The Prion Challenge to the `Central Dogma' of Molecular Biology, 1965–1991

Pergamon

Stud. Hist. Phil. Biol. & Biomed. Sci., Vol. 30, No. 2, pp. 181–218, 1999  1999 Published by Elsevier Science Ltd. All rights reserved Printed in Great Britain 1369-8486/99 $ - see front matter

The Prion Challenge to the ‘Central Dogma’ of Molecular Biology, 1965–1991 Part II: The Problem with Prions Martha E. Keyes* Since the 1930s, scientists studying the neurological disease scrapie had assumed that the infectious agent was a virus. By the mid 1960s, however, several unconventional properties had arisen that were difficult to reconcile with the standard viral model. Evidence for nucleic acid within the pathogen was lacking, and some researchers considered the possibility that the infectious agent consisted solely of protein. In 1982, Stanley Prusiner coined the term ‘prion’ to emphasize the agent’s proteinaceous nature. This infectious protein hypothesis was denounced by many scientists as ‘heretical’. This two-part essay asks why the concept of an infectious protein was considered controversial. Some biologists justified their evaluation of this hypothesis on the grounds that an infectious protein contradicted the ‘central dogma of molecular biology’. Others referred to vague theoretical constraints such as molecular biology’s ‘theoretical structure’ or ‘framework’. Examination of the objections raised by researchers reveals exactly what generalizations were being challenged by a protein model of infection. This two-part survey of scrapie and prion research reaches several conclusions: (1) A theoretical framework is present in molecular biology, exerting its influence in hypothesis formation and evaluation; (2) This framework consists of several related, yet separable, generalizations or ‘elements’, including Francis Crick’s Central Dogma and Sequence Hypothesis, plus notions concerning infection, replication, protein synthesis, and protein folding; (3) The term ‘central dogma’ has stretched beyond Crick’s original 1958 definition to encompass at least two other ‘framework elements’: replication and protein synthesis; and (4) From the study of scrapie and related diseases, biological information has been delineated into at least two classes: sequential and what I call ‘conformational’. In Part I of this essay, a brief review of the central dogma was given, and the developments in scrapie research from 1965 to 1972 were traced. This section summarized many of the puzzling, non-virus-like properties of the scrapie agent. Alternative hypotheses to the viral explanation were presented, including early versions of a protein-only hypothesis. Part II of this essay will follow the developments in scrapie and * 5725 Vesper Avenue, Van Nuys, CA 91411, U.S.A. Received 17 October 1997; in revised form 22 June 1998.

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prion research from the mid-1970s through 1991. The growing prominence of a protein-only model of infection will be countered by continued objections from many researchers to a pathogen devoid of nucleic acid. These objections will help illuminate those generalizations in molecular biology that were indeed challenged by a proteinonly model of infection.  1999 Published by Elsevier Science Ltd. All rights reserved

1. From ‘Scrapie Agent’ to ‘Prion’ By the late 1960s, a once obscure neurological disease afflicting sheep had captured the attention of a wide range of biologists. The keen interest in this fatal disorder, commonly known as ‘scrapie’, went well beyond concern for Europe’s livestock. Scrapie, and related diseases such as kuru and Creutzfeldt–Jakob disease,1 captured the imagination of scientists because the disease-causing agent could not be easily categorized. The pathogen exhibited several non-virus-like properties, and researchers began to consider several alternatives to a viral explanation, including the possibility that the scrapie agent was a viroid, a replicating polysaccharide or even a proteinaceous pathogen lacking nucleic acid entirely. The wealth of hypotheses and conjectures surrounding scrapie research throughout the 1960s and 1970s was made possible by the lack of direct evidence regarding the molecular constitution of the scrapie agent.2 Researchers struggled with questions related to the propagation of this disease without any assurance as to the kind of infectious agent with which they were dealing. Doubts about the presence of a scrapie-specific nucleic acid within the agent had pervaded scrapie research since the publication of Tikvah Alper’s reports in the mid 1960s (Alper et al., 1966, 1967). No single rival hypothesis was in a position to replace the viral one, however, as long as the chemical nature of the scrapie agent remained elusive. The molecular constitution of the agent, in turn, would remain obscure as long as preparations of the scrapie agent continued to be riddled with impurities. Beginning in the mid 1970s, major strides in purification techniques were made by Stanley Prusiner and colleagues at the University of California at San Francisco.3 By 1981, these improvements had culminated in the discovery of a unique 1 This category of related ‘prion diseases’ (with commonly infected species in parentheses) includes: scrapie (sheep and goat); transmissible mink encephalopathy (mink); chronic wasting disease (mink); bovine spongiform encephalopathy (cattle); kuru (humans); Creutzfeldt–Jakob disease (humans); Gerstmann–Stra¨ussler–Scheinker syndrome (humans); and fatal familial insomnia (humans). 2 Please refer to Part I of this essay for my discussion of scrapie research from the 1960s through the early 1970s. 3 Stanley Prusiner came into scrapie research by way of his interest in Creutzfeldt–Jakob disease (CJD), a rare neurological disease afflicting humans. Like kuru, a disease once rampant among the cannibals of Papua New Guinea, CJD shares many characteristics with scrapie. All three diseases are fatal, slow-acting disorders of the central nervous system. These diseases are characterized by a vacuolation of brain tissue, which leads to a progressive loss of coordination, dementia (in humans), and eventually death. In addition, all three are transmissible via the injection of diseased brain extracts into the brains of healthy animals.

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protein thought to be a major, if not sole, component of the scrapie agent. Prusiner and his UCSF team found their highly purified preparations of scrapie agent to be enriched ‘100–1000-fold with respect to protein’ (Prusiner et al., 1981, p. 6675). They then pointed to ‘five separate and distinct lines of evidence now clearly show[ing] that the scrapie agent contains a protein that is required for infectivity’ (Prusiner et al., 1981, p. 6677). The first line of evidence was the demonstration that scrapie infectivity was reduced 99.9% after prolonged exposure to proteinase K, an enzyme used to ‘digest’ proteins by breaking the peptide bonds linking amino acids. Secondly, the scrapie agent was inactivated by diethylpyrocarbonate (DEP), a compound known to chemically (and reversibly) modify proteins. Scrapie infectivity was also destroyed by the denaturing detergent sodium dodecyl sulfate (SDS). The fourth line of evidence was the inactivation of the agent by a very low concentration of chaotropic ions (e.g., guanidinium and SCN−), which regularly altered proteins, particularly enzymes. It normally took a concentration four to five times greater than that used on the scrapie agent to damage nucleic acids. Finally, Prusiner and colleagues pointed to the fact that the scrapie agent was rendered inactive by treatment with phenol, an organic solvent known to denature most proteins. Together, they concluded, these five sets of experimental results made ‘a compelling case for a protein within the scrapie agent’ (Prusiner et al., 1981, p. 6677).4 Prusiner and colleagues also searched for a scrapie-specific nucleic acid within the agent; the results of this search were published in the same 1981 paper. Purified samples of the scrapie agent were exposed to DNase and RNase, enzymes which break down DNA and RNA, respectively. When put in solution with either of these enzymes, however, the scrapie agent did not lose its ability to infect. The conclusion that the scrapie agent depended—at least in part—on a protein for infectivity represented ‘the most convincing identification so far of a macromolecule within the agent’ (Prusiner et al., 1981, p. 6675). The continued failure to detect a nucleic acid within the scrapie agent, however, raised central questions as to how the agent could replicate and infect: Now that the presence of a protein has been demonstrated, an intensive search must be made for the genome of the agent. Does a genome coding for the scrapie agent protein reside within the infectious agent itself or is the genome part of the cellular genetic material? Will the unusual properties of the scrapie agent, which distinguish it from conventional viruses, reveal unprecedented mechanisms of replication? (Prusiner et al., 1981, p. 6679)

The work of Prusiner and his colleagues represented a major step in elucidating

4 The authors of this 1981 paper did allow for the possibility of more than one protein within the agent. When commenting on the hydrophobic properties of the protein they believed to be contained within the agent, Prusiner and colleagues remarked that the protein required for infectivity may not have been identical to the protein exhibiting the hydrophobic characteristics (Prusiner et al., 1981, p. 6675).

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the chemical nature of the scrapie agent but its means of infection and replication remained a mystery. When referring to the scrapie agent, Prusiner and his collaborators initially endorsed the use of the term ‘unusual slow virus-like agent’ over what they considered to be the misleading ‘slow virus’ label (Prusiner et al., 1981, p. 6675). By 1982, however, Prusiner had coined his own term, stressing the protein requirement for infection: . . . the molecular properties of the scrapie agent differ from those of viruses, viroids, and plasmids . . . Its resistance to procedures that attack nucleic acids, its resistance to inactivation by heat, and its apparent small size all suggest that the scrapie agent is a novel infectious entity. Because the dominant characteristics of the scrapie agent resemble those of a protein, an acronym is introduced to emphasize this feature. In place of such terms as ‘unconventional virus’ or ‘unusual slow virus-like agent’, the term ‘prion’ (pronounced pree-on) is suggested. Prions are small proteinaceous infectious particles which are resistant to inactivation by most procedures that modify nucleic acids. The term ‘prion’ underscores the requirement of a protein for infection (Prusiner, 1982, p. 141).

2. Hypotheses and Frameworks The establishment of a protein within the agent necessary for infection, along with earlier experimental evidence, allowed Prusiner to narrow his field of alternate hypotheses for the scrapie agent (prion) down to two ‘possible models’. The first was that of a small piece of nucleic acid, ‘buried within a tightly packed protein shell’, which would make it virtually undetectable by conventional experiments designed to isolate or destroy nucleic acids (Prusiner, 1982, p. 141). This model, Prusiner estimated, ‘might seem the most plausible’ from a theoretical standpoint considering the restrictions laid down by the central dogma (Prusiner, 1982, p. 141). The experimental evidence gathered thus far, however, had given no indication of a nucleic acid within the agent. Nevertheless, Prusiner admitted in 1982 that ‘current knowledge does not allow exclusion of a small nucleic acid within the interior of the particle’ (Prusiner, 1982, p. 141). The second possible model for the scrapie agent, an infectious protein devoid of nucleic acid, faced the reverse criticism. Although this second model, according to Prusiner, was ‘consistent with the experimental data’, the idea of an infectious protein was considered ‘clearly heretical’ (Prusiner, 1982, p. 142). The branding of a protein model of infection as ‘heretical’ clearly underscored the community’s insistence on the need for nucleic acid for replication of an infectious agent.5 5 The Editors of a 1982 issue of Chemical and Engineering News interpreted this model as a challenge to an even greater biological principle: the definition of life itself. In their article, entitled ‘Possible New Life Form Tinier Than Virus’, the authors implied that infectious agents are, by definition, living beings. The authors wrote: ‘A life form that does not contain nucleic acid, Prusiner admits, is clearly heretical. It is a central tenet of biology that nucleic acids—DNA and RNA—are the universal bearers of the genetic code and, hence, are necessary for life’ (Anonymous, 1982).

