IN-VITRO ASSEMBLY OF INFECTIOUS VIRIONS OF DOUBLE-STRANDED DNA-PHAGE PHI-29 FROM CLONED GENE-PRODUCTS AND SYNTHETIC NUCLEIC-ACIDS

Up to 6 x 10(7) PFU of infectious virions of the double-stranded DNA b acteriophage phi 29 per mi were assembled in vitro, with 11 proteins d erived from cloned genes and nucleic acids synthesized separately. The genomic DNA-gp3 protein conjugate was efficiently packaged into a pur ified recombinant procapsid with the aid of a small viral RNA (pRNA) t ranscript, a DNA-packaging ATPase (gp16), and ATP. The DNA-filled caps ids were subsequently converted into infectious virions after the addi tion of four more recombinant proteins for neck and tail assembly. Ele ctron microscopy and genome restriction mapping confirmed the identity of the infectious phi 29 virions synthesized in this system. A nonstr uctural protein, gp13, was indispensable for the assembly of infectiou s virions. The overproduced tail protein gp9 was present in solution i n mostly dimeric form and was purified to homogeneity. The purified gp 9 was biologically active for in vitro phi 29 assembly. Higher-order c oncentration dependence of in vitro phi 29 assembly on gp9 suggests th at a complete tail did not form before attaching to the DNA-filled cap sid, a result contrary to earlier findings for phages T4 and X. The wo rk described here constitutes an extremely sensitive assay system for the analysis of components in phi 29 assembly and dissection of functi onal domains of structural components, enzymes, and pRNA (C.-S. Lee an d P. Guo, Virology 202:1039-1042, 1995). Efficient packaging of foreig n DNA in vitro and synthesis of viral particles from recombinant prote ins facilitate the development of phi 29 as an in vive gene delivery s ystem. The finding that purified tail protein was able to incorporate into infectious virions might allow the construction of chimeric phi 2 9 carrying a tail fused to ligands for specific receptor of human cell s.