Receptor-Targeted Nipah Virus Glycoproteins Improve Cell-Type Selective Gene Delivery and Reveal a Preference for Membrane-Proximal Cell Attachment
Fig 5
Expanding the system to additional target receptors.
(A) Surface expression of NiV G proteins (blue line) targeted to four different receptors was compared to that of the corresponding MV H protein counterparts (red line). All expression plasmids encoding the different constructs were transfected into HEK-293T cells. Surface expression was analyzed after 48 hours using a His-tag-specific antibody (PE-labeled). Mock transfected cells (filled curves) served as negative control. One representative out of three experiments is shown. For quantitative data see S3 Fig. (B) Western blot analysis of NiVmutEpCAM-LV, NiVmutCD8-LV, NiVmutCD20-LV, and NiVmutHer2-LV. For generation of the vectors used for Western blot analysis, F protein C-terminally tagged with the AU1 immunological tag was used to allow detection via the anti-AU1 antibody. Incorporation of the G variants was detected via an anti-His antibody. 2.5x1010 particles per sample were used. Mock transfected cells (mock) as well as bald particles without glycoproteins (bald-LV) served as controls. In addition, particles pseudotyped with full-length His-tagged G and AU1 tagged F (GHis-LV) as well as particles pseudotyped with Gc∆34His/Fc∆22-AU1 (NiVwt-LV) were used. For quantitative data see S4 Fig. (C) Titers of receptor-targeted NiV-LVs and their MV-LV counterparts. Unconcentrated stocks of EpCAM-targeted vectors were titrated on CHO-EpCAM cells, CD20-targeted vectors on Raji, CD8 targeted-vectors on Molt4.8 and Her2/neu-targeted vectors on SK-OV-3 cells. (n = 4; mean ± standard deviations (SD) are shown; *, P<0.1; ***, P<0.001; ****, P<0.0001 by unpaired t-test). (D) Fold change in titers (t.u./ml) determined by normalizing the titers of NiV glycoprotein based LVs to those of the corresponding MV glycoprotein based LVs. (E) Concentration of vector stocks from (C) by centrifugation. (F) Number of transducing units per 108 physical particles of NiV and MV glycoprotein based LVs. Particle numbers were determined by single nanoparticle tracking analysis (NTA) (n = 4; mean ± standard deviations (SD) are shown; *, P<0.1; ***, P<0.001; ns, not significant by unpaired t-test).