The px Region of the Bovine Leukemia Virus Is Transcribed as a 2.1-Kilobase mrna

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JOURNAL OF VROLOGY, May 1985, p. 625-629 22-538X/85/5625-5$2./ Copyright C 1985, American Society for Microbiology Vol. 54, No. 2 The px Region of the Bovine Leukemia Virus s Transcribed as a 2.

1 JOURNAL OF VROLOGY, May 1985, p X/85/5625-5$2./ Copyright C 1985, American Society for Microbiology Vol. 54, No. 2 The px Region of the Bovine Leukemia Virus s Transcribed as a 2.1-Kilobase mrna R. Z. MAMOUN,' T. ASTER-GN,' R. KETTMANN,2* J. DESCHAMPS,2 N. REBEYROTTE,' AND B. J. GULLEMAN' nstitut National de la Sante et de la Recherche Medicale, Unite 117, 3376 Bordeaux Cedex, France,1 and Universite Libre de Bruxelles, Departement de Chimie Biologique, 164 Rhode-Ste-Genese, Belgium2 Received 26 October 1984/Accepted 31 January 1985 The bovine leukemia virus mrnas expressed in cultured bovine cells of various origins are a 9.-kilobase genomic RNA, a 5.1-kilobase env RNA, and a newly detected 2.1-kilobase RNA corresponding to the transcription of px sequences located in between the env gene and the 3' end of the provirus. The bovine leukemia virus (BLV), an exogenous competent retrovirus, is known to be the etiological agent of the enzootic form of the B-type cell bovine leukemia (7-9, 13, 19, 21). The provirus is found integrated in the tumor cells in which it remains unexpressed (1, 9, 24). The models known to be applicable for virus leukemogenesis cannot explain cell transformation after BLV infection for the following reasons. (i) Because BLV sequences are entirely exogenous, the mink cell focus-forming mechanism is excluded, for such virus arise at least partially via recombination between endogenous proviral sequences. (ii) A virus-coded oncogene is not implicated because proviral sequences encountered in tumor cells have no homology with normal cell sequences (3); furthermore, no evidence of onc or onc-bearing viral proteins specific for the tumorous state could be detected by immunoprecipitation techniques (15). (iii) The downstream promotion (2) of an onc cellular gene in the tumor does not seem to be likely, for BLV does not integrate at specific chromosomal sites or regions (5, 12), and no cellular mrna containing viral long terminal repeat (LTR) is synthetized (11). 5' ss u 3 R i5 2 A P P B Bg 6 3 B X 4C could play an important role in tumor genesis. Moreover, an indication of a major role of this region resides in its constant conservation in tumors in which the BLV provirus is deleted (1). To investigate the BLV products, we established a number of cell lines expressing BLV (i.e., normal cells, 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 are referred to only gives an indication 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.- kilobase [kb]) and env (5.1-kb) RNAs, a 2.1-kb mrna is also transcribed from the px region. Polyadenylated RNAs were prepared from a noncultured tumor (T31), three BLV-infected cells from cultured tumors (LB59Ly, 31gg, and 3894gg), non-blv-infected cells from cultured tumor (LB44K), normal cells (3894c and 31c), 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 X B E SS 3' Proviral - D N A 4 B 4 A 1 B 1A Probes FG. 1. Localization of the cloned BLV fragment probes versus the map of BLV provirus (3) obtained with restriction enzymes Sacl (S), Pst (P), BamH (B), Bgl (Bg), Xba (X), and EcoR (E). These probes were subcloned with the pbr322 and pat153 plasmids. Restriction of the cloned BLV DNAs were generated by the specific restriction endonuclease digestions. Each fragment was further purified by preparative agarose gel electrophoresis. DNAs were labeled by nick translation in the presence of [32P]dCTP (6 Ci/mmol), with 5 U of DNA polymerase as described by Maniatis (17). Probes were isolated from triphosphates by centrifugation through Sephadex G-5. The final specific activity was ca. 18 cpm/,ug. All of these findings also apply to the human T-cell leukemia virus (HTLV) as described in studies by both Burny and Gallo (nternational Conference on Tumor Virus in RNA, in press); thus, the bovine system is quite appropriate as an experimental model. nterestingly, the analysis of the sequence at the 3' end of the env gene of both HTLV and BLV revealed the existence of open reading frames, the putative product of which was tentatively called px and * Corresponding author. Northern blotting, the RNAs were hybridized with the probes as detailed in Fig. 1. These were subclones of the 8.3-kb Sac clone containing the BLV information, except for 14 bases missing because the LTR of this virus contains two Sac sites (3). The 1A probe (Fig. 1) represents the 3' end sequence located between the EcoR and Sacl sites and including the entire U3 and the first 45 bases of the R region. t thus contains the 3' part of the so-called px region of the BLV genome. Figure 2 shows a representative pattern indicating a 625

probe 1 A probe 1 B3 -s s c. - CT X e t C" u) c t co to m - m m -fl in 2 in co i:13 J o C: a Cl) C), ",.:L..'T -., -n....:.." f-n a_ -1 a) 9 FG. 2. Northern blot of polyadenylated RNAs hybridized with probes 1A and 1B.

