residue, L110, is shown as a red sphere.
Fig 2.
Hexameric Assembly of NiV-F glycoprotein trimers.
The six NiV-F copies are colored in blue, green, pink, yellow, grey and purple, respectively. The C-terminal helical bundles of the NiV-F trimers are colored in red. Six NiV-F trimers assemble into a hexagonal ring around a three-fold crystallographic symmetry axis. The hexameric assembly of NiV-F is presented in cartoon views from the top (A), bottom (B) and side (C). The inset in panel C shows a hydrophobic patch of the hexameric interface between two neighboring NiV-F trimers (interface 1). Specifically, the cathepsin-L cleavage site-containing loop of one F trimer inserts into a hydrophobic pocket of the adjacent F trimer. Residue R109 is embedded into a pocket defined by P52, L53, Y248, T250, L256, F282, P283 and I284. The surrounding residues V108, A111, Q393 and G398 also contribute to the interaction. In the two interface inserts, the blue, green, pink, and purple text marks residues in the monomers of the corresponding colors.
Fig 3.
Formation of F hexamers-of-trimers in solution.
(A) 100μg of 50 mg/mL sF cross-linked with 0.08% glutaraldehyde was subjected to 10–25% sucrose gradient ultracentrifugation and fractionated. 10μL of each fraction was analyzed on Blue Native PAGE followed by western blotting. F oligomers were probed by monoclonal mouse anti-F specific antibody. The bottom and top gradient fractions are indicated. Arrows indicate higher-order oligomers formed from F trimers. (B) Hexameric sF assemblies imaged with negative stain EM of pooled fractions 1–6 from panel (A). It shows the cross-linked NiV-F particles boxed out from raw images. The majority of NiV-F oligomers appear as hexamers-of-trimers. (C) A small density slab (7.4 nm thick) from an electron tomogram of NiV-F decorated VLP showing the hexameric arrangement of the NiV-F spikes on the VLP. The individual NiV-F spikes are ~8–10 nm long with a ~1–2 nm stalk, consistent with the electron microscopy studies of soluble PIV5-F in its pre-fusion state [59]. The hexameric assembly revealed in NiV-sFGCNt crystal structure was shown next to it as reference.
Fig 4.
Cell-cell fusion and mAb binding activities of NiV-F mutants.
Four mutants of NiV-F within the trimer-trimer interface were tested for their ability to promote cell-cell fusion when co-expressed with NiV-G in a β-Gal reporter cell-cell fusion assay using HEK293T cells as the target population. (A) The data shown are the mean percentage of WT fusion levels measured for each mutant calculated from four separate experiments. The data was normalized for cell surface expression of WT F measured by flow cytometry, using a F-specific mAb that recognizes total F surface expression. The bars represent the range from multiple experiments. WT: wild type F. Statistical analysis probes activity deviation relative to WT. *: p<0.01; **: p<0.001; p = 0.3668 for R109L; p = 0.1488 for Q393L. (B) Expression of NiV-F mutants and NiV-G in HeLa USU cells from (A). Equal amount of remaining cells from fusion were lysed and clarified by centrifugation followed by immunoprecipitation by polyclonal rabbit anti F and G serum and protein G Sepharose. The precipitated products were analyzed on SDS PAGE followed by western blotting. F and G were probed by monoclonal mouse anti F (upper panel) and G (lower panel) specific antibody. (C) Cleared lysates of NiV-F mutants expressed in 293T cells were immunoprecipitated with 3 different competition groups of neutralizing anti F mAb as indicated. All 3 mAbs efficiently immunoprecipitated WT F. The precipitated complexes were analyzed on SDS-PAGE followed by western blotting and F was probed using polyclonal rabbit anti-F antisera.
Fig 5.
Viral entry is affected by mutations in the hexameric interface.
(A) Relative entry levels of NiV/VSV-rluc virions containing wt NiV-G and wt NiV-F (solid black line) or mutant L53D, V108D, R109L or Q393L NiV-F (dotted red line). Vector alone (pcDNA3)/VSV is shown as a dotted gray line. RLU for lysates of infected Vero cells were quantified 18–24 h post infection and plotted against the number of viral genomes/ml over 3 logs of viral input. Data shown are averages ± SEM from at least three independent experiments (n = 3). (B) Representative Western blot analysis of NiV/VSV-rluc virions shown under Fig 5A. 4x108 NiV/VSV pseudotyped virions (genome copies) were separated by denaturing 10% SDS–PAGE and probed against NiV-G (rabbit anti-HA, Bethyl) and NiV-F (mouse anti-AU1, Covance).
Fig 6.
Schematic representation of the proposed NiV-F activation model.
Upon viral attachment, the ephrin receptor-mediated re-arrangements of NiV-G exert a triggering disturbance at an exposed “priming site” (presumably via direct G-F priming-site interactions) that is sufficient to activate one F glycoprotein trimer and trigger its transition from a pre-fusion to a post-fusion conformation. The transformation of a single F trimer within the hexameric assembly would disrupt its interactions with both of its neighbors, unlocking their “priming sites” and facilitating their pre-to-post fusion transitions. Thus a single ephrin/NiV-G/F trimer interaction would result in the synergetic switch from a pre-fusion to a post-fusion conformation in all six F trimers within the hexameric assembly. The resulting eighteen copies of the six-helical post-fusion F bundle would form a stable fusion pore allowing virus entry into the host cell.