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Mouse-adapted scrapie infection of SN56 cells: greater efficiency with microsome-associated versus purified PrP-res
Gerald S Baron1, Ana C Magalhães, Marco A M Prado
1Rocky Mountain Laboratories, Laboratory of Persistent Viral Diseases, NIAID, NIH, Hamilton, MT 59840, USA. gbaron@niaid.nih.gov
Abstract:
The process by which transmissible spongiform encephalopathy agents, or prions, infect cells is unknown. We employed a new differentiable cell line (SN56) susceptible to infection with three mouse-adapted scrapie strains to gain insight into the cellular infection process. The effect of disease-associated PrP (PrP-res) association with microsomal membranes on infection efficiency was examined by comparing sustained PrP-res production in cells treated with either scrapie brain microsomes or purified, detergent-extracted PrP-res. When normalized for quantity of input PrP-res, scrapie brain microsomes induced dramatically enhanced persistent PrP-res formation compared to purified PrP-res. Infected SN56 cells released low levels of PrP-res into the culture supernatant, which also efficiently initiated infection in recipient cells. Interestingly, microsomes labeled with a fluorescent marker were internalized by SN56 cells in small vesicles, which were subsequently found in neuritic processes. When bound to culture wells to reduce internalization during the infection process, scrapie microsomes induced less long-term PrP-res production than suspended microsomes. Long-term differentiation of infected SN56 cells was accompanied by a decrease in PrP-res formation. Our observations provide evidence that infection of cells is aided by the association of PrP-res with membranes and/or other microsomal constituents.
Insights
Prion infection of cells is enhanced when disease-associated prion protein (PrP-res) associates with cell membranes and microsomal components. This membrane association aids in sustained prion formation and infectivity.
Area of Science:
- Neuroscience
- Cell Biology
- Infectious Diseases
Background:
- The cellular mechanisms underlying transmissible spongiform encephalopathy (TSE) agent, or prion, infection remain largely unknown.
- Understanding prion entry and replication is crucial for developing therapeutic strategies against neurodegenerative diseases.
Purpose of the Study:
- To investigate the role of disease-associated prion protein (PrP-res) association with cellular membranes in prion infection efficiency.
- To elucidate the cellular uptake and intracellular trafficking of prions.
Main Methods:
- Utilized a novel differentiable SN56 cell line susceptible to mouse-adapted scrapie strains.
- Compared prion infection efficiency using scrapie brain microsomes versus purified PrP-res.
- Assessed PrP-res formation, release into culture supernatant, and internalization via fluorescently labeled microsomes.
Main Results:
- Scrapie brain microsomes significantly enhanced persistent PrP-res formation compared to purified PrP-res when normalized for input quantity.
- Infected cells released infectious PrP-res into the supernatant, capable of infecting recipient cells.
- Microsomal uptake occurred via vesicles, with accumulation in neuritic processes; reduced internalization decreased long-term PrP-res production.
Conclusions:
- PrP-res association with membranes and/or microsomal constituents aids cellular prion infection.
- Cellular uptake and intracellular localization within vesicles and neuritic processes are important for prion propagation.
- Membrane association appears critical for sustained prion formation and infectivity.
