Fig. 5

Reduction of the copy number of SeVdp genome by Shield1-responsive Csy4. A Structure of SeVdp vectors. P450: rat cytochrome P450 2B1, ATG Csy4RS: Csy4 recognition sequence inserted immediately after the start codon of the L gene. B Inhibition of viral replication and transcription to change EGFP expression. NIH3T3 cells were infected with the indicated vector and selected with G418. The vector-infected cells were cultured with or without 300 nM Shield1 for 6 days. Scale bars, 100 μm. C Relative copy numbers of the vector genome after inhibition of viral replication and transcription. Cells prepared as B were cultured with or without 300 nM Shield1. At indicated date, RNA was extracted from the cells, and the amount of SeVdp genomic RNA was determined by RT-qPCR. Data are represented as the means ± SEM of three independent experiments. D Selection of SeVdp vector-free cells using P450-based negative selection. NIH3T3 cells infected with the indicated vector were cultured with 300 nM Shield1 for 20 days to eliminate the vector from the cells, and then residual vector-harboring cells were removed by exposure with 1 mM CPA for 2 or 4 days. Scale bars, 100 μm. E Crystal violet assay of vector-free cells. NIH3T3 cells infected with the indicated vector were cultured with or without 300 nM Shield1 for 9 or 20 days, followed by exposure with 1 mM CPA for 7 days. The cells were subjected to Crystal violet assay. Data are represented as the means ± SEM of three independent experiments. *p < 0.05 and ***p < 0.001 versus SeV(Csy4/P450)-infected cells cultured without Shield1. ^#p < 0.05. ^##p < 0.01. ^###p < 0.001. F Determination of the relative copy numbers of the vector genome in cells after Shield1 and CPA treatments. NIH3T3 cells infected with SeV(Csy4/P450/ATG-RS-L) were cultured with 300 nM Shield1 for 20 days and then with 1 mM CPA for additional 7 days (27 days) followed by cell culture without Shield1 and CPA for 20 days (47 days). The copy number of SeV genomic RNA in the cells was determined by RT-qPCR. Data are represented as the means ± SEM of three independent experiments. mock: uninfected cells, n.d: not detected