Isolation and characterization of a virus-specific ribonucleoprotein complex from reticuloendotheliosis virus-transformed chicken bone marrow cells.

0022-538X/78/0028-0034$2.00/0

Copyright ( 1978 AmericanSociety forMicrobiology Printed inU.S.A.

Isolation and Characterization of

a

Virus-Specific

Ribonucleoprotein Complex

from Reticuloendotheliosis

Virus-Transformed

Chicken Bone Marrow Cells

TIMOTHY C. WONGANDC. YONG KANG*

Departmentof Microbiology, University of Texas Health Science CenteratDallas, Southwestern Medical School, Dallas,Texas75235

Receivedforpublication26April1978

Chicken bone marrow cells transformed by reticuloendotheliosis virus (REV)

produce in the cytoplasm a ribonucleoprotein (RNP) complex which has a

sedimentation value ofapproximately 80 to lOOS and a density of 1.23 g/cm3.

This RNPcomplexis not derived from the mature virion. Anendogenous

RNA-directed DNA polymerase activity is associated with the RNP complex. The

enzymeactivitywascompletely neutralizedby anti-REV DNApolymerase

anti-bodybut notbyanti-avianmyeloblastosisvirus DNApolymerase antibody. The

DNAproduct from the endogenous RNA-directedDNA polymerase reaction of the RNPcomplex hybridizedtoREV RNAbut not toavianleukosis virusRNA.

The RNA extracted fromthe RNPhybridizedonlytoREV-specific

complemen-tary DNAsynthesized fromanendogenousDNApolymerase reaction of purified

REV. The sizeoftheRNAinthe RNPis 30 to35S, whichrepresentsthe subunit

size of the genomic RNA. No 60S mature genomic RNA was found within the RNP complex. The significance of finding the endogenous DNA polymerase activityinthe viralRNP ininfectedcells and thematurationprocess of60S virion RNAof REVarediscussed.

Reticuloendotheliosis viruses (REVs) are a

group of avian retroviruses which are distinct

from theavian leukosis-sarcomaviruses(12, 17,

20, 22, 29). Thisgroupof viruses consists of four

members: reticuloendotheliosis virus strain T

(REV-T), duck

spleen

necrosisvirus, duck infec-tious anemia virus, and chick syncytial virus

(25). These areC-typeparticleswhich possess a

single-strandedRNA genome(17,20,25)and an

endogenous RNA-directedDNApolymerase

ac-tivity(15, 24, 31).

The four members ofREVs are very closely

related in terms of nucleotide sequence of the RNA(17, 18),antigenicityof their DNA

polym-erase (22, 23), group-specific antigens (21), and

theirmorphology (19).

Althoughmuchinformation has accumulated

regarding the mature virions of REV, verylittle

is known about the intracellular stage of the

virusprecursor before budding. We used

REV-T-transformed chicken bone marrow cells (BMC)tostudy theintracellularviral precursor.

This cell line has been cloned and shown by

biochemical assay and electron microscopic

study to produce REV-T continuously under

normal culture conditions (9, 15, 19). The

hap-loid genome of BMC contains approximately

fivecopies of REV genome(manuscript in

prep-aration). The REV-T

produced

from the BMC contains no detectable avian leukosis virus

(ALV) by hybridization studies (15), and the

endogenous RNA-directedDNApolymerase

ac-tivity in theREV-TfromBMCis notneutralized by antiserum against the DNA polymerase of

Rous sarcoma virus-Rous-associated virus-0

[RSV- (RAV-0)] (31).Theseproperties, together with theabilitytogrow insuspension cultureto

high density, make the BMC suitable for the

study ofintracellular viralprecursor of REV.

A search forintracytoplasmicviral precursor

structures by thin-section electron microscopy

using BMCrevealed no viral substructure in the

cells. Usingbiochemical methods,we have

iso-lated from the cytoplasm of the BMC a

virus-specific

ribonucleoprotein (RNP) complex

which has properties markedly different from,

and which appears to be the precursor of, the

mature virion. This report describes the

char-acterization of this complex with regard to its

physical properties,the size andspecificityof its

geneticcontent, anditsassociatedDNA

polym-eraseactivity.

MATERIALS AND METHODS

Cells and viruses. REV-T-transformed chicken

BMC were a kind

gift

from H. Bose, University of 34

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RNP COMPLEX FROM REV-TRANSFORMED CELLS

Texas at Austin. The BMC were derived from an infected bird andhave been cloned and characterized (9).Thesecells were grown in suspension in Dulbecco-modified Eagle medium, pH 6.9 (GIBCO), supple-mented with 7.5% fetal calf serum. The BMC produce REV-T under normal culture conditions. REV-T was harvested every 12h, concentrated by ultracentrifu-gation, and purifiedby equilibrium density gradient centrifugation in 10to65%(wt/vol)sucrosegradients. B77 strainof aviansarcomavirus and Rous-associated

virus-61 (RAV-61) were obtainedfrom H. M. Temin.

Primary chickenembryofibroblasts (CEF) from 12-day-old chickenembryos(SPAFAS,Norwich,Conn.) werepreparedby standardtechniques previously de-scribed (1). The CEF cells were grown in Temin-modified Eaglemedium (Intemational Scientific In-dustries) supplemented with 20% tryptose phosphate broth and 5%fetal calfserum.

Endogenous RNA-directed DNA polymerase

assay. Theendogenous RNA-directed DNA

polym-erasereaction wascarriedout inareaction mixture

containing 80 uM dATP, dCTP, and dGTP, 60,M

ATP, 80 ug of pyruvate kinase/ml, 20 ,ug of

phos-phoenol pyruvate/ml, 2.5 mM MnCl2, 40 mM KCl,

and about 2.75 nM [3H]dTTP (58 Ci/mmol)

(Schwarz/Mann)in 20 mMTris-hydrochloridebuffer

(pH 8.0)containing5mMdithiothreitol and100Agof

actinomycin D/ml.Samplesfrom fractions ofsucrose

gradient analysisofvirus-specificcomponentsinBMC

wereassayed inafinal volume of 100

pl

at37°Cfor60

min, unless otherwise stated in the figure legends.

