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3D Analysis of HCMV Induced-Nuclear Membrane Structures by FIB/SEM Tomography: Insight into an Unprecedented Membrane Morphology.

Villinger C, Neusser G, Kranz C, Walther P, Mertens T - Viruses (2015)

Bottom Line: We found that the previously described infoldings of the inner nuclear membrane, which are unique among its kind, form an extremely complex network of membrane structures not predictable by previous two-dimensional studies.Only 0.8% proved to be enveloped capsids which were exclusively detected in 1st order infoldings (perinuclear space).Distribution of the capsids between 1st, 2nd and 3rd order infoldings is in complete agreement with the envelopment/de-envelopment model for egress of HCMV capsids from the nucleus and we confirm that capsid budding does occur at the large infoldings.

View Article: PubMed Central - PubMed

Affiliation: Electron Microscopy Facility, Ulm University, Albert-Einstein-Allee 11, 89081 Ulm, Germany. thomas.mertens@uniklinik-ulm.de.

ABSTRACT
We show that focused ion beam/scanning electron microscopy (FIB/SEM) tomography is an excellent method to analyze the three-dimensional structure of a fibroblast nucleus infected with human cytomegalovirus (HCMV). We found that the previously described infoldings of the inner nuclear membrane, which are unique among its kind, form an extremely complex network of membrane structures not predictable by previous two-dimensional studies. In all cases they contained further invaginations (2nd and 3rd order infoldings). Quantification revealed 5498HCMV capsids within two nuclear segments, allowing an estimate of 15,000 to 30,000 capsids in the entire nucleus five days post infection. Only 0.8% proved to be enveloped capsids which were exclusively detected in 1st order infoldings (perinuclear space). Distribution of the capsids between 1st, 2nd and 3rd order infoldings is in complete agreement with the envelopment/de-envelopment model for egress of HCMV capsids from the nucleus and we confirm that capsid budding does occur at the large infoldings. Based on our results we propose the pushing membrane model: HCMV infection induces local disruption of the nuclear lamina and synthesis of new membrane material which is pushed into the nucleoplasm, forming complex membrane infoldings in a highly abundant manner, which then may be also used by nucleocapsids for budding.

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FIB/SEM tomography of an HCMV infected nucleus five days post infection. (A–D) Volume 1; (E–F) Volume 2; (A,E) Single FIB/SEM micrographs. High contrast allows identification of membranes and nuclear capsids. Nuclear membrane structures (infoldings) are visible close to the nuclear envelope (see also Movies S1 and S2). nu nucleoplasm, cy cytoplasm. Dimensions of each volume: 6.4 µm × 5.5 µm × 5 µm; (B) Xz view of the tomogram of volume 1. The dashed line marks the position of the FIB/SEM image shown in (A). (C,D,F) Three-dimensional reconstructions of the inner nuclear membrane with infoldings (blue, semi-transparent) and capsids (nucleoplasm: A capsids pink; B capsids blue; C capsids white; infoldings: A capsids purple; B capsids orange; C capsids green). See also Movies S3 and S4. (C and D) Volume 1 contains 4160 capsids. (E) The dashed line marks a replication center. (F) Infolding D has no connection to the inner nuclear membrane. Infolding E consists of tubular and spherical segments. Volume 2 contains 1338 capsids.
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viruses-07-02900-f003: FIB/SEM tomography of an HCMV infected nucleus five days post infection. (A–D) Volume 1; (E–F) Volume 2; (A,E) Single FIB/SEM micrographs. High contrast allows identification of membranes and nuclear capsids. Nuclear membrane structures (infoldings) are visible close to the nuclear envelope (see also Movies S1 and S2). nu nucleoplasm, cy cytoplasm. Dimensions of each volume: 6.4 µm × 5.5 µm × 5 µm; (B) Xz view of the tomogram of volume 1. The dashed line marks the position of the FIB/SEM image shown in (A). (C,D,F) Three-dimensional reconstructions of the inner nuclear membrane with infoldings (blue, semi-transparent) and capsids (nucleoplasm: A capsids pink; B capsids blue; C capsids white; infoldings: A capsids purple; B capsids orange; C capsids green). See also Movies S3 and S4. (C and D) Volume 1 contains 4160 capsids. (E) The dashed line marks a replication center. (F) Infolding D has no connection to the inner nuclear membrane. Infolding E consists of tubular and spherical segments. Volume 2 contains 1338 capsids.

Mentions: With the three-dimensional imaging approach we could reveal an unpredicted high complexity of the intranuclear membrane structures in HCMV-infected cells with respect to their external appearance (Figure 3) and internal structure (Figure 4 and Figure 5).


