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Fig. 3. Transduction of N2a58/22L cells with the MoPrPQ167R and MoPrPQ218K virions. (A) GFP analysis after transduction of N2a58/22L cells. (A1) non-transduced cells, (A2) cells transduced with GFP vector, (A3) MoPrPQ167R or (A4) MoPrPQ218K PrP mutants. (B) Inhibition of endogenous MoPrP^Sc levels by dominant negative mutants in N2a58/22L cells. Proteinase K digested cell lysates were analyzed by immunoblotting and the wild-type MoPrP^Sc levels revealed with SAF mix. N2a58/22L cells expressing the MoPrPQ218K mutant showed a strong inhibition of the PrP^Sc level at day 8 (D8) and day 12 (D12) after transduction, whereas cells expressing MoPrPQ167R mutants showed a slight diminution. (C) Kinetic analysis of PrP^Sc inhibition in N2a58/22L cells by western blot analysis, at 8, 12 and 15 days (lanes 1, 2 and 3, respectively) after transduction. (NT) non-transduced cells, (GFP) cells transduced with virions carrying GFP only, or virions carrying (Q167R) MoPrPQ167R or (Q218K) MoPrPQ218K. These results are representative for several independent experiments. (D) Immunofluorescence of the PrP^Sc accumulation using SAF61 anti-PrP antibody 20 days after transduction. D5 non-transduced, D6: GFP, D7: MoPrPQ167R, D8: MoPrPQ218K. D1, D2, D3, D4 correspond to the Hoechst nuclear staining of the cells presented in figure D5, D6, D7, D8 respectively. Scale bars, 8 µm.

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