Retargeting HIV-1 Gag to Endosomes

<p>(A) Schematic representation of retargeted Gag proteins in which the globular head of matrix is replaced by specific membrane-binding domains.</p> <p>(B) Distribution of retargeted Gag proteins in 293T cells. Cells expressing FYVE-Gag-GFP, PX-Gag-GFP, or C2-Gag-GFP (green) were fixed at 24-h post-transfection. Scale bars indicate 8 μm (left panel) or 4 μm (center and right panels).</p> <p>(C) FYVE-Gag-GFP targets late endosomes. 293T cells expressing FYVE-Gag-GFP (green) were fixed at 24-h post-transfection and stained with anti–Lamp-1 or anti-CD63 antibodies (red). Alternatively, cells were co-transfected with mRed-Hrs or CherryFP-Rab5aQ79L (red). Insets show expanded views of individual FYVE-Gag-GFP accumulations. Scale bars indicate 4 μm.</p

HIV-1 Particles Are Not Released When Assembly Is Targeted to Endosomes

<p>(A) Electron microscopy analysis of 293T cells expressing CherryFP-Rab5aQ79L together with WT-Gag-Pol (left), in which a portion of the PM is shown, or FYVE-Gag-Pol (right), in which mature virions and assembly intermediates are found in intracellular vesicles. Scale bars indicate 200 nm.</p> <p>(B) Western blot analysis of 293T cells expressing WT (wild type) or retargeted Gag-Pol or Gag-GFP proteins. Samples were collected at 24-h post-transfection. Cell and virion lysates were probed with anti–HIV-1 CA antibodies. Numerical values below the blots indicate VLP Gag-GFP or p24CA signal intensities, derived by densitometry.</p> <p>(C) Infectivity measurements using supernatant collected from 293T cells expressing WT, G2A mutant, or retargeted Gag-Pol proteins along with a packageable HIV-1 GFP expression vector and VSV-G. The percentage of infected (GFP⁺) TE671 target cells is plotted as a function of inoculum volume.</p

Efficient HIV-1 Particle Formation in the Absence of Microtubules

<p>(A and B) Western blot analysis of 293T cells expressing HIV-1 Gag-Pol in the absence (A) or presence (B) of nocodazole, added immediately after transfection. Samples were collected every hour, as indicated, and cell and virion lysates were probed with anti–HIV-1 CA antibodies. Numerical values below the blots indicate VLP p24CA signal intensities, derived by densitometry; absence of a value indicates undetectable signal.</p> <p>(C) 293T cells expressing Gag-GFP (green) in the absence or presence of nocodazole were fixed 14-h post-transfection. Samples were stained with anti–α-tubulin antibodies (red) and with Hoechst 33258 (blue). Scale bars indicate 8 μm.</p> <p>(D) 293T cells expressing VSV-G in the absence or presence of nocodazole were fixed 14-h post-transfection. Samples were stained with anti–VSV-G antibodies (green) and with Hoechst 33258 (blue). Scale bar indicates 8 μm.</p

HIV-1 Gag Accumulation at the PM Precedes Accumulation in Primary Human Macrophage Endosomes

<p>(A) Distribution of Gag-GFP in macrophages. Cells expressing Gag-GFP (green) were fixed at 24-h post-transfection and stained with Hoechst 33258 (blue). Examples of cells exhibiting diffuse Gag only (left), PM accumulations (center), or PM + internal (Int; right) accumulations are shown. Insets show individual Gag-GFP puncta at the PM coverslip interface. Scale bar indicates 10 μm.</p> <p>(B) Western blot analysis of macrophages expressing Gag-GFP. Samples were collected every 2 h, from 4- to 12-h post-transfection. Cell and extracellular particle lysates were probed with anti–HIV-1 CA antibodies. Numerical values below the blots indicate VLP signal intensities, derived by densitometry.</p> <p>(C) Quantification of Gag-GFP distribution in macrophages. The proportion of cells (of 100 counted at each time point) in which Gag-GFP was observed in accumulations only at the PM, as internal and PM accumulations, or as a diffuse cytoplasmic signal only were counted.</p> <p>(D) Intracellular Gag-GFP is observed on early and late endosomes in macrophages. Cells expressing Gag-GFP (green) were fixed at 24-h post-transfection and stained with anti-EEA1 (red). Alternatively, cells were co-transfected with mRed-Hrs (red). Inset in the bottom right panel shows an individual EEA1⁺ endosome and associated Gag-GFP. Scale bars indicate 10 μm (top panel) and 4 μm (bottom panel).</p