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Theoretical considerations aside, Prusiner acknowledged that there was still not enough known about the molecular composition of the agent to rule in favor of either a virus or protein-only model of infection: There seems to be little advantage in championing one model over another . . . Rigid categorization of the scrapie agent at this time would be premature. Determination of its molecular structure will be required prior to deciding whether prions represent a distinct subgroup of extraordinarily small viruses or a completely different type of pathogen which lacks a nucleic acid genome (Prusiner, 1982, p. 142).

In his 1982 review paper, Prusiner explored some hypothetical modes of replication for both cases: (1) prions containing undetected nucleic acid and (2) prions devoid of nucleic acid entirely. Assuming that prions did hold small bits of nucleic acid under their protective protein coats, the first possibility was that the nucleic acid contained within the prion could code for the protein shell and replication of the entire prion could be carried out independently of the host genome. The ‘exceptionally small’ size of the scrapie agent (Alper et al., 1967), however, required that the segment of nucleic acid be so small that, ‘if such oligonucleotides exist, they must have a function other than that of a template directing the synthesis of scrapie coat proteins’ (Prusiner, 1982, p. 141). In this case, unlike viral infections, the small segment of nucleic acid would serve ‘as a regulatory element instead of a coding template’ (Prusiner, 1982, p. 141). The nucleic acid fragment would somehow play the role of an inducer, triggering the production of the prions’ protein(s) encoded by the host’s genome. Replication mechanisms for the second model considered by Prusiner were more troublesome: The second possibility is that prions are, in fact, devoid of nucleic acid. If this is the case, then alternative modes of replication for these infectious proteins must exist. The macromolecular information required for the synthesis of prions must be contained either in the host cell or in the prion itself (Prusiner, 1982, p. 142).

In the case where the host cell carried the gene for the infectious protein(s), Prusiner postulated that the infectious prions themselves would assume the role of the inducer upon infection, switching on the production of the prion protein(s) in the host cell which were normally repressed. The prion-producing gene in this case, Prusiner remarked, would be unusual due to the fact that it would have to be ‘highly regulated, not readily activated, and present in various mammalian cells ranging from mice to monkeys’ (Prusiner, 1982, p. 142). Prusiner raised two areas of concern with regard to this mechanism for prion replication. First, no instance of an inducer acting on a specific gene which coded for its own synthesis had ever been found. The second problem posed by this method of replication was how one could account for the selection and preservation of genes that produced particles lethal to the organism. Prusiner suggested that perhaps, ‘under normal regulation’ (in the absence of harmful prions), these same

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genes could code for some ‘related protein or proteins’ which were not harmful but quite necessary for the survival of the organism.6 The final mode of replication for a scrapie agent (prion) devoid of nucleic acid required that the ‘macromolecular information required for the synthesis of prions’ somehow be contained within the proteinaceous agent itself. Without the concept of a transfer of conformational information from one protein to another, the difficulties with this last proposal were obvious: This hypothesis contradicts the ‘central dogma’ of molecular biology. Unorthodox mechanisms such as reverse translation or protein-directed protein synthesis would allow prions to replicate. We have no precedents for either of these synthetic processes in biology (Prusiner, 1982, p. 142).

Here Prusiner melds the two issues that I argue have been considered jointly throughout the biology literature from (at least) the early 1960s through the 1980s: replication and information transfer. The first ‘unorthodox’ mechanism, reverse translation, was clearly a violation of Francis Crick’s original constraints for the transfer of sequential information outlined by his Central Dogma (Crick, 1958). Reverse translation required not only that proteins direct their own synthesis, but that the linear order of amino acids constituting the protein be somehow ‘read back’ and translated into a corresponding sequence of nucleic acid bases. The resultant nucleic acid would then encode the sequential information necessary to ‘reconstruct’ or synthesize the particular prion protein(s) necessary for infection. This proposal explicitly contradicted Crick’s original definition of the Central Dogma, which prohibited the transfer of sequential information from proteins back into nucleic acids. ‘Protein-directed protein synthesis’, the second ‘unorthodox’ mechanism of prion replication discussed by Prusiner, also required that the protein code for its own synthesis. Although Prusiner did not specify the mechanism by which this synthesis would occur, it is likely that he was considering the kind of protein synthesis once envisioned by Crick. In that instance, a protein could hypothetically unfold and act as the physical template upon which an identical string of amino acids was formed (Crick, 1964). This method of protein synthesis would also violate both Crick’s and James Watson’s versions of the central dogma.7 In order for this kind of protein synthesis to occur, sequential information would have to be transmitted

6 This explanation is strikingly similar to the present account, which postulates that scrapie prions are simply proteins produced under normal conditions with a lethal change of shape. At this point, however, there is no indication that Prusiner was considering the possibility of prions conferring a change of shape upon normal cellular proteins. 7 Crick’s Central Dogma stated that sequential genetic information could flow from nucleic acid to nucleic acid, or from nucleic acid to protein. The transfers of sequential information forbidden by Crick’s Central Dogma were from protein to protein, or from protein back into nucleic acids. Watson, on the other hand, equated the unidirectional flow of sequential information from DNA to RNA to protein with the synthesis of nucleic acids and proteins (Watson, 1965). Please refer to Part I of this essay for a more complete discussion of Crick and Watson’s versions of the central dogma.

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from protein to protein, and the template for the formation of the new protein would be another protein rather than messenger RNA. ‘Unorthodox’ for Prusiner did not mean impossible. If the still undetermined structure of the prion macromolecule was found not to include nucleic acid, Prusiner acknowledged that ‘alternative mechanisms of replication and information transfer must then be entertained’ (Prusiner, 1982, p. 143). Such a conclusion would not be easily drawn, however. Charges to be leveled against such a challenge to the central dogma, described in this one paper alone as ‘heretical’, ‘unorthodox’, and ‘forbidding’, would testify that this hypothesis of Crick’s had indeed been elevated to a higher status. The publication of Prusiner’s 1982 review article provoked a variety of ardent responses. For I. H. Pattison, the discoveries of Prusiner and colleagues allowed him—‘after a long time in the dog-house’—to reassert his own protein hypothesis for scrapie infectivity (Pattison, 1992, p. 21). Pattison’s hypothesis, which allowed for the host genome to code for the scrapie agent, or at least the protein required for infectivity, was based on a set of experiments performed in the 1960s. Pattison and his colleague Katherine Jones had inoculated mice with preparations from both scrapie-infected and healthy mice. The samples of normal brain tissue were fractionated and prepared in exactly the same manner as the scrapie-infected tissue. Remarkably, Pattison and Jones found that scrapie sometimes occurred in control mice injected with healthy tissue (Pattison and Jones, 1968). The possibility arose, therefore, that fractionation procedures alone were capable of isolating or ‘releasing’ a scrapie-producing agent present in all brain tissue. This proposal that the scrapie agent might be an ever-present, albeit ‘inhibited’, product of normal brain tissue would explain several of the puzzling properties of the agent. These properties included the apparent lack of a scrapie-specific nucleic acid, absence of host antibodies, and Pattison’s earlier detection (Pattison, 1974) of the spontaneous onset of the disease in sheep: If for a moment, it be accepted that we did indeed detect scrapie agent in normal tissue, an explanation can be offered for the apparent enigma of replication without nucleic acid that faced us initially, and now faces Prusiner. The agent may not, in fact, replicate as the disease progresses; instead—as we suggested in 1968—replication may be simulated by an unmasking process of a particle already present. Again, if scrapie agent were a component of normal tissue, there would be a simple explanation for the widely observed but obscure phenomenon that the scrapie agent does not apparently stimulate antibody. If the agent were ‘self’ no antibody would be expected (Pattison, 1982, p. 200, my emphasis).

Pattison made an interesting clarification in this passage which is useful to keep in mind as hypotheses concerning the nature of the scrapie agent continue to unfold. If, in fact, scrapie were caused by an ‘unmasking’ or activation of a particle already present in the host organism, this would not constitute ‘replication’ in the true sense. Genuine biological replication, Pattison implied, required the presence of nucleic acid.

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Other reactions to the recent findings in scrapie research were more cautious. Richard Kimberlin, for one, warned that scientists need not rush outside the bounds of ‘conventional thinking’ in order to account for the behavior of the scrapie agent (Kimberlin, 1982, p. 107). One of Kimberlin’s first points in his critique of the work of Prusiner and others was that the existence of a scrapie-specific protein contained within the agent had yet to be demonstrated (Kimberlin, 1982, p. 107). He noted that the agent’s loss of infectivity, caused by treatments which usually denature or destroy most proteins, made it ‘reasonable to conclude’ that infectivity somehow involved a protein (Kimberlin, 1982, p. 107). However, Kimberlin continued, ‘the unresolved issue is whether such a protein is “contained” in the agent as an integral part of its structure or merely associated with it’ (Kimberlin, 1982, p. 107). Kimberlin also argued that the existence of a nucleic acid within the agent could not be ruled out. In fact, he maintained that a scrapie-specific nucleic acid was necessary in order to account for the transmission of various stable strains of the disease to other animals both within and outside their own species (Dickinson and Outram, 1979). Kimberlin reviewed all three of Prusiner’s ‘highly unorthodox models’ that excluded a scrapie-specific nucleic acid, and argued that none could account for such phenomena. First, in the case of protein-directed protein synthesis, Kimberlin found it hard to imagine how a protein could transmit such specificity, either by reverse translation or by acting as its own template: It is difficult to conceive how proteins could code for their own synthesis although scrapie models of this kind were proposed as long ago as 1967 . . . But since then the requirements of a scrapie model have become more stringent; the copying process must have a precision compatible with the existence of several different, genetically stable strains of scrapie (Kimberlin, 1982, p. 108).

Kimberlin next considered Prusiner’s two other possible models for an agent devoid of scrapie-specific nucleic acid. One model postulated that the protein contained within the infectious prion acted as an inducer, triggering the host gene that coded for the agent’s synthesis. Prusiner’s other model allowed for a small piece of nucleic acid to be contained within the prion. In order to fit inside the exceptionally small agent, the oligonucleotide could not possibly be large enough to code for the prion. Instead, Prusiner postulated, it too could act as inducer or ‘regulatory element’, initiating the production of the scrapie agent coded for by the host genome. In both of these models, the host gene(s) encoded the scrapie-specific information necessary to reproduce the scrapie agent. Synthesis of the agent via the transcription and translation of the sequential information encoded by nucleic acids was certainly a more conventional proposal than that of reverse translation or protein-directed protein synthesis. However, as Kimberlin pointed out, these two models still failed to offer an explanation for the existence of multiple strains:

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[A] host nucleic acid that encodes scrapie-specific information . . . is certainly conceivable but difficult to reconcile with evidence that scrapie agents behave like pathogens with their own coded information: the natural disease is infectious, various strains of the agent can be transmitted experimentally to many different species, some strains give rise to mutants and strain selection can occur on serial passages of mixtures. To explain these properties in terms of host coding introduces a complexity that seems unnecessary in the absence of data which necessitate it (Kimberlin, 1982, p. 108).

