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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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Electron tomography of PML cages reveals the cross-linking of VZV capsids by an electron-dense meshwork.Melanoma cells that express doxycycline-induced PML IV were infected with VZV for 48 h and then high pressure frozen, freeze-substituted and embedded in epoxy-resin. 80 nm sections (A–E) or 300 nm sections (F–L) were investigated by dual-axis electron tomography. (A) A representative tomographic slice shows the periphery of the nucleus with the electron dense heterochromatin (blue bottom area) and part of the PML cage (light green area) containing numerous VZV capsids. A light electron-dense fibrous meshwork (grey) is visible within the PML-domain. These fibers are directly associated with capsids (arrows) and can cross-link them. (B) The area in the black square in A is shown at higher magnification in inverted mode, e.g. electron dense structures appear bright. Arrows depict fibrous material associated with VZV capsids. (C) Same image as in B but with traces for 3D reconstruction shown: capsids (yellow) were traced manually; electron dense meshwork (green) was traced automatically by thresholding. See also Video S10. (D and E) show 3D models of the VZV capsids (yellow) associated with the electron-dense meshwork (green). Scale bars are 200 nm. See also Video S11. (F) Volume view with inverted contrast of a reconstruction from a dual-axis tomogram of a 300 nm section. The arrangement of VZV capsids within a PML cage is visible. (G) Same reconstruction as in F but in orthoslice mode that reveals the arrangement of capsids in the interior of the reconstructed volume. (H) Volume view of a part of the tomographic reconstruction that was then traced and segmented (I) to reveal the position of capsids and the electron dense meshwork in a 3D model. (I) Traces on one representative digital tomographic slice: immature capsids (yellow), mature capsids (red), electron dense fibers and meshwork (green). (J) 3D model shows the packaging of capsids (protein meshwork is green/transparent for unobscured view of capsids). (K) 3D model shows capsids with associated electron-dense meshwork (green) at higher magnification. (L) Representative tomographic slice images that show protein fibers (green arrows) associated with VZV capsids. Scale bars are 200 nm (A–D and F–J) and 100 nm (E, K and L).
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ppat-1002740-g007: Electron tomography of PML cages reveals the cross-linking of VZV capsids by an electron-dense meshwork.Melanoma cells that express doxycycline-induced PML IV were infected with VZV for 48 h and then high pressure frozen, freeze-substituted and embedded in epoxy-resin. 80 nm sections (A–E) or 300 nm sections (F–L) were investigated by dual-axis electron tomography. (A) A representative tomographic slice shows the periphery of the nucleus with the electron dense heterochromatin (blue bottom area) and part of the PML cage (light green area) containing numerous VZV capsids. A light electron-dense fibrous meshwork (grey) is visible within the PML-domain. These fibers are directly associated with capsids (arrows) and can cross-link them. (B) The area in the black square in A is shown at higher magnification in inverted mode, e.g. electron dense structures appear bright. Arrows depict fibrous material associated with VZV capsids. (C) Same image as in B but with traces for 3D reconstruction shown: capsids (yellow) were traced manually; electron dense meshwork (green) was traced automatically by thresholding. See also Video S10. (D and E) show 3D models of the VZV capsids (yellow) associated with the electron-dense meshwork (green). Scale bars are 200 nm. See also Video S11. (F) Volume view with inverted contrast of a reconstruction from a dual-axis tomogram of a 300 nm section. The arrangement of VZV capsids within a PML cage is visible. (G) Same reconstruction as in F but in orthoslice mode that reveals the arrangement of capsids in the interior of the reconstructed volume. (H) Volume view of a part of the tomographic reconstruction that was then traced and segmented (I) to reveal the position of capsids and the electron dense meshwork in a 3D model. (I) Traces on one representative digital tomographic slice: immature capsids (yellow), mature capsids (red), electron dense fibers and meshwork (green). (J) 3D model shows the packaging of capsids (protein meshwork is green/transparent for unobscured view of capsids). (K) 3D model shows capsids with associated electron-dense meshwork (green) at higher magnification. (L) Representative tomographic slice images that show protein fibers (green arrows) associated with VZV capsids. Scale bars are 200 nm (A–D and F–J) and 100 nm (E, K and L).

