Cell culture and reagents
HEK 293T/17 cells (ATCC, Manassas, VA) and TZM-bl cells were cultured at 37°C with 5% of CO2 in complete media containing 90% of DMEM and 10% of FBS (HyClone, Laboratories, Logan, UT). Rev-CEM cells and SUP-T1 cells were maintained in RPMI 1640 supplemented with 10% FBS. Throughout, all cell lines were discarded after 10 passages and new aliquots of frozen cells were thawed to improve reproducibility of virion production and infection experiments. Dynasore (Sigma-Aldrich) stock solution was prepared in DMSO. Control experiments showed that DMSO has no effect on HIV-1 infectivity. T20 (Roche) stock solution was prepared in distilled and deionized milliQ water. Inhibitor stock solutions were aliquoted, flash-frozen, and stored in -80°C freezer.
Production of single-cycle HIV-1 virions
To produce single cycle HIV-1 virions, 106 293T cells in a 2 ml culture volume were seeded overnight in a 35 mm dish before transfection using the TransIT LT-1 transfection reagent (Mirus Bio, Madison, WI). For each dish, 1 mg of the provirus-containing plasmid pNL4-3E- was used to make the transfection reagent mixture, together with 1 mg envelope expression plasmid for NL4-3 or HXB2 as indicated in the text. The transfection reagent mixture was incubated at room temperature for 15 min before drop wise addition to the culture media. At 6 h post transfection, the culture media together with the transfection reagents was replaced with fresh complete media and the incubation was continued at 37°C with 5% CO2. At 24 h post transfection, the entire culture media containing single-cycle HIV-1 viruses was collected and filtered through a 0.45 µm syringe filter (Millex-HV PVDF, Millipore) in less than 10 min on average. The filtrate was then aliquoted on ice, flash-frozen in liquid nitrogen and stored in a -80°C freezer. Control experiments showed that virion infectivity remains constant during incubation on ice for up to 8 h and flash-freezing in complete media leads to no loss of virion infectivity. The single cycle virions pseudotyped with either VSV-G or HXB2 envelope were produced using the exact procedures above except that the envelope plasmid for NL4-3 was replaced with that of VSV-G or HXB2. To produce single-cycle virions tagged with mCherry, the same procedures were used as described above except the input DNA used during transfection. In detail, HIV virions carrying free mCherry were generated by transfection of 293T cells with 0.5 mg pNL4-3E- plasmid, 0.5 mg pNL4-3E-MA-mCherry-CA plasmid and 0.1 mg NL4-3 envelope plasmid in 2 ml volume in a 35 mm dish. The pNL4-3E-MA-mCherry-CA plasmid encodes a provirus in which the mCherry protein was inserted between the matrix and capsid domains of the Gag protein, and the mCherry protein sequence was flanked by 2 HIV protease cleavage sites.
Infection assays in each cell line
For infection assay in TZM-bl cell line, 8×104 TZM-bl cells in a 1 ml culture volume were seeded in each well of a 12 well plate 1 day prior to infection. On the next day, virus stocks taken out of -80°C freezer were placed in a room temperature water bath until just thawed, and serially diluted in complete media containing 20 mg/ml DEAE-dextran. 100 ml of viruses at each dilution were layered on top of the cell and the infection was continued for 2 h at 37°C with gentle rocking every 30 min. For spinoculation in TZM-bl cells, upon mixing the virus with cells in the absence of DEAE-dextran, the mixture in a 12 well plate was centrifuged at 1200 g for 2 h at 25°C. At the end of 2 h, 1 ml of complete media was added to each well and the incubation was continued at 37°C for 48 h with 5% CO2. At the end of 48 h, cells were fixed in 2% gluteraldehyde at room temperature for 5 min. After fixation, the cells were washed 3 times with PBS, and stained for 50 min at 37°C using cell staining solution provided in the beta-galactosidase staining kit (Mirus Bio, Madison, WI). After incubation, the cells were washed 3 times with milliQ water and the number of blue cells in each well was counted with a Nikon TS100-F inverted microscope. For infection assay in Rev-CEM or SUP-T1 cell lines, 200 µl of cells were incubated with 100 µl of 50% free mCherry-labeled virus in 1.5 ml polypropylene test tube for 2 h at 37°C with continuous gentle rocking on a nutator in the presence of DEAE-dextran at designated concentrations. After 2 h, free virions were removed by washing with complete media. The cells were further incubated for 5 days at 37°C in 2 ml of complete media in a 12 well plate. At the end of 5 days, cells were fixed with 2% paraformaldehyde for 5 min at room temperature and washed with PBS. For both Rev-CEM cells and SUP-T1 cells, infected cells express mCherry protein and are quantitated with a flow cytometer using 561 nm laser (iCyt Synergy, Sony). For each cell line, the concentration of DEAE-dextran was optimized to obtain the highest infectious virus concentration for a given batch of virus in each cell line, which resulted in 20, 5 and 10 mg/ml, for TZM-bl, Rev-CEM and SUP-T1 cells, respectively. At these concentrations of DEAE-dextran for each cell line, the fractions of live cells as quantitated by trypan blue assay 16, 17 were all above 95%.
