Properties of the scrapie agent-endomembrane complex from hamster brain.

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Properties of the Scrapie Agent-Endomembrane Complex

from

Hamster Brain

J. S. SEMANCIK,* R. F. MARSH, J. L. M. C. GEELEN, AND R. P. HANSON

Departments ofPlant Pathology and CellInteraction Group, University of California, Riverside, California 92502,*and Department of Veterinary Science, University of Wisconsin, Madison, Wisconsin 53706

Received for publication 2 September 1975

Subcellular fractionation of scrapie-infected hamster brain indicatedthe

asso-ciation of the scrapie agent with a component of the endomembrane system.

Characterization by equilibrium density gradient centrifugation, electron

mi-croscopy, and markerenzymessuggestedaprimary association with rough and

smooth endoplasmic reticulum and a possible incorporation into the plasma

membrane. DNA polymerase activity demonstrated a direct correlation with

regions of scrapie activity from thegradientfractions. Ascrapie-related product

wasdetected after [3H]TMP incorporationand analysison 2.2% polyacrylamide

gels. Analysis of nucleic acid species extracted from subcellular fractions

re-sulted in a greater quantity fromhealthybrain; however, noqualitative

distinc-tions weredetected.

Thetransmissible agent of scrapie disease of

sheep has unusualproperties ofthermal

stabil-ity (8) and a small radiation target size of 2 x 105 daltons (2). Similar characteristics have also been reportedfor"viroid"agentsproducing infection ofplants (5, 18-20). No nucleic acid

species analogousto the low-molecular-weight

pathogenic RNA, characterized as the causal

agentofviroid diseases (21), has beenisolated

from scrapie-infected tissue or any other

ani-mal system (16). Nevertheless, high titers of

biologicallyactivescrapieagent canbe demon-strated ie membranepreparations (17). The

as-sociation of the citrus exocortis viroid with a

componentof theendomembranesystem (J. S.

Semancik, D. Tsuruda, L. Zaner, J. L. M. C. Geelen,and L.G.Weathers,Virology,inpress)

further extended the comparative features of these diseases. The characterization of the

scra-pie agent-membrane complex from hamster

brain, employing procedures comparable to

those utilizedinviroid characterization, is the subject of this report.

MATERIALS AND METHODS

Scrapie agents. The scrapie agent usedin these

studies originated from the Chandler strain of

mouse-adapted scrapieasdescribedpreviously (11).

Fractionationexperiments wereperformedonpools

(5 g each) of fresh brain tissue representing the

agent's4thand 5th passagesinoutbred(LVG:LAK) Syrian hamsters with intermittent passages in

inbred hamsters (LHC/LAK), both obtained from the Lakeview HamsterColony, Newfield,N.J.

Ad-ditional pools, containing 50 g each offresh brain

from the 6th scrapie passage or from normal

ani-mals, wereused for fractionation followed by nucleic acid extraction.

Extraction and subcellular fractionation proce-dure. Hamsters, 4monthsold, either uninoculated

orshowing early signs of disease, were killed with chloroform, and their brains were rapidly removed and placed on wet ice. Healthy or scrapie-infected brains (2.5 g) were quartered and immediately ho-mogenized on ice (Polytron blender, 15 s, no. 4 set-ting)in10to 15 ml of a medium composed of 0.4M

sucrose,0.01 Mtricine (pH 7.5), 0.002MMgCl2, 0.02

MKCl,0.1%bovine serumalbumin, 0.001 M dithio-threitol, and 0.04Mmercaptoethanol. The extracts were pooledand centrifuged at 250 xg for 10min, and thepellet was discarded.The250-gsupernatant

wassequentially centrifuged at 1,000 xg(fraction 1 pellet) and 13,000 xg (fraction2pellet)inaSorvall RC-2B for10mineach. The 13,000 xgsupernatant

wasthen centrifuged at 80,000xg for 30min (frac-tion3pellet)inaBeckman SW27 rotor. Finally, the supernatant wascentrifuged at 100,000 x gfor2h (fraction4pellet) in a Beckman 35 rotor. The high-speed supernatant constituted fraction5.Allpellets were suspended by three strokes in a Vitro Teflon homogenizer in a medium containing 15% (wt/wt) sucrose,0.01Mtricine(pH 7.5),0.002 MMgCl2,0.02 MKCl,and 0.001 M dithiothreitol.

