Viral spread in polarized T84 cells.

A and B) Viral spread in T84 cells cultured in transwell chambers. Cells were cultured as described for Fig. 4. Ad vectors were added to the inner chamber at an MOI of 10 vp/cell. A) Confocal microscopy images (XY planes) of the cell surface and of a layer that is 20 µm beneath the cell surface were taken 4 days after infection. Transduced cells express GFP (green). The scale bar of all images is 20 µm. B) Titration of progeny viruses in the inner chamber and in the outer chamber. T84 cells, which had been cultured in 0.4 µm transwell inserts for 16 days, were infected from the apical side with Ad3 vectors at the indicated MOIs for 2 hours. Supernatants were collected from the inner chambers at days 4, 7, 9 and 10 post-infection, and from the outer chambers at days 9 and 10, and titered for GFP-expressing units on 293 cells. The medium were changed at days 4, 7, 9 and 10 post-infection. Shown are data from samples collected at day 9 post-infection. N = 3. The differences between wt-Ad3GFP and mu-Ad3GFP titers in the inner chamber are not significant (n.s.). *P<0.01. Virus titers in inner chamber were about 5 higher on day 7 than on day 4. Viral titers in the inner chamber remained stable from day 7 on (data not shown). Note that, the filter pore size of the chambers is 400 nm and should allow for the passage of 100 nm Ad virions). C) Viral spread in the presence of recombinant PtDd added to the inner chamber. T84 cells were cultured as desrcibed in B) and infected (in triplicate) with mu-Ad3GFP and wt-Ad3GFP at an MOI of 1vp/cell. Recombinant PtDd (5 µg/ml) was added to to the inner chamber on days 3, 5, and 7 post-infection. Medium in both chambers was changed on days 4, 6, 8, and 10 p.i. Day 8 and 10 medium samples from the outer chamber were then titered on 293 cells for progeny virus that has passed through the layer of T84 cells based on GFP epressing/infectious units. N = 6, *- p<0.01, n.s.-not significant.