The Extended Cell Panel Assay Characterizes the Relationship ofPrion Strains RML, 79A, and 139A and Reveals Conversion of 139Ato 79A-Like Prions in Cell Culture

Anja M. Oelschlegel,a Mohammad Fallahi,b Shannon Ortiz-Umpierre,c and Charles Weissmanna

Department of Infectology,a IT Services,b and Animal Resources Centre,c Scripps Florida, Jupiter, Florida, USA

Three commonly used isolates of murine prions, 79A, 139A, and RML, were derived from the so-called Chandler isolate, whichwas obtained by propagating prions from scrapie-infected goat brain in mice. RML is widely believed to be identical with 139A;however, using the extended cell panel assay (ECPA), we here show that 139A and RML isolates are distinct, while 79A and RMLcould not be distinguished. We undertook to clone 79A and 139A prions by endpoint dilution in murine neuroblastoma-derivedPK1 cells. Cloned 79A prions, when returned to mouse brain, were unchanged and indistinguishable from RML by ECPA. How-ever, 139A-derived clones, when returned to brain, yielded prions distinct from 139A and similar to 79A and RML. Thus, when139A prions were transferred to PK1 cells, 79A/RML-like prions, either present as a minor component in the brain 139A popula-tion or generated by mutation in the cells, were selected and, after being returned to brain, were the major if not only componentof the population.

Prions, the causative agents of transmissible spongiform en-cephalopathies, consist mainly or entirely of aggregated iso-

forms of the normal host protein PrPC, designated PrPSc. PrPSc

may occur in a proteinase K (PK)-resistant form, designatedrPrPSc or PrPres, or in a PK-sensitive form, sPrPSc or PrPsen. Inter-estingly, prions may present in the form of different strains, whosePrPSc differ in regard to conformation but not to amino acid se-quence (reviewed in reference 21).

Three commonly investigated isolates of murine prions—79A,139A, and RML—were derived from populations that originatedwhen prions from pooled scrapie-infected sheep brains (SSBP/1)were passaged through goats (the “drowsy goat” line) into mice.Because these isolates were never directly obtained from scrapie-infected sheep, they are believed to have originated in the goatsused for transmission, which suffered from unrecognized “goatscrapie” (5). One goat-to-mouse transfer experiment gave rise to79A prions, and another gave rise to the so-called Chandler iso-late, from which both 79A and 139A prions were obtained (5). TheChandler isolate, transferred to the Rocky Mountain Laboratoryby W. J. Hadlow in the early 1960s, gave rise to the (uncloned)RML isolate, believed to be the same as 139A (11; R. Kimberlin,personal communication). 79A and 139A were classically distin-guished by their very different incubation times in VM (Prnb)mice, but to our knowledge, RML was not characterized by theincubation time method.

The cell panel assay (CPA) distinguishes RML, 22L, ME7, and301C prions by their differential ability to chronically infect thefour cell lines PK1, R33, CAD5, and LD9 (17). The discriminatorypower of the CPA has been potentiated by the introduction of thestrain- and cell line-specific inhibitors swainsonine (Swa), kifu-nensine (Kifu), and castanospermine (1). The resulting extendedcell panel assay (ECPA) has enabled us to demonstrate that, con-trary to previous belief, RML and 139A prions are distinct entities,while 79A and RML prions are not distinguishable. Moreover, wefound that transferring 139A prions from mouse brain to the N2aneuroblastoma PK1 cell line and back to mouse brain reproduc-

ibly gave rise to prions distinct from 139A but indistinguishablefrom 79A or RML.

MATERIALS AND METHODSCells. The isolation of N2a-PK1 cells (here called PK1 cells) (14), CAD5cells (here called CAD cells) (17), LD9 cells (17), and R332H11 cells (18)has been described previously. All cell lines were propagated in OBGS(Opti-MEM [Invitrogen] containing 4.5% bovine growth serum [Hy-Clone, Logan, UT], 50 units penicillin/ml, and 50 �g streptomycin/ml[Invitrogen]). Uninfected cells were maintained for nine serial passages by1:10 splits before being replaced by freshly thawed cells.

Prion strains. RML (RML 1856-II) was obtained from the Prion Unit,University College London; 79A, 139A, and 22L were received from theTSE Resource Centre, Compton, Newbury, United Kingdom. All prionstrains were propagated in C57BL/6 mice (from Charles River Laborato-ries, Wilmington, MA).

