JVI Accepts, published online ahead of print on 7 November 2007

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J. Virol. doi:10.1128/JVI.01593-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

A cis-element between the TATA Box and the Transcription Start Site of the Major Immediate-Early (MIE) Promoter of Human Cytomegalovirus determines Efficiency of Viral Replication

Hiroki Isomura^*, Mark F. Stinski, Ayumi Kudoh, Sanae Nakayama, Takayuki Murata, Yositaka Sato, Satoko Iwahori, and Tatsuya Tsurumi

Division of Virology, Aichi Cancer Center Research Institute, 1-1, Kanokoden, Chikusa-ku, Nagoya 464-8681, Japan, Department of Microbiology, Carver College of Medicine, University of Iowa, Iowa, City, Iowa 52242, USA

^* To whom correspondence should be addressed. Email: hisomura{at}aichi-cc.jp.

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 -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, 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.

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