Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Design and self-assembly of simple coat proteins for artificial viruses

Abstract

Viruses are among the simplest biological systems and are highly effective vehicles for the delivery of genetic material into susceptible host cells1. Artificial viruses can be used as model systems for providing insights into natural viruses and can be considered a testing ground for developing artificial life. Moreover, they are used in biomedical and biotechnological applications, such as targeted delivery of nucleic acids for gene therapy1,2 and as scaffolds in material science3,4,5. In a natural setting, survival of viruses requires that a significant fraction of the replicated genomes be completely protected by coat proteins. Complete protection of the genome is ensured by a highly cooperative supramolecular process between the coat proteins and the nucleic acids, which is based on reversible, weak and allosteric interactions only6,7,8,9. However, incorporating this type of supramolecular cooperativity into artificial viruses remains challenging10,11,12,13,14,15. Here, we report a rational design for a self-assembling minimal viral coat protein based on simple polypeptide domains. Our coat protein features precise control over the cooperativity of its self-assembly with single DNA molecules to finally form rod-shaped virus-like particles. We confirm the validity of our design principles by showing that the kinetics of self-assembly of our virus-like particles follows a previous model developed for tobacco mosaic virus9. We show that our virus-like particles protect DNA against enzymatic degradation and transfect cells with considerable efficiency, making them promising delivery vehicles.

Your institute does not have access to this article

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Design of the minimal viral coat protein C–Sn–B.
Figure 2: Self-assembly of VLPs: AFM and cryo-TEM images of complexes of linear dsDNA with C–Sn–B show morphologies that depend on the size n of the self-assembly block.
Figure 3: Cooperativity of the self-assembly of VLPs.
Figure 4: Formation process of VLPs.

References

  1. Naldini, L. et al. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 272, 263–267 (1996).

    CAS  Article  Google Scholar 

  2. Mastrobattista, E., van der Aa, M. A. E. M., Hennink, W. E. & Crommelin, D. J. A. Artificial viruses: a nanotechnological approach to gene delivery. Nature Rev. Drug Discov. 5, 115–121 (2006).

    Article  Google Scholar 

  3. Comellas-Aragones, M. et al. A virus-based single-enzyme nanoreactor. Nature Nanotech. 2, 635–639 (2007).

    CAS  Article  Google Scholar 

  4. Grigoryan, G. et al. Computational design of virus-like protein assemblies on carbon nanotube surfaces. Science 332, 1071–1076 (2011).

    CAS  Article  Google Scholar 

  5. Douglas, T. & Young, M. Host–guest encapsulation of materials by assembled virus protein cages. Nature 393, 152–155 (1998).

    CAS  Article  Google Scholar 

  6. Klug, A. The tobacco mosaic virus particle: structure and assembly. Phil. Trans. R. Soc. Lond. B 354, 531–535 (1999).

    CAS  Article  Google Scholar 

  7. Caspar, D. L. & Namba, K. Switching in the self-assembly of tobacco mosaic virus. Adv. Biophys. 26, 157–185 (1990).

    CAS  Article  Google Scholar 

  8. Butler, P. J. G. Self–assembly of tobacco mosaic virus: the role of an intermediate aggregate in generating both specificity and speed. Phil. Trans. R. Soc. Lond. B 354, 537–550 (1999).

    CAS  Article  Google Scholar 

  9. Kraft, D. J., Kegel, W. K. & van der Schoot, P. A kinetic zipper model and the assembly of tobacco mosaic virus. Biophys. J. 102, 2845–2855 (2012).

    CAS  Article  Google Scholar 

  10. Percec, V., Heck, J., Johansson, G., Tomazos, D. & Ungar, G. Towards tobacco mosaic virus-like self-assembled supramolecular architectures. Macromol. Symp. 77, 237–265 (1994).

    CAS  Article  Google Scholar 

  11. Remy, J. S., Kichler, A., Mordvinov, V., Schuber, F. & Behr, J. P. Targeted gene transfer into hepatoma cells with lipopolyamine-condensed DNA particles presenting galactose ligands: a stage toward artificial viruses. Proc. Natl Acad. Sci. USA 92, 1744–1748 (1995).

    CAS  Article  Google Scholar 

  12. Aoyama, Y. et al. Artificial viruses and their application to gene delivery. Size-controlled gene coating with glycocluster nanoparticles. J. Am. Chem. Soc. 125, 3455–3457 (2003).

    CAS  Article  Google Scholar 

  13. Wagner, E. Strategies to improve DNA polyplexes for in vivo gene transfer: will ‘artificial viruses’ be the answer? Pharm. Res. 21, 8–14 (2004).

    CAS  Article  Google Scholar 

  14. Lim, Y. B., Lee, E., Yoon, Y. R., Lee, M. S. & Lee, M. Filamentous artificial virus from a self-assembled discrete nanoribbon. Angew. Chem. Int. Ed. 47, 4525–4528 (2008).

    CAS  Article  Google Scholar 

  15. Miyata, K., Nishiyama, N. & Kataoka, K. Rational design of smart supramolecular assemblies for gene delivery: chemical challenges in the creation of artificial viruses. Chem. Soc. Rev. 41, 2562–2574 (2012).

    CAS  Article  Google Scholar 

  16. Whitty, A. Cooperativity and biological complexity. Nature Chem. Biol. 4, 435–439 (2008).

    CAS  Article  Google Scholar 

  17. Namba, K., Pattanayek, R. & Stubbs, G. Visualization of protein–nucleic acid interactions in a virus. Refined structure of intact tobacco mosaic virus at 2.9 Å resolution by X-ray fiber diffraction. J. Mol. Biol. 208, 307–325 (1989).

