The pX region of the bovine leukemia virus is transcribed as a 2.1-kilobase mRNA.

Vol. 54, No. 2 JOURNALOF VIROLOGY, May1985, p.625-629

0022-538X/85/050625-05$02.00/0

CopyrightC 1985, AmericanSociety forMicrobiology

The pX

Region of the Bovine Leukemia Virus Is Transcribed

as a

2.1-Kilobase mRNA

R. Z.

MAMOUN,'

T.

ASTIER-GIN,'

R. KETTMANN,2* J. DESCHAMPS,2 N.

REBEYROTTE,'

AND B. J. GUILLEMAIN'

InstitutNational de la Sante etde la RechercheMedicale, Unite 117, 33076 Bordeaux Cedex, France,1 and Universite Libre deBruxelles, Departement de Chimie Biologique, 1640Rhode-Ste-Genese, Belgium2

Received 26 October1984/Accepted 31 January 1985

Thebovineleukemia virus mRNAs expressedinculturedbovine cells of variousorigins are a9.0-kilobase genomic RNA, a 5.1-kilobase env RNA, and a newly detected 2.1-kilobase RNA corresponding to the transcription ofpX sequenceslocated in betweentheenvgeneand the3' end of the provirus.

The bovine leukemia virus (BLV), anexogenous

compe-tent retrovirus, is known to be the etiological agent ofthe

enzootic formofthe B-type cell bovine leukemia (7-9, 13,

19, 21).Theprovirus is foundintegratedin the tumorcellsin

which itremains unexpressed (1, 9, 24).

The modelsknowntobe

applicable

for virus

leukemoge-nesiscannotexplain cell transformation afterBLVinfection

for the following reasons. (i) Because BLV sequences are

entirelyexogenous, the minkcellfocus-forming mechanism

isexcluded,forsuchvirusarise at leastpartially via

recom-bination between endogenous proviral sequences. (ii) A

virus-coded oncogene is not implicated because proviral

sequences encountered in tumor cells have no homology with normal cellsequences (3);furthermore, noevidence of

onc or onc-bearing viral proteins specific forthe tumorous statecould be detected by immunoprecipitation techniques

(15). (iii)The downstreampromotion (20) ofan onccellular

geneinthetumordoesnot seem tobelikely, forBLVdoes

notintegrateatspecific chromosomal sitesorregions (5, 12),

and no cellularmRNAcontaining viral long terminalrepeat

(LTR) is synthetized (11).

5' ss

u3 R i5

P P B Bg

I II I

6 2 A

could play animportant role in tumor genesis.Moreover,an indication of a major role of this region resides in its constant conservation in tumors in which the BLVprovirusis deleted (10).

Toinvestigate the BLV products, we established a number of cell linesexpressing BLV (i.e., normalcells, normal cells experimentally infected with BLV, and cells free of BLV or expressing BLV originating from a tumor). The way in which these cell lines arereferredtoonly gives anindication

of their origin but does not specify their actual nature, neoplastic or not (14, 15). The analysis of BLV mRNAs in these cells shows that, in addition to the genomic

(9.0-kilobase [kb]) and env (5.1-kb) RNAs, a 2.1-kb mRNA is alsotranscribedfromthe pXregion.

PolyadenylatedRNAs werepreparedfromanoncultured

tumor (T3010), three BLV-infected cells from cultured

tu-mors(LB59Ly,3010gg, and3894gg), non-BLV-infectedcells

from cultured tumor (LB44K), normal cells (3894c and

3010c), in vitro BLV-infected normal cells (FLK-BLV and

BESP-LB59), and non-BLV-infected cells cultured from a

sporadic tumor case (2412Ly). After size separation and

B X

I I

X B E SS 3' Proviral

I

I-

D N A

4C 3

4 B

4A 1B

1A

Probes

FIG. 1. Localization of the clonedBLVfragment probesversusthemapof BLVprovirus (3) obtained with restrictionenzymesSacl(S),

PstI(P),BamHI(B),BglII(Bg),XbaI(X),andEcoRI(E). Theseprobesweresubcloned with the pBR322 and pAT153plasmids.Restriction of the cloned BLV DNAs were generated by the specific restriction endonuclease digestions. Each fragment was further purified by preparativeagarosegelelectrophoresis.DNAs werelabeled by nick translation inthe presenceof[32P]dCTP(600Ci/mmol), with 5 U ofDNA polymerase asdescribed by Maniatis (17). Probes were isolated fromtriphosphates by centrifugation through Sephadex G-50. The final specific activitywas ca. 108cpm/,ug.