Prion propagation in mice. Mice were anesthetized by isoflurane in-halation and inoculated in the prefrontal cortex with 20- to 30-�l samples.In some experiments, volumes or concentrations were adjusted to deliversimilar amounts of infectivity (determined by standard scrapie cell assay[SSCA] on CAD cells) or of PrPres (determined by Western blot analysis).Clinical signs of disease were cessation of nesting, ruffled coat, lateraldeviation with medial pronation of hind limbs, hind limb weakness, my-oclonus, urinary incontinence with lesions in the vaginal area, hunchedback, decreasing activity with increasing periods of lethargy, weight loss,squinty eyes, bruxing, shivering, or trembling.

When clinical signs reached the terminal stage, the animals were eu-thanized by CO2 asphyxiation followed by cervical dislocation. The brainswere collected, and 10% homogenates in phosphate-buffered saline (PBS)were prepared as described previously (18).

Received 22 January 2012 Accepted 22 February 2012

Published ahead of print 29 February 2012

Address correspondence to Charles Weissmann, charlesw@scripps.edu.

Copyright © 2012, American Society for Microbiology. All Rights Reserved.

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Concentrated CM. Prions secreted by infected cells were recoveredfrom conditioned medium (CM). It has been previously shown that theCPA results obtained with prions from cell lysates and those obtained withprions from CM are similar (see supporting online material in reference15). CM was cleared at 500 � g for 5 min, and prions were ultracentri-fuged for 2 h onto a 10-ml sucrose cushion (20% sucrose [wt/wt] in 1�TNE buffer [25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA]), ina Ti45 rotor (Beckman Coulter) at 35,000 rpm and 4°C. The resultingpellet was suspended in OBGS to a 100� to 300� concentration of theoriginal volume.

SSCA. Typically, six serial 1:5 dilutions of the prion preparation (brainhomogenate or concentrated conditioned medium) were added in tripli-cate to 96-well plates and 5,000 cells were added to each well. Triplicatewells with uninfected cells served as background control; another set oftriplicates contained the highest concentration of inoculum used and 10�g pentosan polysulfate/ml (Bene PharmaChem GmbH & Co. KG,Geretsried, Germany) to inhibit prion replication (3) and to assess thepossible persistence of the inoculum. After 4 days, the cells were split 1:5 to1:8, depending on their growth rate. After reaching confluence followingthe third split, 20,000 cells/well were transferred into wells of preactivatedMultiscreen IP 96-well 0.45-�m filter plates (Millipore). Supernatantswere drained by vacuum, the plates were dried at 50°C for at least 1 h, and

the samples were subjected to the PK– enzyme-linked immunosorbentspot (ELISPOT) assay directly or after storage at 4°C.

PK-ELISPOT assay. Samples were incubated for 90 min at 37°C with70 �l of 1 �g proteinase K (Roche)/ml lysis buffer (50 mM Tris-HCl, pH8.0, 150 mM NaCl, 0.5% sodium deoxycholate, 0.5% Triton X-100). Allfurther steps were carried out at room temperature. The samples werewashed twice with PBS and denatured with 120 �l of 3 M guanidiniumthiocyanate in 10 mM Tris-HCl, pH 8.0, for 10 min. After four washeswith distilled water (dH2O), samples were incubated for 1 h with 0.5%nonfat dry milk in TBS (10 mM Tris-HCl, pH 8.0, 150 mM NaCl), fol-lowed by 1 h of incubation with 70 �l of 0.7 �g humanized anti-PrPantibody D18 (22)/ml of 1% nonfat dry milk in TBST (10 mM Tris-HCl,pH 8.0, 150 mM NaCl, 0.1% Tween 20). After four washes with TBST, 70�l of alkaline phosphatase (AP)-conjugated anti-IgG (1:5,000; SouthernBiotechnology Associates, Birmingham, AL) in 0.5% nonfat dry milk inTBST was applied for 1 h. Wells were washed four times with TBST. Then,the whole plate was immersed once in TBS and dried. Signals were visu-alized with the AP conjugate substrate kit (Bio-Rad), and PrPres-positivecells (“spots”) were counted using the Bioreader 5000-Eb cytometer (Bio-Sys).

