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Viruses 2015, 7(11), 5686-5704; doi:10.3390/v7112900

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

1
Electron Microscopy Facility, Ulm University, Albert-Einstein-Allee 11, 89081 Ulm, Germany
2
Institute of Virology, University Medical Center Ulm, Albert-Einstein-Allee 11, 89081 Ulm, Germany
3
Institute of Analytical and Bioanalytical Chemistry, Ulm University, Albert-Einstein-Allee 11, 89081 Ulm, Germany
*
Author to whom correspondence should be addressed.
Academic Editor: Michael Laue
Received: 27 May 2015 / Revised: 22 October 2015 / Accepted: 26 October 2015 / Published: 4 November 2015
(This article belongs to the Special Issue Electron Microscopy in Virus Diagnostics and Research)
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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. View Full-Text
Keywords: FIB/SEM tomography; HCMV; nuclear capsid egress; inner nuclear membrane infoldings; high-pressure freezing; three-dimensional structure FIB/SEM tomography; HCMV; nuclear capsid egress; inner nuclear membrane infoldings; high-pressure freezing; three-dimensional structure
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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MDPI and ACS Style

Villinger, C.; Neusser, G.; Kranz, C.; Walther, P.; Mertens, T. 3D Analysis of HCMV Induced-Nuclear Membrane Structures by FIB/SEM Tomography: Insight into an Unprecedented Membrane Morphology. Viruses 2015, 7, 5686-5704.

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