FIG. 5.

Nuclear import profiles of fluorescently labeled rAAV particles. (a) Labeled rAAV particles were co-infected into HeLa cells as in the experiments outlined in Figs. 2 and 3 (MOI=10⁵ VGC/cell). Cells were fixed at 2 hr post-infection and imaged by confocal fluorescence microscopy. Shown are representative images of cells co-infected with rAAV2/1 (red) and rAAV2/2 (green) (right panels), and with rAAV2/5 (red) and rAAV2/2 (green) (left panels). Nuclei were stained blue with DAPI to allow for co-localization of the virions with the nucleus. (b) Efficiency of nuclear import at 2 hr post-infection, as determined from quantification of data from images in a. Total fluorescence signal intensity of each viral serotype overlapping with blue DAPI signal was calculated for each cell using Metamorph software, and the average integrated intensity overlap was plotted. (c) Uncoating efficiency, indirectly evaluated as the fluorescence intensity of unprocessed nuclear virions that retained label (as depicted in b) divided by the total number of nuclear viral genomes at 2 hr (shown in Fig. 4a) and adjusted to the number of cells per well. (d) Relative transduction units at 24 hr divided by the average nuclear fluorescence intensity of AAV virions at 2 hr (from b) following infection at MOI=100,000 VGC/cell in both assays. For all panels, error bars=mean±SEM, n=4 independent experimental samples. Scale bar=10 μm. Post-test (Tukey's multiple comparison) results: AAV2/1 vs. AAV2/2, *p<0.001; AAV2/2 vs. AAV2/5, ⁺p<0.001; AAV2/1 vs. AAV2/5, ^p<0.001.