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Review
. 2013 Jan 22;5(1):374-405.
doi: 10.3390/v5010374.

Cellular aspects of prion replication in vitro

Andrea Grassmann  1 , Hanna Wolf, Julia Hofmann, James Graham, Ina Vorberg
Affiliations
Review

Cellular aspects of prion replication in vitro

Andrea Grassmann et al. Viruses. .

Abstract

Prion diseases or transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative disorders in mammals that are caused by unconventional agents predominantly composed of aggregated misfolded prion protein (PrP). Prions self-propagate by recruitment of host-encoded PrP into highly ordered b-sheet rich aggregates. Prion strains differ in their clinical, pathological and biochemical characteristics and are likely to be the consequence of distinct abnormal prion protein conformers that stably replicate their alternate states in the host cell. Understanding prion cell biology is fundamental for identifying potential drug targets for disease intervention. The development of permissive cell culture models has greatly enhanced our knowledge on entry, propagation and dissemination of TSE agents. However, despite extensive research, the precise mechanism of prion infection and potential strain effects remain enigmatic. This review summarizes our current knowledge of the cell biology and propagation of prions derived from cell culture experiments. We discuss recent findings on the trafficking of cellular and pathologic PrP, the potential sites of abnormal prion protein synthesis and potential co-factors involved in prion entry and propagation.

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Figures

Figure 1
Figure 1
Localization of PrPC and PrPSc in L929 fibroblast cells. (A) Indirect immunofluorescence (IF) staining of cellular PrP (green) in uninfected L929 cells. PrPC predominantly resides at the cell surface with some intracellular localization. (B) Detection of PrPSc in L929 cells persistently infected with prion strain 22L by IF. In contrast to PrPC, PrPSc (green) primarily localizes intracellularly and partially co-localizes with the lysosomal marker Lamp-1 (red). (A,B) Nuclei were counterstained with Hoechst (blue). Scale bar: 5 µm.
Figure 2
Figure 2
Cell biology of PrP in scrapie-infected cells. PrPC is synthesized in the endoplasmic reticulum (ER) and passes through the secretory pathway to the cell surface, where it resides in lipid rafts. In many cells, PrPC leaves lipid rafts prior to being internalized by clathrin-dependent endocytosis (I). Clathrin-independent raft/caveolae-dependent internalization (II) of PrPC has also been proposed for some cells. PrPC can be degraded by lysosomes or rapidly recycled back to the cell surface by recycling endosomes (RE). In cultured scrapie-infected cells the conversion of PrPC to PrPSc is believed to take place on the cell surface and/or in vesicles along the endolysosomal pathway. After conversion PrPSc can accumulate at the cell surface or in intracellular vesicles (e.g. lysosomes).
Figure 3
Figure 3
Non-neuronal cells and PrP-deficient cells take up PrPSc. Brain homogenate from mice infected with the 22L prion strain is taken up by L929 fibroblast cells (left panel) and PrP-deficient HpL3-4 cells (right panel). Cells were incubated with infected brain homogenate for 18 hours prior to fixation, permeabilization, guanidine hydrochloride treatment and immunofluorescence staining. Cells incubated with uninfected brain homogenate (MOCK ctrl) served as control for specific detection of PrPSc. PrPSc uptake is observed in both fibroblast cells and PrP-deficient cells. Monoclonal anti-PrP antibody: 4H11. Nuclei were counterstained with Hoechst (blue). Scale bar: 5 µm.
Figure 4
Figure 4
Propagation of cytosolic prions derived from the S. cerevisiae Sup35 prion domain NM. N2a cells ectopically express the HA-tagged prion domain NM of Sup35, which is the most well characterized yeast prion. The left image shows aggregated NM-HA (green) after induction with recombinant NM fibrils, the right image shows the soluble NM-HA (green). NM was stained with anti-HA antibody. F-Actin was stained with fluorescently conjugated phalloidin (red). Nuclei were stained with Hoechst (blue). Scale bar: 5 µm.
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