Fig. 3.

Live-cell imaging of PK15 islands during axon-to-cell infection. (A) Schematic representation of the “island” of multiple PK15 cells that seeded in close association with isolated neurite extensions in the far right compartment of compartmentalized neuronal cultures. Axon tracks are highlighted in blue. Approximate cell boundaries marked in black and nuclei annotated as dark gray circles. (B and C) Sequential images from the time-lapse recording are presented. (Scale bar, 100 µm.) (B) At 11.5 h postinfection, mRFP puncta become associated with the nuclei of the PK15 cells. (C) At 14 h postinfection, many of these cells (large white arrowheads) begin to accumulate detectable amounts of farnesylated YFP, indicative of successful infection by the virus. (D and E) Two representative infected cells after virion entry and before expression of viral proteins. A merged mRFP fluorescence and phase image is presented (Left), mRFP fluorescence (Center), and mRFP fluorescence with an overlay of the approximate locations of axons and the infected cell (Right). Approximate cell periphery is outlined in a solid white line; the nucleus is outlined with a dashed white line, axons are blue. (Scale bars, 10 µm.) (D) A single cell with a single mRFP-puncta associated with the nucleus of the cell. (E) A cell infected with a large number of virions, greater than five. (F) The number of virions per infected cell was counted across three experiments for a total of 157 infected cells. The number of cells containing 0, 1, 2, 3, 4, or >5 capsids is expressed as a percent of the total infected cells per experiment and averaged across all three experiments. Error bars represent SD between experiments.