FIG. 2.
Expression of Ad5 fiber in cell lines. (A) Constructs used for fiber expression. In all plasmids, the fiber cDNA is driven by the cytomegalovirus (CMV) immediate-early promoter. pDV61 contains a partial TPL cDNA with no introns and lacking the first 32 nt of the first leader segment. In pDV67, this fragment was replaced by a TPL cassette (see Materials and Methods) which includes all three complete leader segments as well as the native first intron. (B) Nuclear expression of fiber protein. Cells (approximately 50,000/well) were plated on eight-well chamber slides, fixed, and stained with a polyclonal anti-Ad2 fiber serum (which also detects the Ad5 fiber). Line 601 and 633 were generated by transfection of pDV61 and pDV67, respectively. Note the increased and more consistent fiber expression in nuclei of 633 cells. As a control, non-fiber-expressing E1-2a cells were stained in parallel. (C) Increased synthesis of fiber protein from the complete TPL. Proteins extracted from the indicated cell lines (3 × 105 cells/lane) were electrophoresed and immunoblotted as described previously (50), and fiber was detected by using the anti-Ad2 fiber polyclonal antibody. Cont., control. (D) The complete TPL increases fiber content of Ad5.βgal.ΔF particles. Ad5.βgal.ΔF was CsCl purified from either 601 or 633 cells. The purified particles (10 μg) were electrophoresed on a sodium dodecyl sulfate–8 to 16% polyacrylamide gel (Novex) and immunoblotted. Fiber protein was detected with the anti-Ad2 fiber antibody. As a control, 10 μg of purified Ad5.βgal.wt (WT) was run alongside the mutants. To verify equal loading, the blot was reprobed with an antibody against the Ad2 penton base.