Molecular Biotechnology

, Volume 59, Issue 1, pp 9–23 | Cite as

Virus-Like Particles Derived from HIV-1 for Delivery of Nuclear Proteins: Improvement of Production and Activity by Protein Engineering

  • Marc-André Robert
  • Viktoria Lytvyn
  • Francis Deforet
  • Rénald Gilbert
  • Bruno Gaillet
Original Paper

Abstract

Virus-like particles (VLPs) derived from retroviruses and lentiviruses can be used to deliver recombinant proteins without the fear of causing insertional mutagenesis to the host cell genome. In this study we evaluate the potential of an inducible lentiviral vector packaging cell line for VLP production. The Gag gene from HIV-1 was fused to a gene encoding a selected protein and it was transfected into the packaging cells. Three proteins served as model: the green fluorescent protein and two transcription factors—the cumate transactivator (cTA) of the inducible CR5 promoter and the human Krüppel-like factor 4 (KLF4). The sizes of the VLPs were 120–150 nm in diameter and they were resistant to freeze/thaw cycles. Protein delivery by the VLPs reached up to 100% efficacy in human cells and was well tolerated. Gag-cTA triggered up to 1100-fold gene activation of the reporter gene in comparison to the negative control. Protein engineering was required to detect Gag-KLF4 activity. Thus, insertion of the VP16 transactivation domain increased the activity of the VLPs by eightfold. An additional 2.4-fold enhancement was obtained by inserting nuclear export signal. In conclusion, our platform produced VLPs capable of efficient protein transfer, and it was shown that protein engineering can be used to improve the activity of the delivered proteins as well as VLP production.

Keywords

Virus-like particles HIV-1 Gag VLP production Protein delivery Protein engineering Green fluorescent protein Transcription factor 

Notes

Acknowledgements

This work was funded by a NSERC/CIHR jointed grant #315642. M.-A.R. was supported by grants from ThéCell and PROTÉO networks. The authors declare no conflict of interests.

Supplementary material

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Supplementary material 1 (TIFF 45 kb)
12033_2016_9987_MOESM2_ESM.tif (107 kb)
Supplementary material 2 (TIFF 107 kb)
12033_2016_9987_MOESM3_ESM.tif (5 mb)
Supplementary material 3 (TIFF 5150 kb)

