


Professor and Head of
Neuropathology at YaleDepartment of Surgery and
Faculty of Neurosciences and
Virology
Focuses on dementia with
emphasis on TSEs
“We think it is most likely the host
PrP is a required receptor for TSE
viruses, and that viral PrPmembrane interactions ultimately
cause a pathological PrP
response. Ongoing experiments
are designed to test this viral
hypothesis”
Kohtaro Miyazawa, Kaitlin Emmerling, and Laura Manuelidis
Journal of Cellular Biochemistry 112: 3630–3637 (2011)
“Substantial and often ignored evidence indicates that PrP-res is part of
a pathological response to infection, rather than the causal agent that
incites disease. Many viruses require a specific host protein for entry
and replication, and infectious TSE particles similarly require host PrP
for effective spread from ce ll to cell. Merely increasing PrP, even from
a different species, enhances transmission of foreign TSE. Diverse
published data that are inconsistent with an infectious form of PrP “
(i)the variety of unique and mutable agent strains, a property of nucleic acid not
protein
(ii)TSE agents breed true in various tissues, cell cultures, and cross-species
transmissions whereas PrP patterns do not
(iii) a preventable environmental source of infection in which epidemic
outbreaks disappear after infectious material is removed (as in BSE and kuru)
strongly implicates a foreign source of infection rather than a ‘‘spontaneously
generated’’ host PrP self-conversion into an infectious form
(iv) infection elicits early innate immune responses that indicate host
recognition of an invading foreign entity and these responses, not educed by
host PrP, occur well before PrP-res begins to accumulate and can be protective
(v) microglia with barely detectable PrP, and no PrP-res, contain high levels
infectivity
(vi)all detectable forms of PrP are digested in the gastro intestinal tract yet the
invasive infectious particle (as many conventional viruses) is not destroyed.
(vii) infectious particles of viral size ( 25nm diameter) with protected nucleic
acids can be separated from the majority of host PrP and other proteins, and
GdnSCN disruption of these particles into protein and nucleic acid components
reduces infectivity by>99.8%. Some of these released nucleic acids have been
sequenced such as capsid-protected endogenous retroviral RNAs, mitochondrial
DNA, and newly discovered circular Sphinx DNAs of1.8kb with large sequence
regions that are not in the database
(viii) High CJD infectivity remains after the prion protein is destroyed with a
protease.



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.
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


Manuelidis et al (1997)
◦ Several strains, same PrP amino acid sequence
◦ Different phenotypes
 Due to PrP protein or hidden virus?
Experiment
◦ Succeeded in changing 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.
Manueldis, L., Fritch, W., Xi, Y. (1997). Evolution of a strain of CJD that Induces
BSE-Like Plaques. Science. 277(5322): 94-98.

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”



“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

Authors argue “These findings (two strains
that have different phenotypes but same PrPres banding or glycosylation patterns) are
problematic for the prion hypothesis where
abnormal PrP folding or glycosylation, and
hence PrP-res band patterns, are postulated
to encode each agent strain”
◦ PrP(Sc) prion populations with distinct phenotypes but same
amino acid sequence..are distinct strains.
◦ Strain identity encoded by conformation of PrP(Sc)
◦ Brain-derived 22L prions are able to infect R33 cells (R33
competent) AND PK1 cells in the presence of the
swainsonine (swa resistant).
◦ 22L prions retained these characteristic s including swa
resistance when transferred from brain to R33 cells.
◦ However, when transferred from the R33 cells to PK1 cells,
they gradually became R33 incompetent and swa sensitive,
unless the transfer was in the presence of swa, in which
case swa resistance and R33 competence were retained.
Mahal et at. (2010). Transfer of a prion strain to different hosts leads to emergence of
strain variants. PNAS. 107(52): 22653-22658
 PrP(Sc) with swa-resistant/R33-competent and PrP(Sc) swa-


sensitive/R33-incompetent prions had different
conformational stabilities.
When R33-incompetent/swa-sensitive prions were
propagated in brain, their properties gradually reverted to
those of the original brain-derived 22L prions.
Conclusion: 22L prion populations are heterogeneous and
that distinct prion variants are selected in different cellular
environments.
◦ ….viral hypothesis would say….???
Mahal et at. (2010). Transfer of a prion strain to different hosts leads to
emergence of strain variants. PNAS. 107(52): 22653-22658


