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Journal of Virology, August 2004, p. 8026-8035, Vol. 78, No. 15
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.15.8026-8035.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Comprehensive Mutational Analysis of a Herpesvirus Gene in the Viral Genome Context Reveals a Region Essential for Virus Replication{dagger}

Anja Bubeck,1 Markus Wagner,1,{ddagger} Zsolt Ruzsics,1 Mark Lötzerich,1 Margot Iglesias,2 Ila R. Singh,2 and Ulrich H. Koszinowski1*

Max von Pettenkofer Institut für Virologie, Ludwig-Maximilians-Universität München, 80336 Munich, Germany,1 Department of Pathology, Columbia University College of Physicians & Surgeons, New York, New York 100322

Received 17 November 2003/ Accepted 28 January 2004

Essential viral proteins perform vital functions during morphogenesis via a complex interaction with other viral and cellular gene products. Here, we present a novel approach to comprehensive mutagenesis of essential cytomegalovirus genes and biological analysis in the 230-kbp-genome context. A random Tn7-based mutagenesis procedure at the single-gene level was combined with site-specific recombination via the FLP/FLP recognition target site system for viral genome reconstitution. We show the function of more than 100 mutants from a larger library of M50/p35, a protein involved in capsid egress from the nucleus. This protein recruits other viral proteins and cellular enzymes to the inner nuclear membrane. Our approach enabled us to rapidly discriminate between essential and nonessential regions within the coding sequence. Based on the prediction of the screen, we were able to map a site essential for viral protein-protein interaction at the amino acid level.

* Corresponding author. Mailing address: Max von Pettenkofer Institut für Virologie, Pettenkoferstrasse 9a, 80336 Munich, Germany. Phone: 49-89-5160-5203. Fax: 49-89-5160-5292. E-mail: koszinowski{at}m3401.mpk.med.uni-muenchen.de.

{dagger} Supplemental material for this article may be found at http://jvi.asm.org.

{ddagger} Present address: Harvard Medical School, Department of Pathology, Boston, MA 02115.

Journal of Virology, August 2004, p. 8026-8035, Vol. 78, No. 15
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.15.8026-8035.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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