The insistence on strain-specific information within the scrapie agent would keep alternative hypotheses in check throughout the 1980s. Given the apparent necessity of a scrapie-specific nucleic acid to explain not only replication but strain variation (and mutation) as well, Kimberlin chose to endorse yet another hypothesis not considered by Prusiner. The ‘virino hypothesis’ was first introduced in 1979 as a kind of compromise between the virus and proteinonly hypotheses (Dickinson and Outram, 1979). The virino hypothesis satisfied the need for nucleic acid within the agent, while allowing the host (and host proteins) to play a major role in the replication of the agent. In Kimberlin’s opinion, ‘it fit nicely into a biological niche lying between viruses, which specify some of their own proteins, and viroids which need no protein at all’ (Kimberlin, 1982, p. 108). The virino hypothesis was based on two assumptions: (1) a scrapie-specific nucleic acid was necessary; and (2) the scrapie nucleic acid was not translated. Unlike viral nucleic acid, the postulated nucleic acid within the virino did not code for a scrapie protein. As a result, the virino’s nucleic acid could be small enough to conform to the small size calculated for the scrapie agent. Upon infection, this small nucleic acid could replicate with the aid of host enzymes and ‘interact in some strain specific way with the host to produce the disease’ (Kimberlin, 1982, p. 108). One way this could be accomplished was by the binding of the scrapiespecific nucleic acid and a host protein to form the infectious agent. This hypothesis could account for both the existence of multiple strains and the proteinaceous characteristics of the agent. Assuming that the invading nucleic acid were somehow covered by a ‘sticky’ host protein, one would expect that traditional methods designed to detect the nucleic acid would fail. The recruitment of normal host proteins in the production of the agent would also explain the lack of an antigenic response by the host. In Kimberlin’s opinion, the virino hypothesis demonstrated that hypotheses could be constructed to account for all experimental evidence, including multiple strains, without violating accepted notions about the replication of proteins: . . . the main point of these speculations is that we do not yet need to build hypotheses outside the current framework of molecular biology to accommodate the scrapie agent. The real need is to assemble more hard facts (Kimberlin, 1982, p. 108).

Kimberlin’s call was answered that same year when a unique protein was found in highly purified samples of scrapie-infected hamster brain (Prusiner et al., 1982; Bolton et al., 1982). With improvements in purification techniques once again play-

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ing a crucial role, gel electrophoresis revealed a protein with a molecular size of 27,000 to 30,000 daltons, dimensions on the order of the estimated size of the agent. Having already argued that a protein was required for scrapie infectivity (Prusiner et al., 1981; Prusiner, 1982), Prusiner’s next task was to demonstrate that at least one specific protein was a structural component of the agent. The unique protein discovered in 1982 proved to be the perfect candidate. UCSF researchers David Bolton, Michael McKinley and Prusiner pointed to several results which suggested that this protein, dubbed ‘PrP’ for prion protein, was a necessary structural component of the prion and not simply an extraneous protein that copurified with the infectious agent (McKinley et al., 1983). There were several converging lines of evidence: (1) The concentration of the protein (PrP) was directly proportional to the titer of the agent in all scrapie-infected preparations; (2) No PrP could be detected in any of the control samples of non-infected hamster brain; (3) Under nondenaturing conditions, PrP was resistant to hydrolysis by proteinase K. This unusual feature distinguished PrP from all other proteins found in normal brain. Like PrP, the scrapie agent’s infectivity remained unaltered by treatment with various proteases; (4) After prolonged exposure to proteinase K at high temperatures, PrP was eventually degraded. Only following the denaturation of PrP did the scrapie agent suffer a reduction in infectivity; and (5) PrP and prions copurified by two different procedures, indicating that the two molecules were very similar in constitution if not identical. McKinley, Bolton, and Prusiner concluded that these ‘parallel changes’ between PrP and scrapie prions indicated that PrP was the ‘one major protein’ within the infectious agent. Whether or not prions contained additional ‘minor proteins’ remained to be established (McKinley et al., 1983, p. 57). The identification of a unique scrapie-specific protein certainly strengthened the claim that protein played a crucial role in scrapie infection. This assumption implied that PrP also played a central role in the replication of the agent. The continued lack of evidence for a scrapie-specific nucleic acid left researchers with the now familiar dilemma: how could one account for an infectious protein without impugning accepted notions about infection and replication in modern biology? Following the discovery of PrP, Prusiner published an article in Scientific American in which he characteristically reviewed the set of alternative hypotheses for the scrapie agent in light of the new experimental evidence (Prusiner, 1984). This essay represents an important turning point in scrapie research. For the first time, Prusiner began to separate fundamental claims about the nature of information transfer, replication, and the overall role of nucleic acids in biology. Once these clarifications were made, Prusiner was then able to reconsider whether or not the proposal of an infectious protein contradicted the central dogma at all. Prusiner laid out these crucial distinctions at the very beginning of his 1984 essay:

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. . . the replication of the DNA is the crucial event in reproduction. Even viruses, which cannot reproduce independently and whose status as living organisms is therefore questionable, take their identity from a molecule of DNA or RNA . . . The principle that genetic [my italics] information invariably flows from nucleic acids to proteins is called the central dogma of molecular biology (Prusiner, 1984, p. 50).

This passage is significant for two reasons. First, Prusiner made explicit the definition of the Central Dogma given by Crick in 1958.8 Earlier papers on scrapie research either mentioned the term ‘central dogma’ in passing or alluded to it with the use of such phrases as ‘theoretical structure’ (Griffith, 1967) or ‘accepted dogma’ (Lewin, 1972). In his 1982 review, Prusiner himself spoke of protein-directed protein synthesis as a violation of the central dogma but did not include a definition of the central dogma in that paper. Others, like Peter Lewin (1972), provided a definition more stringent than either Crick’s or Watson’s version. A scientist or historian familiar with Crick’s work might not be struck by Prusiner’s inclusion of the ‘correct’ definition of the central dogma. However, as this body of scrapie research attests, the appearance of Crick’s definition of the Central Dogma—in the very literature concerned with a possible exception to it—was not as common as one might think. A second and even more surprising distinction made by Prusiner in this 1984 essay was his separation of the Central Dogma from the insistence on the replication of DNA for reproduction. Again, this is an important clarification rarely found up to this point. By distinguishing the rules for the transfer of information among nucleic acids and proteins (i.e., Crick’s Central Dogma) from the phenomenon of reproduction, Prusiner brought vague terms like ‘theoretical structure’ and ‘intellectual base’ into sharper focus. Once the fundamental claims of molecular biology were delineated, one could then ask which specific claims were threatened by the prion challenge and why. With these distinctions in mind, Prusiner’s analysis continued: It now appears that an infectious agent named a prion may stand out as an exception to the rule that every organism carries nucleic acids defining its own identity. The prion is known to be capable of initiating the production of new prions . . . Moreover, among the molecular components of the prion there is at least one protein, and so one would expect to find a DNA or RNA template specifying the structure of the protein. The evidence gathered so far however, indicates the prion has no nucleic acid at all. Even if some DNA or RNA is ultimately found in the prion, there is probably not enough to encode the structure of the protein. From these facts it does not necessarily follow that the prion violates the central dogma—the latest results favor less heretical hypotheses—but there is little question its mode of reproduction is highly unusual (Prusiner, 1984, p. 50, my emphasis).

8 It is safe to assume here that—as was the case with Crick’s definition—the term ‘genetic information’ can be taken to mean the sequential information stored within the order of nucleic acid bases.

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According to Prusiner, therefore, the concept of an infectious protein challenged at least two generalizations concerning infection and protein synthesis. The first was ‘the rule that every organism carries nucleic acids defining its own identity’. Implicit within this statement was the assumption that an infectious agent was— by definition—an organism. Referring back to the previous excerpt from this paper, Prusiner also stipulated that living organisms must be able to reproduce independently, hence the ‘questionable’ status of viruses. The second major claim contradicted by the proposed infectious protein was the need for nucleic acids in the reproduction of proteins. Even in the case where the host genome did code for the infectious protein and the activation of the prion gene(s) was induced by the agent’s protein or fragment of nucleic acid, it would still constitute a ‘highly unusual’ means of protein reproduction. Unusual methods of reproduction, Prusiner argued, had ramifications for one’s understanding of the term ‘infection’ as well: If the prion is indeed a single protein and the product of a gene native to the host organism, the time may have come for a reconsideration of what is meant by the concept of infection (Prusiner, 1984, p. 59).9

What was not obvious to Prusiner, however, was how the concept of an infectious protein challenged the Central Dogma. Prusiner had recognized two mechanisms for protein replication that plainly contradicted Crick’s Central Dogma. The first was that of reverse translation (Fig. 1f), which stipulated that sequential information would flow from proteins back into nucleic acids. The second ‘heretical’ mechanism was that of ‘protein-directed protein synthesis’ (Fig. 1g). In his 1984 paper, Prusiner better specified this alternative first mentioned in his 1982 review. He described an example of ‘protein-directed protein synthesis’ as ‘the amino acid sequence of PrP . . . serv[ing] directly as a template for the construction of a new protein molecule’ (Prusiner, 1984, p. 58). This definition of proteindirected protein synthesis contradicted both Crick’s and Watson’s versions of the central dogma. These two violations of Crick’s Central Dogma aside, Prusiner argued that there were several ‘less heretical’ hypotheses to account for the prion’s replication, assuming that this particle contained at least one major protein but no scrapiespecific nucleic acid. Although these additional mechanisms for prion replication might have clashed with accepted notions about infection and protein reproduction, these contradictions should not have been taken as evidence that the Central Dogma was somehow compromised. Prusiner proceeded to outline several possible mechanisms for prion replication that did not violate Crick’s Central Dogma. After dismissing the virus hypothesis as ‘quite unlikely’, Prusiner considered the possibility that the host genome carried 9 The scientific community may have indeed formally recognized this new ‘concept of infection’ by awarding Prusiner the 1997 Nobel Prize in Physiology or Medicine. In their announcement, the Karolinska Institute’s Nobel Assembly stated that Stanley B. Prusiner had been awarded the Nobel Prize ‘for his discovery of Prions—a new biological principle of infection’.

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Fig. 1. From ‘Prions’, by Stanley Prusiner, p. 57. Copyright  (1984) Scientific American, Inc. All rights reserved.

