Chapter 3 - Cell biology of prion infection
Introduction
The establishment of in vitro cell culture models of infection can be a daunting task but, once established, they are much more easily manipulated and controlled than in vivo models of infection. As such, they are critical tools for understanding the myriad of processes which underlie how infectious agents infect, and sometimes kill, cells. Data from studies using cell culture models can be used to design and implement studies in animals where the in vitro results can often – although by no means always – be recapitulated. Indeed, in vitro cell-based systems are an important component of translational research and continue to be used as model systems to identify compounds that can be used for prophylactic or therapeutic treatments of a wide variety of infections as well as for the development and propagation of vaccines to prevent infection.
The development of cell culture models of prion infection has, in many ways, been more difficult than that of viruses and bacteria. This is due in part to the unusual nature of the infectious agent and the mechanism by which it replicates. The causative agents of these diseases are known as prions and are distinguished from more conventional viruses and bacteria by the composition of the infectious particle. Prions are composed largely, if not entirely, of an abnormally refolded, insoluble, and protease-resistant form (PrPTSE) of the mammalian prion protein (PrP). PrPTSE itself is a conformationally distinct form of the soluble and protease-sensitive cellular prion protein (PrPC) which is expressed in almost every cell type in the body. As described in greater detail elsewhere in this volume, PrPTSE, and thus prions, replicate via a conversion process called seeded polymerization. In seeded polymerization, aggregates of PrPTSE interact with PrPC in the host cell and, through a poorly understood process, refold the host PrPC into the PrPTSE conformer, thus incorporating it into the PrPTSE aggregate. This process is repeated until PrPTSE accumulates to levels that eventually lead to disease.
Prion replication is thus dependent upon the host's PrPC molecule. One consequence of this is that, once a prion infects a cell, the process of prion replication inevitably leads to the host PrPC molecule being incorporated into the incoming PrPTSE prion. Furthermore, if persistent infection is established, all PrPTSE in the cell will eventually be derived solely from its host PrPC precursor. Studying prion replication in a cell therefore requires being able to distinguish three types of PrP molecules: the exogenous PrPTSE that comprises the infectious prion, the PrPC molecule of the host cell, and the newly made PrPTSE that is derived from the host's own PrPC. Distinguishing these different types of PrP molecules has proven to be very difficult in live cells since the differences are not necessarily dependent upon differences in the amino acid sequence of the PrP molecule but rather on differences in how the protein is folded (i.e., its conformation). Most studies of prion-infected cells have therefore used either fixed cells and denaturing treatments to distinguish PrPTSE from PrPC microscopically or infected cell lysates treated with proteases to distinguish PrPC from PrPTSE biochemically. This has in many ways limited our ability to fully understand the process of prion infection using cell-based models.
Nevertheless, over the last two decades there has been a large increase in the number of cell culture models of prion infection, including the development of established cell lines that do not express PrPC and the identification of highly susceptible cell lines. New techniques have been developed to distinguish host cell PrP from exogenous PrPTSE that have allowed us to look at the earliest stages of prion infection. These approaches have greatly enhanced our understanding of the process of prion infection from the initial contact of prions with the cell to the establishment of persistent infection.
