Creutzfeldt-Jakob disease

Proc. Natl. Acad. Sci. USA Vol. 92, pp. 5124-5128, May 1995 Medical Sciences

Viral particles are required for infection in neurodegenerative Creutzfeldt-Jakob disease (scrapie/molecular dissociation/RNA/nucleic acid-binding proteins/prion protein)

L. MANUELIDIS*, T. SKLAVIADIS, A. AKOWITZ, AND W. FRITCH Yale Medical School, 310 Cedar Street, New Haven, CT 06510

Communicated by Sheldon Penman, Massachusetts Institute of Technology, Cambridge, MA, February 15, 1995

epidemic of an entirely new infection in cows (4). This bovine outbreak indicates that newly emerging virulent strains of scrapie can evolve and decimate a species. Similarly, the recent outbreak of CJD in Slovakia could potentially signify an evolving strain of CJD that is more virulent for humans. The incidence of CJD in one region of Slovakia has grown to >1000-fold that of the typical incidence world-wide, and all tested cases, with afflicted individuals as young as 30 yr old, have been transmissible after a prolonged incubation period in animals (5). Hence there are compelling medical reasons to further characterize these infectious agents. Many different strains of scrapie have been isolated and characterized in inbred mice (for example, see ref. 6). On a biological level, these scrapie strains are different from the more than 15 rodent-passaged CJD isolates started here (7). To further understand the intrinsic properties of the CJD infectious agent, we have systematically approached its isolation using classical virological methods, accompanied by essential viral titrations. Such purification studies have been done with only one established CJD isolate because quantitative infectious bioassays require 150-250 days. Nonetheless, these structural characteristics are probably reasonably representative. Our studies have shown the following attributes of the CJD infectious agent: (i) it has a core-like viral density of 1.27-1.28 g/cc in sucrose, (ii) it has a viral size of 120S and a diameter of "30 nm, and (iii) >99% of starting prion protein (PrP), a host-encoded protein, can be separated from infectivity during fractionation. Typically, 15-20% of the initial brain titer is recovered when these attributes are exploited for agent purification (3). No similar physical characterizations have been reported for scrapie isolates, possibly because a host-encoded protein known as PrP, or some truncated derivative of PrP, has been proposed to be the infectious agent (8). This prion theory has become so dominant that typical self-aggregating and (3-pleated features of PrP (common to a variety of amyloidforming proteins) have been equated with infectivity in scrapie (9, 10). However, all attempts to demonstrate infectivity from purified, recombinant, and amyloid-forming peptides have been negative (1). A recent model of prion conversion was based on the finding that proteolytic resistance of PrP in crude scrapie fractions could be abolished by 3 M guanidine hydrochloride (Gdn-HCl) and then rapidly restored (11). Neither nucleic acid analyses nor essential viral titrations were determined. We show here that less denaturing conditions actually reduce infectivity by >99.5% in CJD. Furthermore, our accompanying analytical data indicate that intact viral-like complexes are required for infection. Although many previous studies have shown that PrP is linked to disease expression and susceptibility to infection (1), PrP may not be an intrinsic agent component. (i) PrP changes