Density gradient centrifugation. Sucrose

solu-tions were prepared in the resuspension buffer. Samples (4to6ml)werelayeredover alinear(25to

40%, wt/wt) sucrosegradientover 4 mlof50% (wt/ wt) and2mlof60% (wt/wt)sucroseand then

centri-fuged for21 hat 25,000 rpm ina Beckman SW27

rotor at 4 C.Fractions (1to 2ml)werecollectedby needle-puncturing the bottom of the tube, and the density of theconsecutive sampleswasdetermined withanAbberefractometer. Aliquotsof these

sam-pleswereemployed for the enzyme assay,analytical analysis, and scrapiebioassay.

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Analytical and enzymaticdetermination. RNA, DNA, and protein were quantitated by theorcinol, diphenylamine, and Folin tests, respectively (22). The plasma membrane (PM) marker enzyme (ATP-ase) and endoplasmic reticulum (ER) marker

en-zyme (reduced nicotinamide adenine dinucleotide [NADH] cytochrome c reductase) were determined by the method of Hodges and Leonard (9). DNA and RNA polymerase activities were monitored by [3H]TMP and [3H]UMP (J. L. M. C. Geelen, L. G. Weathers, and J. S. Semancik, Virology, inpress) incorporation.

Nucleic acid extraction and gel electrophoresis. Nucleic acid preparations were extracted and parti-tioned as reported previously (17) and analyzedon 5

and 2.2% gels with 0.5% agarose (3). Gels were scanned at 260 nm in a Beckman Acta II spectropho-tometer.

Bioassay of scrapie infectivity. Coded fractions were tested for scrapie infectivity by intracerebral inoculation (0.05 ml) of weanling outbred hamsters. Four animals were inoculated with each dilution, beginningat10-5,and end pointswerecalculatedat

the end of 15 weeksbythe method of Spearman and Karber (7).

Electron microscopy.Gradientsamples of subcel-lular fractions were pelleted and immediately placed in 2% glutaraldehyde in 0.01 M potassium phosphate buffer, pH 7.2. After a 2-h fixation, the pelletswererinsed well with buffer andpostfixedin 1%buffered osmium tetroxide for 90 min.After sev-eralbuffer andwaterrinses, specimens were dehy-drated through anacetone series andembedded in

Epon (14). Thin sections were stained with uranyl

acetateand leadcitrate.Specimenswereviewed and photographed with a Hitachi HU-11 microscope.

RESULTS

Subcellular fractionation of hamster brain.

A differential centrifugation sequence similar

tothat employed for detecting theassociationof

thecitrus exocortis viroidwitha componentof

theendomembranesystem (Semanciketal.,in press) was utilized to process polytron

homoge-nized brain tissue. After the250 x g (10 min)

crude pellet was discarded, the series of

frac-tions wastaken, as presented in Table 1, and

assayed for scrapie activity (SA). The SA

de-TABLE 1. Associationof SA with subcellular fraction from infected hamster brain.

Total recoverableSAa Pellets

Expt1 Expt2 Expt3

(1) 1,000 xg, 10 min 10925 10W75 109 (2) 13,000 xg, 10min 109 108.75 109 (3) 80,000 xg,30 min

109i25

108T75

109 (4) 100,000 xg,2h 108'5 108.25 108.75

(5)Supernatant 10775 108-25 108

a Mean lethaldose per total volume of the

resus-pended pellet.

tected in the three fractions collected between 250 and80,000 xg, containingthe bulk of the

endomembrane system, was usually 1 log

greaterthan the final high-speed pelletor su-pernatant. Whensubjectedtoequilibrium

cen-trifugation ina25 to60% (wt/wt) sucrose

den-sity gradient, characteristic light-scattering

patterns were observed in the three fractions,

indicating the presence ofaPM-likezone (37%)

in the 1,000 x g pellet, a heavy mitochondria

zone (39%) in the 13,000 xgpellet, anda more

diffuse ER-like zone at lower densities (30 to

34%) in the 80,000 x g pellet. The latter two

fractions also displayedmorefaintzonesinthe

PM region. Since the levels of SA in the three

fractions were comparable, yet the 13,000 x g

pelletwasgreatly enriched inmitochondria, we

chose to focus on the characterization of the scrapie-associated component in the 1,000 and

80,000 xgpellets, thus using themostdistinct

subcellular membrane fractions not contami-nated with excessive levels of mitochondria.

