Intracellular Virus-Specific Structures and RNAs in Oncornavirus-Producing Human Cells

Copyright © 1974 AmericanSocietyforMicrobiology Printed in U.S.A.

Intracellular

Virus-Specific Structures and RNAs in

Oncornavirus-Producing Human Cells

A. G. BUKRINSKAYA, G. G. MILLER, E. N. LEBEDEVA, AND V. M. ZHDANOV

TheD.I.IvanovskyInstituteof Virologyand the N. F.GamaleyaInstituteofMicrobiologyandEpidemiology, Academyof Medical Sciences, Moscow, U.S.S.R.

Received forpublication 18September1973

Two kinds of virus-specific structures were isolated from the cytoplasm of

Detroit-6 and human amnion cells producing oncornavirus-like particles.These

structures represented A particles with the diameter of 70 to 80 nm and

aggregated strands of nucleocapsids with the diameter of 3 and 6 nm. The

structures were separated from cellular contaminants by isopycnic banding in

linear sucrosegradients and subsequently further purified by sedimentation in

velocity sucrose gradients. Their sedimentation coefficient was 250 and 150S,

respectively. Both structures contain 60, 45, and 35S RNA species, and 150S

structuresalsocontained20SRNA. The 35and 20S RNA from the 150S structure

formed hybrids with DNA enzymatically synthesized on extracellular virions.

The structures displayed endogeneous polymerase activity, DNAproductofthe

reactionbeingpredominantly associated with60SRNA. No 70S RNAwasfound

in the cellstructures ofvarious densities. Also, the virions purifiedfrom tissue

culture fluid contained 70SRNA. These findings are consistent with those on

extracellular maturation ofoncornavirusRNA.

Little information is available concerning the properties of intracellular precursors of

oncor-naviruses and virus-specific RNA in

oncor-navirus-infected cells. Coffin and Temin (4) isolated, from Rous sarcoma-infected cells, vi-rus-specific particles which banded in an equi-librium sucrose density gradient at 1.24 g/ml.

These particles possessed RNA-dependent

DNApolymeraseactivitywhich was resistant to ribonucleasedigestion. However, they were not further studied. Smith and Wivel, and Wivel

and Yang (14, 17) reported the presence of A

particles in cells producing oncornaviruses of

B andCtype. Theseparticles contained,among

otherheterogeneous RNA species, high

molecu-larweight RNA. Intracellular A particles were

described also in monkey cells producing

Ma-son-Pfizeroncornavirus of B type (8).

The data on the properties of intracellular

virus-specific RNA and the site of itssynthesis

are also very scarce. Parental 60 to 70S RNA

wasshown topenetrate the nuclei early in

infec-tion (5), and de novosynthesized heterogeneous

virus-specific RNA including high molecular weight species were isolated from the nuclei and

thecytoplasm(9). On the other hand, Tsuchida

et al. (15, 16) extracted from

oncornavirus-infected cells 35 and 20S RNA species, and did

not find anyhigh molecular weight RNA.

The results mentioned above were obtained

mainly on the systems of animal cells producing

oncornaviruses type C. As forthe cells of human

originproducingoncornavirus typeB, there are

no available dataconcerningthisquestion.

In this investigation we present the data on

the properties of cytoplasmic virus-specific

structuresand RNAextracted fromthese

struc-tures ofDetroit-6 and human amnioncells, two

of humancontinuous lineswhich spontaneously

produce oncornavirus type B. The

characteris-tics of these cell systems have been published

earlier (1, 18).

MATERIALS AND METHODS

Cells. Cell monolayers were grown in 199 medium supplied with 2% calfserum, and within 72to 96 h they were fed with fresh medium containing 3H-uri-dine(2 gCi/ml, specificactivity 16 Ci/mmol) or car-rier-free 32P04 (1 iACi/ml) inthe phosphate-free me-dium. After 24 h of incubation, cell cultures were washed withTris-saline (Tris-hydrochloride, 0.01 M, pH7.4;NaCl,0.1M) andthecellswereremoved from theglass by0.5%trypsin.

Virus. The virus from tissue culture fluid was concentrated as described (3) and subjected to equi-librium densitygradient centrifugation in 20 to 60% sucrose (wt/wt) in TNE buffer (Tris-hydrochloride 0.05M, pH8.3;NaCl,0.15M; EDTA,0.001M). The viruswasrecovered fromdensity gradientfractionsby pelletingandresuspended inthe same buffer.