Polyadenylated RNA was selected on oligodeoxythymidylate-cellulose and denatured with either glyoxal (18) or methylmercuric hydroxide (2).

2 probe 1 A probe 1 B3 -s s c. - CT X e t C" u) c t co to m - m m -fl in 2 in co i:13 J o C: a Cl) C), ",.:L..'T -., -n....:.." f-n a_ -1 a) 9 FG. 2. Northern blot of polyadenylated RNAs hybridized with probes 1A and 1B. RNA was extracted in TNE buffer (.1 M Tris [ph 7.9],.1 M NaCl,.1 M EDTA) containing.5% sodium dodecyl sulfate and.5% Macaloid three times with phenolchloroform satured in TNE. Polyadenylated RNA was selected on oligodeoxythymidylate-cellulose and denatured with either glyoxal (18) or methylmercuric hydroxide (2). Size separations (2 to 5,ug of RNA) were carried out by electrophoresis on 1.2% agarose gels. The RNAs were transferred (Northern blotting) on diazobenzyloxymethyl paper and then hybridized with 32P-labeled DNA probes. Conditions of hybridization and washing were performed as described by Maniatis (17), but the temperature of hybridization was 37 C. The origins of the RNAs were as follows: cultured normal cells (31c and 3894c, normal corneal cells from enzootic lymphosarcoma cells [31 and 3894]); cultured normal cells in vitro infected with BLV (FLK-BLV, reference fetal lamb kidney cells (25); BESP-LB59, bovine embryo spleen cells infected with the BLV produced by LB59Ly cells); cells from a cultured tumor with no BLV expression (2412Ly, leukemic leukocytes from a sporadic lymphosarcoma case [2412]; LB44K, lymphomatous kidney from an enzootic lymphosarcoma case [LB44]); BLV expressing cultured cells from enzootic lymphosarcoma cases LB59, 31, and 3894 (Ly, leukemic leukocytes; gg, leukemic lymph nodes [3894gg has a very low BLV expression]); and T31, noncultured 31 leukocytes..ai 'r\ r n 1, s,b 2. it k 1.) : i. -.j 'Z. - -j il, L' M Lj i.,.46ta tt Alp -A! tall.6 5 K t,nm ua fi 2 1 k b fl.ipf yr Ai FG. 3. Northern blot of the polyadenylated RNAs hybridized with probes 3, 4A, 413, or 4C. See the legend to Fig

3 VOL. 54, 1985 NOTES kb mrna TGA Ss M-12=.- Li R LTR Ub? P P lo" P P B Bg,l, _m ON B x gag / Po/ / " Bam Hi Xba Splice Splice HTLV N"K a env 5.1 kb mrna.aaaa Proviral DNA x B E SS U3 R U5 px? 2.1 kb mrna _ one. AAAAA 'U :::i:.: q : aw-1: D MEE %Fw " %w - if -t l ? Eco R TGA Sacl l l Proviral DNA Kb m-rna px M.W. > 34 Kd. Putative protein FG. 4. (A) The upper part of the figure represents the origin of the mrnas versus the BLV provirus; the restriction enzyme sites are shown as described in the legend to Fig. 1. (B) n the lower part, the px region of the provirus is detailed. The BamH site is considered as point zero of the sequence; HTLV, common sequences between BLV, HTLV-1, and HTLV-2; splice, possible acceptor splice sites; TGA (-877), termination codon of the env gene; TGA (+967), possible end translation codon for the px. The dotted lines of the 2.1-kb mrna means that its origin is located in the central part of the BLV genome. clear hybridization in infected cells of not only the 9.- and 5.1-kb mrnas but also a 2.1-kb mrna species. Such a result is evocative of the avian lymphoma model, in which cellular mrnas often contain viral sequences originating from the 3' end of the viral genome. However, this does not 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 the cell line in which it was detected. Furthermore, the sole contribution of the 45 bases of the R region cannot explain 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 including the 5' part of the px region. Again, all BLV-producing cells presented the same pattern, indicating that such a transcript (2.1 kb) does not originate from a LTR downstream promotion of cellular sequences (Fig. 2). Hybridization was then performed with 4A, 4B, and 4C

628 NOTES probes (Fig. 1). 4A contains all of the open reading frame of 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.