Mentions: The observation by ss-immunoTEM that PML protein was present in the shell and in the center of PML cages, where it was found directly associated with many VZV capsids, suggested that PML protein is not only a structural component of the electron dense shell of PML cages, but may also be involved in the immobilization or cross-linking of sequestered VZV nucleocapsids. To address this hypothesis, we investigated the ultrastructure of PML cages and of sequestered nucleocapsids by electron tomography, which provided a higher resolution than SSA-SEM, albeit at the cost of allowing analysis of only a much smaller (thinner) sample volume. The samples for tomography consisted of HPF/FS-treated and epoxy resin-embedded VZV infected melanoma cells that expressed PML IV together with endogenous PML [22]. We first recorded dual-axis tomograms from 80 nm sections of VZV infected cell nuclei with PML cages (Figure 7A–E). The 3D models were generated by analyzing digital tomogram slices as was done for SSA-SEM, combining manual tracing and automatic threshold-based tracing. EM tomography revealed that all nucleocapsids within PML cages were embedded in an irregular electron dense meshwork with numerous fibrous structures emanating from the nucleocapsids and often cross-linking adjacent capsids (Figure 7A and Video S10). These irregular fibrils were even better visible when the contrast was inverted (Figure 7B, white arrows) and were then traced automatically by applying a threshold (green outline) (Figure 7C) in order to reconstruct a 3D model of the irregular meshwork (green) within PML domains (Figure 7D, E and Video S11). The 3D volume information of tomograms from 80 nm sections is very limited because of the small z-dimension of the section. In order to reveal the precise arrangement and packing of nucleocapsids within the center of the PML cages and to confirm the presence of an irregular electron dense meshwork entrapping VZV nucleocapsids, we next recorded dual-axis tomograms from 300 nm thick sections through PML cages. A volume view representation of a representative tomogram (Figure 7F and Video S12) and an ortho-slice view of the same volume (Figure 7G and Video S12) shows the packing of nucleocapsids in several layers and that, in contrast to paracrystalline inclusion bodies of nucleocapsids observed in some HSV-infected cells [22], those entrapped in PML cages were rather loosely configured, were usually not in direct contact, and the space between them was filled with an irregular electron dense meshwork and fibers. Threshold-aided tracing and 3D reconstructions of the irregular meshwork (green) (Figure 7J–K), and of mature (red) and immature (yellow) VZV nucleocapsids showed that all traced capsids were tightly associated with the irregular meshwork that also cross-linked adjacent capsids (Figure 7I–K and Videos S13 and S14). This cross-linking of adjacent capsids was also visible in the original digital tomogram slices (green arrows) (Figure 7L) and confirmed our observations from the 80 nm tomography reconstructions.


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)

Electron tomography of PML cages reveals the cross-linking of VZV capsids by an electron-dense meshwork.Melanoma cells that express doxycycline-induced PML IV were infected with VZV for 48 h and then high pressure frozen, freeze-substituted and embedded in epoxy-resin. 80 nm sections (A–E) or 300 nm sections (F–L) were investigated by dual-axis electron tomography. (A) A representative tomographic slice shows the periphery of the nucleus with the electron dense heterochromatin (blue bottom area) and part of the PML cage (light green area) containing numerous VZV capsids. A light electron-dense fibrous meshwork (grey) is visible within the PML-domain. These fibers are directly associated with capsids (arrows) and can cross-link them. (B) The area in the black square in A is shown at higher magnification in inverted mode, e.g. electron dense structures appear bright. Arrows depict fibrous material associated with VZV capsids. (C) Same image as in B but with traces for 3D reconstruction shown: capsids (yellow) were traced manually; electron dense meshwork (green) was traced automatically by thresholding. See also Video S10. (D and E) show 3D models of the VZV capsids (yellow) associated with the electron-dense meshwork (green). Scale bars are 200 nm. See also Video S11. (F) Volume view with inverted contrast of a reconstruction from a dual-axis tomogram of a 300 nm section. The arrangement of VZV capsids within a PML cage is visible. (G) Same reconstruction as in F but in orthoslice mode that reveals the arrangement of capsids in the interior of the reconstructed volume. (H) Volume view of a part of the tomographic reconstruction that was then traced and segmented (I) to reveal the position of capsids and the electron dense meshwork in a 3D model. (I) Traces on one representative digital tomographic slice: immature capsids (yellow), mature capsids (red), electron dense fibers and meshwork (green). (J) 3D model shows the packaging of capsids (protein meshwork is green/transparent for unobscured view of capsids). (K) 3D model shows capsids with associated electron-dense meshwork (green) at higher magnification. (L) Representative tomographic slice images that show protein fibers (green arrows) associated with VZV capsids. Scale bars are 200 nm (A–D and F–J) and 100 nm (E, K and L).
