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.
Département de génie chimique, Université Laval, 1065 Avenue de la Médecine, Québec, QC, G1V 0A6, Canada.|National Research Council Canada, 6100 Avenue Royalmount, Montréal, QC, H4P 2R2, Canada.|Regroupement québécois de recherche sur la fonction, l'ingénierie et les applications des protéines, PROTEO, Québec, QC, Canada.|Réseau de thérapie cellulaire et tissulaire du FRQS, ThéCell, Québec, QC, Canada.
Marc-André
MA
Robert
National Research Council Canada, 6100 Avenue Royalmount, Montréal, QC, H4P 2R2, Canada.
Viktoria
V
Lytvyn
National Research Council Canada, 6100 Avenue Royalmount, Montréal, QC, H4P 2R2, Canada.
Francis
F
Deforet
National Research Council Canada, 6100 Avenue Royalmount, Montréal, QC, H4P 2R2, Canada.|Réseau de thérapie cellulaire et tissulaire du FRQS, ThéCell, Québec, QC, Canada.
Rénald
R
Gilbert
Département de génie chimique, Université Laval, 1065 Avenue de la Médecine, Québec, QC, G1V 0A6, Canada. bruno.gaillet@gch.ulaval.ca.|Regroupement québécois de recherche sur la fonction, l'ingénierie et les applications des protéines, PROTEO, Québec, QC, Canada. bruno.gaillet@gch.ulaval.ca.|Réseau de thérapie cellulaire et tissulaire du FRQS, ThéCell, Québec, QC, Canada. bruno.gaillet@gch.ulaval.ca.
Bruno
http://orcid.org/0000-0002-9903-0116
B
Gaillet
D000072283:A549 Cells; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D015497:HIV-1 / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors / Q000235:genetics / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014771:Virion / Q000235:genetics; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000235:genetics*
C000715467:KLF4 protein, human; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus; D049452:Green Fluorescent Proteins
Green fluorescent protein; HIV-1 Gag; Protein delivery; Protein engineering; Transcription factor; VLP production; Virus-like particles