Characterization of membrane components ofsubcellular fractions. Subcellular fractions

of the 250 to1,000 x g and 13,000 to80,000 x g

pellet, designated H-1 and H-3, respectively, were prepared from Polytron-homogenized,

healthy hamster brains.Afterequilibrium

sed-imentation in a 25 to 60% (wt/wt) sucrose

gra-dient, sampleswerecollected andanalyzed for

density (Fig. 1A) protein concentration (Fig.

1B), NADH cytochrome c reductase activity

(Fig. 1C) as a mitochondria and ER marker,

ATPase activity (Fig. 1D) as a PMmarker, and

[3H]TMP incorporation into an acid-insoluble product as an indication of DNA polymerase

activity (Fig.

1E).

Since these experiments

were designed to localize the fractions in the gradient enriched in either ER or PM, the

marker enzyme activities arepresentedas

val-ues relative to other gradient samples. The [3H]UMP incorporatedacrossgradientsamples

wasinsufficientto serve as amarkerfor RNA

polymeraseactivity.

Thehigh level of cytochromec reductase

ac-tivity (Fig. 1C) in sample 4 (-42%)from both

the H-1 and H-3 preparations indicates that

residual mitochondria, as well as aggregated

debris,collect on the high-density(-55%)

cush-ion. The second peak of cytochrome creductase

activityat -37%intheH-1gradient could

rep-resent either larger sheets of rough ER, since

Mg2+ is present in the gradient, or

mitochon-drial membrane fragments. The areaof

reduc-taseactivity at 30 to 35% in the H-3 gradient is

compatible with the density properties of smooth ER.

The prominent ATPase activity (Fig. 1D) at

37% inthe H-1 gradient, indicating the

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VOL. 18, 1976

4 8 12 16 20

F R ACT 0N

FIG. 1. Distribution ofprotein (B), NADH cyto-chromecreductase(C),ATPase(D), and[3H]TMP incorporated (E) by gradient (A) samples after

equi-librium sedimentation (21 h at 25,000 rpm) ofa 1,000 xg,10-min(H-1) pellet( )andan80,000 x g, 30-min (H-3) pellet (-) from healthy

hamster brain.

ble sedimentationposition of PM, demonstrates

that the250to1,000 xgpellet comprisesa

PM-rich fraction. This suggests that the PM-rich

zone ofthe H-1 preparation might display an

equilibrium density comparable withaportion

of the rough ER (Fig. 1C). Other regions of

ATPase activity at 42% andthe disperse 30 to

35% region probably correspond to

mitochon-dria-rich and smooth ER-rich regions,

demon-strating nonspecific phosphatase activities.

In-PROPERTIES OF THE SCRAPIE AGENT 695

corporation of [3H]TMP is principally confined

to the mitochondria region (Fig. 1E) and is in

good agreement with the density indicated by

the cytochrome c reductase and ATPase levels. The equilibrium sedimentations of the 250 to

1,000 xg pellet (S-1) and 13,000 to 80,000 x g

pellet(S-3) from scrapie-infected brains present similar distributions of subcellular components (Fig. 2). Mitochondria and aggregated debris are indicated at about 42%, a PM-rich region is indicated at about 36.5% as noted by the high ATPase activity (Fig. 2D), and an ER-rich

re-gion isindicated atabout 30 to 35% as notedby

the diffuse cytochrome c reductase activity

(Fig. 2C). The[3HITMPincorporation profile of

theS-1 preparation is distinct from the healthy

profile, with a minor peak of activity localized

at about 36 to 37%, corresponding to the

PM-rich region described by the ATPase activity in

Fig. 2D. This putative DNA polymerase

activ-ity might result from mitochondrialfragments;

however, no corresponding cytochrome c

reduc-tase activity was detected (Fig. 2C).

The distribution ofSA inthe S-1preparation

demonstrates coincidence with the peak ATP-ase and [3H]TMP activity at 37%, suggesting

the possible associationofa PM-like constitu-ent with DNA polymerase activity. Asimilar

regionofSA isobservedinthe S-3preparation;

however, significant levels of SA are also

dis-played in the 42% and 30 to 35% regions.

