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Journal of Virology, November 2005, p. 13548-13560, Vol. 79, No. 21
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.21.13548-13560.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

The Herpesvirus Saimiri Replication and Transcription Activator Acts Synergistically with CCAAT Enhancer Binding Protein Alpha To Activate the DNA Polymerase Promoter

Louise Wakenshaw,^1, Matthew S. Walters,^1,, and Adrian Whitehouse^1,2*

Institute of Molecular and Cellular Biology, Faculty of Biological Sciences,¹ Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom²

Received 18 May 2005/ Accepted 1 August 2005

The open reading frame (ORF) 50 gene product, also known as the replication and transcription activator (Rta), is an immediate-early gene which is well conserved among all gamma-2 herpesviruses and plays a pivotal role in regulating the latent-lytic switch. Herpesvirus saimiri (HVS) ORF 50a functions as a sequence-specific transactivator capable of activating delayed-early (DE) gene expression via binding directly to an ORF 50 response element (RE) within the respective promoter. Analysis of the ORF 50 REs have identified two distinct types within HVS gene promoters. The first comprises a consensus sequence motif, CCN₉GG, the second an AT-rich sequence. Here we demonstrate that ORF 50a is capable of transactivating the DE ORF 9 promoter which encodes the DNA polymerase. Deletion analysis of the ORF 9 promoter mapped the ORF 50 RE to a 95-bp region situated 126 bp upstream of the initiation codon. Gel retardation analysis further mapped the RE to a 28-bp fragment, which was able to confer ORF 50 responsiveness on an enhancerless simian virus 40 minimal promoter. Furthermore, sequence analysis identified multiple CCAAT enhancer binding protein alpha (C/EBP) binding sites within the ORF 9 promoter and specifically two within the close vicinity of the AT-rich ORF 50 RE. Analysis demonstrated that the HVS ORF 50a and C/EBP proteins associate with the ORF 9 promoter in vivo, interact directly, and synergistically activate the ORF 9 promoter by binding to adjacent binding motifs. Overall, these data suggest a cooperative interaction between HVS ORF 50a and C/EBP proteins to activate the DNA polymerase promoter during early stages of the lytic replication cycle.

These authors contributed equally to this work.

Present address: Department of Microbiology, Columbia University, New York, NY 10032.

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