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Figure 6 | Retrovirology

Figure 6

From: Inhibition of HIV-1 endocytosis allows lipid mixing at the plasma membrane, but not complete fusion

Figure 6

The effect of dynasore on the HIV-1 fusion-competence and on the curvature of lipid bilayers is minimal. (A) Concentrated stocks of JRFL and HXB2 pseudoviruses (1·107 IU/ml) were pretreated with 80 μM dynasore in DMEM for 20 min at 37°C. Virus inoculum was diluted 20-fold prior to adding to TZM-bl cells and centrifuging at 4°C to aid binding. Cells were washed, and fusion was triggered by shifting to 37°C for 90 min in DMEM/10% FBS and measured by the BlaM assay. (B) Dynasore and MiTMAB were mixed with DiPoPE lipid and the temperature of lamellar-HII transition (TH) was measured by differential scanning calorimetry. Each sample contained 5 mg of DiPoPE and varying amounts of dynasore or MiTMAB suspended in 0.8 ml PIPES buffer (20 mM PIPES, 1 mM EDTA, 140 mM NaCl, pH 7.4). The sample and the same reference buffer were added to separate cells. Temperature scans were made from 15 to 55°C at a 1°/min scan rate. The transition temperature for each mixture was determined using a curve fitting program DA-2 supplied by Microcal, Inc. (Northampton, MA). This transition temperature (TH) is plotted against the mole fraction of drug added. (C) The waiting time from triggering fusion to HXB2 hemifusion events (fast DiD decay) with TZM-bl cells was determined in the presence of dynasore and plotted as the number of events over time (circles). For comparison, the kinetics of hemifusion with the plasma membrane in the absence of dynasore is re-plotted from [23] (triangles).

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