Our earlier analyses of residual nucleic acids in purified fractions of scrapie prions were performed on samples prepared according to the “standard protocol” (31
), which consists of brain homogenization followed by detergent extraction, enzymatic digestions, differential centrifugation, and sucrose gradient centrifugation. To remove the prions from the sucrose, ethanol precipitation was routinely used. To minimize residual nucleic acids, enzymatic digestion and Zn2+
hydrolysis were used followed by DLPC formation with consecutive nuclease digestions. After deproteinization of the samples, the remaining nucleic acids were analyzed by RRGE.
To increase the recovery of prion infectivity and reduce the amount of residual nucleic acid, we investigated the order of nuclease addition, conditions for Zn2+ hydrolysis, and the use of ultracentrifugation in place of ethanol precipitation. In another series of experiments, we substituted ultrafiltration for sucrose gradient centrifugation.
Nucleic acid degradation prior to prion rod formation.
Because nucleic acids might be trapped during prion rod formation and thus protected against nucleic acid degradation, we attempted to degrade nucleic acids before rod formation. Prion rods form when PrPSc
is converted to PrP 27-30 by limited proteolysis in the presence of detergent (44
). Although the recovery of prion infectivity in these experiments was good, the protein concentration was so high that nucleic acid analysis was problematic. Digestion of genomic DNA with benzonase apparently releases chromosomal proteins that were otherwise separated from the prions by a low-speed centrifugation that sediments much of the nuclear DNA. These problems prevented us from pursuing this approach after three experiments.
The “standard protocol” reported earlier included two ethanol precipitations: one after sucrose gradient fractionation and another after Zn2+ hydrolysis. These precipitations were used to change the buffer and volume for subsequent processing. Because bioassays repeatedly indicated a decrease in prion infectivity after ethanol precipitation, ultracentrifugation was used to sediment the prions. In Table , the recoveries of prion infectivity after ethanol precipitation are compared to those after ultracentrifugation at ≈100,000 × g for 6 or 15 h. The recovery of prion infectivity after ultracentrifugation was generally 10-fold better than by ethanol precipitation.
Recovery of prion infectivity in samples subjected to ethanol precipitation or ultracentrifugationa
Zn2+-mediated hydrolysis of polynucleotides.
Zn2+-catalyzed hydrolysis of RNA is a well-documented reaction. In our earlier studies, we employed 2 mM Zn2+ but in the studies described here, we varied the Zn2+ concentration from 4 to 40 mM and incubated the samples from 24 to 72 h. Maximal degradation of RNA occurred after 24 h of incubation at 65°C with 20 mM Zn2+. Under these conditions, prion infectivity remained stable (data not shown). When we performed benzonase incubation subsequent to Zn2+ hydrolysis, the titer also remained unchanged.
We investigated whether ultrafiltration might be substituted for sucrose gradient centrifugation. The retentate fraction after ultrafiltration did not contain sucrose but did possess >99.9% of the initial prion infectivity. Since Sarkosyl in the retentate is incompatible with subsequent nucleic acid degradation by Zn2+ hydrolysis, a second ultrafiltration was performed without Sarkosyl. Unfortunately, this second ultrafiltration step reduced the prion infectivity by a factor of ≈100. This loss in infectivity was unacceptable, and so the protocol using a second ultrafiltration to remove Sarkosyl was abandoned.
Based on the foregoing findings, the P3 fraction was resuspended in a buffer containing 2% Sarkosyl and subjected to ultrafiltration. After reducing the volume of the retentate from 500 to 50 ml, aliquots of 1 to 2 ml were diluted to 35 ml in H2O. The samples were then sedimented at 100,000 × g for 15 h, resuspended in 35 ml H2O and sedimented again at 100,000 × g for 6 h. After these washes, prion infectivity was quantitatively recovered in the sample in the absence of Sarkosyl (Table ).
Retentate fraction analysis for nucleic acid.
For nucleic acid degradation, 20 mM Zn2+ was used as described above followed by benzonase digestion. To remove the free Zn2+ ions from solution prior to benzonase digestion, the Zn2+ was chelated with cyclen. We analyzed three samples KK-D5, KK-D6, and KK-D7 by 9% RRGE. An example of RRGE analysis is shown in Fig. .
FIG. 1. Analysis of residual nucleic acids in preparations of PrP 27-30 by 15% RRGE. The modified protocol was applied to sucrose gradient fractions. Nucleic acids of the following size ranges are refocused: (a) 767 to 103 nucleotides; (b) 102 to 54 nucleotides; (more ...)
Because PrPSc forms amorphous aggregates and does not assemble into prion rods, we analyzed purified prion fractions containing full-length PrPSc (sample KK-D7) for nucleic acids. Dispersion of the prions into DLPCs was omitted. RRGE analysis of this preparation containing full-length PrPSc revealed that the amount of residual nucleic acid was ≈20-fold higher than that in preparations containing PrP 27-30. The prion infectivity of this preparation containing full-length PrPSc was 107.8 ID50 units/ml, which was slightly lower than the titer of preparations containing PrP 27-30. The maximum length for a hypothetical prion-specific nucleic acid in the PrPSc preparation yielded a value of 200 nucleotides. This large number of nucleotides caused us to abandon the use of preparations containing full-length PrPSc.
