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Journal of Virology, June 2007, p. 5508-5517, Vol. 81, No. 11
0022-538X/07/$08.00+0     doi:10.1128/JVI.02796-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Random Screening for Dominant-Negative Mutants of the Cytomegalovirus Nuclear Egress Protein M50{triangledown}

Brigitte Rupp,1,{dagger} Zsolt Ruzsics,1,{dagger} Christopher Buser,2 Barbara Adler,1 Paul Walther,2 and Ulrich H. Koszinowski1*

Max von Pettenkofer Institut für Virologie, Ludwig-Maximilians-Universität München, Pettenkoferstrasse 9a, 80336 Munich, Germany,1 Institut für Mikrobiologie, Abteilung Virologie und Zentrale Einrichtung Elektronenmikroskopie, Universität Ulm, Albert-Einstein-Allee 11, 89069 Ulm, Germany2

Received 19 December 2006/ Accepted 9 March 2007

Inactivation of gene products by dominant-negative (DN) mutants is a powerful tool to assign functions to proteins. Here, we present a two-step procedure to establish a random screen for DN alleles, using the essential murine cytomegalovirus gene M50 as an example. First, loss-of-function mutants from a linker-scanning library were tested for inhibition of virus reconstitution with the help of FLP-mediated ectopic insertion of the mutants into the viral genome. Second, DN candidates were confirmed by conditional expression of the inhibitory proteins in the virus context. This allowed the quantification of the inhibitory effect, the identification of the morphogenesis block, and the construction of DN mutants with improved activity. Based on these observations a DN mutant of the homologous gene (UL50) in human cytomegalovirus was predicted and constructed. Our data suggest that a proline-rich sequence motif in the variable region of M50/UL50 represents a new functional site which is essential for nuclear egress of cytomegalovirus capsids.

* Corresponding author. Mailing address: Max von Pettenkofer Institut für Virologie, Pettenkoferstrasse 9a, 80336 Munich, Germany. Phone: 49-89-5160-5290. Fax: 49-89-5160-5292. E-mail: Koszinowski{at}mvp.uni-muenchen.de

{triangledown} Published ahead of print on 21 March 2007.

{dagger} B.R. and Z.R. made equal contributions to this article.

Journal of Virology, June 2007, p. 5508-5517, Vol. 81, No. 11
0022-538X/07/$08.00+0     doi:10.1128/JVI.02796-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Sam, M. D., Evans, B. T., Coen, D. M., Hogle, J. M. (2009). Biochemical, Biophysical, and Mutational Analyses of Subunit Interactions of the Human Cytomegalovirus Nuclear Egress Complex. J. Virol. 83: 2996-3006 [Abstract] [Full Text]  
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