Expression plasmids with the UL76-77 sequence.

(a) Diagram of expression plasmids with the UL76-77 sequence inserted downstream of the HCMV MIE promoter with or without frame-shifts. (b) Quantity of the UL77 gene transcripts with the expression plasmids. RNAs were analyzed with UL77 specific primers and probe by real-time PCR as described in the Materials and Methods. The assay was performed in triplicate, and the standard error of the mean was determined. RNAs were normalized to G6PD RNA, and each value was relative to the level of pCMVflagUL76-77HA. (c) Western blot analysis of UL76 and UL77 fusion proteins. HeLa cells were transfected with pCMVflagUL76-77HA with or without a frame shift mutation and harvested at 48 h post transfection. To detect the fusion protein with a flag or HA epitope, antibody F3165 (Sigma) or 3F10 (Roche) was used, respectively. Lanes: 1, pCMVflag-frame-shift-UL76-77HA; 2, pCMVflagUL76-77HA; 3, pCMVflagUL76-77-frame-shift-HA.