The conditions employed for sample KK-D6 gave the most effective nucleic acid degradation and thus were repeated two more times to produce samples KK-D8 and KK-D9. The corresponding total prion infectivity levels in the three samples were 108.0, 108.8 and 109.2 ID50 units, respectively. These samples contained ≈100-fold more prion infectivity than previously obtained by ultracentrifugation using the “standard protocol.” From the RRGE analysis, the amounts of nucleic acids within the different size ranges were estimated by comparing the intensities of the bands to those of the marker DNAs (Fig. ). The best quantification was obtained using the original gels; photographic reproduction, as shown in Fig. , was inferior. If a band was not visible, the lowest concentration of a visible marker DNA was taken.
The total amount of residual nucleic acids was in the range of 10 to 20 ng. Taking the titers into account, the P/I ratio for a hypothetical prion-specific nucleic acid could be calculated depending upon the assumed size. Plots are shown in Fig. for the KK-D6, KK-D8, and KK-D9 samples. The line for a P/I ratio of 1 represents the most extreme assumption: one molecule is sufficient for infection. Nucleic acids below the line cannot be essential for infectivity, whereas those above the line cannot be excluded. Although the specific infectivity of retentates after ultrafiltration was approximately 100-fold lower than those from sucrose gradient fractions, the P/I ratios for these experiments were similar. The maximum lengths of putative prion-specific nucleic acids were 45, 54, and 129 nucleotides.
FIG. 2. Particle-to-infectivity ratio plotted as a function of the average length of the residual nucleic acid. (A) Three independent preparations, KK-D6 (black squares); KK-D8 (gray squares), and KK-D9 (white squares), were analyzed, which were prepared according (more ...)
The above calculations assume that all the prions and accompanying nucleic acids remain in the brain of the bioassay animals and initiate infection. Assuming the P/I ratio is not altered upon inoculation, the clearance of prions from the brain will affect the maximum length of the potential prion-specific nucleic acid. As described below, the size of such putative nucleic acids must be adjusted to reflect the clearance from the brain.
Sucrose gradient fraction analysis for nucleic acid.
Encouraged by the foregoing results using the retentate obtained by ultrafiltration, we repeated the nucleic acid degradation procedures using sucrose gradient fractions with high specific infectivity. Since sucrose gradient fractions contain ≈50% sucrose, aliquots were diluted twofold. Both Zn2+-mediated hydrolysis and benzonase digestion were carried out in the presence of 25% sucrose (samples KK-G1 to -G4). Under these conditions, we found that Zn2+ seems to precipitate nucleic acids and thus protects them from further degradation. Based on this finding, we omitted the Zn2+ hydrolysis from subsequent studies. RRGE analysis of the sample labeled KK-G5 is shown in Fig. and the P/I ratios for samples KK-G5 and -G6 experiments are depicted in Fig. . The maximum length of a putative prion-specific nucleic acid in six samples (KK-G1 to KK-G6) was between 53 and 116 nucleotides.
We next compared the prion infectivity in purified fractions derived from sucrose gradient ultracentrifugation and from ultrafiltration. Each step used to reduce nucleic acid in these purified fractions is listed in Table along with the prion titers as measured by bioassay in rodents. Prions in fractions purified by sucrose gradient centrifugation behaved quite differently from those purified by ultrafiltration with respect to infectivity after exposure to Sarkosyl or ethanol. While the total infectivity of the retentate fraction could be reactivated after DLPC formation, the titers of the sucrose gradient fraction continuously declined. This finding contrasts with earlier data in which infectivity in sucrose gradient fractions purified by the “standard protocol” increased ≈10-fold upon dispersion into DLPCs (22
Changes in infectivity during purification for sucrose gradient and retentate fractionsa
Clearance of prions upon inoculation into brain.
The P/I ratio for PrPSc
molecules per ID50
unit was originally estimated using fractions purified by sucrose gradient centrifugation (60
). That analysis indicated that the P/I ratio is ≈105
. This estimate assumed that all of the PrP 27-30 intracerebrally inoculated into bioassay rodents gave rise to prion infectivity; in other words, all of it was retained in the brain.
In the studies presented here on the kinetics of PrPSc
retention in the brain after intracerebral inoculation of Syrian hamsters, we found that less than 10% of PrP 27-30 in the injected inoculum remained in the brain. This result should not have been surprising since earlier studies on India ink particles and bacteriophage showed similar levels of retention in the brains of mice (13
Groups of eight Syrian hamsters were inoculated intracerebrally with Sc237 prions in SHa brain homogenate that contained ≈107.1 ID50 units per 50-μl dose. The hamsters were euthanized at various times after inoculation and their brains removed and homogenized in PBS to create a 10% (wt/vol) suspension. The level of prions in homogenate at each time point was measured by bioassay and level of rPrPSc determined by the CDI.
