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In vitro screening for molecules that affect virus capsid assembly (and other protein association reactions)

Nature Protocols volume 2, pages 490–498 (2007)Cite this article

Abstract

Protein self-assembly is critical for numerous biological processes. Yet, assembly is rarely targeted by therapeutic agents, in part because it is hard to identify molecules that interfere with protein–protein interactions. Here, we describe a simple fluorescence-based screen for self-association and its application to the assembly of hepatitis B virus capsids. These data are analyzed to identify kinetic and thermodynamic effects—both of which are critical for the viral lifecycle and for understanding the mechanism of assembly effectors. Suggestions are made for modification of this protocol so that it can be applied to other self-assembling systems. With manual pipetting, setting up a plate takes about 2 h, the initial reading takes 1 h and the end point reading the following day takes about 5 min.

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Figure 1: Assembly dramatically changes the fluorescence of C150Bo.
Figure 2: Representative assembly kinetics from a single experiment.
Figure 3: Assembly kinetics and extent of assembly for controls and selected experimental compounds.
Figure 4: Absorbance spectra.

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References

  1. Alberts, B. et al. Molecular Biology of the Cell (Garland Publishing, New York, 1994).

    Google Scholar 

  2. Strausberg, R.L. & Schreiber, S.L. From knowing to controlling: a path from genomics to drugs using small molecule probes. Science 300, 294–295 (2003).

    Article  CAS  Google Scholar 

  3. Zlotnick, A. To build a virus capsid. An equilibrium model of the self assembly of polyhedral protein complexes. J. Mol. Biol. 241, 59–67 (1994).

    Article  CAS  Google Scholar 

  4. Zlotnick, A., Ceres, P., Singh, S. & Johnson, J.M. A small molecule inhibits and misdirects assembly of hepatitis B virus capsids. J. Virol. 76, 4848–4854 (2002).

    Article  CAS  Google Scholar 

  5. Prevelige, P.E.J. Inhibiting virus-capsid assembly by altering the polymerisation pathway. Trends Biotech. 16, 61–65 (1998).

    Article  CAS  Google Scholar 

  6. Smith, T.J. et al. The site of attachment in human rhinovirus 14 for antiviral agents that inhibit uncoating. Science 233, 1286–1293 (1986).

    Article  CAS  Google Scholar 

  7. Zhang, Y. et al. Structural and virological studies of the stages of virus replication that are affected by antirhinovirus compounds. J. Virol. 78, 11061–11069 (2004).

    Article  CAS  Google Scholar 

  8. Romero, J.R. Pleconaril: a novel antipicornaviral drug. Expert Opin. Investig. Drugs 10, 369–379 (2001).

    Article  CAS  Google Scholar 

  9. Weber, O. et al. Inhibition of human hepatitis B virus (HBV) by a novel non-nucleosidic compound in a transgenic mouse model. Antiviral Res. 54, 69–78 (2002).

    Article  CAS  Google Scholar 

  10. Deres, K. et al. Inhibition of hepatitis B virus replication by drug-induced depletion of nucleocapsids. Science 299, 893–896 (2003).

    Article  CAS  Google Scholar 

  11. Hacker, H.J., Deres, K., Mildenberger, M. & Schroder, C.H. Antivirals interacting with hepatitis B virus core protein and core mutations may misdirect capsid assembly in a similar fashion. Biochem. Pharmacol. 66, 2273–2279 (2003).

    Article  CAS  Google Scholar 

  12. Stray, S.J. et al. A heteroaryldihydropyrimidine activates and can misdirect hepatitis B virus capsid assembly. Proc. Natl. Acad. Sci. USA 102, 8138–8143 (2005).

    Article  CAS  Google Scholar 

  13. Bourne, C., Finn, M.G. & Zlotnick, A. Global structural changes in hepatitis B capsids induced by the assembly effector HAP1. J. Virol. 80, 11055–11061 (2006).

