Interaction of MEQ protein and C-terminal-binding protein is critical for induction of lymphomas by Marek's disease virus

Abstract

Marek's disease virus (MDV) is an oncogenic herpesvirus that induces fatal T cell lymphomas in chickens. With more than 20 billion doses of vaccine used annually, vaccination constitutes the cornerstone of Marek's disease control. Despite the success of vaccination, evolution of virulence among MDV strains continues to threaten the effectiveness of the current Marek's disease vaccines. MDV-encoded protein MEQ (MDV EcoRI Q) probably acts as a transcription factor and is considered to be the major MDV oncoprotein. MEQ sequence shows a Pro-Leu-Asp-Leu-Ser (PLDLS) motif known to bind C-terminal-binding protein (CtBP), a highly conserved cellular transcriptional corepressor with roles in the regulation of development, proliferation, and apoptosis. Here we show that MEQ can physically and functionally interact with CtBP through this motif and that this interaction is critical for oncogenesis because mutations in the CtBP-interaction domain completely abolished oncogenicity. This direct role for MEQ-CtBP interaction in MDV oncogenicity highlights the convergent evolution of molecular mechanisms of neoplastic transformation by herpesviruses because Epstein-Barr virus oncoproteins EBNA 3A and 3C also interact with CtBP. We also demonstrate that the nononcogenic MDV generated by mutagenesis of the CtBP-interaction domain of MEQ has the potential to be an improved vaccine against virulent MDV infection. Engineering MDV with precisely defined attenuating mutations, therefore, represents an effective strategy for generating new vaccines against this major poultry disease.

Fig. 1.

Interaction of MEQ and chCtBP1. (a) Sequence of MEQ, EBNA3A, and Evi-1 showing the predicted bipartite CtBP-binding motifs (underlined). (b) Alignment of the conserved residues (shown in yellow) of the CtBP sequence from chicken (chCtBP1), human (huCtBP1), mouse (moCtBP1), and Drosophila (doCtBP1). The residues that form the PXDLS peptide-binding cleft are indicated (red). (c) Predicted model of chCtBP1-binding groove with the MEQ peptide PLDLSLG. (d) Immunofluorescence labeling on MSB-1 cells showing chCtBP1 (Upper Left) and MEQ (Upper Right) and their interaction with the overlay of the two images (Lower Left). (Lower Right) DIC- and DAPI-stained nuclei. (Scale bar: 8 μm.)

Fig. 2.

MEQ binds to chCtBP1 through the PLDLS motif and induces transcriptional repression. (a) Illustration of the mutations in the CtBP-binding motifs. (b) Interaction of GST-MEQ_1–170 fusion protein with [³⁵S]methionine-labeled chCtBP1 demonstrated by SDS/PAGE. (c) Extracts of DF-1 cells cotransfected with chCtBP1 and MEQ constructs immunoprecipitated with rabbit (anti-CtBP or unrelated anti-endogenous avian retrovirus) antibodies and detected with anti-MEQ antibody. (d) Mean ± SD luciferase activity (expressed as fold repression over that of the GAL4DBD construct) of extracts of DF-1 cells cotransfected with the firefly luciferase GAL4 reporter, Renilla plasmid, and GAL4DBD-MEQ_1–174 fusion constructs. (e) Western blotting of protein extracts with mouse anti-GAL4DBD antibody.

Fig. 3.

Replication of RB-1B viruses in vitro and in vivo. (a) Schematic illustration of the BAC clones: wild type (pRB-1B, dark blue), MEQ_AVEFT-PDAMA single copy mutant (pRB-1B-Ct10, red), double mutant (pRB-1B-Ct20, light blue), revertant (pRB-1B-Ct22, green), and ΔMEQ mutant (pRB-1B-D2, orange). (b) MEQ and glycoprotein B expression in MDV plaques from transfected BAC DNA. (Scale bar: 20 nm.) (c) TaqMan quantitative PCR showing the mean ± SE of viral genome copy numbers in peripheral blood leukocytes of inbred line P chickens (n = 6) at different days postinfection. (d) Mean ± SE of viral genome copy numbers in the peripheral blood leukocytes of outbred RIR chickens (n = 6). ΔMEQ deletion mutant (pRB-1B-D2) was also included in this experiment.

Fig. 4.

Genetic disruption of the MEQ–CtBP interaction leads to complete attenuation of MDV. (a) Percentage survival in inbred line P birds (n = 15) infected with MDV. (b) Percentage survival in outbred RIR birds (n = 12) infected with MDV. The pRB-1B-Ct20 virus showed complete loss of oncogenicity in both lines of birds. (c) Percentage survival of line P chickens vaccinated with commercial Rispens vaccine or pRB-1B-Ct20 virus (n = 9) after being challenged with virulent RB-1B virus. The minimum number of birds in each experimental group is shown in brackets.