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. 1995 Dec;69(12):7734–7742. doi: 10.1128/jvi.69.12.7734-7742.1995

Expression of the polyomavirus minor capsid proteins VP2 and VP3 in Escherichia coli: in vitro interactions with recombinant VP1 capsomeres.

S E Delos 1, T P Cripe 1, A D Leavitt 1, H Greisman 1, R L Garcea 1
PMCID: PMC189715  PMID: 7494283

Abstract

The polyomavirus VP2 and VP3 capsid proteins were expressed in Escherichia coli. The majority of the expressed proteins were in an insoluble fraction, and they were extracted and initially purified in 8 M urea before renaturation. Soluble VP2 and VP3 were mixed with purified recombinant VP1 capsomeres, and their interactions were assayed by immunoprecipitation and ion-exchange chromatography. Coimmunoprecipitation could be demonstrated with antibodies to either VP1 or VP2/VP3. Mixing recombinant VP1 with VP2 and VP3 modified the recognition of VP1 by domain-specific antipeptide antibodies and altered the chromatographic behavior of the individual proteins. Similar results were observed when a truncated VP1 protein, delta NCOVP1, with 62 amino acids deleted from the carboxy terminus was mixed with VP2/VP3. After the mixing, equilibrium dissociation constants for their binding to either VP1 or delta NCOVP1 were determined to be 0.37 +/- 0.23 microM for VP2 and 0.18 +/- 0.21 microM for VP3. These studies demonstrate that the recombinant VP2 and VP3 proteins interact with VP1 to affect the biochemical properties of VP1 capsomeres and to change the epitope accessibility of VP1 pentamers. These changes may reflect conformational alterations in VP1 capsomeres which are necessary for viral genome encapsidation.