Section snippets
Brain cell culture
The earliest cell models of prion infection were developed in the 1960s and 1970s, decades prior to the identification of the prion protein. At that time, prion diseases were known as TSEs and the infectious agent was believed to be a slow virus. In 1965, Gustafson and Kanitz isolated transmissible spongiform encephalopathy (TSE)-infected cells by taking portions of scrapie-infected sheep or mouse brain, dissociating the cells using trypsin, and adding the resulting suspension to cell growth
Expression of PrPC
Deletion of the prion protein gene (PRNP) in mice renders those mice resistant to prion infection (Bueler et al., 1993) and restoration of the PRNP gene locus restores susceptibility to prion infection (Manson et al., 1994; Weissmann et al., 1994; Sakaguchi et al., 1995; Fischer et al., 1996; Raeber et al., 1997), results which have been recapitulated using cell culture models. There are now several established cell lines where the PRNP gene locus has been ablated (Table 3.1), all of which were
Acute prion infection
For the purposes of this chapter, the process by which prions infect cells in vitro has been broadly divided into three stages. The first, acute stage of infection encompasses the earliest events which must occur for a cell to become infected. These include the initial interaction of PrPTSE with the cell, PrPTSE uptake and trafficking, and the initial conversion of the host PrPC molecule to PrPTSE, i.e., prion replication. In vitro, the acute phase of infection for established cell lines is
Adaptation
Intriguingly, formation of acute PrPTSE does not always lead to persistent PrPTSE formation and chronic infection of a cell (Vorberg et al., 2004a). This suggests that there may be prion strain-specific or cell-specific factors that can control the switch from acute prion infection to persistent prion infection. In N2a cells that are resistant to infection with 22L mouse prions, acute PrPTSE is detectable at 24 hours but then decreases in amount over the next 72 hours. This suggests that prion
Cellular location of PrPTSE formation
Not surprisingly, the cellular compartments where prion replication can occur in chronically infected cells are almost identical to those implicated in acute PrPTSE formation (Fig. 3.2). Early studies in RML prion-infected N2a cells demonstrated that PrPTSE formation was temperature-dependent and was inhibited if the cells were treated with proteases or phosphatidylinositol phospholipase C, which cleaves GPI membrane-anchored proteins at the cell surface (Borchelt et al., 1990, Borchelt et al.,
Acknowledgments
The author would like to thank Anita Mora for technical assistance in preparation of the figures. This work was supported by the National Institutes of Health intramural research program.
References (184)
- et al.
Stimulation of PrP(C) retrograde transport toward the endoplasmic reticulum increases accumulation of PrP(Sc) in prion-infected cells
J Biol Chem
(2002) - et al.
Evidence for synthesis of scrapie prion proteins in the endocytic pathway
J Biol Chem
(1992) - et al.
Mice devoid of PrP are resistant to scrapie
Cell
(1993) - et al.
The anti-prion activity of Congo red. Putative mechanism
J Biol Chem
(1998) - et al.
The scrapie-associated form of PrP is made from a cell surface precursor that is both protease- and phospholipase-sensitive
J Biol Chem
(1991) - et al.
Uptake and degradation of protease-sensitive and -resistant forms of abnormal human prion protein aggregates by human astrocytes
Am J Pathol
(2014) - et al.
Multiplication of scrapie agent in cell culture
Res Vet Sci
(1970) - et al.
Prion infection impairs cholesterol metabolism in neuronal cells
J Biol Chem
(2014) - et al.
Identification of differentially expressed genes in scrapie-infected mouse neuroblastoma cells
Microb Pathog
(1995) - et al.
Characterization of early transient accumulation of PrP(Sc) in immune cells
Biochem Biophys Res Commun
(2013)
The tyrosine kinase inhibitor STI571 induces cellular clearance of PrPSc in prion-infected cells
J Biol Chem
Acute cellular uptake of abnormal prion protein is cell type and scrapie-strain independent
Virology
Stimulating the release of exosomes increases the intercellular transfer of prions
J Biol Chem
Prion infection of differentiated neurospheres
J Neurosci Methods
PrPSc incorporation to cells requires endogenous glycosaminoglycan expression
J Biol Chem
Heparan sulfate is a cellular receptor for purified infectious prions
J Biol Chem
Heparan sulfate and heparin promote faithful prion replication in vitro by binding to normal and abnormal prion proteins in protein misfolding cyclic amplification
J Biol Chem
Transfer of scrapie prion infectivity by cell contact in culture
Curr Biol
Human tonsil-derived follicular dendritic-like cells are refractory to human prion infection in vitro and traffic disease-associated prion protein to lysosomes
Am J Pathol
