FIG. 7.

Intracellular Ad particles interact with human cell line-derived microtubules in a nucleotide-sensitive manner. A549 cells were cultured to 80% confluence as described in Materials and Methods. Cells were washed three times with serum-free DMEM and infected with Ad5 (1,000 particles/cell) for 10 min at 37°C in serum-free DMEM. Cells were washed three times with serum containing DMEM and incubated for 40 min at 37°C. Ad-infected A549 cell lysate was then harvested as previously described. To test the interaction of intracellular Ad capsids with A549-derived microtubules, endogenous tubulin of Ad-infected A549 cell lysate was polymerized into microtubules by the addition of taxol (40 μM) and incubated for 80 min at 22°C. Reactions were centrifuged as described previously. The presence of Ad capsid in the supernatant (S) and pellet (P) was evaluated by SDS-PAGE and Western analysis.