Rapid identification of non-essential genes for in vitro replication of Marek's disease virus by random transposon mutagenesis.

Marek's disease virus (MDV) is a highly oncogenic alphaherpesvirus that induces rapid-onset T-cell lymphomas in poultry. The MDV genome encodes more than 100 genes. However, the role of many of these genes in virus replication is not known. The construction of an infectious bacterial artificial chromosome (BAC) clone of the highly oncogenic RB-1B strain of MDV has been described previously. Virus reconstituted from the BAC clone induced rapid-onset lymphomas in chickens very similar to the wildtype viruses. In this paper, the construction of a high-density random transposon-insertion mutant library of the RB-1B BAC clone using a high throughput in vitro transposon mutagenesis technique is described. Furthermore a PCR screening method, using primers specific for the transposon sequence and the MDV gene(s) of interest, was developed for the rapid identification of specific insertion mutants. The application of the screening method to identify some of the non-essential genes for MDV replication in vitro is described.

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

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.