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Journal of Virology, September 2009, p. 8893-8904, Vol. 83, No. 17
0022-538X/09/$08.00+0     doi:10.1128/JVI.02239-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

The CREB Site in the Proximal Enhancer Is Critical for Cooperative Interaction with the Other Transcription Factor Binding Sites To Enhance Transcription of the Major Intermediate-Early Genes in Human Cytomegalovirus-Infected Cells {triangledown}

Philip Lashmit,1 Shuhui Wang,2 Hongmei Li,1 Hiroki Isomura,3 and Mark F. Stinski1*

Department of Microbiology, Carver College of Medicine, University of Iowa, Iowa City, Iowa 52242,1 Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado,2 Division of Virology, Aichi Cancer Center Research Institute, 1-1, Kanokoden, Chikusa-ku, Nagoya 464-8681, Japan3

Received 23 October 2008/ Accepted 16 June 2009

One of the two SP1 sites in the proximal enhancer of the human cytomegalovirus (HCMV) major immediate-early (MIE) promoter is essential for transcription in human fibroblast cells (H. Isomura, M. F. Stinski, A. Kudoh, T. Daikoku, N. Shirata, and T. Tsurumi, J. Virol. 79:9597-9607, 2005). Upstream of the two SP1 sites to –223 relative to the +1 transcription start site, there are an additional five DNA binding sites for eukaryotic transcription factors. We determined the effects of the various transcription factor DNA binding sites on viral MIE RNA transcription, viral gene expression, viral DNA synthesis, or infectious virus production. We prepared recombinant HCMV bacterial artificial chromosome (BAC) DNAs with either one site missing or one site present upstream of the two SP1 sites. Infectious recombinant HCMV BAC DNAs were transfected into various cell types to avoid the effect of the virion-associated transactivators. Regardless of the cell type, which included human fibroblast, endothelial, and epithelial cells, the CREB site had the most significant and independent effect on the MIE promoter. The other sites had a minor independent effect. However, the combination of the different transcription factor DNA binding sites was significantly stronger than multiple duplications of the CREB site. These findings indicate that the CREB site in the presence of the other sites has a major role for the replication of HCMV.

* Corresponding author. Mailing address: Department of Microbiology, University of Iowa, 51 Newton Rd., Iowa City, IA 52242. Phone: (319) 335-7792. Fax: (319) 335-9006. E-mail: mark-stinski{at}uiowa.edu

{triangledown} Published ahead of print on 24 June 2009.

Journal of Virology, September 2009, p. 8893-8904, Vol. 83, No. 17
0022-538X/09/$08.00+0     doi:10.1128/JVI.02239-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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