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3D reconstruction of VZV infected cell nuclei and PML nuclear cages by serial section array scanning electron microscopy and electron tomography.

Reichelt M, Joubert L, Perrino J, Koh AL, Phanwar I, Arvin AM - PLoS Pathog. (2012)

Bottom Line: Here we report the development of a novel 3D imaging and reconstruction strategy that we term Serial Section Array-Scanning Electron Microscopy (SSA-SEM) and its application to the analysis of VZV-infected cells and these nuclear PML cages.This method allowed a quantitative determination of how many nucleocapsids can be sequestered within individual PML cages (sequestration capacity), what proportion of nucleocapsids are entrapped in single nuclei (sequestration efficiency) and revealed the ultrastructural detail of the PML cages.This SSA-SEM analysis extends our recent characterization of PML cages and provides a proof of concept for this new strategy to investigate events during virion assembly at the single cell level.

View Article: PubMed Central - PubMed

Affiliation: Departments of Pediatrics and Microbiology & Immunology, Stanford University School of Medicine, Stanford, California, United States of America. reichelt@stanford.edu

ABSTRACT
Varicella-zoster virus (VZV) is a human alphaherpesvirus that causes varicella (chickenpox) and herpes zoster (shingles). Like all herpesviruses, the VZV DNA genome is replicated in the nucleus and packaged into nucleocapsids that must egress across the nuclear membrane for incorporation into virus particles in the cytoplasm. Our recent work showed that VZV nucleocapsids are sequestered in nuclear cages formed from promyelocytic leukemia protein (PML) in vitro and in human dorsal root ganglia and skin xenografts in vivo. We sought a method to determine the three-dimensional (3D) distribution of nucleocapsids in the nuclei of herpesvirus-infected cells as well as the 3D shape, volume and ultrastructure of these unique PML subnuclear domains. Here we report the development of a novel 3D imaging and reconstruction strategy that we term Serial Section Array-Scanning Electron Microscopy (SSA-SEM) and its application to the analysis of VZV-infected cells and these nuclear PML cages. We show that SSA-SEM permits large volume imaging and 3D reconstruction at a resolution sufficient to localize, count and distinguish different types of VZV nucleocapsids and to visualize complete PML cages. This method allowed a quantitative determination of how many nucleocapsids can be sequestered within individual PML cages (sequestration capacity), what proportion of nucleocapsids are entrapped in single nuclei (sequestration efficiency) and revealed the ultrastructural detail of the PML cages. More than 98% of all nucleocapsids in reconstructed nuclear volumes were contained in PML cages and single PML cages sequestered up to 2,780 nucleocapsids, which were shown by electron tomography to be embedded and cross-linked by an filamentous electron-dense meshwork within these unique subnuclear domains. This SSA-SEM analysis extends our recent characterization of PML cages and provides a proof of concept for this new strategy to investigate events during virion assembly at the single cell level.

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Three-dimensional distribution of VZV nucleocapsids in host cell nuclei with PML cages.Melanoma cells that express doxycycline-induced PML IV were infected with VZV for 48 h and processed for BSE-SEM imaging. (A) BSE-SEM images at different magnifications of a syncytium of VZV infected melanoma cells. Left panel: low magnification view of a syncytium; middle panel: one nucleus of the same syncytium with two PML cages; right panel: higher magnification view of a PML cage with sequestered VZV capsids. Black squares indicate areas that are shown at higher magnification in the panels to the right. Scale bars are 5 µm. (B) Five representative images (s1, s5, s9, s13, s17) from a series of 18 consecutive sections through the nucleus shown in A, middle panel. See also Video S4. (C and D) 3D models based on tracing and segmentation in all 18 sections of electron dense heterochromatin (blue); nucleocapsids (yellow spheres) and PML cages (green, shown only in D). 1,732 and 1,324 capsids were identified in the upper and lower PML cage, respectively). Scale bars are 5 µm. See also Video S5. (E and F) 3D models of the upper PML cage. Color code as above, but immature capsids (A and B-type capsids) are shown as yellow spheres and mature capsids (C-type) in orange; the PML cage is transparent green (shown only in F). Scale bars are 2 µm.
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ppat-1002740-g004: Three-dimensional distribution of VZV nucleocapsids in host cell nuclei with PML cages.Melanoma cells that express doxycycline-induced PML IV were infected with VZV for 48 h and processed for BSE-SEM imaging. (A) BSE-SEM images at different magnifications of a syncytium of VZV infected melanoma cells. Left panel: low magnification view of a syncytium; middle panel: one nucleus of the same syncytium with two PML cages; right panel: higher magnification view of a PML cage with sequestered VZV capsids. Black squares indicate areas that are shown at higher magnification in the panels to the right. Scale bars are 5 µm. (B) Five representative images (s1, s5, s9, s13, s17) from a series of 18 consecutive sections through the nucleus shown in A, middle panel. See also Video S4. (C and D) 3D models based on tracing and segmentation in all 18 sections of electron dense heterochromatin (blue); nucleocapsids (yellow spheres) and PML cages (green, shown only in D). 1,732 and 1,324 capsids were identified in the upper and lower PML cage, respectively). Scale bars are 5 µm. See also Video S5. (E and F) 3D models of the upper PML cage. Color code as above, but immature capsids (A and B-type capsids) are shown as yellow spheres and mature capsids (C-type) in orange; the PML cage is transparent green (shown only in F). Scale bars are 2 µm.

