Virus-Like Particles Derived from HIV-1 for Delivery of Nuclear Proteins: Improvement of Production and Activity by Protein Engineering
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.
KeywordsVirus-like particles HIV-1 Gag VLP production Protein delivery Protein engineering Green fluorescent protein Transcription factor
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.
- 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.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
- 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
- 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
- 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.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.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
- 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.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
- 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
- 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