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ppat-1002740-g007: Electron tomography of PML cages reveals the cross-linking of VZV capsids by an electron-dense meshwork.Melanoma cells that express doxycycline-induced PML IV were infected with VZV for 48 h and then high pressure frozen, freeze-substituted and embedded in epoxy-resin. 80 nm sections (A–E) or 300 nm sections (F–L) were investigated by dual-axis electron tomography. (A) A representative tomographic slice shows the periphery of the nucleus with the electron dense heterochromatin (blue bottom area) and part of the PML cage (light green area) containing numerous VZV capsids. A light electron-dense fibrous meshwork (grey) is visible within the PML-domain. These fibers are directly associated with capsids (arrows) and can cross-link them. (B) The area in the black square in A is shown at higher magnification in inverted mode, e.g. electron dense structures appear bright. Arrows depict fibrous material associated with VZV capsids. (C) Same image as in B but with traces for 3D reconstruction shown: capsids (yellow) were traced manually; electron dense meshwork (green) was traced automatically by thresholding. See also Video S10. (D and E) show 3D models of the VZV capsids (yellow) associated with the electron-dense meshwork (green). Scale bars are 200 nm. See also Video S11. (F) Volume view with inverted contrast of a reconstruction from a dual-axis tomogram of a 300 nm section. The arrangement of VZV capsids within a PML cage is visible. (G) Same reconstruction as in F but in orthoslice mode that reveals the arrangement of capsids in the interior of the reconstructed volume. (H) Volume view of a part of the tomographic reconstruction that was then traced and segmented (I) to reveal the position of capsids and the electron dense meshwork in a 3D model. (I) Traces on one representative digital tomographic slice: immature capsids (yellow), mature capsids (red), electron dense fibers and meshwork (green). (J) 3D model shows the packaging of capsids (protein meshwork is green/transparent for unobscured view of capsids). (K) 3D model shows capsids with associated electron-dense meshwork (green) at higher magnification. (L) Representative tomographic slice images that show protein fibers (green arrows) associated with VZV capsids. Scale bars are 200 nm (A–D and F–J) and 100 nm (E, K and L).
Mentions: The observation by ss-immunoTEM that PML protein was present in the shell and in the center of PML cages, where it was found directly associated with many VZV capsids, suggested that PML protein is not only a structural component of the electron dense shell of PML cages, but may also be involved in the immobilization or cross-linking of sequestered VZV nucleocapsids. To address this hypothesis, we investigated the ultrastructure of PML cages and of sequestered nucleocapsids by electron tomography, which provided a higher resolution than SSA-SEM, albeit at the cost of allowing analysis of only a much smaller (thinner) sample volume. The samples for tomography consisted of HPF/FS-treated and epoxy resin-embedded VZV infected melanoma cells that expressed PML IV together with endogenous PML [22]. We first recorded dual-axis tomograms from 80 nm sections of VZV infected cell nuclei with PML cages (Figure 7A–E). The 3D models were generated by analyzing digital tomogram slices as was done for SSA-SEM, combining manual tracing and automatic threshold-based tracing. EM tomography revealed that all nucleocapsids within PML cages were embedded in an irregular electron dense meshwork with numerous fibrous structures emanating from the nucleocapsids and often cross-linking adjacent capsids (Figure 7A and Video S10). These irregular fibrils were even better visible when the contrast was inverted (Figure 7B, white arrows) and were then traced automatically by applying a threshold (green outline) (Figure 7C) in order to reconstruct a 3D model of the irregular meshwork (green) within PML domains (Figure 7D, E and Video S11). The 3D volume information of tomograms from 80 nm sections is very limited because of the small z-dimension of the section. In order to reveal the precise arrangement and packing of nucleocapsids within the center of the PML cages and to confirm the presence of an irregular electron dense meshwork entrapping VZV nucleocapsids, we next recorded dual-axis tomograms from 300 nm thick sections through PML cages. A volume view representation of a representative tomogram (Figure 7F and Video S12) and an ortho-slice view of the same volume (Figure 7G and Video S12) shows the packing of nucleocapsids in several layers and that, in contrast to paracrystalline inclusion bodies of nucleocapsids observed in some HSV-infected cells [22], those entrapped in PML cages were rather loosely configured, were usually not in direct contact, and the space between them was filled with an irregular electron dense meshwork and fibers. Threshold-aided tracing and 3D reconstructions of the irregular meshwork (green) (Figure 7J–K), and of mature (red) and immature (yellow) VZV nucleocapsids showed that all traced capsids were tightly associated with the irregular meshwork that also cross-linked adjacent capsids (Figure 7I–K and Videos S13 and S14). This cross-linking of adjacent capsids was also visible in the original digital tomogram slices (green arrows) (Figure 7L) and confirmed our observations from the 80 nm tomography reconstructions.

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