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the gene(s) for the prion protein. This hypothesis allowed for at least three ‘plausible’ mechanisms for prion replication (Prusiner, 1984, p. 58). In the case where the infectious prion contained a small, nongenomic segment of nucleic acid, this oligonucleotide could interact with the host DNA in one of two ways: (1) it could insert itself into the host DNA just ahead of the PrP gene, thereby triggering transcription of mRNA and synthesis of the protein; or (2) the segment of nucleic acid could bind to the host DNA like an inducer, activating the transcription of the PrP gene. The third possible mechanism for prion replication assumed that the prion contained no nucleic acid at all. In that instance, the prion protein itself served as the inducer and initiated its own synthesis, a phenomenon ‘not entirely without precedent’ (Prusiner, 1984, p. 58). Whatever the method for triggering the host gene(s) to code for PrP, the phenomenon of multiple strains still had to be accounted for. As Prusiner himself asked, ‘if replication of the disease agent is nothing more than activation of a host gene, how can the same genetic line of animals serve as hosts to multiple prions?’ (Prusiner, 1984, p. 58). Prusiner suggested that a rearrangement of genes might account for the apparent variation in prion proteins produced, but did not elaborate. Not included in Prusiner’s discussion but present in diagrammatic form was a fourth hypothetical mechanism for prion replication not seen in his earlier papers. In this instance, the host genome did not code for PrP itself, but for a PrP ‘precursor’ (Fig. 1e). The synthesis of this prion precursor occurred via the conventional transcription and translation of the sequential information in the linear order of nucleic acid bases. The prion itself contained no nucleic acid and consisted primarily, if not entirely, of PrP. Upon infection, the PrP interacted not with the host DNA but with the host protein playing the role of the PrP precursor. According to Prusiner’s diagram, the PrP precursor was of a different shape from the prion protein. The prion protein then catalyzed the conversion of the normal host protein into one identical to itself. This last mechanism, although downplayed by Prusiner in 1984, would become increasingly visible as researchers sought an explanation for prion replication that accounted for the experimental evidence without violating the Central Dogma. Before one could choose between the alternative mechanisms for prion replication laid out by Prusiner in 1984, it first had to be determined if the host genome did indeed code for the infectious protein PrP. (The small protease-resistant protein found in 1981 was now designated ‘PrP 27–30’ in reference to its molecular weight.) The first step in locating the PrP 27–30 gene was taken in 1984 by Leroy Hood, Prusiner, and their co-workers at the California Institute of Technology and UCSF. At one end of PrP 27–30, referred to as the N-terminal of the protein, seventeen amino acids were identified (Prusiner et al., 1984). This particular sequence of amino acids did not correspond to any other known N-terminal sequences. In addition to the partial sequencing of PrP 27–30, Prusiner, Hood, and col-

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leagues also examined the ultra-violet absorption spectra of the purified protein. The spectra indicated no covalent links between the prion protein and potential nucleic acid segments. These results suggested that the prion contained neither a large scrapie-specific nucleic acid nor a small, nongenomic oligonucleotide. In light of these findings, the authors concluded that any nucleo-protein model of infection, including that of a small virus, was ‘unlikely’ (Prusiner et al., 1984, p. 132). With the partial amino acid sequence of PrP 27–30 in hand by 1985, researchers began their search for a prion protein gene. Using oligonucleotide probes corresponding to the N-terminal sequence of PrP 27–30, a DNA clone (cDNA) encoding PrP 27–30 was selected from a cDNA library. This radiolabeled cDNA served as a probe to seek out a corresponding segment of DNA in the chromosomes of healthy and scrapie-infected hamsters. In an investigation led by Charles Weissmann and Bruno Oesch of the University of Zu¨rich, researchers found segments of DNA in both healthy and scrapie-infected hamster brain that appeared to encode PrP 27–30 (Oesch et al., 1985).10 PrP-related messenger RNA (mRNA) was also found in both healthy and infected hamster brain tissue, and at the same levels (Oesch et al., 1985; Chesebro et al., 1985). The steady amount of mRNA implied that the prion protein was being produced by the host under normal conditions as well as during infection. The question that arose, of course, was how the prion protein could be regularly manufactured by healthy organisms without causing the onset of the disease. This enigma could be explained if a difference were found between the infectious PrP 27–30 and its non-lethal counterpart. Oesch, Weissmann and colleagues pointed to several lines of evidence suggesting that such a crucial difference did exist. First, gel electrophoresis of both healthy and scrapie-infected hamster tissue, before treatment with proteinase K, isolated larger (i.e., slow moving) proteins of about 33– 35 kilodaltons. Proteins of this weight from healthy and infected samples had an affinity for the same antibodies and were thought to be produced by the PrP mRNA. The difference between these two PrP-related proteins lay in their reaction to proteinase K. Treatment of the 33–35 kilodalton protein found in scrapie-infected tissue (PrP 33–35Sc) yielded the familiar PrP 27–30. These results implied that PrP 27–30, once thought to be the entire infectious prion protein, was actually the protease-resistant core of the original prion protein. Proteinase K, it appeared, partially cleaved PrP 33–35Sc, leaving only the small PrP 27–30 intact.11 Because purification of the scrapie agent now routinely involved treatment with this enzyme,

10 Oesch and colleagues also found similar DNA sequences in murine and human DNA, which suggested that they too coded for the prion protein. That same year, a PrP gene was also found in mice (Chesebro et al., 1985). 11 The conclusion that PrP 27–30 was derived from a larger protein was supported by DNA sequence analysis of the purported PrP gene. The cDNA probe, selected on the basis of the PrP 27–30 N-terminal sequence, had singled out a segment of host DNA with an open-reading frame (coding region) that could potentially code for a protein of at least 240 amino acids in length (Oesch et al., 1985, p. 736). A gene of this ‘size’ could easily code for the 33–35 kilodalton protein discovered in these experiments.

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it was not considered unusual that the larger protein had not been detected in previously prepared samples. Preparations of normal tissue containing PrP 33–35C (i.e. the form found in healthy organisms) were also treated with proteinase K. In contrast to the related protein in scrapie-infected samples, this protein from normal tissue was completely degraded by the enzyme (Oesch et al., 1985, p. 735). The reaction of PrP 33– 35C to proteinase K explained why no PrP 27–30 was ever found in non-infected preparations of hamster brain. Oesch and colleagues outlined several possible explanations to account for the fact that two proteins could be encoded by the same chromosomal gene yet exhibit such dissimilar properties. One explanation proposed that PrP 33–35Sc and PrP 33– 35C differed slightly in their primary structure (i.e., amino acid sequence) due to point mutations or base rearrangements in either the host DNA or RNA. Although the authors did not explicitly mention a difference in conformation between the two prion-related proteins, they did point out that ‘small changes in sequence can be of great significance’ (Oesch et al., 1985, p. 742). Because the linear order of amino acids is generally understood to dictate the folding of a protein, this explanation did allow for a difference in conformation between the two related proteins. Another explanation attributed the lethal difference between the two proteins to a chemical change occurring after translation. The possible post-translational changes included the chemical modifications glycosylation, phosphorylation, or proteolytic cleavage (Oesch et al., 1985, p. 742). Also considered was the possibility that scrapie was simply caused by an abnormally high concentration of PrP 33–35 in brain cells. This alternative did not explain how the same protein could react differently to proteinase K when present in different amounts. The following year, additional experiments were performed to help clarify the difference between the two prion-related proteins (Basler et al., 1986). The chromosomal PrP genes found in both healthy and infected hamsters a year earlier were cloned and sequenced. Comparison of the two base sequences showed that the chromosomal PrP genes were identical in healthy and scrapie-infected organisms, leading the authors to conclude the following: Our results suggest that there is only a single PrP gene, and its sequence and organization make it unlikely that the different properties of the PrP isoforms can be explained by alterations in the amino acid sequence; thus, it seems more probable that the isoforms arise from post-translational modifications or variations in protein conformation (Basler et al., 1986, p. 417).

The change of conformation hypothesis considered in this case was quite different from that proposed a year earlier. In 1985, Oesch and colleagues had postulated a slight variation in amino acid sequence between PrPSc and PrPC to account for the difference in shape (and function) between the two isoforms. This hypothesis was based on the long-standing assumption that the primary sequence of amino acids (itself determined by the linear order of nucleic acid bases) dictated the fold-

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ing of the protein (Crick, 1958). This generalization was brought into question, however, once it was found that the sequence of bases in the host-encoded PrP gene was the same in healthy and scrapie-infected hamsters (Basler et al., 1986). According to Crick’s Sequence Hypothesis (1958), all proteins encoded by the same PrP gene should have the same primary sequence. The assumption that followed was that these proteins would then all fold in exactly the same manner (Crick, 1958; Stryer, 1988). For PrPSc and PrPC to be encoded by the same exact chromosomal gene, yet still exhibit such a dramatic difference in function (hence, shape), something must act on the cellular form of the prion protein to convert it into its lethal shape during infection. If the prion (scrapie agent) were to consist of only PrPSc, the most likely candidate for the modification or conversion of PrPC would be the infectious protein itself. The experimental findings of the scientists at UCSF, Zu¨rich, and elsewhere during the mid 1980s were indeed significant. By 1986, a number of research groups, led by Prusiner at UCSF, supported the following conclusions: (1) A protein was required for scrapie infectivity; (2) A unique protease-resistant protein (PrP 27– 30) was found only in highly purified fractions of scrapie-infected tissue; (3) PrP 27–30 was the protease-resistant core of a larger protein PrP 33–35Sc; (4) The prion protein gene was encoded by the host genome of both healthy and scrapie-infected organisms; and (5) PrP 33–35Sc differed from the prion protein found in healthy organisms (PrP 33–35C) as a result of some kind of post-translational chemical or conformational modification. In conjunction with these results was the continued failure by these researchers to detect a nucleic acid within the scrapie agent (prion). Consequently, the possibility of a prion consisting solely of protein had become increasingly viable throughout the 1980s. 3. ‘Romantics’ vs ‘Non-Heretics’ While Prusiner and colleagues entertained the idea of an infectious protein— whatever its means of replication—others followed a completely different track. Those who continued to champion a virus hypothesis interpreted the results of Prusiner and others in an entirely different light. Virus proponents, I will illustrate, were guided by a theoretical framework which adhered to traditional notions of infection and replication. While the insistence on nucleic acid for protein synthesis and disease agent replication played a major role in this framework, Crick’s Central Dogma did not. In 1985, D. Carleton Gajdusek, a pioneer in the research of the scrapie-like disease kuru, described the division between investigators as follows: . . . the crucial argument revolves around the assumption that the scrapie (and kuru and CJD) virus . . . does or does not contain the information for its replication in an intrinsic nucleic acid genome. Should it prove not to have such a nucleic acid genome, then the ‘romantics’, who have prematurely contended, on insufficient evidence, that it is a totally new form of replicating microbe, a pure protein . . . devoid of DNA or

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RNA, will have been right. The conservatives, ‘non-heretics’, who contend that it must have DNA or RNA although they are unable to produce any demonstration thereof, do so on the basis of their faith in the basic tenets of microbiology . . . (Gajdusek, 1985, pp. 493, 496).

