FIG. 5.

Live-cell imaging of GFP-tagged capsid viruses. Dissociated SCG neurons were plated on glass-bottom MatTek dishes and allowed to differentiate for 2 weeks. Images are merged overlays of differential interference contrast and GFP. A minimum of 30 infected neurons per virus were examined for the presence of capsid puncta moving in the anterograde direction. In order to quantify the number of puncta undergoing axonal transport in neurons infected with PRV GS443, eight different axon shafts were imaged where capsids could be visualized moving >50 μm without dropping in and out of the plane of focus. An equal number of axons were examined for PRV 368 for the same time period. (A) Neurons were infected with PRV GS443 and imaged with the Leica SP5 confocal microscope between 13 and 14 h postinfection. Blue, red, and yellow arrowheads track the anterograde movement of individual fluorescent puncta through the field of view during a 1-min interval (see Movie S1 in the supplemental material). (B) A neuron infected for 13.5 h with PRV 368 (green capsid, Us9 null) shows robust green fluorescence in the soma (black arrowhead) but no presence of capsids in the axon (enlarged image, see Movie S2 in the supplemental material).