The Strain ProblemTaylor Goldbeck

Laura Manuelidis- Who is she? Professor and Head of

Neuropathology at Yale- Department of Surgery and Faculty of Neurosciences and Virology

Focuses on dementia was emphasis on TSEs

“We think it is most likely the host PrP is a required receptor for TSE viruses, and that viral PrP-membrane interactions ultimately cause a pathological PrP response. Ongoing experiments are designed to test this viral hypothesis”

How do viral strains occur?

Prion strain- defined as “infectious isolates that, when transmitted to identical hosts, exhibit distinct prion-disease phenotypes”

Prion strain diversity first seen in goats PrP-c and PrP-sc can be unglycosylated,

monoglycosylated or diglycosylated.

Prion Strains

Aguzzi, A., Heikenwalder, M., Polymenidou, M. (2007). Insights into prion strains and neurotoxicity. Nature. 8:552-561

Aguzzi et al. (2007). Insights into prion strains and neurotoxicity. Nature.8): 552-561

PrP-res ◦ Part of response, not the cause◦ Host protein

“(i) The variety of unique and mutable agent strains, a property of nuclei acid not protein”◦ Initial thoughts on this point?

High CJD Infectivity Remains After Prion Protein is Destroyed

Manuelidis et al (1997) Argue:

◦ Several strains, same PrP amino acid sequence◦ Different phenotypes

Due to PrP protein or hidden virus? Experiment

◦ Change a CJD strain into a strain that produced plaques and cerebellar lesions Evaluated host recognition by responses of microglia

and astrocytes Used inbred mice, guinea pigs, and rats.

2/6 rats showed a response after first passageManueldis, L., Fritch, W., Xi, Y. (1997). Evolution of a strain of CJD that Induces

BSE-Like Plaques. Science. 277(5322): 94-98.

Evidence for Viral Genome

If you were pro protein-only hypothesis, how would you explain the reactive microglia and astrocytes?

Authors state “Species barrier and host responses (reactive microglia, astrocytes, and development of new strain that can produce plaques/lesions from a strain that couldn’t before) to the foreign agent are too complex to just be explained by the host’s PrP sequence”◦ If you were pro protein-only hypothesis, how would

you respond to this?

Your Thoughts-

“TSE strains maintain their identity despite various changes in prion protein. This fact strongly implicates a relatively stable but mutable viral genome” (from Yale bio page)

Wanted to see whether PrP-res itself encoded intrinsic infectivity characteristics

Slow SY strain and fast virulent FU CJD strain infect GT1 cells into various cell lines◦ Kept phenotypes through passage but remained indistinguishable by

PrP-res banding or glycosylation patterns FU had different PrP-res patterns in different cell lines

Still had same incubation time and clinical features

◦ Amount of PrP-res was not quantitatively related to infectivity “It is the biology of these agents: their evolution spread, cell

specificity, latency, virus-like interference capabilities and occusional mutation which continues to indicate a viral causative agent”

◦ Arjona et al. (2004). Two Creutzfeldt-Jakob disease agents reproduce prion protein-independent identities in cell cultures. PNAS. 101(23): 8768-8773

Evidence for Viral Genome

Authors argue “These findings (two strains that have different phenotypes but same PrP-res banding or glycosylation patterns) are problematic for the prion hypothesis where abnormal PrP folding or glycosylation, and hense PrP-res band patterns, are postulated to encode each agent strain”◦ If you were pro- protein-only hypothesis, how

would you respond?

Your Thoughts-

Methods: Exposed cells to various dilutions of prion samples, let the cells propagate, determined the proportion of PrP-sc containing cells by ELISA◦ Strain 22L transferred from brain to PK1 cells ◦ In the presence of swainsonine, 22L-infected PK1 cells

led to drug-resistant variants◦ 22L prions transferred to R33 cell population◦ R33 incompetent/swa-sensitive cells return to brain

Mahal et at. (2010). Transfer of a prion strain to different hosts leads to emergence of

strain variants. PNAS. 107(52): 22653-22658

Evidence for prion protein-only hypothesis

Showed prions can adapt to survive in a new host environment ◦ When transferring from one cell line to another,

prion properties chance “Darwinian Evolution without DNA”

◦ Prions can develop mutations drug resistance ◦ Fold in different ways new strains◦ Transferred to a new host which strain ‘wins’?

Your thoughts◦ If you were Manulidis, how would you respond to

this paper?

“The fact that they behave like viruses doesn’t mean they’re anything like a virus”- Weissman

(coauthor)

Back to PrP glycosylation◦ Study by Cancellotti et al., (2013) showed that the passage of

mice that expressed a PrP that either partially or completely lacked N-glycan affected the phenotypic characteristics of at least one TSE strain

Back to species barrier◦ Quasispecies hypothesis

Several PrP-sc conformations in infectious innoculum. One best suited for new host is selected for Problem- there isn’t a lot of evidence that a large number of

conformations exists in an innoculum Another problem?

Explaining the relationship between PrP-res and infectivity

Poggiolini, I., Saverloni, D., Parchi, P. (2013). Prion Protein Misfolding, Strains, and Neurotoxicity: An Update from Studies on Mammalian Prions. International Journal of Cellular Biology. 1-24

Soto, C and Castilla, J. (2004). The controversial protein-only hypothesis of prion propagation. Nature medicine. 563-567

Other evidence for prion protein-only hypothesis