Please also see my update in the project.

Thanks.

"By all logical estimates, the death-knell to the prion

hypothesis should have occurred with Lasmézas’s 1997

interspecies transmission of mad cow in which more than half

of injected mice had no detectable prions [21]. If this was not

enough, then there was Baker and Manuelidis’s 2002 study on

infectious neurons (microglia) with low prion levels in otherwise

highly infectious material, which only served to support the

concept that pathologic prions were the result of infection rather

than being the actual infectious agent [22].

When Yale’s Laura Manuelidis and Sotirios Botsios revisited this

subject in 2016 they outright stated that CJD and Scrapie require

agent-associated nucleic acids for infection, therefore turning

the entire original prion hypothesis on its head [23]. Manuelidis

called for a re-opening of the entire prion concept to find out

just which genetic material was involved, and its origin. To

Manuelidis this was likely to be a virus, although she admitted

the fundamental mystery remained."

21 Lasmézas CI, Deslys JP (1997) Transmission of the BSE agent

to mice in the absence of detectable abnormal prion protein.

Science 275: 402-405.

22 Baker CA, Martin D, Manuelidis L (2002) Microglia from Creutzfeld–

Jakob disease-infected brains are infectious and show specific

mRNA activation profiles. J Virol 76: 10905-10915.

23 Botsios S, Manuelidis L (2016) CJD and Scrapie Require Agent-

Associated Nucleic Acids for Infection. J Cell Biochem 117:

1947-1958.

Very nice. What about Surewicz et al.  work which clearly showed transmission of prions using recombinant proteins? Increase in stability of prion protein eliminated prion disease in mice as well. The whole prion hypothesis is not so easily dismissed.

The question of prions in CJD or cows may be related to other agents inducing conditions favoring protein aggregation. There might be a virus or other factor which may be essential in some cases but is not in others. To me, the question of prions is open and both sides can have their own arguments.

Re: Surewicz. work.

This is not the first time this group publishes a paper saying recombinant prion protein is infectious alone (see their similar paper from 2010). And it is not the only group to publish such papers. The problem with this type of work?

Good to know that the question is open for you, unlike many for whom the question is closed, but not because of the existing irrefutable evidence, but rather because they close their eye on evidence to contrary.

In early to midst of 20th century many scientists hold a strong believe that Tobacco Mosaic Virus was a self-replicating enzyme (protein). This was before the DNA and RNA era and virus reconstruction experiments were required to demonstrate that its the RNA that determines the strain of the virus and not the protein (http://www.eplantscience.com/index/genetics/chemistry_of_the_gene_nucleic_acids_and_their_structure/experiments_with_tobacco_mosaic_virus_tmv.php)

Today, no one remained who still thinks TMV is a self replicating infectious protein.

The day when it will be clear what exactly causes TSE does not seem to be too far away anymore.

Dear Yervand,

1. Contamination. I worked at that lab and set up protocols for expression, refolding and purification myself. Stating that the labs are contaminated with prions preventing a scientifically sound and controlled experiments is a total BS. '

2. Reproducibility. The conversion could be done in a test tube and I was the first one to show it. Statement that it not reproducible is another BS.

There are people who have doubts and always be. To you, I suggest you keep your mind open if you want to pursue career in science. Otherwise, you may end up "consulting" politicians or another group without any clue what is going on. EU is full of such groups.

Best regards,

Wes Swietnicki, Ph.D.

Dear Dr Swientnicki,

It was not me who raised the issue of contamination to be a problem for "de novo generation" of infectivity using recombinant prion protein. Jiyan Ma himself admitted that he cannot exclude the possibility of contamination for his Science 2010 paper when he was invited to talk at Scripps Florida by Charles Weissmann, who himself underscored that once you use infectious material in a lab then the lab is contaminated once and forever. The contamination was admitted to be the cause of "de novo generation" of infectivity in PMCA reactions published by J.Castilla.

Just to clarify for you - we are not talking about the reproducibility of conversion in a test tube. No, we are talking about the infectivity of the converted product. Not all converted products and not always turn to be infectious, which is precisely the the point that John Collinge makes in the cited paper above:

"Overall, approximately 20,000 unique conditions were examined. While some resulted in apparently infected cell cultures, this was transient and not reproducible."

