BC sustain communication between uninfected and infected MDM. a Assessment by flow cytometry of HIV-1 infection in single culture MDM. b Formation of BC between infected and uninfected MDM labeled for HIV-1 envelope (red) and microtubules (green). Observe presence of HIV-1 Env along the conduit (scale bars, 10 μm, inset, 5 μm). c Frequency of BC formation in MDM uninfected, infected and mixed cultures (equal ratios of uninfected and infected MDM) at 30, 60 and 180 min in adherence (n = 450 cells/group, error bars ± S.E.M). Data are representative of five independent experiments. d Bright field images of conduits connecting MDM in mixed culture. Arrows indicate tethering sites (scale bar, 10 μm). e, f Frequency of conduit length and diameter observed in MDM mixed populations (n = 238 cells). g Time-lapse confocal images showing contact establishment and lipid transfer through BC from infected (DiD-labeled, red) to uninfected (DiO-labeled, green) MDM. h Velocity of lipid transfer through the BC. Graph reflects average velocity values recorded at each time point for 80 particles tracked along the BC (error bars ± S.E.M.). i Intercellular transfer activity in mixed MDM in presence (adherent cultures) and absence (suspension cultures) of conduits. Ratios of cells affected by this process over time (yellow, upper right quadrants) compared to the overall red (DiD-labeled infected MDM, upper left quadrants) and green populations (DiO-labeled, uninfected MDM, lower right quadrants) are shown in bold

Ultrastructural and biochemical characterization of BC. a, b SEM image of macrophage conduit reveals vesicle-like structures sheathed by plasma membrane. c TEM image of a sectioned conduit shows coated and uncoated vesicles of various sizes and electron densities, and large lipid droplets (inset 1 and 2, arrows). d TEM image of BC cross-sections generated from polarized MDM in mixed culture. Cells were immunostained for HIV-1 Gag. Secondary Ab conjugated to 6 nm gold particles was used for detection. Arrows (red) indicate endocytic compartments laden with gold particles. Rare capsid-like dense structures were observed in endocytic vesicles (blue arrow). e SEM image of infected macrophages seeded in the upper chamber of a Boyden chemotaxis apparatus, extending their processes through a polycarbonate porous membrane towards the lower chamber (arrows). f Classification by function of the proteins identified with two or more significant (P < 0.05) and unique peptides in the BC proteome (pooled sample from five independent experiments). Refer also to Table S2 for a list of proteins identified in the conduits

Endocytic compartments carry infectious viral cargo. a Illustration of immunoisolation of endocytic compartments from polarized MDM in mixed culture and their magnet-induced entry in TZM-bl reporter cell line. b TEM of enriched endocytic compartments bound to paramagnetic beads. Arrows indicate high-density endosomes. c HIV-1 viral load (RNA copies) in early endosomes, lysosomes, MVB and recycling endosomes and isotype control Abs. d, e Representative bright field images of uninfected TZM-bl, cells exposed to HIV-1 Tat, cell-free HIV-1, and immune-isolated endocytic compartments expressing different levels of ß-gal (blue) at 72 h post-exposure. Arrows indicate paramagnetic beads (brown) within TZM-bl. f Representative images of TZM-bl showing susceptibility of cell-free and endocytic HIV-1 infection to antiretroviral treatment. g Quantiation of overall endosome infectivity and susceptibility to antiretroviral therapy (efavirenz) in TZM-bl (scale bars 100 μm; error bars, ± S.E.M., n = 400 cells/group, N = 2 independent experiments)

Pathways of macrophage intra- and intercellular HIV-1 trafficking. HIV-1 receptor-mediated entry is regulated by clathrin-coated pits (Miyauchi et al. 2009). Upon inhibition of dynamin by dynasore, viral entry is blocked. Clathrin-coated vesicles containing HIV-1 may undergo Rab5/EEA1-dependant fusion with the early sorting endosome(Bucci et al. 1992; Zerial and McBride 2001). Endosomes can pinch off the vesiculo-tubular network also termed early sorting endosome (Maxfield and McGraw 2004) and undergo the following downstream sorting routes: a fuse with the endocytic recycling compartment (ERC; perinuclear region) and then undergo Tfn-like Rab11-mediated recycling to the BC and plasma membrane (Maxfield and McGraw 2004). Disruption of this processes by brefeldin A results in accumulation of HIV-1 constituents in large cytosolic compartments (Wang et al. 2001). b undergo sorting to the MVB for ILV biogenesis, virus assembly and budding regulated by ESCRT family (Babst et al. 2002; Garrus et al. 2001; Gill et al. 2007). MVB in turn may undergo fusion with Rab11 endosomes to be transported either to the plasma membrane for exosomal release (Simons and Raposo 2009) or intercellular transfer through the conduits. Disruption of MVB biogenesis by wortmannin results in agregation of HIV-1 constituents in large vaccuoles (Gruenberg and Stenmark 2004). Lysosomes may undergo backfusion with MBV and Rab11 compartments to be targeted at the plasma membrane (secretory non-degrading lysosomes; (Blott and Griffiths 2002; Luzio et al. 2005) or the bridging conduits. In parallel to endocytic entry and intercellular trafficking, virion-plasma membrane fusion may also occur followed by uncoating, reverse trancription, formation of pre-integration complexes (PIC), and RNA expression (Brass et al. 2008). Thick blue arrows indicate trafficking routes that target HIV-1 directly to the conduits

HIV-1 constituents are identified in the BC. a Detection by fluorescence in-situ hybridization of viral RNA using Alexa Fluor488-labeled HIV-1 Gag-Pol probes (green). Confocal and differential interference contrast (DIC) images show presence of HIV-1 RNA in the cell body and conduits (insets 1 and 2). b–d HIV-1 Env (green), Gag (Gag^Pr55, p24, p17, matrix; green), RT and RNA Alexa Fluor594 (red, metabolically-labeled with 5-ethynyl uridine) are readily visible in the conduits. (Refer to Table S1 for newly synthesized RNA and viral protein co-localization coefficients). e–h HIV-1 protease (red), integrase (red), Tat and Vif (red) are present in the BC. Staining for cortical actin (phalloidin-Alexa Fluor 488) and nuclei (DAPI, blue) was performed to distinguish MDM connected by the conduits from polarized single cells (scale bar, 10 μm; inset, 5 μm)

Distribution of HIV-1 constituents with BC endocytic compartments. a–k Confocal images of MDM in mixed culture showing distribution of HIV-1 Env (red, panels a, c, d and blue, b), Gag (green, b and red, e–g), metabolically labeled RNA (red) with early endosomes (green), MVB (green), recycling endosomes (green), and lysosomes (green) identified in the conduits (scale bars, 10 μm; insets, 5 μm). l–n, Pearson’s colocalization tests of HIV-1 Env, Gag and RNA distribution with BC endocytic compartments. Pearson’s colocalization coefficients were converted to percent overlap between fluorophores (error bars ± S.E.M., n = 80 cells)

Dependence of viral transfer on integrity of endocytic transport. a, b Distribution of myosin II with HIV-1 Env and Gag, in MDM untreated or exposed to CD, Noc or BBST post-formation of BC (scale bar, 10 μm). c–e Disruption of endosome/viral motility by CD, Noc or BBST. Progressive average movement was determined using the mean velocity (μm•s⁻¹), path length (μm), and displacement from the origin for all analyzed particles (error bars, ± S.E.M., n = 70 particles/group, N = 3 independent experiments) at each time point for 120 s