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Selected References

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  1. Amann E., Brosius J., Ptashne M. Vectors bearing a hybrid trp-lac promoter useful for regulated expression of cloned genes in Escherichia coli. Gene. 1983 Nov;25(2-3):167–178. doi: 10.1016/0378-1119(83)90222-6. [DOI] [PubMed] [Google Scholar]
  2. Barouch D. H., Harrison S. C. Interactions among the major and minor coat proteins of polyomavirus. J Virol. 1994 Jun;68(6):3982–3989. doi: 10.1128/jvi.68.6.3982-3989.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brady J. N., Consigli R. A. Chromatographic separation of the polyoma virus proteins and renaturation of the isolated VP1 major capsid protein. J Virol. 1978 Aug;27(2):436–442. doi: 10.1128/jvi.27.2.436-442.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cai X., Chang D., Rottinghaus S., Consigli R. A. Expression and purification of recombinant polyomavirus VP2 protein and its interactions with polyomavirus proteins. J Virol. 1994 Nov;68(11):7609–7613. doi: 10.1128/jvi.68.11.7609-7613.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chang D., Cai X., Consigli R. A. Characterization of the DNA binding properties of polyomavirus capsid protein. J Virol. 1993 Oct;67(10):6327–6331. doi: 10.1128/jvi.67.10.6327-6331.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chang D., Haynes J. I., 2nd, Brady J. N., Consigli R. A. Identification of a nuclear localization sequence in the polyomavirus capsid protein VP2. Virology. 1992 Dec;191(2):978–983. doi: 10.1016/0042-6822(92)90276-u. [DOI] [PubMed] [Google Scholar]
  7. Christiansen G., Landers T., Griffith J., Berg P. Characterization of components released by alkali disruption of simian virus 40. J Virol. 1977 Mar;21(3):1079–1084. doi: 10.1128/jvi.21.3.1079-1084.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Delos S. E., Montross L., Moreland R. B., Garcea R. L. Expression of the polyomavirus VP2 and VP3 proteins in insect cells: coexpression with the major capsid protein VP1 alters VP2/VP3 subcellular localization. Virology. 1993 May;194(1):393–398. doi: 10.1006/viro.1993.1274. [DOI] [PubMed] [Google Scholar]
  9. Etchison D., Walter G. Subunit interactions in polyoma virus structure. Virology. 1977 Apr;77(2):783–796. doi: 10.1016/0042-6822(77)90499-8. [DOI] [PubMed] [Google Scholar]
  10. Forstová J., Krauzewicz N., Wallace S., Street A. J., Dilworth S. M., Beard S., Griffin B. E. Cooperation of structural proteins during late events in the life cycle of polyomavirus. J Virol. 1993 Mar;67(3):1405–1413. doi: 10.1128/jvi.67.3.1405-1413.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Garcea R. L., Benjamin T. L. Isolation and characterization of polyoma nucleoprotein complexes. Virology. 1983 Oct 15;130(1):65–75. doi: 10.1016/0042-6822(83)90118-6. [DOI] [PubMed] [Google Scholar]
  12. Garcea R. L., Salunke D. M., Caspar D. L. Site-directed mutation affecting polyomavirus capsid self-assembly in vitro. Nature. 1987 Sep 3;329(6134):86–87. doi: 10.1038/329086a0. [DOI] [PubMed] [Google Scholar]
  13. Gharakhanian E., Kasamatsu H. Two independent signals, a nuclear localization signal and a Vp1-interactive signal, reside within the carboxy-35 amino acids of SV40 Vp3. Virology. 1990 Sep;178(1):62–71. doi: 10.1016/0042-6822(90)90379-6. [DOI] [PubMed] [Google Scholar]
  14. Gharakhanian E., Takahashi J., Clever J., Kasamatsu H. In vitro assay for protein-protein interaction: carboxyl-terminal 40 residues of simian virus 40 structural protein VP3 contain a determinant for interaction with VP1. Proc Natl Acad Sci U S A. 1988 Sep;85(18):6607–6611. doi: 10.1073/pnas.85.18.6607. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Goff S. A., Goldberg A. L. Production of abnormal proteins in E. coli stimulates transcription of lon and other heat shock genes. Cell. 1985 Jun;41(2):587–595. doi: 10.1016/s0092-8674(85)80031-3. [DOI] [PubMed] [Google Scholar]
  16. Griffith J. P., Griffith D. L., Rayment I., Murakami W. T., Caspar D. L. Inside polyomavirus at 25-A resolution. Nature. 1992 Feb 13;355(6361):652–654. doi: 10.1038/355652a0. [DOI] [PubMed] [Google Scholar]
  17. Guarente L., Lauer G., Roberts T. M., Ptashne M. Improved methods for maximizing expression of a cloned gene: a bacterium that synthesizes rabbit beta-globin. Cell. 1980 Jun;20(2):543–553. doi: 10.1016/0092-8674(80)90640-6. [DOI] [PubMed] [Google Scholar]
  18. Ishii N., Nakanishi A., Yamada M., Macalalad M. H., Kasamatsu H. Functional complementation of nuclear targeting-defective mutants of simian virus 40 structural proteins. J Virol. 1994 Dec;68(12):8209–8216. doi: 10.1128/jvi.68.12.8209-8216.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kasamatsu H., Nehorayan A. Vp1 affects intracellular localization of Vp3 polypeptide during simian virus 40 infection. Proc Natl Acad Sci U S A. 1979 Jun;76(6):2808–2812. doi: 10.1073/pnas.76.6.2808. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Krauzewicz N., Streuli C. H., Stuart-Smith N., Jones M. D., Wallace S., Griffin B. E. Myristylated polyomavirus VP2: role in the life cycle of the virus. J Virol. 1990 Sep;64(9):4414–4420. doi: 10.1128/jvi.64.9.4414-4420.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Leavitt A. D., Roberts T. M., Garcea R. L. Polyoma virus major capsid protein, VP1. Purification after high level expression in Escherichia coli. J Biol Chem. 1985 Oct 15;260(23):12803–12809. [PubMed] [Google Scholar]
  22. Li M., Delos S. E., Montross L., Garcea R. L. Polyomavirus VP1 phosphorylation: coexpression with the VP2 capsid protein modulates VP1 phosphorylation in Sf9 insect cells. Proc Natl Acad Sci U S A. 1995 Jun 20;92(13):5992–5996. doi: 10.1073/pnas.92.13.5992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Liddington R. C., Yan Y., Moulai J., Sahli R., Benjamin T. L., Harrison S. C. Structure of simian virus 40 at 3.8-A resolution. Nature. 1991 Nov 28;354(6351):278–284. doi: 10.1038/354278a0. [DOI] [PubMed] [Google Scholar]
  24. Lin W., Hata T., Kasamatsu H. Subcellular distribution of viral structural proteins during simian virus 40 infection. J Virol. 1984 May;50(2):363–371. doi: 10.1128/jvi.50.2.363-371.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lin W., Shurgot J. L., Kasamatsu H. The synthesis and transport of SV40 structural proteins. Virology. 1986 Oct 15;154(1):108–120. doi: 10.1016/0042-6822(86)90434-4. [DOI] [PubMed] [Google Scholar]
  26. Montross L., Watkins S., Moreland R. B., Mamon H., Caspar D. L., Garcea R. L. Nuclear assembly of polyomavirus capsids in insect cells expressing the major capsid protein VP1. J Virol. 1991 Sep;65(9):4991–4998. doi: 10.1128/jvi.65.9.4991-4998.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Moreland R. B., Montross L., Garcea R. L. Characterization of the DNA-binding properties of the polyomavirus capsid protein VP1. J Virol. 1991 Mar;65(3):1168–1176. doi: 10.1128/jvi.65.3.1168-1176.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Rayment I., Baker T. S., Caspar D. L., Murakami W. T. Polyoma virus capsid structure at 22.5 A resolution. Nature. 1982 Jan 14;295(5845):110–115. doi: 10.1038/295110a0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Sahli R., Freund R., Dubensky T., Garcea R., Bronson R., Benjamin T. Defect in entry and altered pathogenicity of a polyoma virus mutant blocked in VP2 myristylation. Virology. 1993 Jan;192(1):142–153. doi: 10.1006/viro.1993.1016. [DOI] [PubMed] [Google Scholar]
  30. Salunke D. M., Caspar D. L., Garcea R. L. Self-assembly of purified polyomavirus capsid protein VP1. Cell. 1986 Sep 12;46(6):895–904. doi: 10.1016/0092-8674(86)90071-1. [DOI] [PubMed] [Google Scholar]
  31. Schaffhausen B. S., Benjamin T. L. Deficiency in histone acetylation in nontransforming host range mutants of polyoma virus. Proc Natl Acad Sci U S A. 1976 Apr;73(4):1092–1096. doi: 10.1073/pnas.73.4.1092. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Stamatos N. M., Chakrabarti S., Moss B., Hare J. D. Expression of polyomavirus virion proteins by a vaccinia virus vector: association of VP1 and VP2 with the nuclear framework. J Virol. 1987 Feb;61(2):516–525. doi: 10.1128/jvi.61.2.516-525.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Walter G., Deppert W. Intermolecular disulfide bonds: an important structural feature of the polyoma virus capsid. Cold Spring Harb Symp Quant Biol. 1975;39(Pt 1):255–257. doi: 10.1101/sqb.1974.039.01.033. [DOI] [PubMed] [Google Scholar]

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