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. 1979 Nov;32(2):640–647. doi: 10.1128/jvi.32.2.640-647.1979

In vitro reassembly of infectious polyoma virions.

J N Brady, J D Kendall, R A Consigli
PMCID: PMC353596  PMID: 228081

Abstract

Initial experiments in our laboratory have successfully reassembled infectious polyoma virions from dissociated virion products. Virions treated with ethyleneglycol-bis-N,N'-tetraacetic acid and the reducing agent beta-mercaptoethanol at pH 7.5 were dissociated to a 48S DNA-protein complex and capsomere subunits. The virion dissociation products were not infectious by plaque assay and lacked hemagglutination activity. These virion dissociation products were reassembled to intact virions by overnight dialysis against a reassembly buffer containing CaCl2, dimethyl sulfoxide, and Triton X-100 in phosphate-buffered saline at pH 7.4. The biophysical characteristics of the reassembled virions were identical to those of untreated virions in that the reassembled virions had a sedimentation value of 240S in sucrose gradients and a buoyant density of 1.315 g/cm3 in CsCl isopycnic gradients. The reassembled virions were intact as determined by electron microscopy and were found to be 60% resistant to DNase I treatment. Biologically, the reassembled purified virions were found to partially regain both hemagglutinating activity and plaque-forming ability.

