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Journal of Virology, November 2001, p. 10149-10160, Vol. 75, No. 21
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.21.10149-10160.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

The R3 Region, One of Three Major Repetitive Regions of Human Herpesvirus 6, Is a Strong Enhancer of Immediate-Early Gene U95

Masaya Takemoto,1 Takuya Shimamoto,1,* Yuji Isegawa,1,2 and Koichi Yamanishi1

Department of Microbiology, Osaka University Medical School C1,1 and Division of Advanced Medical Bacteriology, Osaka University Medical School G5,2 Suita, Osaka 565-0871, Japan

Received 19 March 2001/Accepted 20 July 2001

An immediate-early (IE) gene of human herpesvirus 6 (HHV-6), U95, has similarity at the amino acid level to the murine cytomegalovirus (MCMV) IE2 gene and is related to the human cytomegalovirus (HCMV) US22 gene family. Sequence analyses of U95 cDNA clones revealed that the transcription start site was located about 1.6 kbp upstream of the putative initiating ATG and that the transcript consisted of two exons. A single intron extended from nucleotides 142589 to 144229, which contained ORF U94. A protein with a molecular mass of about 120 kDa was translated from this cDNA clone in an in vitro transcription-translation assay. The transcription start site was found to be 220 bp downstream of the R3 region by primer extension analysis. HHV-6 has three repetitive elements, R1, R2, and R3, in or near the IE-A locus. R3 is composed of 24 copies of a 104- to 107-bp sequence element, which contains multiple putative binding sites for cellular transcription factors such as AP2 and NF-kappa B, and its biological significance has yet to be elucidated. The region between -710 and +46 relative to the transcription start site of U95 was analyzed in this study. Deletion from -710 to -396, corresponding to three copies of an R3 unit, decreased the promoter activity by 15-fold, and coexpression of Ikappa Balpha (S32A/S36A) repressed it to almost the same level. Electrophoretic mobility shift assays showed that NF-kappa B family members p50 and c-Rel bound to NF-kappa B sites derived from the R3 region. These results demonstrate that R3 strongly enhances the U95 promoter activity and that NF-kappa B and binding sites for NF-kappa B in the R3 region play an important role in its activation. Because U95 promoter activity correlated with the number of R3 units, which each contained an NF-kappa B site, the repetitive organization of R3 is important for regulating U95 transcription.

* Corresponding author. Mailing address: Department of Microbiology, Osaka University Medical School C1, 2-2 Yamada-Oka, Suita, Osaka 565-0871, Japan. Phone: 81-6-6879-3323. Fax: 81-6-6879-3329. E-mail: simamoto{at}micro.med.osaka-u.ac.jp.

Journal of Virology, November 2001, p. 10149-10160, Vol. 75, No. 21
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.21.10149-10160.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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