Cell-free translation of avian erythroblastosis virus RNA.

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Vol. 34, No. 1 JOURNALOF VIROLOGY, Apr.1980, p. 280-284

0022-538X/80/04-0280/05$02.00/0

Cell-Free Translation of Avian

Erythroblastosis Virus RNA

TONY PAWSON* ANDG. STEVEN MARTIN

Department of Zoology, University ofCalifornia,Berkeley,California94720

Avianerythroblastosis virus (AEV) RNA rescued from nonproducer cells by

superinfection with a helpervirus is translated into three polypeptides in the

messenger-dependent

rabbitreticulocyte lysate. A 75,000 molecular weight

poly-peptide (P75A

V)

is synthesized from 28SRNA and is encoded by the5' section

ofthe AEVRNA, including gag-related andAEV-specificsequences. Thep75AEV synthesized in infected cellsand the

p75AEv

synthesized in the cell-free system areelectrophoretically identical. A44,000molecularweight polypeptide

(p44'V)

is synthesized from 20-24S RNA, apparently from the3' section of the

AEV-specific RNAsequence. Aminor37,000 molecular weight polypeptide

(P37'V)

iS synthesized from 20S AEV RNA. A comparison is drawn between the cell-free

products of MC29 andAEVRNAs. The avianacuteleukemia virusesare agroup

ofhighly oncogenic, transforming RNA tumor

viruseswhichinduceleukemias, sarcomas, and

carcinomas ininfectedchickens (1,7, 12). They have been divided into three classesonthe basis of their oncogenic spectra (1, 2, 4, 6, 11; P. H. Duesberg, K.Bister, and C. Moscovici, Virology, in press). The prototypes of these groups are

avian myelocytomatosis virus (MC29), avian erythroblastosisvirus(AEV), and avian myelo-blastosis virus (AMV). Theyare allreplication defective, and a nondefective helper virus is

thereforerequired to rescue theleukemia viral RNAgenomefromtransformed cells (1,2, 5,11). Such superinfected cells release both virions containingacute leukemia virus RNA and

viri-ons containinghelper virus RNA (2, 11; Dues-berg et al., Virology, in press). Although the defectiveness of theacuteleukemia viruseshas

precludedadetailedgenetic analysis,a biochem-icalinvestigationofseveral avianacuteleukemia viruses has revealeda commongeneticstructure

(2, 4, 6, 11; Duesberget al., Virology, inpress). The genomic RNA measures 5 to 6 kilobases (kb) and contains threeregions. Group-specific regions are located at the 5' and 3' ends and

measure about 1 kb and 1.5 to 2.5

kb,

respec-tively.These sequencesarerelatedtothe repli-cativegenesof nondefective viruses of theavian group,andinthecaseofAEV,areisogenicwith

thenaturallyoccurring, nontransforming helper, avianerythroblastosis-associated virus(AEAV) (2). The 5' group-specific section contains gag

(group-specific antigen)generelatedsequences.

The 3'sectionprobablycontainsenv

(envelope)

gene related sequences and a C or modified C region. The thirdregion is an intemal

specific

sequence of2 to 3kb which isunrelated tothe

replicativesequences ofnondefectivehelper

vi-ruses, to the src (transforming) gene of Rous sarcoma virus (RSV), or to the specific se-quencesof otherclasses ofacuteleukemiavirus

(2,4, 6, 11, 16, 17; Duesbergetal., Virology,in

press). Becauseviruses withasimilaroncogenic spectrum have related specific sequences (1, 2, 4, 6, 7), these intemal specific sequences are

thoughttobelongtothetransforming (onc)gene

of the virus.

Nonproducer cells transformed by the avian

acute leukemia viruses contain virus-specific, nonvirion gag-related proteins (3, 4, 8, 9). A 110,000molecularweight polypeptide

(P11OMc)

can be detected in MC29-infected cells after immunoprecipitation with anti-gag serum (3,

11). This polypeptide contains MC29-specific peptidesequences,andalsop19 andpartofp27

(the twogroup-specific antigens located atthe N-terminus of Pr769`9[18], the primary product of the

gag

gene) (3, 11). We have shown that PlOMc is encoded by MC29 virion RNA and is translated from full-length 28S MC29 RNA, being initiatednearthe 5' end andsynthesized from thegag-related5'group-specific regionand the MC29-specific region (11). It is thereforea

candidate for theMC29-transforming protein.In

addition,a56,000 molecularweightpolypeptide (P56MC)wassynthesized frompolyadenylicacid [poly(A)]-containing 20-24S (3-kb) RNA,anda 37,000 molecular weight polypeptide (p37MC)

froma 15-18S (1.5 kb) RNA. p37MC isenv re-lated and is probably translated from the 3'

group-specificregion.