Preparaton and analysis of cytoplasmic ex-478

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tracts. Thecells were washed at 4 C with Tris-saline and thenwith RSB (Tris-hydrochloride, 0.01 M pH 7.4; NaCl, 0.01 M;MgCl2,0.003 M) and disrupted in RSBby Dounce homogenization. Nuclei and cell rem-nantswere pelleted at 800 x g. Post-nuclear fraction was centrifuged at 15,000 xg to remove mitochondria and microsomes, supernatants (cytoplasmic extracts) inthe volume of5ml were layered over a 25 to 65% (wt/wt) 10-ml linear sucrose gradient prepared on TNEbuffer, and the structures were banded isopycni-callyby centrifugation in anSW 27.1 bucket rotor at 25,000 rpm for 16 to 18 h at 4 C. The upper layer containing the material was removed, the gradients weredivided into fractions, 0.1-volume sample from each was precipitated by 5% trichloroacetic acid along with 25 to 50 gg ofcasein. Radioactivity was countedin aPackard Tri-Carbscintillation spectrom-eterby usinga toluene-based scintillant. The appro-priate fractionscorresponding to the peaks of radio-activity werepooled, twice diluted with thesame buf-fer, and either recentrifuged in velocity sucrose gradients (17 to 40% wt/vol) in an SW 27.1 bucket rotor at 24,000 rpm for1.5hat 4C, orusedforRNA, analysis.

Polymerase assay.Cytoplasmicextractsfrom

un-labeled cells were prepared as described above and

centrifuged in 25 to 65% equilibrium sucrose

gradi-ents.DNApolymeraseactivity wasassayedeither in crudeextractsorinportions ofeach fraction in

reac-tion mixture containingthe following: Tris, pH 8.3,

0.05M;KCl, 0.15M;MgCl2,0.006M; dATP,dCTP,

anddGTP,0.1 mMeach;dithiothreitol (DTT),0.2%;

'H-TTP, 1

;iCi

(specific activity 26 Ci/mmol). The

mixture in avolume of0.5mlwasincubatedat30C for15minandchilled, and acid-insolubleradioactivity

was counted. In some experiments, the material of each fractionwasdivided intotwoparts,onepartwas

used for radioactivity determination, and the other part foranalysis of nucleic acids by velocity sucrose gradient centrifugation.

Extraction of RNA from thecytoplasmic struc-tures and its velocity sucrose gradient centrifu-gation. RNA from sucrose gradient fractions after centrifugation ofcytoplasmicextracts was extracted eitherby1%sodiumdodecyl sulfate (SDS) or by the

SDS-phenol method followed by precipitation with

ethanol. Both methodsyielded thesameresults. RNA precipitates werecollected bycentrifugation and redissolvedinTNEbuffercontaining0.5%SDS. All RNA samples were centrifuged in 15 to 30%

(wt/vol) sucrose gradients prepared on STE

(Tris-hydrochloride,0.01 M, pH 7.4; NaCl, 0.15M; EDTA,

0.001M) containing0.5%SDS in anSW27.1bucket rotor at14,000to 16,000 rpmfor 14 to16 hat25C.

RNA-DNA hybridization. 3H-labeled DNA was obtainedas aproductofreversetranscriptasereaction by usingdetergent-disrupted virions from tissue cul-ture fluid as a template. Polymerase reaction was

performedin a2-ml volume byusingthepolymerase

mixturementioned above andcontaining0.02%NP40 which was incubated for 1 h at 35C. Then the polymerasemixturewaschilled,and thenucleic acids wereextracted twice with SDS-phenol. Purified calf thymus DNA was added as a carrier. The nucleic acidswereprecipitated bytwovolumes ofethanoland

left overnight. The pellet was redissolved in STE, RNA was hydrolyzed by 0.5 MNaOH at 37 C during a 16-h period, and after neutralization, DNA was pre-cipitated by ethanol and left overnight. DNA was then dissolvedin70%formamide, melted at 90 C for 2 min, and chilled andmixed with an equal volume of RNA dissolved in 30% formamidecontaining 0.8 M NaCl. The mixture was incubated at 37 C for 16 to 20 h and the extent ofRNA-DNA hybrid formation was mea-suredby the method of Hehlman, Kufe, and Spiegel-man (7) by using isopycnic centrifugation in pre-formed caesium sulfate gradients in a Ti5O rotor at 35,000 rpm for 60h.