References

  1. 1.
    Mortlock, A., Low, W., & Crisanti, A. (2003). Suppression of gene expression by a cell-permeable Tet repressor. Nucleic Acids Research, 31, e152.CrossRefGoogle Scholar
  2. 2.
    Zhang, H., Ma, Y., Gu, J., Liao, B., Li, J., Wong, J., et al. (2012). Reprogramming of somatic cells via TAT-mediated protein transduction of recombinant factors. Biomaterials, 33, 5047–5055.CrossRefGoogle Scholar
  3. 3.
    Zhou, H., Wu, S., Joo, J. Y., Zhu, S., Han, D. W., Lin, T., et al. (2009). Generation of induced pluripotent stem cells using recombinant proteins. Cell Stem Cell, 4, 381–384.CrossRefGoogle Scholar
  4. 4.
    Kim, D., Kim, C.-H., Moon, J.-I., Chung, Y.-G., Chang, M.-Y., Han, B.-S., et al. (2010). Generation of human induced pluripotent stem cells by direct delivery of reprogramming proteins. Cell Stem Cell, 4, 472–476.CrossRefGoogle Scholar
  5. 5.
    Ramakrishna, S., Kwaku Dad, A. B., Beloor, J., Gopalappa, R., Lee, S. K., & Kim, H. (2014). Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA. Genome Research, 24, 1020–1027.CrossRefGoogle Scholar
  6. 6.
    Zuris, J. A., Thompson, D. B., Shu, Y., Guilinger, J. P., Bessen, J. L., Hu, J. H., et al. (2014). Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nature Biotechnology, 33, 73–80.CrossRefGoogle Scholar
  7. 7.
    Liu, J., Gaj, T., Patterson, J. T., Sirk, S. J., & Barbas, C. F. (2014). Cell-penetrating peptide-mediated delivery of TALEN proteins via bioconjugation for genome engineering. PLoS ONE, 9, e85755.CrossRefGoogle Scholar
  8. 8.
    Liu, J., Gaj, T., Wallen, M. C., & Barbas, C. F. (2015). Improved cell-penetrating zinc-finger nuclease proteins for precision genome engineering. Molecular Therapy Acids, 4, e232.CrossRefGoogle Scholar
  9. 9.
    Lee, C.-Y., Li, J.-F., Liou, J.-S., Charng, Y.-C., Huang, Y.-W., & Lee, H.-J. (2011). A gene delivery system for human cells mediated by both a cell-penetrating peptide and a piggyBac transposase. Biomaterials, 32, 6264–6276.CrossRefGoogle Scholar
  10. 10.
    Järver, P., Fernaeus, S., El-Andaloussi, S., Tjörnhammar, M. L. & Langel, Ü. (2008). Co-transduction of sleeping beauty transposase and donor plasmid via a cell-penetrating peptide: A simple one step method. International Journal of Peptide Research and Therapeutics, 14, 58–63.CrossRefGoogle Scholar
  11. 11.
    Patsch, C., Peitz, M., Otte, D. M., Kesseler, D., Jungverdorben, J., Wunderlich, F. T., et al. (2010). Engineering cell-permeant FLP recombinase for tightly controlled inducible and reversible overexpression in embryonic stem cells. Stem Cells, 28, 894–902.Google Scholar
  12. 12.
    Jo, D., Nashabi, A., Doxsee, C., Lin, Q., Unutmaz, D., Chen, J., et al. (2001). Epigenetic regulation of gene structure and function with a cell-permeable Cre recombinase. Nature Biotechnology, 19, 929–933.CrossRefGoogle Scholar
  13. 13.
    Justesen, S., Buus, S., Claesson, M. H., & Pedersen, A. E. (2007). Addition of TAT protein transduction domain and GrpE to human p53 provides soluble fusion proteins that can be transduced into dendritic cells and elicit p53-specific T-cell responses in HLA-A*0201 transgenic mice. Immunology, 122, 326–334.CrossRefGoogle Scholar
  14. 14.
    Varkouhi, A. K., Scholte, M., Storm, G., & Haisma, H. J. (2011). Endosomal escape pathways for delivery of biologicals. Journal of Controlled Release, 151, 220–228.CrossRefGoogle Scholar
  15. 15.
    van den Berg, A., & Dowdy, S. F. (2011). Protein transduction domain delivery of therapeutic macromolecules. Current Opinion in Biotechnology, 22, 888–893.CrossRefGoogle Scholar
  16. 16.
    Yamaguchi, K., Inoue, M., & Goshima, N. (2011). Efficient protein transduction method using cationic peptides and lipids. Journal of Biomedicine and Biotechnology, 2011, 872065.CrossRefGoogle Scholar
  17. 17.
    Erazo-Oliveras, A., Muthukrishnan, N., Baker, R., Wang, T. Y., & Pellois, J. P. (2012). Improving the endosomal escape of cell-penetrating peptides and their cargos: Strategies and challenges. Pharmaceuticals, 5, 1177–1209.CrossRefGoogle Scholar