There-fore, the DNA polymerase activity does not

totallyrepresentscrapie-specific activity. Since

the S-3 pellet did not resuspendwith

homoge-neousconsistency, theactivity at 42% may

re-flect aggregated debris. Nevertheless, the

membranes sedimenting at 30 to 35% appear to

be well dispersed and presumably represent a

smooth ER-rich region. Since it is difficult to

envision such ahigh concentration of PM-like

membranesat 30 to 35%after equilibrium

sedi-mentationfor21h, the datasuggest an

associa-tionof thescrapie agentwith smooth ERat30

to 35%.Itmightfollow then that the SAatthe

centerof thegradient (37%)could be relatedto

thepresenceofroughER.

Evidence for thePM-rich1,000 xgpellet and ER-rich80,000 xgpellet, aswellasthe

cosedi-mentation of PM androughERmembrane, is

provided by electronmicroscopeexamination of

fractions 6 through 10 from the S-1 and S-3

gradients (Fig. 3). It canbe observedthat this

ATPase-rich region of the S-1 contains

large

PM-likevesicles (Fig. 3A). Nevertheless, even

thoughnocytochromec reductase

activity

was

detected in those samples, a trace amount of rough ER-like vesicles couldbe observed. The

presenceofmitochondrial fragments might

ex-plainthe [3H]TMP incorporationintothis

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tion.

Conversely,

examination of the S-3

prepa-3;A ration (Fig. 3B) indicates smaller vesicles,

3 40 some identifiable as rough ER, with a minor

quantity

of

large

PM-like vesicles.

30

_ Assuming that the scrapie agent might be

a 41.8% 36.5% 32.6% a comprised, in part, of a nucleic acid component,

LO 4 4 the detection of the [3H]TMP incorporation

co-20

inciding

with

SA

lends credence

to

the

possible

o / % B involvement of nucleic acidmetabolism in the

o 4 ' synthesis of a scrapie agent-membrane complex

40 _ orthe

pathogenic expression

of the scrapie

dis-rL

VA/

Vease. A similar interaction was

suggested

pre-Z

viously

for the

pathogenic

(viroid) RNAof

exo-l

\cortis

disease (Semancik et

al.,

in

press).

With

0

these considerations, a 13,000 to 80,000 x g

resuspended pellet

was

subjected

to

equilib-a5

c rium sedimentation in a lighter gradient (-20

to40%)asindicated in

Fig.

4A. In thisway, an

- A

attempt

was madeto

pellet

the

heavy

scrapie-> 3 A

containing

fraction (42%)while

achieving

a

bet-ter definition of the two

lighter

regions,

-37%

1 $

l\and

30 to 35%

(Fig.

4E). When the

gradient

C <

samples

z_ t > \were

_

assayed

for 13H]UMP

(Fig.

4C)

,0

12m

4 ,

,

and

[3H]TMP

(Fig.

4D)

incorporation,

distinct

12 D profiles emerged, suggesting that

contamina-10 - tion by nuclear

fragments

was probably not

F 8_ in v_ ^ _

prevalent.

The distribution of

[3H]TMP

incor-X@6_ s \ | 5 \ _ poration coincided with SA, possibly

implicat-ing a DNA-related phase in scrapie

pathogen-4 -

esis.

2 m Totest for the presence ofa

scrapie-specific

product, gradient fractions from healthy and

infected brains were allowed to incubate over-600 E

night

in

[3H]TMP

followed

by

nucleicacid

isola-tion (by phenol extraction). When analyzed by

EO

electrophoresis

on2.2%

polyacrylamide

gels,

the

400-

incorporation

product

couldbe detected

princi-pally

in

high-molecular-weight

DNAfromboth

healthy

and

scrapie-infected preparations (Fig.

200 - 5). However, avery low,

reproducible

profile of

incorporation

into

low-molecular-weight

DNA

couldbedetectedinboth theS-1andS-3

prepa-,'7r;N

DF

'ration but notin the

comparable

fraction from

109 healthy

tissue.

Nucleic acid

species

recovered

from

subcel-i [////

A/////lular

( s 2fractions.

Quantitative

and

qualitative

o r determinations of nucleic acid

species

extracted

from subcellular fractions were undertaken.

About 50 brainseachfrom healthy and

scrapie-infected hamsters were

Polytron homogenized

o / FIG. 2. Distribution ofprotein (B), NADH

cyto-LLI

///// \\\\ \\\\ ///_ chromec reductase (C), ATPase (D),[3H]TMP

incor-poration (E), and SA (F) by gradient (A) samples

vr

//g;\\\\\\\\/// after equilibriumsedimentation(21hat25,000 rpm)

0 7 / ofa 1,000 x g, 10-mm (S-i) pellet ( ) and an

2 4 6 8 10 12 14 80,000 x g,30-min (S-3) pellet(-) from scrapie-F RA CT 0N infected hamster brain.