Disease-associated prion protein oligomers inhibit the 26S proteasome
Mol Cell
Proteinase-resistant protein in human neuroblastoma cells infected with brain material from Creutzfeldt-Jakob patient
Lancet
Mouse-adapted sporadic human Creutzfeldt-Jakob disease prions propagate in cell culture
Int J Biochem Cell Biol
Autophagy induction by trehalose counteracts cellular prion infection
Autophagy
Persistent propagation of variant Creutzfeldt-Jakob disease agent in murine spleen stromal cell culture with features of mesenchymal stem cells
J Virol
Murine bone marrow stromal cell culture with features of mesenchymal stem cells susceptible to mouse-adapted human TSE agent, Fukuoka-1
Folia Neuropathol
Mouse neuroblastoma cells release prion infectivity associated with exosomal vesicles
Biol Cell
Cultured peripheral neuroglial cells are highly permissive to sheep prion infection
J Virol
Prion strains are differentially released through the exosomal pathway
Cell Mol Life Sci
Two Creutzfeldt-Jakob disease agents reproduce prion protein-independent identities in cell cultures
Proc Natl Acad Sci U S A
The abnormal isoform of the prion protein accumulates in late-endosome-like organelles in scrapie-infected mouse brain
J Pathol
Conversion of raft associated prion protein to the protease-resistant state requires insertion of PrP-res (PrP(Sc)) into contiguous membranes
EMBO J
Mouse-adapted scrapie infection of SN56 cells: greater efficiency with microsome-associated versus purified PrP-res
J Virol
Opposite effects of dextran sulfate 500, the polyene antibiotic MS-8209, and Congo red on accumulation of the protease-resistant isoform of PrP in the spleens of mice inoculated intraperitoneally with the scrapie agent
J Virol
Cell-based quantification of chronic wasting disease prions
J Virol
Scrapie strains maintain biological phenotypes on propagation in a cell line in culture
EMBO J
p62/SQSTM1 forms protein aggregates degraded by autophagy and has a protective effect on huntingtin-induced cell death
J Cell Biol
Scrapie and cellular prion proteins differ in their kinetics of synthesis and topology in cultured cells
J Cell Biol
Cultured cell sublines highly susceptible to prion infection
J Virol
Scrapie-infected murine neuroblastoma cells produce protease-resistant prion proteins
J Virol
Early increase and late decrease of Purkinje cell dendritic spine density in prion-infected organotypic mouse cerebellar cultures
PLoS One
Sulfated polyanion inhibition of scrapie-associated PrP accumulation in cultured cells
J Virol
N-terminal truncation of the scrapie-associated form of PrP by lysosomal protease(s): implications regarding the site of conversion of PrP to the protease-resistant state
J Virol
Fukuoka-1 strain of transmissible spongiform encephalopathy agent infects murine bone marrow-derived cells with features of mesenchymal stem cells
Transfusion
Genetic heterogeneity versus molecular analysis of prion susceptibility in neuroblasma N2a sublines
Arch Virol
GPI anchoring leads to sphingolipid-dependent retention of endocytosed proteins in the recycling endosomal compartment
EMBO J
A specific population of abnormal prion protein aggregates is preferentially taken up by cells and disaggregated in a strain-dependent manner
J Virol
Infection of cell cultures with scrapie agent
Evidence for the multiplication of scrapie agent in cell culture
Nature
Infection of a cell line of mouse L fibroblasts with scrapie agent
Nature
Prion-infected cells regulate the release of exosomes with distinct ultrastructural features
FASEB J
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2021, BioSystemsCitation Excerpt :The key role of PrPC in TSE was undoubtedly demonstrated through deletion of PRNP in mice, which resulted in complete resistance to prion infection, while restoration of PRNP gene also restored the susceptibility to prion infection (Priola, 2018). However, although expression of host PrPC is an utter requirement for the infection, it is not sufficient determinant for a cell to be susceptible to prion infection, as demonstrated by cell lines which express PrPC but can remain refractory to infection (Oelschlegel et al., 2015; Priola, 2018). CJD results in a rapidly evolving neurodegenerative disease characterized by neuronal loss, spongiform degeneration and astrogliosis.
Therapeutic implications of prion diseases
2021, Biosafety and HealthCitation Excerpt :Infectious cellular models are the primary tool for screening of antiprion drugs. To date, at least 20 different cell lines that can propagate certain prion strains have identified [14,15]. Notably, cell infection models have demonstrated that pharmacological treatments can induce strain-specific inhibition of prion replication and produce treatment-resistant prion strains [16,17].
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Cell biology of prion strains in vivo and in vitro
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