Afterincubation,aportion of each mixturewas spot-tedontofilter paperpresaturatedwith0.1Msodium

pyrophosphate. Trichloroacetic acid-precipitable

ra-dioactivitywasthen determined.

Preparation of 3H-labeled complementary DNA([3H]cDNA)productsfrom theendogenous

reaction. TheendogenousDNApolymerase activity

was provided by either Nonidet P-40

(NP-40)-disrupted virionsorthe extracted RNPcomplex

from BMC with theuseof thesameconditionsasin the DNA polymerase assay. Incubation wascarried

out at37°Cfor 1hinafinal volume of5ml. At the

endof theincubation,thelabled DNAproductswere

preparedaspreviously described (17, 18).

Isolation of RNP complex from REV-trans-formed chicken bonemarrowcelis.Amountsof1 x109 to2 x109actively growing REV-T-transformed BMCwere harvested and washed twiceby

centrifu-gationinice-coldphosphate-bufferedsaline(PBS)at

300xgfor10min.Thecellpelletwasresuspendedin

1 ml of reticulocyte standard buffer (0.01 M

Tris-hydrochloride,pH7.4,0.01MNaCl,0.0015MMgCl2).

Cellswerehomogenized by applying15 strokes ina

tight-fitting glass Dounce homogenizer which was

heated in an oven to destroy ribonuclease activity.

Nuclei and cellularmembraneswereseparated from themicrosomal fractionbytwoconsecutive

centrifu-gations at 5,500 x g for 10 min each at 5°C. The

cytoplasmic fractionwasthen treated with EDTAto

afinal concentration three times that of

Mg2e

ionsin

reticulocyte standard buffer todissociatepolysomes

beforebeingoverlaidonto10 to30%(wt/vol) sucrose

gradientsin BeckmanSW41 centrifuge tubes. Sepa-ration ofcytoplasmic components wasaccomplished

by centrifugation at 40,000 rpm for 120 min with a Beckman SW41 rotor. Intracellular RNP complex was detectedby assaying each collected fraction for

endog-enousRNA-directed DNA polymerase activity. The

sedimentation coefficient of the enzyme-associated material was determined by comparing it with the polyribosomalprofile of mouse Lss cells in a parallel gradient, which was monitored by UV absorption. Fractions containing the highest endogenous RNA-directed DNApolymerase activity were pooled for the

synthesisof DNAproductsorforpreparation of RNA

for use inhybridization experiments. To separate the RNPcomplex by density, cytoplasmic extracts from BMCwerelayered onto 30 to 70% sucrose gradients.

Centrifugationwascarried out at 40,000 rpm for 34 h

at5°C. Intracellular RNP was detected by endogenous RNA-directed DNA polymerase assay as described above.

Toinsure that the enzyme-associated RNP complex found in thecytoplasmof BMC was not due to deg-radation of mature virions in the intracytoplasmic vacuoles and/or on the membranesduring the homog-enizationprocedure,equalamountsofpurified REV-T(approximately 200,ugof proteins) were added to about2 x108BMCorthe same number of uninfected normal CEF just before homogenization. The

cyto-plasmic extracts were prepared from both of these

preparations in exactly the same manner as the control BMC. Thecytoplasmicfractionswerethenanalyzed

with10 to30%(wt/vol) sucrose gradients formed on

top of 70% (wt/vol) sucrose cushions in Beckman

SW50 1 centrifugetubes.Centrifugation wascarried

out at49,000 rpmfor100minat5°CwithaSW50 1

rotor. Amounts of 20

id

from eachgradient fraction

wereassayed forendogenous RNA-directed DNA

po-lymeraseactivity in the presence of 0.1% NP-40. The

nuclei and membrane pelletswereresuspended in0.15

mlof PBS containing 0.2% NP-40. Samplesof60,u

from each of these nuclei and membrane fractions

wereassayed for endogenous RNA-directed DNA

po-lymeraseactivitytodetect any membrane-associated

virus.

Antibody neutralization. The anti-REV DNA

polymerase antiserum and the anti-avian

myeloblas-tosisvirus (AMV) DNApolymerase antiserum were

kind gifts from S. Mizutani and H. M. Temin, Univer-sity of Wisconsin (22, 23). Totestfor thespecificity of DNApolymerase in the RNP complex from the

cy-toplasm ofBMC,the isolated RNPcomplexwas

di-vided into

100-pl

portions and treatedseparately with anti-REV DNApolymerase antiserum (50 yg of im-munoglobulin G), anti-AMV DNA polymerase

anti-serum(50

,g

ofimmunoglobulin g),orwithamixture

of both (60 ug of each). Rabbit-anti-rat antiserum madeagainst normal rat kidneycells wasused as a

control.Incubation was carried out at room

tempera-turefor45min.Afterincubation, the RNA-directed

DNA polymerase activity was determined by using theendogenousreaction asdescribedabove.

Nucleic acidhybridization. [3H]cDNAproducts

weresynthesizedfrom eitherpurifiedREV-T or from

theintracellular RNP complex isolated fromBMC, by useof theendogenous RNA-directedDNA polymer-ase reaction. The phenol-chloroform procedure for extraction ofDNA andRNA from cells or purified 35

VOL. 28,1978

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virusfollowed the previously published method (17). Liquid-phase hybridization techniqueswereused for all hybridization experiments (17). The extentof

hy-bridizationwas determinedby Si nuclease digestion (17,28).

