Herpes simplex virus type 1 (HSV1) capsids undergo extensive structural changes during maturation and DNA packaging. As a result, they become more stable and competent for nuclear egress. To further elucidate this stabilization process, we used biochemical and nanoindentation approaches to analyze the structural and mechanical properties of scaffold-containing (B), empty (A), and DNA-containing (C) nuclear capsids. Atomic force microscopy experiments revealed that A and C capsids were mechanically indistinguishable, indicating that the presence of DNA does not account for changes in mechanical properties during capsid maturation. Despite having the same rigidity, the scaffold-containing B capsids broke at significantly lower forces than A and C capsids. An extraction of pentons with guanidine hydrochloride (GuHCl) increased the flexibility of all capsids. Surprisingly, the breaking forces of the modified A and C capsids dropped to similar values as those of the GuHCl-treated B capsids, indicating that mechanical reinforcement occurs at the vertices. Nonetheless, it also showed that HSV1 capsids possess a remarkable structural integrity that was preserved after removal of pentons. We suggest that HSV1 capsids are stabilized after removal of the scaffold proteins, and that this stabilization is triggered by the packaging of DNA, but independent of the actual presence of DNA.atomic force microscopy ͉ nanoindentation ͉ penton ͉ viral structure ͉ virus mechanics H erpes simplex virus type 1 (HSV1) virions encapsidate their 152 kbp double-stranded DNA genome in an icosahedral capsid that is surrounded by an amorphous protein layer, called the tegument, and a lipid-containing envelope. Assembly of herpesviruses is initiated in the nucleus where procapsids selfassemble around a protein scaffold and subsequently mature (1-4). This transformation is characterized by massive conformational changes of the Ϸ200 MDa shell, resulting in stable, mature capsids, and is in many aspects analogous to maturation processes in bacteriophages (5, 6). The scaffold is proteolytically cleaved and removed, whereas the outer shell transforms from a spherical into an icosahedral shape (7, 8). The mature capsid has an outer diameter of 125 nm with an overall shell thickness of Ϸ15 nm (9). Capsomeres converge at their proximal ends, forming a contiguous shell of Ϸ4 nm thickness that is only interrupted by channels passing through all of the 162 capsomeres (10, 11). The capsomeres are hexamers (hexons) and pentamers (pentons) of the major capsid protein VP5 that form a shell with a triangulation number of T ϭ 16. In total, 320 triplexes, each formed by a heterotrimer of 1 VP19c and 2 VP23 molecules, connect these capsomeres (10). One of the 12 capsid vertices is occupied by the pUL6 portal (12). Three particle types can be isolated from the nuclei of infected cells due to their different sedimentation behavior: B capsids, which still have the scaffold inside; the lighter A capsids, which are empty; and the denser C capsids, which contain the DN...

virions. In the topographies the protineaceous features of symmetries 5, 3 and 2 are visualized. Afterwards we performed nanoindentation experiments in T7 mature capsids and virions. Preliminary experiments indicate that the DNA plays a structural role within the capsid, since virions present different mechanical properties than empty capsids.