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. 1997 Jan;71(1):487–494. doi: 10.1128/jvi.71.1.487-494.1997

Approaches to determine stoichiometry of viral assembly components.

M Trottier 1, P Guo 1
PMCID: PMC191076  PMID: 8985375

Abstract

Due to the rapidity of biological reactions, it is difficult to isolate intermediates or to determine the stoichiometry of participants in intermediate reactions. Instead of determining the absolute amount of each component, this study involved the use of relative parameters, such as dilution factors, percentages probabilities, and slopes of titration curves, that can be more accurately quantified to determine the stoichiometry of components involved in bacteriophage phi29 assembly. This work takes advantage of the sensitive in vitro phage phi29 assembly system, in which 10(8) infectious virions per ml without background can be assembled from eight purified components. It provides a convenient assay for quantification of the stoichiometry of packaging components, including the viral procapsid, genomic DNA, DNA-packaging pRNA, and other structural proteins and enzymes. The presence of a procapsid binding domain and another essential functional domain within the pRNA makes it an ideal component for constructing lethal mutants for competitive procapsid binding. Two methods were used for stoichiometry determination. Method 1 was to determine the combination probability of mutant and wild-type pRNAs bound to procapsids. The probability of procapsids that possess a certain amount of mutant and a certain amount of wild-type pRNA, both with an equal binding affinity, was predicted with the binomial equation [EQUATION IN TEXT] where Z is the total number of pRNAs per procapsid, M is the number of mutant pRNAs bound to one procapsid, and (ZM) is equal to [FORMULA IN TEXT]. With various ratios of mutant to wild-type pRNA in in vitro viral assembly, the percent mutant pRNA versus the yield of virions was plotted and compared to a series of predicted curves to find a best fit. It was determined that five or six copies of pRNA were required for one DNA-packaging event, while only one mutant pRNA per procapsid was sufficient to block packaging. Method 2 involved the comparison of slopes of curves of dilution factors versus the yield of virions. Components with known stoichiometries served as standard controls. The larger the stoichiometry of the component, the more dramatic the influence of the dilution factor on the reaction. A slope of 1 indicates that one copy of the component is involved in the assembly of one virion. A slope larger than 1 would indicate multiple-copy involvement. By this method, the stoichiometry of gp11 in phi29 particles was determined to be approximately 12. These approaches are useful for the determination of the stoichiometry of functional units involved in viral assembly, be they single molecules or oligomers. However, these approaches are not suitable for the determination of exact copy numbers of individual molecules involved if the functional unit is composed of multiple subunits prior to assembly.