Mentions: These SSA-SEM results were confirmed by two more 3D models of large volumes of VZV-infected cell nuclei, which were derived by morphological segmentation of 18 consecutive TEM sections of 100 nm thickness (Figures 3F and 3G; Video S3). In the reconstructed nuclear volume in Figure 3F, which accounted for 46.2 µm3, 109 (25.65%) mature and 316 (74.4%) immature nucleocapsids were identified and 102 (7.6%) mature and 1,238 (92.4%) immature capsids were identified in the nuclear volume in Figure 3G (43.4 µm3) (Figure 3G and Video S3). Similar to the nucleus in Figure 3A–E, most nucleocapsids were distributed evenly throughout the reconstructed nuclear volume; no extended clusters of aggregated nucleocapsids were visible. The quantifications of structures shown in Figure 3 are summarized in Table 1.


3D reconstruction of VZV infected cell nuclei and PML nuclear cages by serial section array scanning electron microscopy and electron tomography.

Reichelt M, Joubert L, Perrino J, Koh AL, Phanwar I, Arvin AM - PLoS Pathog. (2012)

Three-dimensional distribution of VZV nucleocapsids in host cell nuclei with PML cages.Melanoma cells that express doxycycline-induced PML IV were infected with VZV for 48 h and processed for BSE-SEM imaging. (A) BSE-SEM images at different magnifications of a syncytium of VZV infected melanoma cells. Left panel: low magnification view of a syncytium; middle panel: one nucleus of the same syncytium with two PML cages; right panel: higher magnification view of a PML cage with sequestered VZV capsids. Black squares indicate areas that are shown at higher magnification in the panels to the right. Scale bars are 5 µm. (B) Five representative images (s1, s5, s9, s13, s17) from a series of 18 consecutive sections through the nucleus shown in A, middle panel. See also Video S4. (C and D) 3D models based on tracing and segmentation in all 18 sections of electron dense heterochromatin (blue); nucleocapsids (yellow spheres) and PML cages (green, shown only in D). 1,732 and 1,324 capsids were identified in the upper and lower PML cage, respectively). Scale bars are 5 µm. See also Video S5. (E and F) 3D models of the upper PML cage. Color code as above, but immature capsids (A and B-type capsids) are shown as yellow spheres and mature capsids (C-type) in orange; the PML cage is transparent green (shown only in F). Scale bars are 2 µm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3369938&req=5

