The EBNA-2 N-Terminal Transactivation Domain Folds into a Dimeric Structure Required for Target Gene Activation

Abstract

Epstein-Barr virus (EBV) is a γ-herpesvirus that may cause infectious mononucleosis in young adults. In addition, epidemiological and molecular evidence links EBV to the pathogenesis of lymphoid and epithelial malignancies. EBV has the unique ability to transform resting B cells into permanently proliferating, latently infected lymphoblastoid cell lines. Epstein-Barr virus nuclear antigen 2 (EBNA-2) is a key regulator of viral and cellular gene expression for this transformation process. The N-terminal region of EBNA-2 comprising residues 1-58 appears to mediate multiple molecular functions including self-association and transactivation. However, it remains to be determined if the N-terminus of EBNA-2 directly provides these functions or if these activities merely depend on the dimerization involving the N-terminal domain. To address this issue, we determined the three-dimensional structure of the EBNA-2 N-terminal dimerization (END) domain by heteronuclear NMR-spectroscopy. The END domain monomer comprises a small fold of four β-strands and an α-helix which form a parallel dimer by interaction of two β-strands from each protomer. A structure-guided mutational analysis showed that hydrophobic residues in the dimer interface are required for self-association in vitro. Importantly, these interface mutants also displayed severely impaired self-association and transactivation in vivo. Moreover, mutations of solvent-exposed residues or deletion of the α-helix do not impair dimerization but strongly affect the functional activity, suggesting that the EBNA-2 dimer presents a surface that mediates functionally important intra- and/or intermolecular interactions. Our study shows that the END domain is a novel dimerization fold that is essential for functional activity. Since this specific fold is a unique feature of EBNA-2 it might provide a novel target for anti-viral therapeutics.

Fig 1. Structure of the EBNA-2 N-terminal dimerization (END) domain.

Schematic representation of important features of the EBNA-2 protein: two dimerization motifs (Dim1/Dim2), N-terminal and C-terminal transactivation domains (N-TAD, C-TAD), repetitive primary sequence motifs like the poly-proline (polyP) and the poly arginine-glycine (polyRG) stretch, the nuclear localization signals (NLS),and the adapter region of EBNA-2, which interacts with CBF1/CSL, are illustrated. (B) NMR solution structure of the END (EBNA-2 N-terminal Dimerization) domain. Left: β-strands are shown in blue, helices in orange, and loops in gray. Right: Monomers highlighted in gray and blue. (C) Dimerization of monomers is stabilized by hydrophobic interactions. The inside of each monomer is lined with numerous hydrophobic residues (left; sticks). A subset of these residues is located at the dimer interface (blue/bold labels). Panels (right) show side views of the END domain and highlight the interface residues of each monomer.

Fig 2. Secondary structure topology of the END domain and sequence alignment.

(A) Calculated secondary chemical shifts, Δδ(¹³Cα-¹³Cβ), of the END domain. Positive (orange) and negative (blue) values indicate propensity for α-helical and β-strand conformation, respectively. (B) Secondary structure elements of the END domain based on the NMR structure. Black rectangles indicate residues included in our mutational analysis (for details see Fig 3A). Green rectangles mark backbone amides protected from solvent exchange in hydrogen-deuterium exchange experiments (Fig D in S1 Text). Blue rectangles show the hydrophobic core residues of the END domain forming the interface between the two dimers (Fig 1C). (C) Multiple sequence alignment of potential EBNA-2 END domains in human and related monkey viruses. The construct of this study was based on type 1 EBV strain B95-8 (P12978). The B95-8 sequence was aligned to several type 1 EBV strains (AKATA: AFY97831.1; GD1: Q3KSV2.1; HKNPC1: AFJ06836.1; MUTU: AFY97916.1), the type 2 EBV strain AG876 (YP_001129441.1), and to the LCV strains from baboon (AAA79034.1) and macaque (YP_067943.1). A residue is conserved and colored if the sequence identity over all displayed sequences is higher than 60%. The color code for the amino acid residues is as follows: hydrophobic (blue: M, F, L, I, V, A), small polar (green: T, Q, S, N), aromatic polar (cyan: Y, H), negatively charged (magenta: D, E), glycine (orange), proline (yellow).