"Collectively, our findings are consistent with the formation of prion infectivity from recombinant mouse PrP being a rare stochastic event and we conclude that systematic generation of prions from recombinant PrP may only become possible once the detailed structure of authentic ex vivo prions is solved."

And no, I do not want to pursue a career in science, this is absolutely not my goal.

All I want is to know what is the nature and the precise structure of the infectious agent in TSEs.

With kind regards,

Yervand

"Protein misfolding, even when the protein is PrP, does not inevitably lead to the development of an infectious TSE disease. It is possible that most in vivo and in vitro produced misfolded PrP is not infectious and that only a specific subpopulation is associated with infectivity and neurotoxicity."

https://www.researchgate.net/publication/313781052_Infectious_prions_and_proteinopathies

https://www.researchgate.net/publication/304821840_PrP_aggregation_can_be_seeded_by_pre-formed_recombinant_PrP_amyloid_fibrils_without_the_replication_of_infectious_prions

Article Infectious prions and proteinopathies

Article PrP aggregation can be seeded by pre-formed recombinant PrP ...

Discussion

Comments:

17 comments

Adriano Aguzzi on 19 June 2009 10:36 UTC

Peer-review is not primarily meant to be a hassle for scientists eager to publish their findings. It is also a mean of protecting authors, e.g. by pointing out logical flaws, missing controls, alternative interpretations, etc. Not only does the process improve the quality of published science, but it also helps authors avoiding potential embarassment.

This paper appears to represent a good case in point. A plausible interpretation is that the traces of infectivity detected by the authors represent residual contamination stemming from the 263K inoculum. There would be valid ways to test for that, however no such efforts were undertaken. Experienced referees would have advised the authors to perform these tests before going public.

Time will tell whether the bypassing of the peer-review process, in this case, will have done any good to science at large and to the authors specifically.

Charles Weissmann on 19 June 2009 17:28 UTC

I agree with Adriano Aguzzi’s comments. In addition I would note that if one wishes to make the case that a cofactor such as RNA is required to generate infectious particles, it would be appropriate to quote the prior work of S.Supattapone and his colleagues: They generated infectious prions from purified PrPC using the PMCA technique and showed that addition of a polyanion such as poly(A) was essential for the generation of infectivity (Deleault et al. PNAS 104, 9741 (2007)). Supattapone’s results suggest that the role of the polyanion is structural rather than informational and there seems to be no urgent reason to resuscitate the virino hypothesis at this stage.

I also object to being misquoted. The authors claim that I dismissed the possibility that an RNA molecule might be part of the prion when they say “The possibility of such RNAs interacting with PrP has been evoked previously, but was immediately rejected because of the absence of data establishing a link between the RNA substrate and the infectious phenomenon 13,14. In fact, in the article quoted (ref. 14; Weissmann C. Nat Rev Microbiol. 2004 :861-71) I state: “The discovery of siRNAs and microRNAs, which would have escaped notice in earlier analyses of prion preparations, owing to both their size and their host origin, provides candidates for the hypothetical co-prion that has been proposed by the UNIFIED THEORY18.”

Hilary Spencer on 19 June 2009 21:06 UTC

(Disclosure: I am the Product Manager for Nature Precedings). Nature Precedings is not designed to bypass the peer-review process, but to act as a complement. The ability of preprint servers to complement the traditional peer review and editorial process has already been demonstrated with the use of the preprint server arXiv in the physical sciences. Many of the articles later peer-reviewed and published in Nature Physics are first posted on arXiv prior to (or concurrently with) journal submission. We believe that the use of arXiv benefits the physics community at large and helps to accelerate the research cycle, and many authors believe feedback they receive from the service benefits their research. Nature Precedings is intended to bring the same benefits to the biomedical community.

One of the benefits is that authors are able to receive (hopefully) helpful comments from others who may not have participated in the traditional peer-review process, thereby leading to more robust (and open) research.

Yervand Karapetyan on 25 June 2009 06:23 UTC

This paper is the first succesful TSE agent reconstitution experiment showing infectivity of RNA isolated form TSE infected tissue, much alike to the virus reconstitution experiments that were required to demonstrate the infectivity of tobacco mosaic virus RNA which (the virus) at some point was beleived to be a protein.