Several models have been proposed for the ABSTRACT infectious agents that cause human Creutzfeldt-Jakob disease (CJD) and sheep scrapie. Purified proteins and extracted nucleic acids are not infectious. To further identify the critical molecular components of the CJD agent, 120S infectious material with reduced prion protein (PrP) was treated with guanidine hydrochloride or SDS. Particulate and soluble components were then separated by centrifugation and molecularly characterized. Conditions that optimally solubilized residual PrP and/or nucleic acid-protein complexes were used to produce subfractions that were assayed for infectivity. All controls retained >90% of the 120S titer (- 15% ofthat in total brain) but lost >99.5% of their infectivity after heat-SDS treatment (unlike scrapie fractions enriched for PrP). Exposure to 1% SDS at 22°C produced particulate nucleic acidprotein complexes that were almost devoid of host PrP. These sedimenting complexes were as infectious as the controls. In contrast, when such complexes were solubilized with 2.5 M guanidine hydrochloride, the infectious titer was reduced by >99.5%. Sedimenting PrP aggregates with little nucleic acid and no detectable nucleic acid-binding proteins had negligible infectivity, as did soluble but multimeric forms of PrP. These data strongly implicate a classical viral structure, possibly with no intrinsic PrP, as the CJD infectious agent. CJDspecific protective nucleic acid-binding protein(s) have already been identified in 120S preparations, and preliminary subtraction studies have revealed several CJD-specific nucleic acids. Such viral candidates deserve more attention, as they may be of use in preventing iatrogenic CJD and in solving a fundamental mystery. Human Creutzfeldt-Jakob disease (CJD) is caused by an infection with viral properties (for review, see ref. 1). Although the infectious agent is not characterized at the molecular level, peripheral iatrogenic infections have revealed an extraordinary latency of >20 yr to the expression of symptomatic disease. In later stages of infection, the virus enters the brain and evokes obvious dysfunction and pathology, a consequence of efficient viral replication at this site. Typically, there is a rapidly progressive dementia, with vacuolar neuronal degeneration but no amyloid plaques or lymphocytic infiltrates. With the passage of human CJD to rodents (2) it has been possible to show that clinical signs and vacuolar changes are evoked only by very high viral titers (3), and the predominant phase of infectious disease is asymptomatic. Asymptomatic but infected people have been used as donors for therapeutic tissue and blood products (1). It therefore becomes important to identify these individuals and to devise improved separation techniques to minimize the inadvertent spread of CJD. Scrapie, an endemic and more frequently symptomatic infection of sheep, is caused by the same type of "slow virus." The sheep virus is thought to be the source for the recent

Abbreviations: CJD, Creutzfeldt-Jakob disease; IAP, intracisternal A particle; PrP, prion protein; Prp-res, "infectious" form of PrP; GFAP, glial fibrillary acidic protein; Gdn-HCl, guanidine hydrochloride. *To whom reprint requests should be addressed.

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Medical Sciences: Manuelidis et at do not correlate with viral titers during incubation but instead are intimately linked to the sudden appearance of vacuoles in the brain. (ii) Overexpression of host PrP alone, induced by transgenic insertion of multiple copies of the PrP gene, leads to vacuolar changes but does not produce significant infectious titers (3, 12). (iii) Most pertinent for the present studies are the methods devised to exclude most PrP from highly infectious CJD preparations. These methods minimize n-sheet aggregates of PrP that are proteolyzed more slowly in test tube assays for the "prion" or presumed "infectious form" of this protein (hereafter designated PrP-res). We find no visible PrP-res after mild disaggregation procedures that maintain complete infectivity (13). However, a small amount of rapidly proteolyzed PrP ('1% of starting brain) still contaminates more purified 120S preparations (14). Thus the present experiments evaluated segregation of PrP molecules that could not be distinguished from normal forms. Because all high-titer preparations of scrapie and CJD contain nucleic acids (15), and both scrapie and CJD agents have a reproducible size of .25 nm by filtration and other criteria (1), we investigated conditions that could be used to separate more completely the different molecular components in infectious preparations with reduced PrP. We were particularly interested in conditions that would partition nucleic acids and capsid-like proteins. Analyzed proteins included PrP and its multimers, nucleic acid-binding proteins that could protect a viral nucleic acid (16), and a Gag protein derived from one cosedimenting endogenous viral complex (14). This retroviral intracisternal A particle (IAP) is known to resist harsh denaturing conditions (e.g., 1% SDS or 6 M Gdn HCl). Thus, simultaneous evaluation of IAP Gag solubilization from this noninfectious complex provided a control for the relative sensitivity of the CJD agent at the molecular level. Pilot studies defined the most effective ways to partition PrP and nucleic acid-protein complexes, with the ultimate aim of identifying intrinsic components necessary for infection. Sedimentation was used to find whether an intact virus-like particle was required for infection. Because multimeric PrP could be separated from intact nucleic acid-protein complexes, we monitored the titer of these subfractions as compared with native and denatured controls. Had infectivity partitioned with partially purified PrP multimers, or with sedimenting aggregates of PrP with little nucleic acid, we would have concluded that the prion hypothesis was valid. However, the data showed that infectivity partitioned instead with particulate nucleic acid-protein complexes. The sedimentation of these complexes was in accord with their previously determined viral size, and the recovery of infectivity in the relevant subfractions was essentially quantitative.

