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Journal of Virology, February 1999, p. 863-870, Vol. 73, No. 2
Department of Microbiology and Molecular Cell
Biology, Eastern Virginia Medical School, Norfolk, Virginia 23501
Received 9 September 1998/Accepted 22 October 1998
The human cytomegalovirus (HCMV) US11 early gene encodes a protein
involved in the down-regulation of major histocompatibility complex
class I cell surface expression in HCMV-infected cells. Consequently,
this gene is thought to play an important role in HCMV evasion of
immune recognition. In this study, we examined the transcriptional
regulation of US11 gene expression. Analysis of deletions within the
US11 promoter suggests that two sequence elements are important for
activation by the viral immediate-early (IE) proteins. Deletion of a
CREB site located at
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Transcriptional Regulation of the Human
Cytomegalovirus US11 Early Gene
83 relative to the cap site resulted in a
reduction in promoter activity to 50% of the wild-type level. Deletion
of an additional ATF site immediately upstream of the TATA box resulted
in abrogation of responsiveness to the IE proteins. To confirm the role
of the CREB and ATF sites within the US11 promoter, mutagenesis of
these two sites, both individually and in combination, was carried out. Results indicate that both the CREB element and the ATF site were required for full promoter activity, with the ATF site critical for
US11 promoter activation. The loss of transcriptional activation correlated with a loss of cellular proteins binding to the mutated US11
promoter elements. In combination with the viral IE proteins, the HCMV
tegument protein pp71 (UL82) was found to up-regulate the US11 promoter
by six- to sevenfold in transient assays. These results suggest that
pp71 may contribute to the activation of the US11 promoter at early
times after infection. Up-regulation by pp71 required the presence of
the CREB and ATF sites within the US11 promoter for full activation.
The role of the ATF and CREB elements in regulating US11 gene
expression during viral infection was then assessed. The US11 gene is
not required for replication of HCMV in tissue culture. This property
was exploited to generate US11 promoter mutants regulating expression
of the endogenous US11 gene in the natural genomic context. We
generated recombinant HCMV that contained the US11 promoter with
mutations in either the CREB or ATF element or both regulating the
expression of the endogenous US11 gene. Northern blot analysis of
infected cell mRNA revealed that mutation of the CREB element reduced
US11 mRNA expression to approximately 25% of that of the wild-type promoter, with identical kinetics of expression. Mutation of the ATF
site alone reduced US11 mRNA levels to 6% of that of the wild-type promoter, with mRNA detectable only at 8 h after infection.
Mutation of both the CREB and ATF elements in the US11 promoter reduced US11 gene expression to undetectable levels. These results demonstrate that the CREB and ATF sites cooperate to regulate the US11 promoter in
HCMV-infected cells.
*
Corresponding author. Mailing address: Department of
Microbiology and Molecular Cell Biology, Eastern Virginia Medical
School, P.O. Box 1980, 700 W. Olney Rd., Norfolk, VA 23501. Phone:
(757) 446-5663. Fax: (757) 624-2255. E-mail:
JAK{at}BORG.EVMS.EDU.
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