Electron microscopy. Cytoplasmic extracts from unlabeled cells mixed with a little portion of the extract from labeled cells was centrifuged in equilib-rium sucrose density gradients. After counting radio-activity in portions, the appropriate fractions were pooled and diluted with TNEbuffer, and the material waspelletedat80,000 x g for30min, dissolved in the same buffer, negatively stained with uranyl acetate, and examined in an electron microscope (JEM 100B).

Forultra-thin sections, the monolayer culture cells were fixed in Dalton's chrome-osmium, dehydrated, embedded in Epon-araldite, and sectioned and stained withuranyl acetate and lead citrate.

RESULTS

Distribution of radioactivity after isopyc-nic centrifugation of cytoplasmic extracts. Cytoplasmic extracts were prepared from the cells labeledwith 3H-uridineor37P0o,and sep-arated in

equilibrium

sucrose

density

gradi-ents. For comparison, cytoplasmic extracts of

an HeLa cell strain, which does not produce virus-like particles (as revealed by electron

microscopy), were analyzed. Figure la shows

that radioactivity in this case is found in the

region ofdensity 1.18 g/ml. According to

pub-lished data (14), thisregioncontains mitochon-dria, microsomes, and cytoplasmic fragments.

An essential part of radioactivity was also

found in this region after isopycnic

centrifuga-tion ofAO and Detroit-6 cytoplasmic extracts.

Besides, some part of radioactivity was

regu-larly revealed in the region of density 1.22 to

1.23 and 1.27 to 1.29

g/ml

(Fig. lb). These

components werereproduciblyobserved in both

cell lines irrespective ofwhether the cells were

labeled with

32po0

or 3H-uridine. The 3-h cen-trifugation yielded the same results as the centrifugation

during

16 h. Addition of 0.4%

DTT to the bufferdid notchangethe

distribu-tion ofradioactivity (data notshown).

Polymerase activity assay of the gradient fractions. To identify the possible precursors

of oncornavirusesin thegradient, each fraction

ofthegradient similar to that shownin Fig. lb

was assayed for endogenous

polymerase

activ-ity in theabsenceofdetergent.Thereactionwas

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performed for a short period of time (15 min) to

detect DNA still associated withthetemplate.

It is seen from Fig. 2a that the highest

polymer-aseactivity is revealed at the density of 1.22 and

1.19

g/ml,

and thelower activity is found also at

1.27to 1.29 and 1.23g/ml.

To identify thereaction as virus specific, the nucleic acids were extracted from the

incuba-a!

.2', 1.21 1.18 1.15

g,

1.29 I.? 1.23 1.20 1.17

FIG. 1. Distribution of radioactivity after equilib-riumdensity centrifugationofcytoplasmicextractsof HeLa (a) and humanamnion (b) cells. Cytoplasmic

extractsfrom3H-labeledHeLa cells (a)and AOcells (b) were centrifuged in a 25 to 65% equilibrium

sucrosegradient prepared on TNE buffer inanSW

27.1 bucketrotor at25,000rpmfor 16 hat4C.After fractionation, radioactivitywascounted in0.1-volume

portions.

tion mixture after polymerase reaction with

crude cytoplasmic extracts as well as from the

gradient fractions corresponding to peaks of

radioactivity and were analyzed in velocity

sucrose gradients. Figure 2b shows that an

essential part of 3H-labeled DNA extracted

fromcytoplasmic extract sediments at 60S. The

similar sedimentation profile was observed for

DNAextractedfromthe 1.22 (Fig.2c) and 1.19

zone of the gradient (data not shown), and the

lesser part of the label was found in the region

lighterthan 60S (Fig. 2c). The60S peak

disap-peared after treatment with RNase (Fig. 2b)

indicating that DNA in this position is associ-ated with RNA.

Sedimentation characteristics of the com-ponents from different

density

regions.

The material from the regions of the gradient dis-playingthepolymerase activitywasfurther

ana-lyzed in velocity sucrose gradients. Figure 3a

shows that an essentialpart oflabeledmaterial

from the 1.22 to 1.23 region of both cell lines

sediments at 250 to 300S and the lesser part of radioactivity is found near the bottom of the gradient. The last component has not been

regularly observed and may be a product of

aggregation of the abovementioned component.

