Fig. 1. ESR spectra of free 5-DSA (A), plasma membranes from intact MOLT-4/C-2 (B) and MT-2 (E) cells, and viral envelopes from intact C-2 (C), CD45-depleted C-2 (D), C-2(MT-2) (F), and CD45-depleted C-2(MT-2) (G) viruses. The outer and inner hyperfine splittings, 2A and 2A were measured as shown by arrows. Asterisks show spectra of free 5-DSA remaining in each sample. Order parameters (S-values) of B, C, D, E, F, and G were calculated at 0.611, 0.729, 0.776, 0.585, 0.704, and 0.721, respectively. Scale of the horizontal axis (magnetic field) is shown as 10 G (gauss).

Fig. 2. Dependence of the fluidity (A) of the plasma membrane from intact MOLT-4/C-2 cells (square) and the viral envelope from intact C-2 viruses (circle) on temperature, and dependence of HIV-1 infectivity (B) on temperature. GHOST/CXCR4 cells in a flat-bottomed 48-well plate were treated with 100 μl/well of NL43-luc virus at room temperature (RT), 37 and 40 °C for 1 h. After washing once with complete medium, the infected cells were cultured at 37 °C for 2 days before measuring luciferase activity. All experiments in (A,B) were carried out in triplicate, and solid lines and bars show the means ± SD.

Fig. 3. Effects of xylocaine (A) and DPPC (B) on HIV-1 infectivity and fluidity of plasma membranes from MT-2 cells (C). (A,B) GHOST/CXCR4 cells in a flat-bottomed 48-well plate were treated with a 100 μl/well mixture of NL43-luc virus with serially diluted xylocaine or DPPC at 37 °C for 1 h. After washing once with complete medium, the cells were cultured at 37 °C for 2 days before assessing luciferase activity. (C) MT-2 cells (7 × 10⁶) were treated with 0.2% xylocaine or DPPC (10 or 200 μg/ml) at 37 °C for 30 min, washed once and resuspended with 1 ml of complete medium. The cells were then labeled with 5-DSA and examined by ESR spectroscopy. All experiments in (A–C) were carried out in triplicate, and solid lines and bars show the means ± SD.

Fig. 4. Relationship between HIV-1 infectivity and membrane fluidity. The infectivity ratio was calculated as follows: luciferase activity in test/luciferase activity in control, and the order parameter ratio as: order parameter in test/order parameter in control. Circles from the left indicate 40 °C, 0.2% xylocaine, 0.05% Tween 20, 200 μg/ml DPPC, 10 μg/ml DPPC, anti-HLA-II and 25 °C.

Fig. 5. Effects of T140 on post-attachment enhancement (PAE) at 40 °C (A) or by 0.2% xylocaine (B). GHOST/CXCR4 cells that had been seeded the previous day on a flat 48-well plate were infected with 100 μl/well of NL43-luc pseudovirus. The viral adsorption took place at room temperature (RT) for 1 h. After washing once with complete medium, cells were incubated at 37 or 40 °C (A), or with or without 0.2% xylocaine (B), for 1 h in the presence or absence of T140 and then washed once. The plate was incubated at 37 °C for 2 days and luciferase activity was measured. All experiments in (A,B) were carried out in triplicate, and solid lines and bars show the means ± SD.

Order parameter and cholesterol/protein (C/P) of plasma membrane and viral envelope