Clone A1-L4 expresses functional levels of L4 proteins. (A) 293TETOFF or Clone A1-L4 cells were transfected with pBiL1-3 and either pCMVFLAG (empty vector; ) or L4-22/33KFLAG or L4-100KFLAG expression plasmids. Cell lysates were separated on 10% polyacrylamide gels and expressed proteins detected by western blot analysis using AbJLB1 anti-late protein antisera. All lanes shown derive from the same exposure of a single blot with irrelevant lanes excised for clarity of presentation. The positions to which proteins of known molecular mass migrated are shown on the left (kDa). (B) 293TETOFF or Clone A1-L4 cells were grown in the presence (g-l) or absence of Dox (a-f & m-x) for 3 days with daily media changes prior to mock infection (a-c) or infection with P2 WT (d-f), Δ47 (g-r) or ΔL4 (s-x) viruses. Cells were fixed and stained 20 h.p.i. for Ad5 DNA binding protein (DBP) (green; b, e, h, k, n, q, t & w) or Ad5 late proteins (red; c, f, i, l, o, r, u & x) and nuclear DNA (DAPI, blue) and visualised using a Leica SP2 confocal microscope. Images a, d, g, j, m, p, s & v are overlays in Leica software of DBP, late protein and DAPI images, collected sequentially to avoid cross-talk between fluors. (C) To confirm the ΔL4 virus genotype, Ad5 23863-27086 bp region was amplified by PCR from DNA isolated from either ΔL4 or wt virus particles. ΔL4A and ΔL4B reactions contained 1 and 4 μg viral genomic DNA respectively as template; -ve, PCR negative control. The positions to which DNA size markers migrated are indicated (kbp).