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Journal of Virology, May 2002, p. 5000-5013, Vol. 76, No. 10
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.10.5000-5013.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Characterization of Interactions between RTA and the Promoter of Polyadenylated Nuclear RNA in Kaposi's Sarcoma-Associated Herpesvirus/Human Herpesvirus 8

Moon Jung Song, Xudong Li, Helen J. Brown, and Ren Sun*

Department of Molecular and Medical Pharmacology, the UCLA AIDS Institute, the Jonsson Comprehensive Cancer Center, and the Molecular Biology Institute, University of California at Los Angeles, Los Angeles, California 90095

Received 11 December 2001/ Accepted 12 February 2002

RTA (replication and transcription activator; also referred to as ORF50, Lyta, and ART), an immediate-early gene product of Kaposi's sarcoma-associated herpesvirus (KSHV)/human herpesvirus 8, disrupts latency and drives lytic replication. RTA activates the expression of polyadenylated nuclear (PAN) RNA (also known as T1.1 or nut-1) of KSHV. This novel noncoding PAN RNA is the most abundant lytic transcript of KSHV; therefore, studying PAN RNA expression serves as a model system for understanding how RTA transactivates target genes during lytic replication. The RTA-responsive element of the PAN promoter (pPAN RRE) was previously identified, and our data suggested direct binding of full-length RTA to the pPAN RRE. Here, we present a detailed analysis of specific interactions between RTA and the PAN promoter. We expressed and purified the DNA-binding domain of RTA (Rdbd) to near homogeneity and measured its affinity for the pPAN RRE. In electrophoretic mobility shift assays (EMSAs), the dissociation constant (Kd) of Rdbd on the pPAN RRE was determined to be approximately 8 x 10-9 M, suggesting a strong interaction between RTA and DNA. The specificity of RTA binding to the PAN promoter was confirmed with supershift assays. The Rdbd binding sequences on the PAN promoter were mapped within a 16-bp region of the pPAN RRE by methylation interference assays. However, the minimal DNA sequence for Rdbd binding requires an additional 7 bp on both sides of the area mapped by interference assays, suggesting that non-sequence-specific as well as sequence-specific interactions between RTA and DNA contribute to high-affinity binding. To better understand the molecular interactions between RTA and the PAN promoter, an extensive mutagenesis study on the pPAN RRE was carried out by using EMSAs and reporter assays. These analyses revealed base pairs critical for both Rdbd binding in vitro and RTA transactivation in vivo of the PAN promoter. The results from methylation interference, deletion analysis, and mutagenesis using EMSAs and reporter assays were closely correlated and support the hypothesis that RTA activates PAN RNA expression through direct binding to DNA.

* Corresponding author. Mailing address: Department of Molecular and Medical Pharmacology, University of California at Los Angeles, Los Angeles, CA 90095-1735. Phone: (310) 794-5557. Fax: (310) 825-6267. E-mail: rsun{at}mednet.ucla.edu.

Journal of Virology, May 2002, p. 5000-5013, Vol. 76, No. 10
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.10.5000-5013.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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