MATERIALS AND METHODS Ten grams of brain from control or clinically ill hamsters was pooled to generate the 120S preparations as described (14). Briefly, crude p215 pellets were exhaustively digested with micrococcal nuclease and disaggregated at pH 8.9. The 120S infectious peak was then separated from 75% of the PrP in a 10-30% sucrose gradient. To concentrate the 120S peak fractions, an equal volume of dilution buffer D (25 mM Tris-HCl, pH 8.9 in 0.05% Sarkosyl) was added before centrifugation at 100,000 X g for 1 hr at 20°C over a 20-s.l cushion of 80% sucrose in buffer D. The bottom 40 ,ul was suspended in buffer D to yield 3 g of the concentrated 120S preparation in 400 ,l. Aliquots of the 120S mixture (0.25 g/200 ,ul each) were bath-sonicated with buffer D and/or additives. Each tube was mixed for -45 min and then bath-sonicated for 30 min (two cycles) to minimize impenetrable aggregates. After 2.5-3 hr, the samples were centrifuged at 100,000 xg for 1 hr at 20°C, conditions classically used to define soluble (supernatant) and

particulate (pelleted) components.

Proc. Natl. Acad. Sci. USA 92 (1995)

5125

Each supernatant and pellet was diluted equivalently in buffer D (g/ml) for titers in the linear range (17). Pellets (in 20 ,ul) were dispersed by pipetting 50 times through a micro tip for "10 min, bath-sonicated for 10 min, redispersed, and then mixed with a 2% normal brain suspension for an additional 1.5-2 hr before intracerebral inoculation (six hamsters per sample). For immunoblots, supernatant proteins were concentrated by addition of 9 vol of EtOH, frozen at - 20°C for 20 hr, and spun at 19,000 x g for 25 min at 4°C with quantitative recovery of proteins >12 kDa. Protein blotting, detection, and densitometry methods were as described (13) with polyclonal antibody dilutions of 1:1000, except for IAP 2 (1:250). Nucleic acid and/or RNA was determined after 32p incorporation (see Fig. 4).

RESULTS Previous experiments showed that high salt concentrations promoted PrP aggregation and insolubility in crude p215 CJD brain fractions (13). We therefore tested different concentrations of Gdn HCl on more purified 120S fractions to find a balance between the denaturing and precipitating effects of this salt. We identified Gdn HCl concentrations that produced the maximal release of residual PrP into the supernatant, as well as those that induced PrP aggregation and sedimentation. Fig. 1 shows a typical pilot experiment, with comparison of PrP in the 100,000 x g pellet and supematant fractions. Essentially all PrP is recovered in the pellet when samples are sonicated in buffer D (lane 0), whereas '70% of PrP is released into the supematant by 2 M Gdn HCl. At concentrations of 4 M or above, however, salting out (aggregating) effects are obvious, as increased amounts of PrP are sedimented. Other optimized studies showed maximal solubilization of PrP at 2.5 M Gdn HCl. The IAP Gag protein and .5000 bases of its cognate viral nucleic acid genome sediments together with the CJD agent during density and size purifications. Although this IAP complex is not CJD-specific, it could potentially encapsidate a CJD nucleic acid (15). Therefore, the IAP Gag protein was evaluated with three antibodies (Fig. 2). At concentrations of 2 M Gdn HCl, antibodies 1 and 3 showed '50% of Gag was solubilized. Antibody 2, however, showed only -20% solubilization. Thus, in accord with other studies (18), .50% of IAP complexes were resistant to 2 and 6 M Gdn-HCl at the molecular level of analysis. Subsequent experiments showed the IAP complex was more resistant to disruption than sedimenting PrP (Fig. 3). The IAP Gag protein in a retroviral complex is also far more resistant to proteolytic digestion than PrP in CJD brain homogenates (3). We additionally analyzed the solubilization of molecular components after sonication in 1% SDS at 200C. Interestingly,

