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Journal of Virology, September 2003, p. 9451-9462, Vol. 77, No. 17
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.17.9451-9462.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Comparative Study of Regulation of RTA-Responsive Genes in Kaposi's Sarcoma-Associated Herpesvirus/Human Herpesvirus 8

Moon Jung Song, Hongyu Deng, and Ren Sun*

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

Received 10 January 2003/ Accepted 3 June 2003

Replication and transcription activator (RTA) (also referred to as ORF50), an immediate-early gene product of Kaposi's sarcoma-associated herpesvirus (KSHV)/(human herpesvirus 8), plays a critical role in balancing the viral life cycle between latency and lytic replication. RTA has been shown to act as a strong transcription activator for several downstream genes of KSHV. Direct binding of RTA to DNA is thought to be one of the important mechanisms for transactivation of target genes, while indirect mechanisms are also implicated in RTA transactivation of certain selected genes. This study demonstrated direct binding of the DNA-binding domain of RTA (Rdbd) to a Kaposin (Kpsn) promoter sequence, which is highly homologous to the RTA-responsive element (RRE) of the PAN promoter. We undertook a comparative study of the RREs of PAN RNA, ORF57, vIL-6, and Kpsn to understand how RTA regulates gene expression during lytic replication. Comparing RNA abundance and transcription initiation rates of these RTA target genes in virus-infected cells suggested that the transcription initiation rate of the promoters is a major determinant of viral gene expression, rather than stability of the transcripts. RTA-mediated transactivation of reporters containing each RRE showed that their promoter strengths in a transient-transfection system were comparable to their transcription rates during reactivation. Moreover, our electrophoretic mobility shift assays of each RRE demonstrated that the highly purified Rdbd protein directly bound to the RREs. Based on these results, we conclude that direct binding of RTA to these target sequences contributes to their gene expression to various extents during the lytic life cycle of KSHV.

* 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, September 2003, p. 9451-9462, Vol. 77, No. 17
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.17.9451-9462.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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