Parvovirus H1 cause NEBD with chromatin escape.

<p><b>A, B:</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. <b>A.</b> 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>B.</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. <b>C, D.</b> 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. <b>C.</b> Overview of chromatin disappearance in nuclei in one microscopical field. Left column: H1, right column: buffer only. Bar = 20 µm. <b>D.</b> 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.