    Article  CAS  Google Scholar 

  14. Stray, S.J. & Zlotnick, A. BAY 41-4109 has multiple effects on hepatitis B virus capsid assembly. J. Mol. Recognit. 19, 542–548 (2006).

    Article  CAS  Google Scholar 

  15. Stray, S.J., Johnson, J.M., Kopek, B.G. & Zlotnick, A. An in vitro fluorescence screen to identify antivirals that disrupt hepatitis B virus capsid assembly. Nat. Biotechnol. 24, 358–362 (2006).

    Article  CAS  Google Scholar 

  16. Ganem, D. & Schneider, R.J. Hepadnaviridae: The Viruses and Their Replication. 4th edn. (Lippincott Williams & Wilkins, Philadelphia, 2001).

    Google Scholar 

  17. Zhou, S. & Standring, D.N. Hepatitis B virus capsid particles are assembled from core-protein dimer precursors. Proc. Natl. Acad. Sci. USA 89, 10046–10050 (1992).

    Article  CAS  Google Scholar 

  18. Crowther, R.A. et al. Three-dimensional structure of hepatitis B virus core particles determined by electron cryomicroscopy. Cell 77, 943–950 (1994).

    Article  CAS  Google Scholar 

  19. Stannard, L.M. & Hodgkiss, M. Morphological irregularities in Dane particle cores. J. Gen. Virol. 45, 509–514 (1979).

    Article  CAS  Google Scholar 

  20. Steven, A.C. et al. Structure, assembly, and antigenicity of hepatitis B virus capsid proteins. Adv. Virus Res. 64, 125–164 (2005).

    Article  CAS  Google Scholar 

  21. Wynne, S.A., Crowther, R.A. & Leslie, A.G. The crystal structure of the human hepatitis B virus capsid. Mol. Cell 3, 771–780 (1999).

    Article  CAS  Google Scholar 

  22. Dryden, K.A. et al. Native hepatitis B virions and capsids visualized by electron cryomicroscopy. Mol. Cell 22, 843–850 (2006).

    Article  CAS  Google Scholar 

  23. Zlotnick, A., Johnson, J.M., Wingfield, P.W., Stahl, S.J. & Endres, D. A theoretical model successfully identifies features of hepatitis B virus capsid assembly. Biochemistry 38, 14644–14652 (1999).

    Article  CAS  Google Scholar 

  24. Ceres, P. & Zlotnick, A. Weak protein-protein interactions are sufficient to drive assembly of hepatitis B virus capsids. Biochemistry 41, 11525–11531 (2002).

    Article  CAS  Google Scholar 

  25. Zlotnick, A. Theoretical aspects of virus capsid assembly. J. Mol. Recognit. 18, 479–490 (2005).

    Article  CAS  Google Scholar 

  26. Adolph, K.W. & Butler, P.J. Assembly of a spherical plant virus. Philos. Trans. R. Soc. Lond. B. 276, 113–122 (1976).

    Article  CAS  Google Scholar 

  27. Prevelige, J., Thomas, D. & King, J. Scaffolding protein regulates the polymerization of P22 coat subunits into icosahedral shells in vitro . J. Mol. Biol. 202, 743–757 (1988).

    Article  CAS  Google Scholar 

  28. Johnson, J.M. et al. Regulating self-assembly of spherical oligomers. Nano Lett. 5, 765–770 (2005).

    Article  CAS  Google Scholar 

  29. Parent, K.N., Zlotnick, A. & Teschke, C.M. Quantitative analysis of multi-component spherical virus assembly: scaffolding protein contributes to the global stability of phage P22 procapsids. J. Mol. Biol. 359, 1097–1106 (2006).

