It is widely believed that host prion protein (PrP), without nucleic acid, converts itself into an infectious form (PrP-res) that causes transmissible encephalopathies (TSEs), such as human sporadic CJD (sCJD), endemic sheep scrapie, and epidemic BSE. There are many detailed investigations of PrP, but proteomic studies of other proteins in verified infectious TSE particles have not been pursued, even though brain homogenates without PrP retain their complete infectious titer. To define proteins that may be integral to, process, or protect an agent genome, we developed a streamlined, high-yield purification of infectious FU-CJD mouse brain particles with minimal PrP. Proteinase K (PK) abolished all residual particle PrP, but did not reduce infectivity, and viral-size particles lacking PrP were ∼70S (vs. 90-120S without PK). Furthermore, over 1,500 non-PrP proteins were still present and positively identified in high titer FU-CJD particles without detectable PrP by mass spectrometry (LC-MS/MS); 114 of these peptides were linked to viral motifs in the environmental-viral database, and not evident in parallel uninfected controls. Host components were also identified in both PK and non-PK treated particles from FU-CJD mouse brain and human sCJD brain. This abundant cellular data had several surprises, including finding Huntingtin in the sCJD but not normal human brain samples. Similarly, the neural Wiskott-Aldrich sequence and multivesicular and endosome components associated with retromer APP (Alzheimer amyloid) processing were only in sCJD. These cellular findings suggest that new therapies directed at retromer-vesicular trafficking in other neurodegenerative diseases may also counteract late-onset sCJD PrP amyloid pathology.

Unlike Alzheimer's and most other neurodegenerative diseases, Transmissible Spongiform Encephalopathies (TSEs) are all caused by actively replicating infectious particles of viral size and density. Different strain-specific TSE agents cause CJD, kuru, scrapie and BSE, and all behave as latent viruses that evade adaptive immune responses and can persist for years in lymphoreticular tissues. A foreign viral structure with a nucleic acid genome best explains these TSE strains and their endemic and epidemic spread in susceptible species. Nevertheless, it is widely believed that host prion protein (PrP), without any genetic material, encodes all these strains. We developed rapid infectivity assays that allowed us to reproducibly isolate infectious particles where >85% of the starting titer separated from the majority of host components, including PrP. Remarkably, digestion of all forms of PrP did not reduce brain particle titers. To ask if TSE agents, as other viruses, require nucleic acids, we exposed high titer FU-CJD and 22L scrapie particles to potent nucleases. Both agent-strains were propagated in GT1 neuronal cells to avoid interference by complex degenerative brain changes that can impede nuclease digestions. After exposure to nucleases that are active in sarkosyl, infectivity of both agents was reproducibly reduced by ≥99%. No gold-stained host proteins or any form of PrP were visibly altered by these nucleases. In contrast, co-purifying protected mitochondrial DNA and circular SPHINX DNAs were destroyed. These findings demonstrate that TSE agents require protected genetic material to infect their hosts, and should reopen investigation of essential agent nucleic acids. J. Cell. Biochem. 117: 1947-1958, 2016. © 2016 Wiley Periodicals, Inc.

Neurodegenerative human CJD and sheep scrapie are diseases caused by several different transmissible encephalopathy (TSE) agents. These infectious agents provoke innate immune responses in the brain, including lateonset abnormal prion protein (PrP-res) amyloid. Agent particles that lack detectable PrP sequences by deep proteomic analysis are highly infectious. Yet these agents, and their unusual resistance to denaturation, are often evaluated by PrP amyloid disruption. To reexamine the intrinsic resistance of TSE agents to denaturation, a paradigm for less resistant viruses and microbes, we developed a rapid and reproducible high yield agent isolation procedure from cultured cells that minimized PrP amyloid and other cellular proteins. Monotypic neuronal GT1 cells infected with the FU-CJD or 22L scrapie agents do not have complex brain changes that can camouflage infectious particles and prevent their disruption, and there are only 2 reports on infectious titers of any human CJD strain treated with chemical denaturants. Infectious titers of both CJD and scrapie were reduced by >4 logs with Thiourea-urea, a treatment not previously tested. A mere 5 min exposure to 4M GdnHCl at 22 C reduced infectivity by >5 logs. Infectious 22L particles were significantly more sensitive to denaturation than FU-CJD particles. A protocol using sonication with these chemical treatments may effectively decontaminate complicated instruments, such as duodenoscopes that harbor additional virulent microbes and biofilms associated with recent iatrogenic infections.