During the first 24 h after intracerebral inoculation, prion infectivity in the brain declined ≈99% and the level of rPrPSc decreased ≈96% (Fig. ). By 96 h, the levels of infectivity and rPrPSc were increasing; this indicates that accumulation of apparently newly formed rPrPSc had begun.
FIG. 3. Parallel clearance of infectivity and levels of rPrPSc after intracerebral inoculation of Sc237 prions in Syrian hamsters. An infectivity titer of 107.05 ID50 units was injected. The data points for infectivity are means ± standard error of the (more ...)
Whether the PrPSc detected at 24 h is a mixture of newly formed and inoculated rPrPSc molecules is unclear. Assuming that all of the rPrPSc in the brain after 24 h comes from the inoculum, we estimate that only 4% of the inoculated rPrPSc remains at this time. The 50-μl inoculum contained 72 ± 3 ng of rPrPSc while the brains (≈1 g) of hamsters sacrificed 24 h after inoculation contained 2.9 ± 2 ng of rPrPSc, as determined by the CDI.
If we assume that between 96 and 99% of the prions in the inoculum exit from the brain within the first 24 h after inoculation, then it is reasonable to reduce the P/I ratio by a factor between 25 and 100. After recalculation, the P/I ratio for rPrPSc molecules per ID50 unit is between 1,000 and 4,000.
Similarly, the number of potential nucleic acid molecules per infectious unit has to be corrected for the clearance effect. If we assume that the residual nucleic acids in the purified inoculum exit from the brain during the first 24 h at the same rate as rPrPSc, then we must adjust the calculation of the maximum size of a polynucleotide present in our purified fractions at an abundance of one per ID50 unit. The interpolating straight lines in Fig. have to be shifted to account for the clearance of 96% of the inoculum (Fig. ). Based on these plots, we estimate that the maximum length of a putative, prion-specific polynucleotide is 25 nucleotides.
FIG. 4. Effect of clearance on the nucleic acid particle/infectious unit ratio in dependence upon the average length of the residual nucleic acid. According to 96% clearance (cf. Fig. ), all original data in Fig. are reduced to (more ...) UV irradiation at 254 nm.
Homogenates and microsomes harboring the Sc237 and 139H strains were irradiated with UV light (254 nm) up to 1,200 kJ/m2. Prion inactivation was measured by bioassay. The dose-effect curves with a logarithmic scale for the titer were nonlinear, whereas a linear relationship is expected for a simple, one-hit inactivation mechanism (Fig. ). After an initial decrease up to about 50 kJ/m2, the curves nearly level off.
FIG. 5. UV inactivation of two scrapie strains and a plasmid as a control. (A) Summary of infectivity data for strains Sc237 and 139H after irradiation up to 239 kJ/m2. (B) Dose reduction from shielding of microsomes and homogenates as used in panel A, analyzed (more ...)
We observed two effects that might be responsible for the nonlinearity. First, the solutions exhibited a fairly high optical density, i.e., the homogenate was even-colored. Due to the high optical density, the samples could not be irradiated homogeneously, which could have been overcome by stirring the sample, but this was not performed for technical reasons. This feature led to particular problems in connection with a second effect, the insolubility of the prion preparations, because the infectious material sedimented during irradiation into regions of higher optical density and consequently lower irradiation intensity. If one takes into account that the high doses were achieved due to long irradiation times, the lower efficacy of the irradiation after 1 h can be explained.
A control experiment was performed measuring damage to the restriction sites in plasmids. The results of this study support the interpretation given above. Plasmid damage was studied in buffer and compared to plasmids added to brain homogenate and microsome fractions, which were prepared like those used in Fig. . The homogenate shielded the plasmids from the UV irradiation by a factor of ≈5, whereas the shielding by microsomes was smaller (Fig. ). We were unable to interpret these results quantitatively, and the UV optical density of the homogenate or microsome fraction might vary too much from preparation to preparation for us to be able to correct its influence on the data in Fig. . Control experiments showed that prolonged irradiation of homogenate or microsomes was difficult to evaluate quantitatively.
If the dose-effect curves were evaluated for only up to 1 h of irradiation time, more reliable results were obtained (Fig. ). These data are affected by a relatively high error rate of every single point, but the errors did not depart from a linear relationship. Fitting the data results in D37
values between 8 and 24 kJ/m2
for the two strains; moreover, no difference in resistance to inactivation by UV irradiation at 254 nm could be detected between the two strains. The range of D37
values is similar to those two of us (J.E.C. and S.B.P.) reported in an earlier study in which the D37
values varied from 17 to 22 kJ/m−2
). In Fig. , the incubation times are depicted as obtained directly from the experiments.