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

Visualization of PML cages and VZV nucleocapsids by confocal microscopy, TEM and SEM.(A–F) Melanoma cells that expressed doxycycline-induced PML IV were infected with VZV for 48 hours. (A) Analysis by confocal microscopy: permeabilized cells on coverslips were immunostained for PML (green) and ORF23 capsid protein (red); nuclei were stained with Hoechst (blue). Scale bar, 10 µm. (B) Immunogold TEM analysis: cells were high-pressure frozen, freeze-substituted, embedded in LR-White resin and then labeled with anti-PML polyclonal rabbit antibody and Protein A conjugated with 15 nm gold particles. Note the dense PML-gold labeling (arrows) in the amorphous layer (surrounded by a green line) that encloses the sequestered capsids. (C and D). Standard TEM for morphological analysis: cells were aldehyde-fixed, ‘en block’ stained for high contrast and then embedded in epoxy-resin. Note the electron dense amorphous PML layer (surrounded by green line) that encloses the clustered capsids. (D) Three types of capsids (A, B, C-type capsids, red arrows) can be distinguished by TEM. (E and F) Scanning EM analysis with a back-scattered electron detector (BSE) of the same sample as in C. Note the electron dense PML layer (surrounded by a green line) that encloses the sequestered capsids. (F) The three types of capsids (A, B, C-type capsids, red arrows) can also be distinguished by BSE-SEM. Scale bars in B–F are 500 nm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3369938&req=5

ppat-1002740-g001: Visualization of PML cages and VZV nucleocapsids by confocal microscopy, TEM and SEM.(A–F) Melanoma cells that expressed doxycycline-induced PML IV were infected with VZV for 48 hours. (A) Analysis by confocal microscopy: permeabilized cells on coverslips were immunostained for PML (green) and ORF23 capsid protein (red); nuclei were stained with Hoechst (blue). Scale bar, 10 µm. (B) Immunogold TEM analysis: cells were high-pressure frozen, freeze-substituted, embedded in LR-White resin and then labeled with anti-PML polyclonal rabbit antibody and Protein A conjugated with 15 nm gold particles. Note the dense PML-gold labeling (arrows) in the amorphous layer (surrounded by a green line) that encloses the sequestered capsids. (C and D). Standard TEM for morphological analysis: cells were aldehyde-fixed, ‘en block’ stained for high contrast and then embedded in epoxy-resin. Note the electron dense amorphous PML layer (surrounded by green line) that encloses the clustered capsids. (D) Three types of capsids (A, B, C-type capsids, red arrows) can be distinguished by TEM. (E and F) Scanning EM analysis with a back-scattered electron detector (BSE) of the same sample as in C. Note the electron dense PML layer (surrounded by a green line) that encloses the sequestered capsids. (F) The three types of capsids (A, B, C-type capsids, red arrows) can also be distinguished by BSE-SEM. Scale bars in B–F are 500 nm.

Mentions: As we have shown previously [22], PML cages in VZV-infected cells appear as ring-like structures that contain ORF23 capsid protein by confocal microscopy using antibodies to PML and ORF23, the small capsid protein (Figure 1A). At the higher resolution obtained by immunogold-TEM, mature (C-type capsids) and immature (A-and B-type capsids) can be identified that are embedded within and surrounded by densely immunogold-labeled PML positive material (Figure 1B). Next, we employed a high-contrast sample preparation protocol in order to be able to identify PML cages solely by their distinct morphology in samples not suitable for immunogold labeling. Similar to the densely labeled PML shell visible by immunoTEM (Figure 1B), a shell of amorphous electron dense material surrounding clusters of VZV nucleocapsids was visible in the high-contrast embedded samples (Figure 1C, green line). Sequestered mature C-type capsids and immature A-and B-type capsids could be distinguished clearly (Figure 1D). Importantly, the electron dense PML-positive shell surrounding sequestered VZV nucleocapsids was also visualized when the same sample was studied using a high-resolution scanning electron microscope (SEM) equipped with a field emission gun (FEG) and a back-scattered electron detector (BSE-detector) (Figure 1E) and the SEM resolution was sufficient to distinguish between mature and immature nucleocapsids (Figure 1F). Therefore, the distinctive morphological profiles of nuclear PML cages that contain sequestered nucleocapsids could be identified unequivocally by TEM as well as SEM. These results made it possible to perform the large volume and high-resolution 3D reconstruction of VZV-infected cell nuclei and PML cages aided by SEM imaging.


