Nuclease-resistant polyadenylated RNAs of significant size are detected by PCR in highly purified Creutzfeldt-Jakob disease preparations

Akowitz, Alfred; Sklaviadis, Theodoros; Manuelidis, Elias E.; Manuelidis, Laura

doi:10.1016/0882-4010(90)90038-r

Cited by 39 publications

(35 citation statements)

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“…§1734 solely to indicate this fact. scrapie preparations, including those treated with Zn2+ and nucleases, invariably show nucleic acids of 500-6000 bp by conventional 32p labeling (6)(7)(8). We considered that nucleic acid binding proteins might be constituents ofthese infectious agents that could protect a viral-like genome from nuclease attack and additionally endow these agents with unique attributes of resistance.…”

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confidence: 99%

“…After disaggregation, Gp34 cosedimenting with infectivity loses its resistance to limited proteolysis, a presumed characteristic of the "infectious prion" form of this host protein (5), and infectivity migrates with reproducible virus-like size and density characteristics in sucrose gradients (7-9). Exhaustive nuclease digestion, which does not reduce infectivity or alter these physical characteristics, is used to substantially increase agent purification (7,8). In sucrose gradients, almost all applied infectivity is recovered within a single gaussian peak centered at 120S (8).…”

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confidence: 99%

“…The supernatant was centrifuged on a linear 10-30% sucrose gradient to resolve the infectious 120S peak, yielding an '=100,000-fold purification of infectivity with respect to starting nucleic acids (8) and >11,000-fold with respect to protein (7). The 120S peak sucrose fractions were diluted =3-fold and were concentrated by pelleting at 215,000 x g for 2 hr with no loss of titer (10).…”

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confidence: 99%

“…The 120S peak sucrose fractions were diluted =3-fold and were concentrated by pelleting at 215,000 x g for 2 hr with no loss of titer (10). Initial homogenization releases only =20% ofthe brain infectivity, almost all ofwhich is recovered in the final concentrates-i.e., 1.5-3 x 107 IU/g (7,8,10 RNA (8). This RNA was reverse transcribed by using random primers, the resultant cDNAs were converted to double-stranded DNA molecules, and then designed PCR adaptor/primers were blunt-end ligated to the cDNA for sequence-independent amplification (12).…”

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confidence: 99%

“…MATERIALS AND METHODS Clarified supernatants from brain homogenized in 10% Sarkosyl were centrifuged at 215,000 x g for 2 hr, and the resuspended pellet, treated with excess micrococcal nuclease and Ca2+ (7,8), was disaggregated at pH 8.9 (9). The supernatant was centrifuged on a linear 10-30% sucrose gradient to resolve the infectious 120S peak, yielding an '=100,000-fold purification of infectivity with respect to starting nucleic acids (8) and >11,000-fold with respect to protein (7).…”

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Nucleic acid binding proteins in highly purified Creutzfeldt-Jakob disease preparations.

Sklaviadis

Akowitz

Manuelidis

et al. 1993

Proc. Natl. Acad. Sci. U.S.A.

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The nature of the infectious agent causing human Creutzfeldt-Jakob disease (CJD), a slowly progressive dementia, is controversial. As in scrapie, no agent-specific proteins or nucleic acids have been identified. However, biological features of exponential replication and agent strain variation, as well as physical size and density data, are most consistent with a viral structure--i.e., a nucleic acid-protein complex. It is often assumed that nuclease treatment, which does not reduce infectious titer, leaves no nucleic acids of > 50 bp. However, nucleic acids of 500-6000 bp can be extracted from highly purified infectious complexes with a mass of approximately 1.5 x 10(7) daltons. It was therefore germane to search for nucleic acid binding proteins that might protect an agent genome. We here use Northwestern blotting to show that there are low levels of nonhistone nucleic acid binding proteins in highly purified infectious 120S gradient fractions. Several nucleic acid binding proteins were clearly host encoded, whereas others were apparent only in CJD, but not in parallel preparations from uninfected brain. Small amounts of residual host Gp34 (prion protein) did not bind any 32P-labeled nucleic acid probes. Most of the minor "CJD-specific" proteins had an acidic pI, a characteristic of many viral core proteins. Such proteins deserve further study, as they probably contribute to unique properties of resistance described for these agents. It remains to be seen if any of these proteins are agent encoded.

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