Receptor-Targeted Nipah Virus Glycoproteins Improve Cell-Type Selective Gene Delivery and Reveal a Preference for Membrane-Proximal Cell Attachment
Fig 2
Mutation of the NiV glycoprotein to ablate natural receptor recognition.
(A) Surface representation of top-view of NiV-G. G is shown as dimer by modeling its monomer crystal structure (Protein Data Bank (PDB) ID: 3D11) on the crystal structure of the Hendra virus G dimer (PDB ID: 2X9M) using PyMOL. The binding site for ephrin-B2 is depicted in blue [26]. Residues mutated in G to screen for their potential to ablate natural receptor tropism are shown in red. (B) Six different single mutations and the combinations E501A+W504A (Gc∆34EpCAMmut2.1), Q530A+E533A (Gc∆34EpCAMmut2.2), or E501A+W504A+Q530A+E533A (Gc∆34EpCAMmut4) were introduced into Gc∆34EpCAM. Unconcentrated vector stocks generated with the mutated G proteins were titrated on CHO-EpCAM (white bars; negative for natural NiV receptors) and U87-MG cells (black bars; positive for NiV receptors). NiV-LVs pseudotyped with Gc∆34/Fc∆22 having the natural NiV tropism (Gc∆34) served as control. Arrows indicate titers <6x102 t.u./ml. Statistics refer to unmutated Gc∆34EpCAM. Titers of EpCAM-targeted LVs on CHO-EpCAM cells were not statistically different. (n = 4; mean ± standard deviations (SD) are shown; *, P<0.1 **, P<0.01 by one-way ANOVA with Dunnett's multiple comparisons test). (C-D) Binding of ephrin-B2 (C) and B3 (D) to NiV-G mutants is shown. HEK-293T cells were transfected either mock or with plasmids encoding the indicated G protein variants and then incubated with 1 μg/ml recombinant Fc-ephrin-B2 or -B3 prior to staining against the Fc-tag using FITC coupled anti-Fc antibody. The binding efficiencies of the different mutants to the receptors are shown as MFI (mean fluorescence intensity) values (n = 3; mean ± standard deviations (SD) are shown). (E) Concentrated vector stocks of Gc∆34EpCAMmut4/Fc∆22 pseudotyped LV vectors were generated and particle size was analyzed via single nanoparticle tracking analysis (NTA). Particle size measurement of one representative out of three independent stocks is shown (black). As control, concentrated vectors stocks of VSV-LV (blue) and NiVwt-LV (red) were analyzed. The mean size ± SD of the main peak out of three measurements of each particle type is indicated. (F) Electron microscopy of concentrated LV particles pseudotyped with Gc∆34EpCAMmut4/Fc∆22 proteins. The white arrowhead points to the NiV glycoproteins on the particle surface. Scale bar: 100 nm.