ECPA. Prion strains can be distinguished by their specific ability toinfect a panel of different cell lines, as monitored by the SSCA (17). Prion-

TABLE 2 Cloning of 79A and 139A prions in PK1 cells

Straina Dilution (109)No. ofcells/well

No. of positivewells/total no.of wellsb Fraction positive P(0)

c md mCe (104)

P(n � 1)f

(104)

79A 1 100 11/240 0.046 0.954 0.047 4.7 110.5 100 4/252 0.016 0.984 0.016 1.6 1.3

139A 1 100 11/240 0.046 0.954 0.047 4.7 110.5 100 7/252 0.028 0.972 0.028 2.8 3.90.5 100 3/462 0.0065 0.994 0.007 0.65 0.21

a PK1 cells were seeded at 100 cells/well in 96-well plates and inoculated with estimated endpoint dilutions of 79A or 139A brain homogenates (10�9 and 5 � 10�10). The cells werepropagated for about 50 doublings, and PrPres-positive cells were determined by the PK-ELISPOT assay.b Wells giving spot numbers of � (background plus 5 SDs) were scored as positive. The probability that a well was infected by more than one prion, P(n � 1) � 1 � P(0) � P(1) �1 � e�m (1 � m), is derived from the Poisson distribution Pn � (e�m · mn)/n!, where m is the average number of prions/well and Pn is the probability of n prions/well.c P(0) � 1 � (positive wells/total wells).d m � ln[1/P(0)] � average number of prions/well.e mC � m/100 � average number of prions/cell.f P(n � 1) � 1 � e�m (1 � m).

TABLE 1 Prion characterization by the ECPAd

Inoculuma

Log RIb Log[RICAD/RIX] Log[RIPK1/RIX]

CAD LD9 2H11 PK1PK1 �Swa

PK1 �Kifu LD9 2H11 PK1

PK1 �Swa

PK1 �Kifu

PK1 �Swa

PK1 �Kifu

C57/PK1[79A] clone 11 6.72 4.62 ��3 5.12 �3.36 ��3 2.1 ��3.77 1.59 �3.35 ��3.77 �1.76 ��2.12C57/PK1[79A] clone 14 6.83 4.5 ��3 4.92 3.25 ��3 2.33 ��3.83 1.9 3.58 ��3.83 1.67 ��1.92C57/PK1[139A] clone 15 6.87 4.55 ��3 4.8 3.13 ��3 2.32 ��3.87 2.07 3.74 ��3.87 1.67 ��1.80C57/PK1[139A] clone 18 6.77 4.63 ��3 4.88 �3.23 ��3 2.14 ��3.77 1.88 �3.54 ��3.77 �1.66 ��1.88C57[RML] 6.84 4.43 ��3 4.68 �3.26 ��3 2.42 ��3.84 2.16 �3.58 ��3.84 �1.41 ��1.68C57[79A]c 6.83 4.42 ��3 4.93 3.28 ��3 2.41 ��3.83 1.9 3.55 ��3.83 1.66 ��1.93C57[139A] 6.79 4.38 ��3 4.84 3.8 4.65 2.41 ��3.79 1.96 3 2.14 1.04 0.18C57[22L] 6.31 5.7 5.38 5.71 5.62 5.22 0.61 0.93 0.61 0.69 1.09 0.08 0.48a The SSCA was performed on CAD, LD9, R332H11, and PK1 cells, the latter in the absence or presence of swainsonine (1 �g/ml) or kifunensine (5 �g/ml). The proportion ofPrPres-positive cells was plotted against the logarithm of the inoculum dilutions as in Fig. 1.b RIs are the reciprocals of the dilutions required to yield 750 PrPres-positive cells per 20,000 cells. The ratio of RIs on pairs of cell lines (or the log of the ratios) characterizes the celltropism pattern of a prion strain. The table shows the averages (geometric means) of log RI values and log[RI ratios] determined in triplicate.c One of the curves for C57[79A] in PK1 cells failed to reach 750 spots; calculations for this sample were therefore only approximate and the assay was not used for statisticalevaluation.d Swa, swainsonine; Kifu, kifunensine.

