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Journal of Virology, February 2001, p. 1581-1593, Vol. 75, No. 4
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.4.1581-1593.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Reactivation of the Human Cytomegalovirus Major Immediate-Early
Regulatory Region and Viral Replication in Embryonal NTera2 Cells:
Role of Trichostatin A, Retinoic Acid, and Deletion of the
21-Base-Pair Repeats and Modulator
Jeffery L.
Meier*
Department of Internal Medicine and Helen C. Levitt Center for Viral Pathogenesis and Disease, University of
Iowa College of Medicine, Iowa City, Iowa 52242
Received 10 July 2000/Accepted 14 November 2000
Inactivity of the human cytomegalovirus (HCMV) major
immediate-early regulatory region (MIERR), which is composed of
promoter, enhancer, unique region, and modulator, is linked to lack of
HCMV replication in latently infected cells and in other nonpermissive cell types, including human embryonal NTera2 carcinoma (NT2) cells. I
refined the embryonal NT2 cell model to enable characterization of the
unknown mechanistic basis for silencing of HCMV MIERR-dependent transcription and viral replication in nonpermissive cells. These infected NT2 cells contain nonreplicating viral genomes with
electrophoretic mobility equivalent to a supercoiled, bacterial
artificial chromosome of comparable molecular weight. MIERR-dependent
transcription is minimal to negligible. Increasing the availability of
positive-acting transcription factors by retinoic acid (RA) treatment
after infection is largely insufficient in reactivating the MIERR. In
contrast, trichostatin A (TSA), a histone deacetylase inhibitor,
reactivates MIERR-dependent transcription. Contrary to prior findings
produced from transfected MIERR segments, deletion of the 21-bp repeats and modulator from the MIERR in the viral genome does not relieve MIERR
silencing. To demonstrate that MIERR silencing likely results from
enhancer inactivity, I examined an HCMV with a heterologous MIERR
promoter that is enhancer dependent but exempt from IE2 p86-mediated negative autoregulation. This heterologous promoter, like
its neighboring native MIERR promoter, exhibits
immediate-early transcriptional kinetics in fibroblasts. In embryonal
NT2 cells, the heterologous MIERR promoter is transcriptionally
inactive. This silence is relieved by TSA but not by RA. Remarkably,
TSA-induced reactivation of MIERR-dependent transcription from
quiescent viral genomes is followed by release of infectious virus. I
conclude that a mechanism of active repression imposes a block to
MIERR-dependent transcription and viral replication in embryonal NT2
cells. Because TSA overcomes the block, viral gene silencing may
involve histone deacetylase-based modification of viral chromatin,
which might account for the covalently closed circular conformation of
quiescent HCMV genomes.
*
Mailing address: Department of Internal Medicine and
Helen C. Levitt Center for Viral Pathogenesis and Disease, University of Iowa College of Medicine, Iowa City, Iowa 52242. Phone: (319) 356-7055. Fax: (319) 335-9006. E-mail:
jeffery-meier{at}uiowa.edu
Journal of Virology, February 2001, p. 1581-1593, Vol. 75, No. 4
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.4.1581-1593.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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