All of these findings also apply to the human T-cell

leukemia virus (HTLV) as described in studies by both

Burny

and Gallo (International Conference on Tumor Virus in RNA, in press); thus, the bovine system is quite

appro-priateas anexperimental model. Interestingly, the analysis

of the sequence at the 3' endofthe envgene of both HTLV and BLVrevealed theexistenceofopenreadingframes,the

putative product of which was tentatively called pX and

* Correspondingauthor.

Northern blotting, the RNAs were hybridized with the

probes as detailed in Fig. 1. These were subclones ofthe

8.3-kb SacI clone containing the BLV

information,

except for 104 basesmissingbecause the LTR of this virus contains twoSacIsites (3).

The 1A probe (Fig. 1) represents the 3' end sequence

locatedbetween the EcoRI and Sacl sites andincludingthe entire U3 and the first 45 bases of the R region. It thus

contains the 3' part of the so-called pX region ofthe BLV

genome.Figure2showsarepresentativepattern

indicating

a

625

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m

-s s c.

-CT It

X e C" u)

c to 0 It co 0 m

- m in in

i:13 J

co 02

-fl

o C:

a

0

Cl)

0

0 C),

9

FIG. 2. Northernblot ofpolyadenylated RNAs hybridized with probes 1Aand1B. RNAwasextracted in TNEbuffer(0.01M Tris[pH 7.9], 0.1 MNaCl, 0.001 M EDTA) containing 0.5% sodium dodecyl sulfate and0.5%Macaloid three times withphenolchloroformsatured in

TNE. Polyadenylated RNA was selected onoligodeoxythymidylate-cellulose and denatured with either glyoxal (18) or methylmercuric hydroxide (2). Size separations (2to5 ,ug of RNA)werecarriedoutby electrophoresis on1.2%agarosegels.The RNAs weretransferred (Northern blotting) on diazobenzyloxymethyl paperand then hybridized with 32P-labeled DNAprobes. Conditions ofhybridization and washing wereperformedasdescribed by Maniatis (17), but thetemperature ofhybridizationwas 37°C. The origins of theRNAs were as

follows: culturednormal cells(3010c and 3894c,normal corneal cells from enzooticlymphosarcomacells[3010and3894]);cultured normal cells in vitro infected withBLV(FLK-BLV, reference fetallambkidneycells(25); BESP-LB59,bovineembryo spleencells infected with the BLV produced by LB59Ly cells); cells from acultured tumorwith no BLV expression (2412Ly, leukemic leukocytesfrom a sporadic lymphosarcomacase [2412]; LB44K,lymphomatous kidney fromanenzootic lymphosarcomacase [LB44]);BLVexpressingcultured cells from enzooticlymphosarcomacasesLB59,3010,and 3894(Ly, leukemic leukocytes;gg,leukemiclymphnodes[3894gghasaverylow BLV expression]);andT3010, noncultured 3010 leukocytes.

'r

.A_{i \ r n 1, s,b .46tAtt}

.60

5 Kt

,NM _fl.iPf

ua fi _{2 1 k b}

Ai yr

FIG. 3. Northernblot ofthe

polyadenylated

RNAs

hybridized

with

probes

3, 4A, 413,or4C. See the

legend

to

Fig.

2.

f-n

I... .:. ." a_

-1 a)

I ", .:L

..'T

-., -n

0:

i.

2.it k1.)

- -.j

'Z. -j il,

LI' M Lji

I.,

Alp_-A!

tall

I

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NOTES 627

9.0 kbmRNA

5.1 kb mRNA

_mON .AAAA

.- -.

?0.-.0lo" -.

P P

P P B Bg

Proviral DNA

x B E SS

I,l0,

I I I

Bx

gag / Po/ /"

I U3 R U5

env I pX ?

I

I

I

2.1 kbmRNA

_ one. AAAAA

Bam Hi

XbaI Splice Splice HTLV

I

N"IK

I

a

aw-1I:I 'U:::i:.:q : D

l1

-8 0 52 124 261

Proviral DNA

Eco RI TGA Sacl

I

I

I

IlI

I

l

723 967 1242 1513

I?

