Mutational analysis of EBNA-1 identifies a transcriptional activation domain within LR1. (A) Derivatives of EBNA-1 depicted schematically. EBNA-1 has two highly charged regions within its amino terminus, LR1 (shaded boxes) and LR2. The nuclear localization signal of EBNA-1 is found adjacent to a putative flexible linker domain or JD and is represented in all derivatives by the hatched boxes. The Gly-Gly-Ala repeats span ∼225 residues in the B95-8 strain of EBV. The derivative used in these studies contains only three Gly-Gly-Ala repeats. wt, wild type. (B) Western blot demonstrating expression levels of the various derivatives relative to that of wild-type EBNA-1. The membrane was simultaneously probed with antibodies to EBNA-1 and β-actin, which served as a loading control. The relative levels of expression of the transfected derivatives of EBNA-1 were corrected for loading error, and OD units obtained from ImageQuant analysis are represented as OD units per transfected cell at the bottom of each lane. (C) Mean luciferase results corrected for transfection efficiency obtained from at least three independent transfections performed in duplicate are depicted graphically. The increases in transcription over empty vector (mean increase [*n*-fold] over BJAB cells lacking integrated FR-TK-luciferase, 10; average number of RLU, 5,425 ± 231) mediated by wt-EBNA-1 (mean increase [*n*-fold] over empty vector, 26 [*P* = 5.3 × 10^{−6}]; average number of RLU, 85,000 ± 3,780), Δ359-369 (mean increase [*n*-fold] over empty vector, 21 [*P* = 0.009]; average number of RLU, 95,900 ± 17,800), shuffled JD (mean increase [*n*-fold] over empty vector, 56 [*P =* 0.05]; average number of RLU, 116,000 ± 10,700), and 2×LR1 (mean increase [*n*-fold] over empty vector, 46.7 [*P =* 0.007]; average number of RLU, 21,2000 ± 31,000) are significant. The apparent increases in transcription over wild-type EBNA-1 mediated by the shuffled JD and 2×LR1 derivatives are not statistically significant (*P* > 0.05). The derivatives Δ65-89 (mean increase [*n*-fold] over empty vector, 1.5 [*P* = 0.85]; average number of RLU, 6,510 ± 491), 2×LR2 (mean increase [*n*-fold] over empty vector, 2.5 [*P =* 0.21]; average number of RLU, 9,169 ± 1,070), and dnE1 (mean increase [*n*-fold] over empty vector, 2.4 [*P* = 0.33]; average number of RLU, 8,957 ± 1,800) were found to have no effect on transcription from the integrated template.

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