FIG 2.

Prolonged inhibition of GBF-1 enhances HAdV infection in A549 cells but not virus binding to cells. (A) Dispersal of the Golgi apparatus upon treatment of A549 cells with the GBF-1 inhibitor GCA for 30 min. Cells were fixed and immunostained with antibodies directed against the Golgi apparatus-associated protein giantin (green), and nuclei (blue) were stained with DAPI. Samples were imaged by confocal fluorescence microscopy. Images show maximum projections of confocal sections. Note that control DMSO-treated cells showed normal perinuclear Golgi apparatus staining. Five micromolar GCA had no effect, 10 μM caused incomplete disruption of the Golgi apparatus, and 20 μM induced efficient disruption of the Golgi apparatus in all cells. Bar, 20 μm. (B) Minor effect of GCA on metabolic activity of A549 cells. Cells were treated with GCA or control DMSO for 5 h, and the metabolic activity in cells was measured by resazurin fluorescence assay (RFU, relative fluorescence units). (C) A 5-h preincubation with GCA is sufficient to enhance HAdV-C5-dE1_GFP infection of A549 cells. Cells were preincubated with GCA or DMSO for 5 h and inoculated with HAdV-C5-dE1_GFP, and infection was carried out in the presence or absence of GCA. Cells were fixed at 18 h p.i., and the mean nuclear intensity of GFP was used to score infection efficiency. (Left) Graphical representation of the experiment; (right) experimental results with mean values from three parallel experiments ± SD. inc., incubation. (D) A 5-h preincubation with GCA enhances HAdV-C5-dE1_GFP, HAdV-C2_dE3B_GFP, and HAdV-B3-dE1_GFP infection in A549 cells. (E) GCA has no effect on a plasmid-mediated CMV promoter-driven GFP expression. A549 cells were transfected with plasmid pMAX-GFP, at 24 h posttransfection were treated with GCA for 5 h, and were analyzed for GFP expression at 5 or 21 h after drug removal. (F) Decrease in atto565-labeled HAdV-C5 attachment to A549 cells upon GCA treatment. Cells were treated with GCA for 12 or 0.5 h, followed by inoculation with atto565-labeled HAdV-C5_wt in the cold for 30 min, washing, and a 5-min pulse at 37°C. The number of virus particles in individual cells was determined from maximum-intensity projections of confocal stacks using a custom-made Matlab script. Each symbol represents one cell. Error bars represent the means ± SEMs, and P values were calculated using the Mann-Whitney test for statistics. (G) Acute inhibition of GBF-1 upon virus addition does not enhance HAdV-C5-dE1_GFP infection in A549 cells. GCA or DMSO was added to the cells at 30 min prior to virus infection or at the indicated times postinfection, and incubation was continued until 18 h p.i., when the cells were analyzed.