Selective inhibition of avian sarcoma virus protein synthesis in 3-deazaadenosine-treated infected chicken embryo fibroblasts.

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0022-538X/81/040173-11$02.00/0

Selective Inhibition of Avian Sarcoma Virus Protein

Synthesis

in

3-Deazaadenosine-Treated

Infected Chicken Embryo

Fibroblasts

C. MARTIN STOLTZFUS'* ANDJOHN A.MONTGOMERY'

Departmentof Microbiology, University of Iowa, Iowa City, Iowa 52242,1 and The Southern Research

Institute,Birmingham,Alabama

352052

Received10October 1980/Accepted5January 1981

Theproduction ofB77aviansarcomavirions wasinhibitedmore than 90%in

infected chicken embryo fibroblasts thatweretreatedwith 100

,uM

3-deazaaden-osine, an inhibitor ofadenosylhomocysteine hydrolase and, for this reason, an

inhibitorofmethylation reactions. This nucleoside analog at aconcentrationof 100

,uM

inhibited theratesof overall cellular protein synthesisandpolyadenylated RNAsynthesis by40 to50%. Rates of viral proteinsynthesis werecompared,and

the results indicated that in infected cells treated with 3-deazaadenosine syntheses of both the precursor of the gag proteins

(pr765¶g)

and the precursor of the reverse transcriptase

(pr180'`9 P"')

were inhibited. Synthesisof theprecursor of

the viral envelope glycoproteins (pr92env) appeared to be affected less by the

analogtreatment.Mostof the hostpolypeptides also continuedto besynthesized in3-deazaadenosine-treatedcells. The fraction ofthe total RNArepresentedby

virus-specific RNA in the 3-deazaadenosine-treated cellswasapproximately40%

of the fraction of the total RNA represented byviral RNA in control cells, as

determined byhybridization kinetics. Therefore,there was aselective inhibition of viral RNAaccumulation in the presence of 3-deazaadenosine. Theamountsof

genome-sized 35S and 38S RNAswerereduced compared withthe amountsof 28S and 21S viral mRNA's. These resultssuggestthatselectiveinhibitionof the

synthesis of viral proteins is due to selective decreases in the amounts of the

mRNA's for these polypeptides.

Bader etal. have shown that the nucleoside analog 3-deazaadenosine,aninhibitor of S-ade-nosylhomocysteine hydrolase, reversibly

in-hibits the reproduction of the Bryan strain of

Rous sarcomavirus and the transformation of chicken embryocells (2). These authors

postu-latedthat this effect is due tothe inhibitionof methylation reactions required for virus repli-cation. They also proposed that inhibition of virus replication and inhibition of oncogenic

transformationarecausedbyaninhibition of 5'

capmethylations (2).However, the biochemical basis of inhibitionwas notinvestigated.Wehave shownpreviouslythat2'-O-methylcap methyl-ations and internal N6-methyladenosine meth-ylations of cellular mRNA and viral genome RNAare inhibitedmore than 90% in the pres-enceofcycloleucine,yet virusproductionunder these conditions is inhibited only slightly (7). Thissuggestedthatthemethylationsblockedin the presence of cycloleucine (i.e., the

2'-O-methyl cap and internal N6-methyladenosine methylations) are not essentialfor virus repli-cation. We have investigated the basis for the

inhibition of virusproduction by 3-deazaadeno-sine and the reasonsfor the differences in the effects of thetwoin vivomethylationinhibitors 3-deazaadenosine and cycloleucineon virus

re-production. In this work we confirmed for

an-other strain of aviansarcomavirus (ASV), B77

ASV, the observation of Baderetal. thatvirus

production is inhibited drastically in the pres-ence of 3-deazaadenosine (2). We then

deter-mined themetaboliceffects of 3-deazaadenosine

onbulk cellular mRNA and protein syntheses and on virus-specific RNA and

protein

syntheses. We found that thesyntheses of the

internal non-glycosylated virion

polypeptides

(gagproteins) are inhibited

selectively

in the presence of 3-deazaadenosine. This inhibition appears toresult,atleast inpart, from selective decreases in theamountsof viral mRNA's.

MATERIALS AND METHODS

Virus and cells. Chicken embryocells were pre-pared from10-day-old embryosandweresubcultured

at3-day intervals. Cellswereinfectedwith B77ASV

at amultiplicityofinfection of2 inthepresenceof2 173

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,ugofPolybrene perml.The infected cellswere pas-saged one ortwotimesin ordertoinsure that allof the cells were infected.Cellsweremaintainedin me-dium 199containing 1%(vol/vol) dimethylsulfoxide. Forexperimentsinwhichthe effect of 3-deazaadeno-sine was tested, Earle minimal essential medium (MEM) wasused, and tryptosephosphatebrothwas notadded. The media containedafinal concentration of 5%(vol/vol)calfserum.

Purification of virus. Viruswaspurifiedfromthe medium of infected cellsbypreviouslydescribed tech-niques (21).Briefly, the virus waspelletedfrom the medium through a 20%(wt/vol) sucroseshelfontoa 70%(wt/vol)sucrosepad.Thepelletedviruswasthen banded to equilibrium in a continuous 20 to 70% sucrosegradient.

Isolation of RNA. Total RNAwasisolatedfrom cells by phenol-chloroform extraction, as described previously (20).Cellswerefractionated into nuclei and cytoplasmby lysiswith 1%(vol/vol)Nonidet P40-1% (wt/vol) deoxycholate,andthe polysomes were iso-latedby discontinuoussucrosegradientsaspreviously

described (8). The fraction containing polyadenylic

acid[poly(A)+]wasisolatedbyoligodeoxythymidylic

acid-cellulosechromatography (20).

