Fig 1.

T3.0 RNA enhances the expression of RTA in cells. (A) Schematic representation of the intron-exon structure of ORF50 mRNA and the T3.0 transcript with its nucleotide positions in the KSHV genome. T3.0-1st mut and T3.0-2nd mut represent two separate point mutations. The positions of the point mutations that changed the initiation codon AUG to AAG are indicated. (B) The T3.0 expression vector and pCR3.1-ORF50 were introduced into 293T cells. RNAs were isolated at 48 h posttransfection and were subjected to Northern blot analysis for T3.0 or ORF50. 28S and 18S rRNAs served as controls to ensure the equal loading of each sample. E, empty vector. (C) 293T cells were cotransfected with 5 μg of either a T3.0 expression vector or an empty construct and 0.5 μg of pCR3.1-ORF50. Cells were analyzed by Western blotting using an anti-RTA antibody. (D) 293T cells were cotransfected with 0.1 μg of the ORF50 expression plasmid and increasing amounts of the T3.0 expression vector (0, 0.1, 2.5, or 5.0 μg). Cells were analyzed as described in the legend to panel C. The total amount of plasmids was maintained at 5.1 μg by the addition of the empty vector pCR3.1. (E) BCBL-1 cells were transfected with 3 μg of either an empty vector or the T3.0 expression vector by nucleoporation (Amaxa). After 24 h, cells were treated with 20 ng/ml of TPA for 4 h and were analyzed for the RTA protein level by Western blotting. (F) The promoter-luciferase reporter plasmids under the control of either the K8 or the ori-Lyt promoter were introduced into 293 cells with pCR3.1-ORF50 along with either an empty vector (RTA + E) or the T3.0 expression vector (RTA + T3.0). The reporter plasmid pRL-TK, encoding Renilla luciferase, was included as an internal control. Cells were lysed, and luciferase activities were measured at 48 h. The activity of firefly luciferase relative to that of the internal control, Renilla luciferase, is shown. Data are means and standard deviations for three experimental replicates.