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Selected References

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

  1. Bancroft J. B., Wagner G. W., Bracker C. E. The self-assembly of a nucleic-acid free pseudo-top component for a small spherical virus. Virology. 1968 Sep;36(1):146–149. doi: 10.1016/0042-6822(68)90126-8. [DOI] [PubMed] [Google Scholar]
  2. Brady J. N., Winston V. D., Consigli R. A. Characterization of a DNA-protein complex and capsomere subunits derived from polyoma virus by treatment with ethyleneglycol-bis-N,N'-tetraacetic acid and dithiothreitol. J Virol. 1978 Jul;27(1):193–204. doi: 10.1128/jvi.27.1.193-204.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brady J. N., Winston V. D., Consigli R. A. Dissociation of polyoma virus by the chelation of calcium ions found associated with purified virions. J Virol. 1977 Sep;23(3):717–724. doi: 10.1128/jvi.23.3.717-724.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brunk C. F., Leick V. Rapid equilibrium isopycnic CsC1 gradients. Biochim Biophys Acta. 1969 Mar 18;179(1):136–144. doi: 10.1016/0005-2787(69)90129-4. [DOI] [PubMed] [Google Scholar]
  5. Christensen M., Rachmeler M. Studies on the in vitro formation of infectious DNA-proteins aggregates from SV40 components. Virology. 1976 Dec;75(2):433–441. doi: 10.1016/0042-6822(76)90041-6. [DOI] [PubMed] [Google Scholar]
  6. Consigli R. A., Minocha H. C., Abo-Ahmed H. Multiplication of polyoma virus. II. Source of constituents for viral deoxyribonucleic acid and protein synthesis. J Bacteriol. 1966 Sep;92(3):789–791. doi: 10.1128/jb.92.3.789-791.1966. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Consigli R. A., Zabielski J., Weil R. Plaque assay for polyoma virus on primary mouse kidney cell cultures. Appl Microbiol. 1973 Oct;26(4):627–628. doi: 10.1128/am.26.4.627-628.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Crawford L. V., Gesteland R. F. Synthesis of polyoma proteins in vitro. J Mol Biol. 1973 Mar 15;74(4):627–634. doi: 10.1016/0022-2836(73)90053-3. [DOI] [PubMed] [Google Scholar]
  9. Crawford L. V. Proteins of polyoma virus and SV40. Br Med Bull. 1973 Sep;29(3):253–258. doi: 10.1093/oxfordjournals.bmb.a071016. [DOI] [PubMed] [Google Scholar]
  10. Drzeniek R., Bilello P. Dissociation and reassociation of infectious poliovirus particles. Nat New Biol. 1972 Nov 22;240(99):118–122. doi: 10.1038/newbio240118a0. [DOI] [PubMed] [Google Scholar]
  11. Fey G., Hirt B. Fingerprints of polyoma virus proteins and mouse histones. Cold Spring Harb Symp Quant Biol. 1975;39(Pt 1):235–241. doi: 10.1101/sqb.1974.039.01.030. [DOI] [PubMed] [Google Scholar]
  12. Fraenkel-Conrat H. Reconstitution of viruses. Annu Rev Microbiol. 1970;24:463–478. doi: 10.1146/annurev.mi.24.100170.002335. [DOI] [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. Frearson P. M., Crawford L. V. Polyoma virus basic proteins. J Gen Virol. 1972 Feb;14(2):141–155. doi: 10.1099/0022-1317-14-2-141. [DOI] [PubMed] [Google Scholar]
  15. Friedmann T., Haas M. Rapid concentration and purification of polyoma virus and SV40 with polyethylene glycol. Virology. 1970 Sep;42(1):248–250. doi: 10.1016/0042-6822(70)90263-1. [DOI] [PubMed] [Google Scholar]
  16. Friedmann T. In vitro reassembly of shell-like particles from disrupted polyoma virus. Proc Natl Acad Sci U S A. 1971 Oct;68(10):2574–2578. doi: 10.1073/pnas.68.10.2574. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Friedmann T. Structural proteins of polyoma virus: proteolytic degradation of virion proteins by exogenous and by virion-associated proteases. J Virol. 1976 Nov;20(2):520–526. doi: 10.1128/jvi.20.2.520-526.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gibson W. Polyoma virus proteins: a description of the structural proteins of the virion based on polyacrylamide gel electrophoresis and peptide analysis. Virology. 1974 Dec;62(2):319–336. doi: 10.1016/0042-6822(74)90395-x. [DOI] [PubMed] [Google Scholar]
  19. Goodman R. M. Reconstitution of potato virus X in vitro. III. Evidence for a role for hydrophobic interactions. Virology. 1977 Jan;76(1):72–78. doi: 10.1016/0042-6822(77)90283-5. [DOI] [PubMed] [Google Scholar]
  20. Hiebert E., Bancroft J. B., Bracker C. E. The assembly in vitro of some small spherical viruses, hybrid viruses, and other nucleoproteins. Virology. 1968 Mar;34(3):492–508. doi: 10.1016/0042-6822(68)90069-x. [DOI] [PubMed] [Google Scholar]
  21. Himes R. H., Burton P. R., Gaito J. M. Dimethyl sulfoxide-induced self-assembly of tubulin lacking associated proteins. J Biol Chem. 1977 Sep 10;252(17):6222–6228. [PubMed] [Google Scholar]
  22. Hohn T. Selfassembly of defective particles of the bacteriophage fr. Eur J Biochem. 1967 Sep;2(2):152–155. doi: 10.1111/j.1432-1033.1967.tb00119.x. [DOI] [PubMed] [Google Scholar]
  23. Hsu C. H., White J. A., Sehgal O. P. Assembly of southern bean mosaic virus from its two subviral intermediates. Virology. 1977 Sep;81(2):471–475. doi: 10.1016/0042-6822(77)90162-3. [DOI] [PubMed] [Google Scholar]
  24. Kocsis J. J., Harkaway S., Snyder R. Biological effects of the metabolites of dimethyl sulfoxide. Ann N Y Acad Sci. 1975 Jan 27;243:104–109. doi: 10.1111/j.1749-6632.1975.tb25349.x. [DOI] [PubMed] [Google Scholar]
  25. Ling C. M., Hung P. P., Overby L. R. Specificity in self-assembly of bacteriophages Q beta and MS2. Biochemistry. 1969 Nov;8(11):4464–4469. doi: 10.1021/bi00839a036. [DOI] [PubMed] [Google Scholar]
  26. Mackay R. L., Consigli R. A. Early events in polyoma virus infection: attachment, penetration, and nuclear entry. J Virol. 1976 Aug;19(2):620–636. doi: 10.1128/jvi.19.2.620-636.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. McMillen J., Center M. S., Consigli R. A. Origin of the polyoma virus-associated endonuclease. J Virol. 1975 Jan;17(1):127–131. doi: 10.1128/jvi.17.1.127-131.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. McMillen J., Consigli R. A. Characterization of polyoma DNA-protein complexes. I. Electrophoretic identification of the proteins in a nucleoprotein complex isolated from polyoma-infected cells. J Virol. 1974 Dec;14(6):1326–1336. doi: 10.1128/jvi.14.6.1326-1336.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. McMillen J., Consigli R. A. Immunological reactivity of antisera to sodium dodecyl sulfate-derived polypeptides of polyoma virions. J Virol. 1977 Mar;21(3):1113–1120. doi: 10.1128/jvi.21.3.1113-1120.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Roberts J. W., Steitz J. E. The reconstitution of infective bacteriophage R17. Proc Natl Acad Sci U S A. 1967 Oct;58(4):1416–1421. doi: 10.1073/pnas.58.4.1416. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Roblin R., Härle E., Dulbecco R. Polyoma virus proteins. 1. Multiple virion components. Virology. 1971 Sep;45(3):555–566. doi: 10.1016/0042-6822(71)90171-1. [DOI] [PubMed] [Google Scholar]
  32. Semancik J. S., Reynolds D. A. Assembly of protein and nucleoprotein particles from extracted tobacco rattle virus protein and RNA. Science. 1969 May 2;164(3879):559–560. doi: 10.1126/science.164.3879.559. [DOI] [PubMed] [Google Scholar]
  33. Smith G. L., Consigli R. A. Transient inhibition of polyoma virus synthesis by Sendai virus (parainfluenza I). I. Demonstration and nature of the inhibition by inactivated virus. J Virol. 1972 Dec;10(6):1091–1097. doi: 10.1128/jvi.10.6.1091-1097.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. VINOGRAD J., HEARST J. E. Equilibrium sedimentation of macromolecules and viruses in a density gradient. Fortschr Chem Org Naturst. 1962;20:373–422. [PubMed] [Google Scholar]

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