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. 2009 Jun 12;5(6):e1000469. doi: 10.1371/journal.ppat.1000469

Figure 1. Two conserved cysteines in EBNA1's UR1 domain are essential for transactivation.

Figure 1

(A) Schematic representation of the EBNA1 derivatives used in this study. The positions of the EBNA1's AT-hook domains (ATH1 & ATH2), UR1, DBD are indicated. The NLS spans from a.a 379–386. The EBNA1 derivatives used in this study contain a shortened glycine-alanine repeat that is 15 a.a. in length. EBNA1Δ(71-88) contains a deletion of a.a. 71–88 within the UR1 region. The HMGA1a-DBD protein contains the full length HMGA1a protein fused to the NLS, and the DBD. UR1-DBD contains a.a. 65–89 fused to a.a. 379–641 of EBNA1. The EBNA1(CC→SS) protein is described in detail below. (B) Transactivation of the FR-TKp-Luciferase reporter plasmid by the derivatives of EBNA1 shown in Figure 1A. C33a cells were individually co-transfected with the FR-TKp-Luciferase reporter plasmid, and expression plasmids for the indicated EBNA1 derivatives as described in the Methods section. Luciferase assays were performed at 48 hours post-transfection. Luciferase activity is expressed as fold over the level observed with when DBD is used as the effector protein. The data represents the average of at least three experiments for each EBNA1 derivative. (C) The UR1 region of EBNA1 is conserved in the EBNA1 orthologs of EBV-like gammaherpesviruses. Amino acids 75–85 of EBNA1 from EBV strain B95-8 (accession no. CA24816) (red) are aligned with the corresponding regions from EBNA1 proteins of other gammaherpesviruses (accession nos. YP_067973, BAB03281, AAA66373) (blue), and a portion of a C4 zinc-finger contained in the catalytic subunit of DNA polymerase δ from Xenopus laevis (accession no. NP_001087694), Danio rerio (accession no. CAM46996), Nematostella vectensis (accession no. XP_001641357), Drosophila mojavensis (accession no. XP_002008314), and Homo sapiens (accession no. P28340), identified by BLAST searches. Three or more alike amino-acids in the aligned sequences are shaded the same color. The Genbank accession number for each protein is indicated adjacent to it. (D) Schematic depiction of EBNA1 and the EBNA1(CC→SS) mutant, in which conserved cysteines at position 79 and 82 are mutated to serine. The sequences of wild-type (WT), and mutant (M) peptides used in this study are also shown.