Abstract
BackgroundIn 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.ResultsTo 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.ConclusionsUsing 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-016-0272-3) contains supplementary material, which is available to authorized users.
Highlights
In HIV-infected macrophages, newly formed progeny virus particles accumulate in intracellular plasma membrane-connected compartments (IPMCs)
A key difference in HIV assembly in these different host cells is that, in mature macrophages, newly formed virions accumulate in characteristic highly interconnected intracellular compartments [4,5,6,7,8,9]. Many of these compartments have been shown to be connected to the plasma membrane, so that they are known as intracellular plasma membrane-connected compartments (IPMCs)
When HIV-infected monocyte-derived macrophages (MDM) interact with autologous CD4+ T cells, viruses contained within IPMCs can be released through virological synapses leading to efficient infection of the T cells [15,16,17,18]
Summary
In HIV-infected macrophages, newly formed progeny virus particles accumulate in intracellular plasma membrane-connected compartments (IPMCs). A key difference in HIV assembly in these different host cells is that, in mature macrophages, newly formed virions accumulate in characteristic highly interconnected intracellular compartments [4,5,6,7,8,9]. HIV-infected monocytes invading the brain differentiate to macrophages that mediate neuroinflammation and contribute to pathogenesis and HIV-associated neurocognitive disorders (reviewed in [20,21,22]) These cells may represent a viral reservoir at this immunologically privileged site [23, 24]
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