The polyomavirus coat protein viral protein 1 (VP1) has the intrinsic ability to self-assemble in vitro into polymorphic capsid-like structures on addition of calcium. In contrast, polyomavirus assembly in vivo is rigorously controlled, such that virions of uniform size are formed only in the cell nucleus. During viral infection, the 72 kDa cellular chaperone heat shock cognate protein (hsc70) binds VP1 posttranslation and colocalizes with VP1 to the nucleus, thereby suggesting a role for Ϸ70-kDa heat shock protein (hsp70) family chaperones in regulating the quality and location of capsid assembly. We found that, after expression of recombinant VP1 in Escherichia coli, the prokaryotic hsp70 chaperone DnaK copurified with the VP1 C-terminal domain that links pentamers in an assembled capsid. When stably bound to VP1, DnaK inhibited in vitro assembly induced by calcium. However, in the presence of ATP, the hsp70 chaperone system comprised of DnaK, DnaJ, and GrpE assembled VP1 into uniform capsids without requiring calcium. Chaperonemediated assembly was similarly catalyzed by the eukaryotic hsc70 protein, in combination with the J-domain function of the simian virus 40 large T-antigen protein. Thus, polyomavirus capsid assembly can be recapitulated with high-fidelity in vitro using either prokaryotic or eukaryotic hsp70 chaperone systems, thereby supporting a role for cellular chaperones in the in vivo regulation of virion assembly. The Polyomaviridae family of small, nonenveloped, icosahedral DNA viruses includes murine polyomavirus and simian virus 40 (SV40). Like most DNA viruses, polyomavirus capsid proteins are synthesized in the cytosol, whereas assembly of virions occurs only in the nucleus. Polyomavirus capsids are comprised of 72 pentamers (capsomeres) of the major capsid viral protein (VP1), which is arranged in a T ϭ 7 icosahedral lattice Ϸ50 nm in diameter (1, 2). One minor capsid protein, either VP2 or VP3, binds in the central 5-fold cavity of each VP1 pentamer (3). The atomic structure of the virion reveals that the C-terminal domain of each VP1 monomer ''invades'' a neighboring pentamer to form the principal interpentamer contacts, and these contacts are stabilized by calcium ions (2). Consistent with the structural data, a recombinant VP1 protein lacking 63 amino acids from the C terminus forms pentamers that are incapable of assembly (4). In vitro capsid assembly of full-length recombinant VP1 pentamers incubated with calcium or at high ionic strength is robust but frequently yields polymorphic assemblies of sizes that are disparate from authentic capsids (5, 6). In contrast to calcium-mediated assembly in vitro, expression of recombinant VP1 in eukaryotic cells results in the formation of uniform virus-like particles (VLPs), exclusively in the cell nucleus (7).Cellular Ϸ70-kDa heat shock protein (hsp70) family chaperones bind newly synthesized proteins and escort the unfolded domains until protein folding is completed (reviewed in ref. 8). The constitutively expressed eukaryotic chaperone Ϸ70-kD...

Hsp70 chaperones play a role in polyoma-and papillomavirus assembly, as evidenced by their interaction in vivo with polyomavirus capsid proteins at late times after virus infection and by their ability to assemble viral capsomeres into capsids in vitro. We studied whether Hsp70 chaperones might also participate in the uncoating reaction. In vivo, Hsp70 coimmunoprecipitated with polyomavirus virion VP1 at 3 h after infection of mouse cells. In vitro, prokaryotic and eukaryotic Hsp70 chaperones efficiently disassembled polyoma-and papillomavirus-like particles and virions in energy-dependent reactions. These observations support a role for cell chaperones in the disassembly of these viruses.The Papillomaviridae and Polyomaviridae are small, nonenveloped DNA viruses that have structurally similar 50-to 55-nm-diameter capsids comprised of 72 capsomeres arranged in a T ϭ 7 icosahedral lattice (2). Each capsomere is a pentamer of the major capsid protein: L1 for papillomavirus and VP1 for polyomavirus. The carboxy terminus of L1 or VP1 mediates interpentameric contacts in the assembled capsid (13,16,17,22). These contacts are stabilized by disulfide bonds for papillomaviruses (15,21,29) or by both disulfide bonds and calcium bridges for polyomaviruses (4,5,14).