Abstract
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.
Highlights
The Epstein-Barr virus (EBV) is a double-stranded DNA-based virus that infects more than 90% of the world’s adult population [1]
We demonstrate that the transactivation domain (TAD) of EBNA2 binds to the pleckstrin homology (PH) domain from the Tfb1/p62 subunit of transcription factor IIH (TFIIH) and determine a three-dimensional structure of a complex between EBNA2 and Tfb1/p62
The structure shows that three hydrophobic residues from the TAD of EBNA2 make key interactions at the complex interface and these same residues play an important role in the binding to CBP/ p300
Summary
The Epstein-Barr virus (EBV) is a double-stranded DNA-based virus that infects more than 90% of the world’s adult population [1]. Upon primary infection of B cells, the EBV activates gene expression of proliferative elements in its latent phase [4]. The host cells are slowly transformed to the point of immortalization, and this helps insure the persistence of the virus [5]. It is in this latent phase that most of the EBV-related diseases, including Hodgkin’s lymphomas [6,7], Burkitt’s lymphomas [8,9], nasopharyngeal carcinomas [10,11] and post-transplant lymphoproliferative disorders [12,13] are manifested
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