Reconstituted virus–nucleus system reveals mechanics of herpesvirus genome uncoating

Figures

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  Fig. 1.. Confocal fluorescence microscopic images show binding of GFP herpes simplex virus type 1 C-capsids (green) to DAPI-stained isolated nuclei (blue), in the presence of cytosol and ATP-regeneration system. The addition of wheat germ agglutinin decreases capsid binding to nuclei for both capsid types, which demonstrates that capsids bind specifically to nuclear pore complexes as opposed to binding anywhere on the nuclear membrane.

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  Fig. 2.. (a,b) and (g,h) show an overview and a zoomed image of two representative nuclei investigated by atomic force microscopic (AFM) imaging (both nuclei and AFM cantilever with focused laser spot, blue, are visible). In (b,h), the imaging area is indicated by the overlayed image. (c) shows the topography recorded on the nucleus shown in (a,b). Capsids bound to the nuclear surface are encircled in red. (d) shows the features marked with an ellipse in (c) at higher resolution. The inset in (d) shows the surface profile of herpes simplex virus type 1 capsids on the nucleus surface along the black solid line. The height of one capsid is measured to be ~122 nm. (e,f) are 3D representations of (c,d). (i) shows the surface topography of the second nucleus reported in (g,h). Virus capsids are encircled in red. Arrows in (i) indicate nuclear pore complexes in the nuclear membrane. (j) shows a close-up view of the capsids marked with the ellipse in (i). (k,l) are 3D representations of (i,j). Note: The colour scales correspond to 500 (c), 300 (d) and 250 nm (i,j).

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  Fig. 3.. (a) A representative 10 × 10 force volume map for an unfixed nucleus with attached C-capsids (corresponding to 100 force–distance curves) acquired over a 200 nm × 200 nm area at the centre of each nucleus. (b) shows representative force–distance (Fz) curve, comprising each force volume map, for nucleus indentation with herpes simplex virus type 1 C-capsids attached (solid blue line). The red line shows Fz curve for atomic force microscopy (AFM)-tip retraction. The black dashed line shows a fit using the Hertz mechanics model for Young’s modulus determination. (c) Young’s moduli for 100 force–distance curves in each force volume map were repeatedly collected for several nuclei as well as for several areas on the same nucleus. All moduli values were collectively plotted in a histogram for nuclei with attached C-capsids. The Gaussian fit to the data in the histograms yields the average value and the standard error.