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Plasma membrane is the site of productive HIV-1 particle assembly.

ABSTRACT: 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.

SUBMITTER: Jouvenet N

PROVIDER: S-EPMC1750931 | biostudies-literature | 2006 Dec

REPOSITORIES: biostudies-literature

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Plasma membrane is the site of productive HIV-1 particle assembly.

Jouvenet Nolwenn N Neil Stuart J D SJ Bess Cameron C Johnson Marc C MC Virgen Cesar A CA Simon Sanford M SM Bieniasz Paul D PD

PLoS biology 20061201 12

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 efficientl ...[more]

PMID: 17147474

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Project description:BackgroundIt has been accepted that HIV buds from the cell surface in T lymphocytes, whereas in macrophages it buds into intracellular endosomes. Recent studies, on the other hand, suggest that HIV preferentially buds from the cell surface even in monocytic cells. However, most studies are based on observations in acutely infected cells and little is known about HIV budding concomitant with reactivation from latency. Such studies would provide a better understanding of a reservoir for HIV.ResultsWe observed HIV budding in latently infected T lymphocytic and monocytic cell lines following TNF-alpha stimulation and examined the upregulation of host factors that may be involved in particle production. Electron microscopy analysis revealed that reactivation of latently infected J1.1 cells (latently infected Jurkat cells with HIV-1) and U1 cells (latently infected U937 cells with HIV-1) displayed HIV particle budding predominantly at the plasma membrane, a morphology that is similar to particle budding in acutely infected Jurkat and U937 cells. When mRNA expression levels were quantified by qRT-PCR, we found that particle production from reactivated J1.1 and U1 cells was accompanied by CD44 upregulation. This upregulation was similarly observed when Jurkat and U937 cells were acutely infected with HIV-1 but not when just stimulated with TNF-alpha, suggesting that CD44 upregulation was linked with HIV production but not with cell stimulation. The molecules in endocytic pathways such as CD63 and HRS were also upregulated when U1 cells were reactivated and U937 cells were acutely infected with HIV-1. Confocal microscopy revealed that these upregulated host molecules were recruited to and accumulated at the sites where mature particles were formed at the plasma membrane.ConclusionOur study indicates that HIV particles are budded at the plasma membrane upon reactivation from latency, a morphology that is similar to particle budding in acute infection. Our data also suggest that HIV expression may lead to the upregulation of certain host cell molecules that are recruited to sites of particle assembly, possibly coordinating particle production.

Project description:BackgroundIn HIV-1-infected human monocyte-derived macrophages (MDMs), virus particles assemble primarily on intracellularly sequestered plasma membrane domains termed intracellular plasma membrane-connected compartments (IPMCs). Despite their clear role in virus formation, little is known of the organization, composition, dynamics or function of these compartments.ResultsWe have used amphipathic membrane dyes to reveal the complex three-dimensional structure of IPMCs in whole MDMs and to visualize connections between IPMCs and the cell surface. The observation of similar IPMC structures in both infected and uninfected cells indicates that these compartments are not induced by virus infection, but are present constitutively in MDMs. By expressing a phospholipase Cδ pleckstrin homology domain linked to green fluorescent protein, we demonstrate that IPMCs contain phosphatidylinositol 4,5-bisphosphate. Live cell imaging of cells expressing this probe shows that IPMCs are dynamic, but relatively stable, sub-domains of the plasma membrane. As recent electron microscopy studies indicated that portions of IPMCs are coated with β2 integrin-containing focal adhesion-like complexes linked to actin, we investigated whether the actin cytoskeleton is required for the organization of IPMCs. In MDMs treated with the actin polymerization inhibitor latrunculin, the normally compact IPMCs dispersed into smaller structures that remained connected to the plasma membrane. Moreover, latrunculin enhanced the release of preformed, mature HIV-1 particles from infected MDMs.ConclusionsIPMCs are constitutive features of MDMs that are continuous with the plasma membrane and are used as unique sites for the assembly of new virions following infection by HIV-1. A functionally intact actin cytoskeleton is required to maintain the organization of the IPMCs and, in HIV-1-infected cells, perturbation of the actin cytoskeleton influences both the organization of the compartment and the release of sequestered virus.

Project description:In HIV-infected macrophages, newly formed progeny virus particles accumulate in intracellular plasma membrane-connected compartments (IPMCs). Although the virus is usually seen in these compartments, it is unclear whether HIV assembly is specifically targeted to IPMCs or whether some viruses may also form at the cell surface but are not detected, as particles budding from the latter site will be released into the medium.To investigate the fidelity of HIV-1 targeting to IPMCs compared to the cell surface directly, we generated mutants defective in recruitment of the Endosomal Sorting Complexes Required for Transport (ESCRT) proteins required for virus scission. For mutants unable to bind the ESCRT-I component Tsg101, HIV release was inhibited and light and electron microscopy revealed that budding was arrested. When expressed in human monocyte-derived macrophages (MDM), these mutants formed budding-arrested, immature particles at their assembly sites, allowing us to capture virtually all of the virus budding events. A detailed morphological analysis of the distribution of the arrested viruses by immunofluorescence staining and confocal microscopy, and by electron microscopy, demonstrated that HIV assembly in MDMs is targeted primarily to IPMCs, with fewer than 5 % of budding events seen at the cell surface. Morphometric analysis of the relative membrane areas at the cell surface and IPMCs confirmed a large enrichment of virus assembly events in IPMCs. Serial block-face scanning electron microscopy of macrophages infected with a budding-defective HIV mutant revealed high-resolution 3D views of the complex organisation of IPMCs, with in excess of 15,000 associated HIV budding sites, and multiple connections between IPMCs and the cell surface.Using detailed quantitative analysis, we demonstrate that HIV assembly in MDMs is specifically targeted to IPMCs. Furthermore, 3D analysis shows, for the first time, the detailed ultrastructure of an IPMC within a large cell volume, at a resolution that allowed identification of individual virus assembly events, and potential portals through which virus may be released during cell-cell transfer. These studies provide new insights to the organisation of the HIV assembly compartments in macrophages, and show how HIV particles accumulating in these protected sites may function as a virus reservoir.

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Plasma membrane is the site of productive HIV-1 particle assembly.

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Plasma membrane is the site of productive HIV-1 particle assembly.

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