FIG. 4.

Repression of gene expression by LANA in a reporter plasmid harboring the TR. (A) Schematic representation of the reporter plasmids used in the transient-transfection assay and a representative structure of the KSHV genome and its genes (top). Part of the TR-containing fragment from cosmid Z6 (EcoRI-BglII) was linked to the RTA-promoter luciferase cassette. Z6, cloning region in cosmid Z6 (NIH AIDS Research and Reference Reagent Program); Amp R, ampicillin resistance gene; Luc, luciferase gene. (B) Transient assay for transcriptional regulation by LANA of a TR-bearing plasmid. The transfection and luciferase assay were performed as described in Materials and Methods. 293 cells were transfected with the effector plasmid (total of 2 μg/well in a 12-well plate) and the reporter plasmid (0.2 μg/well). (C) SUV39H1 enhances the repression by LANA. 293 cells were cotransfected transiently with pTriEX-LANA with pFLAG-SUV39H1 (0.3 and 0.6 μg/well) or pFLAG-SUV39H1ΔN (SUV39ΔN) (0.3 and 0.6 μg/well). The luciferase activities were normalized to the protein concentration and are shown in the upper graph, in which the open bars indicate empty vector (pTriEX1.1) (0.5 μg/well) and the closed bars indicate pTriEX-LANA (0.5 μg/well). Transfection efficiency could not be normalized to a reference plasmid such as a β-galactosidase expression vector due to LANA's modulation of some promoters. The fold repression by LANA was calculated as follows and is shown in the lower graph: [(pTriEXLANA + X)/(pTriEX + X)]⁻¹, where the designations in parentheses show the luciferase units (relative light units [RLU]) for each combination and X is pFLAGCMV1, pFLAG-SUV39H1, or pFLAG-SUV39H1ΔN (SUV39ΔN). The assays shown in panels B and C were performed in triplicate for each set of transfections, and the mean value and standard deviation were calculated. (D) Western blotting (WB) of 293 cells transfected with SUV39H1 and its mutant expression vector. The expression of each construct (0.6 μg/well) transfected with pFLAGCMV1 (lane 1), pFLAG-SUV39H1 (lane 2), and pFLAG-SUV39ΔN (lane 3) in 293 cells was analyzed by Western blotting with an anti-FLAG antibody (Sigma Aldrich).