To further make the point of RNA being infectious and playing informational rather than structural role I suggest authors to do the following experiments:

1. Isolate RNA from Strain 1 and mix it with recPrP and inoculate into the animals brain. Do same thing with RNA isolated from Strain 2. Compare the resulting disease. RNA from strain 1 should give rise to Strain 1 TSE agent and RNA from Strain 2 should generate Strain 2 TSE agent. Taking into account the possibilty of pathology modification observed in the first passage the strain should be evaluated in the second and further passage animals.

Also to ease the strain discrimination such study should be done using other well caracterised TSE models like mouse scrapie strains such as RML,ME7,22L, 301C which are now well defined by IHC and CPA both in C57Bl/6 and Tga20 mice “Prion Strain Discrimination Based on Rapid In Vivo Amplification and Analysis by the Cell Panel Assay” http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005730

2. To demonstrate that recPrP has only structural but not informational role in TSE agent, RNA from infected brain should be mixed with nucleic acid binding proteins other than PrP. My first proposal would be NCP7 – retroviral nucleocapsid protein. Then the infectivity of such complex should be tested.

Ultimately similar experiment could be done by simply mixing RNA with lipofectamine or other trasfecting agents like peptides.

Also SAF preparations generally have less infectivity in them than unprocessed total brain homogenate, so the total RNA isolated from brain before SAF preparation might have even more infectivity.

RNAse A depending on salt concentration can also cleave double stranded RNA. I suggest to use RNAse III that digests only dsRNA to define the RNA species more precisely.

At the end I would like to comment on possible siRNA nature of TSE agent RNA. As I mentioned in my abstract (supplement of Brain Pathology 2006, absrtacts of ICNP, page 54, abstract 117: PRION OR VIRINO OR JUST A SMALL INTERFERING

RNA SILENCING NEURONAL SURVIVAL GENES) the strict homology of

TSE agent specific nucleic acid (siRNA) to host gene(s) that it is targeting and silencing explains

the failure to identify any “foreign” (non-host encoded) nucleic

acid sequences in infected tissues.

I propose satellite viral derivation of such siRNAs (owing to nucleic acid sequence homology with host genes). Satellite RNA replication would be supported by cryptic helper virus dormantly and persistently infecting all TSE susceptible species.

Steve Simoneau on 25 June 2009 13:42 UTC

The publication to Nature Precedings was not our first choice. The data was first submitted to Nature that decided, after almost one month of consideration, to reject the manuscript without sending it for peer-review. During this delay, Nature suggested the possibility that we submit the manuscript in Nature Precedings, an online preprint server that preserves the right of the authors to publish it a peer-reviewed journal subsequently. Because of the sensitivity of the data and to disseminate the results quickly to the whole scientific community we seized this opportunity, which is not so different than presenting data at a scientific conference.

Jean-Guy Fournier on 25 June 2009 13:52 UTC

As corresponding author I would like to make some comments . First of all to thank Nature Precedings to have opened this space of freedom. Freedom is the master word for a researcher. Of course peer-review is useful, but everyone has, at least once, lived the injustice of the emotional reactions of a unfair peer-reviewer and many of us are aware of work published in prestigious journals that came from unreproducible results. Reproductibility represents yet a crucial element in experimental research to validate results.We chose this option for our own work to further legitimate the interpretation of the data. If controls are missing, it is not by intellectual laziness because we carefully surveyed a possible contamination in several critical animal groups during a voluntary uncommon long incubation time 545 (1st experiment) /367 (2nd experiment) days.We though that to be sufficiently convincing for the reader. So I apologize to the colleagues for this truncated information because we have also performed other experiments not described in this pre-print version. Using a different protocol, we obtained a residual contamination that gave a 263K lesion profile. Also we performed second passage experiments where we noted for one animal, an infectious agent clearly different than the 263K suggesting it was born from recombinant PrP. This type of result with recPrP has never been obtained with the PMCA technique and it is not easy to compare our strategy with this approach. Concerning the attractive « UNIFIED THEORY » I would like just to specify that about the co-prion « it (the theory) denies that this nucleic acid is required for infectivity » what is precisely the heart of the debate here

Christopher Baker on 25 June 2009 15:40 UTC

I welcome the forum of Nature Precedings and appreciate its characterization as a digital scientific conference, of sorts. I am very pleased to see these data and have the opportunity to comment.

