Victoria A. Lawson

Genomic Structure of an Attenuated Quasi Species of HIV-1 from a Blood Transfusion Donor and Recipients

A blood donor infected with human immunodeficiency virus-type 1 (HIV-1) and a cohort of six blood or blood product recipients infected from this donor remain free of HIV-1-related disease with stable and normal CD4 lymphocyte counts 10 to 14 years after infection. HIV-1 sequences from either virus isolates or patient peripheral blood mononuclear cells had similar deletions in the nef gene and in the region of overlap of nef and the U3 region of the long terminal repeat (LTR). Full-length sequencing of one isolate genome and amplification of selected HIV-1 genome regions from other cohort members revealed no other abnormalities of obvious functional significance. These data show that survival after HIV infection can be determined by the HIV genome and support the importance of nef or the U3 region of the LTR in determining the pathogenicity of HIV-1.

Transmissible spongiform encephalopathies

Nosologically, transmissible spongiform encephalopathies (TSE or prion diseases) should be grouped with other neurodegenerative disorders such as Alzheimers and Parkinsons diseases, which are all caused by toxic gain of function of an aberrant form of a constitutively expressed protein. Failure to clear these proteins from the brain induces neuronal dysfunction. Transmissibility is the property that separates TSE from other neurodegenerative diseases, and this property seems to reside within the structure of the abnormal protein. The human phenotypic range of these encephalopathies includes Creutzfeldt-Jakob disease and its variant form, kuru, Gerstmann-Sträussler-Scheinker syndrome, and fatal familial insomnia. Notwithstanding the generally low incidence of TSE and their limited infectiousness, major epidemics such as bovine spongiform encephalopathy and kuru arise in situations where intraspecies recycling of the abnormal protein is sustained. Moreover, evidence of chronic subclinical infection in animals offers insights into pathogenesis and prompts re-evaluation of the notion of species barriers and present infection control measures. Since case-to-case transmission is the only known mechanism underlying epidemics of TSE, potential reservoirs of infectivity in the tails of epidemics need continued vigilance.

Packaging of prions into exosomes is associated with a novel pathway of PrP processing.

Prion diseases are fatal, transmissible neurodegenerative disorders associated with conversion of the host‐encoded prion protein (PrPC) into an abnormal pathogenic isoform (PrPSc). Following exposure to the infectious agent (PrPSc) in acquired disease, infection is propagated in lymphoid tissues prior to neuroinvasion and spread within the central nervous system. The mechanism of prion dissemination is perplexing due to the lack of plausible PrPSc‐containing mobile cells that could account for prion spread between infected and uninfected tissues. Evidence exists to demonstrate that the culture media of prion‐infected neuronal cells contain PrPSc and infectivity but the nature of the infectivity remains unknown. In this study we have identified PrPC and PrPSc in association with endogenously expressing PrP neuronal cell‐derived exosomes. The exosomes from our prion‐infected neuronal cell line were efficient initiators of prion propagation in uninfected recipient cells and to non‐neuronal cells. Moreover, our neuronal cell line was susceptible to infection by non‐neuronal cell‐derived exosome PrPSc. Importantly, these exosomes produced prion disease when inoculated into mice. Exosome‐associated PrP is packaged via a novel processing pathway that involves the N‐terminal modification of PrP and selection of distinct PrP glycoforms for incorporation into these vesicles. These data extend our understanding of the relationship between PrP and exosomes by showing that exosomes can establish infection in both neighbouring and distant cell types and highlight the potential contribution of differentially processed forms of PrP in disease distribution. These data suggest that exosomes represent a potent pool of prion infectivity and provide a mechanism for studying prion spread and PrP processing in cells endogenously expressing PrP. Copyright

Glycosylation influences cross-species formation of protease-resistant prion protein

