Cell-to-cell transfer of HIV-1 via virological synapses leads to endosomal virion maturation that activates viral membrane fusion.

Viral Constructs and Preparation of Viral Particles HIV Gag-iGFP and HIV Gag-iCherry, infectious variants of pNL4-3, have a fluorescent protein inserted between the matrix and capsid domains of Gag ( Hübner et al., 2007 ). pEGFP-Vpr, a gift of Dr. Warner Greene (UCSF, San Francisco, CA), and pmm310, a gift of Dr. Michael Miller (Merck Research Laboratories), are available at the AIDS Research and Reference Reagent Program (Cat#11386 and Cat#11444, respectively). HIV Gag-iGFP Pr(−) and HIV Gag-iCherry Pr(−) were made by site-directed mutagenesis and are protease catalytic mutants carrying a double-alanine (D25A/T26A) mutation ( Loeb et al., 1989 ). pNL4-3 MA/p6 was a gift of Christopher Aiken (Vanderbilt University, Nashville, TN). Viral particles were produced using standard calcium phosphate-mediated transfection of 293T cells ( Pear et al., 1993 ). Where noted, viral particles were produced in the presence of 2 μM Indinavir (AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH). Viral supernatants were quantitated by p24 ELISA and routinely yielded p24 concentrations of 400–1,000 ng/ml. Cells and Tissue Culture Human CD4+ T cell lines Jurkat CE6.1 and MT4 were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and grown in Jurkat culture media (RPMI 1640, 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin). Human CD4+ T cells were purified by negative selection using a CD4+ T cell isolation kit II (Miltenyi Biotec, Auburn, CA) from buffy coats obtained from blood donors at the New York Blood Center (Long Island City, NY). Jurkat T cells were nucleofected using program S-18 (Lonza, Walkersville, MD). MT4 cells and primary CD4+ cells were labeled, as noted, by minor modification of the manufacturer's protocol. Incubation of cells at 37°C for 6 min in 0.7 μM DDAO Far Red or 30 min in 10–20 μM CellTracker Blue gave optimal labeling.

Quantitation of Viral Membrane Fusion by the BlaM Assay

Cell-free viral fusion was quantitated as described ( Cavrois et al., 2002 ). Briefly, HIV-1 virus was produced by cotransfecting 293T cells with wild-type proviral DNA (pNL4-3) and a plasmid (pMM310) that encodes β-lactamase fused to the amino terminus of Vpr at a 3:1 ratio. Fusion assays with cell-free virus were performed by adding virus (30 ng) to 2 × 10 5 cells in a volume of 200 μl for the indicated amount of time. Cells were washed and loaded with BLaM substrate CCF2-AM (1.5 μM) for 90 min at 25°C. Cells were then washed and incubated at 18°C in CO 2 -independent media for 12 hr to allow for substrate cleavage. Finally, cells were washed in PBS, fixed in 3.7% formaldehyde, and read on an LSR II flow cytometer (Becton Dickinson, Franklin Lakes, NJ). Cleavage of CCF2-AM was determined by exciting cells with a 405 nm laser and recording emission at 450 nm (+/− 50 nm) and 525 nm (+/− 50 nm). Flow cytometry data was exported and analyzed using FlowJo software (Tree Star, Ashland, OR). Gates were set using untransfected cocultures. All conditions were background-subtracted. In each experiment, control conditions were normalized to 100% and experimental conditions were expressed as a percentage of control. To assess cell-mediated fusion, 7 × 10 6 Jurkat T cells were transfected with pNL4-3/Vpr-BLaM (30 ng) at a 3:1 ratio. After 24 h, transfected Jurkat cells were purified by Ficoll density gradient centrifugation and used as donor cells. These donor cells were mixed with Far Red-labeled CD4+ cells at a 2:1 ratio for the indicated amount of time. Cell cultures were then trypsinized, loaded with CCF2-AM, and processed for flow cytometry as described above.

Viral Particle Maturation Assays as Assessed by p17 Staining Jurkat

T cells were transfected with HIV Gag-iGFP and mixed with Far Red-labeled CD4+ T cells; viral transfer was quantitated as described previously ( Chen et al., 2007 ). To measure the fraction of mature virus in acceptor cells, trypsinized cocultures were fixed in 3.7% formaldehyde, permeabilized in 0.05% saponin, and stained with a monoclonal antibody that recognizes p17 only in the context of mature virus ( Ono et al., 2000 ; Zhou and Resh, 1996 ). p17 staining was detected with a PE-labeled secondary antibody that allowed for simultaneous detection of viral transfer (GFP) and maturation (PE).