4 628 NOTES probes (Fig. 1). 4A contains all of the open reading frame of 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.-kb mrna. Noteworthy is the fact that no 2.1-kb mrna was visualized, indicating that such an mrna is coded by sequences lying in between the LTR Sacl site and the next upstream BamH site. By use of cloned probe (no. 3) containing essentially pol sequences (Fig. 1), a 9.-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 the 5' 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 the pol representative probe. With the gag-specific probe 2A or 5' pol-specific probe 6 (Fig. 1), only the 9.-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 (31c 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 lymphosarcornatous cow (2412Ly) or for non-blv-infected cells derived from an enzootic lymphosarcomatous cow (LB44K). The known absence of BLV expression in noncultured tumor cells was also confirmed with T31 cells. These results were extended for the 2.1-kb mrna. n all experiments performed with mrnas of cells infected with BLVs of lymphosarcomatous animals, the hybridizations corresponded to the three same sizes. t 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 by BLV-infected producing cells are mapped in Fig. 4A. Briefly, three species were observed: a 9.-kb mrna, a 5.1-kb mrna, and a new species, i.e., a 2.1-kb mrna. The 9.-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 the left-hand side of the central BamH site to the 3' end of the provirus. The 2.1-kb mrna was found to be specific for BLV-infected cells. Like the two other mrnas, the 2.1-kb mrna was observed only in cells expressing the viral proteins and was also not detectable 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. The hybridization pattern indicates that such an mrna is representative of two regions because of a splicing event. One region is located in sequences corresponding to probe no. 3, and the other is located in the px sequences. The px regions of BLV, HTLV-1, and HTLV-2 were previously 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 end translation TGA codon (no. +967). With a frame identical to that proposed for HTLVs (21a), one can assume that 1, nucleotides is the minimal coding capacity of the px gene, thus allowing the synthesis of a protein with a molecular weight of at least 34,. Although the protein(s) coded by the px sequences remains as yet unidentified, its role in leukemogenesis can be envisaged because both BLV and HTLV are competent viruses able to code for proteins of the px sequences in addition to all of their structural proteins. However, the px cannot be considered as strictly homologous for a viral oncogene, because it has no cellular proto-oncogene counterpart. n 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. Arguments for such a modification of the px include the following. (i) Only a few of the BLV-infected animals develop persistent lymphocytosis and lymphosarcoma. (ii) As evidenced in our epidemiological surveys, in herds in which a BLV-induced lymphosarcoma occurs, the leukemia risk of the other animals drastically increases (this situation mimics the emergence of an "acute BLV" in tumor cells which then is horizontally transmissible within the herd). (iii) Finally, the role of the px as the "initiator" of cell multiplication by a "hit-and-run" mechanism is supported by the complete absence of BLV expression in tumor cells. We are indebted to J. M. Miller and M. J. Van der Maaten for supplying the FLK-BLV cell line. We thank J. Quesnel for access to a large variety of tumor-bearing cattle. We also acknowledge the technical assistance of C. Bourget and the careful preparation of the manuscript by L. Couillaud. LTERATURE CTED J. VROL. 1. Burny, A., C. Bruck, H. Chantrenne, Y. Cleuter, D. Dekegel, J. Ghysdael, R. Kettmann, M. Leclercq, J. Leunen, M. Mammerickx, and D. Portetelle Bovine leukemia virus: molecular biology and epidemiology, p n G. Klein (ed.), Viral oncology. Raven Press, New York. 2. Chandler, P. M., D. Rimkus, and N. Davidson Gel electrophoretic fractionation of RNAs by partial denaturation with methylmercuric hydroxide. Anal. Biochem. 99: Deschamps, J., R. Kettmann, and A. Burny Experiments with cloned complete tumor-derived bovine leukemia virus information prove that the virus is totally exogenous to its target animals species. J. Virol. 4: Ghysdael, J., R. Kettmann, and A. Burny Translation of bovine leukemia virus virion RNAs in heterologous protein-synthesizing systems. J. Virol. 29: Gregoire, D., D. Couez, J. Deschamps, S. Heuertz, M.-C. Hors-Cayla, J. Szpirer, C. Szpirer, A. Burny, G. Huez, and R. Kettmann Different bovine leukemia virus-induced tumors harbor the provirus in different chromosomes. J. Virol. 5: Haseltine, W. A., J. Sodroski, R. Patarca, D. Briggs, D. Perkins, and F. Wong-Staal Structural of 3' terminal region of type human T lymphotropic virus: evidence for new coding region. Science 225: Kawakami, T. G., A. L. Moore, G. H. Theilen, and R. J. Munn Comparisons of virus-like particles from leukotic cattle to feline leukosis virus. Bibl. Haemat. 36: Kenyon, S. J., and C. E. Piper Properties of density gradient-fractionated peripheral blood leukocytes from cattle infected with bovine leukemia virus. nfect. mmun. 16: Kettmann, R., A. Burny, Y. Cleuter, J. Ghysdael, and M. Manmerickx Distribution of bovine leukemia virus proviral DNA sequences in tissues of animals with enzootic bovine

VOL. 54, 1985 leukosis. Leuk. Res. 2:23-32. 1. Kettmann, R., D. Couez, and A. Burny. 1981. Restriction endonuclease mapping of linear unintegrated proviral DNA of bovine leukemia virus. J. Virol.

