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. 2004 Jan;78(2):568–575. doi: 10.1128/JVI.78.2.568-575.2004

FIG. 2.

FIG. 2.

Optimizing the antiviral vector. (A) PM-1 cells expressing the control vector or antiviral vector M87-Ineo (□) or M87/C46-Ineo (▨) were transduced with a lentiviral vector expressing EGFP pseudotyped with the indicated viral envelope glycoprotein. In HxB2resI and HxB2resII, mutations that confer resistance to C36 (T-20) have been introduced. The inhibitory activity (fold inhibition) is represented by the ratio of the transduction efficiency for PM-1 cells expressing the control vector relative to the cells expressing the antiviral vector. The vector supernatants were used at a dilution that gave between 1 and 10% marking in the control cells, since in this range a linear relation between vector input and marking was observed (data not shown). A total of 105 cells per sample were analyzed by FACS to allow accurate analysis of marking even in the inhibited cultures with fewer than 1% GFP-positive cells. The mean values of two independent selected bulk cultures per vector are given. (B) Wild-type PM-1 (shaded curve) and PM-1 cells expressing M87-Ineo (black line) or M87o-Ineo (gray line) were stained with the human monoclonal antibody 2F5 that detects an epitope in the antiviral peptides and a phycoerythrin-conjugated goat-anti-human serum followed by flow cytometric analysis. (C) G418-selected bulk cultures of PM-1 expressing the control vector MP1neo (•) M87-Ineo (□) or M87o-Ineo (▴) were infected with the T-tropic isolate HIV-1NL4-3 or the dualtropic primary isolate D117-II at an MOI of 0.01 or 0.0001, respectively. HIV-1 p24 antigen production into the supernatant was monitored by ELISA. Experiments were performed in triplicate. (D) M87o and M87oRRE have comparable antiviral activity. PM-1 cells were transduced with M87o, M87oRRE, orMPIN and then sorted for expression of C46 or mock sorted (MP1N) by FACS. Purified bulk cultures were infected with NL4-3 at an MOI of 0.01. HIV-1 production was quantified as described in the text. The assays were performed in triplicates. Mean values are shown. Symbols: •, MP1neo; ✠, M87oRRE; ▴, M87o.