Figure 3

Specific expression of reporter by the Rev-dependent lentiviral vector in HIV-infected primary macrophages. M-CSF-treated monocyte-derived macrophages were infected or were not infected with HIV_AD8, followed by Rev-dependent reporter virus vNL-GFP-RRE(SA). (A). Bright-field image of fixed cells with black bar representing 50 micrometers. Nuclei are prominent. Cells that were found to stain positive for HIV p24 are numbered in this image. B. Red fluorescence of identical field. All cells were stained for anti-p24 antibody, followed by a secondary red fluorescent antibody to detect productively infected cells. Intracellular red fluorescent foci identify productive infection; nine cells were identified in this field (B). We examined macrophage populations with four concentrations of HIV input (36, 72, 169, and 361 ng p24 HIV_AD8/10⁶ cells) followed by a constant input of reporter lentiviral vector (5 ng p24 vNL-GFP-RRE(SA)/10⁶ cells; VSV-G envelope). Independent of the input of HIV, approximately one fifth of the p24-positive cells expressed GFP (23.0 ± 2.8%; mean ± SD; n = 4), as shown by green fluorescence in this same field (C). Magnification was identical on all three images. The reporter virus was demonstrated to function in macrophages from three donors.