Fibroblasts from healthy- normal donors (GM00500, GM09503, GM00409, and GM00499) as well as patients carrying mutations in the NPC1 (GM00110, GM17913, GM17921, GM03123) or NPC2 (GM17910, GM18439, GM18445, GM18455) genes were obtained from Coriell Repositories (Coriell Institute for Medical Research, Camden, NJ). The fibroblasts were maintained in Dulbecco modified Eagle medium (Gibco-BRL/Life Technologies, Gaithersburg, MD) supplemented with 15% defined fetal bovine serum (FBS-D) (HyClone, Logan, UT), L-glutamine, and 10 mM HEPES (pH 7.2). Cell viability was assessed by trypan blue exclusion. TZM-bl HIV-1 indicator cells were obtained from the NIH AIDS Research and Reference Reagent Program (Germantown, MD) and maintained in DMEM supplemented with 10% fetal calf serum (FCS), L-glutamine, and 10 mM HEPES (pH 7.2), and 100 U/ml penicillin and streptomycin (cDMEM). 293 T human embryonic kidney cells were maintained in DMEM supplemented with L-glutamine, and 10 mM HEPES (pH 7.2), and 10% FCS (HyClone, Logan, UT).
Vesicular stomatitis virus envelope glycoprotein G (VSV-G)-pseudotyped HIV-1 was prepared using 293T cells. Cells were co-transfected with pNL4.3-GFP plasmid (kind gift from Dr. Robert Silicano, Johns Hopkins School of Medicine) and the VSV-G expression vector pHEF-VSV-G using the calcium phosphate transfection method. Briefly, at 48 h post-transfection the culture supernatants containing virus particles were collected and filtered through a 0.45 μm filter. The virus was quantified by p24 ELISA and used for direct infection of fibroblasts.
Western blot analysis
Intracellular protein expression was analyzed by standard Western blot using the NuPAGE gel electrophoresis system (Invitrogen, Carlsbad, CA). Briefly, cells were lysed on ice for 30 min in RIPA buffer (50 mM Tris-HCl, Adjust to pH 7.41, 50 mM NaCl, 1 mM PMSF, 1 mM EDTA, 5 μg/ml protinin, 5 μg/ml Leupeptin, 1% Triton x-100, 1% Sodium deoxycholate, 0.1% SDS) supplemented with protease inhibitors (Roche catalogue no. 1873580). Lysates were then clarified of cell debris by centrifugation at 13,000 rpm at 4°C for 20 min. Lysates were run on 4-12% Bis-Tris gels, and then transferred onto nitrocellulose using a semi-dry transfer apparatus (BioRad). Membranes were blocked for 30 min in Superblock (Invitrogen, Carlsbad, CA) before probing with primary antibodies. Following washing 3 times in phosphate buffered saline containing 0.05% Tween-20 (PBS-T), the membranes were probed with HRP conjugated secondary antibodies. Chemiluminescent substrate (ECL, GE Healthcare Life Sciences) was used for detection.
The antibodies used in this study were rabbit polyclonal antibodies: anti-NPC1 (Novus Biologicals), anti-ABCA1 (Novus Biologicals), and anti-β-actin (Sigma-Aldrich). Rabbit polyclonal antibody against NPC2 was a kind gift from Dr. Peter Lobel (UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ). Gag monoclonal anti-p24 was purchased from Millipore (Temecula, CA). Secondary antibodies (HRP conjugated goat anti-rabbit or mouse heavy- and light-chain specific) were purchased from Jackson ImmunoResearch Laboratories (Westgrove, PA).
Infection of fibroblasts
Normal and NPC-deficient fibroblasts were seeded (3 × 105 cells) in 10-cm culture plates in cDMEM. Cells were allowed to grow for 12-16 h and infected with 3 μg of VSV G-NL4.3, normalized for HIV-1 capsid p24. After 24 h, the cells were washed to remove virus and cultured in fresh media. At 96 h post infection, the cells and supernatants were harvested. Supernatants were collected and stored at -80°C for further analysis. Cells were trypsinized, washed 3 times in PBS, and prepared for Western blot or flow cytometry analysis. Infection efficiencies were determined by flow cytometry. Briefly, cells were fixed with 2% paraformaldehyde, permeabilized using 0.1% saponin, and stained with fluorophore-conjugated antibodies. Stained cells were analyzed using the Becton FACSCalibur flow cytometer (Cell Quest software). Virus produced from these cells was quantified by p24 ELISA and normalized to the percentage of cells that were positive for Gag.
Fibroblasts were cultured and infected with VSVG-NL4.3 as previously described. At 24 h post infection, the input virus was removed and the cells were washed 3 times in PBS. Cells were then cultured in medium alone, medium supplemented with 15% Lipoprotein Deficient Serum (LPD-S), or medium containing 5 μM TO-901317 (Sigma-Aldrich, St. Louis, MO). Cells and supernatants were harvested 96 h post infection.
Virus titer determination
Virus released from infected cell cultures was measured using an ELISA developed in our laboratory to measure viral p24 antigen (sensitivity 50-2000 pg/mL).
Cell staining and immunofluorescence assay
Cells were grown on 35-mm glass bottom dishes (MatTek Corporation, Ashland, MA). For LysoTracker Red staining, cells were incubated for 2 h at 37°C with 75 nM LysoTracker Red DND-99 and then fixed for 15 min in 2% paraformaldehyde in PBS. The cells were permeabilized with 5% normal goat serum in BD Cytofix/Cytoperm solution (BD Biosciences, San Diego, CA). Gag and cholesterol staining was performed using KC57-FITC (Beckman Coulter, Inc., Fullerton, CA) and filipin (Sigma-Aldrich, St. Louis, MO) respectively. The reagents were diluted in BD Cytofix/Cytoperm solution and added to the cells for 30 min at room temperature. Cells were washed 2 times in PBS and anti-fade solution was applied before imaging. Photographs were taken using a Nikon TE2000 wide-field microscope (Nikon Instruments, Melville, NY) with a 40X oil objective. The FITCx was utilized to eliminate cross excitation between DAPI and FITC.
After cells were stained and fixed as previously described, the cells were washed in PBS and resuspended in 1 mL FACS buffer (1XPBS, 5% FCS, and 0.1% sodium azide). The cells were then analyzed on a FACSCalibur (Becton Dickson) flow cytometer.
TZM-bl cells were plated in 96-well microtiter plate at a density of 12.5 × 103/100 μl. After 24 h, cells were infected for 12 h with virus generated from infected fibroblasts. The input virus was removed and the cells were washed 3 times in PBS and maintained under normal culture conditions. At 24 h-post infection, cells were washed 3 times in PBS and luciferase assay was performed using a luciferase reporter gene assay system (Luc-Screen kit; Applied Biosystem, Foster City, CA). Relative luciferase units were normalized to input p24 values.
Cellular and virion associated cholesterol was measured using the Amplex®Red Cholesterol Assay Kit (Invitrogen, Carlsbad, CA). Samples were processed in accordance with the manufacturer protocol. To measure unesterified cholesterol, the cholesterol esterase enzyme was eliminated from the reaction mixture. Cholesterol content for cells and virions was normalized to protein and p24 concentration. Protein concentrations of cell lysates were determined by BCA assay (Fisher, Waltham, MA). Virus p24 concentrations were measured by p24 ELISA.
Isolation of lipid rafts
Lipid rafts were isolated from cell lysates as previously described by Popik et al.