1887

Abstract

Previous studies have implicated disulfide bonds between Vp1 molecules in the stabilization of the simian virus 40 (SV40) capsid. To identify the cysteine residues involved in intermolecular disulfide interactions, systematic oligo-directed mutagenesis of cysteine codons to serine codons was initiated. Wild-type and mutant Vp1 proteins were produced in rabbit reticulocyte lysates and were allowed to interact post-translationally. Disulfide-linked Vp1 complexes were assessed via non-reducing SDS–PAGE and via sucrose-gradient sedimentation. Wild-type Vp1 forms 7S pentamers followed by 12S disulfide-linked multi-pentameric complexes in cell-free lysates. Mutagenesis of all seven cysteine codons abolished Vp1 12S complexes, but did not affect pentamer formation. A quadruple Vp1 mutant at Cys, Cys, Cys and Cys continued to form 12S complexes, whereas the major products of the Cys, Cys and Cys triple mutant Vp1 were 7S pentamers. Single and double mutant Vp1 proteins at the three cysteines affected continued to form 12S complexes, but to a lesser extent. Thus, inter-pentamer disulfide bonds at Cys, Cys and Cys are essential and sufficient for stabilization of Vp1 complexes in cell-free lysates. These results are in agreement with previous structural studies of SV40 that implicated the same three residues in disulfide linkage in the capsid. Possible parameters for the involvement of the three cysteines are discussed.

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1999-09-01
2020-01-26
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References

