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. 2010 Dec 23;6(12):e1001236.
doi: 10.1371/journal.ppat.1001236.

Structural basis for apoptosis inhibition by Epstein-Barr virus BHRF1

Marc Kvansakul  1 , Andrew H Wei, Jamie I Fletcher, Simon N Willis, Lin Chen, Andrew W Roberts, David C S Huang, Peter M Colman
Affiliations

Structural basis for apoptosis inhibition by Epstein-Barr virus BHRF1

Marc Kvansakul et al. PLoS Pathog. .

Abstract

Epstein-Barr virus (EBV) is associated with human malignancies, especially those affecting the B cell compartment such as Burkitt lymphoma. The virally encoded homolog of the mammalian pro-survival protein Bcl-2, BHRF1 contributes to viral infectivity and lymphomagenesis. In addition to the pro-apoptotic BH3-only protein Bim, its key target in lymphoid cells, BHRF1 also binds a selective sub-set of pro-apoptotic proteins (Bid, Puma, Bak) expressed by host cells. A consequence of BHRF1 expression is marked resistance to a range of cytotoxic agents and in particular, we show that its expression renders a mouse model of Burkitt lymphoma untreatable. As current small organic antagonists of Bcl-2 do not target BHRF1, the structures of it in complex with Bim or Bak shown here will be useful to guide efforts to target BHRF1 in EBV-associated malignancies, which are usually associated with poor clinical outcomes.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. BHRF1 protects cells from diverse apoptotic stimuli.
(A) Viability of FDC-P1 cells stably overexpressing BHRF1, Bcl-2, Bcl-xL, Bcl-w or vector, treated with 0-10 µM etoposide (left) or 0–30 Gy γ-irradiation (right) and cultured for 48 h. (B) Eµ-myc pre-B-cell tumor cells were stably transfected with BHRF1, Bcl-2 or vector were exposed to 0–10 µM cytosine arabinoside (Ara-C) for 24 h. (C) MEFs overexpressing BHRF1, Bcl-2, Bcl-xL or vector were treated with 0–100 µM etoposide for 24 h. (D) Bcl-2, Bcl-xL, Bcl-w and BHRF1 are expressed at comparable levels in FDC-P1 cells. FLAG-tagged wild-type Bcl-2, Bcl-xL, Bcl-w and BHRF1 were stably expressed in FDC-P1 cells. Protein expression was evaluated using flow cytometry after staining fixed cells with an anti-FLAG antibody, followed by an anti-mouse FITC secondary antibody. Controls (dotted lines) indicate staining of cells expressing empty vector. (E) BHRF1 and Bcl-2 are expressed at comparable levels in Eµ-myc pre-B-cell tumor cells. Pre-B-cell tumor cells derived from Eµ-myc transgenic mice were stably transfected with FLAG-tagged BHRF1, Bcl-2 or an empty control vector. Protein expression was evaluated using flow cytometry after staining fixed cells with an anti-FLAG antibody, followed by an anti-mouse FITC secondary antibody. Controls (dotted lines) indicate staining of cells expressing empty vector. Cell viability was determined by PI exclusion; data shown are means ±1 SEM of 3 independent experiments except for the representative γ-irradiation experiment shown in (A).
Figure 2
Figure 2. BHRF1 inhibits loss of mitochondrial transmembrane potential and Bax/Bak activation.
(A) FDC-P1 cells expressing BHRF1, Bcl-2 or a control vector were treated with 25 nM staurosporine for 24 h. Outer mitochondrial transmembrane potential (Δψm) was assessed by DiOC6(3) uptake. (B) HeLa cells treated with 200 J/m2 UV-irradiation were analyzed at 4 h for Bax translocation and cytochrome c release by immunoblotting after fractionation into soluble (s) and pellet (p) fractions. (C) Bax and Bak activation in UV-irradiated HeLa cells were assessed at 4 h using the conformation-specific mouse anti-Bak clone Ab-1 (Calbiochem) or mouse anti-Bax clone 3 antibodies .
Figure 3
Figure 3. BHRF1 binds a subset of pro-apoptotic Bcl-2 family proteins to counter apoptosis.
(A) The affinity of recombinant BHRF1ΔC31 for BH3 domain peptides (26-mers, except for a Bax 28-mer and a Bid 34-mer) was assessed using isothermal titration calorimetry (ITC). Kd values (in nM) are the means of 3 experiments ± SD. NB: no binding detected. (B) Lysates prepared from 35S-labeled HEK-293T cells co-expressing FLAG-tagged BHRF1 and EE-tagged BH3-only proteins were immunoprecipitated with mouse monoclonal antibodies recognizing the FLAG (FL), EE, or an irrelevant control (C) tag. **Endogenous 14-3-3 associating with Bad . (C) Yeast co-transformed with constructs encoding Bax or Bak and the indicated pro-survival proteins, each under the control of an inducible (GAL) promoter, were spotted onto inducing galactose (“ON”) or repressing glucose (“OFF”) plates as 5-fold serial dilutions. Images are representative of 2 independent experiments.
Figure 4
Figure 4. BH3 peptides bind BHRF1 in a canonical binding groove.
(A–B) BHRF1 (blue) in complex with the Bim BH3 domain (yellow). BHRF1 helices are labeled α1, α1′, α2-8. The view in (A) is into the hydrophobic binding groove formed by helices α3-5, while (B) is rotated by 180° through the vertical axis to illustrate helix α1′. (C) BHRF1 (blue) in complex with the Bak BH3 domain (orange). The view is as in (A). (D) Bcl-xL (cyan) in complex with the Bim BH3 domain . The view is as in (A). (E–F) Unliganded BHRF1 (; in green). The view in (E) is similar to (A), while (F) is comparable to (B). The binding groove is occluded by the near parallel arrangement of helices α3 and α4. (G) Detailed view of the BHRF1:Bim complex interface. The BHRF1 surface, backbone and binding groove are shown in grey, cyan and magenta respectively, while Bim BH3 is shown in yellow. The four key hydrophobic residues of Bim (I58, L62, I65 and F69; numbering based on human BimL) protruding into the binding groove, as well as the conserved salt-bridge formed by Bim D67 and BHRF1 R100 are labeled. (H) Detailed view of the BHRF1:Bak complex interface. Colour scheme is as for (G) with Bak BH3 shown in orange. The four key hydrophobic residues of Bak (V75, L78, I81 and I85) protruding into the BHRF1 binding groove, as well as the conserved salt-bridge formed by Bak D83 and BHRF1 R100 are labeled.
Figure 5
Figure 5. BHRF1 inhibits ABT-737 induced apoptosis and confers chemoresistance in a mouse model of Burkitt lymphoma.
(A) Colony formation of mcl-1−/− MEFs overexpressing BHRF1, Bcl-xL or a control vector, 6 d after treatment with 0-10 µM ABT-737. (B, C) Kaplan-Meier survival plots of mice inoculated with Eµ-myc tumor cells overexpressing BHRF1, Bcl-2 (or an empty vector) and treated on d 4, 5 and 6 (arrows) afterwards with (B) saline or (C) 100 mg/kg cytosine arabinoside (Ara-C; given intra-peritoneally). N = 5 mice in each group. Expression of BHRF1 or Bcl-2 conferred chemoresistance (p = 0.014 and p = 0.049 respectively by log-rank test when compared to saline treated controls). However, there is no significant difference whether or not BHRF1 or Bcl-2 is expressed as a transgene (p = 0.85 by log-rank test).
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