Nothing Special   »   [go: up one dir, main page]

Skip to main content
Journal of Virology logoLink to Journal of Virology
. 1995 Aug;69(8):5018–5023. doi: 10.1128/jvi.69.8.5018-5023.1995

In vitro assembly of infectious virions of double-stranded DNA phage phi 29 from cloned gene products and synthetic nucleic acids.

C S Lee 1, P Guo 1
PMCID: PMC189318  PMID: 7609071

Abstract

Up to 6 x 10(7) PFU of infectious virions of the double-stranded DNA bacteriophage phi 29 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 phi 29 virions synthesized in this system. A nonstructural protein, gp13, was indispensable 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 phi 29 assembly. Higher-order concentration dependence of in vitro phi 29 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 lambda. The work described here constitutes an extremely sensitive assay system for the analysis of components in phi 29 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 phi 29 as an in vivo gene delivery system. The finding that purified tail protein was able to incorporate into infectious virions might allow the construction of chimeric phi 29 carrying a tail fused to ligands for specific receptor of human cells.

Full Text

The Full Text of this article is available as a PDF (689.3 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Abuladze N. K., Gingery M., Tsai J., Eiserling F. A. Tail length determination in bacteriophage T4. Virology. 1994 Mar;199(2):301–310. doi: 10.1006/viro.1994.1128. [DOI] [PubMed] [Google Scholar]
  2. Aoyama A., Hamatake R. K., Hayashi M. In vitro synthesis of bacteriophage phi X174 by purified components. Proc Natl Acad Sci U S A. 1983 Jul;80(14):4195–4199. doi: 10.1073/pnas.80.14.4195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bazinet C., King J. The DNA translocating vertex of dsDNA bacteriophage. Annu Rev Microbiol. 1985;39:109–129. doi: 10.1146/annurev.mi.39.100185.000545. [DOI] [PubMed] [Google Scholar]
  4. Beckett D., Wu H. N., Uhlenbeck O. C. Roles of operator and non-operator RNA sequences in bacteriophage R17 capsid assembly. J Mol Biol. 1988 Dec 20;204(4):939–947. doi: 10.1016/0022-2836(88)90053-8. [DOI] [PubMed] [Google Scholar]
  5. Bjornsti M. A., Reilly B. E., Anderson D. L. In vitro assembly of the Bacillus subtilis bacteriophage phi 29. Proc Natl Acad Sci U S A. 1981 Sep;78(9):5861–5865. doi: 10.1073/pnas.78.9.5861. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bjornsti M. A., Reilly B. E., Anderson D. L. Morphogenesis of bacteriophage phi 29 of Bacillus subtilis: oriented and quantized in vitro packaging of DNA protein gp3. J Virol. 1983 Jan;45(1):383–396. doi: 10.1128/jvi.45.1.383-396.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Black L. W. DNA packaging in dsDNA bacteriophages. Annu Rev Microbiol. 1989;43:267–292. doi: 10.1146/annurev.mi.43.100189.001411. [DOI] [PubMed] [Google Scholar]
  8. Black L. W. In vitro packaging of bacteriophage T4 DNA. Virology. 1981 Aug;113(1):336–344. doi: 10.1016/0042-6822(81)90160-4. [DOI] [PubMed] [Google Scholar]
  9. Blanco L., Bernad A., Lázaro J. M., Martín G., Garmendia C., Salas M. Highly efficient DNA synthesis by the phage phi 29 DNA polymerase. Symmetrical mode of DNA replication. J Biol Chem. 1989 May 25;264(15):8935–8940. [PubMed] [Google Scholar]
  10. Blanco L., Lázaro J. M., de Vega M., Bonnin A., Salas M. Terminal protein-primed DNA amplification. Proc Natl Acad Sci U S A. 1994 Dec 6;91(25):12198–12202. doi: 10.1073/pnas.91.25.12198. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Bowden D. W., Calendar R. Maturation of bacteriophage P2 DNA in vitro: A complex, site-specific system for DNA cleavage. J Mol Biol. 1979 Mar 25;129(1):1–18. doi: 10.1016/0022-2836(79)90055-x. [DOI] [PubMed] [Google Scholar]
