Figure 1

Characterization of epitope-tagged EBNA1 and Tat. (A) Diagrams of epitope-tagged EBNA1 and Tat. EBNA1 is 641 a.a. long and binds 20 sites in the EBV FR through its DNA binding domain (DBD). EBNA1's UR1 domain, essential for transactivation, contains a redox-regulated cys-x-x-cys motif. Tat is 87 a.a. long and binds HIV-1 TAR RNA through its basic region (BR) Tat's redox-regulated cysteine-rich (CRR) is required for transactivation. (B) Epitope-tagged EBNA1 and Tat, expressed in C33a cells, were visualized as described in the materials and methods. (C) Indirect immunofluorescence indicates EBNA1 is primarily nuclear, while Tat is nuclear and cytoplasmic. Proteins were visualized as described in the materials and methods. Bars indicate a scale of 10 μM. (D) Diagram of the transcription reporter plasmids. The minimal TK promoter (TKp) in both reporters has -1 to -80 of the HSV-1 TK promoter. The Tat reporter, TKp-TAR-luciferase, contains the HIV-1 TAR between the promoter and the luciferase gene. The EBNA1 reporter, FR-TKp-luciferase, contains the EBV FR 5' to the TKp. The HSV-1 TK polyadenylation signal (TKpA) was used for polyadenylation. (E) 24 hours post-transfection, epitope-tagged EBNA1 transactivates FR-TKp-luciferase 55-fold over the control (pcDNA3) (left-hand scale). Epitope-tagged Tat transactivates TKp-TAR-luciferase 10-fold over pcDNA3 (right-hand scale). (F) Exposure to 1 μM TPEN, a zinc chelator, reduced transactivation of FR-TKp-luciferase by EBNA1 to 50% of control, as observed for native EBNA1. TPEN did not alter transactivation by Tat. The asterisk indicates statistical significance by the Wilcoxon rank-sum test (p < 0.05) over control conditions.