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Proc. Natl. Acad ScL USA 92

Medical Sciences: Manuelidis et aL

5126

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ation step. Thus, the prion theory would predict infectivity in the Gdn-HCl or SDS supernatants but not in the SDS pellet. Furthermore, residual aggregates of PrP in the Gdn HCl pellet would be expected to be infectious. These predictions were not borne out experimentally. Rather, the presence of sedimenting particles with nucleic acid and nucleic acid-binding proteins was a far more accurate predictor of infectivity (vide infra). Fig. 3 also shows the IAP Gag protein and the glial fibrillary acidic protein (GFAP) in the inoculated subfractions. The solubilized Gag protein approximated that seen after 2 M GdnHCl treatment when assessed with antibody 3 (Figs. 2 and 3). SDS treatment gave a similar profile of Gag solubilization as GdnHCl. Because we found GFAP only in CJD but not in normal 120S preparations, we evaluated this protein after Gdn-HCl and SDS treatments. GFAP, which increases as a pathological response to CJD infection (19), has been proposed as a strong ligand for PrP (20). However, in experiments with Gdn-HCl or SDS, GFAP partitioned differently than PrP (Fig. 3). Notably after SDS, GFAP remained in an insoluble form with intact nucleic acid-protein complexes, whereas PrP was solubilized (Figs. 3 and 4). Had the function of GFAP not

this treatment efficiently released >85% of PrP into the supematant subfraction, whereas most nucleic acid-protein complexes remained intact. Fig. 3 shows the general and antibody-specified protein profiles of SDS and Gdn HCltreated aliquots that were inoculated for viral titration. Gold, used as a general protein stain, showed that most proteins remained in the pellet after Gdn HCl or SDS treatment. In contrast, 80% of PrP was released into the supernatant after 2.5 M Gdn HCl treatment. Dimeric and trimeric forms of PrP are obvious in the Gdn HCl supernatant and are as abundant as in the control pellets. These multimers account for -40% of the total PrP detected in both cases. SDS at room temperature liberated even more PrP, and almost no PrP was retained in the SDS pellet. Quantitative gram equivalent determinations for PrP (3) showed PrP in the SDS pellet was - 1,000 fold less than in starting brain (data not shown). Because dimeric and trimeric forms of PrP were present in both Gdn HCl and SDS supernatants after boiling for denaturing gel electrophoresis, it is likely that even greater amounts of self-associated PrP molecules were present in the inoculated supernatant samples not subjected to this additional denatur-

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DISCUSSION The most parsimonious interpretation of the preceding data is that a specific nucleic acid and one or more binding proteins are intrinsic components of the CJD virus. These two molecular components are not infectious unless they are associated in nuclease-resistant, sedimenting particles of viral size. These components are likely to form a complex that is independent of IAP because particulate IAP complexes retained negligible infectivity after Gdn HCl extraction. None of the above data support the notion that small multimeric or larger sedimenting complexes of PrP are infectious. Indeed, SDS removed almost all traces of PrP with no significant loss of titer. Moreover, larger sedimenting forms of PrP had insignificant levels of infectivity. The residual titer in PrP-enriched Gdn HCl pellets probably derives from a miniscule proportion of incompletely disrupted virus. PrP did not bind nucleic acids in previous blotting studies (16), and the present partitioning results do not indicate a strong affinity for either nucleic acid-binding proteins or viral complexes. It has become increasingly questionable whether any form of host-encoded PrP is an intrinsic agent component, given the accumulated data from diverse experiments (1), as well as the present results. Nonetheless, several investigators continue to search for experimental conditions that will create some altered form of PrP that makes it infectious. Because no specific conformational or posttranslational modifications have been identified, the test tube assay for PrP-res has been used to describe the putative infectious form of this protein. However, without the addition of infected tissue preparations (all of which contain nucleic acids), there is no infection. This assay was recently used to evaluate PrP-enriched but crude scrapie preparations similar to those first used in CJD (21). Addition of 3 M Gdn-HCl abolished PrP-res, but 10% of the starting PrP-res recrudesced after a 2-min incubation in 0.75 M Gdn-HCl (11). Because we used less Gdn HCl and found little infectivity after a 2-h incubation in more dilute Gdn-HCl (