Figure 4
Generation of fluorescent protein expressing rToyo-P-mCherry for live-cell imaging. (A) Schematic representation of the plasmid construction of rToyo-P-mCherry, a recombinant Toyohashi strain carrying the fluorescent reporter mCherry gene at the 3′-terminus of the viral P gene. (B) Sanger sequencing chromatograms of C terminus region of rToyo and rToyo-P-mCherry. (C) Images of fluorescent foci in NA cells at 4 dpi with RABVs. Cells were strained with anti-RABV N antibody (green) and Hoechst 33342 (blue). The scale bars on the images indicate 100 μm. (D) Intracellular trafficking of Toyohashi P protein. SK-N-SH cells were infected with rToyo-P-mCherry and then monitored by live-cell time-lapse imaging from 14 to 26 hpi. Fluorescence signals from P-mCherry were visualized in the red color. Yellow arrowheads indicate fusion between inclusion bodies. Scale bars indicate 20 μm and the time post-infection is displayed in the lower left corner of each panel. All the images have been extracted from Supplementary Movie and are shown at 10-min intervals.
