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	Figure 2. Intra and inter assay variability of chromatin loss in permeabilized HeLa cells incubated with or without H1.Merge of six experiments. The chromatin by PI was quantified in 42 nuclei exposed to H1 and in 67 nuclei exposed to buffer only. A. Each circle represents the relative PI fluorescence at the indicated time. The fluorescence at t = 0 of each cell was set as 100%. The lines connect the fluorescence of one nucleus at different times. Each colour represents one experiment. Y-axis: relative fluorescence in %, x-axis: time in min. B. Same data as in A. but showing the mean fluorescence and the 95% confidence intervals (CI) (bars). The horizontal bar shows the 95% CI for the 50% loss of PI fluorescence (4.6 to 5.4 min). The data show that parvovirus-mediated NEBD exhibits small variations between different assays and between individual nuclei.
	Figure 3. Loss of chromatin and lamin B receptor (LBR) upon H1 exposure to permeabilized NRK cells.Per permeabilized cell 300 H1 were added. A. Green: EYFP-LBR, red PI-chromatin. Bar = 20 µm. B. Quantification of A. The bars depict 95% CIs. Green dotted line: LBR in buffer only cells (n = 9); red dotted line: PI in buffer only cells. Green line: LBR with H1 (n = 9); red line: PI with H1. The figures A and B show that LBR dissociates first from the nuclear envelope before chromatin becomes released. C. 3D reconstruction of PI stained nuclei 15 min after addition of H1 (top) or buffer (bottom). The cover slip is at the bottom of the images, the cell surface on top. Cells are indicated by white arrows. The panels show that at in some cells after 15 min some chromatin stayed attached to the nuclear membrane adjacent to the part of plasma membrane, which is attached to the cover slip.
	Figure 4. NEBD capacity of different PV.Per permeabilized HeLa cell 300 of each PV were added. 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 = 14). Blue line: AAV2 (n = 13), orange line: canine parvovirus (n = 35), red line: H1 (n = 23), black line: AAV2 after acidification to pH 5.2 and subsequent neutralization (n = 10). Acidification to pH 5.2 of H1 did not change the NEBD activity (not shown). The figure shows that NEBD is independent upon the type of PV with a similar kinetic but to a different extent. Acidification was needed for AAV but not for H1 and the canine parvovirus.
	Figure 5. 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).
	Figure 6. 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.
	Figure 7. 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.
	Figure 8. H1-mediated NEBD leads to simultaneous escape of a 100 kDa cargo and chromatin, which depends upon enzymes needed for mitosis.Nuclei of permeabilized cells preloaded with M9-Alexa647-BSA (M9) prior to addition of 300 H1 per permeabilized HeLa cell together with inhibitors. The graphs show quantifications of DAPI and Alexa647 fluorescences with the mean values and CI 95% (bars). Blue dotted line: DAPI with buffer only, no inhibitor (n = 39), pink dotted line: M9, buffer, no inhibitor (n = 39), orange line: DAPI, H1, no inhibitor (n = 21), red line: M9, H1, no inhibitor (n = 21), black line: DAPI, H1, H89 (n = 33), brown blue dashed line: M9, H1, H89 (n = 33), blue dashed line: DAPI, H1, roscovitine (n = 19), grey dashed line: M9, H1, roscovitine (n = 19), cyan dashed line: DAPI, H1, zVAD-fmk (n = 23), grey: M9, H1, zVAD-fmk (n = 23). Please note that some lines overlap. The figure shows that the escape of chromatin and the 100 kDa cargo cannot be separated despite of their different MW, indicating a catastrophic-like event during which the entire NE disintegrates. As all inhibitors entirely inhibited the loss of both cargos the results further indicate that PKC, cdks and caspase-3 are essential for NEBD.
	Figure 9. Cellular PKC and cdk-2 become activated by H1 and PKCα but not PKCβ is required for NEBD.A, B, C: Activation of PKC, cdk-2 and caspase-3 in permeabilized HeLa cells by H1. Y-axis: activity in arbitrary units. The tested activity is indicated on top of each panel. The columns show the mean values of three independent experiments. The variation bars show the range between the highest and the lowest value. 1. permeabilized cells. 2. permeabilized cells+H1. 3. permeabilized cells+H1+H89. 4. permeabilized cells+H1+Roscovitine. 5. permeabilized cells+H1+zVAD-fmk. 6. H1 without cells. 7. permeabilized cells+H1+thapsigargin. 8. non-permeabilized cells. n.d. not determined. The panels show that permeabilization decrease the activity of all three enzymes and that H1 activates the activities of PKC and cdk-2, while an effect on caspase-3 is doubtful. Inhibition of PKC also reduced activity of cdk2 but not of caspase_3 despite of its inhibitor specificity shown in the supporting information. Thapsigargin pre-treatment, leading to Ca++ depletion inhibits PKC and cdk2 implying that a Ca++-dependent PKC is involved. D. PV H1-mediated NEBD is inhibited by PKCα but not PKCβ. Quantification of PI-stained chromatin of permeabilized HeLa cells to which 300 H1 per permeabilized cell were added. The bars depict 95% CI. Red, dotted line: buffer (n = 28); red line: H1 (n = 14); green dashed line: H1 using PKCα-inhibited cells (n = 19); black dashed line: H1 using PKCβ-inhibited cells. Collectively, the data show that Ca++-dependent PKCα is required for NEBD, which is consistent with the PV-mediated activation of a Ca++-dependent PKC. PKCα subsequently activated cdk-2, which was also shown essential for NEBD. Caspase-3 was not significantly activated by PV but its activity was however essential for NEBD.