A key event in the transmissible spongiform encephalopathies (TSEs) is the formation of aggregated and protease‐resistant prion protein, PrP‐res, from a normally soluble, protease‐sensitive and glycosylated precursor, PrP‐sen. While amino acid sequence similarity between PrP‐sen and PrP‐res influences both PrP‐res formation and cross‐species transmission of infectivity, the influence of co‐ or post‐translational modifications to PrP‐sen is unknown. Here we report that, if PrP‐sen and PrP‐res are derived from different species, PrP‐sen glycosylation can significantly affect PrP‐res formation. Glycosylation affected PrP‐res formation by influencing the amount of PrP‐sen bound to PrP‐res, while the amino acid sequence of PrP‐sen influenced the amount of PrP‐res generated in the post‐binding conversion step. Our results show that in addition to amino acid sequence, co‐ or post‐translational modifications to PrP‐sen influence PrP‐res formation in vitro. In vivo, these modifications might contribute to the resistance to infection associated with transmission of TSE infectivity across species barriers.

Prion-infected cells regulate the release of exosomes with distinct ultrastructural features

Exosomes are small membrane‐bound vesicles released from cells and found in vivo in most biological fluids. Functions reported for exosomes include cell–cell communication, roles in modulating immune responses, and roles in the transfer of pathogens such as prions. Here we investigated the molecular characteristics of the structure of exosomes that harbor prion infectivity to determine the native structure of exosomes and whether infected exosomes have a distinct structure. Cryo‐electron tomography revealed the previously unidentified ultrastructural detail of exosomes with high resolution. Exosomes were found to be naturally spherical in shape and to have a diverse population that varies in size and internal structure, such as differences in the number of membrane structures. Exosomes isolated from prion‐infected cells contained a significantly different population of exosomes with distinct structural features compared to control vesicles from mock‐infected cells. Exosomes are highly structured vesicles that can modify their structure on altering their protein cargo. This finding provides further insight into the role that the exosomal protein cargo plays on influencing the structure of the vesicles as well as highlighting the diversity of exosomes and their relationship to biological processes.—Coleman, B. M., Hanssen, E., Lawson, V. A., Hill, A. F. Prion‐infected cells regulate the release of exosomes with distinct ultrastructural features FASEB J. 26, 4160–4173 (2012). www.fasebj.org

N-terminal Truncation of Prion Protein Affects Both Formation and Conformation of Abnormal Protease-resistant Prion Protein Generatedin Vitro

Transmissible spongiform encephalopathy diseases are characterized by conversion of the normal protease-sensitive host prion protein, PrP-sen, to an abnormal protease-resistant form, PrP-res. In the current study, deletions were introduced into the flexible tail of PrP-sen (23) to determine if this region was required for formation of PrP-res in a cell-free assay. PrP-res formation was significantly reduced by deletion of residues 34–94 relative to full-length hamster PrP. Deletion of another nineteen amino acids to residue 113 further reduced the amount of PrP-res formed. Furthermore, the presence of additional proteinase K cleavage sites indicated that deletion to residue 113 generated a protease-resistant product with an altered conformation. Conversion of PrP deletion mutants was also affected by post-translational modifications to PrP-sen. Conversion of unglycosylated PrP-sen appeared to alter both the amount and the conformation of protease-resistant PrP-res produced from N-terminally truncated PrP-sen. The N-terminal region also affected the ability of hamster PrP to block mouse PrP-res formation in scrapie-infected mouse neuroblastoma cells. Thus, regions within the flexible N-terminal tail of PrP influenced interactions required for both generating and disrupting PrP-res formation.