Viral Particle Maturation Assays as Assessed by FRET

For analysis of cell-free virus, 293T cells were cotransfected with HIV Gag-iGFP and HIV Gag-iCherry at a 1:1 ratio. Filtered viral supernatant was spotted onto poly-L-lysine coated glass coverslips and allowed to adhere for 15 min. Viral particles were fixed in 3.7% paraformaldehyde at room temperature for 15 min, rinsed with PBS, and mounted onto glass slides with ProLong Gold (Invitrogen, Carlsbad, CA). To measure particle maturation in acceptor cells following transfer across the VS, Jurkat cells were cotransfected with HIV Gag-iGFP and HIV Gag-iCherry at a 1:1 ratio. These cells were mixed with CellTracker Blue (Invitrogen)-labeled primary CD4+ T cells at a 2:1 donor:acceptor cell ratio in the presence of AMD3100. Intracellular viral particles were identified in an automated fashion based on their colocalization with the cytoplasmic dye CellTracker Blue and confirmed visually in all three dimensions. Viral particles were imaged using a Leica SP5 DMI confocal microscope. Excitation lasers were 488 nm (GFP) and 561 nm (Cherry). Emissions were captured between 500 and 580 nm (donor) and between 590 and 650 nm (acceptor and FRET). Donor and acceptor bleedthrough constants were 2.7% and 10.4%, respectively. Normalized FRET values were calculated as described previously ( Xia and Liu, 2001 ).

Monitoring Viral Membrane Fusion by Live Cell Imaging

For analysis of cell-free fusion, 293T cells were cotransfected with HIV Gag-iCherry and GFP-Vpr at a 3:1 ratio as described above. Approximately 500 ng/ml of filtered viral supernatant was added to adherent HeLa CD4+ cells for 2 hr at 17°C, a temperature that allows for binding but not fusion of the virus ( Markosyan et al., 2005 ). Cells were then washed gently and incubated at 37°C for 2 hr, fixed, and mounted on glass slides as described above. Analysis of viral membrane fusion was performed using Volocity image analysis software (PerkinElmer, Waltham, MA). Viral particles were identified using an automated image segmentation process that identified particles with a mean GFP fluorescence at least 30-fold over background. To quantitate membrane viral fusion in T cell cocultures, Jurkat T cells were cotransfected with HIV Gag-iCherry and GFP-Vpr at a 3:1 ratio and mixed with CellTracker Blue-labeled primary CD4+ T cells. Cells were incubated together for the indicated amount of time, trypsinized, sorted by flow cytometry (FACSAria, Becton Dickinson), and fixed on poly-L-lysine-coated glass coverslips. Viral particles were identified using the same criteria used in cell-free experiments, with the additional requirement that particles had a mean blue fluorescent signal at least 200-fold over background, indicative of their association with the cytoplasm of a target cell. After identification, all particles were visually inspected in three dimensions to confirm localization in acceptor cells. To image live viral fusion in acceptor cells engaged in a virological synapse, Jurkat T cells were cotransfected with HIV Gag-iCherry and GFP-Vpr at a 3:1 ratio and mixed with primary CD4+ T cells in CO 2 -independent media (Invitrogen), supplemented with 10% FBS and IL-2 (10 units/ml). Cells were loaded into Ibidi imaging chambers (Ibidi, Verona, WI) and imaged for up to 1 hr at various time points after initiating coculture. Cells were imaged at 37°C on an inverted optical microscope (iX71 Olympus, Center Valley, PA) equipped with a 60 ×, 1.42 NA oil immersion objective (UPlanApo N, Olympus), piezoelectric z-stage (Mad City Labs, Madison, WI), spinning disk confocal unit (CSU-10, Yokagawa, Japan) using a 405/488/568/647 nm quad-band dichroic mirror (Semrock, Rochester, NY), and an EM-CCD camera (iXon+ 897, Andor Technologies, Ireland) to allow rapid 3D time-lapse confocal imaging. To further reduce 3D imaging time to 1.5–3 s, we reduced the active area of the EM-CCD to the cell-pair region (~ one-quarter of the 512 × 512 pixel area of the EM-CCD). A multiline ArKr ion gas laser (Innova 70C, Coherent, Santa Clara, CA) paired with an acousto-optic tunable filter (AOTF) (Andor Technologies) allowed fluorescence excitation, with microsecond wavelength switching between 488 and 532 nm (for GFP and iCherry, respectively) at < 1 mW. The AOTF further shuttered the laser during the 10–20 ms EM-CCD readout to reduce photobleaching and photo-toxicity. iGFP and iCherry fluorescence emission were separated using an OptoSplit II image splitter (Cairn, Kent, UK) equipped with a 570 nm long-pass dichroic mirror (Chroma, Rockingham, VT), and were filtered by 525/50 and 609/54 nm bandpass filters (Semrock) respectively. Most electronics and acquisition were coordinated by the Andor iQ v1.8 software.