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Parvoviruses cause nuclear envelope breakdown by activating key enzymes of mitosis.

Porwal M, Cohen S, Snoussi K, Popa-Wagner R, Anderson F, Dugot-Senant N, Wodrich H, Dinsart C, Kleinschmidt JA, Panté N, Kann M - PLoS Pathog. (2013)

Bottom Line: Activation and coordination of the different activities is poorly understood and moreover complicated as some factors translocate between cytoplasm and nucleus in preparatory phases.Consistent with Ca⁺⁺ efflux from the lumen between inner and outer nuclear membrane we found that Ca⁺⁺ was essential for nuclear disassembly by activating PKC.PKC activation then triggered activation of cdk-2, which became further activated by caspase-3.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Virology, University of Giessen, Giessen, Germany ; Univ. de Bordeaux, Microbiologie fondamentale et Pathogénicité, UMR 5234, Bordeaux, France ; CNRS, Microbiologie fondamentale et Pathogénicité, UMR 5234, Bordeaux, France.

ABSTRACT
Disassembly of the nuclear lamina is essential in mitosis and apoptosis requiring multiple coordinated enzymatic activities in nucleus and cytoplasm. Activation and coordination of the different activities is poorly understood and moreover complicated as some factors translocate between cytoplasm and nucleus in preparatory phases. Here we used the ability of parvoviruses to induce nuclear membrane breakdown to understand the triggers of key mitotic enzymes. Nuclear envelope disintegration was shown upon infection, microinjection but also upon their application to permeabilized cells. The latter technique also showed that nuclear envelope disintegration was independent upon soluble cytoplasmic factors. Using time-lapse microscopy, we observed that nuclear disassembly exhibited mitosis-like kinetics and occurred suddenly, implying a catastrophic event irrespective of cell- or type of parvovirus used. Analyzing the order of the processes allowed us to propose a model starting with direct binding of parvoviruses to distinct proteins of the nuclear pore causing structural rearrangement of the parvoviruses. The resulting exposure of domains comprising amphipathic helices was required for nuclear envelope disintegration, which comprised disruption of inner and outer nuclear membrane as shown by electron microscopy. Consistent with Ca⁺⁺ efflux from the lumen between inner and outer nuclear membrane we found that Ca⁺⁺ was essential for nuclear disassembly by activating PKC. PKC activation then triggered activation of cdk-2, which became further activated by caspase-3. Collectively our study shows a unique interaction of a virus with the nuclear envelope, provides evidence that a nuclear pool of executing enzymes is sufficient for nuclear disassembly in quiescent cells, and demonstrates that nuclear disassembly can be uncoupled from initial phases of mitosis.

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H1 causes nuclear envelope breaks in Xenopus laevis oocytes after microinjection into the cytoplasm.A. Electron microscopy of the nuclear membrane after microinjection of 2.13×105 pfu./ml, or 2.17×108 genomes/ml H1 into the cytoplasm of Xenopus laevis oocytes. The oocytes were fixed after 1, 2, 4 h prior to preparation of the nuclei and staining. Membrane breaks are indicated by brackets, the nucleus is on the bottom of each panel. MOCK: Tris EDTA, pH 7.8 injection and incubation for 4 h. Middle panels: injection of H1 with the indicated incubation time. Nuclear: nuclear microinjection (4 h RT). Bar = 100 nm. B. Quantification from 30 electron micrographs derived from microinjection into three oocytes per condition. Mock: mock-injected control. Nuc: nuclear microinjection after 4 h. Left panels: the length of the breaks. Right panels: proportion of degraded nuclear envelope. Nuc: nuclear microinjection. In summary these data show that PV-mediated NEBD leads to disruption of inner and outer nuclear membrane in Xenopus laevis oocytes, supporting that PV-mediated NEBD is a evolutionary well conserved process. The breaks in the membrane indicate that Ca++ leaks out of the lumen between the two membranes. The observation that ONM breaks occur with higher frequency than observed for the INM implies that membrane disintegration starts at the ONM.
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ppat-1003671-g007: H1 causes nuclear envelope breaks in Xenopus laevis oocytes after microinjection into the cytoplasm.A. Electron microscopy of the nuclear membrane after microinjection of 2.13×105 pfu./ml, or 2.17×108 genomes/ml H1 into the cytoplasm of Xenopus laevis oocytes. The oocytes were fixed after 1, 2, 4 h prior to preparation of the nuclei and staining. Membrane breaks are indicated by brackets, the nucleus is on the bottom of each panel. MOCK: Tris EDTA, pH 7.8 injection and incubation for 4 h. Middle panels: injection of H1 with the indicated incubation time. Nuclear: nuclear microinjection (4 h RT). Bar = 100 nm. B. Quantification from 30 electron micrographs derived from microinjection into three oocytes per condition. Mock: mock-injected control. Nuc: nuclear microinjection after 4 h. Left panels: the length of the breaks. Right panels: proportion of degraded nuclear envelope. Nuc: nuclear microinjection. In summary these data show that PV-mediated NEBD leads to disruption of inner and outer nuclear membrane in Xenopus laevis oocytes, supporting that PV-mediated NEBD is a evolutionary well conserved process. The breaks in the membrane indicate that Ca++ leaks out of the lumen between the two membranes. The observation that ONM breaks occur with higher frequency than observed for the INM implies that membrane disintegration starts at the ONM.

