Analysis of HIV-1 Assembly on Intracellular Plasma Membrane-connected Compartments of Primary Human Macrophages

In primary human monocyte-derived macrophages (MDM), HIV assembles on complex intracellular plasma membrane-connected compartments (IPMCs). Currently, it is unclear whether, in addition to assembly at IPMCs, HIV uses the cell surface of MDM, as viruses that bud at the cell surface may dissociate and be lost from the samples. As the endosomal sorting complex required for transport (ESCRT) machinery is required for the final scission events that release assembled virus from the plasma membrane, this question was addressed by depleting the ESCRT components Tsg101 and ALIX, or generating mutant HIV-1 strains defective in recruiting the ESCRT machinery, to arrest HIV budding and visualise all budding events. Using siRNA, ALIX and Tsg101 were depleted efficiently in MDM. Although the effects on virus release were minimal, Tsg101 had a greater effect than ALIX for HIV release in MDM. In the second approach, I generated mutants (HIV-1 PTAP−, YP−, PTAP−YP−, Δp6) defective in recruiting the ESCRT machinery, by mutating the sequences that bind Tsg101 and ALIX. The mutants were characterised on HEK 293T cells, and the release of PTAP−, PTAP−YP− and Δp6 was inhibited. Analysis by electron microscopy (EM) showed that the mutants indeed produced arrested viruses. As the mutants are defective in release, I developed a method to rescue HIV-1 PTAP− and PTAP−YP− viruses for infecting MDM. The cells were infected with the rescued mutants, and analysed using immunofluorescence and EM. By confocal microscopy, 77% of the cells had viruses in the IPMCs. Using EM, immature viruses were found predominantly (97%) in the IPMCs. Estimates of membrane area revealed enrichment of HIV in the IPMCs. This study provides the first conclusive evidence that HIV is targeted to IPMCs in MDM. This may shield the virus from immune surveillance during virus assembly, with potential impact on cell-to-cell transmission and disease progression

The intracellular plasma membrane-connected compartment in the assembly of HIV-1 in human macrophages

Background 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. Results 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

3D correlative light and electron microscopy of cultured cells using serial blockface scanning electron microscopy

The processes of life take place in multiple dimensions, but imaging these processes in even three dimensions is challenging. Here, we describe a workflow for 3D correlative light and electron microscopy (CLEM) of cell monolayers using fluorescence microscopy to identify and follow biological events, combined with serial blockface scanning electron microscopy to analyse the underlying ultrastructure. The workflow encompasses all steps from cell culture to sample processing, imaging strategy, and 3D image processing and analysis. We demonstrate successful application of the workflow to three studies, each aiming to better understand complex and dynamic biological processes, including bacterial and viral infections of cultured cells and formation of entotic cell-in-cell structures commonly observed in tumours. Our workflow revealed new insight into the replicative niche of Mycobacterium tuberculosis in primary human lymphatic endothelial cells, HIV-1 in human monocytederived macrophages, and the composition of the entotic vacuole. The broad application of this 3D CLEM technique will make it a useful addition to the correlative imaging toolbox for biomedical research