In vitro modeling of the HIV-macrophage reservoir.
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.
Brown
Amanda
A
Johns Hopkins School of Medicine, Department of Neurology, 600 North Wolfe St., Baltimore, MD 21287, USA. abrown76@jhmi.edu
Zhang
Hao
H
Lopez
Peter
P
Pardo
Carlos A
CA
Gartner
Suzanne
S
D049109:Cell Proliferation; D004279:DNA, Viral / Q000032:analysis; D020869:Gene Expression Profiling; D015754:Genes, nef / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D015658:HIV Infections / Q000276:immunology / Q000821:virology*; D015497:HIV-1 / Q000235:genetics* / Q000276:immunology; D006801:Humans; D008264:Macrophages / Q000276:immunology / Q000821:virology*; D008957:Models, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods
D004279:DNA, Viral; D049452:Green Fluorescent Proteins