Mentions: H1 and acidified AAV2 bound at least to Nup358, which localises on the cytosolic face of the NPC, to Nup153 and probably Nup214, which both localize on both sides of the NPC [40], and to Nup62, which is part of the hydrophobic mesh filling nuclear pore [41]. Nup62 was not expected to be involved in NEBD as anti p62-antibodies do not block nuclear accumulation of AAV2 DNA or uptake of fluorescently labelled AAV2 into nuclei in vitro[42]. The external localization of Nups358 and 153 let us expect that VP1u causes local NEBD on the ONM first, followed by destruction of the INM. To test this idea we injected H1 into Xenopus laevis oocytes and analyzed local NEBD by EM. Figure 7A shows that PV H1-induced local membrane disruptions, which were not observed in buffer (MOCK)-injected oocytes. This observation is similar to the fenestration of the ONM recently observed in oocytes after microinjection of the minute virus of mice [27]. Number and size of the membrane breaks increased with the incubation time but their maximal size of ∼190 nm remained similar (Fig. 7B). We hypothesize that the difference between cell lines, which show larger breaks and the oocytes with restricted fenestrations by differences between somatic and germinal cells but we also considered that the nuclei of Xenopus laevis differ in their stability due to massive amounts of actin, which becomes exported upon fertilization [43].


Parvoviruses cause nuclear envelope breakdown by activating key enzymes of mitosis.

Porwal M, Cohen S, Snoussi K, Popa-Wagner R, Anderson F, Dugot-Senant N, Wodrich H, Dinsart C, Kleinschmidt JA, Panté N, Kann M - PLoS Pathog. (2013)

H1 causes nuclear envelope breaks in Xenopus laevis oocytes after microinjection into the cytoplasm.A. Electron microscopy of the nuclear membrane after microinjection of 2.13×105 pfu./ml, or 2.17×108 genomes/ml H1 into the cytoplasm of Xenopus laevis oocytes. The oocytes were fixed after 1, 2, 4 h prior to preparation of the nuclei and staining. Membrane breaks are indicated by brackets, the nucleus is on the bottom of each panel. MOCK: Tris EDTA, pH 7.8 injection and incubation for 4 h. Middle panels: injection of H1 with the indicated incubation time. Nuclear: nuclear microinjection (4 h RT). Bar = 100 nm. B. Quantification from 30 electron micrographs derived from microinjection into three oocytes per condition. Mock: mock-injected control. Nuc: nuclear microinjection after 4 h. Left panels: the length of the breaks. Right panels: proportion of degraded nuclear envelope. Nuc: nuclear microinjection. In summary these data show that PV-mediated NEBD leads to disruption of inner and outer nuclear membrane in Xenopus laevis oocytes, supporting that PV-mediated NEBD is a evolutionary well conserved process. The breaks in the membrane indicate that Ca++ leaks out of the lumen between the two membranes. The observation that ONM breaks occur with higher frequency than observed for the INM implies that membrane disintegration starts at the ONM.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3814971&req=5