P56Mc

mustbetranslated eitherfromthe 3' endof the specific region, or theadjacent sectionofgroup-specific region,to its3'side.

The genetic structure of AEV has recently

beendetermined,andit appearstodefinea new class of onc gene (2, 17). A 75,000 molecular 280

on November 10, 2019 by guest

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weightpolypeptide (p75AEV) hasbeen identified

innonproducercellstransformedbyAEVafter immunoprec pitation with anti-gag serum (8). Like Pll0MC this polypeptide contains se-quencesin common withPr769'9(namely, p19), andspecificsequencesunrelatedtoany replica-tivegeneproduct (8). Herewewishedto deter-minewhetherp75AEV isdirectlytranslated from the AEV genome, the region from which it is translated, and other translation products of

AEVRNA.

The strategy employed was similar to that describedpreviouslyforMC29(11).We isolated

aclone of AEV-transformed nonproducer cells by infecting chick fibroblasts with strain ES4 AEV(RAV-1) athighdilution and thencloning the transformed cells in methylcellulose (7). Thisclone of AEV-transformed fibroblastswas

identified as a nonproducer by its inability to

release either infectious transforming virus, or

particles with reverse transcriptase activity. This nonproducer clone was labeled with [35S]methionine, lysed,andimmunoprecipitated with antiserum to disrupted AMV (directed mainly against the

gag

proteins) (13, 14). These cells synthesize p75A V but no viral structural proteins (Fig. 1,lane 1). We thensuperinfected thesenonproducercellswithring-neck pheasant virus (RPV) andrepeated the immunoprecipi-tationexperiment. RPV encodesaPr76'agwhich

can be electrophoretically separated from p75AEV.Superinfection with RPV resulted in the synthesis ofPr769'9,theappearanceof p27 and p19, andareduction in thesynthesis ofp75AEV (Fig. 1, lane 3). Medium was harvested from parallel cultures of RPV-superinfected

AEV-transformed cells, the virionswerepurified, and poly(A)-containing,denatured 50-70S RNAwas

isolated and translated in thereticulocyte lysate (15). Thecell-free products of AEV(RPV) RNA (Fig. 1, lane5) were thencompared with those of RPV alone (Fig. 1, lane 6). Although both AEV(RPV) and RPV RNAsaretranslated into Pr769'9,

P180V'-i",

and other products (11, 14),

three polypeptides are synthesized solely from AEV(RPV) RNA and must therefore be

en-codedbyAEV RNA. Thelargest AEV-specific product has a molecular weight of75,000 and

comigrates with

p75AEV

identifiedinAEV

non-producercells(Fig. 1). ThesecondAEV-specific polypeptide has a molecular weight of 44,000

(P44AE

), and the third has amolecularweight

of 37,000(P37

AEV)

The AEV(RPV) virion RNA was then

frac-tionatedby centrifugation througha 15to30% glycerol gradienttoidentifythesize of the RNA

species fromwhich each ofthethree AEV-spe-cificpolypeptidesissynthesized (11). The RNA

281

wasrecovered from eachfraction and translated in the cell-freesystem, and theproducts were

analyzed by sodium dodecyl

sulfate-polyacryl-amidegel electrophoresis (Fig. 2). P75 EV is

syn-1 2 3 4

*.

5 6

130 94

60 53

v.._;'

Om

.:wf

WI

43

36

Pr76

a9

-W Aiv

-4P75

_ - P44

- P37 F..

29

[image:2.508.255.450.132.414.2]

p27x _

FIG. 1.AEVpolypeptides synthesized in cells and invitro.Cells fromaclone ofAEV-transformed non-producer(NP)chickfibroblasts (see text) were labeled with 100uCi of[35S]methionine in methionine-free medium ina 10-cmdish (14). Cells were lysed and immunoprecipitated by using anti-AMV serum or normal goatserum asdescribed previously (13, 14). Lane 1, AEV-NP fibroblasts, anti-AMV; lane 2, AEV-NP fibroblasts, normal serum. Nonproducer cells weresuperinfectedwithRPV, and the immuno-precipitations were repeated: lane 3, AEV(RPV) fi-broblasts, anti-AMV. Lane 4,AEV(RPV) fibroblasts, control serum. Poly(A)-selected, denatured 50-70S RNA isolated from virions released from the AEV(RPV) infected fibroblasts was translated in the messenger-dependent rabbit reticulocyte lysate as de-scribed (11, 15), as was RNA isolated from RPV virions alone.Lane 5, AEV(RPV) RNA cell-free prod-ucts.Lane 6, RPV RNA cell-free products. Polypep-tideswereanalyzed by electrophoresis on a7.5% SDS-polyacrylamide slab gel (11) followed by fluorogra-phy. Coomassie brilliant blue-stained molecular weight markers (11)areindicated by horizontal bars between lanes 4 and 5, with themolecular weight (x10-3) written to the left of lane 1. Polypeptides discussed in thetextareindicated by arrows.

