Limits...
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.

Show MeSH

Related in: MedlinePlus

Large volume-reconstruction of a VZV infected cell nucleus with four PML cages.Melanoma cells that express doxycycline-induced PML IV were infected with VZV for 48 h and processed for BSE-SEM imaging. (A) Five representative BSE-SEM images (s5, s20, s30, s47, s70) from a series of 82 consecutive sections through a VZV nucleus with four PML cages (1–4, black arrows) with sequestered VZV capsids. A valley-like indentation of the nucleus (blue outline and arrow) and the endoplasmic reticulum (ER) are marked. See also Video S6. (B–D and E–G, respectively) show 3D models of the nucleus in two angles (100 degrees rotation to the left). (B and E) Shape of the nucleus based on tracing its outer boundary (grey). (C and F) The shape of the nucleus (transparent grey) is overlaid with the dense heterochromatin (transparent blue). PML cages 1–4 (solid green) and VZV capsids that escaped sequestration (red spheres) are visible in the interior of the nucleus. (D and G) Same view as above, but the nuclear envelope and the PML domains are completely transparent. This reveals the location of all VZV capsids (5,597) identified in this nucleus; red spheres represent free capsids (70) and yellow spheres represent sequestered capsids (5,527). Scale bars are 5 µm. See also Video S7. (H and I) 3D models of PML cages (transparent green) from the same nucleus at higher magnification that reveal the dense packaging of capsids (yellow) and the close association of PML cages with the electron dense heterochromatin (blue). Red spheres represent free capsids. Scale bars are 2 µm. See also Video S8.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3369938&req=5

ppat-1002740-g005: Large volume-reconstruction of a VZV infected cell nucleus with four PML cages.Melanoma cells that express doxycycline-induced PML IV were infected with VZV for 48 h and processed for BSE-SEM imaging. (A) Five representative BSE-SEM images (s5, s20, s30, s47, s70) from a series of 82 consecutive sections through a VZV nucleus with four PML cages (1–4, black arrows) with sequestered VZV capsids. A valley-like indentation of the nucleus (blue outline and arrow) and the endoplasmic reticulum (ER) are marked. See also Video S6. (B–D and E–G, respectively) show 3D models of the nucleus in two angles (100 degrees rotation to the left). (B and E) Shape of the nucleus based on tracing its outer boundary (grey). (C and F) The shape of the nucleus (transparent grey) is overlaid with the dense heterochromatin (transparent blue). PML cages 1–4 (solid green) and VZV capsids that escaped sequestration (red spheres) are visible in the interior of the nucleus. (D and G) Same view as above, but the nuclear envelope and the PML domains are completely transparent. This reveals the location of all VZV capsids (5,597) identified in this nucleus; red spheres represent free capsids (70) and yellow spheres represent sequestered capsids (5,527). Scale bars are 5 µm. See also Video S7. (H and I) 3D models of PML cages (transparent green) from the same nucleus at higher magnification that reveal the dense packaging of capsids (yellow) and the close association of PML cages with the electron dense heterochromatin (blue). Red spheres represent free capsids. Scale bars are 2 µm. See also Video S8.

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)

Large volume-reconstruction of a VZV infected cell nucleus with four PML cages.Melanoma cells that express doxycycline-induced PML IV were infected with VZV for 48 h and processed for BSE-SEM imaging. (A) Five representative BSE-SEM images (s5, s20, s30, s47, s70) from a series of 82 consecutive sections through a VZV nucleus with four PML cages (1–4, black arrows) with sequestered VZV capsids. A valley-like indentation of the nucleus (blue outline and arrow) and the endoplasmic reticulum (ER) are marked. See also Video S6. (B–D and E–G, respectively) show 3D models of the nucleus in two angles (100 degrees rotation to the left). (B and E) Shape of the nucleus based on tracing its outer boundary (grey). (C and F) The shape of the nucleus (transparent grey) is overlaid with the dense heterochromatin (transparent blue). PML cages 1–4 (solid green) and VZV capsids that escaped sequestration (red spheres) are visible in the interior of the nucleus. (D and G) Same view as above, but the nuclear envelope and the PML domains are completely transparent. This reveals the location of all VZV capsids (5,597) identified in this nucleus; red spheres represent free capsids (70) and yellow spheres represent sequestered capsids (5,527). Scale bars are 5 µm. See also Video S7. (H and I) 3D models of PML cages (transparent green) from the same nucleus at higher magnification that reveal the dense packaging of capsids (yellow) and the close association of PML cages with the electron dense heterochromatin (blue). Red spheres represent free capsids. Scale bars are 2 µm. See also Video S8.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3369938&req=5

ppat-1002740-g005: Large volume-reconstruction of a VZV infected cell nucleus with four PML cages.Melanoma cells that express doxycycline-induced PML IV were infected with VZV for 48 h and processed for BSE-SEM imaging. (A) Five representative BSE-SEM images (s5, s20, s30, s47, s70) from a series of 82 consecutive sections through a VZV nucleus with four PML cages (1–4, black arrows) with sequestered VZV capsids. A valley-like indentation of the nucleus (blue outline and arrow) and the endoplasmic reticulum (ER) are marked. See also Video S6. (B–D and E–G, respectively) show 3D models of the nucleus in two angles (100 degrees rotation to the left). (B and E) Shape of the nucleus based on tracing its outer boundary (grey). (C and F) The shape of the nucleus (transparent grey) is overlaid with the dense heterochromatin (transparent blue). PML cages 1–4 (solid green) and VZV capsids that escaped sequestration (red spheres) are visible in the interior of the nucleus. (D and G) Same view as above, but the nuclear envelope and the PML domains are completely transparent. This reveals the location of all VZV capsids (5,597) identified in this nucleus; red spheres represent free capsids (70) and yellow spheres represent sequestered capsids (5,527). Scale bars are 5 µm. See also Video S7. (H and I) 3D models of PML cages (transparent green) from the same nucleus at higher magnification that reveal the dense packaging of capsids (yellow) and the close association of PML cages with the electron dense heterochromatin (blue). Red spheres represent free capsids. Scale bars are 2 µm. See also Video S8.
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