Determination of hybridization kinetics of viral cDNAtocellular RNA. Varyingamountsof RNAfrom3-deazaadenosine-treated and control cells werehybridized to3P-labeledcomplementaryDNA

(cDNA) prepared by the endogenous reverse

tran-scriptase reaction of detergent-disrupted B77 ASV (20).Hybridizationwascarriedoutinasolution con-taining0.3 MNaCl, 0.01 M Tris-hydrochloride (pH

7.5), and0.001MEDTAat680Cfor 18 h. The resist-anceof the 32P-labeled cDNAtoS1 nucleasewasused as a measureof theextentofhybridization.

GelelectrophoresisofRNA,transferto

diazo-benzoxymethyl paper,andhybridizationto 32P-labeledpSal"'DNA. Gelelectrophoresisofpoly(A)+

RNAon 1% agarosegelscontaining 10mM

methyl-mercuryhydroxidewascarriedoutessentiallyby the method ofBaileyand Davidson(3). The RNA in the gelwastransferredto diazobenzoxymethyl paperby theprocedureof Alwineetal.(1).The paper was then

prehybridizedfor24h,andthiswasfollowedby hy-bridization with a 32P-labeled nick-translated DNA probe (5.7 x 107cpm/pig), asdescribedbyAlwineet al.(1). The3P-labeled probe used in the experiments described belowwas pSal'01,arecombinant plasmid DNAwhich contains the entire ASV Prague A strain genome insertedintothe SallsiteofpBR322. Astock culture of this recombinantplasmid wasgenerously provided by J. Thomas Parsons, University of Vir-ginia, Charlottesville. The location of theradioactivity onthepaperwasdeterminedbyautoradiography on Kodak BB-5X-rayfilm,using a Dupont Cronex Light-ning-Plusintensifyingscreen.

Preparation of

[8Hlleucine-labeled

cytoplas-mic extracts. Chicken embryo fibroblasts infected with B77 ASV in100-mmpetri dishes were incubated for17hwitheither10mlof MEM or 10mlofMEM

containing 100 uM 3-deazaadenosine. The medium wasthen changed to either 5 ml of MEM without leucine or 5mlof MEM without leucine containing 100MiM3-deazaadenosine. Aftera1-h incubation, 250

,Ciof

[3H]leucine

wasaddedtoeach plate, andthe

cells were labeled forvarying intervals, as described below. The cellswerethen washed once with 4 ml of asolution containing 140 mMNaCl, 5 mM KCI, 0.6 mMNaHPO4, 5.5 mM glucose, and 25 mM Tris-hy-drochloride(pH 7.2) (TBS) at 4°C, scraped into 2 ml of a solution containing 10 mM Tris-hydrochloride (pH 7.4), 1 mM NaCl, and 1.5 mM MgCl2, and swelled onicefor 10min. After this the cell suspensions were eachbrought to a final concentration of 1% (vol/vol) Nonidet P-40, homogenized by five strokes of a Douncehomogenizer, and centrifuged for 30 min at 10,000 x g to remove nuclei and cell debris. The supernatant was removed and stored in

100-pl

portions at-700C.

Immunoprecipitation of virus-specific pro-teins. Formalin-fixed StaphylococcusaureusCowan Istrainwaspreparedby the method of Kessler (11). Fixedbacteria(100

pl)

at aconcentration of 10%(vol/

vol)weresedimented and then suspended in 400,l of asolutioncontaining 150 mMNaCl, 5 mMEDTA, 50 mMTris-hydrochloride (pH 7.4), and 0.5% (vol/vol) Nonidet P-40 (buffer A). To this suspension

approxi-mately 100

id

of[3H]leucine-labeled cell extract was added, and the suspension was incubated for 30mi at roomtemperature. The bacteria were then pelleted, and we added to thesupernatant an amount of anti-serum determined to be sufficient toquantitatively immunoprecipitate protein from 10 ug of detergent-disrupted virions. The samples were incubated for 30 minatroomtemperature, followed by the addition of 100p1of a 10%(vol/vol) suspension of Formalin-fixed bacteria. In some cases, varying amounts of disrupted nonradioactive viruswerepresentduringthe incuba-tion (seebelow). The bacteriacontaining the immu-noprecipitated proteins were pelleted, the superna-tants were discarded, and the pellets were washed three times with buffer A.Thepellets were suspended in a solution containing 6 M urea and 4% (wt/vol) sodium dodecyl sulfate and heated for 3 min at 1000C. The releasedproteinwasrecoveredafter centrifuga-tion of the fixed bacteria and eitherdirectly counted forradioactivityorappliedtopolyacrylamidegels.

Polyacrylamide gel electrophoresis of pro-teins. Discontinuous sodium dodecyl sulfate-10%

polyacrylamide slab gelswerepreparedessentially by the method of Laemmli (12). The samples were elec-trophoresed at 80 V until thetrackingdye reached the bottomedgeofthegel.Radioactivelylabeled proteins werelocatedbyfluorographyessentially as described by BonnerandLaskey (4). Kodak BB-5 X-ray film was exposed to the dried gels at -70°C and then

developed to visualize the bands produced by the radioactiveproteins.

Materials. Embryonated gs chf chicken eggs wereobtained from SPAFAS, Inc., Norwich, Conn. 3-Deazaadenosine was synthesized by a previously de-scribed procedure (15). L-[methyl-3H]methionine (9

Ci/mmol), [5,6-3H]uridine (41Ci/mmol),

[2-3H]aden-osine(22Ci/mmol),L-[4,5-3H]leucine (149Ci/mmol), and deoxycytidine 5'[a-3P]triphosphate (2,000 Ci/ mmol)wereobtained fromAmersham Corp., Arling-tonHeights, Ill.S1 nuclease was obtained from Miles Laboratories, Inc.,Elkhart,Ind.Aminobenzoxymethyl paperwaspurchased fromSchleicher & Scheull Co., J. VIROL.

