Fig. 1.

Generation of TS SeV vectors and inactivation after temperature-shift treatment. (A) Point mutations were introduced into the polymerase-related genes P (P2: 433, 434, and 437) and/or L (942, 1361, and 1558), as indicated in the schematic structure of the ΔF/SeV vector. Open angles indicate conventional mutations in the previous TS vector; closed angles, newly introduced mutations. (B) Confluent LLC-MK2 cells were transduced with each SeV vector carrying GFP at an MOI of 5 and cultured at the indicated temperatures (32, 35, 37, 38, and 39 °C). Green fluorescence was compared at 3 d after infection. (C) To confirm the irreversible inactivation of gene expression by temperature-shift treatment, infected cells were cultured at 37 °C for 10 d and then split into two groups, one group cultured at 37 °C and the other cultured at 39 °C for 28 d, with cells passaged every 7 d. Similarly, cells infected with a TS vector treated at a nonpermissive temperature of 39 °C for 7 d were also cultured for a further 28 d at 37 °C, with cells passaged every 7 d, to evaluate GFP expression.