Nascent DNA products analysis. Nascent [3H]DNAproducts wereprepared from the intracel-lular RNP complex from the BMC by use of the endogenous RNA-directed DNApolymerasereaction as described above. After incubation for 10 min at

37°C, thereactionwasterminatedby adding sodium dodecyl sulfate and EDTAtothe finalconcentrations of 0.5% and 50 mM, respectively. Phenol-extracted yeast RNA (500 ,tg) was added as carrier, and the products wereextracted twice withphenol and three times with chloroform.3H-labeled DNA productswere

precipitated inethanolovernightat-20°C. After pel-leting bycentrifugationat 8,000 xgfor 10min, 3H-labeled DNA productswereredissolved in TSE buffer (0.1 M NaCl,0.001 MEDTA, in0.02 M

Tris-hydro-chloride, pH 7.3) and eitherdirectly analyzed by

cen-trifugation in10 to30% (wt/vol)sucrosegradientsor

treated with RNase A or sodium hydroxide before analysis in sucrose gradients. Conditions forsucrose

gradient sedimentation analysis are described in the figurelegends.

To show that the size and theintegrity of the RNA template in the intracellular RNP were not altered during the incubation and extraction procedure, a

smallamount(approximately50,ugofproteins)of

NP-40-disrupted REV-T was mixed with the extracted RNP. The 10-min [3H]DNA productsfrom the

mix-ture wereanalyzed insucrosegradients,and

sedimen-tation values were compared to 32P-labeled mature

REV-Tviral RNA.

RESULTS

Isolation ofa

REV-specific

RNP

complex

from

REV-T-transformed

chicken bone

marrow cells.

Preliminary

experiments

showed that the

cytoplasmic

extracts of REV-T-transformedBMC hadanendogenous RNA-directed DNA polymerase activity similar to

that of mature virions. Since REV has been found to mature by

budding

notonly through the cytoplasmic membranes but also into the

intracytoplasmic vacuoles (19), theDNA polym-erase-associated

cytoplasmic

componentswhich

may represent virion precursors must be sepa-rated from the intravacuolar virions by use of

rate-zonal or equilibriumdensity gradients. To

isolate the cytoplasmic viral components, the

cytoplasmic

extracts were prepared from

ac-tively growing REV-T-transformed BMC and

treated with EDTAto dissociatepolyribosomes.

The cytoplasmic components were then

sepa-ratedbycentrifugation ina 10to 30% (wt/vol)

sucrose gradient. Viralcomponents in the

gra-dient were detectedby assayingeach

collected

fraction forendogenousRNA-directed DNA

po-lymerase activity (Fig. 1).

The

majority

of theendogenousDNA

polym-10 20 30 top

FRACTION NUMBER

FIG. 1. RNA-directed DNA polymerase activity in the cytoplasmic fraction of REV-T transformed chickenbonemarrowcells resolvedby sedimentation velocitysucrosegradientcentrifugation. Cytoplasmic fractions from2 x 109 REV-T-transformed chicken

bonemarrowcells and normal chicken embryo

fibro-blasts weretreated with EDTA and centrifuged at

40,000rpm for120minin 10 to 30%s(wt/vol) linear

sucrosegradientsat5° C. Eachcollectedfraction was

assayedfor endogenous RNA-directed DNA

polym-eraseactivityasdescribed in Materials and Methods.

Enzymeactivitywasexpressedascountsperminute

of[3H]dTMP incorporated during 60min of

incu-bation at 37°C. REV-T-transformed chicken bone

marrowcells(-); normal chicken embryo fibroblasts

(A). Arrowsindicatepolyribosome markers of mouse L929 cells in aparallel gradient monitored by UV absorption.

eraseactivitywasfound in the80 to100Sregion.

Alower level of activity sedimentednearthetop

of thegradient. Uninfected normal CEF didnot

contain the enzyme activity which sedimented in the 80 to lOOS region, although there was

some enzyme activity near the top of the

gra-dient. The latter observation is in accordance withprevious observations of uninfected chicken embryo cells (16). Theenzyme

activity

in the80 to

100S

region is not from the intravacuolar virions becausethematurevirion hasa sedimen-tation coefficient of about 600S and therefore

would be

pelleted

atthe bottom of thegradient.

Torule out the possibility that the

enzyme-associated 80 to 100S material was produced from the mature intravacuolar REV-T during

thehomogenization andextraction procedures, purified REV-T was added to the BMC or to

uninfected normalCEFbeforehomogenization.

These were then processed

identically

as the

controlBMC, which were not mixed with virus,

to obtain the cytoplasmic fractions. The

cyto-plasmic fractions were analyzed by

centrifuga-tion in 10 to 30% (wt/vol) sucrose gradients

formedontopof70% (wt/vol)sucrosecushions.

Viral components in the gradients were

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[image:3.500.263.455.54.232.2]

RNP COMPLEX FROM REV-TRANSFORMED CELLS

tored by assaying asample from each fraction by use of the endogenous RNA-directed DNA

polymeraseassayin thepresenceof 0.1%NP-40

(Fig.2).

BMC alone contained the RNA-directed DNApolymerase activity which sedinented in

theregion of80 to100S. Therewas a verysmall

amountofmaturevirionattheinterface above

the 70% sucrose cushion (Fig. 2a). In the

cyto-plasmic fraction fromthe BMC premixed with

purified REV-T, largeamountsofmaturevirions

weredetectedattheinterfaceonthe 70% sucrose

cushion while thepolymerase activity ofthe 80 to 100S material was unchanged (Fig. 2b). In

contrast, when REV-T was added to normal

BMC

10

0

040

b

7;30 :IlMC+REVT

15-zo

9L 5.

CEF

and theextracted cytoplasmic fractionwas

analyzed, no detectable enzyme-associated

ma-terial was sedimented at 80 to 100S.

Further-more, wewere surprised to find very few mature

virions on the 70% sucrose cushion (Fig. 2c).

