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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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PML protein is associated with entrapped VZV capsids inside PML cages.Melanoma cells that express doxycycline-induced PML IV were infected with VZV for 48 hours and then high pressure frozen, freeze-substituted, embedded in LR-White resin and labeled with anti-PML polyconal rabbit antibody and Protein A conjugated with 15 nm gold particles. (A) A representative TEM image from a series of seven consecutive 100 nm sections is shown. The area in the blue square (left panel) is shown at higher magnification in the right panel. PML specific gold labeling (green arrows) identifies the PML cage (surrounded by a green line, left panel) in the nucleus. Scale bars are 500 nm. (B) The 3D model shows the electron dense heterochromatin (blue) and the location of mature capsids (63; red spheres), immature capsids (403; yellow spheres) and all PML-specific gold particles (5,219; small green spheres) that were identified in the serial sections. Entrapped mature capsids with associated PML labeling are shown as red/green spheres and immature capsids with PML labeling are shown as yellow/green spheres. (C) Same 3D model as in B but at higher magnification and in a different angle. See also Video S9.
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ppat-1002740-g006: PML protein is associated with entrapped VZV capsids inside PML cages.Melanoma cells that express doxycycline-induced PML IV were infected with VZV for 48 hours and then high pressure frozen, freeze-substituted, embedded in LR-White resin and labeled with anti-PML polyconal rabbit antibody and Protein A conjugated with 15 nm gold particles. (A) A representative TEM image from a series of seven consecutive 100 nm sections is shown. The area in the blue square (left panel) is shown at higher magnification in the right panel. PML specific gold labeling (green arrows) identifies the PML cage (surrounded by a green line, left panel) in the nucleus. Scale bars are 500 nm. (B) The 3D model shows the electron dense heterochromatin (blue) and the location of mature capsids (63; red spheres), immature capsids (403; yellow spheres) and all PML-specific gold particles (5,219; small green spheres) that were identified in the serial sections. Entrapped mature capsids with associated PML labeling are shown as red/green spheres and immature capsids with PML labeling are shown as yellow/green spheres. (C) Same 3D model as in B but at higher magnification and in a different angle. See also Video S9.

Mentions: The electron density of the PML positive shell of nuclear PML cages allowed tracing and reconstruction of the shape (3D surface view) of this compartment by SSA-SEM; however the 3D distribution of PML protein within PML cages was not revealed using this approach. Therefore we used a serial section immunoTEM (ss-immunoTEM) approach to investigate quantitatively and in three dimensions how PML protein is distributed within the shell and in the core of the PML cages, where the nucleocapsids are entrapped. Seven consecutive sections (100 nm) through HPF/FS-treated and LRwhite embedded cells that contained PML cages with entrapped VZV capsids, were labeled with a PML specific antibody and Protein A conjugated to 15 nm gold particles, and then imaged by TEM (Figure 6A). The results of tracing and modeling of the PML labeling (small green spheres), mature capsids (red spheres) and immature capsids (yellow spheres) and the electron dense heterochromatin (blue) are shown in Figure 6B, C and Video S9. About 5,219 PML gold particles, 63 mature capsids and 403 immature capsids were identified; 272 of the entrapped nucleocapsids were directly associated with PML gold particles (half-green spheres). The 3D reconstruction clearly reveals a ring-shaped ‘cloud’ of dense PML-labeling that corresponds to the electron dense shell of PML cages as seen in the high-contrast embedded samples analyzed by SSA-SEM before. Significant amounts of PML gold labeling were also found in the core of the PML cage (Figure 6A, right panel and Figure 6C) where 58% of the entrapped nucleocapsids were directly associated with PML gold particles (half-green spheres). Therefore PML protein is not only a structural component of the electron dense shell of PML cages but also binds to nucleocapsids entrapped within the core of the PML cages.


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)

PML protein is associated with entrapped VZV capsids inside PML cages.Melanoma cells that express doxycycline-induced PML IV were infected with VZV for 48 hours and then high pressure frozen, freeze-substituted, embedded in LR-White resin and labeled with anti-PML polyconal rabbit antibody and Protein A conjugated with 15 nm gold particles. (A) A representative TEM image from a series of seven consecutive 100 nm sections is shown. The area in the blue square (left panel) is shown at higher magnification in the right panel. PML specific gold labeling (green arrows) identifies the PML cage (surrounded by a green line, left panel) in the nucleus. Scale bars are 500 nm. (B) The 3D model shows the electron dense heterochromatin (blue) and the location of mature capsids (63; red spheres), immature capsids (403; yellow spheres) and all PML-specific gold particles (5,219; small green spheres) that were identified in the serial sections. Entrapped mature capsids with associated PML labeling are shown as red/green spheres and immature capsids with PML labeling are shown as yellow/green spheres. (C) Same 3D model as in B but at higher magnification and in a different angle. See also Video S9.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3369938&req=5

ppat-1002740-g006: PML protein is associated with entrapped VZV capsids inside PML cages.Melanoma cells that express doxycycline-induced PML IV were infected with VZV for 48 hours and then high pressure frozen, freeze-substituted, embedded in LR-White resin and labeled with anti-PML polyconal rabbit antibody and Protein A conjugated with 15 nm gold particles. (A) A representative TEM image from a series of seven consecutive 100 nm sections is shown. The area in the blue square (left panel) is shown at higher magnification in the right panel. PML specific gold labeling (green arrows) identifies the PML cage (surrounded by a green line, left panel) in the nucleus. Scale bars are 500 nm. (B) The 3D model shows the electron dense heterochromatin (blue) and the location of mature capsids (63; red spheres), immature capsids (403; yellow spheres) and all PML-specific gold particles (5,219; small green spheres) that were identified in the serial sections. Entrapped mature capsids with associated PML labeling are shown as red/green spheres and immature capsids with PML labeling are shown as yellow/green spheres. (C) Same 3D model as in B but at higher magnification and in a different angle. See also Video S9.
Mentions: The electron density of the PML positive shell of nuclear PML cages allowed tracing and reconstruction of the shape (3D surface view) of this compartment by SSA-SEM; however the 3D distribution of PML protein within PML cages was not revealed using this approach. Therefore we used a serial section immunoTEM (ss-immunoTEM) approach to investigate quantitatively and in three dimensions how PML protein is distributed within the shell and in the core of the PML cages, where the nucleocapsids are entrapped. Seven consecutive sections (100 nm) through HPF/FS-treated and LRwhite embedded cells that contained PML cages with entrapped VZV capsids, were labeled with a PML specific antibody and Protein A conjugated to 15 nm gold particles, and then imaged by TEM (Figure 6A). The results of tracing and modeling of the PML labeling (small green spheres), mature capsids (red spheres) and immature capsids (yellow spheres) and the electron dense heterochromatin (blue) are shown in Figure 6B, C and Video S9. About 5,219 PML gold particles, 63 mature capsids and 403 immature capsids were identified; 272 of the entrapped nucleocapsids were directly associated with PML gold particles (half-green spheres). The 3D reconstruction clearly reveals a ring-shaped ‘cloud’ of dense PML-labeling that corresponds to the electron dense shell of PML cages as seen in the high-contrast embedded samples analyzed by SSA-SEM before. Significant amounts of PML gold labeling were also found in the core of the PML cage (Figure 6A, right panel and Figure 6C) where 58% of the entrapped nucleocapsids were directly associated with PML gold particles (half-green spheres). Therefore PML protein is not only a structural component of the electron dense shell of PML cages but also binds to nucleocapsids entrapped within the core of the PML cages.

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