696

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PROPERTIES OF THE SCRAPIE AGENT

697

FIG. 3. Electron micrographs ofgradient fractions (7through9)ofa1,000 xg,10-minpellet(A) andof gradient fractions (6 through 10) of an 80,000 xg, 30-minpreparation (B) from scrapie-infectedhamster brain (see Fig. 2). Arrows indicate rough ER vesicles. x39,000. N

and fractionated as described above, followed by nucleic acidisolation by phenol extraction.

Ethanol-precipitable nucleic acids were

parti-tioned by2 M LiCl precipitation of

ribosomal-like RNA and recovery of

high-molecular-weight DNA after addition of 3 volumes of

ethanoltothe 2MLiClsupernatant. Determi-nations of DNA and RNA by diphenylamine and orcinol tests were made on each of the nucleic acid fractions.

A significantly higher level of extractable

nucleic acids was detected in all subcellular fractions from healthy, ascompared with

scra-pie-infected, braintissue. Thismayreflect the widespread cellular degeneration associated

with thescrapie disease process. The

distribu-tion ofnucleic acidspecies reflects the distinct quality of the subcellular fractions. The 1,000 and 13,000 x g pellets contain relatively high concentrations ofDNA (50 and 35%),

presuma-bly resulting from nuclei and mitochondria, whereas the80,000 and100,000 xgpelletsare

rich (80 and 95%) in RNA, reflecting the

pres-ence ofmembrane-bound and free

ribosomes,

respectively. When aliquots fromhealthy and

scrapie-infected preparationswereanalyzed by

electrophoresis on 2.2 or 5% polyacrylamide

gels, no differences in thequality of the various

RNA species wereevident.

DISCUSSION

Previous attempts to describe an infectious

scrapie nucleic acid species, analogous to the

pathogenic (viroid) RNA, have not been

suc-cessful (16, 23). Renewed interest in the

com-parative pathology ofscrapie disease with vi-roidinfectionsinplants wasstimulated by the

finding of amembrane-relatedphase in

exocor-tisdisease (Semancik et al., in press) and the

observation of acytopathic effect onthe

endo-membrane system (Semancik and

Vander-woude, Virology, in press).

Therefore,

pro-ceduresemployed in the characterization of the

exocortis disease agent have been adapted for

use inexaminingthe scrapieagent membrane

complex

and

possibly

a putative nucleic acid

componentofthe scrapie agent. These studies

have been facilitated by the use of the

ham-ster-scrapie model, providingthehighesttiters of scrapie

infectivity

of any known bioassay

system (15).

The concentration of SA in the subcellular

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IV 2 4 6 8 10 12 14 16

FRACTION

FIG. 4. Distribution ofprotein (B), RNA polym-eraseactivityasindicated by [3H]UMPincorporation

fractions

pelleted

inthe 250 to

80,000

xg range

generally

supports

the

scrapie-membrane

hy-pothesis

(8), i.e.,

the

possible

association ofa

small

pathogenic

nucleic

acid,

asdeduced from

the

target

size of -105daltons

(2),

witha

mem-brane component. The primary association

with the PM

(17)

cannot be

fully

reconciled

with the present data.

Samples

taken from

equilibrium density

gradients,

even after

pre-fractionation of subcellular components, are not

sufficiently homogeneous

toascribeSA toa

single

membrane

species.

The

present

data

in-dicatethat

portions

ofthe

rough

ERcosediment

with the PM-rich

regions. Therefore,

the

pre-ferred

explanation

ofthesedimentation

hetero-geneity

ofthe

scrapie

agentin

13,000

to

80,000

x g

(S-3)

pellets

suggests an association with

rough

ERatabouta

density

of 1.17

g/cm3

and

with smooth ER ata

density

of 1.13to 1.15

g/

cm3.

Alternatively,

the

density heterogeneity

of theSA

complex

mayresultfromthe

specialized

nature ofthe brain

tissue,

since the

synaptic

vesicles and external synaptic membranes demonstrate

characteristically

different

sedi-mentationproperties (24).

Also,

theassociation

of the presumptive nucleic acid component in

the

scrapie-membrane

complex may result in

an alteration in the

density

of normal host membranes.

