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Journal of Virology, April 2005, p. 5185-5202, Vol. 79, No. 8
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.8.5185-5202.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

A Novel Assay Allows Genotyping of the Latent Reservoir for Human Immunodeficiency Virus Type 1 in the Resting CD4+ T Cells of Viremic Patients

Daphne Monie,1 Rachel P. Simmons,1,{dagger} Richard E. Nettles,1 Tara L. Kieffer,1 Yan Zhou,1 Haili Zhang,1 Sharon Karmon,1 Roxann Ingersoll,1 Karen Chadwick,2 Hao Zhang,2 Joseph B. Margolick,2 Thomas C. Quinn,1,3 Stuart C. Ray,1 Megan Wind-Rotolo,1 Michael Miller,4 Deborah Persaud,1 and Robert F. Siliciano1,5*

School of Medicine,1 Bloomberg School of Public Health, Johns Hopkins University,2 Howard Hughes Medical Institute, Baltimore,5 National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland,3 Merck Research Laboratories, West Point, Pennsylvania4

Received 26 August 2004/ Accepted 1 December 2004

A latent reservoir for human immunodeficiency virus type 1 (HIV-1) consisting of integrated provirus in resting memory CD4+ T cells prevents viral eradication in patients on highly active antiretroviral therapy (HAART). It is difficult to analyze the nature and dynamics of this reservoir in untreated patients and in patients failing therapy, because it is obscured by an excess of unintegrated viral DNA that constitutes the majority of viral species in resting CD4+ T cells from viremic patients. Therefore, we developed a novel culture assay that stimulates virus production from latent, integrated HIV-1 in resting CD4+ T cells in the presence of antiretroviral drugs that prevent the replication of unintegrated virus. Following activation, resting CD4+ T cells with integrated HIV-1 DNA produced virus particles for several days, with peak production at day 5. Using this assay, HIV-1 pol sequences from the resting CD4+ T cells of viremic patients were found to be genetically distinct from contemporaneous plasma virus. Despite the predominance of a relatively homogeneous population of drug-resistant viruses in the plasma of patients failing HAART, resting CD4+ T cells harbored a diverse array of wild-type and archival drug-resistant viruses that were less fit than plasma virus in the context of current therapy. These results provide the first direct evidence that resting CD4+ T cells serve as a stable reservoir for HIV-1 even in the setting of high levels of viremia. The ability to analyze archival species in viremic patients may have clinical utility in detecting drug-resistant variants not present in the plasma.

* Corresponding author. Mailing address: Johns Hopkins University School of Medicine, Broadway Research Building, Room 879, 733 North Broadway St., Baltimore, MD 21205. Phone: (410) 955-2958. Fax: (443) 287-6218. E-mail: rsiliciano{at}jhmi.edu.

{dagger} Present address: Massachusetts General Hospital, Boston, Mass.

Journal of Virology, April 2005, p. 5185-5202, Vol. 79, No. 8
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.8.5185-5202.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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