3D Analysis of HCMV Induced-Nuclear Membrane Structures by FIB/SEM Tomography: Insight into an Unprecedented Membrane Morphology.

Villinger C, Neusser G, Kranz C, Walther P, Mertens T - Viruses (2015)

FIB/SEM tomography of an HCMV infected nucleus five days post infection. (A–D) Volume 1; (E–F) Volume 2; (A,E) Single FIB/SEM micrographs. High contrast allows identification of membranes and nuclear capsids. Nuclear membrane structures (infoldings) are visible close to the nuclear envelope (see also Movies S1 and S2). nu nucleoplasm, cy cytoplasm. Dimensions of each volume: 6.4 µm × 5.5 µm × 5 µm; (B) Xz view of the tomogram of volume 1. The dashed line marks the position of the FIB/SEM image shown in (A). (C,D,F) Three-dimensional reconstructions of the inner nuclear membrane with infoldings (blue, semi-transparent) and capsids (nucleoplasm: A capsids pink; B capsids blue; C capsids white; infoldings: A capsids purple; B capsids orange; C capsids green). See also Movies S3 and S4. (C and D) Volume 1 contains 4160 capsids. (E) The dashed line marks a replication center. (F) Infolding D has no connection to the inner nuclear membrane. Infolding E consists of tubular and spherical segments. Volume 2 contains 1338 capsids.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4664973&req=5

viruses-07-02900-f003: FIB/SEM tomography of an HCMV infected nucleus five days post infection. (A–D) Volume 1; (E–F) Volume 2; (A,E) Single FIB/SEM micrographs. High contrast allows identification of membranes and nuclear capsids. Nuclear membrane structures (infoldings) are visible close to the nuclear envelope (see also Movies S1 and S2). nu nucleoplasm, cy cytoplasm. Dimensions of each volume: 6.4 µm × 5.5 µm × 5 µm; (B) Xz view of the tomogram of volume 1. The dashed line marks the position of the FIB/SEM image shown in (A). (C,D,F) Three-dimensional reconstructions of the inner nuclear membrane with infoldings (blue, semi-transparent) and capsids (nucleoplasm: A capsids pink; B capsids blue; C capsids white; infoldings: A capsids purple; B capsids orange; C capsids green). See also Movies S3 and S4. (C and D) Volume 1 contains 4160 capsids. (E) The dashed line marks a replication center. (F) Infolding D has no connection to the inner nuclear membrane. Infolding E consists of tubular and spherical segments. Volume 2 contains 1338 capsids.
Mentions: With the three-dimensional imaging approach we could reveal an unpredicted high complexity of the intranuclear membrane structures in HCMV-infected cells with respect to their external appearance (Figure 3) and internal structure (Figure 4 and Figure 5).

Bottom Line: We found that the previously described infoldings of the inner nuclear membrane, which are unique among its kind, form an extremely complex network of membrane structures not predictable by previous two-dimensional studies.Only 0.8% proved to be enveloped capsids which were exclusively detected in 1st order infoldings (perinuclear space).Distribution of the capsids between 1st, 2nd and 3rd order infoldings is in complete agreement with the envelopment/de-envelopment model for egress of HCMV capsids from the nucleus and we confirm that capsid budding does occur at the large infoldings.

View Article: PubMed Central - PubMed

Affiliation: Electron Microscopy Facility, Ulm University, Albert-Einstein-Allee 11, 89081 Ulm, Germany. thomas.mertens@uniklinik-ulm.de.

ABSTRACT
We show that focused ion beam/scanning electron microscopy (FIB/SEM) tomography is an excellent method to analyze the three-dimensional structure of a fibroblast nucleus infected with human cytomegalovirus (HCMV). We found that the previously described infoldings of the inner nuclear membrane, which are unique among its kind, form an extremely complex network of membrane structures not predictable by previous two-dimensional studies. In all cases they contained further invaginations (2nd and 3rd order infoldings). Quantification revealed 5498HCMV capsids within two nuclear segments, allowing an estimate of 15,000 to 30,000 capsids in the entire nucleus five days post infection. Only 0.8% proved to be enveloped capsids which were exclusively detected in 1st order infoldings (perinuclear space). Distribution of the capsids between 1st, 2nd and 3rd order infoldings is in complete agreement with the envelopment/de-envelopment model for egress of HCMV capsids from the nucleus and we confirm that capsid budding does occur at the large infoldings. Based on our results we propose the pushing membrane model: HCMV infection induces local disruption of the nuclear lamina and synthesis of new membrane material which is pushed into the nucleoplasm, forming complex membrane infoldings in a highly abundant manner, which then may be also used by nucleocapsids for budding.

Show MeSH
Related in: MedlinePlus