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INHIBITION OF ASV PROTEIN SYNTHESIS 175 Keene, N.H. Goat antisera directed against

detergent-disrupted avian myeloblastosisvirus (AMV) and p27

wereobtained from the Division of Cancer Cause and

Prevention, National Cancer Institute. Anti-p19

anti-serum wasgenerously supplied by Volker Vogt,

Cor-nellUniversity, Ithaca, N.Y.

RESULTS

Effect of 3-deazaadenosine on the

pro-duction of B77 ASV virions by infected

chickenembryofibroblasts. Totestthe effect

of3-deazaadenosine onthe production of B77 virions,plates of confluent infected chicken em-bryo fibroblastsweretreatedwitheither MEM or MEM containing 100 LM 3-deazaadenosine for14h. The radioactiveprecursors[3H]uridine

and[3H]methionine (toassayfor RNA and

pro-tein,respectively)werethen addedtothe media. Themediawerecollected after12h oflabeling, the viruswaspurified, and theamountsof

radio-activity in the virus bands fromanequilibrium sucrose gradient were determined. Table 1

shows thatboth theincorporation of [3H]aden-osine and the incorporation of[3H]methionine

into virionswere reducedbymorethan 90% in

thepresenceof3-deazaadenosine. These results

indicated that theproductionof B77virionswas

inhibited in the presence of 3-deazaadenosine

and confirmedthe results of Baderetal. (2)for

adifferent ASV strain. Theproductionof

trans-formation-defective virionswas alsoinhibited in

the presence of3-deazaadenosine (Table 1).In agreementwith Baderetal.,wealsofound that

the inhibition of virusproductionwasreversible.

Uponremoval of3-deazaadenosine, therateof virus production, as determined by

incorpora-tion ofradioactivity orbyreversetranscriptase activity,wasrestoredtonormal levelswithin 12 h(datanotshown).

Effect of3-deazaadenosne on host and

viralpolypeptide synthesis.Onepossible ex-planationfor the inhibition of virusproduction by3-deazaadenosine would beageneral

block-ade of host protein synthesis. Therefore, we

tested the effect of thisdrugontheoverallrate

ofprotein synthesis.Cellsweretreated for

vary-ingtimes in the absence and presence of 100

ILM

3-deazaadenosine. Cell cultures were then pulse-labeledwith [3HJleucine for 1 h, the cells were harvested, and the incorporation of [3H]leucine into hot trichloroacetic acid-insoluble material wasmeasured (Table 2). A 35 to 50% decrease inincorporation was obtained in the presence of 3-deazaadenosine. These results agree reasona-bly well with those obtained by Bader et al., who reported aninhibition of approximately 20% in the rate of

[3H]leucine

incorporation after treat-ment of chicken embryo cells with 100

ItM

3-deazaadenosine for24h(2).

To test for a specific effect of 3-deazaadeno-sine on viral protein synthesis, ASV-infected cells were labeled with [3H]leucine for 1 h, cy-toplasmic extracts were prepared, and the virus-specific proteins were detected by incubation with appropriate antisera to avian retrovirus proteins, followed by adsorption of the

antigen-TABLE 2. Rateofincorporation of[3HJleucineinto

proteinsinthepresence of100X jM 3-deazaadenosinea

Time after Radioactivity(cpm)in:

3-deazaa-denosine 3-Deazaadeno- %of treatment Control sine-treated control

(h) culture

18 18,560 12,268 66

22 21,179 10,449 49

26 15,146 8,834 58

Plates (60 mm) of confluent B77 ASV-infected cells were treated with 100jiM3-deazaadenosine in MEM forvarying times. The mediumwas then

re-placedwith theappropriatemediumlackingleucine. After 1h 50juCiof[3H]leucine was added, and the cells were incubated for an additional1h. After this, thecellswerescrapedfrom each dish and suspended in1ml ofabuffercontaining0.1MNaCl,0.01MTris

(pH7.5), 0.001 MEDTA, 1%sodiumdodecylsulfate, and 1% 8-mercaptoethanol. From thesesamples, 0.1

ml-portionswerewithdrawn into 1-mlportionsof 10%

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trichloroacetic acid,and thesampleswereheated for 15minat900C.Thesampleswere then filtered and counted forradioactivity.

TABLE 1. InhibitionofradioactiveB77virionproductioninthepresence of100 M3-deazaadenosinea

Radioactivity (cpm)in:

Virus8lwtopCo3-Deazaadenosine- %Inhibition

Control treatedculture

B77 [3H]methionine 106,000 9,400 91

B77 [3H]adenosine 17,000 800 95

tdB77 [3H]uridine 4,400 500 88

'Plates(100 mm) of confluent B77 ASV-infected cellswerelabeled with800jiCiof[methyl-3H]methionine

per mlor20,iCiof[3H]adenosineper ml for12haftera14-htreatmentwith3-deazaadenosine. Cells infected withtransformation-defective B77 ASV(tdB77)werelabeled with20jiCiof[3H]uridineper ml for12h. The viruswaspurifiedfrom the mediaasdescribedpreviously (21),and theamountofradioactivityinthepeakat adensityof1.15to1.16g/mlwasdetermined.

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176 STOLTZFUS AND MONTGOMERY

antibodycomplexestoFormalin-fixed S.aureus (11). The materials in the immunoprecipitates

werethenanalyzed by sodium dodecyl sulfate-polyacrylamidegelelectrophoresis,andthe

rel-ative intensities of the bandscorresponding to

the viral proteinswerecompared (Fig. 1).Inthe control extracts, the major bands immunopre-cipitated by the AMV antiserum and detectable

onthegelscorrespondedtop27 and the precur-sorof thegagproteins, pr769ag (Fig. 1, lane 5). Figure1also shows thatuponaddition ofexcess

competing unlabeled viral protein during the immunoprecipitation (lane 6), the relative inten-sities of thepr76Wg andp27bandswere dimin-ished, indicating that the immunoprecipitation of these proteins was specific. Much smaller

amountsofpr769ag and p27were immunoprecip-itated fromanequivalentamountof [3H]leucine-labeledproteinextractobtained from 3-deazaa-denosine-treated cells (Fig. 1, lane 2). Similar results were obtained whenanti-p27 antiserum

wasused(datanotshown).

