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Journal of Virology, February 2001, p. 1870-1878, Vol. 75, No. 4
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.4.1870-1878.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Human Cytomegalovirus with IE-2 (UL122) Deleted Fails To Express Early Lytic Genes

Andrew Marchini,1,dagger Haiqing Liu,2 and Hua Zhu2,*

Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544,1 and Department of Microbiology and Molecular Genetics, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, New Jersey 071032

Received 21 August 2000/Accepted 13 November 2000

Much evidence suggests that the major immediate-early (IE) transactivator of human cytomegalovirus (HCMV), IE-2, is likely to be critical for efficient viral replication; however, the lack of an IE-2 mutant HCMV has precluded an experimental test of this hypothesis. As an initial step toward characterizing an IE-2 mutant, we first cloned the HCMV Towne genome as a bacterial artificial chromosome (BAC) and analyzed the ability of transfected Towne-BAC DNA (T-BACwt) to produce plaques following introduction into permissive human fibroblasts. Like Towne viral DNA, transfected T-BACwt DNA was infectious in permissive cells, and the resulting virus stocks were indistinguishable from Towne virus. We then used homologous recombination in Escherichia coli to delete the majority of UL122, the open reading frame encoding the unique portion of IE-2, from T-BACwt. From this deleted BAC, a third BAC clone in which the deletion was repaired with wild-type UL122 was created. In numerous transfections of permissive human foreskin fibroblast cells with these three BAC DNA clones, the rescued BAC and T-BACwt consistently yielded plaques, while the UL122 mutant BAC never generated plaques, even after 4 weeks. Protein and mRNA of other IE genes were readily detected from transfected UL122 mutant BAC DNA; however, reverse transcription-PCR failed to detect mRNA expression from any of five early genes examined. The generalized failure of this mutant to express early genes is consistent with expectations from in vitro assays which have demonstrated that IE-2 transactivates most HCMV promoters. These experiments provide the first direct demonstration that IE-2 is required for successful HCMV infection and indicate that virus lacking IE-2 arrests early in the replication cycle.

* Corresponding author. Mailing address: Department of Microbiology and Molecular Genetics, MSB-F643, UMDNJ-New Jersey Medical School, 185 South Orange Ave., Newark, NJ 07103. Phone: (973) 972-6488. Fax: (973) 972-3644. E-mail: zhuhu{at}umdnj.edu.

dagger Present address: Tribeca Pharmaceuticals, Inc., New York, NY 10014.

Journal of Virology, February 2001, p. 1870-1878, Vol. 75, No. 4
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.4.1870-1878.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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