The similar sedimentation profilewasobtained

when the material from the 1.27 to 1.29 zone wasanalyzed (datanotshown).Thematerialof density 1.19g/ml consistedof two components,

1.260 1.235 1.210 1.185 1.160 5 10 15 20 25

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g rraction number 5 Fraction10 15 nuuiner20 25

FIG. 2. Distributionof polymeraseactivityinequilibriumsucrosegradient fractions (a) after centrifugation

ofcytoplasmic extractsfromDetroit-6cells and analysis of DNA product (b-c). Theendogenouspolymerase activitywasassayedineachfraction ofthegradientsimilartothatshowninFig.lb in thevolume of0.5mlby using 9H-TTPas aradioactiveprecoursor, andradioactivity wascounted in '/8-volume portions (a). b, The

polymerasereaction withcytoplasmicextractswasperformedat30C for15min, andthen thenucleic acidswere

extractedfrom theincubation mixturewithSDS-phenolanddividedintwoparts,onepart(solid circles)was

left untreated, and theotherpart(empty circles)wastreated with 20ugofpancreaticribonucleasepermlat room temperature for20 min. The material was analyzed by centrifugation in 15 to 30% (wt/vol) velocity sucrosegradientpreparedonSTE buffercontaining0.5%SDS inanSW27.1bucketrotorat15,000rpmfor15h

at25C.c,Thefractionscorrespondingtothe peak ofradioactivityat1.22g/mlwerepooled,diluted with STE,

andnucleic acidswereextracted with 1%SDS and analyzedasinb. Nucleicacidwasextracted from purified

32P-labeled Sendai virions by 1% SDS and usedas a marker (50S) run in thesame gradient. 28and 18S ribosomal RNA extracted from Ehrlichtumorcellswerealsorunin thesamegradient andrevealed byoptical

density.

15

10

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ONCORNAVIRUS-PRODUCINGHUMAN CELLS

one of whichsedimentedatabout150Sand the other whichwas in alighterregion of thesucrose gradient (Fig. 3b).

Analysis ofRNA. RNA was extracted with SDS or SDS-phenol from cytoplasmic extracts andsucrose gradientfractionsand wasanalyzed

in 15 to 30% linear sucrosegradients.

Figure4ashows thatbesides28and18SRNA species, total cytoplasmic RNA from AO cells contains a relativelyhigh percentage of 35 and

a) 115s

-3 n

.4J E

I' 2

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Fraction iLimBer Fract-on nwr'8tr

FIG. 3. Velocity sedimentation of cytoplasmic structures from Detroit-6 and AO cells. Cytoplasmic extracts from 32P-labeled Detroit-6 cells and 3H-labeled AO cells were separated in an equilibrium

sucrose gradient prepared on TNE; the fractions

corresponding tothepeak ofradioactivity at1.22to

1.23g/ml (a), and1.19g/ml (b)werepooled, diluted with TNE, andrecentrifugedina17 to 40%(wt/vol)

velocitysucrosedensity gradientat24,000 rpm for1.5

hat4C. Tobaccomosaic virus(185S) was runin the

same gradients and revealed by optical density.

Symbolsfora: 0, 32P;0, 3H.

28s

a) i

5 10 15 20 25

45S RNA (about 10to 15% and 1to 2%of total RNA,

respectively).

The similar results were

obtained

with Detroit-6 cells.

When RNA wasextractedfrom the fractions

of equilibrium sucrose gradient, 28 and 18S RNA specieswere invariably found throughout the gradient. Therefore, to

obtain

more pure material containing virus-specific RNA species,

RNA wasextracted from theappropriate zones

of velocity sucrose gradients after recentrifu-gationofequilibrium sucrose gradient fractions. The 250S structure of density 1.27 to 1.29 and

1.22 to 1.23

g/ml,

the 150S structureofdensity

1.19

g/ml,

and the top component of the same density shown inFig. 3b wereanalyzed.

RNA extracted from the top component of density 1.19 g/ml contained only 28 and 18S

species. Thus, this component apparently

rep-resents a cellularcomponent.

The mainsize-classesofRNA in the 250 and 150S structures were 60, 45, and 35S RNA. Besides this, the150S structures contained 20S RNA. The relationship between these RNA

species varied from experiment to experiment,

but usually60 and 35S dominated in the250S structureand 35 and20S dominatedin the150S structures (Fig. 4b, c). Rapidly sedimenting RNA species were also seen in a number of experiments (Fig. 4b) in accordance with the data

published

(9).

