Dynamic recruitment of Gag puncta to the synapse and viral transfer into a target cell compartment revealed with rapid spinning-disc 3D confocal fluorescence microscopy. (A) Formation of a buttonlike accumulation of Gag at the site of adhesion, z projection at time = 0 (left), selected 3D reconstructions of contact site (arrows) over time (right four images). (B) A zone of Gag depletion, 2 to 3 μm wide, surrounds the synaptic button (dotted yellow line). (C) Patches of synapse-proximal Gag merge into the synapse. (D) A Gag-iGFP puncta moves out of and into the synapse. (E) During a transfer event, Gag puncta emerge from the synapse, separate, and then move to the distal pole. In (C) to (E), (a) selected frames highlight movement of Gag-iGFP puncta (yellow). (b) Object path is overlayed on the initial image. (c) Object distance to the synapse center and relative velocity are graphed over time. (d) Histogram distribution of the tracked objects velocities. (F and G) Immunostaining of Gag puncta requires membrane permeabilization. (F) Nonpermeabilized, anti-Env immunostain (red) does not stain the Gag-iGFP+ puncta (green) within the CD4 target cell (CellTracker Blue CMF2HC, Invitrogen), whereas surface Env-staining at synapse is observed. Three-color intensity profile along the 12-μm line (right). (G) Permeabilization of fixed cells reveals anti-Env immunostain (red) at the GFP puncta (green) within the CD4 target cell (blue). (H) Transmission electron micrograph of vesicles containing corelike structures in a CD4 cell engaged in synapse with an HIV-infected Jurkat cell. Low (left) and high (right) magnification of 70-nm sections.

Cell-associated infection is coreceptor-dependent and actin-dependent and can resist a neutralizing antiserum. (A) HIV NL-GI–expressing Jurkat cells were cocultured with CellTracker Blue CMF2HC–labeled MT4 cells in the absence or the presence of a 0.4-μm transwell barrier between cells. Productive infection (GFP expression) in CellTracker-labeled target cells was measured by flow cytometry at 48 hours. (B) Coreceptor antagonist AMD3100 (10 μg/ml) inhibits infection of target cells by cell-associated X4-tropic virus, HIV NL-GI, at 48 hours. Productive infection in gated target cells indicated by GFP expression and is plotted against forward scatter width (FSC-W). (C) Cell-associated R5-tropic virus infects CCR5-expressing MT4 cells but not CCR5-negative MT4 cells. Jurkat cells expressing R5-tropic HIV NL-GI(JRFL) were donor cells. (D) Cytochalasin D (2.5 μM) inhibits cell-associated infection (top) but fails to block infection with cell-free virus (bottom). (E) A neutralizing antiserum that blocks cell-free infection (bottom) is less effective at blocking homologous cell-associated infection (top). Results representative of at least three independent experiments.

Gag accumulates at synaptic buttons after T cell adhesion. (A) Time-lapse fluorescence imaging of synapse formation between an HIV Gag-iGFP–expressing Jurkat cell and a CD4 T cell. GFP image (top) and GFP/phase contrast overlay (bottom). Cells (a) before stable contact, (b) in stable adhesion (outlined), and (c and d) showing synaptic buttons (arrowheads). (B) Timing of synapse formation following 24 HIV+ Jurkat cells; each line represents an interactive cell. (C) Confocal fluorescence image of an HIV Gag-iGFP-expressing Jurkat T cell (green) synapsed with three primary CD4 T cells [red, labeled with CellTracker Orange CMRA (Invitrogen, Carlsbad, CA)]. Positioning of perpendicular planes marked at edges. (D) Reconstructed 3D view of (C). (E to H) FRET analysis of Gag-iCerulean (donor) and Gag-iVenus (acceptor) fluorophores at the synaptic button. (E) Three-color overlay donor Cerulean (blue, 405-nm excitation), FRET channel (green, 405-nm excitation), and target cells [red, 543-nm excitation stained with CellTracker Orange CMTMR (Invitrogen)]. (F) Emission spectra at synaptic button, point F, pre- and postacceptor photobleaching. (G and H) Normalized FRET (NFRET) signal (13) before and after acceptor photobleaching in boxed area. (I) Transmission electron micrographs of the synaptic junction between HIV Gag-iGFP–expressing donor, D, and target, T, cells. Low (top) and high (bottom) magnification of 70-nm sections.

Productive infection of synapsed cells is visualized by 72-hour imaging of immobilized cells engaged in virological synapse. (A) Donor cells cotransfected with HIV Gag-iGFP to track viral transfer and HIV NL-GI to visualize new early gene expression in target cells. Images show a synapsed pair where the target cell (number 1) separates from donor at 18 hours and expresses increasing levels of diffuse GFP at 32 hours. Top row shows GFP images; bottom, GFP/phase overlays. (B) Four examples of synapsed MT4 target cells that subsequently expressed HIV (numbers 2 to 5). (C) Fluorescence intensity of the target cells 1 to 7. Numbers 6 and 7 are control bystander cells. Duration of cell contact indicated on bottom.