Theprofiles of the total labeled proteins from 3-deazaadenosine-treated and untreated cells

areshown inFig. 1, lanes3and4, andthe two

profileswerequite similar. Since themajorityof thelabeled proteins are cellular, thisresult

in-dicates that thesynthesis ofmostof thecellular proteinswas notinhibitedselectively by the

3-deazaadenosine treatment. An exception ap-pears to be apolypeptide withanapparent

mo-lecular weight of approximately 70,000 (desig-nated hp), which was present in the control

extract.Theposition of thispolypeptideonthe gel didnotcorrespondtotheposition ofanyof the virion proteins. This suggested that 3-dea-zaadenosinemayspecifically inhibit the synthe-sis of some host cell proteins, as well as the synthesis of viral proteins.

It was possible that the viral proteins were

synthesized in thesamerelativeamountsin the 3-deazaadenosine-treated cells but that these proteinsweredegradedrapidlyunderthese

con-ditions. If thiswere the case, wewould expect

thatlabeling forashortertime with [3H]leucine would result in the production of relatively largeramountsof viralproteinsin 3-deazaaden-osine-treatedcells.Therefore, theproductsofa

20-minpulse-labelingwith[3H]leucinewere

an-alyzed (Fig. 2). In these experiments we used

twoantisera of differentspecificities,anti-AMV and anti-p19. It was clear that the anti-AMV antiserum immunoprecipitated both the gag

proteinsand,to alesser extent,theenvproteins gp85and gp35 (compare Fig. 2A,lane 1 [whole disrupted virions] with Fig. 2A, lane 4 [immu-noprecipitate of whole disrupted virions]).The immunoprecipitation of gp85 and gp35 from

vi-FIG. 1. Polyacrylamide gel electrophoresis of viral proteins pulse-labeled for 1 hwith

[3HJleucine

and immunoprecipitated from extracts of3-deazaadenosine-treated and control infected cells.[3H]leucine-labeled

cell extractswereprepared, andimmunoprecipitationwithanti-AMVantiserumto theFormalin-fixed Cowan Istrainof S. aureus and elution from the bacteria were carried out as described in the text. Equal input amountsofradioactivity from the two extracts were used. Polyacrylamide gel electrophoresis was carried out on theelutedsamples in discontinuous sodiumdodecylsulfate-polyacrylamide gels for 4.5 h at 80 V. The gel wasprepared forfluorography by using previously described procedures (4). Lane 1, Disrupted

[3H]leucine-labeled B77virions; lane 2,immunoprecipitation ofproteins from 3-deazaadenosine-treatedcell extract; lane 3,totalproteinsfrom3-deazaadenosine-treatedcellextract; lane 4, total proteins from control cellextract;

lane5, immunoprecipitation of proteins from control cellextract; lane 6, immunoprecipitation of proteins from control cell extract carried out in the presence of 100ug ofnonradioactive disrupted B77 sarcoma virions; lane 7, immunoprecipitation of disrupted[3H]leucine-labeledB77 virions.

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A

1 2 3 4 5 6 7

gp85_

hp

P 27_ Pl19

B

-pr180

gp

85-gp 35-J

P27_ p19

-Pr76

_pr60

FIG. 2. Polyacrylamide gelelectrophoresis ofviral proteins pulse-labeled for20 min with[3H]leucine

andimmunoprecipitated fromextractsof 3-deazaa-denosine-treated and control infected cells by anti-AMVantiserum(A)andanti-p19antiserum(B). The experimentalconditions were identicaltothose de-scribed in thelegendtoFig. 1,exceptthatthelabeling time was 20 min rather than 1 h. (A) Anti-AMV

antiserum. Lane 1, Disrupted B77 virions; lane 2, total proteins from 3-deazaadenosine-treated

ex-tract;lane3,totalproteins fromcontrolextract;lane 4, immunoprecipitation of virionproteins; lane 6, immunoprecipitation ofcontrolextract;lane5,same aslane 6, but in thepresenceof80pgof nonradio-activedisruptedB77sarcomavirions;lane8,

immu-noprecipitation of 3-deazaadenosine-treatedextract;

lane7,same aslane8,but inthepresenceof80pgof nonradioactive disrupted B77sarcoma virions. (B)

Anti-p19antiserum. Lane 1, DisruptedB77virions;

lane2, immunoprecipitation ofvirionproteins; lane

4, immunoprecipitation of proteins fromcontrolcell

extract;lane3,same aslane4,butcarriedoutin the

presence of80pg ofnonradioactive disruptedB77

sarcomavirions;lane6,immunoprecipitation of

pro-teins from 3-deazaadenosine-treated cell

extract-lane5,same aslane6,but carried out inthepresence

of80 pg ofnonradioactivedisrupted B77sarcoma

virions.

rionswasnotobvious in the 1-hpulse shownin

Fig. 2because theexposure time ofthe

fluoro-gramwas not sufficiently long to detect

radio-activity in these peaks. On the otherhand, the

anti-p19 antiserum precipitated primarily the

p19polypeptides, but it alsoprecipitated p27to

some extent; i.e., the antiserum was

contami-nated slightly with antibodies directed toward p27 (Fig. 2B, lanes 1 and 2). There was no

activity directed toward theenvproteingp85. Figure 2 also shows that in the control infected extract anumber ofbands werespecifically

pre-cipitated with the anti-AMV antiserum (Fig.2A, lanes5and6). Asexpected, the antiserum

pre-cipitatedpr769ag and aless intense band

corre-sponding top27. We also detected a

virus-spe-cific polypeptide migrating at the position of

pr180O9"9I)(, the presumptive precursor of the reverse transcriptase (15). In addition, a poly-peptide with a molecular weight of

approxi-mately90,000wasimmunoprecipitated, and this immunoprecipitation wasinhibitedinthe pres-enceofexcessunlabeleddisrupted virions.

