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Journal of Virology, February 2009, p. 1811-1822, Vol. 83, No. 4
0022-538X/09/$08.00+0     doi:10.1128/JVI.02302-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

High-Resolution Functional Profiling of a Gammaherpesvirus RTA Locus in the Context of the Viral Genome ^,

Vaithilingaraja Arumugaswami,¹ Ronika Sitapara,² Seungmin Hwang,¹ Moon Jung Song,³ Tuyet Ngoc Ho,¹ Nancy Qi Su,¹ Eric Y. Sue,¹ Vidhya Kanagavel,¹ Fangfang Xing,¹ Xiaolin Zhang,¹ Minglei Zhao,¹ Hongyu Deng,⁴ Ting-Ting Wu,¹ Sudhakar Kanagavel,⁵ LuLu Zhang,⁶ Sugandha Dandekar,⁶ Jeanette Papp,⁶ and Ren Sun^1,2*

Department of Molecular and Medical Pharmacology, University of California at Los Angeles, Los Angeles, California 90095,¹ Molecular Biology Institute, University of California at Los Angeles, Los Angeles, California 90095,² College of Life Sciences and Biotechnology, Korea University, Seoul 136-713, Korea,³ School of Dentistry, University of California at Los Angeles, Los Angeles, California 90095,⁴ Infosys, 400 Galleria Parkway, Suite 1490, Atlanta, Georgia 30339,⁵ UCLA Genotyping and Sequencing Core, Department of Human Genetics, University of California at Los Angeles, Los Angeles, California 90095⁶

Received 3 November 2008/ Accepted 1 December 2008

Gammaherpesviruses Kaposi's sarcoma-associated herpesvirus and Epstein-Barr virus are associated with multiple human cancers. Our goal was to develop a quantitative, high-throughput functional profiling system to identify viral cis-elements and protein subdomains critical for virus replication in the context of the herpesvirus genome. In gamma-2 herpesviruses, the transactivating factor RTA is essential for initiation of lytic gene expression and viral reactivation. We used the RTA locus as a model to develop the functional profiling approach. The mutant murine gammaherpesvirus 68 viral library, containing 15-bp random insertions in the RTA locus, was passaged in murine fibroblast cells for multiple rounds of selection. The effect of each 15-bp insertion was characterized using fluorescent-PCR profiling. We identified 1,229 insertions in the 3,845-bp RTA locus, of which 393, 282, and 554 were critically impaired, attenuated, and tolerated, respectively, for viral growth. The functional profiling phenotypes were verified by examining several individual RTA mutant clones for transactivating function of the RTA promoter and transcomplementing function of the RTA-null virus. Thus, the profiling approach enabled us to identify several novel functional domains in the RTA locus in the context of the herpesvirus genome. Importantly, our study has demonstrated a novel system to conduct high-density functional genetic mapping. The genome-scale expansion of the genetic profiling approach will expedite the functional genomics research on herpesvirus.

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* Corresponding author. Mailing address: Molecular and Medical Pharmacology, Associate Dean of Graduate Studies, David Geffen School of Medicine, University of California, Los Angeles, 23-120 Center for Health Sciences, Los Angeles, CA 90095-1735. Phone: (310) 794-5557. Fax: (310) 825-6267. E-mail: rsun{at}mednet.ucla.edu

Published ahead of print on 10 December 2008.

Supplemental material for this article may be found at http://jvi.asm.org/.

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Journal of Virology, February 2009, p. 1811-1822, Vol. 83, No. 4
0022-538X/09/$08.00+0     doi:10.1128/JVI.02302-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.