Prion protein glycosylation

The transmissible spongiform encephalopathies (TSE), or prion diseases are a group of transmissible neurodegenerative disorders of humans and animals. Although the infectious agent (the ‘prion’) has not yet been formally defined at the molecular level, much evidence exists to suggest that the major or sole component is an abnormal isoform of the host encoded prion protein (PrP). Different strains or isolates of the infectious agent exist, which exhibit characteristic disease phenotypes when transmitted to susceptible animals. In the absence of a nucleic acid genome it has been hard to accommodate the existence of TSE strains within the protein‐only model of prion replication. Recent work examining the conformation and glycosylation patterns of disease‐associated PrP has shown that these post‐translational modifications show strain‐specific properties and contribute to the molecular basis of TSE strain variation. This article will review the role of glycosylation in the susceptibility of cellular PrP to conversion to the disease‐associated conformation and the role of glycosylation as a marker of TSE strain type.

Correlative studies support lipid peroxidation is linked to PrPres propagation as an early primary pathogenic event in prion disease

To assess whether heightened oxidative stress plays an early and primary pathogenic role in transmissible spongiform encephalopathies (TSE), we undertook detailed correlative studies using a mouse-adapted model of human disease. The spatio-temporal evolution of the abnormal, protease-resistant isoform of the prion protein (PrP(res)) and neuropathological changes were correlated with the occurrence and type of oxidative stress. Heightened oxidative stress was demonstrated, but restricted to elevated levels of free aldehydic breakdown products of lipid peroxidation, affecting all brain regions to varying extents. The increase in lipid peroxidation was highest over the mid-incubation period, with the onset showing close temporal and general topographical concordance with the first detection of PrP(res) with both pre-empting the typical neuropathological changes of spongiform change, gliosis and neuronal loss. Further, prion propagation over the disease course was assessed using murine bioassay. This revealed that the initial rapid increase in infectivity titres was contemporaneous with the abrupt onset and maximisation of lipid peroxidation. The present results are an important extension to previous studies, showing that heightened oxidative stress in the form of lipid peroxidation is likely to constitute an early primary pathogenic event in TSE, associated temporally with the integral disease processes of prion propagation and PrP(res) formation, and consistent with causal links between these events and subsequent typical neuropathological changes.

Mouse-adapted sporadic human Creutzfeldt–Jakob disease prions propagate in cell culture

Cell based models used for the study of prion diseases have traditionally employed mouse-adapted strains of sheep scrapie prions. To date, attempts to generate human prion propagation in cell culture have been unsuccessful. Rabbit kidney epithelial cells (RK13) are permissive to infection with prions from a variety of species upon expression of cognate PrP transgenes. We explored RK13 cells expressing human PrP for their utility as a cell line capable of sustaining infection with human prions. RK13 cells processed exogenously expressed human PrP similarly to exogenously expressed mouse PrP but were not permissive to infection when exposed to sporadic Creutzfeldt-Jakob disease prions. Transmission of the same sporadic Creutzfeldt Jakob disease prions to wild-type mice generated a strain of mouse-adapted human prions, which efficiently propagated in RK13 cells expressing mouse PrP, demonstrating these cells are permissive to infection by mouse-adapted human prions. Our observations underscore the likelihood that, in contrast to prions derived from non-human mammals, additional unidentified cofactors or subcellular environment are critical for the generation of human prions.

Increased Proportions of C1 Truncated Prion Protein Protect Against Cellular M1000 Prion Infection

Prion disease pathogenesis is linked to the cell-associated propagation of misfolded protease-resistant conformers (PrPres) of the normal cellular prion protein (PrPC). Ongoing PrPC expression is the only known absolute requirement for successful prion disease transmission and PrPres propagation. Further typifying prion disease is selective neuronal dysfunction and loss, although the precise mechanisms underlying this are undefined. We utilized a single prion strain (M1000) and a range of neuronal and nonneuronal, PrPC endogenously expressing and transgenically modified overexpressing cell lines, to evaluate whether PrPC glycosylation patterns or constitutive N-terminal cleavage events may be determinants of sustained PrPres propagation. Our data demonstrates that relative proportions offull-length and C1 truncated PrPC are the most important characteristics influencing susceptibility to sustained M1000 prion infection, supporting PrPC &agr;-cleavage as a protective event, which may contribute to the selective neuronal vulnerability observed in vivo.