In this passage, Gajdusek touched on two major ‘tenets’ to which ‘non-heretics’ subscribed. The first was that either DNA or RNA was necessary for the replication of an infectious agent. The second was that this disease-specific genetic information must be contained within the agent itself. The infectious protein considered by Prusiner and colleagues was clearly in violation of both tenets, regardless of whether or not its ‘replication’ could be achieved without violating Crick’s Central Dogma. Gajdusek’s remarks also reflected the frustration felt by the group of ‘conservatives’ contending that the scrapie agent was a virus, albeit an ‘unconventional’ one. Although the virus proponents argued that those supporting a protein-only model of infection did so on ‘insufficient evidence’, these supporters of the traditional virus alternative were ‘unable to produce any demonstrations’ of nucleic acid within the agent. Although proponents of the virus hypothesis were unable to demonstrate that the scrapie agent was indeed a virus, this did not deter them from punching holes in the arguments in favor of an infectious protein. One of the first arguments put forth against a viral model for the scrapie agent was based on its size (Alper et al., 1966 and Alper et al., 1967). The ‘exceptionally small size’ of the scrapie agent, measured by Tikvah Alper and her colleagues during the mid 1960s, was thought to be too small to allow for a nucleic acid molecule. If the agent were somehow able to contain a fragment of nucleic acid, this oligonucleotide could not have been large enough to encode the genetic information for the entire infectious particle. Proponents of the virus hypothesis argued that Alper’s size estimates were inaccurate (Rohwer, 1984; Carp et al., 1985a and Carp et al., 1985b). Calculations derived from the target theory approach, they argued, depended heavily on ‘parameters that are difficult to determine’ (Rohwer, 1984, p. 661). Proponents of the virus hypothesis maintained that the scrapie agent might very well be ‘virus-like in size’ (Rohwer, 1984). Two other major lines of evidence used against the virus hypothesis had been the scrapie agent’s resistance to heating, formalin, and ultra-violet radiation (Pattison, 1965a; Alper et al., 1966 and Alper et al., 1967; Prusiner, 1982). These findings were also subject to scrutiny by virus proponents. They argued that only a small percentage of the ‘scrapie virus’ population actually withstood these treatments, while the majority of the scrapie agent population was inactivated (Adams, 1985; Rohwer, 1984). Prusiner and his colleagues were also criticized for not comparing the properties of the scrapie agent with a wide range of viruses under the same conditions (Adams, 1985). In those instances when a fraction of the scrapie agent population did survive treatment with heat, formalin, and ultra-violet radiation, virus proponents reasoned

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that these results should not be taken as evidence that the agent was devoid of nucleic acid. Viruses, by definition, contained both a protein and a virus-specific nucleic acid. In the case of the scrapie agent, the scrapie protein(s), perhaps in concert with various cellular components, might form an ‘unusually protective microenvironment’ around the nucleic acid (Adams, 1985, p. 396). This protective protein coat could shield the nucleic acid from treatments, thereby preventing degradation and inactivation. The possibility of a thick protein coating surrounding a nucleic acid molecule or fragment had been acknowledged by Prusiner (Prusiner, 1982 and Prusiner, 1984) and remained a popular explanation for the scrapie agent’s resistance to a range of treatments. Because Prusiner could not eliminate the possibility of nucleic acid within the scrapie agent, the term ‘prion’ itself came under attack by virus proponents (Adams, 1985; Carp et al., 1985a and Carp et al., 1985b). Richard Carp and his team at the New York State Institute for Basic Research in Developmental Disabilities characterized the ambiguity surrounding what many believed to be an unnecessary term: The term prion was derived from the words proteinaceous infectious particle . . . Based upon its derivation, the term fails to provide criteria that distinguish the scrapie agent from most other infectious agents, e.g. bacteria, mycoplasmas, and viruses, all of which, of course contain protein . . . The term prion can contribute to the current discourse on the nature of the agent only if its meaning is restricted to the ‘protein only’ possibility. Proponents of the term strongly imply that the scrapie agent is likely to contain only protein. However, the presence of nucleic acid is usually mentioned as a possibility. The attempt to subsume within the single term, prion, both the ‘protein only’ and the ‘protein with nucleic acid’ concepts, has made it difficult to engage in precise dialogue about the term (Carp et al., 1985a, p. 1362).

As Carp and his colleagues suggested, the term ‘prion’ had become synonymous with a protein-only model of infection. Although they could not formally eliminate the possibility of a small piece of nucleic acid within the pathogen, those scientists who adopted the term ‘prion’—in place of such terms as ‘scrapie agent’ or ‘unconventional virus’—were considered advocates of an infectious protein hypothesis. In the same 1985 review article, Carp and colleagues argued that the experimental evidence supporting a protein-only model of infection was ‘far from conclusive’ (Carp et al., 1985a, p. 1362). Although the virus alternative was also plagued by unanswered questions, Carp and colleagues chose to endorse this hypothesis. The protein-only model of infection, they wrote, placed the scrapie agent ‘in a realm that had no precedent in modern molecular biology’ (Carp et al., 1985a, p. 1357). On the other hand, a virus hypothesis—however unconventional— placed the agent ‘within the constraints of molecular virology’ (Carp et al., 1985a, p. 1362). The unreliable inactivation experiments performed thus far, the authors concluded, ‘fail[ed] to establish a need to go outside the bounds of established molecular biology to invoke a “protein-only” scrapie agent’ (Carp et al., 1985a, p. 1364).

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Criticism of experimental design and technique may have been well justified, but the resistance to the idea of an infectious protein was obviously rooted in a deeper, theoretical objection. A remark made by two other scientists in the field of scrapie research best communicates the gravity of these objections: Should the prion’s protein constitute the complete pathogen, prions would contradict all established beliefs of molecular biologists. No wonder that recent findings in prion research are not accepted universally (Maramorosch and McKelvey, 1985, p. xiv).

Responding to attacks on the accuracy and integrity of her experimental work and the work of her colleagues, Tikvah Alper accused virus proponents of being trapped within a framework of ‘conventional wisdom’ which prevented them from considering novel modes of replication. In the following passage, she likened the prejudices of the virus proponents to those of biologists a generation earlier who continued to argue that genes were made of proteins, despite evidence to the contrary: The main thrust of the radiobiological and photobiological evidence was negative. It could not be reconciled with the ‘Central Dogma’ of molecular biology, according to which all biological replication depends on the self-replicating, i.e., template properties, of the nucleic acids. There are interesting parallels between some objections raised to that evidence and early arguments put forward to explain away the evidence against the assumption that genes were proteins . . . Such ‘explanations’ of results conflicting with the conventional wisdom of the time are very likely ones that were used (in the reverse sense) to dispose of the original evidence against the involvement of an essential nucleic acid moiety in the replication of prions (Alper, 1987, p. 145).

This excerpt provides yet another example of how the term ‘central dogma’ was used by biologists. According to Alper, the ‘Central Dogma’ provided the guidelines for ‘all biological replication’. Once again, the intimate connection between the flow of sequential information (i.e., Crick’s Central Dogma) and the replication of proteins is in evidence. The tangible links between Crick’s more abstract ‘flow of information’ and the synthesis of proteins, as Alper pointed out, were the template properties of nucleic acids, which made this transfer of sequential information possible. Because the machinery of protein synthesis (e.g., DNA, mRNA, tRNA, and ribosomal RNA) made this transfer of specificity from the nucleic acid bases to proteins possible, the coupling of information flow and protein synthesis was a natural one. From this connection, it also followed that nucleic acid must be present in order for proteins to be synthesized. In the absence of a nucleic acid within the scrapie agent, the task of biologists was to envision a different mechanism for protein ‘replication’ that did not hinge on either the flow of sequential information or the traditional machinery of protein synthesis. Alper posed the challenge to biologists in the latter half of the 1980s as follows: ‘What is this “new” mode of replication that is not accommodated by the current conventional wisdom of molecular biology?’ (Alper, 1987, p. 146). By abandoning the familiar mechanism for protein synthesis, one might certainly be

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stepping outside the bounds of ‘conventional wisdom’. But, as Prusiner had pointed out, it did not necessarily follow that Crick’s Central Dogma had to be violated (Prusiner, 1984). In order to salvage the Central Dogma, biologists would have to construct a model of protein-directed protein synthesis independent of the transfer of sequential information, while still preserving the specificity inherent in prion protein molecules. 4. A Change of Conformation Solution? The need to account for the specificity of the scrapie agent (prions) was heightened by the phenomenon of multiple strains. Since the late 1970s, it had been known that scrapie existed in several distinct, stable strains (Dickinson and Outram, 1979). The unique behavior of these strains upon transmission suggested that the infectious prions must carry some form of strain-specific genetic information to make the propagation of these distinct properties possible.12 Without this allowance, it seemed impossible to explain how a single host PrP gene could account for multiple strains breeding true and competing with one another within a single organism or family of organisms. The existence of scrapie strains provided virus proponents with further evidence that the scrapie agent must contain its own strain-specific information, which replicated independently of the host genome (e.g., Carp et al., 1985a and Carp et al., 1985b). The virino hypothesis, which postulated that the scrapie agent consisted of a small, scrapie-specific nucleic acid and protein recruited from the host organism, also offered an explanation for scrapie strains (Kimberlin, 1982). The key assumption made by both virus and virino proponents was that this strain-specific information was contained within nucleic acid. By the late 1980s, however, many researchers began to acknowledge that this assumption could no longer be made. In 1987, two proponents of the virino model of infection discussed the necessity of an ‘informational molecule’ within the scrapie agent to account for the various strains. They concluded that ‘the nature of this replicating informational molecule is not yet known, but a nucleic acid structure remains the most likely possibility’ (Bruce and Dickinson, 1987, p. 80). The use of the term ‘informational molecule’ in the literature of this period is significant. It marked the beginning of a crucial delineation between two different kinds of information where before there was only one. For the first time in scrapie literature, researchers like Bruce and Dickinson had to specify nucleic acid as the type of informational molecule they believed 12 Strain phenomena to be accounted for included: (1) distinct strains of scrapie bred true when transmitted to the same host strain; (2) one scrapie strain interfered or competed with another strain during co-infection: some reports indicated that—given a time delay between inoculation with strains #1 and #2—the first strain to be transmitted would usually ‘block’ replication of the second strain (Dickinson and Outram, 1979; Kimberlin and Walker, 1985); and (3) some scrapie strains could suddenly change or ‘mutate’ in the course of a single passage from organism to another. This new strain would then remain stable through subsequent transmissions (Dickinson and Outram, 1979).

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was carried by the scrapie agent. Earlier arguments insisting that the agent contain scrapie-specific genetic information were all synonymous with arguments in favor of a nucleic acid within the agent. This followed from the fact that the only type of genetic information previously entertained was that of sequential information stored in the linear order of nucleic acid bases. The need to specify the kind of molecule carrying the strain-specific information did not arise until proponents of a protein-only model of infection proposed that the infectious proteins themselves might be capable of transmitting that strain specificity. Research groups considering a protein-only model of infection explored this alternative mode of information transfer. The choice of ‘informational molecule’, they argued, was not always obvious—particularly in the case of scrapie: Scrapie agent contains a proteinaceous component as well as an ‘informational’ molecule (suggested by the existence of distinct strains of scrapie). These operationally defined entities may be the same molecule, an infectious protein, or distinct, in which case a nucleic acid might encode the genetic information (Oesch et al., 1988, p. 209).

