Plasma Membrane Is the Site of Productive HIV-1 Particle Assembly

Recently proposed models that have gained wide acceptance posit that HIV-1 virion morphogenesis is initiated by targeting the major structural protein (Gag) to late endosomal membranes. Thereafter, late endosome-based secretory pathways are thought to deliver Gag or assembled virions to the plasma membrane (PM) and extracellular milieu. We present several findings that are inconsistent with this model. Specifically, we demonstrate that HIV-1 Gag is delivered to the PM, and virions are efficiently released into the extracellular medium, when late endosome motility is abolished. Furthermore, we show that HIV-1 virions are efficiently released when assembly is rationally targeted to the PM, but not when targeted to late endosomes. Recently synthesized Gag first accumulates and assembles at the PM, but a proportion is subsequently internalized via endocytosis or phagocytosis, thus accounting for observations of endosomal localization. We conclude that HIV-1 assembly is initiated and completed at the PM, and not at endosomal membranes

Antiretroviral Activity and Vif Sensitivity of Rhesus Macaque APOBEC3 Proteins▿

The inability of human immunodeficiency virus type 1(HIV-1) to replicate in rhesus macaque cells is in part due to the failure of HIV-1 Vif to counteract the restriction factor APOBEC3G. However, in this study we demonstrate that several rhesus macaque APOBEC3 (rhAPOBEC3) proteins are capable of inhibiting HIV-1 infectivity. There was considerable variation in the ability of a panel of Vif proteins to induce degradation of rhAPOBEC3 proteins, and mutations within HIV-1 Vif that render it capable of degrading rhAPOBEC3G did not confer activity against other antiviral rhAPOBEC3 proteins. These findings suggest that multiple APOBEC3 proteins can contribute to primate lentivirus species tropism

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

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

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