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Journal of Virology, January 2008, p. 849-858, Vol. 82, No. 2
0022-538X/08/$08.00+0     doi:10.1128/JVI.01593-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

A cis Element between the TATA Box and the Transcription Start Site of the Major Immediate-Early Promoter of Human Cytomegalovirus Determines Efficiency of Viral Replication{triangledown}

Hiroki Isomura,1* Mark F. Stinski,2 Ayumi Kudoh,1 Sanae Nakayama,1 Takayuki Murata,1 Yoshitaka Sato,1 Satoko Iwahori,1 and Tatsuya Tsurumi1

Division of Virology, Aichi Cancer Center Research Institute, 1-1, Kanokoden, Chikusa-ku, Nagoya 464-8681, Japan,1 Department of Microbiology, Carver College of Medicine, University of Iowa, Iowa, City, Iowa 522422

Received 20 July 2007/ Accepted 23 October 2007

The promoter of the major immediate-early (MIE) genes of human cytomegalovirus (HCMV), also referred to as the CMV promoter, possesses a cis-acting element positioned downstream of the TATA box between positions –14 and –1 relative to the transcription start site (+1). We determined the role of the cis-acting element in viral replication by comparing recombinant viruses with the cis-acting element replaced with other sequences. Recombinant virus with the simian CMV counterpart replicated efficiently in human foreskin fibroblasts, as well as wild-type virus. In contrast, replacement with the murine CMV counterpart caused inefficient MIE gene transcription, RNA splicing, MIE and early viral gene expression, and viral DNA replication. To determine which nucleotides in the cis-acting element are required for efficient MIE gene transcription and splicing, we constructed mutations within the cis-acting element in the context of a recombinant virus. While mutations in the cis-acting element have only a minor effect on in vitro transcription, the effects on viral replication are major. The nucleotides at –10 and –9 in the cis-acting element relative to the transcription start site (+1) affect efficient MIE gene transcription and splicing at early times after infection. The cis-acting element also acts as a cis-repression sequence when the viral IE86 protein accumulates in the infected cell. We demonstrate that the cis-acting element has an essential role in viral replication.

* Corresponding author. Mailing address: Division of Virology, Aichi Cancer Center Research Institute, 1-1, Kanokoden, Chikusa-ku, Nagoya 464-8681, Japan. Phone: 81-52-762-6111, x7032. Fax: 81-52-763-5233. E-mail: hisomura{at}aichi-cc.jp

{triangledown} Published ahead of print on 7 November 2007.

Journal of Virology, January 2008, p. 849-858, Vol. 82, No. 2
0022-538X/08/$08.00+0     doi:10.1128/JVI.01593-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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