5 VOL. 54, 1985 leukosis. Leuk. Res. 2: Kettmann, R., D. Couez, and A. Burny Restriction endonuclease mapping of linear unintegrated proviral DNA of bovine leukemia virus. J. Virol. 38: Kettmann, R., J. Deschamps, Y. Cleuter, D. Couez, A. Burny, and G. Marbaix Leukemogenesis by bovine leukemia virus: proviral DNA integration and lack of RNA expression of viral long terminal repeat and 3' proximate cellular sequences. Proc. Natl. Acad. Sci. U.S.A. 79: Kettmann, R., J. Deschamps, D. Couez, J.-J. Claustriaux, R. Palm, and A. Burny Chromosome integration domain for bovine leukemia provirus in tumors. J. Virol. 47: Kettmann, R., D. Portetelle, M. Mammerickx, Y. Cleuter, D. Dekegel, M. Galoux, J. Ghysdael, A. Burny, and H. Chantrenne Bovine leukemia virus: an exogenous RNA oncogenic virus. Proc. Natl. Acad. Sci. U.S.A. 73: Mamoun, R. Z., T. Astier, and B. Guillemain Establishment and propagation of a bovine leukaemia virus-producing cell lines derived from the leukocytes of a leukaemic cow. J. Gen. Virol. 54: Mamoun, R. Z., T. Astier, B. Guillemain, and J. F. Duplan Bovine lymphosarcoma: expression of BLV-related proteins in cultured cells. J. Gen. Virol. 64: Mamoun, R. Z., T. Astier, B. Guillemain, and J. F. Duplan Bovine lymphosarcoma: processing of bovine leukaemia virus coded proteins. J. Gen. Virol. 64: Maniatis, T., E. F. Fritsch, and J. Sambrook Molecular cloning, a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 18. McAster, G. K., and G. G. Garmichael Analysis of single and double stranded nucleic acids on polyacrylamide and agarose NOTES 629 gels by using glyoxal and acridine orange. Proc. Natl. Acad. Sci. U.S.A. 74: Miller, J. M., L. D. Miller, C. Olson, and K. G. Gillette Virus-like particles in phytohemagglutinin-stimulated lymphocyte cultures with reference to bovine lymphosarcoma. J. Natl. Cancer nst. 43: Neel, B. G., W. S. Hayward, H. L. Robinson, J. Fang, and S. M. Astrin Avian leukosis virus-induced tumors have common proviral integration sites and synthesize discrete new RNAs: oncogenesis by promoter insertion. Cell 23: Paul, P. S., K. A. Pomeroy, A. E. Castro, D. W. Johnson, C. C. Muscoplat, and D. K. Sorensen Detection of bovine leukemia virus in B-lymphocytes by the syncytia induction assay. J. Natl. Cancer nst. 59: a.Rice, N. R., R. M. Stephens, D. Couez, J. Deschamps, R. Kettmann, A. Burny, and R. V. Gilden The nucleotide sequence of the env gene post-env region of bovine leukemia virus. Virology 138: Seiki, M., S. Hattori, Y. Hirayama, and M. Yoshida Human adult T-cell leukemia virus: complete nucleotide sequence of the provirus genome integrated in leukemia cell DNA. Proc. Natl. Acad. Sci. U.S.A. 8: Stephenson, J. R. (ed.) Type C structural and transformation-specific proteins, p n Molecular biology of RNA tumor viruses. Academic Press, London. 24. Stock, N. D., and J. F. Ferrer Replicating C-type virus in phytohemagglutinin-treated buffy-coat cultures of bovine origin. J. Natl. Cancer nst. 48: Van der Maaten, M. J., and J. M. Miller Replication of bovine leukemia virus in monolayer cell cultures. Bibl. Haemat. 43: Downloaded from on January 22, 219 by guest

Even Transcriptionally Competent Proviruses Are Silent in Bovine Leukemia Virus Induced Tumor Cells*

Even Transcriptionally Competent Proviruses Are Silent in Bovine Leukemia Virus Induced Tumor Cells* A. van den Broeke 1, Y. Cleuter 2, G. Chen 3, D. Portetelle 3, M. Mammerickx\ D. Zagury5, M. Fouchard