Whentheanti-p19 antiserumwas used,both

pr7W6Ma and a band correspondingtopr609'9, a

previously described intermediate (23) in the processing of

pr769"¶,

werespecifically immuno-precipitatedfrom the extract. (Fig. 2B, lanes3

and 4). Although not visible in Fig. 2, a band correspondingto pr1809'9P"wasalso detected inlongerexposures.However,the90,000-dalton polypeptide was not immunoprecipitated. We believe that thelatterpolypeptide corresponds

to the envelope protein precursor (pr92en") for the following reasons: (i) the polypeptide was

immunoprecipitated by the anti-AMV

antise-rum, which contained antibodies directed againstenvproteins,butwas not immunoprecip-itated bytheanti-p19antiserum,whichdidnot;

(ii) thepolypeptide had theapproximate appar-ent molecular weight of the env precursor, as

reported byotherinvestigators (14); and (iii)the

immunoprecipitation of this protein was in-hibited in the presence ofdisrupted virions,

in-dicating that it was related structurally to a

virionprotein.

Inthe extracts prepared from 3-deazaadeno-sine-treatedcells,wedetected littleor nopr769'9

ortheputative

pr1809h

P)o polypeptideineither the anti-AMV immunoprecipitates (Fig. 2A, lanes 7 and8) or the anti-p19

immunoprecipi-tates (Fig. 2B, lanes 5 and 6). This result was

similar to the resultobtained inthe 1-h pulse-labeling experiment (Fig. 1) andsuggestedthat

thesyntheses ofthese proteinswereindeed

in-hibited in the presence of 3-deazaadenosine

rather thantheproteinsbeingrapidly degraded.

Incontrast, thepresumptive

pr92env

polypeptide appearedtobe present in the anti-AMV

immu-noprecipitates (Fig. 2A, lane 8). These results

suggested that detectable amounts of

pr92env

were synthesized in 3-deazaadenosine-treated cells, whereas comparable amounts of pr769`9

werenot.

Wecomparedtheprofilesof the totallabeled

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proteins from 3-deazaadenosine-treated and control extracts (Fig. 2A, lanes 2 and 3). As

observedwith the 1-h labeled extracts, the

pro-files appeared to be identical, except for the presence inthe control extract ofa heavily la-beled band having a molecular weight of

ap-proximately 70,000 (band hp), which appeared

tobemissingintheextractsfrom the 3-deazaa-denosine-treated cells.

Effect of 3-deazaadenosine on host and viral RNAsynthesis.Wenexttestedwhether the selectiveinhibition of viralprotein synthesis

might be due to a selective inhibition of viral

RNAsynthesis.Ithasbeenreported previously

that a 24-h treatmentwith100-,iM

3-deazaaden-osine results in an inhibition ofapproximately 30%intherateof[3H]uridineincorporationinto

the totalRNA of chicken embryo cells (2). To

test the effect of the analog specifically on

mRNA synthesis, cells were treated for 15 h

with3-deazaadenosine and labeled with [3H]ur-idine for12h,and the totalpoly(A)+RNAwas

isolated fromthe cells. Theamountof radioac-tive RNA per cell was determined. There was

an inhibition in the accumulation of both poly(A)+ RNA andpoly(A)-RNA (34and 22% inhibition, respectively) in the presence of

3-deazaadenosine.

Todeterminewhetherthepoly(A)+ RNA

syn-thesized in thepresenceof3-deazaadenosinewas

associated with polysomes, analog-treated and controlcellswerelabeledforvaryingtimeswith

[3H]uridine.

Afterthe labeling period,the cells

werelysedandseparated into nucleiand

cyto-plasm. The cytoplasmic fractions were further

separated into polysomal and nonpolysomal fractions bycentrifugationondiscontinuous su-crose gradients. The amount ofpoly(A)+ RNA

in eachfractionwasthen determined. Figure 3

shows that there was a decrease of

approxi-mately 50% in the rate of

[3H]uridine

incorpo-rationintonuclearRNA (Fig.3A).Therewas a

corresponding decrease in the rate of incorpo-ration of

[3H]uridine

intopolysomalRNA (Fig.

3B). The size distributions of the polysomes

were not significantly different in treated and controlcells (datanot shown). The decrease in the observed rate ofprotein synthesis shown in Table 2 was similar in magnitude to the de-creased rate at which mRNA associated with

polysomes. This result suggested that the inhi-bition ofprotein synthesis in

3-deazaadenosine-treatedcellswas due to lowered levels of mRNA in polyribosomes. Intheexperimentsshown in

Fig.3 andTable 2, the results were not corrected forpossible effects of3-deazaadenosine on cell growth rate.However,weobservedthatthe cell

densities under the two conditions remained comparable over the time periods of the

experi-_x0. B

C3

2-1 2 3 4

HR

FIG. 3. Kineticsofpoly(A) RNA synthesis in 3-deazaadenosine-treated and control infected cells. Confluentmonolayersin100-mmplasticpetridishes ofB77ASV-infectedchickenembryofibroblastswere incubatedfor15h with either MEMorMEM

con-taining100,uM3-deazaadenosine. At the endofthis time, the medium wasremoved, andfresh medium withorwithout100

liM

3-deazaadenosinecontaining

20,uCiof[3HJuridineper mlwasadded. The cells wereharvestedafter varyinglabeling periods by first washingtheplateswith5mlofTBS and then resus-pending the cells in 5 ml ofTBS. The cells were collected by centrifugation, disrupted by detergent lysis, and separated into nuclear and cytoplasmic fractions. The cytoplasmwasseparated into polyso-mal andnonpolysomal fractions by discontinuous

sucrosegradientsas describedpreviously (8). Poly-somal andnuclear RNAs wereisolated by phenol-chloroform extraction, and thepoly(A)+ RNA was recovered by oligodeoxythymidylic acid-cellulose

chromatography. Symbols: 0, 3-deazaadenosine-treatedcells;0,control cells. (A)Nuclear RNA.(B)

Polysomal RNA.

ments.

