The molecular basis of strain variation in scrapie diseases is unknown. The only identified component of the agent is the posttranslationally modified host prion protein (PrPSc). The biochemical and physical properties of PrP from two strains of transmissible mink encephalopathy (TME), called hyper (HY) and drowsy (DY), were compared to investigate if PrP heterogeneity could account for strain diversity. The degradation rate of PrPThE digested with proteinase K was found to be strain specific and correlated with inactivation of the TME titer. Edman protein sequencing revealed that the major N-terminal end of HY PrPTmE commenced at least 10 amino acid residues prior to that of DY PrPTmE after digestion with proteinase K. Analysis of the brain distribution of PrPTME exhibited a strain-specific pattern and localization of PrPTmE to the perikarya of specific neuron populations. Our findings are consistent with HY and DY PrPTME having distinct protein conformations and/or strain-specific ligand interactions that influence PrPTME properties. We propose that PrP .E conformation could play a role in targeting TME strains to different neuron populations in which strain-specific formation occurs. These data are consistent with the idea that PrPTME protein structure determines the molecular basis of strain variation.
Experimental transmission of the Stetsonville, Wisconsin, U.S.A. source of transmissible mink encephalopathy (TME) to outbred Syrian golden hamsters resulted in two distinct syndromes, termed hyper (HY) and drowsy (DY), that diverge by the third hamster passage. The syndromes differed with respect to clinical signs, incubation period, brain titre, brain lesion profile and pathogenicity in mink. HY hamster TME had an incubation period of 65 + 1 days and was characterized by clinical signs of hyperaesthesia and cerebeUar ataxia. Lethargy and the absence of hyperexcitability or cerebellar ataxia were representative of DY hamster TME which had an incubation period of 168 + 2 days. At endstage, HY and DY infected animals had brain titres of 109.5 LD5o/g and 10 TM LDso/g of tissue, respectively, indicating that the replication kinetics of these two strains is different. Hamster TME passaged back into mink revealed that only DY retained mink pathogenicity. This suggests that the DY agent is the major mink pathogen in the Stetsonville TME source that is also pathogenic in hamsters after a long incubation period. The HY agent is likely to be a minor component of the original TME mink brain that replicates more rapidly than DY agent in hamsters, but alone is non-pathogenic in mink. The presence of the HY and DY strains of agent that retain their biological characteristics on repeated hamster passage in the Stetsonville TME source requires that the informational molecule encoding these transmissible agents has the capacity to account for this biological diversity.
Transmissible mink encephalopathy (TME) has been transmitted to Syrian golden hamsters, and two strains of the causative agent, HYPER (HY) and DROWSY (DY), have been identified that have different biological properties. During scrapie, a TME-like disease, an endogenous cellular protein, the prion protein (PrPC), is modified (to PrPSc) and accumulates in the brain. PrPSC-is partially resistant to proteases and is claimed to be an essential component of the infectious agent. Purification and analysis of PrP from hamsters infected with the HY and DY TME agent strains revealed differences in properties of PrPTME sedimentation in N-lauroylsarcosine, sensitivity to digestion with proteinase K, and migration in polyacrylamide gels. PrPc and HY PrPTME can be distinguished on the basis of their relative solubilities in detergent and protease sensitivities. PrPTME from DY-infected brain tissue shared solubility characteristics of PrP from both uninfected and HY-infected tissue. Limited protease digestion of PrPTME revealed strain-specific migration patterns upon polyacrylamide gel electrophoresis. Prolonged proteinase K treatment or N-linked deglycosylation of PrPTIE did not eliminate such differences but demonstrated the PrPTME from DY-infected brain was more sensitive to protease digestion than HY PrPThE. Antigenic mapping of PrPT E with antibodies raised against synthetic peptides revealed strain-specific differences in immunoreactivity in a region of the amino-terminal end of PrPTME containing amino acid residues 89 to 103. These findings indicate that PrPTME from the two agent strains, although originating from the same host, differ in composition, conformation, or both. We conclude that PrPTME from the HY and DY strains undergo different posttranslational modifications that could explain differences in the biochemical properties of PrPITE from the two sources. Whether these strain-specific posttranslational events are directly responsible for the distinct biological properties of the HY and DY agent strains remains to be determined.
Eighty-three percent of hamsters inoculated at birth with JC virus, a human papovavirus isolated from brain tissue of a case of progressive multifocal leukoencephalopathy, developed malignant gliomas within 6 months. Three brain tumors have been serially transplanted as subcutaneous tumors. JC virus was isolated from five of seven tumors tested. Cells from four tumors were cultivated in vitro. These cells contained an intranuclear antigen with the characteristics of a T antigen, and this antigen was antigenically related to SV40 T antigen. Although virus was not recovered from extracts of serially cultured tumor cells, JC virus was rescued when one tumor cell line was fused with permissive cells.