-aP180gclg-po.

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282 NOTES

thesizedpredominantly from 28SRNA (fraction

11),which representsthefull-length5.5-kb AEV

genomic RNA (2). In contrast, the RPV-encoded Pr769'9andP180VG'-PO aresynthesizedprimarily

from34SRNA (fractions 7, 8). Immunoprecipi-tation of fraction11 with asuccessionofantisera

raised againstproducts of thereplicativegenes

(Fig. 3) showsthatp75AEViSrecognizedby anti-gag serum. The synthesis of p75AEV from28S RNA suggests that itissynthesizedfrom the 5'

section of theAEV RNAbetween5.5 and 3 kb,

since in eucaryotesproteinsynthesisisinitiated at or nearthe 5' end of the mRNA (10). This

conclusion is also supportedby thepresenceof

gag-relatedpeptidesinp75AEV(8),and ofa

gag-relatedoligonucleotide in the 1-kb group-specific sequence at the 5' end of AEV RNA (2). The presence ofspecific peptides in the p75AEV

pro-tein (2;unpublished data) suggeststhatits cod-ingregion extends into the AEV-specificsection,

which extends from4.5 to 1.5kb from the 3' end.

p44AEV

is

synthesized from arange of RNA

size classes,butprimarilyfrom20-24S

(approx-imately

3kb)sizeRNA (Fig. 2,fractions15, 16).

Ifp44

AEV

is initiatednearthe5' end of this 3-kb

AEV-specificRNA, it must beencodedbythe 3'

half of the AEV-specific region between 3 and 1.5kb from the 3' end. This is consistent with

the observation that

p44AEV

synthesized from 20S (fraction 16) RNA is not immunoprecipi-tated by antiseraraisedagainst the gag,pol,or envgeneproducts, suggestingthat it isunrelated

toanyreplicativegeneproduct (Fig. 3). This is

alsotrueof

p44AEV

synthesizedfrom

AEV(RAV-2) RNA(data notshown).

Tryptic

peptide

map-ping showsthat

p75AEV

and

p44AEV

arelargely unrelated (datanotshown).

p37AEV is synthesized primarily from 20S AEV-specific RNA (Fig. 2). Its identity is not yetclear, butpreliminaryevidence suggests that itmaybeenvrelated.

These experiments have been repeated with

AEVrescued by RAV-2orby AEAV (data not

shown).

In each case

p44AEV

and

p37AEV

are

28S 185

+ 4

4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

P180

_9 ..

Pr7690

P75AE'V _{._I.I...w}

..mN

...~. %. ..

*4" - 'I'li...~- l' ...s

[image:3.508.148.387.316.588.2]

... _ ..

FIG. 2. TranslationoffractionatedAEV(RPV)virionRNA. A25-pgamountofpoly(A)-selected,denatured

50-70SAEV(RPV)virionRNAwasboiled in 10 mMTris-hydrochloride(pH7.5)-0.1%sodiumdodecyl sulfate

andsedimentedona12-ml,15to 30%glycerol gradientasdescribedpreviously (11, 14).Atotalof25fractions

werecollected,and the viralRNAwasrecoveredaftertheadditionofcarrieryeasttRNA. 10%oftheRNAin eachfractionwasthentranslated in 10

IlI

ofmessenger-dependentrabbitreticulocyte lysate,and theproducts

wereanalyzed by electrophoresison a7.5%sodiumdodecylsulfate-polyacrylamidegelandautoradiography.

The numberofeachfraction fromwhich the translated RNAwasrecovered isindicated above eachlane,as arethepositions of 18Sand28SRNA markerssedimented inparalleL

AE%.

P44

P37ALE

a'

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Fraction 11 (283)

A B C D

Fraction 16(20S)

A B C D

130-

94.-VW gW

60

.-53

M.

