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First published online 19 October 2004
doi: 10.1242/jcs.01484

Journal of Cell Science 117, 5591-5597 (2004)
Published by The Company of Biologists 2004
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Inhibition of PrPSc formation by lentiviral gene transfer of PrP containing dominant negative mutations

Carole Crozet1, Yea-Lih Lin2, Clément Mettling2, Chantal Mourton-Gilles3, Pierre Corbeau2, Sylvain Lehmann1,4 and Véronique Perrier1,*

1 Laboratoire de Biologie des Encéphalopathies Spongiformes, CNRS UPR 1142, 141 rue de la Cardonille, 34396 Montpellier CEDEX 5, France
2 Laboratoire Lentivirus et Transfert de Gènes – Institut de Génétique Humaine, CNRS UPR 1142, 141 rue de la Cardonille, 34396 Montpellier CEDEX 5, France
3 BIO-RAD, Centre de Pharmacologie et Biotechnologies pour la Santé, CNRS UMR 5094, 15 avenue C. Flahault, 34060 Montpellier CEDEX 2, France
4 Laboratoire de Biochimie. Hopital St Eloi, 80 avenue A. Fliche 34295 Montpellier CEDEX 5, France



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  		Fig. 1. Characteristics of the lentiviral vectors. The packaging vector, was derived from the prototypic FIV-14-Petaluma genome (AIDS Research and Reference Reagent Program), in which the 5' LTR is replaced by a CMV promoter and the 3' LTR by a poly-A signal. The transfer vector was also obtained from FIV-14-Petaluma by replacing the U3 region of the 5' LTR with a CMV promoter in order to enable the expression of the vector in cells. Moreover, the gag gene was truncated at position 1243 and a frameshift introduced at position 926. The pol, vif, orfA, env and rev genes were replaced by a cassette containing the mutated PrP sequences driven by the phosphoglycerate kinase promoter. The PrP sequence is followed by an IRES sequence and the enhanced GFP marker gene. The third vector pMD.G, used for pseudotyping, contains the VSV-G envelope gene, which encodes the envelope G protein of the vesicular stomatitis virus. PGK, phosphoglycerate kinase promoter; CTE, constitutive transport element; cPPT CTS, central DNA flap sequence; SD, splice donor site.

 


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  		Fig. 4. In vivo delivery of FIV lentiviral vectors. (A-E) Brain slices of mice intracerebrally injected with FIV lentiviral vectors carrying the ß-galactosidase gene after X-Gal staining (blue coloration) 1 week after injection. (A, B) According to the site of injection (red arrow), the blue coloration was observed in the mediodorsal thalamic nucleus. (C-E) X-Gal staining was also observed along the needle passage: (C) the cortex as well as in the fibers of the corpus callosum, (D) the wall of the ventricules and (E) the anteroventral thalamic nucleus. (F-K) After intraventricular injection, close immunohistochemical examination using anti-ß-galactosidase antibody, allowed the detection of the ß-galactosidase expression (red staining) 3 weeks after injection in (I) the cortex, (J) the striatum, and (K) the cerebellum. No staining was observed in non-injected mice used as control. F, cortex, G, striatum, H, cerebellum). (L-Q) Sections of spleen and liver from intravenously injected mice were analysed by immunocytochemistry. Positive cells were detected in the vicinity of the blood vessels, in (M-N) the spleen, (Q) the liver and (P) some isolated cells in the liver. No staining was observed in non-injected mice (L, spleen; O, Liver). (Cx, cortex; Hp, hippocampus; MDTh, mediodorsal thalamic nucleus; CC, corpus callosum; LV, lateral ventricule; vhc, ventral hippocampal commissure; AV Th, anteroventral thalamic nucleus; BV, blood vessel. Scale bars in A-E, 1 mm; in F-N, 10 µm.

 


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  		Fig. 2. Transduction of Prnp–/– cells with lentivirus carrying the PrP dominant negative mutants MoPrPQ167R and MoPrPQ218K. (A) Transduction efficacy of Prnp–/– cells with lentiviral vectors assessed by GFP fluorescence. Images A1, A2, A3 and A4 correspond to non-transduced, GFP, MoPrPQ167R and MoPrPQ218K transduced cells, respectively. (B) Detection of PrPC by immunoblotting using SAF32 anti-PrP antibody (C) SAF32 immunocytofluorescence of the PrPC in Prnp–/– cells: no staining is visible in (C5) non-transduced and (C6) GFP lentiviral transduced cells, whereas cells transduced with the (C7) MoPrPQ167R, or (C8) MoPrPQ218K lentivirus exhibit a strong rhodamine fluorescence. C1, C2, C3, C4 correspond to the Hoechst nuclear staining of the cells represented in figure C5, C6, C7, C8 respectively. Scale bars, 8 µm.

 


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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 MoPrPSc levels by dominant negative mutants in N2a58/22L cells. Proteinase K digested cell lysates were analyzed by immunoblotting and the wild-type MoPrPSc levels revealed with SAF mix. N2a58/22L cells expressing the MoPrPQ218K mutant showed a strong inhibition of the PrPSc level at day 8 (D8) and day 12 (D12) after transduction, whereas cells expressing MoPrPQ167R mutants showed a slight diminution. (C) Kinetic analysis of PrPSc 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 PrPSc 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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© The Company of Biologists Ltd 2004