We next tested whether all or part of the action of3-deazaadenosine onvirusproduction

was due to selective inhibition of viral RNA

synthesis.Thefractions ofvirus-specificRNA in

analog-treatedandcontrol cellswerecompared by determining the kinetics of annealing of a

viral cDNAprobetoRNA isolated frominfected cells (Fig. 4). The

Crtl/2

values for the RNAs

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0 ~ ~ 0 0

C,t

FIG. 4. Kinetics of RNA from 3-deazaadenosine-treated and control infected cells hybridized to viral cDNA. Confluent monolayer cultures of B77-infected cells were incubated witheither MEM or MEM con-taining1(0) jtM 3-deazaadenosine for 18 h. After this, [8HJadenosine (final concentration, 20yCi/ml) was added, and the incubation was continued for an additional 12 h. Total RNA wasisolated from the cells by phenol-chloroform extraction, DNase diges-tion, LiCIprecipitation, and oligodeoxythymidylic acid-cellulose chromatography, according to previ-ously described techniques (21). Hybridization of RNA at varying concentrations to approximately

1,000)cpm of 32P-labeledcDNA was carried out in a solutioncontaining 0.3 MNaCI,0.01 M Tris-hydro-chloride (pH 7.5), and 0.001 M EDTA for 18 at

68°C. The extent ofhybridization was assayed by the sensitivity of the 32P-labeled cDNA to Si nuclease digestion. Symbols: 0, 3-deazaadenosine-treated cells; 0, control cells.

from the control and 3-deazaadenosine-treated cells were 4.7 and 11.5 mol.s/liter, respectively. This result indicated that the fraction of the total RNA represented by virus-specific RNA in 3-deazaadenosine-treated cells was 40% of the fraction of virus-specific RNA present in control cells. Therefore, there appeared to be a selective decrease in the amount of viral RNA in the analog-treated cells. Since the overall poly(A)-RNA synthesis was inhibited somewhat less than the overall poly(A)+ RNA synthesis, we also directly tested the poly(A)+ RNA from 3-deazaadenosine-treated cells for the fraction of virus-specific RNA byhybridization kinetics. In this case we obtained a value of 50% of the control value (data not shown). We concluded that there was a selective decrease in the steady-state levels of viral RNA in the 3-deazaadeno-sine-treated cells.

We then analyzed the RNA in polyribosomes for individual species of virus-specific mRNA. Chicken embryofibroblasts infected with ASVs reportedly contain subgenomic 285 and 21S mRNA's, as well as 38S genomic RNA (10, 24). The 285 and 21SmRNA's are thought to serve as messengers for theprecursor of the envelope

proteinsgp85 andgp35 and for the protein

re-quired for transformation

(pp6e)'c),

respectively

(17-19). Onthe otherhand, at least part of the 38SgenomicRNA serves asthemRNA for the gag protein presursor (pr769"9) and the

pre-sumptive reverse transcriptase precursor

(prl80V'gPO') (16). The 28S and 21S mRNA's

probably arisefrom the 38S RNA by a process

in whichsequencesin the 3' halfof the genome

RNAaresplicedtoa5'-terminal leadersequence

(6, 13,20).Totestfor thepresence and relative amountsof the various mRNA species,we

elec-trophoresed poly(A)+RNAsfromthe polysomal

fractions of 3-deazaadenosine-treated and

un-treated cells on agarosegels containing 10 mM

methylmercury hydroxide. The separatedRNA

speciesweretransferredtodiazobenzoxymethyl

paperby the procedure of Alwineetal. (1), and

the virus-specific species were detected by

hy-bridization of theblotto aplasmidpreparation

containing the entire ASV Prague A genome

(pSal'01)

which had been labeled with 32P by

nick translation (Fig. 5). Qualitatively, it

ap-peared from the gels that the intensities ofthe

38S and35SgenomeRNA bands inthe

polyso-mal RNA from 3-deazaadenosine-treated cells

werereducedrelativetotheamounts of28Sand

21S mRNA's when compared with equal amounts of polysomal RNAs from the control

cells.(The 35S RNAwasderived from the pres-ence of transformation-defective deletion mu-tants which contaminated the B77 ASV stock used in this experiment). To approximate the

relative amounts of the various virus-specific RNA species presentin the 3-deazaadenosine-treated and control infectedcells,the

autoradi-ograms werescanned with adensitometer, and

the areasunder thepeakswerecompared. The

sizes ofareasunderthepeaks correspondingto

the 21S, 28S, and 35S-38S RNAs were 47, 40,

and12%of the sizes of theareasunder thesame

peakswhenequalamountsof control RNAwere

used. These differenceswereobserved when the gelswereexposed for several different intervals. Therefore, there appeared to be a relatively larger decrease intheamountof 35S-38S RNA than in theamountsof thesubgenomic 28Sand

21S mRNA species in the 3-deazaadenosine-treatedcells.

Further evidence for a decrease in the relative amount of the 35S-38S RNA in

3-deazaadeno-sine-treated cells compared with control cells

was obtained by hybridization of nuclear and

polysomal RNAs from glycerol gradient frac-tionstoradioactiveviral cDNA(Fig. 6).Clearly,

theresolutionof RNAspeciesinthesegradients

wasnotnearlyasgoodasin themethylmercury hydroxide gels shown in Fig. 5. However, the

data obtained from this hybridization of the

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.Y,0

N

0

.0

0.

