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. 2023 Jan 16;14(1):e03114-22. doi: 10.1128/mbio.03114-22

FIG 3.

FIG 3

A point mutation in the CDV-OP receptor-binding protein (A393T) prevents infectivity in Vero and P114 cells. (A) Assessment of LRP6-HA protein expression via Western blotting analyses, using an anti-HA antibody in P114-LRP6KO cells (P114-L2) reconstituted with cLRP6-HA (P114-L2/L-HA) or not (P114-L2/empty). (B) IF staining of indicated cells, using a monoclonal anti-HA MAb. (C) Quantitative assessment of fluorescence emission from OPneon-infected cells, using an MOI of 1. Images were taken at 3 dpi. The means and standard deviations (SD) of data from three independent experiments are shown. Statistical significance was determined using an unpaired t test, using GraphPad Prism v.9 to analyze the differences between the P114-L2/L-HA and P114-L2/empty values (****, P < 0.0001). (D) The viral titers of OPneon in P114-L2/L-HA and P114-L2/empty cells are displayed in Log10 TCID50/mL. The means and standard deviations (SD) of data from six independent experiments are shown. Statistical significance was determined using an unpaired t test, using GraphPad Prism v.9 to analyze the differences between the values obtained from the indicated cells (**, P < 0.01). (E) Cell-cell fusion induced by cells transiently expressing the indicated H-proteins together with FOP and GFP. Schematic representation of the various H-proteins with the amino acid numberings of the exchanged H-fragments are shown. OP-derived fragments are colored-coded in white, and wt-derived fragments are highlighted in gray. Images were taken 2 days posttransfection (dpt). (F) Cell-cell fusion induced by cells transiently expressing the indicated H-protein variants harboring single (or double) point mutation(s) together with FOP and GFP. Images were taken at 2 dpt. (G) Infection of various cell lines with OPneon carrying a single point mutation (A393T) in the H-protein (OPneonHOPA393T). Images were taken at 2 or 3 dpi for the Vero or other cell lines, respectively. (H) Quantitative assessment of the fluorescence emission from the OPneonHOPA393T-infected cells. The means and standard deviations (SD) of data from three independent experiments are shown. Statistical significance was determined using a one-way ANOVA followed by Dunnett’s multiple-comparison test, using GraphPad Prism v.9 to analyze the differences between the values obtained from the Vero-cSLAM cells and those from other cell types (****, P < 0.0001).