Thisinitially puzzling phenomenonwasresolved

when the nuclei and membrane pellets were

assayed for RNA-directed DNA polymerase

ac-tivity (Fig. 2d). Most of the purified REV-T

addedtothe

normal

CEFwasrecoveredin the

membrane fraction(represented by bar c' in Fig. 2d), whereasalargeamountof theREV-Tadded

totheBMCwasrecovered from the interfaceof

the 70% sucrose (Fig. 2b) but very few of the virions became associated with the

cellular

FIG. 2. Sedimentation pattern ofRNA-directed DNA polymerase activity in the cytoplasm of

REV-T-transformedchicken bonemarrowcells(BMC)andnormalchickenembryofibroblasts (CEF)mixedwith

purified REV-T. Cytoplasmicextracts wereprepared from2x 108BMCorCEFasdescribed in Materials andMethodsafter purifiedREV-T(approximately200pgof proteins) wasmixed with the cellsjustbefore

homogenization. Thecytoplasmic fractionsweretreated withEDTA andcentrifugedat49,000rpmfor100

minat5°Cin 10to30%(wt/vol)sucrosegradientsmadeontopof70%(wt/vol)sucrosecushions. Asampleof

20,d fromeach collectedfractionwasassayed for endogenousRNA-directed DNApolymerase activityinthe

presenceof 0.1% NP-40. (a) control BMC without additionofREV-T.(b)BMCmixed with REV-T. (c)CEF

mixed with REV-T. Inpanel d,the nuclei and membranepellets fromBMC(a'),BMC+REV-T(b'),and CEF

+REV-T(c')wereresuspendedinphosphate-bufferedsaline with 0.2% NP-40. A60-,lI sample from eachof thesefractionswas assayedforendogenousRNA-directed DNApolymerase activity. Enzyme activity was

expressedas countsperminuteofCH]dTMPincorporated during60minofincubationat37°C.

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[image:4.500.131.365.245.554.2]

membrane (bar b'). This suggests that the cell

surface of the BMC is already saturated with

viralproteins, whereas the normalCEF provides

freevirus-bindingsites for the added virions.

These experiments conclusively showed that

the 80to 100Senzyme-associatedmaterial from

thecytoplasmof theBMCwasnotderivedfrom

matureREV-T duringthehomogenization

pro-cedure, butrepresentedadistinctentity.

The density of the DNA

polymerase-associ-ated intracytoplasmic material wasdetermined

by equilibrium density gradient centrifugation

in a 30 to70% (wt/vol)sucrosegradient (Fig. 3).

The endogenous RNA-directed DNA

polymer-aseactivityinthe totalcytoplasmicextract was

resolved intotwopeaks,onehavingadensityof

1.23g/cm3and the otherhavingadensityof1.16

g/cm3 (Fig. 3a). The 1.23 g/cm3 band

corre-spondstothedensityofnucleocapsid of mature

virion. The 1.16g/cm3material may well

repre-o e

° le

4c

0

a. x

s 10

at-Ji

a

P1.3

12

e

pl X

;IIII Q

z

0

5

e

t1-10 20 30

[image:5.500.62.252.287.500.2]

FRACTION NUMBER

FIG. 3. RNA-directed DNApolymerase activity in

cytoplasmicfraction of REV-T-transformedchicken

bone marrow cells resolved by equilibrium density

sucrosegradientcentrifugation. Cytoplasmicextract

from 2x 109chicken bonemarrowcellswastreated

withEDTAandcentrifugedat40,000rpmfor 34 h in

a30 to 70%(wt/vol) sucrosegradient at5°C. Each

collectedfractionwasassayed forendogenous

RNA-directed DNApolymeraseactivity(a).Alternatively,

the RNP complex waspooled from the 80to 1XJS

region fromavelocitysucrosegradient and

recentri-fuged at40,000rpmfor 34 h ina30to70%(wt/vol)

sucrosegradient. Eachfractionwasassayedfor

en-dogenous RNA -directed DNApolymeraseactivity (b).

Enzyme activitywasexpressedascountsperminute

of[3H]dTMP incorporated during60 min of incu-bationat37°C (0);densityingrams percubic centi-meter(0).

sentmaturevirionstrappedbetween the cellsor

in the intracytoplasmic vacuoles. Most of this

low-densitymaterial could be removedby

wash-ing

thecellsthreeorfour timesbefore

homoge-nization. When thepooled fractions of the 80to

lOOS material from the velocity gradient as

shown inFig.1wereanalyzedin theequilibrium

density gradient, only one homogeneous peak

correspondingto 1.23 g/cm3wasobserved (Fig.

3b). This indicated that the enzyme-associated

80tolOOS materialwasidenticaltothematerial

havinga density of 1.23 g/cm3 and appearedto

be an RNP complex present within the

cyto-plasmofthetransformed cells.

REV-specific endogenous RNA-directed

DNApolymerase activity in the RNP. Since

the genome of normalCEF containsendogenous

sequences of avianleukosis virus (ALV) (29), it

is necessary to demonstrate that the

RNA-di-rectedDNApolymerase activity detected in the

RNP complex in the BMC is specific for REV

andnotduetotheexpression of theendogenous

ALV sequences. The 80 to 100S RNPcomplex

wasisolated fromREV-T-transformed BMC by

use of rate-zonal centrifugation in sucrose

gra-dient. The RNP complex was divided into four

portions which were treated separately with

anti-REV DNA polymerase antiserum,

anti-AMV DNApolymerase antiserum,or amixture

of anti-REV and anti-AMV DNA polymerase

antisera. Rabbit anti-rat antiserum (made

against normal rat kidney cells) was used as a

control.The treated fractions were then assayed

for endogenousRNA-directed DNA polymerase

activity. Kinetics show thatanti-REV DNA

po-lymerase antiserum can specifically neutralize

theRNA-directed DNA polymeraseactivity

as-sociated with the RNPcomplex (Fig. 4). Neither

the anti-AMV DNA polymerase antiserum nor

theanti-rat antiserumaffectsthe enzyme

activ-ity. Results from the reciprocal experiments

showed that the anti-AMV polymerase

antise-rum completely neutralized thepolymerase

ac-tivity of B77 avian sarcoma virus (data not

shown). Since the anti-AMV DNA polymerase

antiserum completelyneutralized the

polymer-ase activity of the endogenous RAV-0 (22) and

B77 avian sarcoma virus DNApolymerase

activ-ity but not the DNA polymerase activity of

REV, theendogenous DNApolymeraseactivity

found in theRNP is not due to theexpressionof

ALV.

