Evidence that DNA is present in abnormal tubulofilamentous structures found in scrapie.

Abstract

Abnormal tubulofilamentous structures have been identified in electron micrographs of thin sections and negatively stained impression grids prepared from brains of animals with scrapie and other spongiform encephalopathies, and we showed that such tubules contain a core of rdamentous structures resembling scrapie-associated fibrils (SAF). We treated impression grids from brains of scrapie-infected hamsters with several substances that bind to or cleave proteins and nucleic acids to see if they had any effect on the abnormal tubuloframentous structures. Treatment with three proteolytic enzymes reduced the caliber of the tubules from about 50 nm to 30 nm; subsequent treatment of the 30-nm tubules with DNase I left many typical SAF as well as transitional forms in which twisted SAF emerged from tubules. DNase treatment of the original thicker tubules had no effect, and no SAF were seen on grids. Treatment of the 30-nm tubules with any of three other nucleases (micrococcal, mung bean, and BAL-31) also produced SAF. However, treatment with RNase A had no effect either on the original 50-nm tubules or on the 30-nm tubules produced by proteolysis. Detergent treatment of any of the preparations produced SAF. Treatment with ethidium bromide resulted in staining of the tubules that was inhibited by magnesium ions. The data suggest that the abnormal tubulofilamentous particles found in spongiform encephalopathies may consist of an outer cylinder of protein, an inner cylinder of DNA, and an innermost core of SAF.We recently described a simple method of negative-stain transmission electron microscopy using grids touched to the freshly cut surface of brain tissue. In brains infected with the agents ofthe spongiform encephalopathies, we found abnormal tubular structures (1) that generally resembled tubulovesicular particles often seen in thin sections of plastic-embedded brain specimens (2,3). We demonstrated that treatment of those tubules with detergent released inner filaments that were morphologically identical to scrapie-associated fibrils (SAF) (4) and reacted with antisera prepared against the proteaseresistant sialoglycoprotein "prion protein [27][28][29][30] (5, 6) of which SAF are composed (7,8). We report here a study of the effects of several proteases and nucleases and of ethidium bromide on the abnormal tubules. The abnormal tubules appear to consist of at least three layers: (i) an outer coat of protease-sensitive material, (ii) an intermediate layer that is digested by a DNase and several other nucleases but not by an RNase, and (iii) an inner core of SAF. MATERIALS AND METHODSScrapie Virus. The 263K strain of scrapie agent (9) was used at the fourth passage level as described (1 20,10,5, 2.5, and 0.25 units/ml diluted in buffer recommended by the supplier; and (ix) diluent alone. Grids were immersed under 0.5 ml of solution and incubated in a moist chamber. Grids were removed at intervals from 30 min to 120 min. After a 30-min incubation, grids treated with collagenase, trypsin, or proteinase K were...

Antibody to DNA detects scrapie but not normal prion protein

“…These findings suggest that the capture of PrP Sc by OCD4 and g5p depends on nucleic acid and that PrP Sc forms a complex with nucleic acid. The sequence of the nucleic acid associated with PrP Sc , and whether the PrP Sc -nucleic acid complexes are present in vivo as previously suggested (26,(35)(36)(37)(38), or form only in the homogenized tissue, remain to be determined.…”

Structural and Biochemical Evidence that Scrapie-associated Fibrils Assemble in vivo

“…Supporting evidence is the observation of SAF on washed electron microscope impression grids taken directly from scrapie-infected brain sections (Narang et al, 1988). This conclusion contradicts the opinion of Meyer et al (1986) who suggest that fibril formation only occurs in vitro after the dissolution of membrane with detergents.…”

Binding of Recombinant but Not Endogenous Prion Protein to DNA Causes DNA Internalization and Expression in Mammalian Cells

“…In vivo derived PrP Sc does contain DNA (47), but the PrP Sc is much more PK resistant, and it is the N terminus of the PrP Sc that is sensitive to PK digestion (44). Accumulated evidence suggests that cellular factors, such as GAG or nucleic acid may facilitate the conversion of PrP C to PrP Sc (32).…”