In vitro assembly of infectious virions of double-stranded DNA phage f29 from cloned gene products and synthetic nucleic acids

Up to 6 3 107 PFU of infectious virions of the double-stranded DNA bacteriophage f29 per ml were assembled in vitro, with 11 proteins derived from cloned genes and nucleic acids synthesized separately. The genomic DNA-gp3 protein conjugate was efficiently packaged into a purified recombinant procapsid with the aid of a small viral RNA (pRNA) transcript, a DNA-packaging ATPase (gp16), and ATP. The DNA-filled capsids were subsequently converted into infectious virions after the addition of four more recombinant proteins for neck and tail assembly. Electron microscopy and genome restriction mapping confirmed the identity of the infectious f29 virions synthesized in this system. A nonstructural protein, gp13, was indispens-able for the assembly of infectious virions. The overproduced tail protein gp9 was present in solution in mostly dimeric form and was purified to homogeneity. The purified gp9 was biologically active for in vitro f29 assembly. Higher-order concentration dependence of in vitrof29 assembly on gp9 suggests that a complete tail did not form before attaching to the DNA-filled capsid, a result contrary to earlier findings for phages T4 and l. The work described here constitutes an extremely sensitive assay system for the analysis of components in f29 assembly and dissection of functional domains of structural components, enzymes, and pRNA (C.-S. Lee and P. Guo, Virology 202:1039–1042, 1995). Efficient packaging of foreign DNA in vitro and synthesis of viral particles from recombinant proteins facilitate the development of f29 as an in vivo gene delivery system. Th