Analysis of UL76 to UL78 gene transcripts after infection with the wt and recombinant virus.

(a) Diagram of the recombinant BAC DNAs of wt and RdlUL76+F. UL76 was replaced with the kanamycin resistant gene (KanR), and then KanR was excised by FLP-mediated recombination, leaving only 34 bp of the FRT sequence (F). (b) Southern blot analysis of parental and recombinant BAC-DNAs of wt and RdlUL76+F. BAC DNAs were digested with restriction endonuclease BstB I, fractionated by electrophoresis in 0.6% agarose, and subjected to hybridization with a 32P-labeled probe. Standard molecular size markers are indicated in base pairs. Lanes: 1 and 3, RdlUL76+F; 2 and 4, wt; 1 and 2, UL75 probe; 3 and 4, UL76 probe. (c, d, and e) Analysis of UL76 to 78 gene transcripts after infection with the wt or RdlUL76+F. HFFs were infected with an MOI of 1, and cytoplasmic RNA was harvested 6, 24, 48, and 72 h. p.i. as described in the Materials and Methods. 28S and 18S rRNA served as controls for equal amounts of RNA loading. (c), UL76 probe; (d), UL77 probe; (e), UL78 probe. Lanes: 1 and 5, 6 h.p.i.; 2 and 6, 24 h.p.i.; 3 and 7, 48 h.p.i.; 4 and 8, 72 h.p.i.; 1 to 4, wt; 5 to 8, RdlUL76+F.