DNA-RNA hybridization. The 35 and 20S RNAs extracted from the 150S component of density 1.19

g/ml

were annealed with

3H-DNA

productenzymatically synthesizedon the

extra-5 10 15 20 25

Ft-action number

c)

28318S

2

aoS

5 10 15 20 25

FIG. 4. Sedimentation analysis oftotal cytoplasmic RNA (a) and RNA extracted from the cytoplasmic

structures (b, c) of Detroit-6 and AG cells. a, RNA from cytoplasmic extracts ofAO cells extracted by SDS-phenol; b,RNAextractedfromthe250Sstructuresof density1.22 to1.23g/ml.Cytoplasmic extractsfrom 32P-labeledAOcells and 3H-labeled Detroit-6 cellswereseparatedinequilibriumsucrosegradients,fractions correspondingtothepeak ofradioactivityat 1.22 to 1.23g/mlwerepooledandrecentrifugedinavelocity

su-crosegradientasinFig. 3a,fractions corresponding tothe250Speak werepooledanddiluted withSTE,and RNAwasextractedwith 1% SDS.Symbols:0,32p;0,3H.c,RNAextractedfrom the150Sstructures ofdensity

1.19g/ml. Cytoplasmicextractsfrom'H-labeled AOcellswerecentrifuged, the150Sstructurewasobtainedas described inFig.3b, theappropriate fractions werepooledanddiluted, and theRNA wasextracted with 1% SDS. RNAwasanalyzedin 15to30%sucrosedensitygradients preparedonSTEcontaining0.5%SDSin anSW 27.1 bucketrotor at 16,000rpmfor14 to16 hat 25 C. ThesymbolsareasinFig.2.

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[image:4.493.97.391.432.554.2]

cellular virions, and the hybridizationreaction

wasevaluated by centrifugation in a preformed

Cs2SO4 gradient. RNA extractedfrom the cyto-plasm of normal human fibroblasts was used as

a control.

The product synthesized duringl h of

polym-erase reaction ontheextracellularvirions

repre-sented DNA-RNA hybrids that banded at the

density of 1.54 g/ml in Cs2SO4 gradients, and

after alkaline hydrolysis a predominant

compo-nent was one-stranded DNA banded at 1.46

g/ml (Fig. 5a). Also, an essential part ofDNA

radioactivity shifted to the regions of higher

density after the hybridization reaction (Fig.

5b, c). No detectable hybridization was

ob-servedbetweenthe DNA product and the

cellu-larRNAfromfibroblast(Fig. 5d).

Properties of 60S RNA and RNA extracted

from the virions. To characterize further 60S

RNA, it was dissociated into its subunits. With

this purpose, RNA from the 1.23 cytoplasmic

structures was centrifuged, and fractions

con-taining 60S RNA were pooled, precipitated in

ethanol, and dissolved inSTE.

When recentrifuged immediately, 60S RNA

exhibited a rather broad peak ofradioactivity

at55 to65S(Fig. 6a). After heating at 90 C for 2

min, it dissociated into components with

sedi-mentation coefficients of 35, 28, and 20S (Fig.

6b). When recentrifugation was not performed immediately and the samples were left over-night in 1% SDS, 60S RNA spontaneously dissociated into 35 and 28S components (Fig. 6c).

Virions from tissue culture fluid werepurified

bycentrifugation on an equilibrium sucrose

gra-dient; RNA was extracted from the 1.17 peak

and analyzed similarly. High molecular weight

RNA was found in the heavier region of the

sucrose gradientthan 60SRNA extracted from

thecells, the components 35, 28, and 4Sbeing

possible products of its dissociation (Fig. 7a). The sedimentation profileofrecentrifuged cyto-plasmic60S RNA from the 1.19 zone of

equilib-rium sucrosegradient run under the similar

con-ditions is shown forcomparison(Fig. 7b). It can

be seen that radioactivity of RNA extracted

from the virions doesnot coincide with that of

RNA extracted from the cytoplasmic

struc-tures.

