Abstract
HIV-1-containing internal compartments are readily detected in images of thin sections from infected cells using conventional transmission electron microscopy, but the origin, connectivity, and 3D distribution of these compartments has remained controversial. Here, we report the 3D distribution of viruses in HIV-1-infected primary human macrophages using cryo-electron tomography and ion-abrasion scanning electron microscopy (IA-SEM), a recently developed approach for nanoscale 3D imaging of whole cells. Using IA-SEM, we show the presence of an extensive network of HIV-1-containing tubular compartments in infected macrophages, with diameters of ∼150–200 nm, and lengths of up to ∼5 µm that extend to the cell surface from vesicular compartments that contain assembling HIV-1 virions. These types of surface-connected tubular compartments are not observed in T cells infected with the 29/31 KE Gag-matrix mutant where the virus is targeted to multi-vesicular bodies and released into the extracellular medium. IA-SEM imaging also allows visualization of large sheet-like structures that extend outward from the surfaces of macrophages, which may bend and fold back to allow continual creation of viral compartments and virion-lined channels. This potential mechanism for efficient virus trafficking between the cell surface and interior may represent a subversion of pre-existing vesicular machinery for antigen capture, processing, sequestration, and presentation.
Highlights
Conventional transmission electron microscopy is a powerful tool for investigation of HIV pathogenesis and subcellular organization of viral compartments in infected cells [1]
Because of the incompleteness of data obtained from cell sections, the probability of visualizing a full and accurate representation of the cytoarchitecture is minimal. This limitation is overcome with the use of ion abrasion scanning electron microscopy (IA-SEM), a newly developed approach for 3D imaging of large mammalian cells and tissues in their entirety [2,3,4] at in-plane spatial resolutions of,6 nm and z-axis resolutions of,30 nm
Complementary information on subcellular architecture was obtained from electron tomography of thin sections from fixed, plastic-embedded HIV-1 infected macrophages, which provides a view of the interior of the cell (Figure 2 and Video S1)
Summary
Conventional transmission electron microscopy is a powerful tool for investigation of HIV pathogenesis and subcellular organization of viral compartments in infected cells [1]. Insights into cellular ultrastructure using this approach have generally been obtained using thin sections derived from fixed, plastic-embedded cells or tissues. Because of the incompleteness of data obtained from cell sections, the probability of visualizing a full and accurate representation of the cytoarchitecture is minimal. This limitation is overcome with the use of ion abrasion scanning electron microscopy (IA-SEM), a newly developed approach for 3D imaging of large mammalian cells and tissues in their entirety [2,3,4] at in-plane spatial resolutions of ,6 nm and z-axis resolutions of ,30 nm
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