FIG 3.

Inhibition of GBF-1 by GCA enhances HAdV-C early and late gene expression, as well as virus production in A549 cells. (A) A 5-h preincubation with GCA accelerates E1A expression from HAdV-C5_wt in A549 cells, as indicated by Western blotting of infected cell lysates. (Top) E1A forms encoded by the differentially spliced E1A transcripts are indicated. (Bottom) Results for the β-tubulin loading control. (B) GCA does not increase E1A levels in uninfected HEK293T cells, which express E1A from a chromosomal copy. Cell extracts were prepared after 5 h of incubation with DMSO or GCA, and E1A levels were determined by immunoblotting using β-tubulin as a loading control. (C) GCA boosts HAdV-C5-dE1_GFP infection in HEK293T cells. Cells were preincubated with GCA for 5 h, inoculated with virus, and analyzed for GFP expression 18 h p.i. (D) GBF-1 inhibition enhances expression of the late protein protein VI in A549 cells. Cells were preincubated with GCA for 5 h, infected with HAdV-C5_wt, and analyzed for protein VI expression at 18 h p.i. (Left) Representative images. Green, protein VI signal; blue, DAPI signal. Bar, 100 μm. (Right) Quantification of average nuclear protein VI signal. (E) Inhibition of GBF-1 accelerates the production and release of HAdV-C2-dE3B_GFP in A549 cells 40 h p.i. Cells were preincubated with DMSO or GCA for 5 h and inoculated with the virus (MOI, 0.008), and at 40 h p.i., progeny particles were collected from the cells and culture supernatants. Titers of the cell-associated and supernatant fractions were determined on HeLa-ATCC cells by counting the number of GFP-positive cells at 18 h p.i.