Cells infected with scrapie and Creutzfeldt–Jakob disease agents produce intracellular 25-nm virus-like particles

Abstract

We had repeatedly found ≈25-nm-diameter virus-like particles in highly infectious brain fractions with little prion protein (PrP), and therefore we searched for similar virus-like particles in situ in infected cell lines with high titers. Neuroblastoma cells infected with the 22L strain of scrapie as well as hypothalamic GT cells infected with the FU Creutzfeldt–Jakob disease agent, but not parallel mock controls, displayed dense 25-nm virus-like particles in orthogonal arrays. These particles had no relation to abnormal PrP amyloid in situ , nor were they labeled by PrP antibodies that faithfully recognized rough endoplasmic reticulum membranes and amyloid fibrils, the predicted sites of normal and pathological intracellular PrP. Additionally, phorbol ester stimulated the production of abnormal PrP gel bands by >5-fold in infected N2a + 22L cells, yet this did not increase either the number of virus-like arrays or the infectious titer of these cells. Thus, the 25-nm infection-associated particles could not be prions. Synaptic differentiation and neurodegeneration, as well as retroviruses that populate the rough endoplasmic reticulum of neuroblastoma cells, were not required for particle production. The 25-nm particle arrays in cultured cells strongly resembled those first described in 1968 in synaptic regions of scrapie-infected brain and subsequently identified in many natural and experimental TSEs. The high infectivity of comparable, isolated virus-like particles that show no intrinsic PrP by antibody labeling, combined with their loss of infectivity when nucleic acid–protein complexes are disrupted, make it likely that these 25-nm particles are the causal TSE virions that induce late-stage PrP brain pathology.

Rapid chemical decontamination of infectious CJD and scrapie particles parallels treatments known to disrupt microbes and biofilms

“…Both strains induced large PrP-res fibril accumulations within the GT1 cell cytoplasm ultrastructurally, and by Western blot. 3,14 The amount of residual PrP and PrP-res in p18 particles of both strains was also the same. Thus total PrP-res levels did not account for this strain-specific sensitivity.…”

“…Pertinent molecular components are shown in lines B, C and D. The first 500 g nuclear pellet contains only 5-10% of the total infectivity, some of which is likely due to incompletely lysed cells. Large PrP amyloid aggregates, identified ultrastructurally in the cytoplasm of GT1 infected cells, 14 can also sediment at these low g forces. More than 85% of the starting whole cell infectivity is routinely recovered in the p18, with negligible infectivity in the s18 supernatant.…”

“…Urea is a non-charged protein denaturant that disrupts noncovalent protein bonds and unfolds them to reveal their hydrophobic cores. It can progressively separate specific sets of membrane proteins, 47,48 and because normal PrP is predominantly a membrane protein, as demonstrated by ultrastructural PrP antibody labeling, 14 it was relevant to evaluate the effects of different urea concentrations on both protein structure and particle infectivity. Thiourea (ThU), an alternative and more powerful denaturant, 49 was also tested, along with 5% b-ME that reduces S-S bonds, including those of PrP that contribute to PrP-res misfolding and amyloid aggregation.…”

“…PrP-res amyloid is also ultrastructurally separate from virus-like 20-25 nm dense particles. 9,[14][15][16][17] Additionally, destruction of PrP further underscores a biochemical separation between PrP-res and the infectious agent; protease or PK digestion of all detectable PrP did not significantly reduce either BSE or FU-CJD brain titers. [18][19][20] A variety of other proteases also has shown little detrimental effect on 263K or RML scrapie agent brain titers in different material contexts.…”

Establishment of a Cell Line Persistently Infected with Chronic Wasting Disease Prions

“…It was previously reported that phorbol 12-myristate 13-acetate (PMA) was applied to enhance PrP C and PrP Sc production in scrapie 22L-affected N2a cells [9]. After 6 hr of CWD prion infection, the cells were treated with 100 ng/ml of PMA for 4 days, and then PrP Sc accumulation was assessed using the standard scrapie cell assay (SSCA) [8].…”