An obvious but time-consuming experiment is to perform secondary passage of this material. Only then could the reconstituted material be characterized as infectious. We have already seen transgenic/recombinant forms of PrP that evoke pathology without giving rise to a bona fide infectious agent.

Much time has elapsed since the authors isolated these SAF-associated RNAs and the lack of data as to their sequences (or even whether they are double stranded) also makes me uncomfortable.

Yervand Karapetyan on 26 June 2009 00:58 UTC

This refers to the above mentioned virus reconstitution experiments which are very relevant to the current TSE agent reconstitution experiments.

Virus reconstitution and the proof of the existence of genomic RNA. http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=10212937

laura manuelidis on 30 June 2009 20:28 UTC

This paper presents interesting data that deserves attention and follow-up. Also refreshing to see Nature permitting a non-prion interpretation in its (electronic) pages.

Heino Diringer on 01 July 2009 17:03 UTC

Heino Diringer

The principal strategy to characterize an infectious agent remains purification and identification of components, followed by an analysis of their relationship to the transmissibiliy of disease. This has been carried out in the publication by Simoneau et al. which, although not apparently peer-reviewed, provides convincing evidence supporting a novel hypothesis.

The experimental results prove that two small RNA fractions are necessary for the transmission of transmissible spongiform encephalopathies (TSEs). This is the first time that a reconstitution experiment, involving a specific nucleic acid fraction from hamster scrapie and recombinant ‘so-called’ prion protein, has resulted in TSE transmission without the necessity for unnatural sonication.

The clear methodology, the inclusion of appropriate controls, the expected low transmission rate, the fact that the pathology of the induced hamster scrapie was distinct from the starting strain (a crucial control) and the reproduction of the biochemical results in two independent experiments in combination indicate that the conclusions are soundly based.

The paper would be improved by a more detailed presentation of the purification protocol for SAF and treatment with RNase and DNase later in the purification process may have yielded a better starting material for the extraction of nucleic acid. Nevertheless the results warrant priority publication as the data can be easily tested by other groups and may lead to a more defined understanding of the transmissibility of TSEs. The authors should be congratulated to their interesting results.

It seems to me that this kind of communication of new data may sometimes be preferable to the publication of a peer-reviewed sensation (see Science 216: 136 -144, 1982), which turned out to be a false interpretation of an extraordinarily badly designed biochemical experiment (see Bioscience Reports 3: 563 -568, 1983).

Alan Dickinson on 24 July 2009 10:40 UTC

COMMENTS ON THE FRENCH PAPER

I regard the French paper as very interesting. Of course, the possibility of contamination has always to be considered. However, the paper is incomplete until the authors include a section on how they interpret PMCA data in the light of their conclusions. Their work is well conceived and carefully conducted and will become very important in rebalancing TSE research – Laura Manuelidis makes a very valid point because numerous prion sceptics are known to have been coerced by editors of various journals either to write in prionese or to withdraw their document.

—————————————————————————————————————————————————————————-

Charles Weissmann’s reaction that “there seems to be no urgent reason to resuscitate the virino hypothesis at this stage.” requires a response at this juncture. As the originator of the virino hypothesis, it is essential to correct the confusion about virinos in his TSE papers and their diagrams (1). The term virino has never implied that an informational nucleic acid (a ‘viro’) separates from its associated protecting PrP for entrance into host cells or that it is detached from PrP for replication. The virino hypothesis has not yet involved the question, if or when, the viro, during its lifecycle, may separate from its donor-coded dimeric PrP. The viro is probably a small RNA.

The virino concept originated during the 1970s from genetical and pathogenesis experiments that initially yielded a replication site hypothesis in which different TSE strains compete when injected at different times (2). A numerically limiting stage in the process of agent replication appears to be involved, but a protein-only, prion copying cascade, is open ended without any bottleneck. A prion explanation of competition is still awaited.

