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From: TSS ()
Cells infected with scrapie and Creutzfeldt–Jakob disease agents produce intracellular 25-nm virus-like particles Laura Manuelidis*, Zhoa-Xue Yu, Nuria Banquero, and Brian Mullins Yale Medical School, 333 Cedar Street, New Haven, CT 06510 Communicated by Sheldon Penman, Massachusetts Institute of Technology, Cambridge, MA, December 11, 2006 (received for review October 10, 2006) 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 N2a22L cells, yet this did not increase either the number of virus-like arrays or the infectious titer of these cells. Thus, the 25-nm infectionassociated 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. snip. In summation, all of this data provides a clear, consistent, substantive, and logical alternative to the accepted prion hypothesis. The causative TSE agent is most consistent with an exogenous 25-nm virion without intrinsic host PrP. The stimulation of host innate immune responses by these agents, a complex set of molecular reactions that precedes the elaboration of pathologic PrP (9) and one that is not provoked by PrP-res itself (25), also point to a foreign pathogen rather than some unpredictably spontaneous mutation in the host’s PrP without cause. The presence of these particles in many different species infected with a wide variety of TSE strains is in accord with Koch’s first requirement (1). It is also improbable that an identical virus-like structure would be a contaminant or a secondary coincidental feature of all these different TSE models. Nevertheless, a more detailed molecular analysis of these particles will be required to substantiate their causal nature. Purification of these 25-nm particles from productive tissue cultures should be informative if the essential infectivity assays are performed systematically with parallel ultrastructural and molecular analyses. Animal titrations of infectivity are expensive and prolonged. However, sustained and reproducible infection of indicator GT cells by a variety of TSE agents already has shown that they can rapidly authenticate the presence of agent in disrupted samples as well as in living cells (4, 17). GT cells also may be used for testing infectivity of viral nucleic acids as well as PrP conformers. Rapid assays of infectivity in culture should facilitate the isolation of infectious particles from host components, and treatments that modify the production of these particles in culture may resolve further the infectious structure from the pathological disease processes it initiates. Materials and Methods......snip......end........TSS http://www.pnas.org/cgi/content/abstract/0610999104v1
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