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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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(Serial-)TEM images of nuclear infoldings in HCMV infected fibroblasts at five days post infection. (A,B) The sample used for FIB/SEM tomography was objected to serial ultrathin sectioning. The images are captured from two different nuclei. (A) Three subsequent images through an infolding show a tubular segment with a 2nd order infolding (arrowheads); (B) The lower image shows an opening of a 2nd order infolding towards the nucleoplasm (arrow); (C) A standard TEM image of an independent experiment also shows the opening of a 2nd order infolding towards the nucleoplasm.
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viruses-07-02900-f007: (Serial-)TEM images of nuclear infoldings in HCMV infected fibroblasts at five days post infection. (A,B) The sample used for FIB/SEM tomography was objected to serial ultrathin sectioning. The images are captured from two different nuclei. (A) Three subsequent images through an infolding show a tubular segment with a 2nd order infolding (arrowheads); (B) The lower image shows an opening of a 2nd order infolding towards the nucleoplasm (arrow); (C) A standard TEM image of an independent experiment also shows the opening of a 2nd order infolding towards the nucleoplasm.

Mentions: Standard and serial TEM images of more than 40 nuclei consistently showed nuclear infoldings with nested membranes (Figure 7). The high resolution in the z direction by FIB/SEM allowed a completely new insight into these complex intranuclear structures and enabled us to finally interpret two-dimensional TEM images.


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)

(Serial-)TEM images of nuclear infoldings in HCMV infected fibroblasts at five days post infection. (A,B) The sample used for FIB/SEM tomography was objected to serial ultrathin sectioning. The images are captured from two different nuclei. (A) Three subsequent images through an infolding show a tubular segment with a 2nd order infolding (arrowheads); (B) The lower image shows an opening of a 2nd order infolding towards the nucleoplasm (arrow); (C) A standard TEM image of an independent experiment also shows the opening of a 2nd order infolding towards the nucleoplasm.
© Copyright Policy
Related In: Results  -  Collection

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

viruses-07-02900-f007: (Serial-)TEM images of nuclear infoldings in HCMV infected fibroblasts at five days post infection. (A,B) The sample used for FIB/SEM tomography was objected to serial ultrathin sectioning. The images are captured from two different nuclei. (A) Three subsequent images through an infolding show a tubular segment with a 2nd order infolding (arrowheads); (B) The lower image shows an opening of a 2nd order infolding towards the nucleoplasm (arrow); (C) A standard TEM image of an independent experiment also shows the opening of a 2nd order infolding towards the nucleoplasm.
Mentions: Standard and serial TEM images of more than 40 nuclei consistently showed nuclear infoldings with nested membranes (Figure 7). The high resolution in the z direction by FIB/SEM allowed a completely new insight into these complex intranuclear structures and enabled us to finally interpret two-dimensional TEM images.

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