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INFORMATION FOR

TSE Infectious Agents

Prions have extraordinary biological charateristics, not the least of which is their possible non-existence.

The force of Prions: Lancet 2000

Concepts and Facts

Agent Hypothesis

PrP

We also elucidate the structure of the infectious agent by subcellular fractionation and electron microscopy. Our new tissue culture models have revealed virus-like arrays (at right) that correspond to isolated infectious brain particles of 20-25nm that are seen only in infectious cells. These arrays were not detectable in uninfected cells. They do not bind PrP antibodies, unlike PrP amyloid, a misfolded form of host PrP that is commonly believed to be the infectious agent or “prion”. We think misfolded PrP is a pathological response to infection in PNAS".(PMID: 26773845)

Dominant belief:

  • Normal host prion protein (PrP) spontaneously converts to an infectious amyloid form, called a “prion” and the prion requires no nucleic acid for infection: “It is not a virus”.

Alternative view:

  • TSE agents are environmental pathogens (Ref 4)
  • An essential nucleic acid encodes different agent strains
  • Host PrP is the cellular receptor required for establishment of infection and exponential replication of the infectious agent
  • PrP amyloid is a late defense response to high levels of infectious agent (Ref 2 and see below).

Current Status

Attempts to fabricate an infectious PrP in a test tube:

  1. No reproducible infectivity for any pure PrP, including recombinant PrP (rPrP)
  2. More than 100 independent experiments have failed to generate any infectivity from pure recombinant PrP in an amyloid form

Reproducible data that contradicts the prion hypothesis:

  1. When “infectious” PrP amyloid increases, infectivity falls by 10,000 fold. PMID: 22509412, and PrP amyloid can trap and delete agent as part of a host innate immune response.
  2. Removal of PrP from highly infectious brain does not reduce infectivity. PMID: 24933657; PMID: 21793041
  3. All infectious particle preparations from brain and cell cultures contain circular DNAs of >500nt by standard molecular analyses PMID: 21165784
  4. Digestion of infectious particle nucleic acids reduces infectivity by 3 logs (>99.8%) but leaves PrP amyloid intact PMID: 26773845.
  5. Major new antiviral responses revealed in differentiating neurons with high infectivity http://www.growkudos.com/publications/10.1371%25252Fjournal.pone.0323825/reader and see additional data on CJD agents

Are “established” Prion claims accurate?

Table of widely accepted major prion claims show key facts that contradicts the definitive infectious protein (prion) concept.

Claims, Experiments, and Comments
Claim Experiment Comment
1) “PrPSc (PrP-res amyloid) is proportional to titer” FALSE diverse data from many labs falsify
2) “Procedures that hydrolyze PrPSc inactivate prions” TRUE These treatments also inactivate viruses
3) “No evidence exists for a virus-like particle” FALSE ~25nm viral particles in infected brain and cells
4) “Transmissible particles are devoid of nucleic acid” FALSE all infectious material contains long nucleic acids
5) “PrP gene mutations cause formation of PrPSc” FALSE toxic pathology, but not “infectious PrPSc”
6) “PrP gene mutations cause transmissible disease” FALSE Contamination with lab scrapie and irreproducible
7) “Prion diversity is enciphered by PrPSc” folding FALSE unfolding PrP-res has no effect on strain or titer
9) “No immune response to foreign agent” FALSE Early innate immune responses well before PrP-res
10) “PrPSc associated with pathology” TRUE PrPSc is a late response to infection
11) Protein X binds PrP to make it infectious X not found PrP itself not infectious; X is probably a virus
12) “Prions defy the rules of protein structure” TRUE and of thermodynamics
13) CJD infectious agent arises spontaneously No evidence An old & disproven concept of infection
14) recombinant PrP can be made infectious FALSE Irreproducible: numerous failed attempts

For details see: A 25nm virion is the likely cause of Transmissible Spongiform Encephalopathies. J Cell Biochem. 100, 897-915, 2006, PMID: 17044041).

Infectivity versus prion protein characteristics:

FU-CJD in wt mice

Right panel shows undigested CJD brain and Left panel shows the same brain samples without PK digestion from 50-130 days after brain inoculation. The agent produces 100,000x (5 logs) of infectivity before any PrP-res (the infectious form of the prion protein is detected. Same pattern in 3 species

  1. PrP amyloid (PrP-res) is detected late while infectivity increases by 5 logs during the latent clinically silent phase.
  2. PrP is converted but not replicated exponentially, unlike the infectious agent.
  3. Complete destruction of all PrP (including PrP-res) wih Proteinase K (PK) at 250mg/ml does not reduce infectivity as shown in corresponding protein (left) and infectivity (right) red dots in both panels below (from ref 4).
  1. In contrast, obliteration of all proteins with NAP destroys 3 logs (99.9%) of infectivity, i.e., non-PrP molecules probably protect and/or act as an essential part of the infectious particle.
Nucleases effect on PrP

Nucleases have no effect on PrP but destroys 2 logs of infectivity. Red asteriks show the PrP gel and graphed smapes (- & + nuclease)

5. Nucleic acid digestion destroys 3 logs (99.98%) of infectivity while preserving PrP and PrP-res in both CJD and scrapie (ref 6). These data strongly underscore a nucleic acid (DNA or RNA) is an essential component of the infectious replicating agent particle.

References:

  1. Kipkorir T, Tittman S, Botsios S, Manuelidis L. Highly infectious CJD particles lack prion protein but contain many viral-linked peptides by LC-MS/MS. J Cell Biochem. 2014 Jun 16. doi: 10.1002/jcb.24873. PMID: 24933657
  2. Manuelidis, L. Infectious particles, stress and induced prion amyloids: A unifying perspective. Virulence 2013 Jul 1;4(5):373-83. doi: 10.4161/viru.24838. PMID: 23633671
  3. Miyazawa K, Kipkorir T, Tittman S, Manuelidis L. Continuous production of prions after infectious particles are eliminated: implications for Alzheimer's disease. PLoS One. 2012;7(4):e35471, PMID: 22509412
  4. Miyazawa, K, Emmerling, K Manuelidis, L High CJD infectivity remains after prion protein is destroyed. J Cell Biochem. 112: 3630-3637, 2011 PMID: 21793041
  5. Manuelidis, L, Chakrabarty, T, Miyazawa, K, Nduom, N-A, and Emmerling, K. The kuru infectious agent is a unique geographic isolate distinct from Creutzfeldt–Jakob disease and scrapie agents. Proc. Natl. Acad. Sci. USA 106: 13529-13534, 2009. PMID: 19633190
  6. Botsios, S and Manuelidis, L, CJD and scrapie require agent-associated nucleic acid for infection. J. Cell. Biochem. 117(8) 2016 Jan 16. PMID:26773845