HIV-1 Nef inhibits ruffles, induces filopodia, and modulates migration of infected lymphocytes

Abstract

The HIV-1 Nef protein is a pathogenic factor modulating the behavior of infected cells. Nef induces actin cytoskeleton changes and impairs cell migration toward chemokines. We further characterized the morphology, cytoskeleton dynamics, and motility of HIV-1-infected lymphocytes. By using scanning electron microscopy, confocal immunofluorescence microscopy, and ImageStream technology, which combines flow cytometry and automated imaging, we report that HIV-1 induces a characteristic remodeling of the actin cytoskeleton. In infected lymphocytes, ruffle formation is inhibited, whereas long, thin filopodium-like protrusions are induced. Cells infected with HIV with nef deleted display a normal phenotype, and Nef expression alone, in the absence of other viral proteins, induces morphological changes. We also used an innovative imaging system to immobilize and visualize living individual cells in suspension. When combined with confocal "axial tomography," this technique greatly enhances three-dimensional optical resolution. With this technique, we confirmed the induction of long filopodium-like structures in unfixed Nef-expressing lymphocytes. The cytoskeleton reorganization induced by Nef is associated with an important impairment of cell movements. The adhesion and spreading of infected cells to fibronectin, their spontaneous motility, and their migration toward chemokines (CXCL12, CCL3, and CCL19) were all significantly decreased. Therefore, Nef induces complex effects on the lymphocyte actin cytoskeleton and cellular morphology, which likely impacts the capacity of infected cells to circulate and to encounter and communicate with bystander cells.

FIG. 1.

HIV-1 alters the shape of T lymphocytes through Nef expression. Shown are representative images of NI or HIV WT- or Δnef-infected Jurkat cells. (A) SEM images. HIV virions were stained with anti-Env MAb coupled to gold particles (appearing as white dots). Insets, higher magnifications of cellular regions showing virus-like particles. (B) 3D reconstruction of confocal images. Cells were stained with phalloidin-rhodamine (red) and anti-Gag MAb (green). (C) Cells without protrusions or with ruffles or filopodia were scored by visual counting under a fluorescence microscope. The data are means plus standard deviations (SD) of four independent experiments, with a total of 415 NI, 337 HIV WT-, and 296 HIV Δnef-infected cells scored. Significance was assessed by the Mann-Whitney test (*, P < 0.05). (D) Nef accumulates in filopodia. Shown is a 3D reconstruction of a representative confocal image. HIV-infected cells were stained with phalloidin-rhodamine (red) and anti-Nef MAb (green).

FIG. 2.

Automated quantification of T-cell protrusions. Uninfected (NI) or HIV WT- or Δnef-infected Jurkat cells were stained with anti-Gag MAbs and with phalloidin. Nuclei were visualized with DAPI. Automated quantification of cell shape changes on gated cells was performed using ImageStream technology. (A) Uninfected (Gag⁻) or infected (Gag⁺) cells, 40.7% for HIV WT and 35.6% for HIV Δnef, were gated and analyzed by symmetry 3 and circularity features, as detailed in the supplemental material (Sup. Fig. 1 at http://www.pasteur.fr/ip/easysite/go/03b-00003g-063/virus-and-immunity/supplemental-material). When plotted together, these two features enable identification and gating of circular cells versus cells with protrusions. (B) Representative images of circular cells and cells with protrusions, identified in panel A. (C) Quantitative analysis of images from these two populations was performed. The percentages of cells with protrusions and circular cells were quantified automatically with IDEAS software. The data represent means and SD of five independent experiments.

FIG. 3.

Nef inhibits ruffles and increases filopodia. (A and B) Representative images of NT Jurkat cells (left) and of cells transduced with lentiviral expression vectors encoding the WT (middle) or the G2A mutant (right). (A) SEM images. (B) 3D reconstruction of confocal images. Cells were stained with phalloidin-rhodamine (red) and anti-Nef MAb (green). (C) Cells without protrusions, with ruffles, or with filopodia were scored by visual counting under a fluorescence microscope, with a total of 235 NT and 235 Nef WT- and 242 Nef G2A-expressing cells analyzed. The data are means and SD of four independent experiments. Significance was assessed by the Mann-Whitney test (*, P < 0.05). (D) High-resolution 3D imaging of living cells in suspension. Actin-GFP Jurkat cells transduced with WT- or G2A Nef-expressing vectors were analyzed. Nontransduced cells were used as controls. Representative images of cells with or without filopodia are shown. The data are means and SD of three independent experiments, with about 60 cells analyzed for each condition. Significance was assessed by the Mann-Whitney test (*, P < 0.05).

FIG. 4.

Nef impairs T-cell adhesion to fibronectin. (A) Quantification. Jurkat cells, NT or transduced with Nef WT or GFP, were left adhering to a fibronectin-coated surface for 30 min. The number of bound cells was determined with an automated system (Opera). The percentages of adherent cells are shown. The data are means and SD of four independent experiments. (B) Visualization of cells by time-lapse videomicroscopy. Control (left) and Nef-expressing actin-GFP (right) Jurkat cells were seeded on fibronectin-coated surfaces. The images are snapshots taken from the supplemental movies (http://www.pasteur.fr/ip/easysite/go/03b-00003g-063/virus-and-immunity/supplemental-material) and are representative of three independent experiments.

FIG. 5.

Nef alters T-cell intrinsic motility and chemotaxis to CXCL12 (SDF-1). Shown are transwell chemotaxis assays. Jurkat cells were placed in the upper chamber of a transwell and medium or chemokines in the lower chamber. The percentages of T cells attracted to the medium after 4 h or to CXCL12 after 2 h were calculated. (A and B) Representative experiments. (A) Cells transduced with control GFP, Nef WT, or Nef G2A. (B) Cells infected with HIV or HIV Δnef. (C) The data were normalized to the percentage of migration of noninfected cells (100%). The means and standard errors of the mean of four independent experiments are shown. Significance was assessed by a Mann-Whitney test (*, P < 0.05).

FIG. 6.

Nef inhibits chemotaxis of primary T cells to CXCL12, CCL19, and CCL3 in transwell chemotaxis assays. Blasts were placed in the upper chamber of a transwell and medium or chemokines in the lower chamber. The percentages of T cells attracted to the lower chamber after 1 h are shown. For each condition, the data were normalized to the percentage of migration of noninfected cells (100%). The medians and interquartile ranges of 7 independent experiments are shown for all conditions except CCL3. For CCL3, the data are means and SD of two independent experiments. Significance was assessed by a Mann-Whitney test (*, P < 0.05).