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Journal of Virology, September 2000, p. 7720-7729, Vol. 74, No. 17
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Fast Screening Procedures for Random Transposon Libraries of Cloned Herpesvirus Genomes: Mutational Analysis of Human Cytomegalovirus Envelope Glycoprotein Genes

Urs Hobom, Wolfram Brune, Martin Messerle, Gabriele Hahn, and Ulrich H. Koszinowski*

Lehrstuhl für Virologie, Max von Pettenkofer-Institut, Ludwig-Maximilians-Universität München, 80336 Munich, Germany

Received 28 December 1999/Accepted 23 May 2000

We have cloned the human cytomegalovirus (HCMV) genome as an infectious bacterial artificial chromosome (BAC) in Escherichia coli. Here, we have subjected the HCMV BAC to random transposon (Tn) mutagenesis using a Tn1721-derived insertion sequence and have provided the conditions for excision of the BAC cassette. We report on a fast and efficient screening procedure for a Tn insertion library. Bacterial clones containing randomly mutated full-length HCMV genomes were transferred into 96-well microtiter plates. A PCR screening method based on two Tn primers and one primer specific for the desired genomic position of the Tn insertion was established. Within three consecutive rounds of PCR a Tn insertion of interest can be assigned to a specific bacterial clone. We applied this method to retrieve mutants of HCMV envelope glycoprotein genes. To determine the infectivities of the mutant HCMV genomes, the DNA of the identified BACs was transfected into permissive fibroblasts. In contrast to BACs with mutations in the genes coding for gB, gH, gL, and gM, which did not yield infectious virus, BACs with disruptions of open reading frame UL4 (gp48) or UL74 (gO) were viable, although gO-deficient viruses showed a severe growth deficit. Thus, gO (UL74), a component of the glycoprotein complex III, is dispensable for viral growth. We conclude that our approach of PCR screening for Tn insertions will greatly facilitate the functional analysis of herpesvirus genomes.

* Corresponding author. Mailing address: Max von Pettenkofer-Institut, Pettenkofer Str. 9a, 80336 Munich, Germany. Phone: 49 89 5160 5290. Fax: 49 89 5160 5292. E-mail: koszinowski{at}m3401.mpk.med.uni-muenchen.de.

Journal of Virology, September 2000, p. 7720-7729, Vol. 74, No. 17
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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