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Journal of Virology, December 2000, p. 11129-11136, Vol. 74, No. 23
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

The Major Immediate-Early Gene ie3 of Mouse Cytomegalovirus Is Essential for Viral Growth†

Ana Angulo,1,* Peter Ghazal,1 and Martin Messerle2,*

Department of Immunology and Molecular Biology, Division of Virology, The Scripps Research Institute, La Jolla, California 92037,1 and Max von Pettenkofer-Institut für Hygiene und Medizinische Mikrobiologie, Genzentrum der Ludwig-Maximilians-Universität München, D-81377 Munich, Germany2

Received 9 June 2000/Accepted 30 August 2000

The significance of the major immediate-early gene ie3 of mouse cytomegalovirus (MCMV) and that of the corresponding ie2 gene of human cytomegalovirus to viral replication are not known. To investigate the function of the MCMV IE3 regulatory protein, we generated two different MCMV recombinants that contained a large deletion in the IE3 open reading frame (ORF). The mutant genomes were constructed by the bacterial artificial chromosome mutagenesis technique, and MCMV ie3 deletion mutants were reconstituted on a mouse fibroblast cell line that expresses the MCMV major immediate-early genes. The ie3 deletion mutants failed to replicate on normal mouse fibroblasts even when a high multiplicity of infection was used. The replication defect was rescued when the IE3 protein was provided in trans by a complementing cell line. A revertant virus in which the IE3 ORF was restored was able to replicate with wild-type kinetics in normal mouse fibroblasts, providing evidence that the defective growth phenotype of the ie3 mutants was due to disruption of the ie3 gene. To characterize the point of restriction in viral replication that is controlled by ie3, we analyzed the pattern of expression of selective early (beta ) and late (gamma ) genes. While we could detect transcripts for the immediate-early gene ie1 in cells infected with the ie3 mutants, we failed to detect transcripts for representative beta  and gamma  genes. These data demonstrate that the MCMV transactivator IE3 plays an indispensable role during viral replication in tissue culture, implicating a similar role for the human CMV ie2 gene product. To our knowledge, the ie3 deletion mutants represent the first MCMV recombinants isolated that contain a disruption of an essential gene.

* Corresponding authors. Mailing address for Martin Messerle: Max von Pettenkofer-Institut, Genzentrum, Feodor-Lynen-Strasse 25, D-81377 Munich, Germany. Phone: 49 89 2180 6850. Fax: 49 89 2180 6898. E-mail: messerle{at}lmb.uni-muenchen.de. Mailing address for Ana Angulo: Department of Immunology and Molecular Biology, The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037. Phone: (858) 784-9933. Fax: (858) 784-9272. E-mail: angulo{at}scripps.edu.

dagger Publication 13327-IMM from The Scripps Research Institute.

Journal of Virology, December 2000, p. 11129-11136, Vol. 74, No. 23
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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