Figure 6. Ultrathin-sectioning EM visualization of complete osmotic suppression of DNA ejection from HSV-1 capsids into reconstituted cell nuclei when capsid pressure is 'turned off' by 18 atm osmotic pressure generated by PEG 8 kDa.

Negative control at 4°C without added PEG and without ATP-regenerating system, shows that no ejection from nuclei bound C-capsids occurs. Positive control at 37°C shows complete DNA ejection from C-capsids bound to isolated cell nuclei supplemented with cytosol and ATP-regenerating system. EM images show that capsids can bind to the nuclear membrane as individual capsids or in multilayer clusters. Consequentially, only capsids in the first layer that are bound to the NPCs are able to eject their DNA. EM shows that the addition of 30% PEG 8 kDa to reconstituted capsid-nuclei system inhibits DNA ejection from HSV-1 C-capsids into host nuclei through the NPC. In all samples, capsids and nuclei were incubated for 40 min. Thin arrows show DNA-filled capsids, and bold arrows show empty capsids that ejected DNA. 1. Bar 500 nm. 2. Bar 90 nm. Representative EM images are shown. At least 100 capsids bound to NPCs were counted for each sample’s statistical analysis, shown in the table below.

Figure 6—figure supplement 1. Calculated DNA pressures in herpesvirus capsids.

The analytical expression used to calculate pressures inside virus capsids is based on a theoretical model from refs. (Grayson et al., 2006; Purohit et al., 2005; Purohit et al., 2003; Tzlil et al., 2003) that involves forces generated by DNA-DNA electrostatic repulsive force and DNA bending stress, which depend on the DNA packing density. DNA packing density in the capsid was estimated with values for packaged DNA length and inner capsid radius derived from previous Cryo-EM data (Booy et al., 1991; Germi et al., 2012).