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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 (
) and late (
) 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
and
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.

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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