When consideringthe high specific

infectiv-ity

ofthe scrapie agent inthe absence of any

recognizableagent,onemustbe extremely cau-tious in

ascribing

a

specific

association to a

single

component of the endomembrane sys-tem.

Furthermore,

iftheagentbecomes

associ-ated with theendomembrane system at

early

developmental periods,

it may be possible for

the agent to retain biological activity

during

andafterthe process ofmembrane

differentia-tion,

thus

resulting

insomeSAintegratednot

only

with the ER but also with the PM. A

further considerationcenters ontheassociation

ofthe SA with some scrapie-directed de novo

membranestructure orpossibleartifacts of

sub-cellularfractionation which cosediments with

either PM or ER but is not discernible as a

distinct component ofthe membrane popula-tion.

The data are compatible with the

hypothesis

presentedfor the pathogenesis ofthecitrus

exo-cortis viroid (Semancik et al., in press), in

which both a nuclear (19) and an

endomem-brane

phase

havebeen

postulated

forthe

possi-(C), DNA polymerase activity as indicated by

[3H]TMPincorporation (D), and SA (E) bygradient

(A) samples after equilibrium sedimentation (21 h

at25,000 rpm) ofan80,000 xg,30-min (S-3)pellet fromscrapie-infected hamster brain.

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AGENT

I I

B

50~~~~~~~~~~~~

0 10 20 30

F R ACTI0N

FIG. 5. Electrophoresis of[3H]TMPincorporation products of subcellular fractions 1,000 x g,10 min (A) and80,000xg,30min(B) from healthy (0)and

scrapie-infected (@) hamster brain in 2.2% poly-acrylamide gels with 0.5%agarose after4 h and6

mA/gel. Migrationwasfromlefttoright, with3-mm

slices treatedinOmnifluor-toluenewith 3% Protosol

for48hat40C.

ble siteof synthesis and site of accumulation of

the pathogenic RNA. The scrapie-membrane

complex may reflect a synthetic end point or

accumulation ofthe scrapie agent. Sincewedo

not know the molecular nature of the scrapie

agent, much less the template and pathway by

which the agent is synthesized, the most

cau-tious interpretation of the present data would

be thatthescrapieagentappearstobe

primar-ily associated with ER-like, and possiblysome

PM-like, membranes which may represent

sitesofaccumulation of scrapieprogeny.

The correlation of [3H]TMP incorporation or

DNApolymerase activity with SAcanbe taken

onlyas averypreliminary indication that this

site of accumulationmayalsorepresentasite of

some synthetic activity of the scrapieagent. A

low-molecular-weight DNA species has also

been reported in scrapie-infected mousebrain

after the intracerebral inoculation of

[3H]thy-midine (10). These data, together with the

dynamics of [3H]thymidineturnoverin

scrapie-infectedmousebrain (1,12)and the correlation

between cell division and scrapie infectivity

(4), are compatible with the suggestion of a

DNAcomponentinthe scrapie agent.

The characteristics ofscrapiediseaseand its transmissible agent present interesting paral-lels to the plant disease caused by the citrus exocortis viroid. The association of the

patho-genic(viroid) RNA of the exocortisdisease with

the endomembrane system appears to result in

a vesicular proliferation or disruption of PM

(Semancik and Vanderwoude, in press). Since the overt pathology of scrapie disease also in-volves aberrations of the PM (13), these cellu-lar phenomena, coupled with the estimates of

molecular size and stability, reinforcethe

possi-bleviroid nature of the scrapie agent (6).

Com-parative aspectsof the two diseases project the

possibility of a nucleic acid component in the scrapie agent, as well as a nuclear phase

in-volved in the replication process. The hamster

scrapie model presents an excellent system to exploit in the study of membrane-"viroid"

inter-actions,eventhoughamoleculardescriptionof

the scrapie agent, fundamental to the

under-standing of the disease, has yet to be estab-lished.

ACKNOWLEDGMENTS

These investigations weresupported byaNational Sci-enceFoundation grant (GB 39605) to J. S. Semancik andby Public Health Service grant AI 11250 fromthe National Institute of Allergy and Infectious Diseases.

Wewish to acknowledge theexcellenttechnical contri-butions of Deanna Tsuruda, Kazue Matsumoto, and D. A. Reynolds,and thankW.VanDerWoudeandR.Leonard for advice and consultation.

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7. Dougherty, R. M. 1964. In R. J. C. Harris (ed.), Tech-niques in experimental virology, p. 183. Academic PressInc.,NewYork.

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