Expression plasmids with the UL76-77 Luc sequence.

(a) Diagram of expression plasmids with UL76-77 Luc sequence inserted downstream of the HCMV MIE promoter with or without a stop codon or a frame-shift in the UL76 ORF. (b) Western blot analysis of UL76 protein in 12.5% SDS-PAGE. HeLa cells were transfected with pCMVflagUL76-77Luc with or without insertion of a stop codon in the UL76 ORF and harvested at 48 h post transfection. To detect fusion protein with a flag epitope, antibody F3165 (Sigma) was used. Lanes: 1, pCMVflagUL76-77Luc; 2, pCMVflagUL76stop1-77Luc (stop1); 3, pCMVflagUL76stop3-77Luc (stop3); 4, pCMVflagUL76stop4-77Luc (stop4); 5, pCMVflag-frame-shift-UL76-77Luc (frame-shift). (c) Western blot analysis of UL76 protein in 5–20% SDS gradient gel electrophoresis. Lanes: 1, stop1; 2, stop3; 3, frame-shift. (d) Quantity of the UL77 gene transcripts with the expression plasmids. RNAs were analyzed with UL77 specific primers and probe by real-time RT-PCR. 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 pCMVflagUL76-77Luc. (e) Western blot analysis of UL77 luciferase fusion protein. To detect fusion protein with a luciferase protein, antibody PM016 (MBL, Nagoya, Japan) was used. Lanes: 1, wt; 2, stop1; 3, stop3; 4, stop4; 5, frame-shift. (f) Effects of UL76 ORF translation on the luciferase activity. Hela cells were transfected with the expression plasmids and pCMV-Renilla luc for standardization of the transfection efficiency and the cells were harvested 48 h posttransfection. The relative luciferase activity (ratio of Firefly to Renilla luciferase activity) was calculated. Each transfection was performed in triplicate. Statistical analyses were done using STATA version 10 (Stata Corporation, http://www.stata.com/).