  1. Baker, T. S., Drak, J. & Bina, M. ( 1988; ). Reconstruction of the three-dimensional structure of simian virus 40 and visualization of the chromatic core. Proceedings of the National Academy of Sciences, USA 85, 422-426.[CrossRef]
    [Google Scholar]
  2. Benerza, R. ( 1994; ). An intermolecular disulfide bond stabilizes E2A homodimers and is required for DNA binding at physiological temperatures. Cell 79, 1057-1067.[CrossRef]
    [Google Scholar]
  3. Brady, J. N., Lavialle, C. & Salzman, N. P. ( 1980; ). Efficient transcription of a compact nucleoprotein complex isolated from purified simian virus 40 virions. Journal of Virology 35, 371-381.
    [Google Scholar]
  4. Brady, J. N., Winston, V. D. & Consigli, R. A. ( 1977; ). Dissociation of polyoma virus by the chelation of calcium ions found associated with purified virions. Journal of Virology 23, 717-724.
    [Google Scholar]
  5. Brady, J. N., Winston, V. D. & Consigli, R. A. ( 1978; ). Characterization of a DNA–protein complex and capsomere subunits derived from polyoma virus by treatment with ethyleneglycol-bis-N,N′-tetraacetic acid and dithiothreitol. Journal of Virology 27, 193-204.
    [Google Scholar]
  6. Chang, D., Fung, C., Ou, W., Chao, P., Li, S., Wang, M., Huang, Y., Tzeng, T. & Tsai, R. ( 1997; ). Self-assembly of the JC virus major capsid protein, Vp1, expressed in insect cells. Journal of General Virology 78, 1435-1439.
    [Google Scholar]
  7. Chinenov, Y., Schmidt, T., Yang, X. & Martin, M. E. ( 1998; ). Identification of redox-sensitive cysteines in GA-binding protein-α that regulate DNA binding and heterodimerization. Journal of Biological Chemistry 273, 6203-6209.[CrossRef]
    [Google Scholar]
  8. Clontech (1993). TransformerTM Site-Directed Mutagenesis Kit: Product Protocol, 2nd edn. Clontech Laboratories, Inc., Palo Alto, CA, USA.
  9. Fasching, C. (1998). The specific role of cysteine residues in the nuclear localization of the SV40 Vp1 protein. MS thesis, California State University, Long Beach, CA, USA.
  10. Gharakhanian, E. (1988). Studies on the nuclear transport and the protein–protein interactions of SV40 structural proteins. PhD thesis, University of California, Los Angeles, CA, USA.
  11. Gharakhanian, E., Sajo, A. K. & Weidman, M. K. ( 1995; ). SV40 Vp1 assembles into disulfide-linked postpentameric complexes in cell-free lysates. Virology 207, 251-254.[CrossRef]
    [Google Scholar]
  12. Gharakhanian, E., Takahashi, J., Clever, J. & Kasamatsu, H. ( 1988; ). 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. Proceedings of the National Academy of Sciences, USA 85, 6607-6611.[CrossRef]
    [Google Scholar]
  13. Kosukegawa, A., Arisaka, F., Takayama, M., Yajima, H., Kaidow, A. & Handa, H. ( 1996; ). Purification and characterization of virus-like particles and pentamers produced by the expression of SV40 capsid proteins in insect cells. Biochimica et Biophysica Acta 1290, 37-45.[CrossRef]
    [Google Scholar]
  14. Liddington, R. C., Yan, Y., Moulai, J., Sahli, R., Benjamin, T. L. & Harrison, S. C. ( 1991; ). Structure of simian virus 40 at 3·8-Å resolution. Nature 354, 278-284.[CrossRef]
    [Google Scholar]
  15. Lin, W. L., Hata, T. & Kasamatsu, H. ( 1984; ). Subcellular distribution of viral structural proteins during simian virus 40 infection. Journal of Virology 50, 363-371.
    [Google Scholar]
  16. Meyer, M., Schreck, R. & Baeuerle, P. A. ( 1993; ). H2O2 and antioxidants have opposite effects on activation of NF-κB and AP-1 in intact cells: AP-1 as secondary antioxidant-responsive factor. EMBO Journal 12, 2005-2015.
    [Google Scholar]
  17. Monastryskaya, K., Booth, T., Nel, L. & Roy, P. ( 1994; ). Mutation of either of two cysteine residues or deletion of the amino or carboxy terminus of the non-structural protein NS1 of bluetongue virus abrogates virus-specified tubule formation in insect cells. Journal of Virology 68, 2169-2178.
    [Google Scholar]
  18. Ng, S. C. & Bina, M. ( 1981; ). Disulfide bonds protect the encapsidated chromosomes of simian virus 40. FEBS Letters 130, 47-49.[CrossRef]
    [Google Scholar]
  19. Pognonec, P., Kato, H. & Roeder, R. G. ( 1992; ). The helix-loop-helix/leucine repeat transcription factor USF can be functionally regulated in a redox-dependent manner. Journal of Biological Chemistry 267, 24563-24567.
    [Google Scholar]
  20. Rayment, I., Baker, T. S. & Caspar, D. L. D. ( 1982; ). Polyoma virus capsid structure at 22·5 Å resolution. Nature 295, 110-115.[CrossRef]
    [Google Scholar]
  21. Sandalon, Z. & Oppenheim, A. ( 1997; ). Self-assembly and protein–protein interactions between the SV40 proteins produced in insect cells. Virology 237, 414-421.[CrossRef]
    [Google Scholar]
  22. Schreck, R., Rieber, P. & Baeuerle, P. A. ( 1991; ). Reactive oxygen intermediates as apparently widely used messengers in the activation of the NF-κB transcription factor and HIV-1. EMBO Journal 10, 2247-2258.
    [Google Scholar]
  23. Stehle, T., Gamblin, S. J., Yan, Y. & Harrison, S. C. ( 1996; ). The structure of simian virus 40 refined at 3·1 Å resolution. Structure 4, 165-182.[CrossRef]
    [Google Scholar]
  24. Volpers, C., Schirmacher, P., Streeck, R. E. & Sapp, M. ( 1994; ). Assembly of the major and minor capsid protein of human papillomavirus type 33 into virus-like particles and tubular structures in insect cells. Virology 200, 504-512.[CrossRef]
    [Google Scholar]
  25. Walter, G. & Deppert, W. ( 1974; ). Intermolecular disulfide bonds: an important structural feature of the polyoma virus capsid. Cold Spring Harbor Symposia on Quantitative Biology 39, 255-257.[CrossRef]
    [Google Scholar]
  26. Weidman, M. K. (1994). Studies on the role of cysteine residues in simian virus 40 Vp1 oligomerization in vitro. MS thesis, California State University, Long Beach, CA, USA.
  27. Xanthoudakis, S., Miao, G., Wang, F., Pan, Y. C. & Curran, T. ( 1992; ). Redox activation of Fos–Jun DNA binding activity is mediated by a DNA repair enzyme. EMBO Journal 11, 3323-3335.
    [Google Scholar]
  28. Yan, Y., Stehle, T., Liddington, R. C., Zhao, H. & Harrison, S. C. ( 1996; ). Structure determination of simian virus 40 and murine polyomavirus by a combination of 30-fold and 5-fold electron-density averaging. Structure 4, 157-164.[CrossRef]
    [Google Scholar]
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