REV-specific RNA in the RNP isolated

from the BMC. Todemonstrate the presence

ofREV-specific RNA in theintracellularRNP,

weextracted RNAfrom pooled 80tolOOSRNP

complex and hybridized the RNA with [3H]-cDNA synthesized frompurified REV-T, using

the liquid phase hybridization technique

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RNP COMPLEX FROM REV-TRANSFORMED CELLS

N

'o 25 ANTI-RAT

a.

o 20-

TI-C ~~~~~~~~~~AMVpol

'5

I-. pF-a ANTI-REVANTI-REVPol

I ~~~~~~ANTI-REV+ANTl-AMV,,

01

0 5 1S 30 60

TIME(m;rO

FIG. 4. Antiserum neutralization of the

endoge-nousRNA-directed DNA polymerase activity associ-ated with the intracellular RNP from REV-T-transformed chicken bone marrow cells. Viral RNPwasisolatedfrom cytoplasmic fraction of

REV-T-transformedchicken bone marrow cells by velocity

sucrosegradient centrifugationsimilartothat in Fig.

1.Equal portions of the isolated RNP were treated withdifferent antibodies for 45 min at room temper-ature and then assayed for endogenous RNA-di-rected DNA polymerase activity. Enzyme activities

wereexpressedas countsperminuteof[3H]dTMP

incorporated at various times. Anti-rat antiserum

(0);anti-AMV DNApolymerase antiserum(0) anti-REVDNApolymerase antiserum (A); mixture ofboth anti-REV DNApolymerase and anti-AMV DNA

po-lymeraseantisera(A).

scribed in Materials and Methods.Asshown in Fig. 5,

approximately

30% of the DNA

hybrid-ized at a

Crt

value of2 x

101

mol s/liter, indi-cating that the80 tolOOS RNP

complex

contains

REV-specific

RNA.Acomparison of the kinetics ofhybridization of REV-T

[3H]-

cDNAtoviral RNA and RNP RNA shows that onlya small fraction of the total RNA in the 80 to

100S

material is

REV-specific

RNA.Adirect

labeling

experiment revealed

that the vast

majority

of RNA in the 80 to lOOS

region

of the

gradient

representsrRNA's because monosomes

cosedi-mentedatthisregion(datanotshown). To rule

outnonspecific

hybridization,

thesameREV-T cDNAprobewas

hybridized

with RAV-61 RNA. No

hybridization

wasobserved.

Furthermore,

no

hybridization

was observed when the RNA ex-tracted from RNP was incubated with cDNA

madefromRAV-61. This shows that the

hybrid-ization between the RNP RNA and REV-T

cDNA wasspecific.

To further substantiate that the 80 to lOOS RNP

complex

is REV

specific,

we

prepared

[3H]cDNA

from the endogenous DNA

polym-erasereactionof the 80tolOOSRNP.This RNP

[3H]cDNA

was

hybridized

tothe REV-T viral

RNA. Maximal hybridization is approximately 40% at aCrt value of2 x

10'

mol-s/liter (Fig. 6). Since the virion RNA used in the hybridiza-tionwasextractedfromvirusproduced by

REV-T-transformed BMC, itwasnecessary to

dem-onstrate that the REV-T was free from the

endogenousALV contamination. RAV-61 [3H]-cDNA was hybridized with the RNA from the REV-Tand showednohybridization, indicating that therewas nodetectableamountof ALV in the viruspreparation. The results demonstrate that the RNP

complex

contains RNA whichcan

be transcribed

by

theRNP-associated DNA

po-lymerase into cDNA. This cDNA

specifically

hybridizestopurified REV-T RNA.

Itisnoteworthy that the hybridization using

0 w a 30

:

420

$

10

Crt (mole-sec/liter)

FIG. 5. Kinetics of hybridization of

['HJcDNA

synthesizedfromREV-TtoRNA extractedfromthe

intracellularRNP.

[3H]cDNA

waspreparedfrom a

1-hproductof endogenousDNApolymerasereaction

of purifiedREV-T in the presenceof100 pgof

acti-nomycin D/ml.Amountsof2,0fJ0 cpm persampleof

theREV-T[3H]cDNA wereannealed with 6pg of REV-T viral RNA/ml (0), 6 pg ofRAV-61 viral

RNA/ml, (0), and36pgofRNA/mlfromthe RNP

isolated from cytoplasm of REV-T-transformed

chicken bonemarrowcells

(0).

Inaddition, 2,000cpm

persample ofRAV-61

[3H]cDNA

washybridizedto

120pg of RNA/ml fromthe RNP isolatedfrom

REV-T-transformedchicken bone marrowcells (A). The

hybridizationwasperformedat63°Cin 25tl of

DNA-RNA annealing buffer (0.5 M NaCl, 0.1% sodium

dodecylsulfate,0.001MEDTA, 2% phenol,and 0.05 M

This-hydrochloride,

pH7.3)sealed in25-IlI

micro-pipettes. Sampleswerewithdrawnatdifferenttimes

and theextentof hybridizationwasdeterminedbySi

nucleasedigestion.The resultswereexpressedasthe proportionof total

[3H]cDNA

hybridizedat agiven valueof

C,t

in 0.5M salt.Approximately8.5%ofthe

REV-T

[3H]cDNA

and4%oftheRAV-61

[3H]cDNA

were resistant to S1 nuclease in a control sample

incubated without RNA. These have been subtracted

fromtheplottedvalues.

