Figure 5

Specific targeting of HIV-1-infected macrophages by the lentiviral vector carrying anlO. (A) Schematic representation of the three constructs used to generate the Rev-dependent AnlO lentivirus. HEK293T cells were cotransfected with pNL-AnlO-GFP-RRE-SA, pCMVΔ8.2, and the M-tropic envelope construct, pCAGGSSF162gp160, in the presence of 6-BOCD. Viruses were harvested, concentrated, and used to infect human macrophages. (B) Specific killing of HIV-1-positive macrophages by the Rev-dependent AnlO lentiviral vector. Human macrophages were derived from peripheral monocytes. Cells were not infected (Cell) or infected with NL4-3.HSA.R+E-(VSV-G) (m.o.i. 0.1). Following HIV infection, at 24 hours, HIV-1-infected cells were super-infected with the lentivirus vNL-AnlO-RRE-SA (approximate m.o.i. 0.5 – 1) or with the same lentivirus using a 10-fold higher dosage (*). HIV-1-infected cells were stained with a PE-labeled rat monoclonal antibody against mouse CD24 (HSA) and analyzed by flow cytometry at 10 days post infection with HIV-1. (C) Undetectable cytolytic activity of the Rev-dependent AnlO lentiviral vector in un-infected macrophages. To determine whether vNL-AnlO-RRE-SA can non-specifically kill un-infected macrophages, cells were similarly infected with highly concentrated virus (m.o.i. 5 – 10). Following infection for two weeks, cells were harvested at different times and analyzed by propidium iodide (PI) staining and flow cytometry for cytolysis. As controls, cells were also mock infected with medium, or the same dose of an empty vector virus, vNL-RRE-SA. Cells were also treated with puromycin (500 ng/ml) to induce non-specific cytolysis for the validation of PI staining and flow cytometry analysis.