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The SV40 capsid is composed primarily of 72 pentamers of the VP1 major capsid protein. Although the capsid also contains the minor capsid protein VP2 and its amino-terminally truncated form VP3, their roles in capsid assembly remain unknown. An in vitro assembly system was used to investigate the role of VP2 in the assembly of recombinant VP1 pentamers. Under physiological salt and pH conditions, VP1 alone remained dissociated, and at pH 5.0, it assembled into tubular structures. A stoichiometric amount of VP2 allowed the assembly of VP1 pentamers into spherical particles in a pH range of 7.0 to 4.0. Electron microscopy observation, sucrose gradient sedimentation analysis, and antibody accessibility tests showed that VP2 is incorporated into VP1 particles. The functional domains of VP2 important for VP1 binding and for enhancing VP1 assembly were further explored with a series of VP2 deletion mutants. VP3 also enhanced VP1 assembly, and a region common to VP2 and VP3 (amino acids 119 -272) was required to promote VP1 pentamer assembly. These results are relevant for controlling recombinant capsid formation in vitro, which is potentially useful for the in vitro development of SV40 virus vectors. Viral capsids are highly organized protein complexes that assemble and disassemble depending on environmental conditions during the viral life cycle. An elucidation of the mechanisms of assembly and disassembly of viral capsids may greatly help in understanding these highly organized protein complexes.SV40 is a small, nonenveloped DNA tumor virus that belongs to the family Polyomaviridae. Its capsid is formed by 72 copies of pentamers of the VP1 major capsid protein (360 molecules in total) and 72 molecules of the VP2 or VP3 minor capsid proteins. VP1 pentamers are arranged in a T ϭ 7d icosahedral lattice, and the carboxyl-terminal arm of VP1 mediates inter-pentameric contacts that hold the capsid together (1-4). VP3 is an amino-terminally truncated form of VP2. One molecule of either minor capsid protein binds to the center of a VP1 pentamer through their common carboxyl-terminal region inside the virion (5-7).However, the precise molecular mechanism for virion assembly is not known.We and others have shown that the VP1 proteins of SV40 (8 -12) and of closely related viruses such as JC virus (13, 14), murine polyomavirus (8,(15)(16)(17)(18)(19), and papillomavirus (20, 21) form virus-like particles (VLPs) 2 when expressed in insect Sf9 cells from baculoviral vectors. The properties of VP1-VLPs are very similar to those of wild type virions in that they dissociate into pentamers following treatment with a calcium-chelating agent (EGTA) under reducing conditions in vitro (22-24). Previously, we prepared highly purified SV40 VP1 pentamers (9, 25, 26), which assembled into morphologically heterogeneous particles under various nonphysiological conditions (26). Such particles include T ϭ 7d (triangulation number 7 dextro) spherical particles, small T ϭ 1 (triangulation number 1) particle composed of 12 pentamers, and long tubu...

The simian virus 40 (SV40) particle is mainly composed of the major capsid protein termed VP1. VP1 self-assembles into virus-like particles (VLPs) of approximately 40 nm in diameter when over-expressed in bacteria or in insect cells, but purified VP1 does not form such a structure under physiological conditions, and thus, the mechanism of VP1 assembly is not well understood. Using a highly purified VP1 assembly/disassembly system in vitro , here we provide evidence that DNA is a factor that contributes to VP1 assembly into 40-nm spherical particles. At pH 5, for example, VP1 preferentially assembles into 40-nm particles in the presence of DNA, whereas VP1 assembles into tubular structures in the absence of DNA. Electron microscopic observations revealed that the concentration of DNA and its length are important for the formation of 40-nm particles. In addition, sucrose gradient sedimentation analysis and DNase I-sensitivity assays indicated that DNA of up to 2000 bp is packaged into the 40-nm particles under the conditions examined. We propose that DNA may facilitate the formation of 40-nm spherical particles by acting as a scaffold that increases the local concentration of VP1 and/or by acting as an allosteric effector that alters the structure of VP1.

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