SEQUENTIAL INTERACTIONS OF STRUCTURAL PROTEINS IN PHAGE PHI-29 PROCAPSID ASSEMBLY

The mechanism of viral capsid assembly is an intriguing problem becaus e of its fundamental importance to research on synthetic viral particl e vaccines, gene delivery systems, antiviral drugs, chimeric viruses d isplaying antigens or ligands, and the study of macromolecular interac tions. The genes coding for the scaffolding (gp7), capsid (gp8), and p ortal vertex (gp10) proteins of the procapsid of bacteriophage phi 29 of Bacillus subtilis were expressed in Escherichia coli individually o r in combination to study the mechanism of phi 29 procapsid assembly. When expressed alone, gp7 existed as a soluble monomer, gp8 aggregated into inclusion bodies, and gp10 formed the portal vertex Circular dic hroisin spectrum analysis indicated that gp7 is mainly composed of at helices. When two of the proteins were coexpressed, gp7 and gp8 assemb led into procapsid-like particles with variable sizes and shapes, gp7 and gp10 formed unstable complexes, and gp8 and gp10 did not interact. These results suggested that gp7 served as a bridge for gp8 and gp10. When gp7, gp8, and gp10 were coexpressed, active procapsids were prod uced. Complementation of extracts containing one or two structural com ponents could not produce active procapsids, indicating that no stable intermediates were formed. A dimeric gp7 concatemer promoted the solu bility of gp8 but was inactive in the assembly of procapsid or procaps id-like particles. Mutation at the C terminus of gp7 prevented it from interacting with gp8, indicating that this part of gp7 may be importa nt for interaction with gp8. Coexpression of the portal protein (gp20) of phage T4 with phi 29 gp7 and gp8 revealed the lack of interaction between T4 gp20 and phi 29 gp7 and/or gp8. Perturbing the ratio of the three structural proteins by duplicating one or another gene did not reduce the yield of potentially infectious particles. Changing of the order of gene arrangement in plasmids did not affect the formation of active procapsids significantly. These results indicate that phi 29 pr ocapsid assembly deviated from the single-assembly pathway and that co existence of all three components with a threshold concentration was r equired for procapsid assembly. The trimolecular interaction was so ra pid that no true intermediates could be isolated. This finding is in a ccord with the result of capsid assembly obtained by the equilibrium m odel proposed by A. Zlotnick (J. Mol. Biol. 241:59-67, 1994).