The challenge immediately put to Prusiner, Weissmann, and their colleagues was the following: if the ‘informational molecule’ and infectious protein were one and the same, how could a protein ‘encode the genetic information’ and propagate this strain-specific information within the host? Moreover, how could this replication and propagation of information be achieved without violating Crick’s Central Dogma, which stated that once sequential information entered a protein, ‘it cannot get out again’ (Crick, 1958, p. 153). The solution was to consider a second kind of genetic information—what I will call ‘conformational information.’13 During the course of infection, conformational information could be transferred from infectious protein to normal cellular protein, thereby converting PrPC into PrPSc. Although this hypothesis still represented a highly unusual means of infection and protein ‘replication’, it could account for much of the experimental evidence, or lack thereof, while preserving Crick’s guidelines for the transfer of sequential information among nucleic acids and proteins. One of the earliest appearances of what I call the ‘change of conformation hypothesis’ was in Prusiner’s 1984 essay review for Scientific American. At that time, Prusiner and his colleagues suspected that the prion might consist solely of PrP. What they did not know was where to find the ‘instructions’ for making this protein. Amidst his discussion of alternative hypotheses for the scrapie agent, Prusiner slipped in a diagram (not discussed in the text) depicting the conversion of cellular proteins into PrP by the infectious proteins themselves (Fig. 1e). This hypothetical mechanism for prion replication was set apart from those of reverse translation and ‘protein-directed protein synthesis’, two alternatives which Prusiner maintained were in ‘clear violation of the central dogma’ (Prusiner, 1984, p. 58). 13 Some scientists and historians have commented that the concept of ‘conformational information’ is not new to molecular biology. The term, however, did not appear in the scrapie and prion literature that I studied for this period. Hence, I ‘introduce’ the term in this paper.

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(By ‘protein-directed protein synthesis’, Prusiner meant that the amino acid sequence of PrP ‘would serve directly as a template for the construction of a new protein molecule’ (Prusiner, 1984, p. 58)). The discovery that the infectious protein PrPC and the normal cellular protein PrPC were encoded by the same chromosomal gene implied that the two isoforms had an identical amino acid sequence. This finding led researchers to conclude that the two proteins must differ as a result of some post-translational chemical modification or a variation in protein conformation (Basler et al., 1986). The continued lack of evidence for a nucleic acid within the prion, and the need to account for the strain phenomena led these researchers to explore this change of conformation alternative further. In 1988, a possible mode of interaction between PrPSc and PrPC was introduced called the ‘direct action model’ (Oesch et al., 1988). According to this model, the PrPSc acted directly on PrPC, converting the isoform into a molecule identical to itself. This newly converted protein would then convert other PrPC into PrPSc. The demands for specificity on this model were challenging indeed: How can one account for distinct, stable strains of scrapie agent within the framework of the ‘protein only’ model? In the ‘direct action’ model, each strain of scrapie agent would be represented by a different variant of the PrPSc molecule. Each such variant would be able to modify the same precursor (there is evidence for only one PrP gene in homozygous animals!) such that the product resembles the incoming agent . . . It is difficult to imagine a molecular basis for this kind of specificity (Oesch et al., 1988, pp. 211, 213).

It was (and still is) difficult for researchers to imagine that a protein could somehow recognize its own primary sequence in a different conformation and then confer its own shape and specificity upon this cellular counterpart. The existence of multiple strains, the ‘blocking’ of one strain by another, and mutation of strains made this kind of transfer of information even more difficult to envision. Nevertheless, this proposal, whether it turned out to be correct or not, did succeed in pushing the concept of information beyond that of a linear sequence of nucleic acid bases or protein amino acids. Clues to the delineation of the concept of information were even more evident in a paper by David Bolton and Paul Bendheim given at a 1988 symposium on novel infectious agents. In summarizing the state of scrapie/prion research, the authors pointed to three ‘currently supported’ models for the scrapie agent: the virus, virino, and ‘modified host protein’ models. While admitting that the virus model was ‘attractive for historical and biological reasons’, Bolton and Bendheim argued that the experimental evidence for which it could not account (e.g., lack of host immune response, small size, resistance to treatments that inactivate most viruses) far outweighed the ease with which this model could explain multiple strains (Bolton and Bendheim, 1988, p. 165). The virino hypothesis, which postulated that the infectious agent contained a scrapie-specific nucleic acid protected by a host-encoded protein, was judged ‘intellectually intriguing’ by the authors.

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However, as was the case with viruses, no scrapie-specific nucleic acid had been detected despite attempts with cDNA probes in highly purified preparations of the agent. Bolton and Bendheim instead proposed what they termed the ‘modified host protein’ model. This was another protein-only model of infection similar to that discussed by Prusiner, Oesch and colleagues that same year. Although Bolton and Bendheim agreed that both the virino and modified host protein models had ‘no precedents in microbiology’, they clearly considered their protein model to be the more controversial of the two: The modified host protein model presents the most radical deviation from known microbiological systems. In essence the model proposes that an abnormally modified protein, perhaps in concert with other unidentified host components, is capable of inducing the disease and directing the manufacture of more of the abnormal protein. The model satisfies the absence of an immune response (the protein is ‘self’) and the apparent lack of a specific nucleic acid. Explaining scrapie strains with the model is more complex; we postulate that strain ‘information’ is stored and transmitted in the form of protein modifications (Bolton and Bendheim, 1988, p. 165, my emphasis).

This radical deviation from known biological systems was two-fold. The first claim, long held suspect in scrapie research, was that proteins could direct their own synthesis—superseding the role of nucleic acids. The second, and more recent proposal made by Bolton and Bendheim (among others) was that proteins could transmit their strain-specific ‘information’ to other proteins in the form of protein modifications. Consequently, the proteins within the scrapie agent ‘made’ more of themselves, not by synthesizing proteins from scratch, but by conferring changes upon already-existing cellular proteins. The ‘information’ stored within the scrapiecausing proteins would instruct PrPC how to refold into molecules identical to PrPSc. Once again, information transfer and protein synthesis were intimately related. In this case, however, the information considered was not sequential and the ‘synthesis’ of scrapie-causing proteins involved the conversion of pre-existing proteins into another form. The extent to which this process of protein modification constituted true replication remained a matter of debate. The delineation of the concept of information into two distinct classes—‘sequential’ and ‘conformational’—removed the protein-only model of infection from the bounds of Crick’s Central Dogma. According to Bolton and Bendheim, proteins need not transfer sequential information to other proteins in order to ‘make’ more of themselves, as was the case with reverse translation and ‘protein-directed protein synthesis’. Instead, strain-specific information could be transferred in the form of ‘protein modifications’. This proposal’s ‘radical deviation’, therefore, was not from Crick’s Central Dogma. Instead, it contradicted the assumption that infection depended upon a pathogen-specific nucleic acid. Also at stake was the assumption that a protein’s amino acid sequence determined its shape, and, therefore, its function. Bolton and Bendheim considered two types of protein modifications under their

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modified host protein model. The first category was covalent (chemical) modifications. In these instances, PrPSc interfered at some post-transcriptional level and inhibited the normal processing of PrPC. The resulting protein would be identical to the infectious PrPSc. Examples of possible covalent modifications to cellular proteins included glycosylation, partial proteolysis, and amino or carboxy-terminal blocking. The authors also considered a second type of protein modification involving non-covalent changes in PrPC. These changes would occur after the cellular protein had already been synthesized. The example of a non-covalent modification discussed by Bolton and Bendheim was the refolding of PrPC into a shape identical to PrPSc. According to their schematic diagram, PrPC would bind to PrPSc, at which point PrPSc ‘directs an abnormal refolding event in the cellular protein’ (Bolton and Bendheim, 1988, p. 172). The PrPSc dimer, they postulated, could either ‘dissociate into two monomers to repeat the process (replication) or assemble into larger aggregates’ (Bolton and Bendheim, 1988, p. 172). Various strains of scrapie, they argued, could be accounted for by a wide range of strain-specific modifications. In the case of a ‘refolding event’, each strain would be represented by uniquely shaped molecules of PrPSc. By the late 1980s, the various protein-only models of scrapie infection were becoming viable alternatives to the virus and virino hypotheses. The credibility of these models was bolstered by two sets of experiments that provided additional evidence that prion diseases were caused by infectious proteins. Since 1985, researchers had been devising experiments to demonstrate that PrP molecules were the sole transmitters of prion diseases. An early plan had been to trigger the PrP gene to generate pure copies of PrP. If infection ensued after injecting these pure PrP molecules into healthy organisms, researchers would have proven that PrP alone caused the disease. By 1986, it was evident that this experiment would never materialize. Even if it were possible to induce the PrP gene on command, the only product would have been PrPC—an apparently harmless protein already present in the organism. Since researchers did not know how PrPC was converted into PrPSc, a different type of experiment would have to be designed to explore the role of PrP and the PrP gene in the spread of scrapie and other prion diseases. A turning point came in 1988 when Prusiner and UCSF colleague Karen Hsiao found that several people suffering from the prion disease Gerstmann–Stra¨ussler– Scheinker syndrome (GSS) carried a point mutation in their PrP gene (Hsiao et al., 1989). Prusiner and coworkers discovered that several GSS patients, many of whom were related, carried the same mutation. These findings, they argued, demonstrated a genetic link between the mutation of the PrP gene and the onset of the disease. These results also strongly implied that mutation was the cause of the disease. Reflecting on the significance of these findings, Prusiner remarked that ‘the discovery of the mutation gave us a way to eliminate the possibility that a nucleic acid was travelling with prion proteins and directing their multiplication’ (Prusiner, 1995, p. 51).

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Another landmark experiment performed in 1990 provided further evidence against the need for a nucleic acid within the infectious prion. The enigmatic feature of prion diseases spotlighted in these investigations was a phenomenon known as ‘the species barrier’. The difficulty in transmitting scrapie from one species to another was first noted in the 1960s by Ian Pattison when he had trouble transmitting scrapie between sheep and mice (Pattison, 1965b, 1966). The ease with which prion diseases could be transmitted from one species to another become a major concern in the late 1980s when cows in Great Britain fell prey to their own ‘prion disease’, bovine spongiform encephalopathy (BSE). The disorder, commonly referred to as ‘mad cow disease’, was believed to be caused by scrapie-infected sheep meal used as a food supplement. If this disorder could leap the species barrier between sheep and cattle, the possibility existed that it might also make the transition from cattle to humans. Prusiner and colleagues suspected that the key to the species barrier might be in the difference between the PrP gene sequences of different species. If PrPSc in the inoculum were encoded by a gene with a sequence different from that of the host PrP gene, they postulated, the infectious PrPSc might not be capable of converting the host’s PrPC into itself, rendering infection impossible. This proposal was supported by the fact that mice, whose PrP gene differs from the PrP gene of hamsters at 16 codons out of 24, rarely acquired scrapie when inoculated with prions from infected hamsters. To test this hypothesis that a difference in sequence barred infection, Prusiner and UCSF’s Michael Scott engineered transgenic (Tg) mice (Scott et al., 1989). The DNA of these rodents carried both the naturally occurring gene for mouse prion protein (MoPrP) and the gene for Syrian hamster prion protein (SHaPrP). Before inoculation, therefore, both mouse PrPC and hamster PrPC were present in the transgenic mice. Researchers then inoculated these transgenic mice with prions from infected hamsters and the mice began to synthesize hamster prions. Conversely, transgenic mice inoculated with prions from infected mice synthesized mouse prions. These results demonstrated that infectious prions did not randomly convert just any host prion proteins, but interacted only with those cellular proteins encoded by the same PrP gene. The barrier, they claimed, arose from a difference in amino acid sequence between the PrP of one species and the PrP of another. The more similar the sequence between the injected PrPSc and the PrPC of the host, the more likely it was that the two would interact and initiate the propagation of PrPSc throughout the host. The details of the ‘species-specific interaction’ presumed to occur between the injected PrPSc and the host PrPC had yet to be resolved but a change of PrPC’s conformation emerged as a strong possibility: Whether this conversion of PrPC or a precursor into PrPSc involves the addition or deletion of a chemical group, a tightly bound ligand, or only a conformational change remains to be established. To date, there is evidence for neither a chemical modification nor a ligand that is unique to the PrPSc isoform. These observations raise the

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possibility that the difference between PrPC and PrPSc is only conformational (Prusiner et al., 1990, p. 681).