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FIG 1 Prion characterization by the ECPA. CAD cells (red), PK1 cells (blue), LD9 cells (purple), R332H11 cells (green), and PK1 cells in the presence ofswainsonine (dashed light blue) or kifunensine (dashed gray) were exposed to serially diluted prion samples as indicated. The proportion of PrPres-positive cellswas plotted against the logarithm of the inoculum dilutions. The cells’ response to a prion sample is defined by the response index (RI), the reciprocal of thedilution required to yield an arbitrary number, here 750, of infected cells per 20,000 cells. The RI ratios or the logarithm of the RI ratios on different cells definesthe cell tropism pattern of the sample. (A) C57[RML] and C57[79A] prions show similar cell tropism patterns; in particular, both strains are unable to infectR332H11 cells (R332H11 incompetent) and are strongly inhibited by swainsonine and kifunensine. C57[139A] prions are also R332H11 incompetent, but they areless inhibited by swainsonine and completely unaffected by kifunensine. For comparison, C57[22L] prions can infect R332H11 cells (R332H11 competent); they areunaffected by swainsonine or kifunensine. 79A and 139A prions that were cloned and propagated in cell culture and then returned to C57BL/6 mice are Swasensitive and kifunensine sensitive; the cell tropism pattern is indistinguishable from that of C57[RML] and C57[79A] but very different from that of C57[139A]prions. Log RIs and log[RI ratios] of the complete ECPA are shown in Table 1. (B) The log[RI ratios] � SDs of PK1 and PK1 plus swainsonine (light blue) andof PK1 and PK1 plus kifunensine (gray) are shown in a bar chart. *, one of the curves representing C57[79A] in PK1 cells failed to reach 750 spots for technicalreasons; calculations for this sample were therefore only approximate and the assay was not used for statistical evaluation.

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and cell line-specific inhibitors like swainsonine, kifunensine, or castano-spermine (1) complement and extend the original panel of cell lines. Inthe work described here, the ECPA includes CAD cells, LD9 cells, R332H11

cells, and PK1 cells, as well as PK1 cells in the constant presence of swain-sonine (1 �g/ml) or kifunensine (5 �g/ml). The cells’ response to prions isdetermined by the response index (RI), the reciprocal of the dilutionrequired to yield a certain number of positive cells (spots) per 20,000 cells.The ratio of RIs is characteristic for different prion strains (Table 1). Forcalculating statistics, the log values of the RIs and their ratios were used.

Endpoint dilution cloning in cell culture. PK1 cells were seeded at100 cells/well in 96-well plates and inoculated with highly diluted 79A- or139A-infected brain homogenates (final concentration, 10�9 and 5 �10�10). The cells were repeatedly grown to confluence and split threetimes 1:3 followed by eight 1:10 splits; after a total of about 50 doublings,20,000 cells from each well were subjected to the PK-ELISPOT assay andsamples containing PrPres-positive cells (spot numbers � [backgroundplus 5 standard deviations {SDs}]) were scored as positive. The probabilitythat under the conditions chosen a well was infected by more than oneprion, P(n � 1) � 1 � e�m (1 � m), where Pn is the probability of findingn prions/well and m is the average number of prions/well, was 10�3 or less,

as shown in Table 2. Five 79A or 139A prion clones were expanded; con-ditioned media were harvested, concentrated, and inoculated intoC57BL/6 mice. The brain homogenates from terminally ill mice werecharacterized by ECPA.

For comparison, uncloned PK1[139A] prions were prepared by inoc-ulating PK1 cells seeded at either 100 cells/well or 5,000 cells/well with lowdilutions of 139A brain homogenate (final concentrations, 5 � 10�5 and4 � 10�5, respectively). After about 50 and 14 cell doublings, respectively,three distinct populations were expanded, concentrated conditioned me-dium was inoculated into mice, and the brains were processed as de-scribed above.

Infectivity determination by SSCA. Infectivity determinations wereperformed by SSCA on CAD cells, relative to control brain homogenate,whose titer was determined by the mouse bioassay (18).

Western blot analysis. To detect PrPres by Western blot analysis, sam-ples (3 mg total protein/ml in PBS, 0.5% Triton X-100) were digested with25 �g proteinase K (Roche)/ml at 37°C for 1 h. Undigested controls wererun in parallel. The digestion was stopped with 20 �l phenylmethylsulfo-nyl fluoride (PMSF) (100 mM)/ml, and the samples were denatured bybeing boiled in XT-MES sample buffer (Bio-Rad) at 100°C for 10 min.Electrophoretic separation (4 to 12% Criterion gel; Bio-Rad) and wettransfer (Bio-Rad) to polyvinylidene difluoride (PVDF) Immobilonmembranes (Millipore) were performed by standard procedures. All fur-ther procedures were done at room temperature. Membranes were incu-bated for 1 h in blocking solution (5% nonfat dry milk in PBST [PBS, 0.1%Tween 20]) and immunostained with 0.7 �g humanized anti-PrP anti-body D18 (22)/ml in 1% nonfat dry milk in PBST, followed by threewashes with PBST and 1 h of incubation with horseradish peroxidase(HRP)-conjugated anti-IgG antibody (Southern Biotechnology Associ-

