FIG. 3.
Mutation of the conserved PKA recognition site in UR1 does not affect transactivation by EBNA1. (A) The wild-type version of EBNA1 used in these studies contains five copies of the Gly-Gly-Ala repeat and is depicted schematically, indicating domains of EBNA1. The sequence of the conserved portion of UR1 is shown with serine 78 indicated in bold. The sequence of the S78D, S78A, and S78T substitution mutations constructed for this study are also shown with the substituted amino acids shown in bold. (B) Immunoblot of 5 × 105 C33a cells transfected with expression plasmids for EBNA1, DBD, S78D, S78A, and S78T. Proteins were detected using the K67.3 or 2638 antibodies against the DBD of EBNA1 and were visualized by chemiluminescence. The migration of prestained markers of known molecular sizes are indicated by the arrowheads. (C) C33a cells were transfected with an expression plasmid for EBNA1, S78D, S78A, or S78T along with an FR-TKp-luciferase reporter. Assays for luciferase activity were performed at 48 h posttransfection. At this time, EBNA1 transactivates FR-TKp-luciferase approximately 80-fold over the DBD alone, and this value was set to 100%. Data from three independent transfections indicate that replacing the conserved serine in UR1 with alanine, aspartic acid, or threonine does not significantly affect transactivation (P > 0.05 for pairwise comparisons with EBNA1).