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40

a MNP CDNA

w +

N REV-TvRNA

5

30-420

z

i RNPcDNA

+

1 YEASTRNA RAV-61 cDNA

REV-T vRNA

0

10

Ie

10

Crt (mole-sec/liter)

FIG. 6. Kineticsofhybridizationoff[H]cDNA

syn-thesized from intracellular RNP from

REV-T-transformedchicken bonemarrowcellstoREV-T

viralRNA.[3H]cDNAwasprepared froma1-h

prod-uct of endogenous DNA polymerase reaction of pooledintracellular RNP in thepresenceof100pgof actinomycin D/ml.Amountsof 2,000cpm persample

oftheRNP[3H]cDNA wereannealed with 6pgof

REV-T viral RNA/ml(0),or6 pgofyeastRNA/ml

(0).Inaddition,2,000cpm persample of[3H]cDNA synthesized fromRAV-61washybridized with6pgof

REV-T viral RNA/ml (A).Conditionsof

hybridiza-tionanddetermination oftheextentofhybridization

werethesame asinFig.5.Approximately7.8%of the

RNP[3H]cDNAwasresistanttoSInucleasein the

controlsampleincubatedby itselfand has been

sub-tractedfromplotted values. Approximately 90% of

3P-labeled RAV-61 RNA became resistantto

ribo-nucleasedigestion after annealing toabout30-fold

excessofRA V-61DNAproducts.

[3H]cDNA prepared from theRNPwouldonly

proceed to40% of themimum when

hybrid-ized withhomologous viral RNA. This apparent

lowefficiency in hybridization isnotduetothe

presence of DNA ofopposite polarities in the [3H]cDNA preparations, because hybridization

stillproceededtoonly about 40% when thesame

[3H]cDNA probes made from the RNP were

hybridizedtocellular DNA ofREV-transforned

BMC (data not shown). Similar results were

observed when [3H]cDNA synthesized from

REV-T was hybridized to REV-T RNA (15).

Thesignificance of this is still unclear.

Size of virus-specific RNA in the RNP

complex. To determine the size of the RNA

template in the RNP complex, RNA was

ex-tractedfrom the 80to100S RNP andseparated

ina10to30%(wt/vol)sucrosegradient by

rate-zonal centrifugation. A sample from each

col-lected fraction of thegradientwashybridizedto

a constant amount of [3H]cDNA made from

purified REV-T. Figure 7 shows a prominent

peak ofvirus-specific RNA that sedimented at

approximately30 to35S.Inaddition,small

pro-portionsof other RNA specieswereobservedto

sediment both faster and slower than the

major

RNAspecies. This resultsuggeststhat the size of the majorspecies of RNA in the RNPcomplex

is30 to35S.

Analysis of the nascent DNA

products

from

the

endogenous

DNA

polymerase

re-action

of the

RNP. To determine the size of theRNAtemplate in the RNP complex,wealso analyzed the

RNA-template-associated

nascent

DNAproducts.

[3H]DNA

productswere

synthe-sized for 10 min from the 80 to 100S RNP by

use of the

endogenous

DNA

polymerase

reac-tion. After

phenol-choloroform extraction,

the

nascent

[3H]cDNA products

recovered from the

aqueous phase were analyzed in a 10 to 30%

(wt/vol)

sucrose

gradient by

rate-zonal centrif-ugation. Under

nondenaturing

conditions,a dis-tinct

population

of the nascent DNAproducts sedimented in the30 to35S

region,

and thevast

majority

of DNA sedimentednearthetopof the gradient

(Fig.

8a). The 30 to35S

peak

could be removed when the initial

products

weretreated with RNase A or0.5 N

NaOH (Fig.

8b and c). This indicates that the DNA sedimenting in the

30

1 30418

IC

z

0

10 20 30 40top

FRACTION NUMBER

FIG. 7. Sedimentation pattern of REV-specific RNA in the intracellular RNP from

REV-T-transformedchicken bonemarrowcells.

Intracel-lularRNPwasisolatedfromcytoplasmicfractionof

REV-T-transformed chicken bone marrow cells by

use of sucrose gradient sedimentation velocity

cen-trifugationsimilartothat shown inFig.1.RNAwas

extractedfrom thepooled RNPmaterialand sub-jectedtocentrifugation at49,000rpmfor 90 min at 5°C ina10 to30%(wt/vol)sucrosegradient in TSE buffer. An equal samplefromeachcollectedfraction washybridized with 1,000 cpm of[3H]cDNA synthe-sizedfrompurified REV-Tin a

50-Al

final volume of DNA-RNA hybridization buffer. Hybridization was

performedat630Cfor100h. Extentof hybridization

wasdetermined by digestion with SInuclease.

Ap-proximately3%of the[3H]cDNAwasresistanttoS,

nucleaseinacontrolsample incubated without RNA

andwas subtractedfrom theplotted values.

["4C]-rRNAwasusedasmarkers(arrow).

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RNP COMPLEX FROM REV-TRANSFORMED CELLS

N

4T

28.

IV

IC

+NOOH

3 2

10 20 30 40 top

FRACTION NUMBER

FIG. 8. Sedimentation analysis ofnascent DNA productsfrom the endogenous DNA polymerase re-action carried out withintracellularRNPfrom

REV-T-transformedchicken bone marrow cells.

Intracel-lular RNP was isolatedfrom REV-T-transformed

chicken bonemarrowcellsbyuseofsucrosegradient

sedimentationvelocity centrifugation similartothat shown inFig. 1. ThepooledRNP was usedfor

en-dogenousRNA-directed DNApolymerase reaction.

