Fig. 1

Construction of lentivirus constructs LV15sol and LV15Rc. Diagram of the lentiviruses (LVs) pDy.tpa-mIL15 (LV15sol) and pDY.mIL-15Rc (LV15Rc). Both LVs contain LTR, HIV long terminal repeat; Ψ, human immunodeficiency virus packaging sequence; SD, 5′ splice donor; ΔGAG, truncated group antigen sequence; RRE, Rev. response element; SA, 3′ splice acceptor; cPPT, central polypurine tract; EF-1α, elongation factor-1 alpha promoter; WPRE, woodchuck hepatitis virus post-transcriptional regulatory element; SIN/LTR, self-inactivating HIV long terminal repeat. LV15sol: The signal sequence (s.s.) and pro-peptide of tissue plasminogen activator (tPA) (amino acids 1–35 as predicted by Uniprot bioinformatic analyses) replaced the endogenous signal sequence and pro-peptide (amino acids 1–48 as predicted by Uniprot bioinformatic analyses) of mouse IL-15. A DNA cassette comprising a Kozak consensus sequence and this IL-15sol cDNA was synthesized by Genscript (Piscataway, USA) and subcloned into the lentiviral backbone pDY.cPPT-EF1α.WPRE downstream of the EF-1a promoter. LV15Rc: The first 98 amino acids of mouse IL-15Rα, including its signal peptide and sushi domain as identified by Prosite bioinformatic analysis (SIB Swiss Institute of Bioinformatics), were fused to mouse IL-15 amino acids 49–162 (signal and pro-peptide removed by Uniprot bioinformatic analyses) by a linker (SGGSGGGGSGGGSGGGGSLQ). A DNA cassette comprising a Kozak consensus sequence upstream of this IL-15Rc cDNA was synthesized by Genscript (Piscataway, USA) and subcloned into the 3′SIN, HIV-1-based, lentiviral backbone pDY.cPPT-EF1α WPRE, downstream of the EF-1α promoter. Both vectors were verified by restriction enzyme digestion and DNA sequencing. Lentiviral particles were produced at the University Health Network Vector Production Facility