title: Production of HIV-1 integrase fusion protein-carrying lentiviral vectors for gene therapy and protein transduction.
abstract: Lentiviral vectors have broad target cell tropism and efficient machinery to integrate transgenes into the host genome. Modification of these vectors by incorporating heterologous proteins into virions has relied mostly on the fusion of proteins into the HIV-1 accessory protein Vpr. Vpr expression can be harmful for cells and its gene has been deleted from third-generation vector production plasmids. We therefore developed a direct integrase fusion protein strategy as an alternative way to package heterologous proteins into vectors. The method was tested by creating two different integrase fusion proteins, IN-p53 and IN-mCherry, cloned into the 3' end of pol in the packaging plasmid. Lentiviral vectors were produced by conventional methods, using the modified packaging plasmids. Vector-incorporated fusion proteins were correctly processed from Gag-Pol, retained the ability to catalyze transgene integration, and showed fusion protein-specific activity by being fluorescent or inducing apoptosis. Functional third-generation lentiviral vectors containing IN-fusion proteins can thus be produced by standard production protocols independent of Vpr expression. Our results suggest that this packaging method is useful for lentiviral vector-mediated protein transduction, such as intranuclear meganuclease, transposon, or zinc finger protein delivery, intracellular imaging of vector particles, and generation of modified lentiviral vectors that contain both toxic and nontoxic IN-fusion proteins.
authors.affiliation: Department of Biotechnology and Molecular Medicine, A.I. Virtanen Institute for Molecular Sciences, University of Kuopio, FIN-70211 Kuopio, Finland.
authors.forename: Diana
authors.initials: D
authors.lastname: Schenkwein
authors.forename: Vesa
authors.initials: V
authors.lastname: Turkki
authors.forename: Hanna-Riikka
authors.initials: HR
authors.lastname: Kärkkäinen
authors.forename: Kari
authors.initials: K
authors.lastname: Airenne
authors.forename: Seppo
authors.initials: S
authors.lastname: Ylä-Herttuala
mesh_terms: D015751:Genes, pol / Q000187:drug effects; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000187:drug effects; D019427:HIV Integrase; D015497:HIV-1 / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D006801:Humans; D019426:Integrases / Q000235:genetics*; D010957:Plasmids / Q000187:drug effects; D011506:Proteins / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics; D019076:Transgenes / Q000187:drug effects; D014771:Virion / Q000187:drug effects / Q000235:genetics; D054325:vpr Gene Products, Human Immunodeficiency Virus
chemical_list: D011506:Proteins; D011994:Recombinant Proteins; D054325:vpr Gene Products, Human Immunodeficiency Virus; C514912:vpr protein, Human immunodeficiency virus 1; D019427:HIV Integrase; D019426:Integrases; C514801:p31 integrase protein, Human immunodeficiency virus 1