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. 2011 Nov 3;118(18):4841-52.
doi: 10.1182/blood-2010-09-305417. Epub 2011 May 11.

HIV-1 activates Cdc42 and induces membrane extensions in immature dendritic cells to facilitate cell-to-cell virus propagation

Damjan S Nikolic  1 , Martin Lehmann, Richard Felts, Eduardo Garcia, Fabien P Blanchet, Sriram Subramaniam, Vincent Piguet
Affiliations

HIV-1 activates Cdc42 and induces membrane extensions in immature dendritic cells to facilitate cell-to-cell virus propagation

Damjan S Nikolic et al. Blood. .

Abstract

HIV-1 cell-to-cell transmission confers a strong advantage as it increases efficiency of transfer up to 100-fold compared with a cell-free route. Mechanisms of HIV-1 cell-to-cell transmission are still unclear and can in part be explained by the presence of actin-containing cellular protrusions. Such protrusions have been shown to facilitate cell-to-cell viral dissemination. Using fluorescence microscopy, electron tomography, and ion abrasion scanning electron microscopy we show that HIV-1 induces membrane extensions in immature dendritic cells through activation of Cdc42. We demonstrate that these extensions are induced after engagement of DC-SIGN by HIV-1(env) via a cascade that involves Src kinases, Cdc42, Pak1, and Wasp. Silencing of Cdc42 or treatment with a specific Cdc42 inhibitor, Secramine A, dramatically reduced the number of membrane protrusions visualized on the cell surface and decreased HIV-1 transfer via infectious synapses. Ion abrasion scanning electron microscopy of cell-cell contact regions showed that cellular extensions from immature dendritic cells that have the appearance of thin filopodia in thin section images are indeed extended membranous sheets with a narrow cross section. Our results demonstrate that HIV-1 binding on immature dendritic cells enhances the formation of membrane extensions that facilitate HIV-1 transfer to CD4(+) T lymphocytes.

