Identification of candidate gammaherpesvirus 68 genes required for virus replication by signature-tagged transposon mutagenesis

Journal of Virology
Nathaniel J MoormanSamuel H Speck

Abstract

Current methods for determining the role of a given gene product in the gammaherpesvirus 68 (gammaHV68) life cycle require generation of a specific mutation by either homologous recombination in mammalian cells or bacterial artificial chromosome-mediated mutagenesis in Escherichia coli. The mutant virus is then compared to wild-type virus, and the role of the gene in the viral life cycle is deduced from its phenotype. This process is both time-consuming and labor intensive. Here we present the use of random, transposon-mediated signature-tagged mutagenesis for the identification of candidate viral genes involved in virus replication. Pools of viral mutants, each containing a random insertion of a transposon, were generated with a transposon donor library in which each transposon contains a unique sequence identifier. These pools were transfected into mammalian cells, and the ability of each mutant to replicate was assessed by comparing the presence of virus in the output pool to that present in the input pool of viral genomes. With this approach we could rapidly screen up to 96 individual mutants simultaneously. The location of the transposon insertion was determined by sequencing individual clones with a common primer specific...Continue Reading

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Citations

Dec 2, 2006·Nature Reviews. Genetics·Piotr MazurkiewiczDavid W Holden
Oct 11, 2005·Viral Immunology·Philip G Stevenson, Stacey Efstathiou
Mar 21, 2008·Journal of Virology·Inga DryGeorge C Russell
Aug 24, 2012·Journal of Virology·Lucy ThorneIan Goodfellow
Sep 28, 2007·Journal of Virology·Laurent Gillet, Philip G Stevenson
Dec 17, 2008·Journal of Virology·Vaithilingaraja ArumugaswamiRen Sun
Sep 29, 2005·Journal of Virology·Andrew J Davison, Nigel D Stow
Mar 30, 2005·Journal of Virology·Janet S MayPhilip G Stevenson
Jan 19, 2012·PloS One·Daniel L GlauserPhilip G Stevenson
Mar 2, 2005·Proceedings of the National Academy of Sciences of the United States of America·Moon Jung SongRen Sun
Jan 12, 2011·Annual Review of Immunology·Erik BartonSamuel H Speck
Apr 8, 2011·The Journal of General Virology·Ricardo MilhoPhilip G Stevenson
Jul 20, 2010·Cell Host & Microbe·Samuel H Speck, Don Ganem
Sep 2, 2016·Frontiers in Microbiology·Brandon CieniewiczLaurie T Krug
Mar 24, 2005·The Journal of General Virology·Janet S MayPhilip G Stevenson
Feb 5, 2021·Journal of Virology·Guoxun WangTiffany A Reese

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