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Journal of Virology, November 2008, p. 10709-10723, Vol. 82, No. 21
0022-538X/08/$08.00+0     doi:10.1128/JVI.01012-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Identification of Direct Transcriptional Targets of the Kaposi's Sarcoma-Associated Herpesvirus Rta Lytic Switch Protein by Conditional Nuclear Localization{triangledown}

Wei Bu ,1,{dagger},{ddagger} Diana Palmeri,1,{dagger} Raghu Krishnan,1 Roxana Marin,1 Virginie M. Aris,2 Patricia Soteropoulos,2 and David M. Lukac1*

Department of Microbiology and Molecular Genetics, Graduate School of Biomedical Sciences,1 Center for Applied Genomics, Public Health Research Institute, University of Medicine and Dentistry of New Jersey/New Jersey Medical School, Newark, New Jersey2

Received 14 May 2008/ Accepted 14 August 2008

Lytic reactivation from latency is critical for the pathogenesis of Kaposi's sarcoma-associated herpesvirus (KSHV). We previously demonstrated that the 691-amino-acid (aa) KSHV Rta transcriptional transactivator is necessary and sufficient to reactivate the virus from latency. Viral lytic cycle genes, including those expressing additional transactivators and putative oncogenes, are induced in a cascade fashion following Rta expression. In this study, we sought to define Rta's direct targets during reactivation by generating a conditionally nuclear variant of Rta. Wild-type Rta protein is constitutively localized to cell nuclei and contains two putative nuclear localization signals (NLSs). Only one NLS (NLS2; aa 516 to 530) was required for the nuclear localization of Rta, and it relocalized enhanced green fluorescent protein exclusively to cell nuclei. The results of analyses of Rta NLS mutants demonstrated that proper nuclear localization of Rta was required for transactivation and the stimulation of viral reactivation. RTA with NLS1 and NLS2 deleted was fused to the hormone-binding domain of the murine estrogen receptor to generate an Rta variant whose nuclear localization and ability to transactivate and induce reactivation were tightly controlled posttranslationally by the synthetic hormone tamoxifen. We used this strategy in KSHV-infected cells treated with protein synthesis inhibitors to identify direct transcriptional targets of Rta. Rta activated only eight KSHV genes in the absence of de novo protein synthesis. These direct transcriptional targets of Rta were transactivated to different levels and included the genes nut-1/PAN, ORF57/Mta, ORF56/Primase, K2/viral interleukin-6 (vIL-6), ORF37/SOX, K14/vOX, K9/vIRF1, and ORF52. Our data suggest that the induction of most of the KSHV lytic cycle genes requires additional protein expression after the expression of Rta.

* Corresponding author. Mailing address: 225 Warren St., ICPH E350C, Newark, NJ 07101. Phone: (973) 972-4483, ext. 0907. Fax: (973) 972-8981. E-mail: Lukacdm{at}umdnj.edu

{triangledown} Published ahead of print on 20 August 2008.

{dagger} These authors contributed equally to this work.

{ddagger} Present address: HIV DRP Retroviral Replication Laboratory, National Cancer Institute, Frederick, MD.

Journal of Virology, November 2008, p. 10709-10723, Vol. 82, No. 21
0022-538X/08/$08.00+0     doi:10.1128/JVI.01012-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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