[3H]cDNA synthesiswasallowed toproceedfor10

min at37°Cin thepresenceof100ugof actinomycin

D/ml. The 10-min DNAproducts were either

ana-lyzed directly with 10 to 30% (wt/vol) sucrose

gra-dients inTSEbuffer centrifugedat49,000 rpmfor90 min(a),treated with 50 pgofRNase A per mlat37C

for 30 min before sedimentation analysis (b), o'r

treated with 0.5 N NaOH at63°Cfor60minbefore

sedimentationanalysis (c). ["C]rRNAwas used as

markers(arrows).

30 to35S

region

of the

gradient

wasthe

hybrid

ofRNA

template

and DNA

product.

We

con-cludethattheRNA

template

in the RNP

com-plex has a sedimentation value of about 30 to 35S, which is consistent withtheresultfromour

hybridization

study

shown in

Fig.

7. Since the

subunit size of the REV RNAis about 35S (12),

it is therefore conceivable that theRNPcomplex

contains only the subunit size of the viral

ge-nome, which may represent the intracellular

precursorof thegenomic RNA.

Todemonstrate that the30 to35S RNAwas

[image:8.500.50.252.55.413.2]

FRACTION NUMBER

FIG. 9. Sedimentation analysis ofnascent DNA products from intracellular RNPmixed with dis-rupted REV-T. Nascent [3H]cDNA products were

synthesized as described in Fig. 8, except that the

RNP wasmixed withpurifiedREV-Tdisruptedwith

0.2% NP-40. The 10-min DNAproductswere either

analyzed directly with 10 to 30% (wt/vol) sucrose

gradients in TSEbuffer centrifugedat49,000 rpmfor 90min(a)ortreatedwith50pgofRNase A per mlat

37°Cfor30 min beforesedimentation analysis (b).

3P-labeledpurified REV-T viral RNAwasanalyzed

in aparallelgradient (c).

['4C]rRNA

wascentrifuged

in anotherparallel gradient to serve as markers

(arrows).

VOL. 28,1978 41

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42 WONG AND KANG

actually the immature form and not derived

froma degradative product of themature viral

genome, NP-40-disrupted purified REV-Twas

added to the intracellular RNP, and 10-min

DNA products weresynthesizedfrom the

mix-ture by use of the endogenous RNA-directed

DNA polymerasereaction. The[3H]DNA

prod-ucts were extracted and analyzed by sucrose

gradients similar to those shown inFig. 9. The

nascent DNAsynthesized inthisreaction

sedi-mented at three distinct places (Fig. 9). The

hybrids that sedimented between 50 and 60S

were undoubtedly produced from the mature

REV-Tgenomethatwas added because ithad

a sedimentation value similar to that of

32p-labeled REV-Tviral RNA (Fig. 9c). This peak

was not observed in DNA synthesis when the

intracellularRNP alonewasused.Therefore,we

conclude that the 30 to 35S RNA template in

the RNP complexis notderived from

degrada-tion of the mature virion RNA. Identicalresults

wereobtainedwith the1.23g/cm3RNPcomplex

isolated from equilibrium density sucrose

gra-dients instead of from velocity sedimentation

gradients (datanotshown).

DISCUSSION

We have isolated anRNP complex fromthe

cytoplasmofREV-T-transformedchickenBMC

whichcontainsendogenousRNA-directed DNA

polymerase activity.This RNPcomplexisfound

only in the REV-transformed cells and not in

thenormal cells. The RNP complex hasa

den-sity of 1.23 g/cm3 and sediments in the 80 to

100S regioninsucrosegradients. These

proper-ties indicate that it isdistinct from themature

virion.Thesedimentationpatternof theRNP is

notalteredbymixingtheBMCwith virusduring

homogenization. Also, the RNP peakcannotbe

created by homogenizing normal CEF mixed

withpurifiedREV-T. These resultsstrongly

in-dicatethat the RNP isnotadegradative product

resulting from the homogenization and

extrac-tionprocedures. Previous studiesshowthat the

coreisolated from REVhasadensity of1.25to

1.26g/cm3 (4), slightlyheavierthanourdensity

determination of the cytoplasmic viral RNP

complex. The RNP complex possesses

proper-tiesthat suggest that it is ina diffused form. It

has a relatively slow sedimentation rate and

slightly lighter density comparedtothemature

viral core. Also, the absence ofanydiscernible

viral substructurein the cells when studied by

electron microscopyseemstosupportthesame

conclusion (19).

EndogenousRNA-directed DNA polymerase

assay shows that the intracytoplasmic RNP

complexisfully functional and abletosynthiesize

REV-specific complementary DNA in vitro.

However, it isnot yet clear whether the RNP

complex is constantly synthesizing

proviral

DNA inthetransformed cells,wherestably

in-tegratedproviralDNA isalreadypresentinthe cellular genome. If this is indeed the case, the

newly synthesized viral DNA from the RNP

could account for the presence oflinear viral

DNA duplexes characteristic ofacuteinfection

upto several weeksafterinfection in the

cyto-plasmofavian sarcomavirus-infected duckcells

(31), avian sarcoma virus-infected quail tumor

cells (11), and mouse mammary tumor

virus-infected ratcells (26).

Onthe otherhand,ourfindingsareincontrast

tothat ofDavid Baltimore, Massachusetts

In-stituteofTechnology,whofoundthat the

RNA-directed DNA polymerase of murine leukemia

viruswas notfully functional until theviruswas

released from the cell (personal communica-tion).

Since all chicken cells contain endogenous

ALV sequence in their DNA genome, it is

im-portant todemonstrate that the RNP complex

in the cytoplasm of the REV-transformed chicken BMC is specific for REV. Antibody neutralization studies showedthat the endoge-nous RNA-directed DNA polymerase activity associated withthe RNP

complex

couldbe neu-tralizedonlyoyanti-REVDNApolymerase an-tiserumbutnotbyanti-AMV DNApolymerase

antiserum, indicating that the enzyme in the

RNP complex was specific for REV. This is in

accordancewith theprevious findingthat REV-Tpurifiedfromthe BMC containedonly REV-specific polymerase (32).