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Selected References

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  1. Anderson D. L., Hickman D. D., Reilly B. E. Structure of Bacillus subtilis bacteriophage phi 29 and the length of phi 29 deoxyribonucleic acid. J Bacteriol. 1966 May;91(5):2081–2089. doi: 10.1128/jb.91.5.2081-2089.1966. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Anderson D., Bodley J. W. Role of RNA in bacteriophage phi 29 DNA packaging. J Struct Biol. 1990 Jul-Sep;104(1-3):70–74. doi: 10.1016/1047-8477(90)90059-l. [DOI] [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. Becker A., Marko M., Gold M. Early events in the in vitro packaging of bacteriophage lambda DNA. Virology. 1977 May 1;78(1):291–305. doi: 10.1016/0042-6822(77)90100-3. [DOI] [PubMed] [Google Scholar]
  5. Bjornsti M. A., Reilly B. E., Anderson D. L. Bacteriophage phi 29 proteins required for in vitro DNA-gp3 packaging. J Virol. 1984 Jun;50(3):766–772. doi: 10.1128/jvi.50.3.766-772.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Chen C., Guo P. Magnesium-induced conformational change of packaging RNA for procapsid recognition and binding during phage phi29 DNA encapsidation. J Virol. 1997 Jan;71(1):495–500. doi: 10.1128/jvi.71.1.495-500.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cohen F. E., Abarbanel R. M., Kuntz I. D., Fletterick R. J. Turn prediction in proteins using a pattern-matching approach. Biochemistry. 1986 Jan 14;25(1):266–275. doi: 10.1021/bi00349a037. [DOI] [PubMed] [Google Scholar]
  9. Coombs D. H., Pearson G. D. Filter-binding assay for covalent DNA-protein complexes: adenovirus DNA-terminal protein complex. Proc Natl Acad Sci U S A. 1978 Nov;75(11):5291–5295. doi: 10.1073/pnas.75.11.5291. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Grimes S., Anderson D. RNA dependence of the bacteriophage phi 29 DNA packaging ATPase. J Mol Biol. 1990 Oct 20;215(4):559–566. doi: 10.1016/s0022-2836(05)80168-8. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. 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]
  14. 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]
  15. 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]
  16. 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]
  17. 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]
  18. Israel J. V., Anderson T. F., Levine M. in vitro MORPHOGENESIS OF PHAGE P22 FROM HEADS AND BASE-PLATE PARTS. Proc Natl Acad Sci U S A. 1967 Feb;57(2):284–291. doi: 10.1073/pnas.57.2.284. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Lee C. S., Guo P. In vitro assembly of infectious virions of double-stranded DNA phage phi 29 from cloned gene products and synthetic nucleic acids. J Virol. 1995 Aug;69(8):5018–5023. doi: 10.1128/jvi.69.8.5018-5023.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. Murialdo H. Bacteriophage lambda DNA maturation and packaging. Annu Rev Biochem. 1991;60:125–153. doi: 10.1146/annurev.bi.60.070191.001013. [DOI] [PubMed] [Google Scholar]
  23. Prevelige P. E., Jr, Thomas D., King J. Scaffolding protein regulates the polymerization of P22 coat subunits into icosahedral shells in vitro. J Mol Biol. 1988 Aug 20;202(4):743–757. doi: 10.1016/0022-2836(88)90555-4. [DOI] [PubMed] [Google Scholar]
  24. Reid R. J., Bodley J. W., Anderson D. Characterization of the prohead-pRNA interaction of bacteriophage phi 29. J Biol Chem. 1994 Feb 18;269(7):5157–5162. [PubMed] [Google Scholar]
  25. Thomas C. A., Jr, Saigo K., McLeod E., Ito J. The separation of DNA segments attached to proteins. Anal Biochem. 1979 Feb;93(1):158–166. [PubMed] [Google Scholar]
  26. Thomas D., Prevelige P., Jr A pilot protein participates in the initiation of P22 procapsid assembly. Virology. 1991 Jun;182(2):673–681. doi: 10.1016/0042-6822(91)90608-e. [DOI] [PubMed] [Google Scholar]
  27. Trottier M., Zhang C., Guo P. Complete inhibition of virion assembly in vivo with mutant procapsid RNA essential for phage phi 29 DNA packaging. J Virol. 1996 Jan;70(1):55–61. doi: 10.1128/jvi.70.1.55-61.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Tsuprun V., Anderson D., Egelman E. H. The bacteriophage phi 29 head-tail connector shows 13-fold symmetry in both hexagonally packed arrays and as single particles. Biophys J. 1994 Jun;66(6):2139–2150. doi: 10.1016/S0006-3495(94)81009-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Turnquist S., Simon M., Egelman E., Anderson D. Supercoiled DNA wraps around the bacteriophage phi 29 head-tail connector. Proc Natl Acad Sci U S A. 1992 Nov 1;89(21):10479–10483. doi: 10.1073/pnas.89.21.10479. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Villanueva N., Lázaro J. M., Salas M. Purification, properties and assembly of the neck-appendage protein of the Bacillus subtilis phage phi 29. Eur J Biochem. 1981 Jul;117(3):499–505. doi: 10.1111/j.1432-1033.1981.tb06365.x. [DOI] [PubMed] [Google Scholar]
  31. Waterman M. S., Gordon L., Arratia R. Phase transitions in sequence matches and nucleic acid structure. Proc Natl Acad Sci U S A. 1987 Mar;84(5):1239–1243. doi: 10.1073/pnas.84.5.1239. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. White J. H., Cozzarelli N. R., Bauer W. R. Helical repeat and linking number of surface-wrapped DNA. Science. 1988 Jul 15;241(4863):323–327. doi: 10.1126/science.3388041. [DOI] [PubMed] [Google Scholar]
  33. Wichitwechkarn J., Bailey S., Bodley J. W., Anderson D. Prohead RNA of bacteriophage phi 29: size, stoichiometry and biological activity. Nucleic Acids Res. 1989 May 11;17(9):3459–3468. doi: 10.1093/nar/17.9.3459. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Zhang C., Garver K., Guo P. Inhibition of phage phi 29 assembly by antisense oligonucleotides targeting viral pRNA essential for DNA packaging. Virology. 1995 Aug 20;211(2):568–576. doi: 10.1006/viro.1995.1439. [DOI] [PubMed] [Google Scholar]
  35. 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]
  36. Zhang C., Tellinghuisen T., Guo P. Confirmation of the helical structure of the 5'/3' termini of the essential DNA packaging pRNA of phage phi 29. RNA. 1995 Dec;1(10):1041–1050. [PMC free article] [PubMed] [Google Scholar]
  37. 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]
  38. Zuker M. On finding all suboptimal foldings of an RNA molecule. Science. 1989 Apr 7;244(4900):48–52. doi: 10.1126/science.2468181. [DOI] [PubMed] [Google Scholar]

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