TABLE 3 Inoculation of C57BL/6 mice with brain-derived orcell-propagated 79A and 139A prions

Inoculum Preparation

Doseb (10�2

IU or PrPres

units)DPId

(avg � SD)

No. of miceaffected/no.inoculated

Expt 1a

C57[79A] 10�3 BH 37 160 � 2 4/4C57[139A] 10�2 BH 432 156 � 2 4/4PK1[79A] clone 15 100� CM 3.3 167 � 3 4/4PK1[79A] clone 14 100� CM 104 154 � 12 4/4PK1[79A] clone 13 100� CM 123 156 � 14 4/4PK1[79A] clone 12 100� CM 16 156 � 2 4/4PK1[79A] clone 11 100� CM 78 156 � 3 4/4PK1[139A] clone 12 100� CM 113 156 � 3 4/4PK1[139A] clone 13 100� CM 254 153 � 0 4/4PK1[139A] clone 15 100� CM 303 153 � 8 4/4PK1[139A] clone 18 100� CM 113 149 � 7 4/4PK1[139A] clone 17 100� CM 106 161 � 4 4/4

Expt 2c

C57[139A]BH1 6.9 · 10�4 BH 100 167 � 20 4/4C57[139A]BH2 8.2 · 10�4 BH 99 173 � 11 4/4PK1[139A]2F3 50� CM 57 157 � 8 4/4PK1[139A]6F11 50� CM 197 148 � 11 4/4PK1[139A]1D6 50� CM 42 156 � 1 4/4PK1[139A]wp/5000 50� CM 67 157 � 14 4/4PK1[139A]wp/strong 50� CM 128 161 � 7 4/4PK1[139A]wp/weak 50� CM 4 158 � 9 4/4PK1-1 (uninfected

cells)50� CM �436 0/4

PK1-2 (uninfectedcells)

50� CM �436 0/4

a C57BL/6 mice were inoculated intracerebrally with either 20 �l homogenate of brains(BH) infected with “authentic” brain-derived 79A or 139A (C57[79A] or C57[139A])or with 20 �l of 100-fold-concentrated conditioned medium (CM) of PK1 cells infectedwith endpoint-diluted 79A or 139A prions (PK1[79A]cloneX and PK1[139A]cloneY),respectively.b For experiment 1, infectivity was determined by the SSCA on CAD cells. Doses areshown as infectivity units (IU). For experiment 2, doses are shown as arbitrary PrPres

units, determined by Western blot assay.c C57BL/6 mice were inoculated with 139A brain homogenates (C57[139A]BH1 andC57[139A]BH2) or with 50-fold-concentrated conditioned medium (CM) fromdifferent lines of 139A-infected PK1 cells: PK1[139A]2F3, PK1[139A]6F11, andPK1[139A]1D6 were infected with endpoint-diluted 139A and represent 139A cloneswhile PK1[139A]wp/5000, PK1[139A]wp/strong, and PK1[139A]wp/weak were infected athigh multiplicities and represent 139A populations (wp, whole population); CMs fromuninfected PK1 cells were injected as negative controls.d DPI, days after inoculation, when terminal stage was reached.

TABLE 4 139A prions cloned in PK1 cells and passaged through braindiffer from the original 139A prions but are indistinguishable frombrain-derived 79A or 79A prions cloned in cell culture and thenpassaged through brain

