Figure 2

Specificity of the Rev-dependent lentiviral vector in HIV-1- positive T cells and macrophages. (A) Schematic representation of the three constructs used to generate the Rev-dependent GFP lentivirus. HEK293T cells were cotransfected with pNL-GFP-RRE-SA, pCMVΔ8.2, and the VSV-G construct. Viruses were harvested, concentrated, and used to infect a human T cell line, CEM-SS, as well as primary human macrophages. (B) Specificity of the Rev-dependent lentiviral vector in HIV-1-positive T cells. CEM-SS cells were not infected (a) or infected with NL4-3.HSA.R+E-(VSV-G) (d, 500 ng p24 per million cells), a VSV-G pseudotyped HIV-1 strain with the murine heat-stable antigen CD24 (HSA) gene inserted into the nef region that allows HIV-1-positive cells to be monitored by surface staining of HSA. At 24 hours, cells were superinfected with lentivirus vNL-GFP-RRE-SA (d, m.o.i. 10). For comparison, cells were also singly infected with either vNL-GFP- RRE-SA (b) or NL4-3.HSA.R+E-(VSV-G) (c). At 72 hours, cells were harvested, stained with a PE-labeled rat monoclonal antibody against mouse CD24 (HSA), and then analyzed on a flow cytometer for both HSA and GFP expression. Isotype staining was not shown. (C) Specificity of the Rev-dependent lentiviral vector in HIV-1-positive macrophages. Human macrophages were derived from peripheral monocytes by culturing in 10 ng/ml M-CSF for two weeks. Cells were not infected (e) or infected with HIV- 1(AD8) (h, 380 ng p24 per million). At 24 hours, cells were superinfected with lentivirus vNL-GFP-RRE-SA (h, m.o.i. 10). For comparison, cells were also singly infected with either vNL-GFP-RRE-SA (f) or HIV-1(AD8) (g). At 72 hours, cells were harvested and analyzed on a flow cytometer for GFP expression. (D) Fluorescent microscopy of GFP expression in macrophages infected with HIV-1 and the Rev-dependent GFP lentiviral vector. Cells in (C, h) were also examined with fluorescent microscope. The left and right panels show the bright and green fluorescent fields of the same cells. Red arrows indicate an HIV-1-infected cell expressing the GFP protein.