  12. Dube P., Tavares P., Lurz R., van Heel M. The portal protein of bacteriophage SPP1: a DNA pump with 13-fold symmetry. EMBO J. 1993 Apr;12(4):1303–1309. doi: 10.1002/j.1460-2075.1993.tb05775.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fraenkel-Conrat H., Williams R. C. RECONSTITUTION OF ACTIVE TOBACCO MOSAIC VIRUS FROM ITS INACTIVE PROTEIN AND NUCLEIC ACID COMPONENTS. Proc Natl Acad Sci U S A. 1955 Oct 15;41(10):690–698. doi: 10.1073/pnas.41.10.690. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gaddipati J. P., Siegel A. Study of TMV assembly with heterologous RNA containing the origin-of-assembly sequence. Virology. 1990 Feb;174(2):337–344. doi: 10.1016/0042-6822(90)90087-8. [DOI] [PubMed] [Google Scholar]
  15. Grimes S., Anderson D. In vitro packaging of bacteriophage phi 29 DNA restriction fragments and the role of the terminal protein gp3. J Mol Biol. 1989 Sep 5;209(1):91–100. doi: 10.1016/0022-2836(89)90172-1. [DOI] [PubMed] [Google Scholar]
  16. Guo P. X., Bailey S., Bodley J. W., Anderson D. Characterization of the small RNA of the bacteriophage phi 29 DNA packaging machine. Nucleic Acids Res. 1987 Sep 11;15(17):7081–7090. doi: 10.1093/nar/15.17.7081. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Guo P. X., Erickson S., Anderson D. A small viral RNA is required for in vitro packaging of bacteriophage phi 29 DNA. Science. 1987 May 8;236(4802):690–694. doi: 10.1126/science.3107124. [DOI] [PubMed] [Google Scholar]
  18. Guo P. X., Erickson S., Xu W., Olson N., Baker T. S., Anderson D. Regulation of the phage phi 29 prohead shape and size by the portal vertex. Virology. 1991 Jul;183(1):366–373. doi: 10.1016/0042-6822(91)90149-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Guo P. X., Rajagopal B. S., Anderson D., Erickson S., Lee C. S. sRNA of phage phi 29 of Bacillus subtilis mediates DNA packaging of phi 29 proheads assembled in Escherichia coli. Virology. 1991 Nov;185(1):395–400. doi: 10.1016/0042-6822(91)90787-c. [DOI] [PubMed] [Google Scholar]
  20. Guo P., Grimes S., Anderson D. A defined system for in vitro packaging of DNA-gp3 of the Bacillus subtilis bacteriophage phi 29. Proc Natl Acad Sci U S A. 1986 May;83(10):3505–3509. doi: 10.1073/pnas.83.10.3505. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Guo P., Peterson C., Anderson D. Initiation events in in-vitro packaging of bacteriophage phi 29 DNA-gp3. J Mol Biol. 1987 Sep 20;197(2):219–228. doi: 10.1016/0022-2836(87)90120-3. [DOI] [PubMed] [Google Scholar]
  22. Guo P., Peterson C., Anderson D. Prohead and DNA-gp3-dependent ATPase activity of the DNA packaging protein gp16 of bacteriophage phi 29. J Mol Biol. 1987 Sep 20;197(2):229–236. doi: 10.1016/0022-2836(87)90121-5. [DOI] [PubMed] [Google Scholar]
  23. Hagen E. W., Reilly B. E., Tosi M. E., Anderson D. L. Analysis of gene function of bacteriophage phi 29 of Bacillus subtilis: identification of cistrons essential for viral assembly. J Virol. 1976 Aug;19(2):501–517. doi: 10.1128/jvi.19.2.501-517.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Hohn T. Packaging of genomes in bacteriophages: a comparison of ssRNA bacteriophages and dsDNA bacteriophages. Philos Trans R Soc Lond B Biol Sci. 1976 Nov 30;276(943):143–150. doi: 10.1098/rstb.1976.0105. [DOI] [PubMed] [Google Scholar]
  25. Hohn T., Wurtz M., Hohn B. Capsid transformation during packaging of bacteriophage lambdaDNA. Philos Trans R Soc Lond B Biol Sci. 1976 Nov 30;276(943):51–61. doi: 10.1098/rstb.1976.0097. [DOI] [PubMed] [Google Scholar]
  26. Kaiser D., Masuda T. In vitro assembly of bacteriophage Lambda heads. Proc Natl Acad Sci U S A. 1973 Jan;70(1):260–264. doi: 10.1073/pnas.70.1.260. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Katsura I. Determination of bacteriophage lambda tail length by a protein ruler. Nature. 1987 May 7;327(6117):73–75. doi: 10.1038/327073a0. [DOI] [PubMed] [Google Scholar]
