Nuclease treatment results in high specific purification of Creutzfeldt-Jakob disease infectivity with a density characteristic of nucleic acid-protein complexes

Abstract

Representative preparations of partially purified Creutzfeldt-Jakob disease (CJD), including disaggregated density gradient fractions, were treated with a variety of nucleases. RNases as well as exhaustive digestions with micrococcal nuclease did not significantly diminish infectivity, but resulted in an approximately 7,000-fold specific purification of infectivity with respect to nucleic acid. Protected nucleic acids included species of up to 2,000 bases in length. After nuclease treatment, infectivity co-migrated with nucleic acid-protein complexes at a density of 1.27 g/cm3 in sucrose. Substantial specific protein purification were also achieved in the gradient step (approximately 11,000-fold), where 70% the host Gp34 ("prion protein") as well as other free proteins separated from infectivity. These CJD purifications are better than those previously attained in scrapie, and may be useful for further studies of non-host protein and nucleic acid species. The data are consistent with the hypothesis that CJD-like agents are composed of nucleic acid-protein complexes.

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

“…§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.…”

“…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).…”

“…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).…”

“…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).…”

“…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).…”