ppat-1002740-g004: Three-dimensional distribution of VZV nucleocapsids in host cell nuclei with PML cages.Melanoma cells that express doxycycline-induced PML IV were infected with VZV for 48 h and processed for BSE-SEM imaging. (A) BSE-SEM images at different magnifications of a syncytium of VZV infected melanoma cells. Left panel: low magnification view of a syncytium; middle panel: one nucleus of the same syncytium with two PML cages; right panel: higher magnification view of a PML cage with sequestered VZV capsids. Black squares indicate areas that are shown at higher magnification in the panels to the right. Scale bars are 5 µm. (B) Five representative images (s1, s5, s9, s13, s17) from a series of 18 consecutive sections through the nucleus shown in A, middle panel. See also Video S4. (C and D) 3D models based on tracing and segmentation in all 18 sections of electron dense heterochromatin (blue); nucleocapsids (yellow spheres) and PML cages (green, shown only in D). 1,732 and 1,324 capsids were identified in the upper and lower PML cage, respectively). Scale bars are 5 µm. See also Video S5. (E and F) 3D models of the upper PML cage. Color code as above, but immature capsids (A and B-type capsids) are shown as yellow spheres and mature capsids (C-type) in orange; the PML cage is transparent green (shown only in F). Scale bars are 2 µm.
Mentions: These SSA-SEM results were confirmed by two more 3D models of large volumes of VZV-infected cell nuclei, which were derived by morphological segmentation of 18 consecutive TEM sections of 100 nm thickness (Figures 3F and 3G; Video S3). In the reconstructed nuclear volume in Figure 3F, which accounted for 46.2 µm3, 109 (25.65%) mature and 316 (74.4%) immature nucleocapsids were identified and 102 (7.6%) mature and 1,238 (92.4%) immature capsids were identified in the nuclear volume in Figure 3G (43.4 µm3) (Figure 3G and Video S3). Similar to the nucleus in Figure 3A–E, most nucleocapsids were distributed evenly throughout the reconstructed nuclear volume; no extended clusters of aggregated nucleocapsids were visible. The quantifications of structures shown in Figure 3 are summarized in Table 1.

Bottom Line: Here we report the development of a novel 3D imaging and reconstruction strategy that we term Serial Section Array-Scanning Electron Microscopy (SSA-SEM) and its application to the analysis of VZV-infected cells and these nuclear PML cages.This method allowed a quantitative determination of how many nucleocapsids can be sequestered within individual PML cages (sequestration capacity), what proportion of nucleocapsids are entrapped in single nuclei (sequestration efficiency) and revealed the ultrastructural detail of the PML cages.This SSA-SEM analysis extends our recent characterization of PML cages and provides a proof of concept for this new strategy to investigate events during virion assembly at the single cell level.

View Article: PubMed Central - PubMed

Affiliation: Departments of Pediatrics and Microbiology & Immunology, Stanford University School of Medicine, Stanford, California, United States of America. reichelt@stanford.edu

ABSTRACT
Varicella-zoster virus (VZV) is a human alphaherpesvirus that causes varicella (chickenpox) and herpes zoster (shingles). Like all herpesviruses, the VZV DNA genome is replicated in the nucleus and packaged into nucleocapsids that must egress across the nuclear membrane for incorporation into virus particles in the cytoplasm. Our recent work showed that VZV nucleocapsids are sequestered in nuclear cages formed from promyelocytic leukemia protein (PML) in vitro and in human dorsal root ganglia and skin xenografts in vivo. We sought a method to determine the three-dimensional (3D) distribution of nucleocapsids in the nuclei of herpesvirus-infected cells as well as the 3D shape, volume and ultrastructure of these unique PML subnuclear domains. Here we report the development of a novel 3D imaging and reconstruction strategy that we term Serial Section Array-Scanning Electron Microscopy (SSA-SEM) and its application to the analysis of VZV-infected cells and these nuclear PML cages. We show that SSA-SEM permits large volume imaging and 3D reconstruction at a resolution sufficient to localize, count and distinguish different types of VZV nucleocapsids and to visualize complete PML cages. This method allowed a quantitative determination of how many nucleocapsids can be sequestered within individual PML cages (sequestration capacity), what proportion of nucleocapsids are entrapped in single nuclei (sequestration efficiency) and revealed the ultrastructural detail of the PML cages. More than 98% of all nucleocapsids in reconstructed nuclear volumes were contained in PML cages and single PML cages sequestered up to 2,780 nucleocapsids, which were shown by electron tomography to be embedded and cross-linked by an filamentous electron-dense meshwork within these unique subnuclear domains. This SSA-SEM analysis extends our recent characterization of PML cages and provides a proof of concept for this new strategy to investigate events during virion assembly at the single cell level.

Show MeSH
Related in: MedlinePlus