Scientific Report 2005

Scripps Florida

Infectology

Generation and Transmission of Prions

C. Weissmann, C.A. Baker, S.P. Mahal, C. Demczyk, A. Sherman

The agents that cause transmissible spongiform encephalopathies such as Creutzfeldt-Jakob disease in humans, bovine spongiform encephalopathy in cattle, and scrapie in sheep are termed prions. The unusual resistance of prions to radiation led early on to the proposal that prions might be devoid of nucleic acid and consist only of protein. The discovery of PrPSc, a protease-resistant protein found only in organisms with transmissible spongiform encephalopathies; the cloning of PrP cDNA and its gene; the recognition that the gene encodes a normal host protein, PrPC, from which PrPSc is derived by conformational rearrangement; and the linkage between the PrP gene and familial prion disease supported the suggestion that an abnormal conformer of PrPC (generically designated as PrP*) is the main or perhaps only constituent of prions. The essential role of PrP in prion diseases was established by the finding that mice lacking the PrP gene were resistant to disease and incapable of propagating prions.

The “protein only” hypothesis proposes that the infectious, abnormal conformer of PrPC is propagated autocatalytically; a specific mechanism is suggested by the “seeding hypothesis.” Intriguingly, distinct prion strains, which generate different disease phenotypes, may be associated with the same PrP sequence, suggesting that the phenotypes are encoded by different PrP conformations.

Infectivity of prions is classically measured in a bioassay that takes many months to complete and requires large numbers of animals. We created a cell-based assay (scrapie cell assay, SCA) that can be carried out in less than 2 weeks, is at least as sensitive and accurate as the animal assay, and allows the simultaneous processing of hundreds of samples by a semiautomated procedure. It has been proposed that prions consist mainly or entirely of the protease-resistant conformer PrPSc and that they are resistant to heating at 80°C for 60 minutes. Using the SCA, we found, in collaboration with F. Properzi, MRC Prion Unit, London, England, that standard protease digestion or heating at 80°C eliminated more than 90% of prion infectivity, indicating that the essential component is a conformer of PrP other than PrPSc. Again using the SCA, in collaboration with S. Supattapone and N. Deleault, Dartmouth Medical School, Hanover, New Hampshire, we found that a cell-free system containing PrPC and primed with prion-infected brain homogenate increased prion infectivity 6-fold within 8 hours, showing that infectivity can be rapidly amplified in a cell-free system.

Only a few cell lines are susceptible to persistent infection by prions, for as yet unknown reasons. The high susceptibility of the subclone N2aPK1 developed for the SCA is unstable, and even after repeated cloning, susceptible populations consist of both highly susceptible and almost resistant cells; susceptibility appears to be determined epigenetically. Similarly, populations of cloned, chronically infected N2aPK1 cells consist of infected and uninfected cells. Interestingly, a nondividing subpopulation is the most prolific in producing and secreting prions, while a dividing subpopulation generates very low levels of prions, perhaps because of the emergence of only a small number of “producer cells” from the population.

N2aPK1 cells are, surprisingly, susceptible to a particular murine prion strain (139A) but not to any others tested so far. We are now searching for cell lines that are sensitive to other strains and are striving to identify the features that enable susceptibility to prions in general and to certain strains in particular. A panel of cell lines with distinct susceptibilities will greatly facilitate the currently arduous task of typing of prion strains.

J-Binding Protein of Leishmania as a Potential Drug Target

C. Weissmann, P. Subramaniam, P. Wentworth,* D. Millar,** R. Sabatini,*** P. Borst****

*Department of Chemistry, Scripps Research
** Department of Molecular Biology, Scripps Research
*** Marine Biological Laboratory, Woods Hole, Massachusetts
**** Netherlands Cancer Institute, Amsterdam, the Netherlands

Leishmaniasis consists of a group of diseases caused by parasitic protozoans of the genus Leishmania. The pathogens are transmitted by sand flies and can infect skin, mucous membranes, and certain internal organs. Three major types of leishmaniasis occur in humans: cutaneous, mucocutaneous, and visceral. According to estimates, 1.5 million to 2 million new cases occur annually, mainly in the tropics and subtropics, including the Middle East. An effective, well-tolerated, and inexpensive therapy is greatly needed.

The DNA of Leishmania organisms, as well as that of other kinetoplastids, contains a modified base, J (β-D-glucosyl-hydroxymethyluracil), that does not occur in higher eukaryotes. Leishmania species contain a protein, J-binding protein (JBP), that binds to the modified base within the context of a nucleotide sequence and plays a role in the conversion of thymine to J. Deletion of JBP is lethal for the organisms. Therefore, most likely a compound that interferes with the binding of JBP to J would be detrimental to the growth or survival of Leishmania organisms. Because J and JBP do not occur in the host, such a compound might lead to a therapeutic drug.

Using a JBP-based approach, members of our broadly based consortium have set out to search for a lead compound, and if proof of principle is achieved, to develop a drug. A fluorescence polarization assay for the binding of a J-containing, fluorescently labeled oligonucleotide to recombinant JBP has been developed and will be optimized for high-throughput screening of libraries of various compounds at Scripps Florida.