Ion-Abrasion Scanning Electron Microscopy Reveals Surface-Connected Tubular Conduits in HIV-Infected Macrophages

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.

3D visualization of HIV transfer at the virological synapse between dendritic cells and T cells

“…3A, Fig. S2, and Movie S3), similar to those recently described in HIV-infected macrophages (22,23). At the synapse, cell-cell contact involves extensive interdigitation of the respective cell membranes, with filopodial extensions from the T cell penetrating into the recesses of the virion-rich surface folds of the dendritic cell.…”

Macrophage Internal HIV-1 Is Protected from Neutralizing Antibodies

“…In addition, it has been reported that macrophages harbor infectious HIV-1 over a long period of time (39) and that the virus has evolved strategies to inhibit the acidification of endosomal compartments, thereby preventing viral degradation (21). However, the formation and dynamics of the VCCs are still elusive, and it has also been proposed that these correspond to internally sequestered plasma membrane domains, which are connected to the cell surface via small microchannels (4,9,22,45).…”

HIV-1 activates Cdc42 and induces membrane extensions in immature dendritic cells to facilitate cell-to-cell virus propagation

“…25 Sections with nominal thicknesses of 100 nm and 175 nm were prepared using a Leica Ultracut microtome and imaged in a Tecnai 12 electron microscope (FEI Company) operated at 120 kV, and equipped with a 4K ϫ 4K Gatan charge-coupled device. For tomography, images were recorded over an angular range of Ϯ 70 degrees, and reconstructed to obtain a 3D volume using weighted back projection as implemented in the image processing package IMOD.…”

“…Because DC-SIGN has previously been identified as a factor promoting DC-T cell infectious synapse formation, 6,25 we investigated whether HIV-1 could trigger a signaling program that induces actin-based protrusions at the surface of DCs, thereby facilitating an anterograde viral transfer from DCs to CD4 ϩ T cells across infectious synapses. Our results demonstrate a 2-step model for HIV-1 transfer from immature DCs to T cells that involves HIV-1 env engagement of the DC-SIGN receptor, leading to Cdc42 activation and formation of membrane extensions, followed by the Cdc42-dependent transfer of virus to the T-cell.…”