  18. 18.
    Cicalese, M. P., & Aiuti, A. (2015). Clinical applications of gene therapy for primary immunodeficiencies. Human Gene Therapy, 26, 210–219.CrossRefGoogle Scholar
  19. 19.
    Oldham, R. A., Berinstein, E. M., & Medin, J. A. (2015). Lentiviral vectors in cancer immunotherapy. Immunotherapy, 7, 271–284.CrossRefGoogle Scholar
  20. 20.
    Bayart, E., & Cohen-Haguenauer, O. (2013). Technological overview of iPS induction from human adult somatic cells. Current Gene Therapy, 13, 73–92.CrossRefGoogle Scholar
  21. 21.
    Luo, T., Douglas, J. L., Livingston, R. L., & Garcia, J. V. (1998). Infectivity enhancement by HIV-1 Nef is dependent on the pathway of virus entry: implications for HIV-based gene transfer systems. Virology, 241, 224–233.CrossRefGoogle Scholar
  22. 22.
    Cai, Y., & Mikkelsen, J. G. (2014). Driving DNA transposition by lentiviral protein transduction. Mobile Genetic Elements, 4, e29591.CrossRefGoogle Scholar
  23. 23.
    Carlson, L. A., Briggs, J. A. G., Glass, B., Riches, J. D., Simon, M. N., Johnson, M. C., et al. (2008). Three-dimensional analysis of budding sites and released virus suggests a revised model for HIV-1 morphogenesis. Cell Host Microbe, 4, 592–599.CrossRefGoogle Scholar
  24. 24.
    Bell, N. M., & Lever, A. M. L. (2013). HIV Gag polyprotein: processing and early viral particle assembly. Trends in Microbiology, 21, 136–144.CrossRefGoogle Scholar
  25. 25.
    Jalaguier, P., Turcotte, K., Danylo, A., Cantin, R., & Tremblay, M. J. (2011). Efficient production of HIV-1 virus-like particles from a mammalian expression vector requires the N-terminal capsid domain. PLoS ONE, 6, e28314.CrossRefGoogle Scholar
  26. 26.
    Crist, R. M., Datta, S. A. K., Stephen, A. G., Soheilian, F., Mirro, J., Fisher, R. J., et al. (2009). Assembly properties of human immunodeficiency virus type 1 Gag-leucine zipper chimeras: implications for retrovirus assembly. Journal of Virology, 83, 2216–2225.CrossRefGoogle Scholar
  27. 27.
    Accola, M. A., Strack, B., & Göttlinger, H. G. (2000). Efficient particle production by minimal Gag constructs which retain the carboxy-terminal domain of human immunodeficiency virus type 1 capsid-p2 and a late assembly domain. Journal of Virology, 74, 5395–5402.CrossRefGoogle Scholar
  28. 28.
    Lee, S. K., Potempa, M., & Swanstrom, R. (2012). The choreography of HIV-1 proteolytic processing and virion assembly. Journal of Biological Chemistry, 287, 40867–40874.CrossRefGoogle Scholar
  29. 29.
    Aoki, T., Shimizu, S., Urano, E., Futahashi, Y., Hamatake, M., Tamamura, H., et al. (2010). Improvement of lentiviral vector-mediated gene transduction by genetic engineering of the structural protein Pr55 Gag. Gene Therapy, 17, 1124–1133.CrossRefGoogle Scholar
  30. 30.
    Aoki, T., Miyauchi, K., Urano, E., Ichikawa, R., & Komano, J. (2011). Protein transduction by pseudotyped lentivirus-like nanoparticles. Gene Therapy, 18, 936–941.CrossRefGoogle Scholar
  31. 31.
    Uhlig, K. M., Schülke, S., Scheuplein, V. A., Malczyk, A. H., Reusch, J., Kugelmann, S., et al. (2015). Lentiviral protein transfer vectors are an efficient vaccine-platform inducing strong antigen-specific cytotoxic T cell response. Journal of Virology, 89, 9044–9060.CrossRefGoogle Scholar
  32. 32.
    Cai, Y., Bak, R. O., Krogh, L. B., Staunstrup, N. H., Moldt, B., Corydon, T. J., et al. (2014). DNA transposition by protein transduction of the piggyBac transposase from lentiviral Gag precursors. Nucleic Acids Research, 42, e28.CrossRefGoogle Scholar
  33. 33.
    Miyauchi, K., Urano, E., Takizawa, M., Ichikawa, R., & Komano, J. (2012). Therapeutic potential of HIV protease-activable CASP3. Scientific Reports, 2, 1–7.CrossRefGoogle Scholar
  34. 34.
    Müller, B., Daecke, J., Fackler, O. T., Dittmar, T., Zentgraf, H., Kräusslich, H., et al. (2004). Construction and characterization of a fluorescently labeled infectious human immunodeficiency virus type 1 derivative construction and characterization of a fluorescently labeled infectious human immunodeficiency virus type 1 derivative. Journal of Virology, 78, 10803–10813.CrossRefGoogle Scholar