Fig 3. Amino acid substitutions of interface or surface residues within the END domain affect dimerization differentially.

(A) Mutated interface (blue) and surface (red) residues highlighted as spheres on the structure of the END domain. (B) Schematic illustration of EBNA-2 and EBNA-2 mutants used in subsequent experiments. (The orange box represents the position of the α-helix). (C-E) HA-tagged EBNA-2 (E2 wt) or HA-tagged END domain mutants were co-expressed with FLAG-tagged EBNA-2 fragments truncated at aa199 (F199) in EBV negative DG75 B cells. Protein complexes were immunoprecipitated using HA-specific antibodies. The precipitates were detected in western blots either by EBNA-2 specific antibodies (E2) recognizing the EBNA-2 C-terminus (upper panel) or FLAG-specific antibodies recognizing F199 (middle panel) or CBF1/CSL specific antibodies recognizing endogenous protein (lower panel). Total lysates (L) correspond to 15% of the sample used for immunoprecipitation (IP). The following EBNA-2 mutants were used: (C) alanine or aspartic acid substitution mutants of residues Leu16 and Ile50 (L16A, L16D and I50A, I50D) residing in the hydrophobic interface of the END domain; (D) N-terminal deletion mutants Δ3–30 and Δ3–52; (E) alanine substitution of residues His15 or Phe51 (H15A and F51A) or deletion of the α-helix at position 35–39 (Δα1) on the surface of the END domain.

Fig 4. LMP1 activation by EBNA-2 requires dimerization, the surface residue His15, and the protruding α1-helix.

1x10⁷ EBV positive but EBNA-2 negative Eli-BL cells were transfected with 5 μg expression constructs for EBNA-2 wt, N-terminal deletion mutants (A), END interface (B) or END surface (C) mutants or the corresponding vector controls (pSG5). 30 μg of whole cell lysates of transfected cells were analyzed on western blots using EBNA-2, LMP1, EBNA-1 and GAPDH specific antibodies. Staining for EBNA-1 and GAPDH was used as loading controls. EBV negative (DG75: 30 μg of total cell lysate) and EBV infected LMP1 positive B cells (721: 5 μg total cell lysate) were used as controls. (D) The chemilumiscence signals were quantified by digital imaging using the Fusion Fx7 and the data are shown as % signal intensity relative to EBNA2 wt (100%). The bars represent the mean values of 4 independent experiments. Standard deviations are shown as error bars.

Fig 5. Transcriptional activation of endogenous viral and cellular target genes by END domain mutants.

1x10⁷ Eli-BL cells were transfected with expression constructs for EBNA-2 wt, N-terminal deletion mutants, END domain mutants or the corresponding control vectors (pSG5). Relative transcript levels of the viral LMP1 and LMP2A gene or the cellular CD23 or CCL3 genes were determined by real-time RT-PCR. Transcript levels were normalized to actin transcript levels. EBNA-2 activation was set to 100% and the data are shown as mean values of four independent experiments. Error bars indicate the standard deviations.

Fig 6. GAL4 DNA-binding fusion proteins of the END domain surface mutants H15A and ΔΔ1 have lost the capacity to activate GAL4-responsive and CBF1-responsive promoters.

5x10⁶ EBV negative DG75 cells were co-transfected with 5 μg of expression constructs for the GAL4 DNA-binding domain fused to EBNA-2 (GAL4-E2 wt) or EBNA-2 END domain mutants with either 5μg GAL4-responsive or CBF1-responsive promoter luciferase constructs plus 0.5 μg of Renilla luciferase construct. EBNA-2 activation of the reporter constructs was set to 100% and the data are shown as the mean of three independent experiments done in triplicates. Error bars indicate the standard deviation.