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Published online before print August 21, 2006

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© by The Society for Leukocyte Biology
Journal of Leukocyte Biology, doi:10.1189/jlb.0206126

Received for publication February 28, 2006.
Revised June 6, 2005.
Accepted for publication June 12, 2006.

Article

In vitro modeling of the HIV-macrophage reservoir

Amanda Brown ^*@, Hao Zhang , Peter Lopez , Carlos A. Pardo ^*, and Suzanne Gartner ^*

Johns Hopkins School of Medicine, *Department of Neurology and Center for Flow Cytometry, School of Public Health, Baltimore, Maryland; and Aaron Diamond AIDS Research Center, The Rockefeller University, New York, New York

^@ To whom correspondence should be addressed. E-mail: abrown76{at}jhmi.edu.

	Abstract

Macrophages are recognized as a putative reservoir for HIV-1, but whether HIV can establish latent infection in this cell type is not known. An in vitro model using long-term cultured primary human monocyte-derived macrophages (MDM) infected with an M-tropic, enhanced green fluorescent protein (EGFP) tagged reporter virus was developed to test the hypothesis that HIV can establish a latent infection of this cell type. The EGFP-IRES-Nef cassette allowed detection of early gene transcription. The expression of GFP+ MDM was followed with time and the GFP- population was purified and analyzed for evidence of latent infection. Interestingly, in MDM cultures propagated for over two months, distinct subpopulations of infected GFP+ cells were observed and quantitated. In particular, infected MDM that displayed a high level of transcription, characterized as the GFP hi group, yet produced low levels of the late viral gene product, p24, increased with time and represented 10% of the GFP+ population in long-term cultures. The high level production of early genes such as Nef, a protein that can facilitate viral immune escape, but low level of structural proteins such as p24 in the GFP hi population suggests that a subset of infected MDM can exhibit an alternative mode of replication. The GFP- MDM population obtained by a two-step purification protocol using flow cytometry and laser ablation contained integrated provirus as assessed by Alu-LTR real-time PCR analyses. A subset of these, were replication competent as shown by their ability to express GFP and/or p24 antigen after reactivation with IL-4.

Key Words: GFP • latency • laser capture • flow cytometry

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