Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2015 Nov 4;7(11):5686-704.
doi: 10.3390/v7112900.

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

Clarissa Villinger  1   2 , Gregor Neusser  3 , Christine Kranz  4 , Paul Walther  5 , Thomas Mertens  2
Affiliations

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

Clarissa Villinger et al. Viruses. .

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.

Keywords: FIB/SEM tomography; HCMV; high-pressure freezing; inner nuclear membrane infoldings; nuclear capsid egress; three-dimensional structure.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Sample manipulation by the focused ion beam (FIB) and image acquisition with the scanning electron microscope (SEM) beam. Sample preparation is described in [39]. Embedded HCMV infected fibroblasts are located directly under the surface of the approximately 1 mm high Epon block. The sample is coated with a thin platinum layer prior to mounting into the dual beam microscope. The area of interest is chosen at 10 kV acceleration voltage. (A) The sample is then tilted to 52° and a trench is FIB-milled into the Epon block, generating a new surface (block face); (B) Removal of the next thin layer (“slice”) of the sample generates a new block face. After every “slice” step the generated block face is imaged by the scanning electron beam (“view”). The “slice and view” cycle is repeated to gain a three-dimensional dataset of the volume of interest. Reprinted from [38] Figure 1 with kind permission from Springer Science+Business Media.
Figure 2
Figure 2
Methodology and resolution of FIB/SEM tomography. (A) The Epon embedded cells are first visualized by SEM at an acceleration voltage of 10 kV. A region of interest can be easily chosen for the subsequent “slice and view” process. The boxes show the positions of the volumes chosen for FIB/SEM tomography; (B) Images of human cytomegalovirus (HCMV) particles acquired from an ultrathin section by transmission electron microscopy (TEM) and from the block face by FIB/SEM. The high resolution of the FIB/SEM image allows clear visibility of the two leaflets of the lipid bilayer. The secondary electron SEM image was acquired with an acceleration voltage of 5 kV.
Figure 3
Figure 3
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.
Figure 4
Figure 4
Internal structure of nuclear infoldings. Left and middle panels: FIB/SEM micrographs of the reconstructed details. See also Movies S1 and S2. Right panels: Three-dimensional reconstructions. Nuclear infoldings (semi-transparent blue) and capsids inside the infoldings (A capsids purple; B capsids orange; C capsids green). (A) Infolding A. Cross sections through two tubular segments (1st order infoldings) with 2nd order infoldings and a 3rd order infolding. Three-dimensional imaging reveals the tubular shape of the 2nd order infoldings and the spherical shape of the 3rd order infolding; (B) Connection of infolding B with the inner nuclear membrane (black arrows). Vesicles (2nd order infoldings) and three enveloped capsids are visible within the 1st order infolding. The 2nd order infolding contains two 3rd order infoldings (black arrowheads); (C) Infolding E with 1st, 2nd and 3rd order infoldings, a budding capsid, enveloped capsids in 1st order infoldings and non-enveloped capsids in 2nd order infoldings. nu nucleoplasm, cy cytoplasm.
Figure 5
Figure 5
Infolding E. (A) Two large spherical segments (diameters 1 µm) are connected by a tubular segment (diameter ~180 nm). A smaller spherical segment at the front branches out into two tubular segments. The boxes mark the position of the details depicted in Figure 4C and Figure 5C–E; (B) Presentation of the capsids alone shows the cloud-like structure of the nucleoplasmic capsids; (C) A 3rd order infolding (arrowhead) is visible in a sequence of more than ten images (see Movie S4). The images also show an invagination within the tubular segment (asterisk). Its connection with the nucleoplasm is evident in image 128. INM inner nuclear membrane, ONM outer nuclear membrane, nu nucleoplasm, cy cytoplasm; (D,E) The yz view of the three-dimensional model shows the opening of the invagination (asterisk). The model is depicted without capsids.
Figure 6
Figure 6
Summary of the architecture of intranuclear membrane structures revealed by FIB/SEM tomography. (A) Model describing infolding formation: The hierarchy of the compartments (1st, 2nd, and 3rd order infolding) could be explained by invagination of membrane into the respective compartment and subsequent membrane fission. Thus, the lumen of 1st and 3rd order infoldings equals the perinuclear space (PNS, blue) and the lumen of 2nd order infoldings the nucleoplasm (white). Non-enveloped capsids were only found in the nucleoplasm and 2nd order infoldings, enveloped capsids only in the lumen of 1st order infoldings; (1a.) It seemed obvious that enveloped capsids result from capsid budding into 1st order infoldings. (1b.) Capsid envelopes might be able to fuse with membranes of 2nd order infoldings, resulting in non-enveloped capsids in 2nd order infoldings; (2.) Alternatively, non-enveloped capsids in 2nd order infoldings could result from capsid trapping during formation of invaginations; (B) We never found tubular segments with capsids. Tubular segments always contained 2nd order infoldings.
Figure 7
Figure 7
(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.
Figure 8
Figure 8
TEM images of nuclear infoldings in two HCMV infected fibroblast nuclei at three days post infection.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Buser C., Walther P., Mertens T., Michel D. Cytomegalovirus Primary Envelopment Occurs at Large Infoldings of the Inner Nuclear Membrane. J. Virol. 2007;81:3042–3048. doi: 10.1128/JVI.01564-06. - DOI - PMC - PubMed
    1. Homman-Loudiyi M., Hultenby K., Britt W., Soderberg-Naucler C. Envelopment of Human Cytomegalovirus Occurs by Budding into Golgi-Derived Vacuole Compartments Positive for gB, Rab 3, Trans-Golgi Network 46, and Mannosidase II. J. Virol. 2003;77:3191–3203. doi: 10.1128/JVI.77.5.3191-3203.2003. - DOI - PMC - PubMed
    1. Jiang X.J., Adler B., Sampaio K.L., Digel M., Jahn G., Ettischer N., Stierhof Y.-D., Scrivano L., Koszinowski U., Mach M., et al. UL74 of human cytomegalovirus contributes to virus release by promoting secondary envelopment of virions. J. Virol. 2008;82:2802–2812. doi: 10.1128/JVI.01550-07. - DOI - PMC - PubMed
    1. Meissner C.S., Suffner S., Schauflinger M., von Einem J., Bogner E. A Leucine Zipper Motif of a Tegument Protein Triggers Final Envelopment of Human Cytomegalovirus. J. Virol. 2011;86:3370–3382. doi: 10.1128/JVI.06556-11. - DOI - PMC - PubMed
    1. Schauflinger M., Fischer D., Schreiber A., Chevillotte M., Walther P., Mertens T., von Einem J. The Tegument Protein UL71 of Human Cytomegalovirus Is Involved in Late Envelopment and Affects Multivesicular Bodies. J. Virol. 2011;85:3821–3832. doi: 10.1128/JVI.01540-10. - DOI - PMC - PubMed

Publication types

MeSH terms