ppat-1003671-g007: H1 causes nuclear envelope breaks in Xenopus laevis oocytes after microinjection into the cytoplasm.A. Electron microscopy of the nuclear membrane after microinjection of 2.13×105 pfu./ml, or 2.17×108 genomes/ml H1 into the cytoplasm of Xenopus laevis oocytes. The oocytes were fixed after 1, 2, 4 h prior to preparation of the nuclei and staining. Membrane breaks are indicated by brackets, the nucleus is on the bottom of each panel. MOCK: Tris EDTA, pH 7.8 injection and incubation for 4 h. Middle panels: injection of H1 with the indicated incubation time. Nuclear: nuclear microinjection (4 h RT). Bar = 100 nm. B. Quantification from 30 electron micrographs derived from microinjection into three oocytes per condition. Mock: mock-injected control. Nuc: nuclear microinjection after 4 h. Left panels: the length of the breaks. Right panels: proportion of degraded nuclear envelope. Nuc: nuclear microinjection. In summary these data show that PV-mediated NEBD leads to disruption of inner and outer nuclear membrane in Xenopus laevis oocytes, supporting that PV-mediated NEBD is a evolutionary well conserved process. The breaks in the membrane indicate that Ca++ leaks out of the lumen between the two membranes. The observation that ONM breaks occur with higher frequency than observed for the INM implies that membrane disintegration starts at the ONM.
Mentions: H1 and acidified AAV2 bound at least to Nup358, which localises on the cytosolic face of the NPC, to Nup153 and probably Nup214, which both localize on both sides of the NPC [40], and to Nup62, which is part of the hydrophobic mesh filling nuclear pore [41]. Nup62 was not expected to be involved in NEBD as anti p62-antibodies do not block nuclear accumulation of AAV2 DNA or uptake of fluorescently labelled AAV2 into nuclei in vitro[42]. The external localization of Nups358 and 153 let us expect that VP1u causes local NEBD on the ONM first, followed by destruction of the INM. To test this idea we injected H1 into Xenopus laevis oocytes and analyzed local NEBD by EM. Figure 7A shows that PV H1-induced local membrane disruptions, which were not observed in buffer (MOCK)-injected oocytes. This observation is similar to the fenestration of the ONM recently observed in oocytes after microinjection of the minute virus of mice [27]. Number and size of the membrane breaks increased with the incubation time but their maximal size of ∼190 nm remained similar (Fig. 7B). We hypothesize that the difference between cell lines, which show larger breaks and the oocytes with restricted fenestrations by differences between somatic and germinal cells but we also considered that the nuclei of Xenopus laevis differ in their stability due to massive amounts of actin, which becomes exported upon fertilization [43].

Bottom Line: Activation and coordination of the different activities is poorly understood and moreover complicated as some factors translocate between cytoplasm and nucleus in preparatory phases.Consistent with Ca⁺⁺ efflux from the lumen between inner and outer nuclear membrane we found that Ca⁺⁺ was essential for nuclear disassembly by activating PKC.PKC activation then triggered activation of cdk-2, which became further activated by caspase-3.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Virology, University of Giessen, Giessen, Germany ; Univ. de Bordeaux, Microbiologie fondamentale et Pathogénicité, UMR 5234, Bordeaux, France ; CNRS, Microbiologie fondamentale et Pathogénicité, UMR 5234, Bordeaux, France.

ABSTRACT
Disassembly of the nuclear lamina is essential in mitosis and apoptosis requiring multiple coordinated enzymatic activities in nucleus and cytoplasm. Activation and coordination of the different activities is poorly understood and moreover complicated as some factors translocate between cytoplasm and nucleus in preparatory phases. Here we used the ability of parvoviruses to induce nuclear membrane breakdown to understand the triggers of key mitotic enzymes. Nuclear envelope disintegration was shown upon infection, microinjection but also upon their application to permeabilized cells. The latter technique also showed that nuclear envelope disintegration was independent upon soluble cytoplasmic factors. Using time-lapse microscopy, we observed that nuclear disassembly exhibited mitosis-like kinetics and occurred suddenly, implying a catastrophic event irrespective of cell- or type of parvovirus used. Analyzing the order of the processes allowed us to propose a model starting with direct binding of parvoviruses to distinct proteins of the nuclear pore causing structural rearrangement of the parvoviruses. The resulting exposure of domains comprising amphipathic helices was required for nuclear envelope disintegration, which comprised disruption of inner and outer nuclear membrane as shown by electron microscopy. Consistent with Ca⁺⁺ efflux from the lumen between inner and outer nuclear membrane we found that Ca⁺⁺ was essential for nuclear disassembly by activating PKC. PKC activation then triggered activation of cdk-2, which became further activated by caspase-3. Collectively our study shows a unique interaction of a virus with the nuclear envelope, provides evidence that a nuclear pool of executing enzymes is sufficient for nuclear disassembly in quiescent cells, and demonstrates that nuclear disassembly can be uncoupled from initial phases of mitosis.

Show MeSH
Related in: MedlinePlus