    Article  CAS  Google Scholar 

  30. Rapaport, D.C. Self-assembly of polyhedral shells: a molecular dynamics study. Phys. Rev. E. 70, 051905 (2004).

    Article  CAS  Google Scholar 

  31. Hagan, M.F. & Chandler, D. Dynamic pathways for viral capsid assembly. Biophys. J. (2006).

  32. Zhang, T. & Schwartz, R. Simulation study of the contribution of oligomer/oligomer binding to capsid assembly kinetics. Biophys. J. 90, 57–64 (2006).

    Article  CAS  Google Scholar 

  33. Reddy, V.S. et al. Virus Particle Explorer (VIPER), a website for virus capsid structures and their computational analyses. J. Virol. 75, 11943–11947 (2001).

    Article  CAS  Google Scholar 

  34. Riddles, P.W., Blakeley, R.L. & Zerner, B. Reassessment of Ellman's reagent. Methods Enzymol. 91, 49–60 (1983).

    Article  CAS  Google Scholar 

  35. Bergstrom, F. et al. Dimers of dipyrrometheneboron difluoride (BODIPY) with light spectroscopic applications in chemistry and biology. J. Am. Chem. Soc. 124, 196–204 (2002).

    Article  Google Scholar 

  36. Vogel, M., Diez, M., Eisfeld, J. & Nassal, M. In vitro assembly of mosaic hepatitis B virus capsid-like particles (CLPs): rescue into CLPs of assembly-deficient core protein fusions and FRET-suited CLPs. FEBS Lett. 579, 5211–5216 (2005).

    Article  CAS  Google Scholar 

  37. Endres, D., Miyahara, M., Moisant, P. & Zlotnick, A. A reaction landscape identifies the intermediates critical for self-assembly of virus capsids and other polyhedral structures. Protein Sci. 14, 1518–1525 (2005).

    Article  CAS  Google Scholar 

  38. van Holde, K.E., Johnson, W.C. & Ho, P.S. Principles of Physical Biochemistry. (Prentice-Hall, Upper Saddle River, New Jersey, 1998).

    Google Scholar 

  39. Pace, C.N., Vajdos, F., Fee, L., Grimsley, G. & Gray, T. How to measure and predict the molar absorption coefficient of a protein. Protein Sci. 4, 2411–2423 (1995).

    Article  CAS  Google Scholar 

  40. Stray, S.J., Ceres, P. & Zlotnick, A. Zinc ions trigger conformational change and oligomerization of hepatitis B virus capsid protein. Biochemistry 43, 9989–9998 (2004).

    Article  CAS  Google Scholar 

  41. Lakowicz, J.R. Principles of Fluorescence Spectroscopy. 2nd edn. (Kluwer Academic/Plenum Publishers, New York, 1999).

    Book  Google Scholar 

  42. Ceres, P., Stray, S.J. & Zlotnick, A. Hepatitis B virus capsid assembly is enhanced by naturally occurring mutation F97L. J. Virol. 78, 9538–9543 (2004).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank Dr MG Finn and Dr Seijun Lee (TSRI) for the B compounds used to demonstrate the assay in this protocol. This work was supported by a grant from the National Institutes of Health (R01-AI 67417-01) to AZ and an American Cancer Society-Mary Horton post-doctoral fellowship (PF-05-237-01-GMC) to CRB.

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, 73104, Oklahoma, USA

    Adam Zlotnick, Angela Lee, Christina R Bourne & Jennifer M Johnson

  2. Department of Technology Development, GlaxoSmithKline, Research Triangle Park, North Carolina, 27713, USA

    Paul L Domanico

  3. Department of Microbiology, University of Mississippi Medical Center, Jackson, 39216, Mississippi, USA

    Stephen J Stray

Authors
  1. Adam Zlotnick
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  2. Angela Lee
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Correspondence to Adam Zlotnick.

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Zlotnick, A., Lee, A., Bourne, C. et al. In vitro screening for molecules that affect virus capsid assembly (and other protein association reactions). Nat Protoc 2, 490–498 (2007). https://doi.org/10.1038/nprot.2007.60

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