SV40 assembles in the nucleus by addition of capsid proteins to the minichromosome. The VPl(5)VP2/3 capsomer is composed of a pentamer of the major protein VPl complexed with a monomer of a minor protein, VP2 or VP3. In the capsid, the capsomers are bound together via their flexible carboxy-terminal arms. Our previous studies suggested that the capsomers are recruited to the packaging signal ses via avid interaction with Spl. During assembly Spl is displaced, allowing chromatin compaction. Here we investigated the interactions in vitro of VPl(5)VP2/3 capsomers with the entire SV40 genome, using mutant VPl deleted in the carboxy-arm that cannot assemble, but retains DNA-binding capacity. EM revealed that VPl(5)VP2/3 complexes bind non-specifically at random locations around the DNA. Spl was absent from mature virions. The findings suggest that multiple capsomers attach simultaneously to the viral genome, increasing their local concentration, facilitating rapid, concerted assembly reaction and removal of Spl. (c) 2006 Elsevier Inc. All rights reserved.