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First published online 8 December 2005
doi: 10.1242/jcs.02705

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The genome of HSV-1 translocates through the nuclear pore as a condensed rod-like structure

¹ Institute of Physiology II, University of Münster, Robert-Koch-Str. 27b, 48149 Münster, Germany
² Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1PD, UK
³ Institute of Medical Microbiology, University of Münster, Domagkstr. 10, 48149 Münster, Germany
⁴ Interdisciplinary Center of Clinical Research (IZKF), Domagkstr. 3, 48149 Münster, Germany

View larger version (16K): [in a new window]   Fig. 1. Idealized force-distance curve describing a single approach-retract cycle of the AFM tip (modified from Shahin et al., 2005). The AFM tip is approaching the sample surface (A). The initial contact between the tip and the surface is mediated by the attractive van der Waals forces (contact) that lead to an attraction of the tip toward the sample (B). Hence, the tip applies a constant and default force upon the sample surface that leads to sample indentation and cantilever deflection (C). Subsequently, the tip tries to retract and to break loose from the surface (D). Various adhesive forces between the sample and the AFM tip, however, hamper tip retraction. These adhesive forces can be taken directly from the force-distance curve (E). The tip withdraws and loses contact with the sample upon overcoming the adhesive forces (F).

View larger version (60K): [in a new window]   Fig. 2. Prepared detergent-treated herpes simplex virus type-1 (HSV-1) capsids. Samples were characterized structurally by electron microscopy (EM) (A) and atomic force microscopy (AFM) (B-D). EM analysis of negatively stained capsid preparations shows that treatment of HSV-1 virions with 0.2% Triton X-100 renders capsids (which are 125 nm in diameter) free of envelopes. AFM analysis of capsid preparations adsorbed onto a poly-L-lysine-coated surface reveals spherical particles (B) with a height distribution exhibiting a sharp maximum between 120 and 130 nm (C,D) (n=200).

View larger version (58K): [in a new window]   Fig. 3. Representative AFM images of both the cytoplasmic and nucleoplasmic faces of the nuclear envelope 30 minutes after injecting solvent (nuclear isolation medium, NIM) into Xenopus laevis oocytes. Upon NIM injection, the cytoplasmic face of the nuclear envelope contains nuclear pore complexes (NPCs) appearing as cargo-free smooth rings exhibiting a diameter of 98.3±0.53 nm (n=200). The nucleoplasmic face of the nuclear envelope also reveals cargo-free NPCs, surrounded throughout by lamins, characterized by their prominent filamentous structure.

View larger version (61K): [in a new window]   Fig. 4. Representative AFM images of the cytoplasmic face of the nuclear envelope 30 minutes after injecting HSV-1 capsids into X. laevis oocytes. Areas indicated by dashed lines in A are shown at higher magnification in panels C and E. On local patches, the NPC rim is found surrounded by various subviral structures with heights between 20 and 130 nm (B), 15% of which obviously represent capsids (120-130 nm in diameter) (C,D). The remaining viral structures (C,E) exhibit heights between 20 and 120 nm. It is striking that approximately 40% of all viral substructures seen at the cytoplasmic side of the nuclear envelope had a uniform, rod-like shape, with a diameter of 35-40 nm and an estimated length of 130-160 nm or more (F).

View larger version (59K): [in a new window]   Fig. 5. Representative AFM images of the nucleoplasmic face of the nuclear envelope 30 minutes after injecting HSV-1 capsids into X. laevis oocytes. Areas indicated by dashed lines in A are shown at higher magnification in panels C and E. Only the rod-shaped subviral structures, also found at the cytoplasmic face of the nuclear envelope, with a diameter of 35-40 nm are visualized at the nucleoplasmic side, indicating that other subviral structures were excluded from the translocation (B-F).

View larger version (56K): [in a new window]   Fig. 6. Analysis of the behavior of recorded force-distance curves of given points (white or black circles) on each AFM-scanned sample surface, that correspond to either NPCs (C,D) or HSV-1 capsids (A,B) and rod-like structures (E,F) (HSV-1 genome).

View larger version (36K): [in a new window]   Fig. 7. Proposed model for nuclear delivery of the HSV-1 genome through NPCs. NE, nuclear envelope.