Robert A. Somerville

A single amino acid alteration (101L) introduced into murine PrP dramatically alters incubation time of transmissible spongiform encephalopathy

A mutation equivalent to P102L in the human PrP gene, associated with Gerstmann–Straussler syndrome (GSS), has been introduced into the murine PrP gene by gene targeting. Mice homozygous for this mutation (101LL) showed no spontaneous transmissible spongiform encephalopathy (TSE) disease, but had incubation times dramatically different from wild‐type mice following inoculation with different TSE sources. Inoculation with GSS produced disease in 101LL mice in 288 days. Disease was transmitted from these mice to both wild‐type (226 days) and 101LL mice (148 days). In contrast, 101LL mice infected with ME7 had prolonged incubation times (338 days) compared with wild‐type mice (161 days). The 101L mutation does not, therefore, produce any spontaneous genetic disease in mice but significantly alters the incubation time of TSE infection. Additionally, a rapid TSE transmission was demonstrated despite extremely low levels of disease‐associated PrP.

Immunodetection of PrPSc in spleens of some scrapie-infected sheep but not BSE-infected cows

The development of diagnostic tools for transmissible spongiform encephalopathies (TSEs) would greatly assist their study and may provide assistance in controlling the disease. The detection of an abnormal form of the host protein PrP in noncentral nervous system tissues may form the basis for diagnosis of TSEs. Using a new antibody reagent to PrP produced in chickens, PrP can be readily detected in crude tissue extracts. PrP from uninfected spleen had a lower molecular mass range than PrP from brain, suggesting a lower degree of glycosylation. A simple method for detecting the abnormal form of the protein, PrPSc, in ruminant brain and spleen has been developed. PrPSc was detected in sheep spleen extracts from a flock affected by natural scrapie and was also found in spleens from some, but not all, experimental TSE cases. In spleens from cattle with bovine spongiform encephalopathy (BSE) no PrPSc was detected. It is therefore suggested that there is differential targeting of PrPSc deposition between organs in these different types of TSE infection which, with other factors, depends on strain of infecting agent.

Characterization of Thermodynamic Diversity between Transmissible Spongiform Encephalopathy Agent Strains and Its Theoretical Implications

Some transmissible spongiform encephalopathy (TSE) (or “prion”) strains, notably those derived from bovine spongiform encephalopathy, are highly resistant to total inactivation by heat. When three TSE strains derived from sheep with scrapie were heated, little inactivation took place at low temperatures, but at higher temperatures, considerable inactivation occurred. The temperature at which substantial inactivation first occurred varied according to TSE strain, and it was calculated to be 70 °C for the 22C strain, 84 °C for ME7, and 97 °C for 22A by fitting the data to a model based on competition between a destructive and a protective reaction. However, PrPSc from mice infected with a range of TSE strains retained similar resistance to proteinase K digestion after heating to below or above these temperatures, showing that the properties of PrPSc responsible for proteinase resistance do not correlate with those conferring thermostability on the TSE agent. The simplest explanation of these data is that the causal agent contains a macromolecular component that is structurally independent of the host, that it varies covalently between TSE strains, and that it is protected by other macromolecular components. The model is in accord with the virino hypothesis, which proposes a host-independent informational molecule protected by the host protein PrP.

Straining the prion hypothesis

Aguzzi and Weissmann in their News and Views feature correctly state that research on the molecular genetics of PrP protein has contributed greatly to our knowledge of the transmissible spongiform encephalopathies (TSEs). But their firm belief that these diseases are caused by rogue proteins (‘prions’) leads them to misrepresent alternative hypotheses of the nature of the agent, dismissing all non-believers as “the die-hard pro-virus faction”. In fact, the prion hypothesis is far from proven: the precise nature of a prion still eludes identification and the prion hypothesis has yet to explain satisfactorily the many strains of TSE. The alternative ‘virino’ hypothesis is not a conventional virus hypothesis, but it addresses the diversity of biological properties of the TSEs. It proposes an agent-specific replicable informational molecule, yet to be identified, bound to a protective host protein, PrP.

Effect of Sinc genotype, agent isolate and route of infection on the accumulation of protease-resistant PrP in non-central nervous system tissues during the development of murine scrapie.

