Fig. 6. The presence of UL14 protein increases luciferase activity in a dose-dependent manner when cells yield low luciferase activity. HEp-2 cells were cotransfected with pGL3-p (0.25 µg) and pcDNA3-UL14 (1, 0 µg; 2, 0.50 µg; 3, 1.0 µg) and an empty vector pcDNA3, to a total of 1.25 µg DNA per assay. The results are shown as the percentage of relative luciferase activity. The increase in the expression of UL14 protein correlates with the rise in luciferase activity (up to 14-fold). Each assay was done in triplicate, and each point represents the average±s.d. of three experiments. The same samples were subjected to western blotting and detected for luciferase showing that the level of protein is almost constant contrary to its rise in activity. (B) Luciferase activity is increased in cell lines expressing UL14 protein without a change in the level of luciferase protein. HEp-2, 14/HEp-2, Vero and 14/Vero cells were each transfected with 0.5 µg of luciferase-expressing plasmid pGL3-p and luciferase activity was measured 24 hours later. In each cell line, constitutive expression of UL14 protein increased luciferase activity. Luciferase activity was measured as in A. Western blots show that the amount of expressed luciferase is almost unchanged. (C) UL14 protein existence substitutes for the loss in luciferase activity induced by transfection with antisense oligomer to cellular Hsp70 mRNA. HEp-2 and 14/HEp-2 cells were cotransfected with: (1) 0.5 µM of either sense or antisense (a.s.) oligomers and 0.1 µg pGL3-p; (2) 0.5 µM of antisense oligomer, 0.1 µg pGL3-p and 0.25 µg pCDNA3-UL14, - UL14R(60,64)A, or -UL14D(51-90). 14HEp-2 cells were cotransfected with 0.5 µM of either sense or antisense oligomers and 0.1 µg pGL3-p. Luciferase activity was assayed as above and relative activity was compared. The results show that the drop in luciferase activity on transfection of antisense oligomers is less in 14/HEp-2 cells (35%) than in HEp-2 cells (55%). In addition, luciferase coexpressed with UL14 mutant proteins showed lower activity, suggesting that UL14 wt protein compensates for the loss of cellular chaperone activity. Each assay was done in triplicate, and each point represents the average±s.d. of three experiments.