2.1 Kb m-RNA

pX M.W. > 34 Kd. Putative protein

FIG. 4. (A) Theupperpartof thefigurerepresentstheorigin of the mRNAsversusthe BLVprovirus; the restrictionenzymesitesare

shownasdescribed in thelegendtoFig. 1. (B) In the lowerpart,thepX region of the provirus is detailed. The BamHI site is consideredas

pointzeroof the sequence;HTLV, commonsequencesbetweenBLV, HTLV-1,andHTLV-2; splice, possibleacceptorsplice sites;TGA (-877), termination codon of the envgene;TGA(+967), possible end translation codon for the pX. The dotted lines of the 2.1-kb mRNA

meansthat itsorigin is located in the centralpartof theBLVgenome.

clearhybridization in infected cells ofnotonlythe 9.0- and 5.1-kb mRNAs but also a 2.1-kb mRNA species. Such a resultis evocative ofthe avian lymphoma model, in which

cellular mRNAs often contain viral sequences originating

from the3' endofthe viralgenome. However, thisdoesnot fit the BLV system, for although BLV has no specific

integration site, the size of the small mRNA observed in these experiments remains constant (2.1 kb) regardless of thecellline in which itwasdetected. Furthermore,the sole

contribution of the 45 bases of the Rregion cannotexplain

the clear hybridization observed at this size. We therefore undertook the same investigations, but with a fragment probe (1B) 1 kb distal from the R region (Fig. 1) and

includingthe 5' partof thepX region. Again, all BLV-pro-ducingcellspresentedthesamepattern, indicatingthatsuch a transcript (2.1 kb) does not originate from a LTR down-stream promotionof cellular sequences (Fig. 2).

Hybridization was then performed with 4A, 4B, and 4C Ss

LiR Ub

LTR

TGA

-877 -382

M-12=.-

II I I I I I

I II I 0 MEE I I %Fw " %w-I

I I if I -t I I I I I

VOL.54,1985

I

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[image:3.612.119.489.71.567.2]

probes (Fig. 1). 4A contains all of the open reading frameof the env gene (gp5l and gp35); 4B is located in between the open reading frames of env and pX; 4C is on the 5' side of 4A. With the same transfer (Fig. 3), one can essentially see the putative env mRNA (5.1 kb) in addition to the genomic 9.0-kb mRNA. Noteworthyis the fact that no 2.1-kb mRNA was visualized, indicating that such an mRNA is coded by sequences lying in between the LTRSacl site and the next upstreamBamHI site.

By use of cloned probe (no. 3) containing essentially pol sequences (Fig. 1), a 9.0-kb mRNA and two faint bands corresponding to 5.1 and 2.1 kb were obtained (Fig. 3). Because the 5.1-kb mRNA is dimly recognized, this suggests that only a small portion located at the 3' end of the probe contains sequences of the5'end of the env gene. As a 2.1-kb mRNA is also dimly recognized, this RNA should contain sequences of the 3' end of thepol representative probe. With the gag-specific probe 2A or 5'pol-specific probe 6 (Fig. 1), only the 9.0-kb mRNA is recognized (data not shown), confirming that this RNA is the gag-pol messenger.

All of these hybridizations, as shown in Fig. 2 and 3, were also performed with mRNAs originating from normal, non-BLV-infected cells (3010c and 3894c). As no hybridization could be observed, this confirms the viral nature of the mRNAs described above. The same findings and the same conclusions hold true for cells from a sporadic lymphosar-cornatous cow (2412Ly) or for non-BLV-infected cells de-rived from an enzootic lymphosarcomatous cow (LB44K). The known absence of BLV expression in noncultured

tumor cells was also confirmed with T3010 cells. These

results were extended for the 2.1-kb mRNA.

In all experiments performed with mRNAs of cells in-fected with BLVs of lymphosarcomatous animals, the hy-bridizations corresponded to the three same sizes. It is interesting to notice that the three mRNAs detected in FLK-BLV cells,which are known to contain a BLV isolated from a nontumorous cow, were of a smaller size than that reported above.

The viral mRNAs synthesized byBLV-infected producing cells are mapped in Fig. 4A. Briefly, three species were observed: a 9.0-kb mRNA, a 5.1-kb mRNA, and a new species, i.e., a2.1-kb mRNA. The 9.0-kb mRNA transcribed from the entire BLV provirus may be compared with that of other retroviruses (23) and thus is responsible for gag-pol gene translation (4, 16). The 5.1-kb mRNA is the transcript of the env part of the BLV provirus, extending from a few nucleotides on theleft-hand side of the centralBamHI site to the 3' end of the provirus.