I

1216

I I \\I \

[image:8.494.280.440.62.345.2]

4 8 12 16 20 24 Fraction

FIG. 5. Electrophoresis ofviral RNAon 1%

aga-rosegels containing10 mMmethylmercury hydrox-ide. Thefractionation of cytoplasmic extracts into

polysomesandpostpolysomal cytoplasmwascarried outasdescribedin the text.Approximately 15 pgof RNAfromeachofthepolysome fractionswas

elec-trophoresed on 1% agarosegels containing 10 mM

methylmercury hydroxide for6 hat50 V. The loca-tions ofthe virus-specific RNA bands were deter-mined by procedures described in the text. The ex-posuretimewas24 h. LaneA, PolysomalRNAfrom controlASV-infected cells;laneB, polysomalRNA

from3-deazaadenosine-treatedASV-infectedcells.

RNAs from the gradient fractions allowed an independent quantitative comparisonof the

rel-ative amounts of genomic and subgenomic RNAs. Hybridization values in therangeof0to

40%canbe assumedtoberoughly proportional

toCrtand, therefore, foraconstanthybridization time proportional to viral RNA concentration (9). These dataaresummarized in Table3.We

observedthat therewasadecrease in the ratio

ofgenomic RNA tosubgenomic RNA in both thenuclear RNA andthepolysomal RNA from 3-deazaadenosine-treatedcells.Therefore, these

datasupportthe data inFig.5andsuggestthat the relative concentration of the 35S-38S RNA

FIG. 6. Glycerol gradient sedimentation of

virus-specific nuclear RNA (A) andpolysomalRNA (B)

from 3-deazaadenosine-treated and control cells. Four100-mmplatesofconfluent cellsinfected with B77ASVwere treated with either MEMorMEM

containing100

AM

3-deazaadenosine for19h. The cells were fractionated into nuclei and cytoplasm,

and thepolysomes were isolated on discontinuous sucrosegradients. The RNAsfrom the nuclear and

polysomalfractionswerepurifiedasdescribed

pre-viously(8). Thepoly(A)J RNAwasisolatedby

oligo-deoxythymidylic acid-cellulose chromatography. Ap-proximately equalamountsof RNA were applied to 5 to30%glycerol gradients. Thegradientswere sed-imentedbycentrifugationat24,000 rpmfor16h ina BeckmanSW41 rotor. Thegradientswere

fraction-ated, and the RNA in eachfractionwasprecipitated with2volumesof95% ethanol. Theprecipitateswere collected and dissolved in sterile distilledwater(100 and50AIfor the polysomal RNAs from thecontrol and3-deazaadenosine-treatedcells,respectively; 50 and25Alfor the nuclear RNAsfromthe controland 3-deazaadenosine-treated cells, respectively). Sam-ples (10

#1)

fromeachfractionwerehybridized for42 hat68oCto32P-labeledB77ASVcDNA. Agradient containing 38S viral genome RNA and 28S and 18S rRNA'swas runinparallel,and thepositions of the peaksaremarkedonthefigure. Symbols: O,

3-dea-zaadenosine-treatedcells;0,control cells.

wasindeedreducedin3-deazaadenosine-treated cells.

DISCUSSION

Weshow in thispaperthat,in the presence of 100 uM 3-deazaadenosine, there is a selective

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TABLE 3. Distributionofvirus-specificRNA in genomic 4nd subgenomic size classes from

3-deazaadenosine-treated and controlcellsa

Control

3-Deazaadenosine-treated culture

Fraction %of virus- %of

virus-specific specific

RNAin: G/SG RNA in: G/SG

G SG G SG

Nuclei 57 43 1.33 33 67 0.49

Polysomes 55 45 1.22 36 64 0.56

aThe values for percent hybridization obtained

from theresults in Fig. 6 were assumed to be propor-tional to concentration. This is only an approximate relationship and is valid between values of 0 and50%

hybridization (9). Genomic (G) and subgenomic (SG) RNAs were defined as the material sedimenting be-tweenapproximately 32Sand 408 and between 18S and 30S,respectively.

inhibition of the synthesis of the ASV proteins pr769'a andpr1809`9

1".

Thesynthesis ofa poly-peptide presumed to be pr92env appears to be affected less. In general, host cell protein

syn-thesis is also less sensitive to inhibition. It ap-pears that the inhibition of viral gag protein synthesis, coupled withadecrease of30 to50% in the rate of overall protein synthesis, could explain the inhibitionof more than 90% inthe production of virions previously observed by Baderetal. (2) and confirmedbyushere.

We found that atleast part of the selective effect of 3-deazaadenosine treatment on viral protein synthesis is dueto aselective decrease in therelative amounts of viral mRNA's. The

Crtl/2

values for therateofhybridizationof viral cDNA toinfected cell RNA indicated that the concentration of viral RNA in treated cellswas

to approximately 40% of the concentration in

control cells. If the inhibition of the rate of synthesis ofradioactivelylabeled cellular RNA (=40 to 50%) is also reflected in a decreased steady-state level of cellular RNA in 3-deazaa-denosine-treated cells,the absoluteamountsof viral RNAmaybereducedtolevels whichare aslittle as20% of theamounts incontrol cells.

Itshould be stated that the cDNA used in the hybridization kinetics experiments (Fig. 4) was

not a representative copy of the viral genome, and therefore we cannot be certain from this experiment whether the concentrations of all

viralsequences are reducedequally.From anal-yses of the viral RNA species onagarose gels containing methylmercury hydroxide and on

glycerolgradients,itappearedthat in

3-deazaa-denosine-treated cells, there were relatively larger decreases in the concentrations of the genome 38S and 35S mRNA's than in the

con-centrations of the 28S env and 21S src mRNA's. These relative decreases in the levels of the putative mRNA's for pr76Wg and prl80gYg P' mayexplainthe greatereffectof the

3-deazaa-denosine treatment on the synthesis of these

polypeptides compared with the presumptive

pr92env polypeptide (Fig.2).

