Figure 1.
IF and EM imaging of BLT mouse GALT.
(A) Histological overview, indicating primary GALT-containing regions. (B) Tissue sections from the small intestine of uninfected or HIV-1–infected humanized BLT mice, stained with antibodies recognizing human CD3 and CD4, CD68, and DC-SIGN (blue = DAPI nuclear stain). The top two panels are longitudinal sections of villi, showing the lamina propria (LP); the bottom four panels are cross-sections showing crypts. Staining for human CD4 revealed depletion of CD4 T cells in both LP and crypts of HIV-1–infected BLT mice, and staining for HIV-1 p24 localized virions to the crypts. Inset: An infected cell co-labeled for CD4 and HIV-1 p24. (C) Left: EM overview of the lower portion of a crypt from BLT-mouse colon. Middle: A pool of free virions (red arrowhead) between two cells. Right: A tomographic slice of the pool with modeled virions (blue, membrane; purple, cores; average diameter = 99.3+/−4.7 nm; n = 50). Figure S1 shows HIV-1 in GALT substructures.
Figure 2.
Virion structures in HIV-1–infected GALT.
(A, B) Slices from tomographic reconstructions of immature budding virions extending from filopodia in positively-stained (A) and negatively-stained (B) HIV-1–infected jejunum. (C, D) Details from tomographic slices, showing mature and immature (insets) virions in positively-stained (C) and negatively-stained (D) samples. (E, F) Immunolabeling (projection images) of virions labeled with antibodies against the HIV-1 capsid (anti-p24) (E) or the HIV-1 envelope (anti-Env) (F), which localized to the expected regions of the virions: anti-p24 to the interior and anti-Env to the exterior. Figure S3 shows immunoEM of human cell markers.
Figure 3.
Structural details in negatively-stained images of HIV-1 in infected GALT.
(A, B) Tomographic reconstructions of budding HIV-1 virions, showing Gag layers (A; slice through equator) and hexagonal lattice (B; slice through surface). Hexagonal symmetry was confirmed by Fourier transformation of the Gag lattice region in a single tomographic slice (upper inset in B). A similar transform of a region of cytoplasm adjacent to the bud (lower inset) shows only the inherent Friedel symmetry of a Fourier transform. (C, D) Tomographic slice (C) and model (D) of a budding profile. The identities of the layers in the budding profile cannot be definitively assigned, but a proposed assignment of features visible in tomogram is as follows: black, plasma membrane; light blue, MA; red, CA-NTD; orange, CA-CTD; magenta dots, NC; gold, RNA genome; green, ESCRT. The white space between the plasma membrane and first Gag layer (see also panels A–C) is an artifact of preservation. Figure S4 shows a gallery of Fourier transforms and a comparison with cryoET.
Figure 4.
(A) Tomographic slice of a GALT region near the edge of a crypt. Two pools of mature HIV-1 virions, indicated in gray boxes, occupied dilated regions of the intercellular space (IS) between two cells (N, nucleus). Collagen fibrils (Coll) were visible at the outer boundary of the crypt. (B) Montaged overview of four cells within GALT. Red dashed lines demark the intercellular spaces. Pools of virions within GALT were defined as a population within an intercellular space that was continuous throughout a given volume of a tomographic reconstruction. Virus pools within intercellular spaces that did not connect within the volume were considered distinct. (C) Region shown in B with modeled HIV virions in three pools containing 59, 17 and 38 virions, respectively. (D) Pool of HIV-1 in a dilated domain associated with a thin channel (red arrowheads) that opened to the mucosa. (E) Segmented model of the microchannel shown in D. The width of the channel remained relatively constant through an ∼600 nm volume, suggesting that morphological changes would be necessary for virions to escape. (A: jejunum; B–E; colon).
Figure 5.
Intercalating infected cell with a budding profile contacting an adjacent cell.
(A) Tomographic slice (9 nm) from a six-frame montaged tomogram near the edge of a crypt in colon. The field contained two cells (N, nucleus) with an intercalating HIV-1–infected cell (presumably a dendritic cell; green outline). Four HIV-1 budding profiles were forming from the presumptive dendritic cell at different positions in the volume (magenta dots and black arrow). Dots indicate the approximate position of free mature virions at different positions in the volume (upper cell: light blue; lower cell magenta); the black arrow indicates a budding profile potentially involved in a virological synapse. (B) Eight tomographic slices (9 nm each) detailing the approach of the bud in panel A (black arrows) to the adjacent cell. Red arrowheads indicate the points of contact with the adjacent cell. (C) Immunolocalization of ICAM-1 near a presumptive virological synapse. A budding virion (arrow) is shown projecting from an infected cell and contacting the surface of an adjacent cell across an intercellular space (IS). The surfaces of both cells labeled for ICAM-1 (arrowheads). (D) Immunolocalization of LFA-1 near a presumptive forming virological synapse. The budding profile (arrow) extending from the infected cell was nearing the surface of the adjacent cell. LFA-1 (arrowheads) was present on the surface and proximal underlying compartments of the infected cell. Details of this interaction are shown in the latter part of Movie S1. Other examples of potential virological synapses are shown in Figure S6.
Figure 6.
ImmunoEM of ESCRT pathway proteins at sites of HIV-1 budding in GALT.
(A–C) Immunolabeling (projection images) of budding virions using antibodies against CHMP1B (A), CHMP2A (B), and ALIX (C). Antibodies localized to the necks of budding virions or to the adjacent plasma membrane. (D) Cluster of spoke-like projections (red arrowheads) radiating from a common origin (see also Figure 3C,D). These types of striations, suggested to represent components of ESCRT-I and/or ESCRT-II, were only seen when the neck diameter was more than half of the diameter of the bud. Figure S8 shows galleries of electron dense structures from both “early” and “late” budding HIV-1 virions.
Figure 7.
Electron dense striations in tomograms of the scission regions of budding virions.
(A–E) Parallel electron dense striations (red arrowheads) circumscribing the necks of budding virions in positively-stained, plastic-embedded samples (A,B,D) and in negatively-stained samples (C,E). Parallel striations, suggested to be portions of the polymerized ESCRT-III complex, were observed only when the diameter of the neck was half or less than the diameter of the bud. (B) Higher magnification view of the bud in (A), rotated to optimize visualization of the striations. (F) Tomographic slice of a positively-stained budding profile displaying five dense spots (red arrows) in the neck region that may correspond to VPS4 complexes recruited to facilitate scission of the bud. Ribosomes in cytoplasm distal from the budding virion appeared slightly smaller and typically less electron dense than the presumptive VPS4 structures. (G) Galleries of HIV-1 budding profiles bearing presumptive VPS4 spots (row 1) and individual presumptive VPS4 spots and cytoplasmic ribosomes, extracted from tomograms and viewed at high magnification (rows 2 and 3, respectively). The VPS4 spots were pleomorphic and solidly dense, with an average width of 13.3±0.8 nm; n = 10). The spots appeared to be slightly larger than ribosomal densities (11.9±0.8 nm; n = 10), which were less dense and often showed a characteristic “groove” between the 30S and 50S subunits. Note that the ribosomes in this and previous ET studies involving positively-stained, plastic embedded samples [72] appear smaller than their 25–30 nm diameter.