Limits...
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.

Show MeSH

Related in: MedlinePlus

PV interact directly with nucleoporins required for NEBD.A. WGA does not inhibit NEBD upon addition of 300 H1 per permeabilized cell. The figure shows the quantification of the PI stain as in figure 2B, and the 95% CIs at each time point. Red dotted line: buffer only (n = 8), red line: H1, green dashed line: H1 in the presence of 1 mg/ml WGA (n = 4). B. Parvoviruses bind directly to nucleoporins. Co-precipitated Nups were detected by Western blot using the mAb414, which interacts with different FG repeat-containing Nups including Nup358, 214,153 and 62. The MW is given on the left, the Nups are indicated on the right. 1: AAV2 (acidified and neutralized) without Nups, 2: same AAV2+Nups, 3: H1, 4: H1+Nups, 5: beads+Nups, 6: 12 µg, 7: 36 µg Nups directly loaded on the gel. Nup153 migrates at ∼170 kDa, as it was described elsewhere [70]. C. Blocking the NPCs by hepatitis B virus capsids inhibits H1-mediated NEBD. Conditions and read-out as in A. Red dotted line: buffer only (n = 14), red dashed line: H1 after pre-incubation of the NPCs with an excess (1200 ng) of in vitro phosphorylated capsids of the hepatitis B virus in the presence of transport receptors (n = 18), green dashed line: same treatment with a mutant of the hepatitis B virus capsid, which lacks the C terminus that is need for NPC interaction (n = 9), red line: H1 (n = 7).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3814971&req=5

ppat-1003671-g005: PV interact directly with nucleoporins required for NEBD.A. WGA does not inhibit NEBD upon addition of 300 H1 per permeabilized cell. The figure shows the quantification of the PI stain as in figure 2B, and the 95% CIs at each time point. Red dotted line: buffer only (n = 8), red line: H1, green dashed line: H1 in the presence of 1 mg/ml WGA (n = 4). B. Parvoviruses bind directly to nucleoporins. Co-precipitated Nups were detected by Western blot using the mAb414, which interacts with different FG repeat-containing Nups including Nup358, 214,153 and 62. The MW is given on the left, the Nups are indicated on the right. 1: AAV2 (acidified and neutralized) without Nups, 2: same AAV2+Nups, 3: H1, 4: H1+Nups, 5: beads+Nups, 6: 12 µg, 7: 36 µg Nups directly loaded on the gel. Nup153 migrates at ∼170 kDa, as it was described elsewhere [70]. C. Blocking the NPCs by hepatitis B virus capsids inhibits H1-mediated NEBD. Conditions and read-out as in A. Red dotted line: buffer only (n = 14), red dashed line: H1 after pre-incubation of the NPCs with an excess (1200 ng) of in vitro phosphorylated capsids of the hepatitis B virus in the presence of transport receptors (n = 18), green dashed line: same treatment with a mutant of the hepatitis B virus capsid, which lacks the C terminus that is need for NPC interaction (n = 9), red line: H1 (n = 7).

Mentions: To identify the trigger of NEBD we analyzed the contact site for PV at the nuclear envelope. Addition of wheat germ agglutinin, which blocks the attachment of nuclear import receptors to NPCs and which prevents active nuclear import at this concentration [34], did not prevent NEBD (Fig. 5A). This finding is in agreement with observations in Xenopus laevis oocytes in which wheat germ agglutinin has no effect on MVM-mediated pore formation [27]. Cohen et al. concluded that NPCs are not involved in nuclear envelope degradation we considered a direct interaction of the PV capsids with the proteins of the nuclear pore. In fact this hypothesis is consistent with our observation that soluble cytosolic proteins were not required for PV-mediated NEBD. We thus performed coprecipitations of H1 and AAV2 (after acidification) using a purified preparation of Nups (Supporting Information, Fig. S4). The preparation was devoid of importin α and importin β was reduced by 50fold compared to intact cells, which is consistent with the observation that purified nuclei - the first step of Nup preparation - are incompetent for active nuclear import. The changed abundance of the Nups in the preparation compared to intact cells further indicated that the NPCs were dissociated into Nups upon preparation.


