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Journal of Virology, November 2002, p. 10942-10950, Vol. 76, No. 21
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.21.10942-10950.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

A Sensitive, Quantitative Assay for Human Immunodeficiency Virus Type 1 Integration

Una O'Doherty,1* William J. Swiggard,2 Deepa Jeyakumar,1 David McGain,1 and Michael H. Malim3

Departments of Pathology and Laboratory Medicine,1 Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania,2 Department of Infectious Diseases, Guy's, King's and St. Thomas' School of Medicine, King's College London, London, England3

Received 26 April 2002/ Accepted 15 July 2002

Quantitative methods to measure human immunodeficiency virus type 1 (HIV-1) integration promise to be important tools in dissecting the mechanisms whereby latent reservoirs of provirus are established, most notably in the resting T cells of patients receiving antiretroviral therapy. Here we describe a fluorescence-monitored, nested PCR assay that is able to quantify the relatively rare integration events that occur within these cells. Following DNA extraction, a nonkinetic preamplification step is performed with primers that bind genomic Alu elements and HIV-1 gag sequences, under conditions where primers, deoxynucleoside triphosphates, and enzyme are not limiting. This is followed by a kinetic PCR that quantitates HIV-1 long terminal repeat sequences. A T-cell-based integration standard which reflects the randomness of HIV-1 integration is also described. The assay is 10 to 100 times more sensitive than previously reported quantitative Alu PCR-based integration assays. It is specific for integration events, since no proviruses are detected in cells infected either in the presence of an integrase inhibitor or with an integrase-deficient virus. This method promises to provide important new insights into the processes underlying the accumulation and persistence of latent HIV-1 reservoirs and may eventually be useful clinically in monitoring the eradication of latent virus by novel therapies.

* Corresponding author. Mailing address: Transfusion Medicine Division, Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, 402C Johnson Pavilion/6060, 3610 Hamilton Walk, Philadelphia, PA 19104-6060. Phone: (215) 573-7273. Fax: (215) 573-4856. E-mail: unao{at}mail.med.upenn.edu.

Journal of Virology, November 2002, p. 10942-10950, Vol. 76, No. 21
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.21.10942-10950.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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