Longitudinal imaging of HIV-1 spread in humanized mice with parallel 3D immunofluorescence and electron tomography

  1. Collin Kieffer
  2. Mark S Ladinsky
  3. Allen Ninh
  4. Rachel P Galimidi
  5. Pamela J Bjorkman  Is a corresponding author
  1. California Institute of Technology, United States
12 figures and 4 videos

Figures

HIV-1 infection and Imaging of hu-mice.

(A) Mean blood p24 levels from three HIV-1–infected PBMC-NSG mice assessed at 0, 12, and 21 days PI. Error bars represent standard deviation. (B) IF of GALT from HIV-infected animal with highest …

https://doi.org/10.7554/eLife.23282.002
Tissue clearing and IF imaging of lymphoid tissues.

(A) Representative images of uncleared (left) and cleared (right) lymphoid tissues from uninfected hu-mice. (B, C) Confocal images of cleared lymphoid tissues from uninfected (panel B) and …

https://doi.org/10.7554/eLife.23282.004
Figure 3 with 1 supplement
Longitudinal quantification of GALT infectivity.

(A) Timeline of immune cell reconstitution, HIV-1 infection, and tissue harvest from hu-mice. Top numbers depict days PI, bottom numbers show number of animals per timepoint. Single uninfected …

https://doi.org/10.7554/eLife.23282.005
Figure 3—figure supplement 1
Validation of automated quantification of nuclei.

(A) Representative confocal slice of colon sample. Nuclei are white. 881 nuclei were manually counted. (B) Automated quantification of the image shown in panel A resulting in 842 nuclei. Low numbers …

https://doi.org/10.7554/eLife.23282.009
Spatial distribution of HIV-1 infection in GALT over time.

(A) Representative confocal slice from 45 μm Z-stack depicting the distribution of human CD4+ T-cells (magenta), HIV-1 p24 (green), human CD8+ T-cells (red) and nuclei (blue) in colon at 12-days PI. …

https://doi.org/10.7554/eLife.23282.010
Electron tomography of longitudinal HIV-1 spread in GALT.

(A, B) Montaged overview EM projection images (panel A) and zoomed tomographic slices (panel B) of HIV-1–infected colon crypts from hu-mice from early (5 days), peak (10–16 days), and late (>21 …

https://doi.org/10.7554/eLife.23282.015
Figure 6 with 1 supplement
Parallel IF and ET of HIV-1 infection in spleen.

(A) Confocal IF images of cleared spleen showing human CD3+ T-cells (magenta), HIV-1 p24 (green), and nuclei (blue) from an uninfected (-HIV) or infected (+HIV) animal at 12-days PI. Yellow boxes …

https://doi.org/10.7554/eLife.23282.017
Figure 6—figure supplement 1
Architecture of the spleen in wildtype, NSG, and PBMC-NSG mice.

Bright-field microscopy images (left panels) of sections of fixed spleen from wildtype, NSG, PBMC-NSG, and HIV-1–infected PBMC-NSG mice. White pulp and red pulp are lighter colored and dark red …

https://doi.org/10.7554/eLife.23282.018
ET and modeling of HIV-1 Infection in spleen.

(A and B) EM projection images of HIV-1-infected cells from regions of spleen from an HIV-1-infected animal 12-days PI (top panels). Collagen fibrils (white arrows) and pools of virions (black …

https://doi.org/10.7554/eLife.23282.019
Localization of HIV-1 budding machinery.

(A) Densities localized to the base of budding virions. (B) Anti-VPS4A immuno-EM revealing localization patterns consistent with densities in panel A. (C) Tomographic slice of HIV-1–infected spleen …

https://doi.org/10.7554/eLife.23282.021
Comparison of virion-associated densities, cytoplasmic densities, and densities corresponding to cytoplasmic ribosomes.

(A) Representative tomographic slices of densities associated with budding virions (top), cytoplasmic pools near budding virions (middle), and cytoplasmic ribosomes. (B) Gallery of images with the …

https://doi.org/10.7554/eLife.23282.022
Author response image 1
Equivalent IF confocal slices of colon crypts from HIV-1-infected BLT (left) or PBMC-NSG (right) hu-mice.

The density and localization of human CD3+ T cells (green) are similar in both models (blue = DAPI nuclear stain).

https://doi.org/10.7554/eLife.23282.024
Author response image 2
Equivalent tomographic slices of colon crypt regions from HIV-1-infected BLT (left) or PBMC-NSG (right) hu-mice.

Free virions are visible at similar levels and locations in both models (red arrows = pools of free virions).

https://doi.org/10.7554/eLife.23282.025
Author response image 3
Equivalent tomographic slices of brush border regions of gut epithelial cells from wild-type mice that were optimally preserved for EM imaging by high-pressure freezing/freeze substitution (HPF/FS) fixation (left) or optically cleared using CLARITY prior to HPF/FS (right).

Clearing caused drastic loss of ultrastructural detail, likely due to extraction of lipids from the tissue (Term. web = terminal web; ICS = intercellular space).

https://doi.org/10.7554/eLife.23282.026

Videos

Video 1
Spatial Distribution of HIV-1 and T-cells in GALT

360˚ rotation of a 45 μm thick Z-stack depicting the spatial distribution of human CD4+ T-cells (magenta), HIV-1 p24 (green), human CD8+ T-cells (red) and nuclei (blue) in colon at 12-days PI. …

https://doi.org/10.7554/eLife.23282.014
Video 2
ET of a pool of virions in GALT at peak infectivity

Tomographic reconstruction of a volume of two consecutive 400 nm serial sections from colon of a PBMC-NSG animal 10-days PI (Figure 5C, upper right panel). A pool of virions resides in the center …

https://doi.org/10.7554/eLife.23282.016
Video 3
Modeling of an infected cell and individual virions in spleen

Tomographic reconstruction of a volume of two consecutive 400 nm serial sections of an HIV-1–infected cell from spleen of a PBMC-NSG animal 12-days PI. Virions are modeled as mature (green), …

https://doi.org/10.7554/eLife.23282.020
Video 4
ET of densities associated with virus budding

Tomographic reconstruction of an accumulation of cytoplasmic densities associated with prolific virus release. Densities were present in cytoplasm immediately adjacent to a region of plasma membrane …

https://doi.org/10.7554/eLife.23282.023

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