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J Virol, February 1998, p. 1153-1159, Vol. 72, No. 2
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Overexpression of Nonconvertible PrPcDelta 114-121 in Scrapie-Infected Mouse Neuroblastoma Cells Leads to trans-Dominant Inhibition of Wild-Type PrPSc Accumulation

Christina Hölscher,1 Hajo Delius,2 and Alexander Bürkle1,*

Abteilung 06101 and Abteilung 0686,2 Deutsches Krebsforschungszentrum, Forschungsschwerpunkt Angewandte Tumorvirologie, D-69120 Heidelberg, Germany

Received 14 July 1997/Accepted 22 October 1997

One hallmark of prion diseases is the accumulation of the abnormal isoform PrPSc of a normal cellular glycoprotein, PrPc, which is characterized by a high content of beta -sheet structures and by its partial resistance to proteinase K. It was hypothesized that the PrP region comprising amino acid residues 109 to 122 [PrP(109-122)], which spontaneously forms amyloid when it is synthesized as a peptide but which does not display significant secondary structure in the context of the full-length PrPc molecule, should play a role in promoting the conversion into PrPSc. By using persistently scrapie-infected mouse neuroblastoma (Sc+-MNB) cells as a model system for prion replication, we set out to design dominant-negative mutants of PrPc that are capable of blocking the conversion of endogenous, wild-type PrPc into PrPSc. We constructed a deletion mutant (PrPcDelta 114-121) lacking eight codons that span most of the highly amyloidogenic part, AGAAAAGA, of PrP(109-122). Transient transfections of mammalian expression vectors encoding either wild-type PrPc or PrPcDelta 114-121 into uninfected mouse neuroblastoma cells (Neuro2a) led to overexpression of the respective PrPc versions, which proved to be correctly localized on the extracellular face of the plasma membrane. Transfection of Sc+-MNB cells revealed that PrPcDelta 114-121 was not a substrate for conversion into a proteinase K-resistant isoform. Furthermore, its presence led to a significant reduction in the steady-state levels of PrPSc derived from endogenous PrPc. Thus, we showed that the presence of amino acids 114 to 121 of mouse PrPc plays an important role in the conversion process of PrPc into PrPSc and that a deletion mutant lacking these codons indeed behaves as a dominant-negative mutant with respect to PrPSc accumulation. This mechanism could form a basis for a new gene therapy and/or a prevention concept for prion diseases.

* Corresponding author. Mailing address: Deutsches Krebsforschungszentrum, Forschungsschwerpunkt Angewandte Tumorvirologie, Abt. 0610, Im Neuenheimer Feld 242, D-69120 Heidelberg, Germany. Phone: 49-6221-424982. Fax: 49-6221-424962. E-mail: a.buerkle{at}dkfz-heidelberg.de.




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