Inoculuma

Log RIPK1

LogRIPK1�Swa

Log[RIPK1/RIPK1�Swa] SDAvg SDb Avg SD

C57[79A] 5.91 0.11 3.73 0.16 2.18 0.19C57[139A] 5.41 0.30 4.46 0.26 0.95 0.40C57/PK1[79A] clone 15 6.00 0.03 3.81 0.13 2.18 0.13C57/PK1[79A] clone 14 6.03 0.10 3.68 0.35 2.35 0.36C57/PK1[79A] clone 13 5.68 0.07 3.63 0.12 2.05 0.14C57/PK1[79A] clone 12 5.92 0.03 3.66 0.08 2.25 0.09C57/PK1[79A] clone 11 6.05 0.18 3.95 0.18 2.10 0.25C57/PK1[139A] clone 12 5.82 0.19 3.73 0.13 2.09 0.23C57/PK1[139A] clone 13 6.12 0.17 3.93 0.15 2.19 0.23C57/PK1[139A] clone 15 5.83 0.08 3.78 0.12 2.05 0.15C57/PK1[139A] clone 18 5.75 0.14 3.25 0.17 2.51 0.22C57/PK1[139A] clone 17c 5.61 0.17 3.07 0.03 2.55 0.17

a 79A and 139A prions were cloned by endpoint dilution in PK1 cells. Five clones ofeach were expanded; concentrated conditioned medium was prepared from each andinoculated into C57BL/6 mice. Brains were harvested from terminally ill mice, and theprions were characterized by triplicate SSCA on PK1 cells in the absence or presence of1 �g swainsonine (Swa)/ml.b RIs are the reciprocals of the dilutions required to yield 750 PrPres-positive cells per20,000 cells. Average log RI is the average (geometric mean) of the log RI values; SD isthe standard deviation. Inhibition is expressed as the logarithm of the ratio of averageRIs, in the absence and presence of swainsonine, in the column“Log[RIPK1/RIPK1�swa]” � SD. One-way analysis of variance (ANOVA) with Tukey’smultiple comparison test of the log[RI ratios] � SDs showed that C57[139A] prionsthat had been passaged in PK1 cells and subsequently in C57BL/6 mice weresignificantly (***, P � 0.001) more susceptible to inhibition by swainsonine than werethe original C57[139A] prions and thus resembled C57[79A] and C57/PK1[79A] in thatway.c One of the three curves representing C57/PK1[139A] clone 17 in PK1 plusswainsonine did not reach 750 spots. Calculations for this sample were therefore onlyapproximate and were not statistically evaluated.

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ates, Birmingham, AL; 1:15,000 in 0.5% nonfat dry milk in PBST). Afterthree washes with PBST, chemiluminescence was induced by ECL-Plus(Pierce) and recorded by charge-coupled device (CCD) imaging (Bio-Spectrum AC Imaging System; UVP).

RESULTS

Many murine prion strains used in laboratories throughout theworld were cloned by endpoint dilution in mice at the Neuro-pathogenesis Unit of the Institute for Animal Health (NPU). Inthis procedure, mice are injected with prion preparations dilutedto the extent that only 1 in 10 or more mice becomes infected, andthis procedure is repeated once or twice more. The strains 79A and139A were derived from the so-called Chandler isolate, whichoriginated from scrapie-infected goats; 79A, but not the 139A thatwe used, had been cloned at the NPU in Edinburgh, United King-dom (H. Baybutt, personal communication). RML was also de-rived from the Chandler isolate and propagated for many decades

in the Rocky Mountain Laboratory but was never cloned. There arecontradictory opinions as to whether or not RML is similar or iden-tical to 79A or 139A; several authors consider RML similar to oridentical with 139A (11, 19), while Tremblay et al. (20) found differ-ent PrPres brain deposition patterns for these two strains.

We compared RML, 139A, and 79A prions by the ECPA andfound that, as described earlier (17), all three strains could chron-ically infect CAD, PK1, and LD9 cells but not R332H11 cells. How-ever, swainsonine (1 �g/ml) and kifunensine (5 �g/ml) stronglyinhibited chronic infection of PK1 cells by both RML and 79Aprions, while infection by 139A was less inhibited by swainsonineand almost unaffected by kifunensine (Fig. 1; Table 1). Thus, 139Aand RML are clearly different, while 79A and RML were indistin-guishable by the criteria that we used.

We next undertook to clone 79A and 139A by endpoint dilu-tion in cultured cells rather than in mice. PK1 cells were distrib-

FIG 2 “Authentic” brain-derived 139A prions differ from 139A prions propagated in PK1 cells and returned to brain. (A) The averages (geometric means) � SDsof the log[RI ratios] of PK1 and PK1 plus Swa of three C57[RML] and four C57[79A], C57[139A], and C57/PK1[139A] brain homogenates were used to analyzeand compare the different prion samples by t test evaluation: the inhibitory effects of swainsonine on prion replication in PK1 cells were the same for C57[79A],C57[RML], and C57/PK1[139A] prions but were significantly different from that on C57[139A] prions. (B) The log[RIPK1/RIPK1�Swa] values for clonedC57/PK1[139A]clone and uncloned C57/PK1[139A]wp were indistinguishable but differed significantly from that of “authentic” C57[139A]. See Table 5 fordetails. ns, not significant.

