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First published online 17 June 2008
doi: 10.1242/jcs.032284

Journal of Cell Science 121, 2287-2292 (2008)
Published by The Company of Biologists 2008
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Exceptional mechanical and structural stability of HSV-1 unveiled with fluid atomic force microscopy

Ivan Liashkovich1,*, Wali Hafezi2,3,*, Joachim E. Kühn2,3, Hans Oberleithner1, Armin Kramer1 and Victor Shahin1,{ddagger}

1 Institute of Physiology II, University of Münster, Robert-Koch-Str. 27b, 48149 Münster, Germany
2 Institute of Medical Microbiology, University of Münster, Domagkstr. 10, 48149 Münster, Germany
3 Interdisciplinary Center of Clinical Research (IZKF), University of Münster, Domagkstr. 3, 48149 Münster, Germany


Figure 1
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  		Fig. 1. Shape and surface topography of the HSV-1 capsids studied by EM and AFM. (A) EM image of the capsid preparation. (B) HSV-1 capsids adsorbed to the surface of poly-L-lysine-coated mica (1.60x1.38 µm). (C,D) A single capsid with the typical hexagonal outline (C) and its cross section (D). (E) Two capsids with apparent icosahedral symmetry and clearly distinguishable triangular faces (510x560 nm). (F) A fragment of the HSV-1 surface with clearly distinguishable capsomeres (100x100 nm). (G,H) A penton surrounded by five nearest neighboring hexons (G) and a hexon (H). Pentons and hexons images are 40x40 nm.

 

Figure 2
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  		Fig. 2. Structural integrity of HSV-1 capsids on various surfaces. AFM images (A,C,E) and cross-sectional graphs (B,D,F). HOPG (A,B) and poly-L-lysine-coated mica (C,D) are capable of adsorbing intact capsids. (E,F) On the surface of poly-L-lysine-coated glass, capsids undergo complete disassembly which yields ~40-nm-high spherical structures. Coiled genomic material also adsorbs to the surface. The AFM images are 3.5x3.5 µm.

 

Figure 3
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  		Fig. 3. Simultaneous AFM (A) and fluorescent (B) imaging of disassembled HSV-1 capsid on the surface of poly-L-lysine-coated glass. Areas of coiled material on the surface of glass correspond to light fluorescent spots. Labeling was performed with 0.1 µM YO-PRO1. Images are 10x10 µm.

 

Figure 4
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  		Fig. 4. Frequency distribution of the measured capsid stiffness at a loading force of 2.2 nN. At this load, linear elastic response of the particle and indentation values below the shell thickness (15 nm) have been observed.

 

Figure 5
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  		Fig. 5. Mechanical probing of HSV-1 capsids. (A) A linear elastic response of capsids is observed when forces below 4.4 nN are applied (curves in light and dark blue). (B) The threshold loading force of 7 nN causes mechanical failure of the capsids. The linear curve (dark blue) was taken when the capsid was still intact, the next round of probing (red curve) has caused mechanical failure of the capsid. (C) Application of the loading force exceeding mechanical limits of the capsids. Linearity is lost at high force (curve in dark green), whereas at low force linear behavior is restored (curve in light green). Nevertheless, the stiffness of mechanically damaged capsids is dramatically reduced. (D) Application of guanidine hydrochloride induces genome release and consequently leads to decreased stiffness of the capsids (curve in light blue is taken from an intact capsid; that in light green from GuHCl-treated capsid). h1 to h8 represent the indentation values.

 

Figure 6
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  		Fig. 6. Structural consequences of mechanical (A,B) and chemical (C,D) treatment of the capsids. Overall shape and dimensions of the majority of the capsids remains intact although the height of several capsids is decreased. AFM was performed at the loading force of 2 nN. A and B as well as C and D are paired experiments, although A and B represent the same area whereas C and D represent different areas of the same sample. (E) Measured stiffness values of intact, mechanically damaged and GuHCl-treated capsids. DNA contribution to the overall stiffness of the nucleocapsid evaluated by mechanical probing of empty capsids appears to be significant (data are shown as mean ± s.e.m.). The AFM images are 3.5x3.5 µm.

 

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© The Company of Biologists Ltd 2008