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Rapid identification of essential and nonessential herpesvirus genes by direct transposon mutagenesis

Nature Biotechnology volume 17, pages 360–364 (1999)Cite this article

Abstract

Herpesviruses are important pathogens in animals and humans. The large DNA genomes of several herpesviruses have been sequenced, but the function of the majority of putative genes is elusive. Determining which genes are essential for their replication is important for identifying potential chemotherapy targets, designing herpesvirus vectors, and generating attenuated vaccines. For this purpose, we recently reported that herpesvirus genomes can be maintained as infectious bacterial artificial chromosomes (BAC) in Escherichia coli . Here we describe a one-step procedure for random-insertion mutagenesis of a herpesvirus BAC using a Tn1721-based transposon system. Transposon insertion sites were determined by direct sequencing, and infectious virus was recovered by transfecting cultured cells with the mutant genomes. Lethal mutations were rescued by cotransfecting cells containing noninfectious genomes with the corresponding wild-type subgenomic fragments. We also constructed revertant genomes by allelic exchange in bacteria. These methods, which are generally applicable to any cloned herpesvirus genome, will facilitate analysis of gene function for this virus family.

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Figure 1: Construction of pTsTM plasmids.
Figure 2: Strategy for transposon mutagenesis and analysis of the cloned mCMV genome.
Figure 3: Analysis of Tn mutants by restriction enzyme digestion and direct sequencing.
Figure 4: Characterization of mutants Tc9, Tc10, Tc13, and Tc14.
Figure 5: Rescue of nonviable mutants by complementation and construction of revertants.
Figure 6: Analysis of BAC stability.

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Acknowledgements

We thank A. Hegele and A. Colomar for excellent technical assistance, R. Haas for providing the TnMax system, and J. Heesemann for helpful suggestions. This work was supported by grants from the Deutsche Forschungsgemeinschaft, the Boehringer Ingelheim Fonds, the Bundesministerium für Bildung und Forschung, and the European Union.

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Authors and Affiliations

  1. Department of Virology, Max von Pettenkofer-Institut Ludwig-Maximilians-Universität München, Pettenkoferstraβe 9a, Munich, 80336, Germany

    Wolfram Brune, Carine Ménard, Urs Hobom, Martin Messerle & Ulrich H. Koszinowski

  2. Department of Bacteriology, Max von Pettenkofer-Institut Ludwig-Maximilians-Universität München, Pettenkoferstraβe 9a, Munich, 80336, Germany

    Stefan Odenbreit

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  1. Wolfram Brune
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  6. Ulrich H. Koszinowski
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Correspondence to Ulrich H. Koszinowski.

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Brune, W., Ménard, C., Hobom, U. et al. Rapid identification of essential and nonessential herpesvirus genes by direct transposon mutagenesis. Nat Biotechnol 17, 360–364 (1999). https://doi.org/10.1038/7914

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