Infection with the Epstein-Barr virus (EBV) can lead to a number of human 
diseases including Hodgkin's and Burkitt's lymphomas. The development of these 
EBV-linked diseases is associated with the presence of nine viral latent 
proteins, including the nuclear antigen 2 (EBNA2). The EBNA2 protein plays a 
crucial role in EBV infection through its ability to activate transcription of 
both host and viral genes. As part of this function, EBNA2 associates with 
several host transcriptional regulatory proteins, including the Tfb1/p62 
(yeast/human) subunit of the general transcription factor IIH (TFIIH) and the 
histone acetyltransferase CBP(CREB-binding protein)/p300, through interactions 
with its C-terminal transactivation domain (TAD). In this manuscript, we examine 
the interaction of the acidic TAD of EBNA2 (residues 431-487) with the Tfb1/p62 
subunit of TFIIH and CBP/p300 using nuclear magnetic resonance (NMR) 
spectroscopy, isothermal titration calorimeter (ITC) and transactivation studies 
in yeast. NMR studies show that the TAD of EBNA2 binds to the pleckstrin 
homology (PH) domain of Tfb1 (Tfb1PH) and that residues 448-471 (EBNA2₄₄₈₋₄₇₁) 
are necessary and sufficient for this interaction. NMR structural 
characterization of a Tfb1PH-EBNA2₄₄₈₋₄₇₁ complex demonstrates that the 
intrinsically disordered TAD of EBNA2 forms a 9-residue α-helix in complex with 
Tfb1PH. Within this helix, three hydrophobic amino acids (Trp458, Ile461 and 
Phe462) make a series of important interactions with Tfb1PH and their importance 
is validated in ITC and transactivation studies using mutants of EBNA2. In 
addition, NMR studies indicate that the same region of EBNA2 is also required 
for binding to the KIX domain of CBP/p300. This study provides an atomic level 
description of interactions involving the TAD of EBNA2 with target host 
proteins. In addition, comparison of the Tfb1PH-EBNA2₄₄₈₋₄₇₁ complex with 
structures of the TAD of p53 and VP16 bound to Tfb1PH highlights the versatility 
of intrinsically disordered acidic TADs in recognizing common target host 
proteins.