Electronmicroscopy. Examination of

ultra-thin sections of Detroit-6 cells revealed alarge

number of A and B particles in intercellular

spaces, and some of them were in the process of budding from the cell surface (Fig. 8a).Ahigh magnification ofa budding Aparticle is shown

in Fig. 8b. Ofspecialinterest are the

filamen-toushelicalstructuresseenunder the cell

mem-brane and showninFig.8c and d. The diameter

of these filaments is about 6 nm. Similar

a)

IOS OSs 80

uS5 28sWS

i

1'

b) 28s'1S C)

50S I 5OS 285

I I

5 ...101.5 20 25 301 5 10 I5 20 25 5 iO i5 210 25

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fraction numBer

FIG. 6. Dissociation of60S cytoplasmic RNA from Detroit-6 cells. Fractions correspondingtothepeakat 60Sfrom the gradients similartothat shown inFig. 4b werepooled, diluted with STE, divided in twoparts, and onepart (b) washeated to 90 C for2 min and quickly chilled. Both samples were recentrifuged in 15 to 30% sucrosegradientsasinFig. 4. c, 60S RNA from thesame zoneof the gradientasarecentrifuged

onethefollowing dayas described in Fig.4.

b a) )c d)

8a4

112

1.61 156 151 146 1.,1 162 1.55 1.4s 141 163 155 i.47 141 163 158 i.52 146

FIG. 5. Hybridization of the 3H-DNA product of the endogenous virion DNA polymerase system with 20 and 35S RNA extracted from the 150Sstructures ofAO cells. RNA was extracted by SDSfrom the 150S structuresof density 1.19g/ml, and 20 and35Sspecieswere obtained as described in Fig. 4. Cytoplasmic RNA extracted byphenol-SDS from theprimary tissue culture ofhumanfibroblasts wasused as a control. The hybridization experiment was set as described (2), and all samples were analyzed by Cs2SO4 equilibrium density gradient centrifugationinTi5Orotor at35,000 rpmfor72h at 5 C.a,3H-DNA;b,c,d,reaction mixture after annealing with20SRNA, 35S RNA, and RNA ofhumanfibroblasts, respectively.

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5 ,0 ,5 20 25 5 10 I5 "0 25

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fraction

numBer

FIG. 7. Sedimentation analysis ofRNA extracted from virions (a) and from cytoplasmic structures of density 1.19g/ml (b). a, RNA was extracted by 1%

SDS from the virions purified by equilibriumsucrose

density centrifugation, and high molecular weight RNAwasrecentrifuged in 15to30%osucrosegradient

containing 0.5% SDS as in Fig. 4. b, Cytoplasmic RNA was extracted by 1% SDS from the region of

density1.19g/ml,andhigh molecular weightRNAwas

recentrifuged asin Fig. 4.

filamentous helical structures ofthe same

di-ameter are seen inside some extracellular

vi-rions. Figure 8e shows one of the A particles

regularly seeninthe hyaloplasm.

Figure 9 shows the material of equilibrium

sucrose gradient fractions correspondingto the peaks of radioactivity.

The material from the 1.22 to 1.23 region

contained a high concentration of A particles

with the diameter of 78 to 80nm (Fig. 9a, b).

Partly disaggregated A particles with released

RNP strands of diameter 6 and 3 nm were

also observed. Morphologically similar

struc-tureswere seeninthe material of density 1.22to

1.23 and 1.27 to 1.29 g/ml from AO cells (data

notshown).

The fractions from 1.19 zone of the gradient

contained rather homogeneous material which

represented clusters of aggregated filamentous

heliceswith the diameter of6nm(Fig. 9c-f). In

some partsthese helices were seen as unwound

threads of diameter 3 nm (Fig. 9d). The

scattered membraneous structures were also

seen inthis material.

The fractions ofdensity 1.18 g/ml from

De-troit-6, AO and HeLa cells contained

mem-braneousstructures ofvarious sizesbutdidnot

contain filaments (Fig. 9g).

DISCUSSION

Detroit-6 and AO cellsbelongtothegroupof

cell strains of human origin which

spontane-ously produce oncornavirus particles of B type

(1, 18). We made an attempt to isolate the

intracellular precursors ofoncornavirus and to

investigate their RNA. The high concentration

of virus-specific structures was achieved by equilibrium sucrose density centrifugation of cytoplasmicextractsfollowedby centrifugation

in velocity sucrose gradients. Intracytoplasmic

A particles similar to that found in ultra-thin

sections were revealed in the region of density

1.22 to 1.23

g/ml

and 1.27 to 1.29 after

centrifu-gation of cytoplasmic extracts, in accordance with the data of Smith and Wivel (14). Clusters ofaggregated filamentous heliceswere found in

the region of the gradient with the density of

1.19

g/ml.

