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. 2007 Oct 24;82(1):77–85. doi: 10.1128/JVI.01484-07

FIG. 4.

FIG. 4.

Activity of the inhibitory element in transiently transfected cells. (A) Schematic diagram of constructs used in luciferase assays from transfected Vero cells highlighting the differences in the 5′ ends of these constructs. pGL3-MCS is a negative-control construct that contains the MCS of pBluescript as its 5′ leader. pGL3-pol5′UTR contains the complete 208-base pol 5′ leader upstream of the luciferase ORF. pGL3-pol1-57 and pGL3-pol58-208 contain bases 1 to 57 and 58 to 208 of the pol 5′ leader, respectively, upstream of the luciferase ORF. pGL3-pol-uORFmut contains the complete pol 5′ leader upstream of the luciferase ORF, but the AUG codon of the pol uORF has been mutated to AAG. (B) Luciferase assay data from transfected Vero cells. Vero cells were transfected with the constructs in panel A, each together with pRL-SV40, and infected with KOS at an MOI of 10 at 18 h posttransfection. At 6 hpi (24 h posttransfection), lysates were prepared and luciferase activity was determined. Results are presented as firefly luciferase activity normalized to Renilla luciferase activity to control for transfection and lysis efficiency and then normalized to firefly luciferase transcript levels that were previously normalized to Renilla luciferase transcript levels. Transcript levels were determined by real time RT-PCR as described in Materials and Methods (for data, see Supplemental Fig. 1 and 2 and Supplemental Table 1 at http://coen.med.harvard.edu/).