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)

PV interact directly with nucleoporins required for NEBD.A. WGA does not inhibit NEBD upon addition of 300 H1 per permeabilized cell. The figure shows the quantification of the PI stain as in figure 2B, and the 95% CIs at each time point. Red dotted line: buffer only (n = 8), red line: H1, green dashed line: H1 in the presence of 1 mg/ml WGA (n = 4). B. Parvoviruses bind directly to nucleoporins. Co-precipitated Nups were detected by Western blot using the mAb414, which interacts with different FG repeat-containing Nups including Nup358, 214,153 and 62. The MW is given on the left, the Nups are indicated on the right. 1: AAV2 (acidified and neutralized) without Nups, 2: same AAV2+Nups, 3: H1, 4: H1+Nups, 5: beads+Nups, 6: 12 µg, 7: 36 µg Nups directly loaded on the gel. Nup153 migrates at ∼170 kDa, as it was described elsewhere [70]. C. Blocking the NPCs by hepatitis B virus capsids inhibits H1-mediated NEBD. Conditions and read-out as in A. Red dotted line: buffer only (n = 14), red dashed line: H1 after pre-incubation of the NPCs with an excess (1200 ng) of in vitro phosphorylated capsids of the hepatitis B virus in the presence of transport receptors (n = 18), green dashed line: same treatment with a mutant of the hepatitis B virus capsid, which lacks the C terminus that is need for NPC interaction (n = 9), red line: H1 (n = 7).
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1003671-g005: PV interact directly with nucleoporins required for NEBD.A. WGA does not inhibit NEBD upon addition of 300 H1 per permeabilized cell. The figure shows the quantification of the PI stain as in figure 2B, and the 95% CIs at each time point. Red dotted line: buffer only (n = 8), red line: H1, green dashed line: H1 in the presence of 1 mg/ml WGA (n = 4). B. Parvoviruses bind directly to nucleoporins. Co-precipitated Nups were detected by Western blot using the mAb414, which interacts with different FG repeat-containing Nups including Nup358, 214,153 and 62. The MW is given on the left, the Nups are indicated on the right. 1: AAV2 (acidified and neutralized) without Nups, 2: same AAV2+Nups, 3: H1, 4: H1+Nups, 5: beads+Nups, 6: 12 µg, 7: 36 µg Nups directly loaded on the gel. Nup153 migrates at ∼170 kDa, as it was described elsewhere [70]. C. Blocking the NPCs by hepatitis B virus capsids inhibits H1-mediated NEBD. Conditions and read-out as in A. Red dotted line: buffer only (n = 14), red dashed line: H1 after pre-incubation of the NPCs with an excess (1200 ng) of in vitro phosphorylated capsids of the hepatitis B virus in the presence of transport receptors (n = 18), green dashed line: same treatment with a mutant of the hepatitis B virus capsid, which lacks the C terminus that is need for NPC interaction (n = 9), red line: H1 (n = 7).
Mentions: To identify the trigger of NEBD we analyzed the contact site for PV at the nuclear envelope. Addition of wheat germ agglutinin, which blocks the attachment of nuclear import receptors to NPCs and which prevents active nuclear import at this concentration [34], did not prevent NEBD (Fig. 5A). This finding is in agreement with observations in Xenopus laevis oocytes in which wheat germ agglutinin has no effect on MVM-mediated pore formation [27]. Cohen et al. concluded that NPCs are not involved in nuclear envelope degradation we considered a direct interaction of the PV capsids with the proteins of the nuclear pore. In fact this hypothesis is consistent with our observation that soluble cytosolic proteins were not required for PV-mediated NEBD. We thus performed coprecipitations of H1 and AAV2 (after acidification) using a purified preparation of Nups (Supporting Information, Fig. S4). The preparation was devoid of importin α and importin β was reduced by 50fold compared to intact cells, which is consistent with the observation that purified nuclei - the first step of Nup preparation - are incompetent for active nuclear import. The changed abundance of the Nups in the preparation compared to intact cells further indicated that the NPCs were dissociated into Nups upon preparation.

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