Suppressor of RNAi B2 protein increases titer and viral genome integrity of lentiviral vectors harboring bidirectional gene expression cassettes. (A) pLV-dt/ΔEGFP and pLV-dt/EGFP are the proviral vector plasmids used in production of these lentiviral vectors. TripZ-B2 is a lentiviral vector used to make the 293T-B2 cell line that expresses B2 following doxycycline treatment. The box legend to the right highlights the components of these proviral plasmids. Definitions are as follows: 5’LTR, 5’ long terminal repeat; ψ, psi, viral packaging signal sequence; RRE, rev response element, where Rev protein binds; cPPT, central polypurine tract, recognition site for proviral DNA synthesis; STOP, translation stop sequence; EGFP-nls, EGFP with nuclear localization signal; ΔCBAp, deletion of chicken beta-actin promoter (CBA); CBAp, CBA promoter in reverse orientation to the viral genomic transcript; PGKp, mouse phosphoglycerate kinase 1 promoter; TRE, tetracycline response element; UBCp, ubiquitin C promoter; rtTA, reverse tetracycline trans-activator; IRES, internal ribosomal entry site; WPRE, Woodchuck hepatitis virus post-transcriptional regulatory element for increasing nuclear export; 3’LTR (ΔU3), 3’ long terminal repeat with deletion in unique 3’ sequence that is necessary for activating viral genome transcription. (B) Comparison of LV-dt/EGFP titers produced by 293T-B2 with doxycycline treatment, 293T-B2 with vehicle treatment, and 293T with doxycycline treatment. Data show the mean+/-SEM for N = 6 viral preparations. (C) Comparison of lentiviral vector LV-dt/ΔEGFP transduction unit per milliliter (Tu/mL) produced by 293T-B2 with doxycycline treatment, 293T-B2 with vehicle (water) treatment, and 293T with doxycycline treatment. Data show the mean+/-SEM for N = 6 viral preparations. (D) Comparison of the percentage of cells positive for EGFP only among LV-dt/EGFP infected cells produced by 293T-B2 with doxycycline treatment, 293T-B2 with vehicle treatment, and 293T with doxycycline treatment. Data show the mean+/-SEM for N = 6 viral preparations. (B,C) Statistical comparisons were made by one-way ANOVA, Bonferroni’s multiple comparison test. ****, p < 0.0001. **, p < 0.01.