The 2.1-kb mRNA was found to be specific for BLV-in-fected cells. Like the two other mRNAs, the 2.1-kb mRNA was observed only in cellsexpressing the viral proteins and was also notdetectable in noncultured tumor cells. Neither is it specific for the tumoral state, as cultured cells from a sporadic case of lymphosarcoma do not express such a 2.1-kb mRNA. Thehybridization pattern indicates that such an mRNA is representative of two regions because of a splicing event. One region is located in sequences corre-sponding to probe no. 3, and the other is located in the pX sequences. The pX regions of BLV, HTLV-1, and HTLV-2 werepreviously sequenced (6, 21, 22; Burny, in press); the compiled results (Fig. 4B) support previous findings that the splicing occurs at one of the two splicing acceptor sites at positions -8 and +52. Under these conditions, the four common sequences to the three viruses (between positions +124 and +261) ate present in the mRNA. Thus, it is likely that the transduction of the pX region starts before the

splicing

sites with an open

reading

frame extending to the

endtranslationTGA codon (no. +967). With aframe

iden-ticaltothatproposed forHTLVs(21a),onecan assumethat

1,000 nucleotides is the minimalcoding capacity ofthe pX

gene, thusallowingthe synthesis ofaproteinwitha

molec-ular weight ofat least 34,000.

Although the protein(s) coded by the pX sequences

re-mains as yetunidentified, its role in leukemogenesis can be

envisaged because both BLV and HTLV are competent

viruses able to code for proteins ofthe pX sequences in

addition

toallof their structural proteins. However, the

pX

cannot be considered as strictly homologous for a viral oncogene, because ithas nocellularproto-oncogene

coun-terpart. In this case, the pX would have to be modified

(for

example, by mutation), and its role would reside in the

initiation of the lymphoproliferative process. Argumentsfor

suchamodification of the pX include the following. (i)

Only

afew of the BLV-infected animals develop persistent

lym-phocytosis and lymphosarcoma. (ii) As evidenced in our

epidemiological surveys, in herds in which a BLV-induced

lymphosarcoma occurs, the leukemia risk ofthe other

ani-mals drastically increases (this situation mimics the

emer-gence of an "acute BLV" in tumor cells which then is

horizontally transmissible within the herd). (iii) Finally, the

role ofthe pX as the "initiator" ofcell multiplication by a

"hit-and-run" mechanism is supported by the complete

absence ofBLV

expression

in tumor cells.

We are indebted to J. M. Miller and M. J. Van der Maaten for

supplyingthe FLK-BLV cell line. We thank J. Quesnel foraccess to alarge variety of tumor-bearing cattle. We also acknowledge the

technicalassistance of C. Bourget and thecareful preparationofthe manuscript by L. Couillaud.

LITERATURECITED

1. Burny, A., C. Bruck, H.Chantrenne, Y. Cleuter, D.Dekegel, J. Ghysdael, R. Kettmann, M. Leclercq, J. Leunen, M. Mam-merickx, and D. Portetelle. 1980. Bovine leukemia virus: mole-cularbiology and epidemiology, p. 231-289. In G. Klein (ed.), Viral oncology. Raven Press, NewYork.

2. Chandler, P. M., D. Rimkus, and N. Davidson. 1979. Gel electrophoretic fractionation of RNAs by partial denaturation with methylmercurichydroxide. Anal. Biochem. 99:200-206. 3. Deschamps, J., R. Kettmann, and A. Burny. 1981. Experiments

with cloned complete tumor-derived bovine leukemia virus information prove thatthe virus is totallyexogenoustoitstarget animals species. J. Virol.40:605-609.

4. Ghysdael, J., R. Kettmann, and A. Burny. 1979. Translation of bovine leukemia virus virionRNAsinheterologous protein-syn-thesizing systems. J. Virol. 29:1087-1098.

5. Gregoire, D., D. Couez, J. Deschamps, S. Heuertz, M.-C. Hors-Cayla, J. Szpirer, C. Szpirer, A. Burny, G. Huez, and R. Kettmann. 1984. Different bovine leukemia virus-induced tu-mors harbor the provirus in different chromosomes. J. Virol. 50:275-279.

6. Haseltine, W. A., J. Sodroski, R.Patarca, D.Briggs,D.Perkins, andF.Wong-Staal.1984.Structuralof 3' terminal region of type II humanTlymphotropic virus: evidence fornewcoding region. Science 225:419-421.

7. Kawakami, T. G., A.L.Moore, G.H.Theilen,and R. J. Munn. 1970. Comparisons ofvirus-likeparticles from leukoticcattle to feline leukosis virus. Bibl. Haemat. 36:471-475.

8. Kenyon, S. J., and C. E. Piper. 1977. Properties of density gradient-fractionated peripheral blood leukocytes from cattle infected with bovine leukemiavirus. Infect. Immun. 16:898-903. 9. Kettmann, R., A. Burny, Y. Cleuter, J. Ghysdael, and M. Manmerickx. 1978. Distribution of bovine leukemia virus pro-viral DNAsequences in tissues of animals with enzootic bovine

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NOTES 629 leukosis. Leuk.Res.2:23-32.