Borrowing a term once reserved by biologists for discussions of the genetic code and sequential information transfer, Prusiner and colleagues envisioned that PrPSc ‘might act as a template for the conversion of PrPC into a second PrPSc molecule’ (Prusiner et al., 1990, p. 681). This refolding could potentially obscure previously exposed protease cleavage sites, explaining why PrPC was completely degraded by proteinase K while the core of PrPSc (PrP 27–30) was preserved. According to this model, the various strains of scrapie could be manifest in a range of stable PrPSc conformations. The transfer of this strain-specific conformational ‘information’ would have to follow some reliable pathway if each PrPC molecule were to be re-folded into the exact shape of the strain-specific PrPSc. This shape would then have to be maintained through repeated passage of the strain to other organisms. Several researchers spoke of ‘replication sites’ on the PrPSc molecules (Kingbury et al., 1987; Dickinson and Outram, 1979), but the mechanism by which this conversion might take place remained unknown (Prusiner, 1992, p. 551). Prusiner postulated that ‘foldases’ or chaperones (proteins that help other proteins fold) might assist in the re-folding of PrPC upon its PrPSc template (Prusiner, 1991, p. 1520).14 Proponents of a protein-only model for prions (scrapie agent) agreed that the change of conformation proposal—although ‘consistent with observations’— remained ‘unorthodox’ (Prusiner, 1991, p. 1520). The proposal that a protein could self-replicate by recognizing a protein of similar amino acid sequence and refolding this isoform into its own lethal shape was unprecedented. There had been much debate as to whether or not this phenomenon represented true ‘self-replication’ or ‘infection’. Since this model of prion replication held no resemblance to conventional notions of protein synthesis or infection, Prusiner himself doubted the appropriateness of these terms for several years. As late as 1989, he maintained that the conversion of pre-existing cellular proteins by infectious PrPSc might be best characterized by the term ‘amplification’ (Prusiner, 1989, p. 363). The results of the transgenic mice experiments, obtained the following year, led Prusiner to rethink his qualifications for biological ‘replication’: Studies with Tg(SHaPrP) mice argue that prion synthesis involves ‘replication’, not merely ‘amplification’ . . . Assuming prion biosynthesis simply involves amplification of post-translationally altered PrP molecules, we might expect Tg(SHaPrP) mice to produce both SHa and Mo prions after inoculation with either prion since these mice produce both SHa and MoPrPC. However, Tg(SHaPrP) mice synthesize only those 14 The change of conformation hypothesis was a novel proposal and should not be confused with normal allosteric regulation of proteins. Allosteric proteins (e.g., hemoglobin), undergo a regulated and reversible change of shape, initiated by the attachment of molecules different from themselves to specific sites on the protein. The change of conformation hypothesis for prion proteins proposed a permanent, irreversible change of a cellular protein’s shape, initiated by a protein of similar amino acid sequence, i.e., ‘self’. It is important to note, however, that both allosteric proteins and the proposed prion protein do demand an expanded notion of specificity in molecular biology that extends well beyond the specific linear sequence of bases and amino acids in nucleic acids and proteins, respectively.

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prions present in the inoculum . . . These results argue that the incoming prion and PrPSc interact with the homologous PrPC substrate to replicate more of the same prions . . . (Prusiner, 1992, p. 551).

The act of infectious PrPSc seeking out only those cellular proteins of similar amino acid sequence and interacting directly with those PrPC to convert them into PrPSc’s own shape, in Prusiner’s opinion, constituted protein ‘replication’. As was the case with the traditional model of protein synthesis, the ‘new’ protein postulated by this change of confirmation model was formed on a ‘template’ that conferred specificity upon the protein. The similarities with conventional protein synthesis ended there. According to this protein-only model of infection, the specificity transmitted to the newly formed PrPSc was not that of a linear order of amino acids. That primary structure had already been determined by the base sequence of its respective gene (Crick’s Sequence Hypothesis). The ‘higher order’ specificity communicated to PrPC was that of conformation. This phenomenon proposed by the change of conformation hypothesis contradicted a ‘central principle of molecular biology’ which states that the amino acid sequence of a protein determines it higher order structure (i.e., shape) (Stryer, 1988, p. 33). The idea of an infectious, ‘self-replicating’ protein challenged more than the rules for protein folding, however. The central generalization at stake was proteins’ dependence on nucleic acids for replication. Proponents of a protein-only model of the scrapie agent (prion) were just as quick to acknowledge the universality of this claim as virus and virino proponents (Alper, 1987; Prusiner, 1984). In defense of this generalization, one might deny that the conversion of a pre-existing protein into a different shape constituted true ‘replication’. Even if this point were taken, however, the idea of an infectious protein remained ‘unorthodox’. A protein-only model of prion infection required that the infectious agent carried strain-specific information in an ‘informational molecule’ other than nucleic acid. This proposal—that the information needed for the propagation of the strain-specific agent could be stored and transmitted by proteins rather than nucleic acids—remained unprecedented. In his recent molecular genetics textbook, Benjamin Lewin addressed the difficulty biologists had in reconciling the change of conformation hypothesis with their expectations for protein synthesis. He characterized the framework under which biologists worked as follows: ‘One might describe the current paradigm of molecular biology in simplistic terms as “DNA makes RNA makes protein, which makes another DNA make RNA make protein” . . .’ (Lewin, 1990, p. v). This ‘paradigm’, of course, was Watson’s famous slogan, which integrated Crick’s flow of sequential information (i.e., the Central Dogma) with the standard machinery of protein synthesis.15 Given this theoretical framework, Lewin considered the ramifications of an infectious, ‘self-replicating’ protein:

15 Lewin’s remark also took into account the role of enzymes in the semi-conservative replication of DNA, hence the ‘additional’ phrase ‘. . . protein . . . makes another DNA . . .’

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If PrP is indeed the infectious agent of scrapie, it must in some way modify the synthesis of its normal cellular counterpart so that it becomes infectious instead of harmless. Such an arrangement would comply with the formal restrictions of the central dogma, but certainly this idea would be at odds with the spirit of the paradigm (Lewin, 1990, p. 108).

The guiding conceptual framework of molecular biology, therefore, consisted of more than Crick’s Central Dogma. Several other related claims have emerged in this study of the prion challenge to molecular biology: proteins should not be capable of making other proteins; proteins should not be capable of storing and transmitting strain-specific information (necessary for infection) to other proteins; infectious agents should not consist of proteins alone; and proteins encoded by the same chromosomal gene (and, therefore, composed of the same amino acid sequence) should have the same shape and function. The introduction of an alternative kind of information preserved Crick’s Central Dogma, but conflicts with accepted notions of infection, replication, protein synthesis, and protein folding remained. 5. Conclusion The proposal of an infectious, self-replicating protein would not have been branded ‘heretical’ if biology were merely a descriptive, fact-collecting enterprise. On the contrary, this two-part survey of scrapie and prion research has highlighted several major generalizations that extended beyond the bounds of any one discipline or field of modern biology. This network of fundamental claims forms an overarching theoretical framework, which is ever-present in molecular biology but not always obvious—even to those ‘dwelling’ in it.16 The credibility and influence of this theoretical framework is made apparent once one or more of its generalizations is challenged by a more specific problem. In the case of scrapie and prion research, this framework has been shown to affect hypothesis formation and evaluation. Various hypotheses concerning the nature of the scrapie agent have been evaluated and ranked by scientists in terms of the degree to which they conformed with elements of the theoretical framework. Despite the importance of these ‘elements’, they have not been clearly stated by scientists. In their struggle to explain experimental findings in scrapie research, biologists have referred to this theoretical framework with such vague terms as ‘theoretical structure’ (Griffith, 1967), or ‘conventional thinking’ (Kimberlin, 1982). If a specific term was used, it was often ‘the central dogma of molecular biology’ (Alper, 1987). The challenge posed by the proposal of an infectious protein, however, goes well beyond Crick’s original rules for the transfer of sequential information among nucleic acids and proteins to challenge several additional generalizations in molecular biology. The term ‘central dogma’, therefore, has served 16 My thanks to Hans-Jo¨rg Rheinberger for the phrases ‘dwelling in’ and ‘living in’ a theoretical framework.

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as an umbrella under which several related, yet separable, generalizations have been subsumed. The study of the prion challenge to this cluster of generalizations has brought some elements of this vague theoretical framework into sharper focus. The proposal of an infectious protein clearly contradicted something. Examination of scientists’ reactions and objections to this proposal provided insight into exactly what these challenged generalizations were. It has been shown in other work (Darden, 1991 and Darden, 1995) that anomalies can drive the articulation of a theory’s separate components. In my study of the prion challenge to the central dogma, I have found a similar phenomenon at work. Just as anomalies may force the delineation of a theory into its respective components, the prion challenge to molecular biology has forced a delineation of the concept of biological information and highlighted specific elements of its theoretical framework. Crick’s Central Dogma is only one of a number of assumptions and generalizations belonging to this framework. Additional framework elements articulated throughout the scrapie/prion case study include accepted notions concerning infection, replication, protein synthesis, and protein folding, as well as the Sequence Hypothesis. Established notions of infection, replication, and protein synthesis were all challenged by the concept of an infectious protein. According to researchers, infectious agents must contain the disease-specific information, which ‘defin[ed] its own identity’ and allowed for the replication of the agent (Prusiner, 1984, p. 50). This ‘informational molecule’ was assumed to be nucleic acid for two reasons: (1) only nucleic acids could carry and transmit genetic and pathogen-specific information; and (2) the replication of nucleic acids was the ‘crucial event’ in the reproduction of the agent (Prusiner, 1984, p. 50). Alper and colleagues summarized the expectations for the chemical nature of the agent as follows: ‘Since the scrapie agent multiplies in the host animal, it has been assumed that nucleic acid must be a part of its structure’ (Alper et al., 1966, p. 283). With the introduction of a proteinonly model of infection, scientists were faced with ‘the apparent enigma’ of replication without nucleic acid (Pattison, 1982, p. 200). The proposed self-replication of proteins was a blatant violation of the claim that ‘all biological activity depends on the self-replicating, i.e., template properties, of the nucleic acids’ (Alper, 1987, p. 145). This challenge to the conventional understanding of protein synthesis was prompted by the influence of ‘Watson’s version’ of the central dogma, which can be characterized by the following statements: ‘DNA makes RNA makes protein’ (Judson, 1979, p. 337); and ‘DNA was the template upon which RNA chains were made. In turn, RNA chains were the likely candidates for the templates for protein synthesis’ (Watson, 1968, p. 89). Because James Watson’s interpretation equated the flow of information with the manufacture of proteins, protein reproduction in the absence of nucleic acid was regarded as impossible. Even the suggestion that a protein could simply initiate its own production by activating its own gene was

