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Journal of Virology, November 2002, p. 11763-11769, Vol. 76, No. 22
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.22.11763-11769.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Deletion of Epstein-Barr Virus Regulatory Sequences Upstream of the EBNA Gene Promoter Wp1 Is Unfavorable for B-Cell Immortalization

Lina I. Yoo,{dagger} Josh Woloszynek, Steven Templeton, and Samuel H. Speck*

Division of Microbiology and Immunology, Yerkes Regional Primate Research Center, Emory University, Atlanta, Georgia 30329

Received 5 June 2002/ Accepted 9 August 2002

Transcription of the six Epstein-Barr virus (EBV) EBNA genes is coordinately regulated, being driven by either the Cp promoter, which is encoded within the unique region just upstream of the EBV major internal repeat (IR-1), or by the Wp promoter, which is encoded within the IR-1 repeat and thus present in multiple copies. Previous analyses of Cp- and Wp-initiated transcription have identified a shared cis-regulatory element mapping to the region extending from -169 to -369 bp upstream of the Wp transcription initiation site (M. T. Puglielli, N. Desai, and S. H. Speck, J. Virol. 71:120-128, 1997). To assess the impact of this regulatory region on Cp and Wp activity in the context of the viral genome, we attempted to delete this regulatory region upstream of the first copy of Wp (Wp1). While 10 recombinant viruses were obtained in which this deletion was incorporated in the interior of the IR-1 repeat, only a single lymphoblastoid cell line (LCL) immortalized by a recombinant EBV harboring the deletion upstream of Wp1 was recovered. In contrast, using a control targeting vector in which the Wp regulatory sequences were intact but which contained a sequence tag within the W0 exon, we demonstrated that of the five recombinant viruses analyzed in which the crossover event had occurred upstream of the Wp sequence tag, four had incorporated the tagged sequences into Wp1 of the virus. Taken together, these results indicate that deletion of the regulatory sequences from -369 to -169 bp upstream of Wp1 is unfavorable for EBV-driven B-cell immortalization but is tolerated within the interior of the IR-1 repeat. Analysis of promoter usage in the clone 9-60 LCL, in which the W enhancer sequences were deleted upstream of Wp1, revealed the following: (i) the level of Cp-initiated transcription was significantly diminished compared to that of wild-type LCLs; (ii) the decreased Cp-initiated transcription was not efficiently compensated by transcription initiation from Wp1; and (iii) transcription initiation from downstream Wp promoters was detectable. This is the first report of an LCL in which transcription initiation from a Wp downstream of Wp1 has been documented.

* Corresponding author. Mailing address: Division of Microbiology and Immunology, Yerkes Regional Primate Research Center, Emory University, 954 Gatewood Rd. NE, Atlanta, GA 30329. Phone: (404) 727-7665. Fax: (404) 727-7768. E-mail: sspeck{at}rmy.emory.edu.

{dagger} Present address: Department of Cell Biology, Harvard Medical School, Boston, MA 02115.

Journal of Virology, November 2002, p. 11763-11769, Vol. 76, No. 22
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.22.11763-11769.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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