Fig 7.

Association of AAV2-containing endosomes with MTs and delayed endosomal acidification upon MT disruption. (A) AAV2 particles migrate on MTs in membranous structures. AAV2 particles before (panel a) and after (panel b) labeling of nanogold were demonstrated by TEM. Bar, 20 nm. A micrograph showing the direct association of AAV-containing endosome with MTs at 1 h after pulse infection of nanogold-labeled AAV2 (panel c) is simplified in a cartoon diagram (panel d). In the cartoon, MTs are indicated by arrows, the endosome membrane is indicated by closed line, and virus is indicated by arrowheads. Bar, 200 nm. (B) Acidification of virus-containing endosomes is inhibited by lysosomotropic chemicals at various time points after pulse infection of AAV2 (panel a). At 1 h after pretreatment with anti-MT drugs (nocodazole and vinblastine), chloroquine was added at 0 h or 3 h after pulse infection of AAV2. To calculate the inhibition of viral transduction, the expression level of GFP transgene was measured with a flow cytometer and normalized to corresponding treatments without chloroquine (panel b).