Nikolaus Osterrieder, Amanda K. Bruskin, Jeremy P. Kamil, Daniel Schumacher, B. Karsten Tischer, Sascha Trapp, Jens von Einem
Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY U.S.A.
Marek's disease virus (MDV) genomics has trailed behind those of related viruses because efficient mutant virus generation was impossible until recently. With the advent of the cloning of three MDV strains (RB-1B, CVI988, 584Ap80C) as bacterial artificial chromosomes (BAC), and one strain as a set of overlapping cosmid clones (Md5), efficient mutant virus generation is now possible (2-4). By using the powerful recombination machinery available in Escherichia coli, we have generated more than 30 virus mutants based on the 584Ap80C-derived BAC20 clone (1). Both targeted and random mutagenesis methods were applied to begin a comprehensive mutagenesis of the MDV genome. While targeted deletions of entire MDV genes were done predominantly by Red recombination or E/T cloning (Figure 1), virus variants harbouring single point mutations as well as revertant viruses were obtained using shuttle (RecA) mutagenesis protocols (5) (Figure 2).
Among the genes targeted by site-directed mutagenesis are the UL1, UL10, UL11, UL20, UL22, UL27, UL31, UL34, UL35, UL41, UL43-UL49.5, UL51, UL53, US2, US3, and US7-US8 genes.
More recently, a transposon-based approach that allows random inactivation of MDV genes has been established. Based on transposon Tn1721 (6), EGFP- and LacZ sequences were randomly introduced into the BAC20 genome and the resulting mutants were analyzed by restriction enzyme analysis and partially by virus reconstitution in both chicken embryo cells and permanent SOgE cells. Preliminary results on the use of the Tn1721-based system have shown that the transposon is randomly inserted into open reading frames and that both EGFP and LacZ are efficiently expressed in the reconstituted mutant viruses. In addition, the use of EGFP did not appear to have a detrimental effect on virus growth in those mutants, in which non-essential viral sequences had been targeted. Therefore, Tn1721-based BAC20 mutagenesis coupled with virus reconstitution and genetic analysis will certainly be a powerful tool for high-throughput MDV gene analysis.
References
1. Osterrieder, N., D. Schumacher, S. Trapp, M. Beer, J. von Einem, and K. Tischer. 2003. Generation and exploitation of infectious bacterial artificial chromosome (BAC) clones of animal herpesviruses. Berl Munch. Tierarztl. Wochenschr. 116:373-380.
2. Osterrieder, N. and J. F. Vautherot. 2004. The genome content of Marek's disease-like viruses Marek's disease. In: Marek's Disease: An evolving problem (Eds. Davison and Nair) Elsevier, London. Pp. 17-31.
3. Petherbridge, L., A. C. Brown, S. J. Baigent, K. Howes, M. A. Sacco, N. Osterrieder, and V. K. Nair. 2004. Oncogenicity of Marek's disease virus cloned as bacterial artificial chromosomes: Deletion of the region encoding meq-vIL8 genes interferes with replication and oncogenicity. Journal of Virology 78, 13376-80
4. Reddy, S. M., B. Lupiani, I. M. Gimeno, R. F. Silva, L. F. Lee, and R. L. Witter. 2002. Rescue of a pathogenic Marek's disease virus with overlapping cosmid DNAs: Use of a pp38 mutant to validate the technology for the study of gene function. Proceedings of the National Academy of Sciences of the United States of America 99:7054-7059.
5. Tischer, B. K., Schumacher, D., and N. Osterrieder. 2004. Expression of Marek's disease virus glycoprotein C is detrimental to virus growth in cultured cells. submitted.
6. Yu, D., M. C. Silva, and T. Shenk. 2003. Functional map of human cytomegalovirus AD169 defined by global mutational analysis. Proceedings of the National Academy of Sciences of the United States of America 100:12396-12401.
From Proceedings of the "7th International Symposium on Marek's disease", St. Catherine's College, Oxford, United Kingdom.