Infection assays in the presence of inhibitors
First, to determine the toxic concentrations of each inhibitor on cell growth, TZM-bl, Rev-CEM, or SUP-T1 cells were pretreated with various concentrations of dynasore or T20 for 30 min at 37°C, and further incubated for 2 or 5 days, following the infection assay conditions but without virus infection. Cells were then collected and incubated with 0.4% w/v trypan blue dye for 5 min at room temperature and the fraction of live cells were counted under a bright-field microscope. T20 didn’t show any apparent toxicity within the range of concentrations used for all cell lines. However, high concentrations of dynasore did show toxicity. For virus infection in the presence of dynasore, all cells were pretreated with a certain concentration of dynasore in the absence of serum for 30 min at 37°C, and then incubated with the virus. The concentrations of dynasore are reflective of the level reported in literature. The virus incubation also included dynasore at desired concentrations, although there was no difference in the inhibition on viral infection between the absence and presence of dynasore during virus incubation for all three cell lines (data not shown).
Transferrin uptake in each cell line
For transferrin uptake assay in TZM-bl cells, 3×104 TZM-bl cells in 1 ml complete media were seeded in each well in a 12 well plate and incubated overnight. On the second morning, the cells were pretreated with dynasore at various concentrations for 30 min at 37°C and then incubated with 20 µg/ml of Alexa-488 conjugated transferrin prepared in DMEM without FBS (Invitrogen) for 5 min at 37°C followed by at 4°C for 20 min. Controls were incubated with the same concentration of transferrin conjugates for 25 min at 4°C throughout. All the subsequent procedures were carried out at 4°C. First, cells were washed with pre-chilled PBS 6 times and incubated with pre-chilled pH 2.0 buffer for 5 min at 4°C followed by pre-chilled PBS washing twice to remove transferrin bound on cell surface. The pH 2.0 buffer was made using 500 mM NaCl and 0.2 N glacial acetic acid in milliQ water. Next, TZM-bl Cells were trypsinized, fixed with 4% paraformaldehyde for 10 min at room temperature, washed with PBS 3 times, and analyzed with a flow cytometer (FACS Canto II) using a 488 nm laser 3. For transferrin uptake in Rev-CEM or SUP-T1 cells, the exact procedures as above were followed except the following modifications: cells and transferrin were incubated in 1.5 ml test tube instead of 12 well plate; no trypsinization was performed for these suspension cells, and washing of the cells were done by mixing with each solution and centrifuge at 1000 g for 5 min, followed by resuspension of the cells in designated buffers, all carried out at 4°C.
Confocal imaging of TZM-bl cells
For confocal imaging of transferrin uptake in TZM-bl cells, cells were seeded on Poly-L-Lysine coated coverslip in a 12 well plate and incubated overnight at 37°C with 5% CO2. On the second morning, the cells were pretreated with 0 or 200 µM dynasore for 30 min at 37°C and then incubated with 20 µg/ml of Alexa-488 conjugated transferrin prepared in DMEM without FBS (Invitrogen) for 5 min at 37°C followed by at 4°C for 20 min. The cells were then washed with pre-chilled PBS 6 times and incubated with pre-chilled pH 2.0 buffer for 5 min at 4°C followed by pre-chilled PBS washing twice to remove transferrin bound on cell surface. The cells on coverslip were then fixed with 4% paraformaldehyde for 10 min at room temperature, and washed with Tris buffered saline. The cells on coverslip were then stained with Cholera Toxin Subunit B conjugated with Alexa-555 (Invitrogen) at a concentration of 1 µg/ml in PBS for visualization of plasma membranes. The coverslip was then washed with PBS and mounted onto a glass cover slide with 3 µl mounting media, sealed with nail polish, and imaged using an Olympus FluoView 500 Laser Scanning Confocal Microscope. Confocal images were collected using 100× oil immersion objective. The fluorophores were excited using 488 nm laser for Alexa-488 conjugated transferrin and 543 nm laser for Alexa-555 conjugated Cholera Toxin Subunit B.
Live cell imaging of transferrin uptake in TZM-bl cells
Exponentially growing TZM-bl cells were trypsinized and seeded onto chambered coverglass for at least 6 h before experiments. The cells were pretreated with 0 or 108 µM dynasore for 30 min at 37°C. At time 0, 5 µg/ml of Alexa-594 conjugated transferrin diluted in DMEM was added to cells, incubated for 5 min, and then replaced immediately with warm complete media without transferrin. For live cell imaging, the chambered coverglass was mounted onto a Tokai Hit stage incubator installed on an Olympus IX71 inverted fluorescence microscope (Olympus, Center Valley, PA). The cells were maintained at 37°C with a supply of 5% CO2 directly to the stage incubator. Live cell fluorescence images were collected with a 100× oil immersion objective (Olympus, N.A. 1.4) and imaged onto an iXon3 897 back-illuminated EMCCD camera (Andor Technology, Belfast, Northern Ireland). The Alexa-594 conjugated transferrin was excited under epifluorescence illumination by a linearly polarized 592 nm laser line (VFL-P-1000; MPB Communications Inc., Montreal, Canada) with an irradiance of 20 W/cm2. The excitation filter was 588/30 nm and the emission filter was 629/40 nm. Fluorescence images were captured at an exposure time of 100 ms and the movies were taken at a rate of 1 frame per second. Stage drift was negligible during this time frame as revealed from images of fiducial markers before and after the movies. The movies shown were taken at 20 min after the addition of Alexa-594 conjugated transferrin to both dynasore treated and untreated TZM-bl cells.