  35. 35.
    Voelkel, C., Galla, M., Maetzig, T., Warlich, E., Kuehle, J., Zychlinski, D., et al. (2010). Protein transduction from retroviral Gag precursors. Proceedings of the National Academy of Sciences USA, 107, 7805–7810.CrossRefGoogle Scholar
  36. 36.
    Kaczmarczyk, S. J., Sitaraman, K., Young, A. H., Hughes, S. H., & Chatterjee, D. K. (2011). Protein delivery using engineered virus-like particles. Proceedings of the National Academy of Sciences, 41, 16998–17003.CrossRefGoogle Scholar
  37. 37.
    Wu, D. T., & Roth, M. J. (2014). MLV based viral-like-particles for delivery of toxic proteins and nuclear transcription factors. Biomaterials, 35, 8416–8426.CrossRefGoogle Scholar
  38. 38.
    Broussau, S., Jabbour, N., Lachapelle, G., Durocher, Y., Tom, R., Transfiguracion, J., et al. (2008). Inducible packaging cells for large-scale production of lentiviral vectors in serum-free suspension culture. Molecular Therapy, 16, 500–507.CrossRefGoogle Scholar
  39. 39.
    Massie, B., Mosser, D. D., Koutroumanis, M., Vitté-Mony, I., Lamoureux, L., Couture, F., et al. (1998). New adenovirus vectors for protein production and gene transfer. Cytotechnology, 28, 53–64.CrossRefGoogle Scholar
  40. 40.
    Cressman, D. E., O’Connor, W. J., Greer, S. F., Zhu, X. S., & Ting, J. P. (2001). Mechanisms of nuclear import and export that control the subcellular localization of class II transactivator. The Journal of Immunology, 167, 3626–3634.CrossRefGoogle Scholar
  41. 41.
    Mullick, A., Xu, Y., Warren, R., Koutroumanis, M., Guilbault, C., Broussau, S., et al. (2006). The cumate gene-switch: a system for regulated expression in mammalian cells. BMC Biotechnology, 6, 43.CrossRefGoogle Scholar
  42. 42.
    Güttler, T., Madl, T., Neumann, P., Deichsel, D., Corsini, L., Monecke, T., et al. (2010). NES consensus redefined by structures of PKI-type and Rev-type nuclear export signals bound to CRM1. Nature Structural & Molecular Biology, 17, 1367–1376.CrossRefGoogle Scholar
  43. 43.
    Gaillet, B., Gilbert, R., Broussau, S., Pilotte, A., Malenfant, F., Mullick, A., et al. (2010). High-level recombinant protein production in CHO cells using lentiviral vectors and the cumate gene-switch. Biotechnology and Bioengineering, 106, 203–215.Google Scholar
  44. 44.
    Robert, M. A., Lin, Y., Bendjelloul, M., Zeng, Y., Dessolin, S., Broussau, S., et al. (2012). Strength and muscle specificity of a compact promoter derived from the slow troponin I gene in the context of episomal (gutless adenovirus) and integrating (lentiviral) vectors. The Journal of Gene Medicine, 14, 746–760.CrossRefGoogle Scholar
  45. 45.
    Chabaud, S., Sasseville, A. J.-M., Elahi, S. M., Caron, A., Dufour, F., Massie, B., et al. (2007). The ribonucleotide reductase domain of the R1 subunit of herpes simplex virus type 2 ribonucleotide reductase is essential for R1 antiapoptotic function. Journal of General Virology, 88, 384–394.CrossRefGoogle Scholar
  46. 46.
    Alain, R., Nadon, F., Seguin, C., Payment, P., & Trudel, M. (1987). Rapid virus subunit visualization by direct sedimentation of samples on electron microscope grids. Journal of Virological Methods, 16, 209–216.CrossRefGoogle Scholar
  47. 47.
    Finkelshtein, D., Werman, A., Novick, D., Barak, S., & Rubinstein, M. (2013). LDL receptor and its family members serve as the cellular receptors for vesicular stomatitis virus. Proceedings of the National Academy of Sciences USA, 110, 7306–7311.CrossRefGoogle Scholar
  48. 48.
    Aiken, C. (1997). Pseudotyping human immunodeficiency virus type 1 (HIV-1) by the glycoprotein of vesicular stomatitis virus targets HIV-1 entry to an endocytic pathway and suppresses both the requirement for Nef and the sensitivity to cyclosporin A. Journal of Virology, 71, 5871–5877.Google Scholar
  49. 49.