Actin Influences HIV-1 Gag Localization but Not Particle Release in Primary Human Macrophages

<p>(A) Disruption of microfilaments in macrophages. Macrophages treated with DMSO or cytochalasin-D were fixed 12 h after treatment and stained with phalloidin (red) and Hoechst 33258 (blue). Scale bars indicate 20 μm.</p> <p>(B) Western blot analysis of macrophages expressing Gag-GFP in the presence of DMSO or cytochalasin-D. Samples were collected every 2 h, from 4- to 12-h post-transfection. Cell and virion lysates were probed with anti–HIV-1 CA antibodies. Numerical values below the blots indicate VLP signal intensities, derived by densitometry.</p> <p>(C) Quantification of Gag-GFP distribution in macrophages in the absence of microfilaments. Macrophages expressing Gag-GFP were treated with DMSO or cytochalasin D for the entire duration of Gag-GFP synthesis. Cells were fixed at 12-h post-transfection, and the proportion of cells (of 100 counted) in which Gag was found at the PM, or at both internal sites and the PM was counted.</p

Efficient HIV-1 Particle Formation Does Not Require Late Endosome Motility

<p>(A) U18666A induces the collapse of late, but not early, endosomes toward a perinuclear region of 293T cells. Cells treated with U18666A or DMSO for 14 h were fixed and stained with anti-Lamp1 or anti-EEA1 antibodies (red) and Hoechst 33258 (blue). Scale bars indicate 8 μm.</p> <p>(B) 293T cells expressing Gag-GFP (green) in the presence or absence of U18666A, which was added immediately after transfection. Samples were fixed at 14-h post-transfection and stained with Hoechst 33258 (blue). Scale bar indicates 4 μm.</p> <p>(C) Western blot analysis of 293T cells expressing Gag-Pol in the absence or presence of U18666A, which was added immediately after transfection. Samples were collected and analyzed as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040435#pbio-0040435-g001" target="_blank">Figure 1</a>A. Numerical values below the blots indicate VLP p24CA signal intensities, derived by densitometry; absence of a value indicates undetectable signal.</p

Accumulation of Gag at the PM Precedes Endosomal Accumulation, Which Can Be Prevented by Blocking Endocytosis

<p>(A) Distribution of Gag-GFP in 293T cells at 24-h post-transfection. Examples of cells exhibiting diffuse Gag only (left), PM accumulations (center), or PM + internal (Int; right) accumulations are shown. Samples were stained with Hoechst 33258 (blue). Scale bars indicate 4 μm.</p> <p>(B) Quantification of Gag-GFP distribution in 293T cells. The number of cells in which Gag-GFP accumulation was observed at the PM, as internal and PM accumulations, or as a diffuse cytoplasmic signal only was counted. Approximately 100 cells were evaluated at each time point.</p> <p>(C) Gag-GFP accumulates at both early and late endosomes. 293T cells expressing Gag-GFP (green) were fixed at 24-h post-transfection and stained with anti-CD63, anti-Lamp1, or anti-EEA1 antibodies (red). Alternatively, cells were co-transfected with CherryFP-Rab5a (red). Cells were stained with Hoechst 33258 (blue). Insets show expanded views of individual Gag puncta. Scale bars, from top to bottom, indicate 4 μm, 2 μm, 8 μm, and 2 μm.</p> <p>(D) Quantification of Gag-GFP distribution in 293T in the presence of endocytosis inhibitors. 293T cells expressing Gag-GFP together with the indicated proteins were fixed at 24-h post-transfection and the proportion of cells (out of 100 counted) in which Gag was found at the PM, or both at internal accumulations and at the PM was determined.</p> <p>(E) Western blot analysis of 293T cells expressing HIV-1 Gag-Pol in the presence of endocytosis inhibitors. Samples were collected at 24-h post-transfection as indicated, and cell and virion lysates were probed with anti-HIV-1 CA antibodies. Numerical values below the blots indicate VLP p24CA signal intensities, derived by densitometry.</p