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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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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.
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viruses-07-02900-f006: 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.

Mentions: Tubular segments of 1st order infoldings always contained one or more tubular 2nd order infoldings (Figures 4A and 5C), i.e., invaginations, which always partially overlapped with each other (Movies S1 and S2). In five cases, the invaginations were found to be open at one end (Figure 5E and Figure 6A top) and in six cases they were closed at both ends, showing no connection to the nucleoplasm (Figure 6A bottom), but they were never open on both ends (Movies S1 and S2 in supplemental file). Some of the tubular segments contained 3rd order infoldings but never capsids. The general architecture of the infoldings is schematically summarized in Figure 6.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

viruses-07-02900-f006: 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.
Mentions: Tubular segments of 1st order infoldings always contained one or more tubular 2nd order infoldings (Figures 4A and 5C), i.e., invaginations, which always partially overlapped with each other (Movies S1 and S2). In five cases, the invaginations were found to be open at one end (Figure 5E and Figure 6A top) and in six cases they were closed at both ends, showing no connection to the nucleoplasm (Figure 6A bottom), but they were never open on both ends (Movies S1 and S2 in supplemental file). Some of the tubular segments contained 3rd order infoldings but never capsids. The general architecture of the infoldings is schematically summarized in Figure 6.

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