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Figure 4 | Retrovirology

Figure 4

From: Selective killing of HIV-1-positive macrophages and T cells by the Rev-dependent lentivirus carrying anthrolysin O from Bacillus anthracis

Figure 4

Inhibition of AnlO-mediated cytolysis of producer cells by β-cyclodextrin derivatives. (A) Structure of the membrane pore blocker 6-boc orthinine amide-β-cyclodextrin (6-BOCD). (B) Inhibition of AnlO-mediated cytolysis by 6-BOCD. HEK293T cells were cotransfected with pCMVΔR8.2 and either pNL-GFP-RRE-SA (labeled as GFP) or pNL-AnlO-GFP-RRE-SA (labeled as AnlO-GFP) in the absence or presence of various doses of 6-BOCD. Increases in viable GFP cells were measured by flow cytometry. (C) Lack of effect of 6-BOCD on α-hemolysin. Cells were similarly cotransfected with pCMVΔR8.2 and pNL-α-HL-GFP-RRE-SA (labeled as α-HL-GFP) in the absence or presence of various doses of 6-BOCD. (D) No inhibition of 6-BOCD on viral infectivity. Lentiviral particles, vNL-GFP-RRE-SA, were generated by cotransfection of HEK293T cells with pNL-GFP-RRE-SA, pCMVΔR8.2, and pCMV-VSV-G in the absence or presence of different concentrations of 6-BOCD. The resulting viral particles were used to infect an HIV-1-positive cell, J1.1 (using an equal p24 level of viruses, 150 ng p24 per million cells). The percentage of GFP positive J1.1 cells was used as an indicator for viral infectivity. (E) The Rev-dependent AnlO lentiviral vector is suicidal in HIV-1-positive cells. The HIV-1-positive cell, J1.1, was infected with vNL-AnlO-RRE-SA (300 ng p24 per million cells). As a control, HIV-1-negative Jurkat cells were identically infected. Following infection, cells were harvested at different times and total cellular DNA was extracted and PCR amplified with primers for the anlO gene (AnlO). As a control, the DNA was also amplified with primers for the cellular β-actin pseudogene (β-actin) to ensure that the same number of cells was used.

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