Prion Strain Switch in Cultured Cells

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uted into 96-well plates at 100 cells/well, infected with 79A or139A brain homogenate at a high dilution (10�9 or 5 � 10�10),and propagated for about 50 doublings, and the individual wellswere assayed by PK-ELISPOT assay. Table 2 shows that theaverage number of prions per cell in the various experimentsranged from 0.65 � 10�4 to 4.7 � 10�4 and that the probabilitythat a well was infected by more than one prion was between10�3 and 10�4 for 79A and between 10�3 and 10�5 for 139A.

C57BL/6 mice inoculated intracerebrally (i.c.) with concen-trated conditioned medium of 79A- or 139A-infected PK1 clones(designated PK1[79A] and PK1[139A], respectively) (Table 3 )succumbed to scrapie disease with the symptoms usually seen inanimals infected with RML, 79A, or 139A (see Materials andMethods). The ECPA patterns of brain homogenates from miceinoculated with original brain-derived C57[79A] and from miceinoculated with PK1[79A]-derived prion populations were indis-tinguishable (Fig. 1 and Tables 1 and 4). Unexpectedly, however,the brains infected with conditioned medium of PK1-passaged139A (C57/PK1[139A] prions) presented an ECPA pattern thatwas distinctly different from that of the original brain-derived139A (C57[139A]) prions: their propagation on PK1 cells wasstrongly inhibited by kifunensine, while that of the originalC57[139A] prions was virtually unaffected (Fig. 1; Table 1). More-over, C57/PK1[139A] prions showed enhanced Swa sensitivitycompared to that of C57[139A] prions (Fig. 1 and 2; Tables 1, 4,and 5). Thus, the 139A prions that had been cloned in PK1 cellsand subsequently propagated in brain differed from the original139A prions and resembled brain-derived RML or 79A prions. Toascertain whether the clones that we picked might have been ab-errant, we infected PK1 cells with high multiplicities of C57[139A]prions, expanded three distinct cell populations, and inoculatedfour mice each with conditioned media of the resulting prion pop-ulations (Table 3). As in the first experiment, the C57/PK1[139A]prions resembled RML or 79A prions and not 139A prions (Fig.2B and Table 5).

DISCUSSION

The ECPA clearly shows that RML and 139A strains are distinctentities but does not distinguish between RML and 79A, whichmay be the same or not. In view of our finding that 139A prionsswitch to a strain indistinguishable from 79A or RML when pas-saged through PK1 cells and returned to mouse brain, it seemsprobable that the Chandler sample transferred to the RockyMountain Laboratory—whatever it originally contained— endedup consisting of 79A prions, either because these were selectedfrom a mixture of 79A and 139A or because 139A converted to79A in the course of multiple vertical transfers. Conversion of139A to 79A has been mentioned, but the details are not accessibleto us (6), as quoted in references 12 and 8; Dickinson and Outramconsidered “139A” a mixture of strains (7). Although 139A hasbeen cloned by endpoint dilution in mice (13), the sample that weobtained was uncloned.

It was recognized early on that prion strains can adapt to newhosts as a consequence of mutation and selection (2, 12, 13), mu-tation at that time being considered a change in the nucleic acidsequence of a virus-like entity, rather than a conformationalchange of PrPSc, as it is now (4, 21). More recently, it was shownthat prions can undergo adaptation even when transferred fromone cell type to another within the same species (15, 18, 21). Thus,when 22L prions were transferred from brain to PK1 cells, they

gradually changed their properties, losing their ability to infectR33 cells and becoming sensitive to inhibition by swainsonine;these changes were reversed when the prions were returned tobrain (15, 16, 18). We believe that prion populations are quasispe-cies (9, 10), consisting of a multitude of variants, the ones fittestfor a particular environment predominating (4, 15, 21), and wehave shown that variants arise in cloned prion populations in thecourse of propagation (15, 16, 18), explaining how quasispeciesarise.