The mechanism by which the steady-state

levels of viral RNAarereduced in

3-deazaaden-osine-treated cells remains obscure. Sucha de-creasecould be causedeither by apreferential decrease in therate ofvirus RNA synthesisor

byapreferential increaseintheturnoverof viral RNA. Baderet al. have proposed that since

3-deazaadenosineis an inhibitor of S-adenosylho-mocysteine hydrolase and since the intracellular concentration of S-adenosylhomocysteine

in-creases dramatically in treated cells,

methyla-tion reacmethyla-tions are inhibited. In particular, the failure to methylate viral RNA may render it

nonfunctional (2). We have shown previously

that another in vivo inhibitor of methylation,

cycloleucine, inhibits the internal N6-methyla-denosine and 2'-O-methyl ribose cap methyla-tions of both viral genome RNA and cellular mRNA'sby80 to90%.However, the production ofASV under these conditions is inhibited only slightly. This ledus to proposethat the

meth-ylations inhibited by the cycloleucinetreatment are notessential for virusreplication (7). There-fore, if the effectsonviral RNA levelsaredueto

inhibition of RNA methylations, we would ex-pect that those methylations which are not

blocked in thepresenceofcycloleucine(i.e., the

5'-terminal N7-methylguanosine methylations) would be affected. We have tested for suchan

effectonthe bulk cellular poly(A)+ RNA from 3-deazaadenosine-treated ASV-infected chicken embryo fibroblasts and have found little or no

inhibition ofN7-methylguanosine methylations (Stoltzfus, unpublished data). Wehavenotyet

tested fora possible specific effect on the N7-methylguanosine methylation of the various

viralmRNA's. However, it would be surprising

ifsuchwerethe case, sincecellularmethylating

enzymes arepresumablyusedtomethylateviral RNA.

As analternativehypothesis,wepropose that

3-deazaadenosine may selectively inhibit the

synthesis of viral RNA. It has been shown that theproductionof avian retrovirions isextremely

sensitive to the effects of thetranscription inhib-itor actinomycin D. Even at concentrations of

actinomycin D as low as 0.1 ,ug/ml, virus pro-duction is inhibitedmorethan 90%(22). Under theseconditions,thesynthesisof hostpoly(A)+

RNA is inhibited only about 30% (Stoltzfus, unpublished data). Whether this sensitivity to

lowconcentrationsofactinomycinD isdue

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tirelytoeffectsonviral RNAsynthesis hasnot

been established yet. We have also obtained preliminary evidence that treatment of ASV-infectedcells with a-amanitin (a specific

inhibi-torof RNApolymerase II)at aconcentrationof

10,ug/ml inhibits cellularpoly(A)+RNA synthe-sis by approximately 30%, whereas virion

pro-duction is inhibited by about 70% (Stoltzfus, unpublished data).Forbothinhibitors,theeffect

onvirus production and presumablythe effect

on viral RNA transcription are considerably

greater than the effect onthe synthesis of the bulk cellular mRNA. We have shown that

3-deazaadenosine inhibits bulk cellular poly(A)+ RNAsynthesis by40 to50%.Therefore, it would

not be surprising if the 3-deazaadenosine inhi-bitionof viral RNAlevelsweredueto aselective inhibition of viral RNA synthesis. Additional studiesonthekinetics of viral RNAsynthesis in 3-deazaadenosine-treated cells will be required

to testthishypothesis.

The reasonfor the selective decrease in the

amountof35S-38S RNA relativetotheamounts

of 28S and 21S mRNA's in 3-deazaadenosine-treatedcells is alsonotclear. Itisthought that the38S RNA isaprecursorofthe 28S and 21S mRNA's and that thesesubgenomic RNAsare

derivedbyasplicingprocess(6, 13, 20).Ifthis is indeed thecaseand ifa constant amountof viral 38S RNA issplicedperunittime,adecrease in the rate of transcription of 38S RNA would result inalarger proportion ofsplicedRNA and

anincrease in the ratio ofsubgenomic RNAto

genomic RNA.Therefore, the alteration inthe steady-state levels of the various viral RNA species observed in 3-deazaadenosine-treated cellsmayreflect the effect ofthe analogon the rate ofviralRNA transcription.

Finally,wepointoutthatthepartial reversion

ofcellsinfected with the Bryan strain ofRous sarcomavirus fromatransforned phenotypeto amorenormalphenotypeupon treatmentwith 3-deazaadenosine, as reported by Bader et al. (2), could also be explained by inhibition ofviral

RNA transcription. This would result in the

production of less src mRNA and, therefore, presumably would lead to a reduction in the

intracellular levels of the protein which is

re-quired for transformation, pp60' (5). We have

not yet determined the rate of synthesis of

pp6OBrc

in3-deazaadenosine-treatedcells.Based

onacomparisonof the relative amounts of the

putative 21S src mRNA in

3-deazaadenosine-treated and control cells (Fig. 5), we anticipate

that thereduction in the amount ofpp60O' is not asprofoundasthereduction in the amount

of

pr76g'.

To our knowledge, 3-deazaadenosine is the only reported example ofa substance that

ex-hibits selectivity in the inhibition of retroviral RNA andproteinsyntheses. Forthisreason,it

maybeausefultool foralteringthe intracellular levels of retroviral mRNA's and proteins and determining theconsequencesof these changes

oncellularand viralfunctions.

ACKNOWLEDGMENTS

Wethank PaulaSnyderandElizabeth Lanshe for technical assistance.

This research was supported by Public Health Service grant CA-28051 fromthe National Cancer Institute. C.M.S. was supported by Public Health Service Research Career Development Award CA-00659 from the National Cancer Institute.

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