Past research States Prion hypothesis and past research of no
past viral genetic material detected States the fact that
mitochondrial DNA has been found in subsequent studies States that
long RNAs cores were also detected Very similar to retroviral cores
Endogenous Viral Complexes with long RNA cosediment with the agent
of Ceutzfeldt-Jacob Disease
Slide 3
CJD Retroviral Theory Strain variation, exponential
replication, tissue specificity, latency, resistance to treatment,
and non- inflammatory response LTRs detection experience found LTRs
present in infectious samples Endogenous retroviral intracisternal
A particle (IAP) genome were identified by using cDNAs that were
created from the LTRs of infectious factions Endogenous Viral
Complexes with long RNA cosediment with the agent of
Ceutzfeldt-Jacob Disease
Slide 4
IAP RNA cosediement found (The RNA that is being detected) IAP
are a class of retrovirus In Infectious sample 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
Isolation of this gag protein would help solidify the idea of virus
particle, not just random IAP RNA Endogenous Viral Complexes with
long RNA cosediment with the agent of Ceutzfeldt-Jacob Disease
Slide 5
Source of materials and purification of CJD infectivity Syrian
hamster IAP genome Polyclonal antibodies that bind the IAP gag
protein 12 CJD infected hamster brains Purification: samples made
devoid of PrP-res Samples also treated with nuclease (allows for
isolation of nuclease resistant genetic material) Extraction of RNA
Elimination of DNA in sample via various methods (DNAase)
Endogenous Viral Complexes with long RNA cosediment with the agent
of Ceutzfeldt-Jacob Disease
Slide 6
RNA/PCR amplification of IAP sequences Usage of various IAP
primers for the production of subsequent cDNA syntheses IAP RNA
from cells -> cDNA-> DNA amplification via IAP primer Probe
hybridization to Southern Blots DNA probes Generated from
recombinant clone of Syrian Hamster IAP Western blot Polyclonal
rabbit anti-mouse IAP gag antibodies Endogenous Viral Complexes
with long RNA cosediment with the agent of Ceutzfeldt-Jacob
Disease
Slide 7
Slide 8
Slide 9
Discussion: Was able to isolate both IAP RNA and RNA gag in
infectious samples Both are resistance to degradation of nuclease,
which shows characteristic of retroviral Results refuted the
assumption of prion infection being devoid of nucleic acid ~6kb IAP
sequences No clear evidence of a CJD specific sequence of IAP RNA
If IAPs are involved with the CJD nucleic acid, it is either
co-packed in the core or uses IAP products to proliferate Second
theory: Completely independent CJD viral complex, only similar to
IAP Supported by presence of gag-like proteins
Slide 10
States the assumption of virus infectivity theory
Characteristic of the CJD infectious agent 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 Primary investigation: Separation and isolation of the
primary components of the viral protein (testing different methods)
PrP Nucleic acid-binding proteins Gag protein Viral Particles are
required for infection in neurodegenerative Creuzfeldt-Jakob
disease
Slide 11
Method Centrifuge isolation of supernatant and pellets after
elimination of the majority of PrP Immunoblots (Western blot method
used for protein isolation) Polyclonal antibodies Viral Particles
are required for infection in neurodegenerative Creuzfeldt-Jakob
disease
Slide 12
Slide 13
Slide 14
Slide 15
Slide 16
Results show that a specific nucleic acid and one or more
binding proteins are intrinsic components of the CJD virus
Independently have low infectivity, but together form a complex
that is the main infective agent Infectivity is not Prp dependent,
SDS treatment eliminates the majority of Prp, but infectivity is
still high Gdn-HCI treatment who greatly lower infectivity due to
elimination of the viral components There is still retention of PrP
in Gdn-HCI samples Viral Particles are required for infection in
neurodegenerative Creuzfeldt-Jakob disease
Slide 17
Begins paper by stating examples of virus that have similar
and/or the same characteristics of CJD viral infection States
theory involving PrP as the viral receptor and that after
interaction with virus it causes the formation of PrP-res Lists the
common flaws in Prion Hypothesis Nuclease resistant circular DNAs
copurify with infectivity in scrapie and CJD
Slide 18
Primary goal: Isolation and identification of circular ssDNA
via generalized detection method 29 polymerase to enhance the
replication of the circular ssDNA Circular DNA hypothesis is based
off CJDs similarity with Torgue tenovirus, which has circular DNA
It is also based on Circular DNAs ability to cause physical
generation like the formation of mouse tumors Nuclease resistant
circular DNAs copurify with infectivity in scrapie and CJD
Slide 19
Infectious material: Three source that underwent PrP depletion
procedures Murine N2a neuroblastoma (22L) Hamster brain (263K)
Japanese FU-CJD patient (FU-CJD) Nucleic acid extractions Usage of
Proteinase K for digestion of endogenous or added nucleases Used
purification method for digested genetic material Nuclease
resistant circular DNAs copurify with infectivity in scrapie and
CJD
Slide 20
29 polymerase and ssDNA chromatography Allow for the
amplification of circular ssDNA from the digested nucleic acid RNA
and RNAase inhibited the effects of 29 polymerase ssDNA was
isolated from these factors and dsDNA before replication
Restriction enzyme analysis, PCR and sequencing Digestion of the 29
polymerase product -> PCR proliferation -> sequenced Nuclease
resistant circular DNAs copurify with infectivity in scrapie and
CJD
Slide 21
Slide 22
Slide 23
Slide 24
Slide 25
Slide 26
Demonstration of two new circular DNAs that is in high
concentration in TSE particles All three strains tested strongly
positive for Sphinx 1.8kb and 2.4kb, which show a clear viral
plasmid structure Both are passed down to daughter cells Both are
also present in uninfected brains, but the concentration in
infected brain is x2,500 higher Positive control: Mitochondrial DNA
was used In essence, Sphinx DNA is viral, their actual role is up
for further investigation Nuclease resistant circular DNAs copurify
with infectivity in scrapie and CJD