These helices had a diameter of 6 nm; unwound threads of diameter 3 nm were

also seen in these preparations. The identical

filamentous helices were revealed in ultra-thin

sections ofthese cells. The similarity of these

structures totheinner component of the cores of

oncomaviruses (11) suggests a virus-specific

or-iginand allowsone to assume that these

struc-tures represent helical nucleocapsids of

oncor-naviruses. It is not clear why their density was

significantly lower than that expected for ribo-nucleoprotein structures. Apparentlythey were

either associated with membraneous material

whichwas seen inthese gradient fractions when observed in electron microscope, or contained lipids as aconstituent.

TheAparticleswereshownto cohtain 60and 35S RNA andthe 150Sstructurecontained 60, 45, 35, and 20S RNA. These RNA specieswere described as virus specific in the systems pro-ducing oncornaviruses ofC type (3, 10, 15, 16).

It must be mentioned that, unlike in that

system, a

significantly highet

percentage of 35

and 45S RNA (about 10 to 15% and 1 to 2%, respectively, ofthe totalcytoplasmic RNA) was found inthecell lines used.

Both kinds of

virus-specific

structures, (A

particles andfilaments) possessed endogeneous polymerase activity in the absence of

deter-gents. This result makes it evident that the

structures lack virus proteins which block the

polymerase activityinthe mature virions.Since the DNA product of polymerase reaction was predominantly assocated with 60S RNA, it is suggested that this RNA is a much better template for DNA polymerization than other RNA

species.

The

significance

of these

struc-tures in virus

reproduction

and their

interrela-tion in the cells requires further investigation.

Since35and 20SRNAextractedfromthe

150S

structures were shownto possess sequences

ho-mologous to those found in extracellular virion

RNAtheymay be

regarded

asits

possible

intra-cellular precursors.

ThesameRNAspecies, 35and

20S,

werealso

foundinthecell nucleibeingextractedat60C.

They also possessed a sequence homologous to

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BUKRINSKAYA ETAL.

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FIG. 8. Ultra-thin sections of Detroit-6 cells. a, Low magnification of the cell surface showing virions in intercellularspaces and budding A and B virions (arrows). x135,000. b, Highermagnification of theabove. Budding A particle is seen. x390,000. c, Sections of the cell surface showing the clusters offilamentous nucleocapsids underthecell membrane. x190,000.d,Higher magnification of the above. x390,000.e, Atypical nucleoid ofAparticle inthehyaloplasm. x390,000.

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-FIG. 9. Negatively stained preparations of equilibrium sucrose gradient fractions of different density. The material from Detroit-6 cells. a, aggregated A particles in the 1.23 band. x 135,000. b, Highmagnification of the above. x390,000. c-f. Clusters of filamentous nucleocapsids in the 1.19 band. c, x 135,000; e, x65,000; d, f, x390,000. g, Membraneous structures in the 1.18 band. x135,000.

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ET AL.

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FIG. 9g.

thatinextracellularvirionRNA,indicatingthat

nuclei play someessential role in oncornavirus

multiplication (manuscript in preparation).

Our results show that eitherno70S RNAor a very small portion exists in the cells, and that

intracellular high molecular weight RNA

sedi-ments slower than RNA extracted from extra-cellular virions. These data are consistent with

the data obtained on oncornaviruses oftype C about extracellular maturation of virion RNA

(2, 3).

ACKNOWLEDGMENTS

Wewishtoexpress ourthankstoNatalyVorkunova for her excellent technical assistance.

ADDENDUM IN PROOF

We haverecently found that RNA extracted from purifiedintracytoplasmic A particles doesnotcontain

the sequences homologous to extracellular virus

ge-nome. When RNA extractedfrom the 250 and 150S

intracellular structures was hybridized to excess of DNAenzymatically synthesized on the extracellular

viriontemplateinthepresenceofactinomycin D,the percentage ofhybridizationasrevealedbyRNA

frac-tion resistant to RNase hydrolysis was 0 and 80 to 90%, respectively. We assume, therefore, that the 150Sstructures butnotAparticlesaret!heprecursors

oftheextracellularvirions.Aparticlesmayrepresent

aseparate oncornavirus orits precursor(manuscript inpreparation).

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