10. Kettmann, R., D. Couez, and A. Burny. 1981. Restriction endonuclease mapping of linear unintegrated proviral DNA of bovine leukemiavirus.J. Virol.38:27-33.

11. Kettmann,R., J. Deschamps, Y.Cleuter, D. Couez, A. Burny, and G. Marbaix. 1982. Leukemogenesis by bovine leukemia virus: proviral DNA integration and lack of RNA expression of viral longterminalrepeatand3'proximatecellular sequences. Proc. Natl. Acad. Sci. U.S.A. 79:2465-2469.

12. Kettmann, R., J. Deschamps, D. Couez, J.-J. Claustriaux, R. Palm,and A. Burny. 1983.Chromosomeintegration domain for bovineleukemia provirus intumors.J. Virol. 47:146-150. 13. Kettmann, R., D. Portetelle, M. Mammerickx, Y. Cleuter, D.

Dekegel, M. Galoux, J. Ghysdael, A. Burny, and H. Chantrenne. 1976.-Bovine leukemia virus: an exogenous RNA oncogenic virus. Proc. Natl. Acad. Sci. U.S.A. 73:1014-1018.

14. Mamoun, R. Z., T. Astier, and B. Guillemain. 1981. Establish-ment and propagation ofabovine leukaemia virus-producing cell lines derived from the leukocytes ofaleukaemic cow. J. Gen.Virol. 54:357-365.

15. Mamoun, R. Z., T. Astier, B. Guillemain, and J. F. Duplan. 1983. Bovinelymphosarcoma: expression of BLV-related pro-teins in culturedcells. J. Gen. Virol. 64:1895-1905.

16. Mamoun, R. Z., T. Astier, B. Guillemain, and J. F. Duplan. 1983. Bovinelymphosarcoma: processing of bovine leukaemia viruscodedproteins. J. Gen.Virol. 64:2791-2795.

17. Maniatis, T., E. F. Fritsch, andJ.Sambrook. 1982.Molecular cloning,alaboratory manual. Cold Spring HarborLaboratory, ColdSpring Harbor,N.Y.

18. McAster,G.K., andG. G. Garmichael. 1977.Analysis ofsingle anddouble stranded nucleic acidsonpolyacrylamide andagarose

gels byusingglyoxal andacridineorange. Proc.Natl. Acad.Sci. U.S.A. 74:4835-4838.

19. Miller, J. M., L. D. Miller, C. Olson, and K. G. Gillette. 1969. Virus-like particles in phytohemagglutinin-stimulated lympho-cytecultures withreferencetobovinelymphosarcoma.J. Natl. CancerInst.43:1297-1305.

20. Neel, B. G., W. S. Hayward, H. L. Robinson,J. Fang, and S. M. Astrin. 1981. Avian leukosis virus-induced tumors have com-mon proviral integration sites and synthesize discrete new RNAs: oncogenesis bypromoterinsertion. Cell 23:323-334. 21. Paul, P.S.,K. A.Pomeroy, A. E.Castro, D. W. Johnson, C. C.

Muscoplat, and D. K. Sorensen. 1977. Detection of bovine leukemia virus in B-lymphocytes by the syncytia induction assay.J. Natl.CancerInst.59:1269-1272.

21a.Rice, N. R., R. M. Stephens, D. Couez, J. Deschamps, R. Kettmann, A. Burny, and R. V. Gilden. 1984. The nucleotide sequence ofthe env gene post-env region of bovine leukemia virus. Virology 138:82-93.

22. Seiki, M., S. Hattori, Y. Hirayama, and M. Yoshida. 1983. Human adult T-cell leukemia virus: complete nucleotide se-quenceof theprovirusgenomeintegratedinleukemia cellDNA. Proc. Natl. Acad. Sci. U.S.A.80:3618-3622.

23. Stephenson, J. R. (ed.). 1980. Type C structural and transfor-mation-specific proteins, p. 245-297. In Molecular biology of RNA tumorviruses. Academic Press, London.

24. Stock,N.D., andJ. F. Ferrer. 1972.ReplicatingC-type virus in phytohemagglutinin-treated buffy-coat cultures of bovine origin. J. Natl. CancerInst.48:985-996.

25. Van derMaaten, M.J., andJ.M. Miller. 1976. Replication of bovineleukemia virusin monolayer cell cultures. Bibl.Haemat. 43:360-362.

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