title: Comprehensive mutational analysis of a herpesvirus gene in the viral genome context reveals a region essential for virus replication.
abstract: 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.
authors.affiliation: Max von Pettenkofer Institut für Virologie, Ludwig-Maximilians-Universität, 80336 Munich, Germany.
authors.forename: Anja
authors.initials: A
authors.lastname: Bubeck
authors.forename: Markus
authors.initials: M
authors.lastname: Wagner
authors.forename: Zsolt
authors.initials: Z
authors.lastname: Ruzsics
authors.forename: Mark
authors.initials: M
authors.lastname: Lötzerich
authors.forename: Margot
authors.initials: M
authors.lastname: Iglesias
authors.forename: Ila R
authors.initials: IR
authors.lastname: Singh
authors.forename: Ulrich H
authors.initials: UH
authors.lastname: Koszinowski
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015723:Gene Library; D016679:Genome, Viral; D051379:Mice; D008969:Molecular Sequence Data; D018146:Muromegalovirus / Q000235:genetics* / Q000502:physiology; D009154:Mutation; D041681:NIH 3T3 Cells; D016366:Open Reading Frames; D014779:Virus Replication