When cDNA synthesized from the

endoge-nousreaction of RNPwashybridizedwith viral

RNAextractedfrompurified REV-T,upto40%

oftheDNAprobewashybridized. Thekinetics

of thishybridization weresimilar to that when

theprobewasmade frompurified REV-T (15).

In bothcasesthe maximal levelsofhybridization

were about

40%.

In contrastto the endogenous

polymerase reaction, the cDNA probe made

fromanexogenousreactionusingpurified REV

RNA and

purified

ALVDNApolymerase could

anneal completely to REV RNA (17). The

in-completehybridizationisnotduetosomecDNA

which is in thesamepolarity asthe virion RNA

since annealing with DNA from infected cells

does not increase the extent of hybridization

(data not shown). We also know that the

non-annealing portion of thecDNAdoesnot

hybrid-ize withanexcessamountof virion RNA.

Inthree separate sets ofhybridization

exper-imentsbetween the cDNA made from the

REV-T and REV-T virion RNA at RNA

concentra-tionsof 6, 12, and 20yg/ml, the maximal levels

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RNP COMPLEX FROM REV-TRANSFORMED CELLS

ofhybridizationwerethesameasthat shown in Fig.6.

Itis possible that the cDNA containsa signif-icantproportion of short DNA transcripts.Since the melting points of hybrids between virion RNA and the short cDNA should be low, the

extentofhybridizationmay notreach100%

un-der stringenthybridization conditions. Another

possibility

is that the

probe

contains some ho-mopolymers synthesized byaterminal transfer-ase-like activity. This

possibility

is, however, highly unlikely because the endogenous RNA-directed DNA

polymerase

reactionrequires all four

nucleoside

triphosphates

and very little DNAis synthesized in the absence ofany one

nucleoside

triphosphate

(15).

To study the size of

intracytoplasmic

virus-specific

RNA,

weusedtwomethods. In the first

approach, REV-specific

RNA was detected by hybridization after the RNA isolated from the RNP was

subjected

to

velocity

centrifugation. This method gave an estimated sedimentation coefficient of about 30 to 35S for the major species of REV-specific RNA in the RNP

com-plex.

Study

of the size of the RNA in the RNP complex had been complicated by the fact the RNPcosedimented with80Sribosomes. Label-ing experiments with

[3H]uridine

and

32p;

showed that the RNA in the80 to

100S

material containedamajority of 28S and 18S rRNA (data

not

shown).

Thus,

the virusspecific RNA could be detected

only by

the

hybridization

method. We

found

someother

sizes

of RNAs which

hy-bridized

with REV cDNA in addition tothe30 to 35S RNA. Of

these,

we often observed two

minor

peaks

atabout 27S and 21S. Thesemay

representcontaminationby subsets of

mRNA's

comparable

to those found in avian sarcoma

virus- and murine

leukemia

virus-infected

cells

(2, 7, 8, 10, 14,27, 30). More difficulttoexplain

arethe

fast-sedimenting

RNAsbetween40and 50S. The

significance

ofthese isstillnot clear,

although virus-specific

RNA of

larger

than sub-unitsize has been found in cells infected

by

Rous

sarcoma virus (14) and murine

leukemia

virus

(13).

In the second

approach,

the sedimentation value of RNA-cDNA complex, which

repre-sented the RNA template-nascent DNA

prod-uct, was determined. The nascent DNAproducts

werefoundtobeinshort

fragments

of lessthan

10S insedimentation value.

Therefore,

the

sed-imentation value of the RNA-cDNA complex wouldbelargely determined bythe RNA

moi-ety. The RNA-cDNA complex was found to

have asedimentation valueof30 to 35Sbased

on19separatedeterminations. Sincethe subunit

size of REV genome isabout 35S(12),it isthus

concluded that the size oftheRNA in the

intra-cellular RNPcomplexisequivalentto one

sub-unit of the RNAgenomeof the virus.Studieson

the maturation of Roussarcomavirusand

mu-rine leukemia virus have shown that viral RNA foundinthecytoplasm of the infectedcellsand innewly budded virions consistsmainly of30 to

35S RNAsubunits, whereasmaturevirions

con-tain the 60 to 70S genomic RNA (5-8, 14, 30). Our findings with REV-T-transformed cells

seemedtobe inaccordance withthese findings.

Our results suggest that the REV-T-transformed chicken BMCproduce an RNP complex which contains an endogenous RNA-directed DNA

polymerase

activity. This RNP

complex containsREV-specific DNA

polymer-aseandREV-specific RNA template, andmay

represent the virion precursor. The size of the

RNA in the RNP complex isequivalentto the subunit size of the virion

genomic

RNA. No60S

maturegenomic RNAwasfound inthe

intracel-lular RNPcomplex.

Thus,

the formation of ma-tureviralgenomic RNAseemsto occurafterthe

budding,

probably

during

the condensation of the nucleoid. Morerecent data onthe protein composition of the RNP complex indicate that it contains several

virus-specific

proteins,

includ-ingsome notfound inthematurevirion, which

may represent the precursor and intermediate

proteins(unpublisheddata).

Moreover,

virus

as-sembly

appears to

begin

when the 30 to 35S RNA is inthe

cytoplasm.

Intemalproteins such

as the polymerase can

complex

with the viral

RNA at this stage to form a functional RNP

complex which also contains the

primer

mole-cule since it is

capable

of

endogenous

transcrip-tion.

ACKNOWLEDGMENTS

Wethank S.Mizutani,H. M.Temin,and H.R. Bose for

kindgiftsofexperimental materials and E. D.Rosenblum, J.

A. Shadduck, G. Smith, B. Ozanne, and D. Baltimore for

helpfulcomments onthemanuscript.

Thisinvestigationwassupportedby Public Health Service

grantsCA 16479 and CA 20012 from the National Cancer Institute.

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