The murine gene, sinc (syn. Prnp), exerts exceptionally precise control of the incubation period of the many scrapie strains (>20) and the complex differences between strains, including reversal of allelic effects, This precision was interpreted as a short, direct pathway between its protein (PrP) and agent replication, and thence the strict incubation dynamics. Our 1971 conjecture that this protein could be the agent’s bonding and replication site, could also imply that a host-coded informational molecule normally occupies the bonding site and that the intruder displaces it (3).

The recurrent objection to the virino hypothesis has been that no nucleic acid has yet been isolated from infective PrP preparations. There is a well established likely explanation. Several groups have shown that it is difficult to detect NAs associated with PrP when using standard NA methods (4). The very positive breakthrough, effectively endorsing the virino hypothesis has been provided by the group led by Jean-Luc Darlix, using a very unexpected approach. They proved that PrP is an RNA chaperone protein that could be interchanged with the NCp7 nucleocapsid protein of Aids virus, where it provided the requisite wide repertoire of nucleic acid processing properties (5).

The following quotation is from Charles Weissmann’s 2004 Nature review of TSEs : ”... the finding that a particular prion strain can be transmitted between two species without changing strain-specific properties, even though the PrP sequences of the two species are very different, is unexpected because it implies that the same conformation can be imposed on PrP molecules with different amino acid sequences.” (1) If he is still able to accept this as compatible with a prion hypothesis, it is akin to adding another epicycle layer to prion theory, rather than accepting its end. The discovery that PrP is an RNA chaperone appears to herald the validation of virinos and the obituary of prions. The real virino hypothesis is very much alive, but not noisy.

(1) Weissmann, C. (2004) The state of the prion. Nature Reviews Microbiology 2, 861 (2) Dickinson, A.G. et al.(1975) Extraneural competition between different scrapie agents leading to loss of infectivity. Nature 253, 556.

(3) Dickinson, A.G. & Meikle, V. M. (1971) Host-genotype and agent effects in scrapie incubation: change in allelic interaction with different strains of agent. Molec. gen. Genet. 112, 73.

(4) Silva, J.L. et al. (2008) Intriguing nucleic-acid-binding features of mammalian prion protein. Trends biochem. Sci. 552,1.

(5) Gabus, C. et al. (2001) The prion protein has RNA binding and chaperoning properties characteristic of nucleocapsid protein NCp7 of HIV-1 J. biol. Chem. 276, 19301

HUGH FRASER on 03 August 2009 11:21 UTC

Hugh Fraser. This is an important paper. The protein-only hypothesis has become stale. The causative agent (& the many strains)of the TSEs obey the rules of biology:- genetic information/diversity, mutation, exclusion competition between strains, (all Dickinson’s work); the protein-only hypothesis fails on all of these criteria, whereas the “virino” meets these challenges. This paper opens new and exciting possibilities on the chemistry of the informational molecule that has been elusive for so long.

George Outram on 03 August 2009 15:42 UTC

I haven’t worked in this field for more than twenty years so I don’t feel qualified to judge the quality of this paper. But it seems to me that if its findings are verified and extended they good go a long way towards vindicating the assertion of Alan Dickinson and his team (with which I was privileged to work) that what ever proteins might be implicated in the pathogenesis of this class of diseases there must also be the involvement of a replicating informational molecule to account for the facts of a considerable number of transmission studies.

George Outram

Dear Yervand,

I leave the discussion to people who work actively in the prion field. There was no technical flaw in the demonstrated conversion in Surewicz lab and converting one protein conformation to another is pretty easy in many cases. Propagation of the conversion can also be done in a test tube.

As to what is the nature of the infectious agent in TSE, there is an open question. Protein-only was sufficient to cause TSE in mice in a lab (Surewicz group) and I know it could be demonstrated. Is it sufficient to have 0.000...1% to show reproducibility or do you need 50% + success to claim the hypothesis is correct?

In biology, there is plenty of templates/viruses/nucleic acids which could facilitate the process in vivo and give you a positive answer. Maybe those factors could be key components to see high (50%+) PrPc->PrPSc conversion in the field? Considering 15+ years for  the symptoms to appear in humans/large animals, it will be always a contentious issue unless you use mice.

 I cannot vouch for other labs and what they do. Every once in a while, there is a "secret sauce" which makes it go and I have seen plenty of them in my career.