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Journal of Virology, August 2001, p. 6786-6799, Vol. 75, No. 15
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.15.6786-6799.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

DNA Binding by Kaposi's Sarcoma-Associated Herpesvirus Lytic Switch Protein Is Necessary for Transcriptional Activation of Two Viral Delayed Early Promoters

David M. Lukac,dagger Lilit Garibyan, Jessica R. Kirshner, Diana Palmeri,dagger and Don Ganem*

Howard Hughes Medical Institute and Department of Microbiology, University of California Medical Center, San Francisco, California 94143-0414

Received 9 October 2000/Accepted 27 April 2001

Kaposi's sarcoma-associated herpesvirus (KSHV; also known as human herpesvirus-8) establishes latent and lytic infections in both lymphoid and endothelial cells and has been associated with diseases of both cell types. The KSHV open reading frame 50 (ORF50) protein is a transcriptional activator that plays a central role in the reactivation of lytic viral replication from latency. Here we identify and characterize a DNA binding site for the ORF50 protein that is shared by the promoters of two delayed early genes (ORF57 and K-bZIP). Transfer of this element to heterologous promoters confers on them high-level responsiveness to ORF50, indicating that the element is both necessary and sufficient for activation. The element consists of a conserved 12-bp palindromic sequence and less conserved sequences immediately 3' to it. Mutational analysis reveals that sequences within the palindrome are critical for binding and activation by ORF50, but the presence of a palindrome itself is not absolutely required. The 3' flanking sequences also play a critical role in DNA binding and transactivation. The strong concordance of DNA binding in vitro with transcriptional activation in vivo strongly implies that sequence-specific DNA binding is necessary for ORF50-mediated activation through this element. Expression of truncated versions of the ORF50 protein reveals that DNA binding is mediated by the amino-terminal 272 amino acids of the polypeptide.


* Corresponding author. Mailing address: Howard Hughes Medical Institute and Department of Microbiology, University of California Medical Center, San Francisco, CA 94143-0414. Phone: (415) 476-2826. Fax: (415) 476-0939. E-mail: ganem{at}cgl.ucsf.edu.

dagger Present address: University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Department of Microbiology and Molecular Genetics, Newark, NJ 07103.


Journal of Virology, August 2001, p. 6786-6799, Vol. 75, No. 15
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.15.6786-6799.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.



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