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Figures

Figure 1
Figure 1
HIV-1 induces membrane extensions and Cdc42 activation in DCs. (A) Quantification by confocal microscopy of the number of membrane extensions or nanotubes in DCs either nonstimulated (NS) or stimulated with X4 HIV-1, R5 HIV-1, HIV-1Δenv, gp120IIIb, H-200, and X4 HIV-1 plus secramine A. Data are mean ± SD of 3 independent counts (left panel). Representative confocal images of DCs (right panel). Bar represents 5 μm. (B) Transmission electron microscopy images of HIV-1–treated DCs. Quantitation of the normalized number of membrane extensions on DCs after HIV-1 treatment. Data are mean ± SD of 2 independent counts (left panel) and corresponding representative images (right panel). Arrows indicate membrane extensions. *HIV-1 viral particles. Bar represents 2 μm. (C) Pull-down assay for activated Cdc42 in DC. Cdc42 expression by Western blot band quantitation. Data are mean ± SD of 3 independent pull-down experiments. (D) Western blot analysis for Phospho-Src (Φ-Src)/Src (left panel), Phospho-Pak1 (Φ-Pak1)/Pak1 (middle panel), and Phospho-Wasp (Φ-Wasp)/Wasp (right panel) detection in DCs. One representative Western blot per condition is represented. *P < .05 (Student t test).
Figure 2
Figure 2
Cdc42 is required for HIV-1 transfer across DC-CD4+ T cell infectious synapses. (A) Impact of silencing in DCs of cytoskeletal rearrangement proteins. (Left panel) Western blots for Cdc42 and Rac1 protein expression. (Middle panel). DC-CD4+ T cell infectious synapses counts. (Right panel). HIV-1 infection transfer to Jurkat CD4+ T cells. Data are mean ± SD of 5 independent experiments. (B) Confocal microscope images of DC-Jurkat CD4+ T cell infectious synapses. White thin arrows indicate viral particles on membrane extensions. *“Hairy” appearance of the DC surface. Thick white arrows indicate the presence of viral aggregates at the infectious synapse. **Loss of membrane extensions at the surface of the Cdc42-depleted DCs. Bar represents 5 μm. (C) Quantitation of the number of thin membrane extensions, dendrites, and lamellipodia in DCs after Cdc42 and Rac1 silencing. White arrows point to membrane extensions; and double-white asterisks, the DC-surface devoid of membrane extensions. The plot at right shows quantitation of membrane extensions, dendrites, and cell surface covered with lamellipodia (right panel). Data are mean ± SD of 4 independent experiments. (D) Impact of Cdc42 mutant nucleofection in DCs on HIV-1 transfer to resting autologous CD4+ T lymphocytes. Data are mean ± SD of 2 independent experiments. (E) Quantitation of the number of membrane extensions in DCs after Cdc42 mutant nucleofection. Data are mean ± SD of 3 independent experiments. *P < .05 (Student t test). Bonferroni test with an α-error value of 5% has been applied to all panels with multiple comparisons.
Figure 3
Figure 3
HIV-1 is localized on membrane extensions at the DC-CD4+ T cell infectious synapse. Transmission electron microscopy of contacts between immature DC and T cells. (A-C) Projection electron microscopy images of a 100-nm-thick section from fixed, plastic-embedded cocultures of T cells and immature DCs either in the absence of added HIV-1 (A), after exposure to HIV-1 for 1 hour (B), and after treatment with the cdc42 inhibitor secramine A, followed by exposure to HIV-1 (C). (D-E) A 5-nm tomographic slice from a 3D image of a 175-nm-thick section prepared from fixed, plastic-embedded cocultures of T cells and DCs exposed to HIV-1 (as in B) showing viruses riding on the membrane extensions from the DCs. (F) Schematic rendering of tomographic slice in panel E highlighting contact between T cells (green) and immature DCs (blue) in the presence of HIV (red). Scale bars, original magnifications: (A-C) 2 μm; (D-E) 1 μm. (G) Representative image of membrane extensions on DCs with HIV-1 near extension tips. Bar represents 1 μm. (H) Quantification of fused HIV-1 viral particles in target CD4+ T lymphocytes after nucleofection of Cdc42 mutants in DCs. All values are normalized to a 100% value assigned to the nontreated condition. Arrows indicate HIV-1 viral particles on membrane extensions on DCs. Data are mean ±SD of 3 independent experiments. *P < .05 (Student t test). Bonferroni test with an α-error value of 5% has been applied to all panels with multiple comparisons.
Figure 4
Figure 4
Immature DCs do not form wrapping sheets around T lymphocytes in the context of the infectious synapse. IA-SEM analysis of immature and mature DC-T cell infectious synapses. (A-D) 2D images of immature DC-T cell infectious synapses from the IA-SEM image stack corresponding to slices at progressively lower locations in the image stack. Red arrows point to some of the HIV-1 particles visible in the image. (E-H) Surface representations of the image stack with top surfaces corresponding to the images shown in panels A to D, respectively. (I-J) Top slice and front surface view of IA-SEM image stack showing the entire imaged area; the boxed region corresponds to the region highlighted in panels A to D. The green spheres represent the location of HIV-1. (K-L) Comparison of the infectious synapse between mature DCs (K) pulsed with HIV-1 and CD4+ T cells and of immature DCs (I) pulsed with HIV-1 and CD4+ T cells. (K) Arrows indicate the location of the sheet-like protrusions from the mature DCs that surround the T cells. Bar represents 2 μm.
Figure 5
Figure 5
Cdc42 silencing in DCs prevents HIV-1 fusion in target T lymphocytes. (A) Confocal microscope analysis of HIV-1 fusion in target CD4+ T lymphocytes. Nontreated (left panels) and siCdc42 (right panels) conditions are represented. Fused viral particles (green only) are shown by arrows. Bar represents 5 μm. (B) Live imaging analysis of HIV-1 transfer across DC-CD4+ T cell infectious synapses. (Left panel) Infectious synapse analyzed in supplemental Video 1. *DCs. **CD4+ T cells. Bar represents 2 μm. Right panel demonstrates time points during HIV-1 transfer across DC-CD4+ T cell infectious synapse shown in supplemental Video 1. White arrow indicates a static point along HIV-1 transfer trajectory. (C) Different examples of HIV-1 transfer trajectories across DC-CD4+ T cell infectious synapses. *DCs. **CD4+ T cells. *P < .05 (Student t test).
Figure 6
Figure 6
Cdc42 is required for HIV-1 transfer from DCs to autologous resting CD4+ T cells in the presence of sAg. (A) HIV-1 infection transfer to resting autologous CD4+ T lymphocytes after sAg stimulation. (B) HIV-1 infection transfer in DC-resting autologous CD4+ T lymphocyte cocultures. DC/T ratio 5:1 (left panel) and DC/T ratio 1:5 are shown (right panel). Data are mean ± SD of 3 independent experiments. (C) Fold decrease of HIV-1 transfer in DCs: resting autologous CD4+ T lymphocyte ratio conditions 5:1 (left panel) and 1:5 (right panel). Data are mean ± SD of 3 independent experiments. (D) Impact of Cdc42 or Larg silencing in MyDCs on HIV-1 transfer to Jurkat CD4+ T lymphocytes (left panel). Data are mean ± SD of 4 independent experiments. Two representative images of MyDCs-CD4+ T cell infectious synapses are shown (middle and right panels). Bar represents 5 μm. *P < .05 (Student t test). Bonferroni test with an α-error value of 5% has been applied to all panels with multiple comparisons.
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