In vitro assembly of infectious virions of the double-stranded DNA bacteriophage phi20 exclusively from cloned gene products and synthetic pRNA
Abstract
Viral assembly has attracted increasing attention due to its fundamental importance to researches on viral vaccines, gene delivery, anti-viral drugs, chimeric virus displaying antigens or ligands, and the mechanism of macromolecular interactions. A system for the in vitro assembly of zero to 6 $\times\ 10\sp7$ plaque-forming units per ml infectious virions of phage $\phi$29 was developed with synthetic nucleic acids and eleven proteins from cloned genes. The high efficiency of assembly with no background constitutes a sensitive assay for structural components, enzymes, and pRNA in $\phi$29 assembly. DNA-gp3 concentration dependence in assembly was first order, while gp16 and tail protein gp9 were higher order; indicating one DNA, but multiple copies of gp16 and gp9 are required for the assembly of one virion. Higher order concentration dependence on purified gp9 suggests that a complete tail did not form before attaching to the DNA-filled capsid, a result contrary to the findings in phages T4 and $\lambda.$ The non-structural protein gp13 was required for assembly. The sequential interaction of structural proteins in procapsid assembly was also examined. When expressed alone, scaffolding protein (gp7) was a monomer, capsid protein (gp8) aggregated, and portal protein (gp10) formed the portal vertex. When two components were co-expressed, gp7 and gp8 assembled into procapsid-like particles with variable size and shape, gp7 and gp10 formed unstable complexes, and gp8 and gp10 did not interact. The results suggested that gp7 served as a bridge for gp8 and gp10. When gp7, gp8 and gp10 were co-expressed, active procapsids were produced. Complementation of one or two structural components could not produce active procapsids. Dimeric gp7 promoted the solubility of gp8, but was inactive in procapsid assembly. Mutation at C-terminus of gp7 abolished the interaction with gp8. Co-expression of the portal protein of phage T4 with $\phi$29 gp7 and gp8 revealed the lack of interaction between T4 portal protein and $\phi$29 gp7 and/or gp8. Alteration of temporal expression or unbalanced overproduction of structural proteins did not affect the rate of procapsid assembly. These results indicate that $\phi$29 procapsid assembly deviates from the single assembly pathway and that the co-existence of all three components with a threshold concentration would be required for procapsid assembly. The triple interaction was so rapid that no true intermediates could be isolated.
Degree
Ph.D.
Advisors
Guo, Purdue University.
Subject Area
Molecular biology|Microbiology
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