Protease sensitivity and nuclease resistance of the scrapie agent propagated in vitro in neuroblastoma cells

Neary, K; Caughey, Byron; Ernst, Darwin; Race, Richard E.; Chesebro, Bruce

doi:10.1128/jvi.65.2.1031-1034.1991

Cited by 23 publications

(9 citation statements)

References 30 publications

(28 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…2). The activity of the proteinase K was confirmed by the fact that, with prolonged treatment (50 min), the PrP-res staining was decreased as we have documented previously (39). Presumably the slow digestion of the epitope (residues 89 to 103),…”

Section: Resultssupporting

confidence: 71%

“…Like PrP-res, the scrapie infectivity in detergent extracts of neuroblastoma cells is also relatively resistant to proteinase K and can be pelleted by ultracentrifugation (39). The fact that PrP-res and scrapie infectivity share these properties when derived from tissue culture cells, as well as from brain tissue (6,23,24,43), provides further suggestive evidence that the protein is physically associated with the transmissible agent, if not the agent itself.…”

Section: Discussionmentioning

confidence: 98%

See 1 more Smart Citation

N-terminal truncation of the scrapie-associated form of PrP by lysosomal protease(s): implications regarding the site of conversion of PrP to the protease-resistant state

Caughey

Raymond

Ernst

et al. 1991

J Virol

Self Cite

389

124

View full text Add to dashboard Cite

Scrapie and related transmissible spongiform encephalopathies result in the accumulation of a proteaseresistant form of an endogenous brain protein called PrP. As an approach to understanding the scrapieassociated modification of PrP, we have studied the processing and sedimentation properties of proteaseresistant PrP (PrP-res) in scrapie-infected mouse neuroblastoma cells. Like brain-derived PrP-res, the neuroblastoma cell PrP-res aggregated in detergent lysates, providing evidence that the tendency to aggregate is an intrinsic property of PrP-res and not merely a secondary consequence of degenerative brain pathology. The PrP-res species had lower apparent molecular masses than the normal, protease-sensitive PrP species and were not affected by moderate treatments with proteinase K. This suggested that the PrP-res species were partially proteolyzed by the neuroblastoma cells. Immunoblot analysis of PrP-res with a panel of monospecific anti-PrP peptide sera confirmed that the PrP-res species were quantitatively truncated at the N terminus. The metabolic labeling of PrP-res in serum-free medium did not prevent the proteolysis of PrP-res, showing that the protease(s) involved was cellular rather than serum-derived. The PrP-res truncation was inhibited in intact cells by leupeptin and NH4Cl. This provided evidence that a lysosomal protease(s) was involved, and therefore, that PrP-res was translocated to lysosomes. When considered with other studies, these results imply that the conversion of PrP to the protease-resistant state occurs in the plasma membrane or along an endocytic pathway before PrP-res is exposed to endosomal and lysosomal proteases.

show abstract

Section: Resultssupporting

confidence: 71%

Section: Discussionmentioning

confidence: 98%

N-terminal truncation of the scrapie-associated form of PrP by lysosomal protease(s): implications regarding the site of conversion of PrP to the protease-resistant state

Caughey

Raymond

Ernst

et al. 1991

J Virol

Self Cite

389

124

View full text Add to dashboard Cite

show abstract

“…To minimize complex neurodegenerative brain changes that might impede nuclease digestions, and to ensure that the majority of the infectious agent was being analyzed, another group digested whole homogenates of neuroblastoma cells infected with Chandler‐RML scrapie agent [Neary et al, ]. These cells have an ∼100‐fold lower titer (2e7/gm) than 263 K scrapie hamster brain.…”

mentioning

confidence: 99%

CJD and Scrapie Require Agent‐Associated Nucleic Acids for Infection

Botsios¹,

Manuelidis²

2016

J of Cellular Biochemistry

View full text Add to dashboard Cite

Unlike Alzheimer's and most other neurodegenerative diseases, Transmissible Spongiform Encephalopathies (TSEs) are all caused by actively replicating infectious particles of viral size and density. Different strain-specific TSE agents cause CJD, kuru, scrapie and BSE, and all behave as latent viruses that evade adaptive immune responses and can persist for years in lymphoreticular tissues. A foreign viral structure with a nucleic acid genome best explains these TSE strains and their endemic and epidemic spread in susceptible species. Nevertheless, it is widely believed that host prion protein (PrP), without any genetic material, encodes all these strains. We developed rapid infectivity assays that allowed us to reproducibly isolate infectious particles where >85% of the starting titer separated from the majority of host components, including PrP. Remarkably, digestion of all forms of PrP did not reduce brain particle titers. To ask if TSE agents, as other viruses, require nucleic acids, we exposed high titer FU-CJD and 22L scrapie particles to potent nucleases. Both agent-strains were propagated in GT1 neuronal cells to avoid interference by complex degenerative brain changes that can impede nuclease digestions. After exposure to nucleases that are active in sarkosyl, infectivity of both agents was reproducibly reduced by ≥99%. No gold-stained host proteins or any form of PrP were visibly altered by these nucleases. In contrast, co-purifying protected mitochondrial DNA and circular SPHINX DNAs were destroyed. These findings demonstrate that TSE agents require protected genetic material to infect their hosts, and should reopen investigation of essential agent nucleic acids. J. Cell. Biochem. 117: 1947-1958, 2016. © 2016 Wiley Periodicals, Inc.

show abstract

“…have shown that the destruction or denaturation of PrP-res in cell or tissue extracts by using proteinase K or sodium dodecyl sulfate (SDS) can also be accompanied by a decrease in scrapie infectivity (15,18,20). The interpretation of these earlier studies with SDS and proteinase K was complicated by the fact that these harsh treatments are broad in effect and can be expected to affect most proteins and infectious agents nonspecifically.…”

mentioning

confidence: 99%

Congo red inhibition of scrapie agent replication

Caughey

Ernst

Race

1993

J Virol

Self Cite

122

View full text Add to dashboard Cite

Congo red inhibits the accumulation of protease-resistant PrP in scrapie-infected mouse neuroblastoma cells. Here we show that Congo red also inhibits the replication of scrapie infectivity in these cells. This observation is consistent with the idea that protease-resistant PrP is a vital component of the scrapie agent or that agent replication depends on the presence of protease-resistant PrP in the cell.

show abstract

Protease sensitivity and nuclease resistance of the scrapie agent propagated in vitro in neuroblastoma cells

Cited by 23 publications

References 30 publications

N-terminal truncation of the scrapie-associated form of PrP by lysosomal protease(s): implications regarding the site of conversion of PrP to the protease-resistant state

N-terminal truncation of the scrapie-associated form of PrP by lysosomal protease(s): implications regarding the site of conversion of PrP to the protease-resistant state

CJD and Scrapie Require Agent‐Associated Nucleic Acids for Infection

Congo red inhibition of scrapie agent replication

Contact Info

Product

Resources

About