Identification of essential and non-essential genes of the guinea pig cytomegalovirus (GPCMV) genome via transposome mutagenesis of an infectious BAC clone

Alistair McGregor, Fenyong Liu, Mark R. Schleiss

Research output: Contribution to journalArticlepeer-review

21 Scopus citations

Abstract

We report application of a transposition methodology that allows the easy characterization and mutation of genes encoded on an infectious bacterial artificial chromosome (BAC) clone. We characterized mutants generated by transposome (Tn) mutagenesis of a BAC clone of guinea pig cytomegalovirus (GPCMV). A pool of Tn mutant GPCMV BACs were screened initially by restriction profile analysis to verify they were full-length, and subsequently GPCMV BAC DNA from individual mutants was transfected onto guinea pig lung fibroblast cells in order to generate virus. Tn GPCMV BAC mutants were classed as either essential or non-essential gene insertions, depending upon their ability to regenerate viable, replication-competent virus. Representative mutants were more fully characterized. Analysis by sequencing the Tn insertion site on the mutated BACs, and by regeneration of virus using transfection of guinea pig fibroblasts (GPL), demonstrated that a recombinant with a Tn insertion in the UL35 homolog gene (GP35) was a non-essential gene for viral replication in tissue culture. A mutant with an insertion in the UL46 homolog (GP46) was nonviable, a phenotype which could be rescued by homologous recombination of BAC DNA with wild-type UL46 sequences, suggesting an essential role of this putative capsid gene in virus replication.

Original languageEnglish (US)
Pages (from-to)101-108
Number of pages8
JournalVirus research
Volume101
Issue number2
DOIs
StatePublished - May 2004

Bibliographical note

Funding Information:
We acknowledge the technical assistance of Jarone Lee. We thank P. Ioannou and U.H. Koszinowski for their respective gifts of pGETrec and pTsTM8 (TnMAX8) plasmids. This work was supported by National Institutes of Health HD044864-01 and HD38416-01, and March of Dimes Birth Defects Foundation awards (to FL and MS).

Keywords

  • Bacterial artificial chromosome
  • Guinea pig cytomegalovirus
  • Human cytomegalovirusis

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