  28. Kerr C., Sadowski P. D. Packaging and maturation of DNA of bacteriophage T7 in vitro. Proc Natl Acad Sci U S A. 1974 Sep;71(9):3545–3549. doi: 10.1073/pnas.71.9.3545. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kikuchi Y., King J. Genetic control of bacteriophage T4 baseplate morphogenesis. III. Formation of the central plug and overall assembly pathway. J Mol Biol. 1975 Dec 25;99(4):695–716. doi: 10.1016/s0022-2836(75)80180-x. [DOI] [PubMed] [Google Scholar]
  30. Lee C. S., Guo P. A highly sensitive system for the in vitro assembly of bacteriophage phi 29 of Bacillus subtilis. Virology. 1994 Aug 1;202(2):1039–1042. doi: 10.1006/viro.1994.1434. [DOI] [PubMed] [Google Scholar]
  31. Lee C. S., Guo P. Sequential interactions of structural proteins in phage phi 29 procapsid assembly. J Virol. 1995 Aug;69(8):5024–5032. doi: 10.1128/jvi.69.8.5024-5032.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Miyazaki J. I., Fujisawa H., Minagawa T. Biological activity of purified bacteriophage T3 prohead and proheadlike structures as precursors for in vitro head assembly. Virology. 1978 Dec;91(2):283–290. doi: 10.1016/0042-6822(78)90376-8. [DOI] [PubMed] [Google Scholar]
  33. Molla A., Paul A. V., Wimmer E. Cell-free, de novo synthesis of poliovirus. Science. 1991 Dec 13;254(5038):1647–1651. doi: 10.1126/science.1661029. [DOI] [PubMed] [Google Scholar]
  34. Méndez J., Blanco L., Esteban J. A., Bernad A., Salas M. Initiation of phi 29 DNA replication occurs at the second 3' nucleotide of the linear template: a sliding-back mechanism for protein-primed DNA replication. Proc Natl Acad Sci U S A. 1992 Oct 15;89(20):9579–9583. doi: 10.1073/pnas.89.20.9579. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Nelson R. A., Reilly B. E., Anderson D. L. Morphogenesis of bacteriophage phi 29 of Bacillus subtilis: preliminary isolation and characterization of intermediate particles of the assembly pathway. J Virol. 1976 Aug;19(2):518–532. doi: 10.1128/jvi.19.2.518-532.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Olkkonen V. M., Gottlieb P., Strassman J., Qiao X. Y., Bamford D. H., Mindich L. In vitro assembly of infectious nucleocapsids of bacteriophage phi 6: formation of a recombinant double-stranded RNA virus. Proc Natl Acad Sci U S A. 1990 Dec;87(23):9173–9177. doi: 10.1073/pnas.87.23.9173. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Poteete A. R., Jarvik V., Botstein D. Encapsulation of phage P22 DNA in vitro. Virology. 1979 Jun;95(2):550–564. doi: 10.1016/0042-6822(79)90508-7. [DOI] [PubMed] [Google Scholar]
  38. Rosenberg A. H., Lade B. N., Chui D. S., Lin S. W., Dunn J. J., Studier F. W. Vectors for selective expression of cloned DNAs by T7 RNA polymerase. Gene. 1987;56(1):125–135. doi: 10.1016/0378-1119(87)90165-x. [DOI] [PubMed] [Google Scholar]
  39. Salas M. Protein-priming of DNA replication. Annu Rev Biochem. 1991;60:39–71. doi: 10.1146/annurev.bi.60.070191.000351. [DOI] [PubMed] [Google Scholar]
  40. Scholz E., Zhang C., Guo P. Transactivation of the early SV40 promoter by avian infectious laryngotracheitis virus in avian hepatoma cells. J Virol Methods. 1993 Dec 31;45(3):291–301. doi: 10.1016/0166-0934(93)90114-7. [DOI] [PubMed] [Google Scholar]
  41. Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. doi: 10.1016/0076-6879(90)85008-c. [DOI] [PubMed] [Google Scholar]
  42. Tosi M., Anderson D. L. Antigenic properties of bacteriophage phi 29 structural proteins. J Virol. 1973 Dec;12(6):1548–1559. doi: 10.1128/jvi.12.6.1548-1559.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Zhang C., Lee C. S., Guo P. The proximate 5' and 3' ends of the 120-base viral RNA (pRNA) are crucial for the packaging of bacteriophage phi 29 DNA. Virology. 1994 May 15;201(1):77–85. doi: 10.1006/viro.1994.1267. [DOI] [PubMed] [Google Scholar]
  44. Zhang C., Trottier M., Guo P. Circularly permuted viral pRNA active and specific in the packaging of bacteriophage phi 29 DNA. Virology. 1995 Mar 10;207(2):442–451. doi: 10.1006/viro.1995.1103. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES