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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 cell nuclei without PML cages.Melanoma cells were infected with VZV for 48 h and processed for SSA-SEM (A–E) or serial section TEM (F and G). A) BSE-SEM images of four representative sections (s20, s30, s40, s50) from a series of 50 consecutive 100 nm sections are shown. A nuclear indentation is outlined and marked with a blue arrow. See also Video S1. (B) 3D model of the shape of the VZV infected nucleus (grey). Upper panel (front view): the cross section plane and a deep invagination (blue arrow) of the nucleus are visible. The middle panel (side view) and bottom panel (rear view) reveal the irregular shape of the nucleus with numerous indentations. (C) View of the same nucleus at different angles in transparent mode. Color code: transparent grey, boundary of the nucleus; transparent blue, electron dense heterochromatin; brown, nucleolus; red spheres (mature capsids, C-type) and yellow spheres (immature capsids, A and B-type). A total of 4,223 capsids were identified and visualized. (D) Higher magnification view; color code as in C, but nuclear envelope not shown. The dense heterochromatin (solid dark blue) hides nucleocapsids that are located deeper in the nuclear volume. (E) Same view as in D, but with transparent heterochromatin: the distribution of capsids throughout the nucleus is revealed. See also Video S2. (F and G) Two different nuclei that were reconstructed from serial sections imaged by TEM. The color code is the same as above. Insets show representative images from the TEM series. The 3D models show the distribution of 425 (F) and 1,340 capsids (G), respectively. See also Video S3. All scale bars are 5 µm.
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ppat-1002740-g003: Three-dimensional distribution of VZV nucleocapsids in cell nuclei without PML cages.Melanoma cells were infected with VZV for 48 h and processed for SSA-SEM (A–E) or serial section TEM (F and G). A) BSE-SEM images of four representative sections (s20, s30, s40, s50) from a series of 50 consecutive 100 nm sections are shown. A nuclear indentation is outlined and marked with a blue arrow. See also Video S1. (B) 3D model of the shape of the VZV infected nucleus (grey). Upper panel (front view): the cross section plane and a deep invagination (blue arrow) of the nucleus are visible. The middle panel (side view) and bottom panel (rear view) reveal the irregular shape of the nucleus with numerous indentations. (C) View of the same nucleus at different angles in transparent mode. Color code: transparent grey, boundary of the nucleus; transparent blue, electron dense heterochromatin; brown, nucleolus; red spheres (mature capsids, C-type) and yellow spheres (immature capsids, A and B-type). A total of 4,223 capsids were identified and visualized. (D) Higher magnification view; color code as in C, but nuclear envelope not shown. The dense heterochromatin (solid dark blue) hides nucleocapsids that are located deeper in the nuclear volume. (E) Same view as in D, but with transparent heterochromatin: the distribution of capsids throughout the nucleus is revealed. See also Video S2. (F and G) Two different nuclei that were reconstructed from serial sections imaged by TEM. The color code is the same as above. Insets show representative images from the TEM series. The 3D models show the distribution of 425 (F) and 1,340 capsids (G), respectively. See also Video S3. All scale bars are 5 µm.

Mentions: Using SSA-SEM we first analyzed a VZV-infected melanoma cell nucleus in which endogenous PML was expressed (Figure 3A–E and Video S1). The shape of the infected cell nucleus and the nuclear volume were determined by tracing the outer boundary of the nucleus in all 50 consecutive sections, encompassing a total thickness of about five microns and a nuclear volume of about 95 µm3. The 3D reconstruction revealed an irregular shape of the nucleus characterized by several indentations and deep invaginations (Figure 3B and Video S2). If visualization was limited to the original two-dimensional sections, these invaginations might be misinterpreted as ‘vesicles’ or ‘vacuoles’ within the nuclear matrix (Figure 3A and Video S1). Morphological tracing and 3D modeling revealed the location and distribution of the electron dense heterochromatin, which is located primarily at the periphery of the nucleus (Figure 3C–E, blue); also seen is the nucleolus in the lower center of the nucleus (Figure 3C–E, brown) and the mature and immature nucleocapsids (Figure 3C–E, red and yellow spheres, respectively). 3,467 (82%) immature capsids and 756 (18%) mature capsids were identified within the serial sections and their positions were precisely modeled in the reconstructed nuclear volume (Figure 3C–E and Video S2). This work revealed that mature and immature capsids were not segregated into different nuclear domains; instead, they were mixed randomly and were evenly distributed within the nuclear volume outside of the heterochromatin and the nucleolus and were excluded from the deep nuclear imaginations (Figure 3E and Video S2).


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 cell nuclei without PML cages.Melanoma cells were infected with VZV for 48 h and processed for SSA-SEM (A–E) or serial section TEM (F and G). A) BSE-SEM images of four representative sections (s20, s30, s40, s50) from a series of 50 consecutive 100 nm sections are shown. A nuclear indentation is outlined and marked with a blue arrow. See also Video S1. (B) 3D model of the shape of the VZV infected nucleus (grey). Upper panel (front view): the cross section plane and a deep invagination (blue arrow) of the nucleus are visible. The middle panel (side view) and bottom panel (rear view) reveal the irregular shape of the nucleus with numerous indentations. (C) View of the same nucleus at different angles in transparent mode. Color code: transparent grey, boundary of the nucleus; transparent blue, electron dense heterochromatin; brown, nucleolus; red spheres (mature capsids, C-type) and yellow spheres (immature capsids, A and B-type). A total of 4,223 capsids were identified and visualized. (D) Higher magnification view; color code as in C, but nuclear envelope not shown. The dense heterochromatin (solid dark blue) hides nucleocapsids that are located deeper in the nuclear volume. (E) Same view as in D, but with transparent heterochromatin: the distribution of capsids throughout the nucleus is revealed. See also Video S2. (F and G) Two different nuclei that were reconstructed from serial sections imaged by TEM. The color code is the same as above. Insets show representative images from the TEM series. The 3D models show the distribution of 425 (F) and 1,340 capsids (G), respectively. See also Video S3. All scale bars are 5 µm.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1002740-g003: Three-dimensional distribution of VZV nucleocapsids in cell nuclei without PML cages.Melanoma cells were infected with VZV for 48 h and processed for SSA-SEM (A–E) or serial section TEM (F and G). A) BSE-SEM images of four representative sections (s20, s30, s40, s50) from a series of 50 consecutive 100 nm sections are shown. A nuclear indentation is outlined and marked with a blue arrow. See also Video S1. (B) 3D model of the shape of the VZV infected nucleus (grey). Upper panel (front view): the cross section plane and a deep invagination (blue arrow) of the nucleus are visible. The middle panel (side view) and bottom panel (rear view) reveal the irregular shape of the nucleus with numerous indentations. (C) View of the same nucleus at different angles in transparent mode. Color code: transparent grey, boundary of the nucleus; transparent blue, electron dense heterochromatin; brown, nucleolus; red spheres (mature capsids, C-type) and yellow spheres (immature capsids, A and B-type). A total of 4,223 capsids were identified and visualized. (D) Higher magnification view; color code as in C, but nuclear envelope not shown. The dense heterochromatin (solid dark blue) hides nucleocapsids that are located deeper in the nuclear volume. (E) Same view as in D, but with transparent heterochromatin: the distribution of capsids throughout the nucleus is revealed. See also Video S2. (F and G) Two different nuclei that were reconstructed from serial sections imaged by TEM. The color code is the same as above. Insets show representative images from the TEM series. The 3D models show the distribution of 425 (F) and 1,340 capsids (G), respectively. See also Video S3. All scale bars are 5 µm.
Mentions: Using SSA-SEM we first analyzed a VZV-infected melanoma cell nucleus in which endogenous PML was expressed (Figure 3A–E and Video S1). The shape of the infected cell nucleus and the nuclear volume were determined by tracing the outer boundary of the nucleus in all 50 consecutive sections, encompassing a total thickness of about five microns and a nuclear volume of about 95 µm3. The 3D reconstruction revealed an irregular shape of the nucleus characterized by several indentations and deep invaginations (Figure 3B and Video S2). If visualization was limited to the original two-dimensional sections, these invaginations might be misinterpreted as ‘vesicles’ or ‘vacuoles’ within the nuclear matrix (Figure 3A and Video S1). Morphological tracing and 3D modeling revealed the location and distribution of the electron dense heterochromatin, which is located primarily at the periphery of the nucleus (Figure 3C–E, blue); also seen is the nucleolus in the lower center of the nucleus (Figure 3C–E, brown) and the mature and immature nucleocapsids (Figure 3C–E, red and yellow spheres, respectively). 3,467 (82%) immature capsids and 756 (18%) mature capsids were identified within the serial sections and their positions were precisely modeled in the reconstructed nuclear volume (Figure 3C–E and Video S2). This work revealed that mature and immature capsids were not segregated into different nuclear domains; instead, they were mixed randomly and were evenly distributed within the nuclear volume outside of the heterochromatin and the nucleolus and were excluded from the deep nuclear imaginations (Figure 3E and Video S2).

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