Why would 139A propagate stably in brain but, after beingpassaged through PK1 cells and returned to brain, propagate sta-bly as 79A/RML-like prions? We consider two possibilities. (i) The139A population contains low levels of 79A/RML prions, which,upon transfer to PK1 cells, are selected to the exclusion of 139A

TABLE 5 Comparison of “authentic” RML, 79A, and 139A and 139Apropagated in PK1 cells and returned to brain

Inoculum SampleAvg logRIPK1

bAvg logRIPK1�Swa

Log[RIPK1/RIPK1�Swa]

Avg log[RIPK1/RIPK1�Swa] SD

Expt 1a

C57[RML] a 5.71 4.03 1.68 1.73 0.21b 4.96 3.41 1.55c 5.32 3.36 1.96

C57[79A] a 5.13 3.40 1.73 1.65 0.14b 5.01 3.46 1.55c 5.70 4.17 1.52d 6.07 4.26 1.81

C57[139A] a 5.17 4.07 1.10 0.83 0.25b 5.11 4.33 0.77c 4.83 3.90 0.92d 5.58 5.06 0.51

C57/PK1[139A] a 4.90 3.15 1.75 1.76 0.12b 5.28 3.50 1.78c 5.44 3.53 1.91d 5.12 3.51 1.61

Expt 2c

C57[139A] a 5.79 4.75 1.05 1.06 0.15b 6.28 5.24 1.05c 6.48 5.25 1.22d 5.97 5.15 0.83e 6.33 5.18 1.15

C57/PK1[139A]clone a 5.91 4.56 1.35 1.51 0.11b 5.86 4.41 1.45c 6.43 4.89 1.53d 6.35 4.74 1.61e 6.53 4.91 1.62

C57/PK1[139A]wp a 6.21 4.73 1.48 1.53 0.05b 6.30 4.73 1.57c 6.55 5.00 1.55

a Brain-derived RML (C57[RML]), 79A (C57[79A]), and 139A (C57[139A]) prionswere compared to prions from brains infected with 139A prions that had beenpropagated in PK1 cells (C57/PK1[139A]) by SSCA in triplicate on PK1 cells with orwithout 1 �g swainsonine (Swa)/ml.b The RI was determined at 500 PrPres-positive cells/20,000 cells and expressed as logRI. Avg log RI, averages (geometric means) of the triplicate log RI values. Theinhibitory effect of swainsonine on the prion replication in PK1 cells is expressed aslog[RIPK1/RIPK1�Swa] or averaged (geometric mean) from the four brains as “Avglog[RIPK1/RIPK1�Swa] � SD.” The values are the same for C57[RML], C57[79A], andC57/PK1[139A] prions but significantly lower for C57[139A] prions (Fig. 2A).c “Authentic” brain-derived C57[139A] prions were compared to C57/PK1[139A]prions, either cloned in PK1 cells by endpoint dilution and returned to brain orpropagated as uncloned whole population (wp) in PK1 cells and then returned to brain.Calculations were performed as described above. The inhibitory effects of swainsonineon the prion replication in PK1 cells are the same for cloned and unclonedC57/PK1[139A] prions, but they are significantly different from those for C57[139A]prions (Fig. 2B).

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prions. (ii) The 139A population propagates stably in brain be-cause it neither contains 79A/RML contaminants nor gives rise tothem by mutation; however, while mutation from 139A to 79A/RML-like prions in brain may be precluded by an insurmountablefree energy barrier, this can be overcome in PK1 cells, where 79A/RML-like prions can arise and be selected. In either case, PK1 cellswould provide a potent selection of 79A/RML-like prions over139A prions.

Conversion of 79A to 139A prions in mouse brain has not beendescribed in the literature and may not occur, either because thefree energy barrier is too high or because any 139A arising is out-competed by 79A.

As mentioned above, 22L prions adapt to cells but, when re-turned to brain, give rise to prions indistinguishable from theoriginal 22L (15, 18). An important conclusion from the workreported here is that transfer of prions from brain to a cell line andback can also result in a permanent strain change, so that theattractive strategy of endpoint cloning of prion strains in cells isnot a viable option.

ACKNOWLEDGMENTS

We thank Alexandra Sherman for training and initial assistance with theanimal work and Emery Smith, Irena Suponitsky-Kroyter, Jason Tread-away, and Joseph Jablonski for assistance with cell splitting.

This work was supported by grants from the National Institutes ofHealth (1RO1NSO59543 and 1RO1NS067214) and from the Alafi FamilyFoundation to C.W.

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