Journal of General Virology (1999), 80, 11–14. Printed in Great Britain .......................................................................................................................................................................................................... SHORT COMMUNICATION Protease-resistant prion protein produced in vitro lacks detectable infectivity Andrew F. Hill, 1 Michael Antoniou 2 and John Collinge 1, 3 1 Department of Neurogenetics, Imperial College School of Medicine at St Mary’s, London W2 1PG, UK 2 Department of Experimental Pathology, United Medical and Dental School, Guy’s Hospital, London SE1 9RT, UK 3 Department of Neurology, St Mary’s Hospital, London W2 1NY, UK The ‘ protein-only ’ hypothesis of prion propagation argues that infectious prions consist of PrP Sc ,a conformational isomer of host-derived prion pro- tein (PrP C ), which can be distinguished from PrP C by its partial resistance to proteases. While protease- resistant PrP has been produced by mixing PrP Sc and recombinant-derived PrP C in vitro, bioassay of any new infectivity has been precluded by the need to use a large molar excess of same species PrP Sc . Transgenic mice expressing a chimaeric hamster– mouse PrP C (MH2M PrP C ) are, unlike conventional mice, highly susceptible to Sc237 hamster scrapie. In addition, they produce MH2M PrP Sc and infec- tivity which is pathogenic for conventional mice. We have therefore attempted to produce MH2M PrP Sc in vitro as any infectivity produced could be distinguished from the hamster PrP Sc used to promote the conversion by bioassay in conventional mice. Although protease-resistant MH2M PrP was produced, no infectivity was detected on bioassay. These results argue that acquisition of protease resistance by PrP C is not sufficient for the propa- gation of infectivity. Prion diseases, such as Creutzfeldt–Jakob disease in humans and scrapie and bovine spongiform encephalopathy in animals, are associated with the accumulation of a disease-specific isoform of cellular prion protein (PrP C ), designated PrP Sc , which appears to be the principal or sole component of the transmissible agent or prion (Prusiner, 1991). PrP Sc is derived post-translationally from its cellular precursor, PrP C , and it is hypothesized that PrP Sc binds cellular PrP C and templates its conversion to further PrP Sc . PrP Sc is distinguished from PrP C by its physico-chemical properties, in particular by its partial Author for correspondence : John Collinge (at Imperial College School of Medicine at St Mary’s). Fax 44 171 706 7094. e-mail j.collingeic.ac.uk resistance to proteolysis. The ‘ protein-only ’ hypothesis for prion propagation implies that it ought to be possible to generate prions in vitro from highly purified, recombinant- derived PrP. An important milestone was the demonstration that protease-resistant PrP could be generated in vitro by mixing hamster PrP C with a large excess of hamster PrP Sc under suitable conditions (Kocisko et al., 1994). In addition, such ‘ conversion ’ shows both the species specificity and strain selection characteristic of prion propagation (Bessen et al., 1995 ; Kocisko et al., 1995). However, while protease resistance is a key marker of prions, a number of experiments appear to uncouple infectivity and protease resistance (Xi et al., 1992; Collinge et al., 1995 ; Lasmezas et al., 1997; Hsiao et al., 1990, 1994). It is therefore crucial to determine by bioassay whether such protease-resistant material produced in vitro is actually infectious. The terminology PrP* has been suggested to designate infectious PrP, which may or may not be protease resistant (Weissmann, 1991). However, the requirement to use a large molar excess of PrP Sc from the same species to drive such in vitro conversion has so far precluded bioassay of any newly produced infectious agent. Conventional mice are typically highly resistant to infection with hamster prions. This ‘ species barrier ’ to infection of mice with hamster prions is ablated in transgenic mice expressing a chimaeric PrP in which the central region of mouse PrP (residues 94–188) is replaced by the corresponding hamster PrP sequence (Scott et al., 1993). This chimaeric PrP is designated MH2M PrP. Such transgenic mice are susceptible to hamster prions and produce chimaeric prions which are pathogenic for wild-type mice as well as for hamsters. We therefore sought to utilize this chimaeric PrP as a means to bridge the hamster–mouse species barrier. If hamster PrP Sc were able to convert MH2M PrP C to MH2M PrP Sc in vitro, then any newly produced chimaeric prions could now be effectively detected in bioassay, as they would be expected to be pathogenic for wild-type mice, unlike the large molar excess of hamster PrP Sc used to promote the conversion. We therefore produced recombinant MH2M PrP in cell culture and subjected it to the same methods established by Kocisko et al. (1994) for in vitro production of protease-resistant hamster PrP. 0001-5812  1999 SGM BB