AE'V

21!-P44 _

43-AEV P37

--

29-FIG. 3. Immunological analysis ofAEV-specific, cell-free products. AEV(RPV)RNAfrom gradient frac-tions 8(34S),11(28S),and 16(20S) (see Fig. 2)wastranslatedin thereticulocyte lysate.A10-,lIamountofthe products ofeachfractionwasanalyzedwithoutfurthertreatmentona7.5% sodiumdodecyl sulfate-polyacryl-amidegel (lanes A).Afurther15

IlI

wasimmunoprecipitatedwithasuccessionofantisera inexcess(14),the

supernatants from the first precipitation being immunoprecipitated with the second antiserum and the supernatantsfromthe secondprecipitation beingimmunoprecipitated bythe thirdantiserum,in thefollowing order: 1, anti-gagserum (lanes B); 2,anti-reversetranscriptaseserum (containing anti-gag activity) (lanes

C);3, anti-RSVPR-Cgp85serum(lanes D).The resultsareshownfortheproducts of28Sand20SAEV(RPV) RNA. Forfraction8(34S), onlytheanti-gagimmunoprecipitateis shown. The mobilitiesofmolecularweight markersaredisplayedasdescribed inFig. 1.

clearly translated specifically from AEV RNA and not from the helper RNA, and from the

sameRNA sizes after RNA fractionationasfrom

AEV (RPV) RNA. The Pr769'9 product of the RAV-2orAEAV RNAs issosimilar in

electro-phoretic mobilitytop75AEV that thetwo cannot be wellseparated. However,whenAEV(AEAV) isfractionated,p75AEVcanbe identifiedasbeing

synthesized from 28S RNA. In the case of

AEV(RAV-2), RNA isolated from virions

re-leased from fibroblasts or erythroblasts was

translated into identical products. Thus, the three cell-free products ofAEV RNAare

syn-thesizedindependentlyofthehelper virusorcell

typeoforigin.

Both p75AEV and p44AEv appear to contain regions translatedfromtheAEV-specific region.

Thus far, only p75AEV has been identified in AEV-transformed cells, and it is not clear if p44AEV is also present in infected cells, or if it

represents anin vitroartifact. Thus,wedonot know yetwhether p75AEVis the sole transform-ing protein of AEV. We are subjecting the

smaller cell-free products of AEV and MC29to

a moredetailedanalysis and investigating their

possible synthesisininfectedcells.

It is striking how similar are the products

synthesized from MC29 and AEVvirion RNAs invitro. Bothcode foralarge gag-related

poly-peptide synthesized from the 5' section of the RNA andincluding specificsequences(P110MC,

p75AEV);a smallerpolypeptide synthesized from

the middle ofthe RNA (P56Mc,p44AEV), proba-bly fromspecific sequences, though thisis less Fr.8

B

P180

130-

UI~60-

53.-

43-

36- 29-VOL. 34,1980

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[image:4.508.133.374.73.378.2]

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284 NOTES

clear for P56MC; and a small, apparently

env-related polypeptide synthesized from the 3'

group-specificregion

(p37Mc,

p37AEV).Thus, al-though theoncgenesof these virusesappear,at least inpart, tobeunrelated, theoverallgenetic

structure and mode ofgene expression of AEV

and MC29seemverysimilar.

Wethank Masae Namba and Louise Roberts fortechnical assistance, P. Duesberg for encouragement anda stock of AEVstrain ES4(AEAV), andT.Grafforagift of AEV strain

ES4 (RAV-1). Anti-PR-C gp85 serum was a gift from D.

Bolognesi, antireverse transcriptaseserum wasfromH.

Op-permann,and inactivatedStaphyloccusaureus werefromL.

Crawford.

This workwassupported by Public Health Servicegrant

NIH CA17542. T.P.wassupported byPublicHealth Service

training grant CA 09041 from the National Institutes of Health.

LITERATURE CITED

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3. Bister, K.,M.J.Hayman, andP. K.Vogt.1977. De-fectiveness of avianmyelocytomatosisvirusMC29: iso-lationoflong-termnonproducerculturesandanalysis ofvirus-specific polypeptide synthesis. Virology 82:431-448.

4. Bister,K.,H.-C.Loliger,and P. H. Duesberg. 1979. Oligoribonucleotidemapandproteinof CMII:detection of conserved and nonconserved genetic elements in avianacuteleukemiavirusesCMII, MC29,and MH2. J.Virol. 32:208-219.

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J. VIROL.

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