    CAS  Article  Google Scholar 

  18. Hernandez-Garcia, A., Werten, M. W., Stuart, M. C., de Wolf, F. A. & de Vries R. Coating of single DNA molecules by genetically engineered protein diblock copolymers. Small 8, 3491–3501 (2012).

    CAS  Article  Google Scholar 

  19. Krejchi, M. T. et al. Chemical sequence control of beta-sheet assembly in macromolecular crystals of periodic polypeptides. Science 265, 1427–1432 (1994).

    CAS  Article  Google Scholar 

  20. Beun, L. H., Beaudoux, X. J., Kleijn, J. M., de Wolf, F. A. & Stuart, M. A. C. Self-assembly of silk-collagen-like triblock copolymers resembles a supramolecular living polymerization. ACS Nano 6, 133–140 (2012).

    CAS  Article  Google Scholar 

  21. Werten, M. W. T., Wisselink, W. H., Jansen-van den Bosch, T. J., de Bruin, E. C. & de Wolf, F. A. Secreted production of a custom-designed, highly hydrophilic gelatin in Pichia pastoris. Protein Eng. 14, 447–454 (2001).

    CAS  Article  Google Scholar 

  22. DeRouchey, J., Walker, G. F., Wagner, E. & Radler, J. O. Decorated rods: a ‘bottom-up’ self-assembly of monomolecular DNA complexes. J. Phys. Chem. B 110, 4548–4554 (2006).

    CAS  Article  Google Scholar 

  23. Osada, K. et al. Quantized folding of plasmid DNA condensed with block catiomer into characteristic rod structures promoting transgene efficacy. J. Am. Chem. Soc. 132, 12343–12348 (2010).

    CAS  Article  Google Scholar 

  24. Ruff, Y., Moyer, T., Newcomb, C. J., Demeler, B. & Stupp, S. I. Precision templating with DNA of a virus-like particle with peptide nanostructures. J. Am. Chem. Soc. 135, 6211–6219 (2013).

    CAS  Article  Google Scholar 

  25. Lecuyer, K. A., Behlen, L. S. & Uhlenbeck, O. C. Mutants of the bacteriophage MS2 coat protein that alter its cooperative binding to RNA. Biochemistry 34, 10600–10606 (1995).

    CAS  Article  Google Scholar 

  26. Williamson, J. R. Cooperativity in macromolecular assembly. Nature Chem. Biol. 4, 458–465 (2008).

    CAS  Article  Google Scholar 

  27. Morrone, S. R. et al. Cooperative assembly of IFI16 filaments on dsDNA provides insights into host defense strategy. Proc. Natl Acad. Sci. USA 111, E62 (2014).

    CAS  Article  Google Scholar 

  28. Rabotyagova, O. S., Cebe, P. & Kaplan, D. L. Protein-based block copolymers. Biomacromolecules 12, 269–289 (2011).

    CAS  Article  Google Scholar 

  29. Zhang, S. G. Fabrication of novel biomaterials through molecular self-assembly. Nature Biotechnol. 21, 1171–1178 (2003).

    CAS  Article  Google Scholar 

  30. King, N. P. et al. A general computational method is used to design protein building blocks that self-assemble into target architectures. Science 336, 1171–1174 (2012).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

The authors thank A. Westphal and I. van Hees for help with the fluorescence correlation spectroscopy experiments and R. Fokkink for help with static light scattering. The authors also thank M.W.P. van de Put for help in performing part of the cryo-TEM experiments and W. Kegel for suggesting the electrophoretic light scattering experiment. A.H-G. is financially supported by the Dutch Polymer Institute (DPI), project #698 SynProt, and by the Consejo Nacional de Ciencia y Tecnología (CONACyT), México. M.E.F. is supported by the Dutch Polymer Institute (DPI, Technology area HTE, project #730). M.C.S. is supported by the European Research Council (advanced grant no. 267254). D.J.K. acknowledges financial support through a Rubicon fellowship (grant no. 680-50-1019) from the Netherlands Organization for Scientific Research (NWO).

Author information

Authors and Affiliations

Authors

Contributions

F.d.W., M.C.S., P.v.d.S. and R.d.V. conceived the initial idea. R.d.V. and A.H-G. conceived and designed the molecules and experiments. A.H-G. produced the proteins, except for C–S2–B, which was produced by A.F.J.J. M.W. contributed to the production of proteins and to writing the technical sections on protein production. Except for the transfection experiments, analytical ultracentrifugation and cryo-TEM imaging, A.H-G. performed all experiments. A.H-G. and R.d.V. interpreted and analysed all experimental data except for the transfection experiments. P.v.d.S. and D.J.K. performed the theoretical analysis of the VLP assembly kinetics. R.B. designed the transfection experiments. M.F. performed and analysed the transfection experiments. D.M.E.T-W. designed, performed and analysed the analytical ultracentrifugation experiments. N.A.J.M.S. designed the cryo-TEM imaging experiments. P.H.H.B. performed the cryo-TEM imaging. A.H-G., D.J.K., P.v.d.S., M.C.S. and R.d.V. wrote the paper. All authors commented on the manuscript.

Corresponding authors

Correspondence to Armando Hernandez-Garcia or Renko de Vries.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary information

Supplementary Information (PDF 3928 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Hernandez-Garcia, A., Kraft, D., Janssen, A. et al. Design and self-assembly of simple coat proteins for artificial viruses. Nature Nanotech 9, 698–702 (2014). https://doi.org/10.1038/nnano.2014.169

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nnano.2014.169

Further reading

Search

Quick links

Find nanotechnology articles, nanomaterial data and patents all in one place. Visit Nano by Nature Research