    Dull, T., Zufferey, R., Kelly, M., Mandel, R. J., Nguyen, M., Trono, D., et al. (1998). A third-generation lentivirus vector with a conditional packaging system. Journal of Virology, 72, 8463–8471.Google Scholar
  50. 50.
    Jacks, T., Power, M. D., Masiarz, F. R., Luciw, P. A., Barr, P. J., & Varmus, H. E. (1988). Characterization of ribosomal frameshifting in HIV-1 gag-pol expression. Nature, 331, 280–283.CrossRefGoogle Scholar
  51. 51.
    Kutner, R. H., Zhang, X.-Y., & Reiser, J. (2009). Production, concentration and titration of pseudotyped HIV-1-based lentiviral vectors. Nature Protocols, 4, 495–505.CrossRefGoogle Scholar
  52. 52.
    Cervera, L., Gutiérrez-Granados, S., Martínez, M., Blanco, J., Gòdia, F., & Segura, M. M. (2013). Generation of HIV-1 Gag VLPs by transient transfection of HEK 293 suspension cell cultures using an optimized animal-derived component free medium. Journal of Biotechnology, 166, 152–165.CrossRefGoogle Scholar
  53. 53.
    Venereo-Sanchez, A., Gilbert, R., Simoneau, M., Caron, A., Chahal, P. S., Chen, W., et al. (2016). Hemagglutinin and neuraminidase containing virus-like particles produced in HEK-293 suspension culture: An effective influenza vaccine candidate. Vaccine, 34, 3371–3380.CrossRefGoogle Scholar
  54. 54.
    Cai, Y., Bak, R. O., & Mikkelsen, J. G. (2014). Targeted genome editing by lentiviral protein transduction of zinc-finger and TAL-effector nucleases. Elife, 3, e01911.CrossRefGoogle Scholar
  55. 55.
    Haffar, O. K., Popov, S., Dubrovsky, L., Agostini, I., Tang, H., Pushkarsky, T., et al. (2000). Two nuclear localization signals in the HIV-1 matrix protein regulate nuclear import of the HIV-1 pre-integration complex. Journal of Molecular Biology, 299, 359–368.CrossRefGoogle Scholar
  56. 56.
    Gamper, A. M., Qiao, X., Kim, J., Zhang, L., De Simone, M. C., Rathmell, W. K., et al. (2012). Regulation of KLF4 turnover reveals an unexpected tissue-specific role of pVHL in tumorigenesis. Molecular Cell, 45, 233–243.CrossRefGoogle Scholar
  57. 57.
    Chen, Z. Y., Wang, X., Zhou, Y., Offner, G., & Tseng, C. C. (2005). Destabilization of Krüppel-like factor 4 protein in response to serum stimulation involves the ubiquitin-proteasome pathway. Cancer Research, 65, 10394–10400.CrossRefGoogle Scholar
  58. 58.
    Wang, Y., Chen, J., Hu, J.-L., Wei, X.-X., Qin, D., Gao, J., et al. (2011). Reprogramming of mouse and human somatic cells by high-performance engineered factors. EMBO Reports, 12, 373–378.CrossRefGoogle Scholar
  59. 59.
    Negrete, A., Pai, A., & Shiloach, J. (2014). Use of hollow fiber tangential flow filtration for the recovery and concentration of HIV virus-like particles produced in insect cells. Journal of Virological Methods, 195, 240–246.CrossRefGoogle Scholar
  60. 60.
    Yang, L., Song, Y., Li, X., Huang, X., Liu, J., Ding, H., et al. (2012). HIV-1 virus-like particles produced by stably transfected drosophila S2 Cells: a desirable vaccine component. Journal of Virology, 86, 7662–7676.CrossRefGoogle Scholar
  61. 61.
    Urano, E., Aoki, T., Futahashi, Y., Murakami, T., Morikawa, Y., Yamamoto, N., et al. (2008). Substitution of the myristoylation signal of human immunodeficiency virus type 1 Pr55Gag with the phospholipase C-δ1 pleckstrin homology domain results in infectious pseudovirion production. Journal of General Virology, 89, 3144–3149.CrossRefGoogle Scholar
  62. 62.
    Gutiérrez-Granados, S., Cervera, L., Gòdia, F., & Segura, M. (2013). Characterization and quantitation of fluorescent Gag virus-like particles. BMC Proceedings, 7, P62.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Département de génie chimiqueUniversité LavalQuébecCanada
  2. 2.National Research Council CanadaMontréalCanada
  3. 3.Regroupement québécois de recherche sur la fonction, l’ingénierie et les applications des protéines, PROTEOQuébecCanada
  4. 4.Réseau de thérapie cellulaire et tissulaire du FRQS, ThéCellQuébecCanada

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