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Journal of Virology, September 2009, p. 8662-8673, Vol. 83, No. 17
0022-538X/09/$08.00+0     doi:10.1128/JVI.00874-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Measurement of Human Immunodeficiency Virus Type 1 Preintegration Transcription by Using Rev-Dependent Rev-CEM Cells Reveals a Sizable Transcribing DNA Population Comparable to That from Proviral Templates

Subashini R. Iyer,¹ Dongyang Yu,¹ Angélique Biancotto,² Leonid B. Margolis,² and Yuntao Wu^1*

Department of Molecular and Microbiology, George Mason University, Manassas, Virginia 20110,¹ Program in Physical Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, Maryland 20892²

Received 30 April 2009/ Accepted 15 June 2009

Preintegration transcription is an early process in human immunodeficiency virus type 1 infection and has been suggested to occur at a low level. The templates have also been suggested to represent a small population of nonintegrated viral DNA, particularly the two-long-terminal-repeat (2-LTR) circles. However, these determinations were made by either using PCR amplification of viral transcripts in bulk cell populations or utilizing the LTR-driving reporter cells that measure the synthesis of Tat. The intrinsic leakiness of LTR often makes the measurement of low-level viral transcription inaccurate. Since preintegration transcription also generates Rev, to eliminate the nonspecificity associated with the use of LTR alone we have developed a novel Rev-dependent indicator cell, Rev-CEM, to measure preintegration transcription based on the amount of Rev generated. In this report, using Rev-CEM cells, we demonstrate that preintegration transcription occurs on a much larger scale than expected. The transcribing population derived from nonintegrated viral DNA was comparable (at approximately 70%) to that derived from provirus in a productive viral replication cycle. Nevertheless, each nonintegrated viral DNA template exhibited a significant reduction in the level of transcriptional activity in the absence of integration. We also performed flow cytometry sorting of infected cells to identify viral templates. Surprisingly, our results suggest that the majority of 2-LTR circles are not active in directing transcription. It is likely that the nonintegrated templates are from the predominant DNA species, such as the full-length, linear DNA. Our results also suggest that a nonintegrating lentiviral vector can be as effective as an integrating vector in directing gene expression in nondividing cells, with the proper choice of an internal promoter.

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* Corresponding author. Mailing address: Department of Molecular and Microbiology, George Mason University, 10900 University Boulevard, MS 4E3, Manassas, VA 20110. Phone: (703) 993-4299. Fax: (703) 993-4288. E-mail: ywu8{at}gmu.edu

Published ahead of print on 24 June 2009.

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Journal of Virology, September 2009, p. 8662-8673, Vol. 83, No. 17
0022-538X/09/$08.00+0     doi:10.1128/JVI.00874-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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