In vitro assembly of Sindbis virus core-like particles from cross-linked dimers of truncated and mutant capsid proteins

A nucleic acid-hound capsid protein dimer was previously identified using a Sindbis virus in vitro nucleocapsid assembly system and cross-linking reag ents. Cross-link mapping, in combination with a model of the nucleocapsid c ore, suggested that this dimer contained one monomer from each of two adjac ent capsomeres. This intercapsomere dimer is believed to be the initial int ermediate in the nucleocapsid core assembly mechanism. This paper presents the purification of cross-linked dimers of a truncated capsid protein and t he partial purification of cross-linked dimers of a full-length assembly-de fective mutant. The assembly of core-like particles from these cross-linked capsid protein dimers is demonstrated. Core-like particles generated from cross-linked full-length mutant CP(19-264)L52D were examined by electron mi croscopy and appeared to have a morphology similar to that of wild-type in vitro-assembled core-like particles, although a slight size difference was often visible. Truncated cross-linked CP(81-264) dimers generated core-like particles as well. These core-like particles could subsequently be disasse mbled when reversible cross-linking reagents were used to form the dimers. The ability of the covalent intercapsomere cross-link to rescue capsid prot eins with assembly defects or truncations in the amino-terminal region of t he capsid protein supports the previous model of assembly and suggests a po ssible role for the amino-terminal region of the protein.