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’?
◦ …..same data different interpretations??
◦ Study by Cancellotti et al., (2013) showed that the
passage through mice that expressed a PrP that
either partially or completely lacked N-glycan
affected the phenotypic characteristics of at least
one TSE strain
◦ Quasi species 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
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





PrP 90-231 expressed and purified from E. coli.
…refolded and kept in solution using 1%SDS
…spontaneously forms fibrils when SDs is diluted to 0.02%
…seeding with different brain extracts results in faster
fibrillation
…go to paper


Evidence of viral presence
Endogenous Viral Complexes with long RNA
cosediment with the infectious agent of
Ceutzfeldt-Jacob Disease




Strain variation, exponential replication, tissue specificity,
latency, resistance to treatment, and non-inflammatory
response…all characteristics of retroviruses
LTRs present in infectious samples
Long terminal repeats (LTRs) are identical sequences of DNA
that repeat hundreds or thousands of times found at either
end of retrotransposons or proviral DNA formed by reverse
transcription of retroviral RNA. They are used by viruses to
insert their genetic material into the host genomes.
Endogenous retroviral intracisternal A particle (IAP) genome
was identified






IAP are a class of retrovirus
Founf in infectious sample but also in non-infectious sample
IAP are highly resistant to forms of treatment like SDS and
chaotropic salts
High resistance is due to IAP gag proteins
gag protein found indicating a virus particle, not just random
IAP RNA
Sensitive to guanidinium chloride treatment
◦ Characteristic of the viral particles
 Core-like viral density of 1.27-1.28 g/cc
 Viral size of 120S and a diameter of ~30nm
 >99% of starting prion protein can be separated from
the viral agent and still have infectivity





Absence of association between the incidence of BK virus and
sporadic Creutzfeldt-Jakob disease. Jeong BH1, Lee JH, Cho
HJ, Kim YS. Intervirology. 2013;56(3):184-9.
OBJECTIVE:
To determine the relationship between BK polyomavirus (BKV)
and sporadic CJD.
MATERIALS AND METHODS:
We investigated the prevalence of BKV in urine samples from
94 sporadic CJD patients and 54 other neurological disease
(OND) patients using polymerase chain reaction.







BK virus:
-member of the polyomavirus family
-first isolated in 1971
-generally asymptomatic except when immunosuppressed
- carried by an estimated 80% of the population
- no treatment except to switch immunosuppression drug
- transmission possibly through saliva and urine




RESULTS:
BKV DNA was detected in 16 (17%) and 9 (16.7%) urine
samples from sporadic CJD and OND patients,
respectively. There was no significant difference in the
incidence of BKV infection between Korean sporadic CJD
and OND patients (p = 0.9558). In order to investigate the
genotypes of BKV, we analyzed 22 BKV isolates obtained
from Korean patients by DNA sequencing and nucleotide
sequence analysis. Three distinct subtypes, namely I, III,
and IV, were found in 66.7, 22.2, and 11.1% of 9 BKV
isolates from OND patients, whereas subtypes I and IV
were detected in 76.9 and 23.1% of 13 BKV isolates from
sporadic CJD patients. Interestingly, subtype III was not
detected in sporadic CJD patients. Significant differences
in the frequency of BKV genotypes were not observed
between sporadic CJD and OND patients.
CONCLUSIONS:
These results indicate that BKV may not play an important
role in the pathogenesis of prion diseases.


Digestion of PrP in sample with protease K
does not reduce infectivity
Manuelidis 2014

CJD killed our son…