Chapter 3 - Cell biology of prion infection

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Abstract

The development of multiple cell culture models of prion infection over the last two decades has led to a significant increase in our understanding of how prions infect cells. In particular, new techniques to distinguish exogenous from endogenous prions have allowed us for the first time to look in depth at the earliest stages of prion infection through to the establishment of persistent infection. These studies have shown that prions can infect multiple cell types, both neuronal and nonneuronal. Once in contact with the cell, they are rapidly taken up via multiple endocytic pathways. After uptake, the initial replication of prions occurs almost immediately on the plasma membrane and within multiple endocytic compartments. Following this acute stage of prion replication, persistent prion infection may or may not be established. Establishment of a persistent prion infection in cells appears to depend upon the achievement of a delicate balance between the rate of prion replication and degradation, the rate of cell division, and the efficiency of prion spread from cell to cell. Overall, cell culture models have shown that prion infection of the cell is a complex and variable process which can involve multiple cellular pathways and compartments even within a single cell.

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.

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