	Figure 10. Quantification of fluorescence intensity of the markers microinjected with or without H1 into U2OS cells.Quantifications of the fluorescences presented as videos in the supporting information. The microinjected markers are indicated on top of each panel (D10-Tx: Dextran 10-Texas Red labelled, D40-FITC: Dextran 40, FITC-labelled, ab-A647: unrelated IgG, Alexa.647-labelled). The grey lines show the cytoplasmic, the red line the nuclear fluorescence. The y-axis depicts the intensity given in arbitrary units, the x-axis the time after microinjection in seconds. A. Microinjection of buffer with marker proteins. D10-Tx equilibrated between cytoplasm and nucleus directly after microinjection while D40-FITC and ab-A647 stayed excluded from nucleus. B. Microinjection of H1 with marker proteins. D40-Tx and ab-A647 entered the nucleus simultaneously 240 seconds after microinjection and reached the equilibrium. C. Microinjection of Ca++ with marker proteins. D40-Tx and ab-A647 entered the nucleus simultaneously 200 seconds after microinjection also reaching the equilibrium. In summery the panels show that both H1 and Ca++ triggered sudden NEBD approx. 2–3 min after microinjection. Dextran 40 and the antibodies entered the nucleus at the same time indicating a catastrophic-like destruction of the barrier as it was also seen for nuclear escape of 100 kDa cargos and chromatin in permeabilized cells.
	Figure 11. Schematic presentation of the parvoviral interaction with the nuclear envelope.A. Upon arrival at the nuclear pores parvoviral capsids (blue icosahedra) interact directly with at least three Nup (Nup358: dark grey; Nup153: light grey, Nup62: middle grey). B. This interaction causes exposure of VP1u on the surface of the virus. C. VP1u exposure allows permeabilization of the nuclear membrane, which is indicated by the dotted line of the NE (black lines). The permeabilization causes efflux of Ca++ indicated by the arrows, causing elevated local Ca++ in the nuclear periphery. D. Ca++ activated PKCα causing exposure of the catalytic domain (orange; PKCα-i: inactive; PKCα-a: active), which than phosphorylates lamins (blue; PKCα phosphorylation indicated by orange P). Further PKCα inactivates indirectly cdk-2, subsequently activating cdk-1 (indirect activations indicated by a dotted circle; cdks in red, cdk-i: inactive, cdk-a: active). Further activation is mediated by caspase-3, likely in an indirect manner. E. Active cdk1 hyper phosphorylates lamins, shown by red P. Hyper phosphorylation subsequently causes lamin depolymerisation. On combination with the spread of membrane disintegration the LBR (green) dissociates from the NE. F. After significant membrane disintegration even larger structures as chromatin (red waved lines) can escape the nucleus.
	Figure 1. Parvovirus H1 cause NEBD with chromatin escape. A, B: Infection of HeLa cells with 1000 H1 per cell. Confocal laser scan microscopy after indirect immune fluorescence. Bar = 10 µm. Chromatin was stained by PI, NPCs by mAb414 and H1 by anti VP2 antibodies. A. Example of a cell in which chromatin passes through the membrane break into the cytoplasm. The NE break is indicated by a bracket, the extruding chromatin is indicated by arrows. B. Magnification of nuclear envelope breaks observed in different cells. Left columns show H1 infected HeLa cells, the right columns uninfected cells. The NPC stain by mAb414 and the H1 stain by anti VP2 antibodies are indicated on top. The arrows indicate the nuclear envelope breaks observed in 102 out of infected cells 913 cells (11%). Uninfected cells showed these discontinuities in only 2 out of 294 cells. Together the results indicate that H1 infection causes local NEBD causing holes partially large enough to allow passage of host chromatin into the cytoplasm. Bar = 10 µm. C, D. Time lapse microscopy of permeabilized HeLa cells incubated in buffer in the absence or in the presence of 300 H1 per permeabilized cell. No soluble cytosolic factors were present in the reaction. Images were taken by confocal microscopy at nine time points in order to exclude bleaching of the chromatin fluorescence by PI. The time points are indicated right (C) or below (D) of the panels. C. Overview of chromatin disappearance in nuclei in one microscopical field. Left column: H1, right column: buffer only. Bar = 20 µm. D. Magnification of chromatin disappearance in one HeLa cell. Bar 0 10 µm. Images C and D show that the PI-stained chromatin was lost in permeabilized HeLa cells, corresponding to the NE breaks observed upon infection despite of the absence of cytosol. No perinuclear chromatin patches as in apoptosis appeared.

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