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considered by many to be a violation of ‘the accepted dogma of present-day biology’—defined by one scientist as ‘DNA and RNA templates control all biological activity’ (Lewin, 1972, p. 748). Infection, replication, and protein synthesis were all major ‘biological activities’ that were expected to depend on nucleic acid. The proposal of an infectious protein, therefore, challenged all three. The Central Dogma is arguably the keystone of molecular biology’s theoretical framework. Crick’s ‘hypothesis’ of 1958 had gained considerable support since the late 1950s. The elucidation of the role of messenger RNA in the early 1960s demonstrated that sequential information was indeed transferred from DNA to proteins via this unstable intermediate. The discovery of reverse transcriptase in 1970 proved that sequential information could also flow from RNA to DNA, another transfer allowed by Crick’s Central Dogma. As Crick stressed in his 1970 paper, no experimental evidence had yet been found for the transfer of sequential information from proteins to other proteins, or from proteins back to nucleic acids. The failure to detect nucleic acid within the ‘unconventional’ scrapie agent was the first real challenge to Crick’s Central Dogma. Because the synthesis of proteins was intimately tied to the flow of sequential information, there were usually only two hypothetical mechanisms considered up through the mid 1980s whereby proteins could make more of themselves: reverse translation and protein-directed protein synthesis. In the case of reverse translation, the protein would transmit sequential information back to nucleic acids. The hypothesis of protein-directed protein synthesis postulated that the protein would unfold and act as a physical template, conferring the same sequential order upon another string of amino acids. Both hypotheses were in violation of Crick’s Central Dogma, which stated that once (sequential) information was passed into proteins, it could not get out again (Crick, 1958). Given the need to preserve this well-established ‘tenet’ of molecular biology (e.g., Gajdusek, 1985), the proposal of an infectious protein was branded ‘heretical’ (e.g., Prusiner, 1982; Carp et al., 1985b; Hunter, 1992). By the late 1980s, however, many researchers began to consider a different kind of information that would allow for the transmission of disease and strain-specific information from protein to protein. Given that this information was not sequential, the proposal of an infectious protein would skirt confrontation with the Central Dogma, while continuing to challenge scientists’ understanding of infection, replication, protein synthesis, and protein folding. The consideration of a different kind of ‘informational molecule’ followed several landmark experiments. One of the most important of these was the discovery that infectious prion proteins (PrPSc) and their normal cellular counterparts (PrPC) were encoded by the same host gene. Researchers inclined to list PrPSc as the sole component of the scrapie agent (prions) took these findings as evidence that no nucleic acid was needed within the infectious agent. This apparent triumph for protein-only/prion proponents was overshadowed by the immediate need to account for the difference between PrPSc and PrPC, as well as the multiple stable strains of the disease. The proposal then

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arose that the difference between proteins, including strains of PrPSc, might be solely conformational. In the words of two researchers, ‘we postulate that strain “information” is stored and transmitted in the form of protein modifications’ (Bolton and Bendheim, 1988, p. 165). According to the change of conformation hypothesis, proteins acted as ‘informational molecules’, a role previously reserved for nucleic acids. The kind of information being considered, however, was not that of sequential information. This distinction between sequential information and what I call ‘conformational information’ needs to be made in order to fully understand the consequences of the change of conformation hypothesis for the Central Dogma. This delineation of the concept of information into two distinct classes was driven by the protein-only model. The proposal that an infectious protein could somehow recognize ‘itself’ (i.e., the same amino acid sequence) in a different conformation, and then recruit these cellular proteins into its own lethal shape has forced a reevaluation of such terms as ‘infection’, ‘replication’, ‘protein synthesis’, and ‘specificity’. The introduction of this new kind of information, however, did remove the infectious protein (prion) proposal from the bounds of Crick’s Central Dogma. Crick’s Sequence Hypothesis, another element of this theoretical framework, stated that ‘the specificity of a piece of nucleic acid is expressed solely by the sequence of its bases, and that this sequence is a (simple) code for the amino acid sequence of a particular protein’ (Crick, 1958, p. 152). The change of conformation hypothesis did not challenge the Sequence Hypothesis per se. Prion proteins encoded by the same chromosomal gene still had the same amino acid sequence. However, it did contradict the assumption that the amino acid sequence of a protein dictated the manner in which the protein folded (Crick, 1958; Stryer, 1988). According to the change of conformation hypothesis, proteins with the same primary structure (i.e., amino acid sequence) could fold into different shapes. The purported differences between PrPSc and PrPC, two proteins believed to have the same primary structure but different shape, illustrated how important a change of protein shape could be for the well-being of an entire organism. In this two-part essay, I have shown that several generalizations or framework elements of molecular biology have been challenged by the proposal of an infectious protein. Contradictions of established notions of infection, replication, protein synthesis, and protein folding, as well as a limited concept of biological ‘information’, have influenced many scientists in their formation and evaluation of possible hypotheses for the scrapie agent. The virus proponents (the ‘non-heretics’) continued to champion the conservative viral model of infection in spite of the scrapie agent’s several ‘bizarre’ properties and the continued failure to detect nucleic acid within the agent. The virus hypothesis remained attractive because it conformed with expectations for infection and replication. Proponents of the radical protein-only model of infection (the ‘romantics’) considered the possibility of an infectious protein despite these contradictions. Although the emerging change of conformation hypothesis avoided a clash with Crick’s Central Dogma, researchers

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still had to reconcile themselves with an unprecedented means of ‘infection’ and protein ‘replication’. Yet another group of scientists opted for a compromise between the ‘heretical’ notion of an infectious protein and the traditional, yet insufficient, viral explanation. The virino hypothesis was conceived as a means of accommodating the unusual nature of the scrapie agent, while adhering to more conventional theoretical constraints for infection and protein replication. Each group of scientists, therefore, sought to account for the unconventional properties of the agent with a particular model of scrapie infection. The endorsement of a particular model depended not only on the experimental evidence, but on the researchers’ willingness to compromise a range of established elements of this theoretical framework. The importance of these framework elements to biologists has emerged clearly in this survey of scrapie research. This is not to imply, however, that molecular biology is merely theory-driven. The researchers mentioned in this thesis did not set out to test the Central Dogma or any of the other generalizations mentioned. Those working on scrapie research merely accepted the framework elements pertaining to the Central Dogma, Sequence Hypothesis, infection, replication, protein synthesis, and protein folding. Only when they found experimental evidence inconsistent with that framework did they begin to question the universality of the framework elements themselves. The hypotheses on the nature of the scrapie agent were bound not only by theoretical constraints, but by experimental ones as well. For those willing to venture beyond the bounds of the virus hypothesis, alternative models of scrapie infection were often shaped by the experimental findings themselves. The wide range of experimental techniques (e.g., microbiological, molecular biological, target theory, and purification techniques) and the various model organisms guided scrapie research as much as the influence of any element of the theoretical framework. The debates over the adequacy of purification techniques alone have divided many researchers as to whether or not the infectious scrapie agent is really devoid of nucleic acid. The effect of different experimental techniques on the course of scrapie research is just one of many issues raised in this essay that deserves further attention. The extent to which scrapie research benefited from, and influenced, studies of related diseases such as kuru and Creutzfeldt–Jakob disease is another vast area in need of investigation. Much more also needs to be said about the concept of information— sequential, conformational, and otherwise—in molecular biology. The topic of ‘communication’ among proteins, both in the case of the change of conformation hypothesis and normal allosteric regulation, is particularly ripe for investigation. The final chapters of the debate over the nature of the scrapie agent remain to be written.17 Arguments continue to this day over whether or not this agent contains 17

This debate extends to the agents of other ‘prion diseases’ as well.

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a nucleic acid necessary for infection. Several experimental results obtained over the past six years appear to weigh in favor the prion/protein-only model of infection. During 1992 and 1993, for example, Charles Weissmann, H. Bu¨eler, and colleagues engineered mice that did not express the PrP gene (Prn-p). When injected with mouse-specific strains of scrapie, these animals remained uninfected (Bu¨eler et al., 1993). For many researchers, the results of these ‘knock-out’ experiments provided ‘the final demonstration’ that scrapie infectivity depended on the presence of host-encoded prion protein (Ridley and Baker, 1993). Other researchers studying these ‘unconventional slow infections’ (which they refused to refer as ‘prion diseases’) do not deny that proteins are necessary for infection. After all, both viruses and the hypothetical virinos include protein as part of the pathogen. What these scientists do dispute is the claim that proteins are the sole component of the infectious agent. Virus and virino proponents (e.g., Bruce et al., 1992 and Carp et al., 1994) doubt that PrPSc could exist in so many different stable conformations and be capable of transmitting that strain specificity (i.e., shape) to a multitude of cellular proteins over several passages. The lack of a mechanism by which this alleged transfer of conformational information takes place does nothing to weaken their arguments. Skeptics of a prion/protein-only model of infection have also offered positive evidence in favor of nucleic acid within agents of this class. In 1995, Laura Manuelidis and colleagues at the Yale University School of Medicine reported that highly infectious fractions of the CJD agent consisted of a protein bound to nucleic acid. Those fractions containing mostly protein had a much lower rate of infectivity (Manuelidis et al., 1995). Manuelidis argued that ‘the simplest explanation’ for this data was that ‘there is a virus that hasn’t been found’ (Anonymous, 1995, p. 383). Some prion proponents have countered with the charge that the purification techniques of Manuelidis and her colleagues were either denaturing the infectious prion protein, or changing its tertiary structure to a non-infectious shape. If nucleic acid was indeed present in infectious samples, one researcher argued, it might actually be helping the prion protein retain its shape. Once inside host cells, enzymes could then remove the nucleic acid, allowing the prion protein to freely convert cellular proteins of similar primary structure (Gomez, 1995). Fortunately, the arguments put forth in this essay do not depend upon the actual nature of the scrapie agent. Whether the scrapie agent is found to be a virus, protein, or even a virino, it has been the challenge to the viral model itself that has illustrated my position. The concept of an infectious protein implied that proteins were capable of ‘making’ more proteins. This ‘heretical’ proposal, which arose from research on relatively obscure diseases like scrapie, kuru, and CJD, served to illuminate generalizations included in the theoretical framework of molecular biology. Crick’s Central Dogma, I have shown, is just one of those framework elements. The change of conformation hypothesis, which arose from research on scrapie

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and related diseases, proposed that proteins could ‘replicate’ and ‘infect’ by conferring a change of shape upon normal cellular proteins with a similar amino acid sequence. This hypothesis eliminated the need for the transfer of sequential information in order for protein ‘synthesis’ to take place. The change of conformation hypothesis also introduced a different kind of biological information that could be stored in a protein’s three-dimensional structure and then transmitted to other proteins. This proposed solution eventually allowed Crick’s rules for the transfer of sequential information to be teased apart from generalizations concerning infection, replication, and protein synthesis. Consequently, the Central Dogma, as originally defined by Crick, remained unchallenged by the prion case. The possibilities concerning the roles of proteins and nucleic acids in protein synthesis, replication, infection, and information transfer have been expanded, however, whether the scrapie agent is found to be an infectious protein or not. Acknowledgements—I am deeply indebted to my former thesis advisor, Lindley Darden, without whom this essay would have never been possible.

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