Mice congenic for the Sinc gene were infected intracerebrally with two scrapie strains, ME7 and 22A. At various times during the incubation period tissues were monitored for the infection-specific form of PrP (PrPSc). PrPSc was found in brain, spleen, lymph nodes, pancreas, submaxillary gland and thymus. After intraperitoneal inoculation PrPSc was found in spleen, lymph nodes, pancreas and submaxillary glands prior to its detection in brain. The kinetics of accumulation of PrPSc in these tissues was dependent on the infecting strain of agent, on the mouse Sinc genotype and on the route of infection. This study supports using the presence of PrPSc as an indicator of infectivity in brain and extraneural tissues and defines some of the parameters which influence when and where PrPSc is first found.

Studies on the removal of a bovine spongiform encephalopathy-derived agent by processes used in the manufacture of human immunoglobulin

Background and Objectives There is still uncertainty over how the agent of variant Creutzfeld‐Jakob disease (vCJD) would partition during the manufacture of plasma derivatives. In this study, a BSE‐derived agent was used as a vCJD model to determine the extent to which infectivity could be removed by selected steps used in the manufacture of intravenous immunoglobulin (IVIG).

Thermostability of mouse-passaged BSE and scrapie is independent of host PrP genotype: implications for the nature of the causal agents

Five experimentally maintained strains of scrapie and BSE agents have been passaged in two PrP genotypes of mice. Brain macerates were autoclaved at 126 degrees C and the levels of surviving infectivity were measured by titration. There was a large difference in the survival properties of transmissible spongiform encephalopathy (TSE) infectivity between TSE strains. PrP genotype had little effect. Phenotypic properties of the TSE strains were not affected with the exception that with one strain (ME7), incubation periods of the heated sample were longer than the controls given equivalent doses. It is concluded that PrP is probably not responsible for differences in thermostability between strains. More likely, a host-independent molecule which differs in covalent structure between strains accounts for these properties.

Differential glycosylation of the protein (PrP) forming scrapie-associated fibrils.

PrP is a glycoprotein found in normal brain. In brain affected by scrapie it forms scrapie-associated fibrils (SAF). PrP from SAF shows considerable heterogeneity of size and charge on two-dimensional gels. It separates into six major regions, the three more acidic regions arising as a result of partial proteolytic degradation. The two more basic higher Mr forms (Mr 34,000 and 29,000) of PrP can be reduced in apparent Mr to a lower Mr form (Mr 25,000) with Peptide-N-glycosidase F. In addition, a series of lectins has been found to bind to PrP. Some bind preferentially to the higher Mr forms whereas others bind more strongly to the lower Mr form. Some of the heterogeneity of PrP is therefore due to differential N-glycosylation. We suggest that one or two N-linked carbohydrate chains are bound to the protein causing some of the differences in Mr. The major cause of heterogeneity of PrP is therefore proteolytic cleavage combined with differential glycosylation at the two potential N-glycosylation sites. The glycolipid moiety attached to PrP may be responsible for some lectin binding to all three bands. Using lectins as a probe to study potential differences in N-glycosylation we have looked at their binding to PrP isolated from SAF, from different strains of scrapie and from different regions of the same brain. No major differences in the N-glycan moieties were found.

Post-translational changes to PrP alter transmissible spongiform encephalopathy strain properties

The agents responsible for transmissible spongiform encephalopathies (TSEs), or prion diseases, contain as a major component PrPSc, an abnormal conformer of the host glycoprotein PrPC. TSE agents are distinguished by differences in phenotypic properties in the host, which nevertheless can contain PrPSc with the same amino‐acid sequence. If PrP alone carries information defining strain properties, these must be encoded by post‐translational events. Here we investigated whether the glycosylation status of host PrP affects TSE strain characteristics. We inoculated wild‐type mice with three TSE strains passaged through transgenic mice with PrP devoid of glycans at the first, second or both N‐glycosylation sites. We compared the infectious properties of the emerging isolates with TSE strains passaged in wild‐type mice by in vivo strain typing and by the standard scrapie cell assay in vitro. Strain‐specific characteristics of the 79A TSE strain changed when PrPSc was devoid of one or both glycans. Thus infectious properties of a TSE strain can be altered by post‐translational changes to PrP which we propose result in the selection of mutant TSE strains.

Distribution of a bovine spongiform encephalopathy‐derived agent over ion‐exchange chromatography used in the preparation of concentrates of fibrinogen and factor VIII

Background and Objectives  The risk of haemophiliacs contracting variant Creutzfeldt‐Jakob disease (vCJD) via treatment with factor VIII concentrates is not known. Therefore, in order to determine the extent to which the vCJD agent might be removed during the preparation of factor VIII concentrate, the partitioning of a bovine spongiform encephalopathy (BSE)‐derived agent was measured over the main purification step used to prepare the Scottish National Blood Transfusion Service high‐purity factor VIII concentrate (Liberate®).