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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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A. Impact of VP1u on NEBD.Quantifications of the PI fluorescences with the mean values and the 95% confidence intervals (bars) after addition of 300 genome-containing wt AAV2 particles and 300 mutant AAV2 per permeabilized HeLa cell. Red dotted line: buffer only (n = 15), blue line: mt AAV2 ΔVP1 (n = 14), blue line: wt AAV2 (not acidified) (n = 20), black line: wt AAV2 (acidified) (n = 11), green line: mt AAV2 ΔVP2 (acidified; n = 18). The panel shows that VP1 is indispensable for NEBD for AAV2. B. PV PLA2 activity increases upon interaction with nucleoporins. 1. Positive control (PLA2 from bee venom), 2. H1, 3. AAV2, 4. AAV2 after acidification, 5. H1+nucleoporins, 6. H1+nucleoporins+Ca++, 7. AAV2+nucleoporins, 8. AAV2 after acidification+nucleoporins, 9. AAV2 after acidification+nucleoporins+Ca++, 10. Nucleoporins. Y-axis: arb. units. The observations indicate that interaction with nucleoporins cause PLA2 activation, which indicates the exposure of VP1u on the surface of the particles. C. PLA2 is not essential for NEBD. Quantification of PI fluorescence as in A. Red dotted line: buffer; black line: AAV2 wt (n = 10), pH-treated; light green line: AAV2 HD/AN mt (n = 10); green line: AAV2 HD/AN mt acidified and neutralized (n = 10). Wt AAV2: 300 per permeabilized cell; mt viruses 120 per permeabilized cell. Collectively the three panels show that PV need VP1 for NEBD and that VP1u becomes exposed upon Nup interaction. The PLA2 activity on VP1u is however not required.
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ppat-1003671-g006: A. Impact of VP1u on NEBD.Quantifications of the PI fluorescences with the mean values and the 95% confidence intervals (bars) after addition of 300 genome-containing wt AAV2 particles and 300 mutant AAV2 per permeabilized HeLa cell. Red dotted line: buffer only (n = 15), blue line: mt AAV2 ΔVP1 (n = 14), blue line: wt AAV2 (not acidified) (n = 20), black line: wt AAV2 (acidified) (n = 11), green line: mt AAV2 ΔVP2 (acidified; n = 18). The panel shows that VP1 is indispensable for NEBD for AAV2. B. PV PLA2 activity increases upon interaction with nucleoporins. 1. Positive control (PLA2 from bee venom), 2. H1, 3. AAV2, 4. AAV2 after acidification, 5. H1+nucleoporins, 6. H1+nucleoporins+Ca++, 7. AAV2+nucleoporins, 8. AAV2 after acidification+nucleoporins, 9. AAV2 after acidification+nucleoporins+Ca++, 10. Nucleoporins. Y-axis: arb. units. The observations indicate that interaction with nucleoporins cause PLA2 activation, which indicates the exposure of VP1u on the surface of the particles. C. PLA2 is not essential for NEBD. Quantification of PI fluorescence as in A. Red dotted line: buffer; black line: AAV2 wt (n = 10), pH-treated; light green line: AAV2 HD/AN mt (n = 10); green line: AAV2 HD/AN mt acidified and neutralized (n = 10). Wt AAV2: 300 per permeabilized cell; mt viruses 120 per permeabilized cell. Collectively the three panels show that PV need VP1 for NEBD and that VP1u becomes exposed upon Nup interaction. The PLA2 activity on VP1u is however not required.

Mentions: Asking which PV domain causes NEBD, we added two mutants of AAV2 to permeabilized cells: AAV2-ΔVP1 is devoid of VP1 including VP1u and AAV2-ΔVP2, which is devoid of VP2 but which comprises VP1u [38]. NEBD was restricted to the VP1u-comprising mutant (Fig. 6A).


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)

A. Impact of VP1u on NEBD.Quantifications of the PI fluorescences with the mean values and the 95% confidence intervals (bars) after addition of 300 genome-containing wt AAV2 particles and 300 mutant AAV2 per permeabilized HeLa cell. Red dotted line: buffer only (n = 15), blue line: mt AAV2 ΔVP1 (n = 14), blue line: wt AAV2 (not acidified) (n = 20), black line: wt AAV2 (acidified) (n = 11), green line: mt AAV2 ΔVP2 (acidified; n = 18). The panel shows that VP1 is indispensable for NEBD for AAV2. B. PV PLA2 activity increases upon interaction with nucleoporins. 1. Positive control (PLA2 from bee venom), 2. H1, 3. AAV2, 4. AAV2 after acidification, 5. H1+nucleoporins, 6. H1+nucleoporins+Ca++, 7. AAV2+nucleoporins, 8. AAV2 after acidification+nucleoporins, 9. AAV2 after acidification+nucleoporins+Ca++, 10. Nucleoporins. Y-axis: arb. units. The observations indicate that interaction with nucleoporins cause PLA2 activation, which indicates the exposure of VP1u on the surface of the particles. C. PLA2 is not essential for NEBD. Quantification of PI fluorescence as in A. Red dotted line: buffer; black line: AAV2 wt (n = 10), pH-treated; light green line: AAV2 HD/AN mt (n = 10); green line: AAV2 HD/AN mt acidified and neutralized (n = 10). Wt AAV2: 300 per permeabilized cell; mt viruses 120 per permeabilized cell. Collectively the three panels show that PV need VP1 for NEBD and that VP1u becomes exposed upon Nup interaction. The PLA2 activity on VP1u is however not required.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1003671-g006: A. Impact of VP1u on NEBD.Quantifications of the PI fluorescences with the mean values and the 95% confidence intervals (bars) after addition of 300 genome-containing wt AAV2 particles and 300 mutant AAV2 per permeabilized HeLa cell. Red dotted line: buffer only (n = 15), blue line: mt AAV2 ΔVP1 (n = 14), blue line: wt AAV2 (not acidified) (n = 20), black line: wt AAV2 (acidified) (n = 11), green line: mt AAV2 ΔVP2 (acidified; n = 18). The panel shows that VP1 is indispensable for NEBD for AAV2. B. PV PLA2 activity increases upon interaction with nucleoporins. 1. Positive control (PLA2 from bee venom), 2. H1, 3. AAV2, 4. AAV2 after acidification, 5. H1+nucleoporins, 6. H1+nucleoporins+Ca++, 7. AAV2+nucleoporins, 8. AAV2 after acidification+nucleoporins, 9. AAV2 after acidification+nucleoporins+Ca++, 10. Nucleoporins. Y-axis: arb. units. The observations indicate that interaction with nucleoporins cause PLA2 activation, which indicates the exposure of VP1u on the surface of the particles. C. PLA2 is not essential for NEBD. Quantification of PI fluorescence as in A. Red dotted line: buffer; black line: AAV2 wt (n = 10), pH-treated; light green line: AAV2 HD/AN mt (n = 10); green line: AAV2 HD/AN mt acidified and neutralized (n = 10). Wt AAV2: 300 per permeabilized cell; mt viruses 120 per permeabilized cell. Collectively the three panels show that PV need VP1 for NEBD and that VP1u becomes exposed upon Nup interaction. The PLA2 activity on VP1u is however not required.
Mentions: Asking which PV domain causes NEBD, we added two mutants of AAV2 to permeabilized cells: AAV2-ΔVP1 is devoid of VP1 including VP1u and AAV2-ΔVP2, which is devoid of VP2 but which comprises VP1u [38]. NEBD was restricted to the VP1u-comprising mutant (Fig. 6A).

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