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)

Visualization of PML cages and VZV nucleocapsids by confocal microscopy, TEM and SEM.(A–F) Melanoma cells that expressed doxycycline-induced PML IV were infected with VZV for 48 hours. (A) Analysis by confocal microscopy: permeabilized cells on coverslips were immunostained for PML (green) and ORF23 capsid protein (red); nuclei were stained with Hoechst (blue). Scale bar, 10 µm. (B) Immunogold TEM analysis: cells were high-pressure frozen, freeze-substituted, embedded in LR-White resin and then labeled with anti-PML polyclonal rabbit antibody and Protein A conjugated with 15 nm gold particles. Note the dense PML-gold labeling (arrows) in the amorphous layer (surrounded by a green line) that encloses the sequestered capsids. (C and D). Standard TEM for morphological analysis: cells were aldehyde-fixed, ‘en block’ stained for high contrast and then embedded in epoxy-resin. Note the electron dense amorphous PML layer (surrounded by green line) that encloses the clustered capsids. (D) Three types of capsids (A, B, C-type capsids, red arrows) can be distinguished by TEM. (E and F) Scanning EM analysis with a back-scattered electron detector (BSE) of the same sample as in C. Note the electron dense PML layer (surrounded by a green line) that encloses the sequestered capsids. (F) The three types of capsids (A, B, C-type capsids, red arrows) can also be distinguished by BSE-SEM. Scale bars in B–F are 500 nm.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1002740-g001: Visualization of PML cages and VZV nucleocapsids by confocal microscopy, TEM and SEM.(A–F) Melanoma cells that expressed doxycycline-induced PML IV were infected with VZV for 48 hours. (A) Analysis by confocal microscopy: permeabilized cells on coverslips were immunostained for PML (green) and ORF23 capsid protein (red); nuclei were stained with Hoechst (blue). Scale bar, 10 µm. (B) Immunogold TEM analysis: cells were high-pressure frozen, freeze-substituted, embedded in LR-White resin and then labeled with anti-PML polyclonal rabbit antibody and Protein A conjugated with 15 nm gold particles. Note the dense PML-gold labeling (arrows) in the amorphous layer (surrounded by a green line) that encloses the sequestered capsids. (C and D). Standard TEM for morphological analysis: cells were aldehyde-fixed, ‘en block’ stained for high contrast and then embedded in epoxy-resin. Note the electron dense amorphous PML layer (surrounded by green line) that encloses the clustered capsids. (D) Three types of capsids (A, B, C-type capsids, red arrows) can be distinguished by TEM. (E and F) Scanning EM analysis with a back-scattered electron detector (BSE) of the same sample as in C. Note the electron dense PML layer (surrounded by a green line) that encloses the sequestered capsids. (F) The three types of capsids (A, B, C-type capsids, red arrows) can also be distinguished by BSE-SEM. Scale bars in B–F are 500 nm.
Mentions: As we have shown previously [22], PML cages in VZV-infected cells appear as ring-like structures that contain ORF23 capsid protein by confocal microscopy using antibodies to PML and ORF23, the small capsid protein (Figure 1A). At the higher resolution obtained by immunogold-TEM, mature (C-type capsids) and immature (A-and B-type capsids) can be identified that are embedded within and surrounded by densely immunogold-labeled PML positive material (Figure 1B). Next, we employed a high-contrast sample preparation protocol in order to be able to identify PML cages solely by their distinct morphology in samples not suitable for immunogold labeling. Similar to the densely labeled PML shell visible by immunoTEM (Figure 1B), a shell of amorphous electron dense material surrounding clusters of VZV nucleocapsids was visible in the high-contrast embedded samples (Figure 1C, green line). Sequestered mature C-type capsids and immature A-and B-type capsids could be distinguished clearly (Figure 1D). Importantly, the electron dense PML-positive shell surrounding sequestered VZV nucleocapsids was also visualized when the same sample was studied using a high-resolution scanning electron microscope (SEM) equipped with a field emission gun (FEG) and a back-scattered electron detector (BSE-detector) (Figure 1E) and the SEM resolution was sufficient to distinguish between mature and immature nucleocapsids (Figure 1F). Therefore, the distinctive morphological profiles of nuclear PML cages that contain sequestered nucleocapsids could be identified unequivocally by TEM as well as SEM. These results made it possible to perform the large volume and high-resolution 3D reconstruction of VZV-infected cell nuclei and PML cages aided by SEM imaging.

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