Interspecies transmission of the transmissible spongiform encephalopathies (TSEs), or prion diseases, can result in the adaptation and selection of TSE strains with an expanded host range and increased virulence such as in the case of bovine spongiform encephalopathy and variant Creutzfeldt-Jakob disease. To investigate TSE strain adaptation, we serially passaged a biological clone of transmissible mink encephalopathy (TME) into Syrian golden hamsters and examined the selection of distinct strain phenotypes and conformations of the disease-specific isoform of the prion protein (PrP Sc ). The long-incubation-period drowsy (DY) TME strain was the predominate strain, based on the presence of its strain-specific PrP Sc following interspecies passage. Additional serial passages in hamsters resulted in the selection of the hyper (HY) TME PrP Sc strain-dependent conformation and its short incubation period phenotype unless the passages were performed with a low-dose inoculum (e.g., 10؊5 dilution), in which case the DY TME clinical phenotype continued to predominate. For both TME strains, the PrP Sc strain pattern preceded stabilization of the TME strain phenotype. These findings demonstrate that interspecies transmission of a single cloned TSE strain resulted in adaptation of at least two strain-associated PrP Sc conformations that underwent selection until one type of PrP Sc conformation and strain phenotype became predominant. To examine TME strain selection in the absence of host adaptation, hamsters were coinfected with hamster-adapted HY and DY TME. DY TME was able to interfere with the selection of the short-incubation HY TME phenotype. Coinfection could result in the DY TME phenotype and PrP Sc conformation on first passage, but on subsequent passages, the disease pattern converted to HY TME. These findings indicate that during TSE strain adaptation, there is selection of a strain-specific PrP Sc conformation that can determine the TSE strain phenotype.
The only known difference between the cellular (PrP C ) and scrapie-specific (PrP Sc ) isoforms of the prion protein is conformational. Because disruption of PrP Sc structure decreases scrapie infectivity, restoration of the disease-specific conformation should restore infectivity. In this study, disruption of PrP Sc (as monitored by the loss of proteinase K resistance) by guanidine hydrochloride (GdnHCl) resulted in decreased infectivity. Upon dilution of the GdnHCl, protease resistance of PrP was restored and infectivity was regained. The addition of copper facilitated restoration of both infectivity and protease resistance of PrP in a subset of samples that did not renature by the simple dilution of the GdnHCl. These data demonstrate that loss of scrapie infectivity can be a reversible process and that copper can enhance this restoration of proteinase K resistance and infectivity. PrP1 is a highly conserved, host-encoded sialoglycoprotein that may play a role in normal synaptic function and circadian rhythms (1, 2). This host protein (PrP C ) is present in the brain and other tissues and is sensitive to proteinase K (PK) digestion (3). During the course of a scrapie infection, PrP C undergoes a post-translational modification to a disease-specific isoform (PrP Sc ) that has increased resistance to limited PK digestion. This modification appears to be solely conformational with PrP Sc having a higher -sheet content than PrP C (4 -7). In vitro cell culture studies have demonstrated that PrP C is the precursor to PrP Sc (8, 9). Inhibition of the migration of PrP C to the cell surface blocks the formation of PrP Sc , confirming that PrP Sc arises via alternate or misprocessing of PrP Sc (8). PrP Sc is resistant to limited PK digestion (10, 11), but upon treatment with GdnHCl, the PrP denatures (12) and becomes sensitive to protease digestion (13). The loss of PK resistance correlates well with loss of infectivity of the PrP Sc -enriched preparation. In this study, we examined the ability of GdnHCl-denatured, PrP Sc -enriched preparations to renature. Renaturation was monitored by Western blot analysis for the presence of PK-resistant PrP and animal bioassay for infectivity. The capacity of PrP to bind copper led us to determine the effect of this transition metal on the renaturation process. EXPERIMENTAL PROCEDURESDenaturation/Renaturation Reactions-Brains were removed from clinically affected hamsters infected with the 263K strain of hamsteradapted scrapie agent, flash frozen, and stored at Ϫ80°C. PrP Sc -enriched fractions were prepared to the P4 pellet step following a modification of Bolton et al. (14) that excludes the PK digestion. The P4 pellet was resuspended in buffered saline containing glycerol (10 mM Tris, pH 7.4, 130 mM NaCl, 5% glycerol), and the protein concentration was determined by the BCA protein assay (Pierce). The reaction conditions for the denaturation and subsequent renaturation were based upon the cell-free conversion reactions described by Kocisko et al. (13). PrP Sc -enriched preparat...
Chronic wasting disease (CWD), a member of the transmissible spongiform encephalopathies (TSEs), was first identified in captive mule and black-tail deer in 1967. Due to the failure to transmit CWD to rodents, we investigated the use of ferrets (Mustela putorius furo) as a small animal model of CWD. The inoculation of CWD into ferrets resulted in an incubation period of 17-21 months on primary passage that shortened to 5 months by the third ferret passage. The brain tissue of animals inoculated with ferret-passaged CWD exhibited spongiform degeneration and reactive astrocytosis. Western blot analysis of ferret-passaged CWD demonstrated the presence of PrP-res. Unlike mule deer CWD, ferret-passaged CWD was transmissible to Syrian golden hamsters (Mesocricetus auratus). Increasing the passage number of CWD in ferrets increased the pathogenicity of the agent for hamsters. This increase in host range of a field isolate on interspecies transmission emphasizes the need for caution when assessing the potential risk of transmission of TSEs, such as bovine spongiform encephalopathy, to new host species.
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