Nef increases infectivity of HIV via lipid rafts Yong-Hui Zheng, Ana Plemenitas, Thomas Linnemann, Oliver T. Fackler, B.Matija Peterlin  Current Biology  Volume 11, Issue 11, Pages 875-879 (June 2001) DOI: 10.1016/S0960-9822(01)00237-8

Figure 1 Nef directs the budding of HIV to lipid rafts. (a) Isolation of lipid rafts. 293T cells were separated into cytosol , C; plasma membrane, PM; and detergent resistant membrane, DRM, lipid rafts fractions. Samples were then normalized to amounts of protein and analyzed for the presence of caveolin, CD45, and GM1 by Western blotting. (b) Determination of amounts of cholesterol in different fractions. Amounts of cholesterol in samples from (a) were determined and are presented in μg/ml. They were normalized to concentrations of protein in these fractions. (c) Analysis of viral proteins in different fractions. 293T cells were transfected with three HIV proviral clones, pNL-43, pNL-ΔN, and pNL-ΔMyrNef. Infected cells were fractionated by sucrose gradient centrifugation. Equal amounts of protein were loaded and analyzed by Western blotting. (d) Quantification of Gag in different fractions. Samples from (c) were normalized to amounts of total cellular protein, and Gag was quantified by p24Gag antigen capture ELISA. (e) Determination of GM1 on the envelopes from different virions. HIV was produced from 293T cells, and viral particles were stained with cholera toxin B and captured by the goat anti-cholera toxin B antibody. Results represent the percentage of cholera toxin B bound compared to the input virus. The slopes represent the following viruses: open squares, pNL-43; open diamonds, pNL-ΔN; and open circles, pNL-ΔMyrNef. The open triangles represent the control experiment in which pNL-43 virus was isolated with the goat IgG after cholera toxin B staining. Standard deviations from the mean are representative of three independent experiments Current Biology 2001 11, 875-879DOI: (10.1016/S0960-9822(01)00237-8)

Figure 2 Cellular cholesterol affects wild-type HIV infectivity. (a) Determination of cellular cholesterol under different culture conditions. 293T cells were cultured in a medium containing 10% FCS in the absence or presence of 25 μM L-659,699 for 6 hr. They were also cultured in the medium containing 10% LPPS in the absence or presence of 25 μM L-659,699 for 6 hr. Cholesterol was extracted and quantified. Results are presented as described in Figure 1b. (b) Determination of viral infectivity by a single round replication assay (MAGI assay). 293T cells were transfected with pNL-43 and pNL-ΔN and grown under the same culture conditions as those described in (a), and virion infectivity was determined. The infectivity of collected viruses was defined as the number of blue cells divided by the amount of p24Gag in the input virus. Results represent relative values, with the infectivity of pNL-ΔN virus from 293T cells grown in LPPS set to 1 Current Biology 2001 11, 875-879DOI: (10.1016/S0960-9822(01)00237-8)

Figure 3 Lipid rafts are essential for Nef to enhance HIV infectivity. (a) Determination of total cellular cholesterol in CHO cells. CHO-K1 and CHO-215 cells were cultured in McCoy's 5A medium containing 10% FCS or LPPS. Cholesterol was extracted and quantified as described in Figures 1 and 2. (b) Determination of levels of free cholesterol in DRM of CHO cells. CHO-K1 and CHO-215 cells were cultured in FCS or LPPS. Their DRM was isolated, and cholesterol levels were determined as in Figure 1b. (c) Determination of GM1 in lipid rafts of CHO cells. DRM from CHO cells was analyzed by Western blotting, and the bands were quantified in arbitrary optical units (OU) by Phospho-Imager. (d) Determination of the infectivity of HIV produced from CHO cells. HIV was produced from CHO-K1 and CHO-215 cells, which were cultured in FCS or LPPS, and viral infectivity was measured by the MAGI assay as in Figure 2b. Results represent relative values, with the infectivity of pNL-ΔN virus from CHO-K1 cells grown in LPPS set to 1 Current Biology 2001 11, 875-879DOI: (10.1016/S0960-9822(01)00237-8)