Structure of a Defective Provirus of Bovine Leukemia Virus

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1 Microbial. Immunol. Vol. 31 (10), , 1987 Structure of a Defective Provirus of Bovine Leukemia Virus Yasuki OGAWA,1,5 Noriyuki SAGATA,2 Junko TSUZUKU-KAWAMURA,2 Hiroyuki KOYAMA,3 Misao ONUMA,*,4 Hisao IZAWA,1 and Yoji IKAWA2 Hokkaido 060, 2Laboratory of Molecular Oncology, The Institute of Physical and Chemical Research, Wako, Saitama , 3Department of Veterinary Microbiology, School of Veterinary Medicine and Animal Science, Kitasato University, Towada, Aomori 034, 4Department of Veterinary Microbiology, College of Dairy Agriculture, Ebetsu, Hokkaido 069, and 5Laboratory of Cell Biology, Basic Research Laboratories, Toray Industries, Inc., Kamakura, Kanagawa 248 (Accepted for publication, June 13, 1987) Abstract Defective proviruses of bovine leukemia virus (BLV) in the genomes of infected cells were investigated by using Southern blotting hybridization analysis with various portions of a cloned BLV DNA as probes. When nine independent tumors of enzootic bovine leukosis with a single proviral copy per cell were examined, a single defective provirus of BLV was found in one tumor and also in a bovine B cell line derived from this tumor. Hybridization analysis of this defective provirus revealed that it underwent deletion between the pol and env genes and contained no major deletion in the other regions. Bovine leukemia virus (BLV), an exogenous and replication-competent retrovirus, causes lymphosarcomas or enzootic bovine leukosis (EBL) in cattle (1, 2) and is related to human T-cell leukemia virus (HTLV) (15, 16) which is responsible for adult T-cell leukemia (ATL) (20). BLV, like HTLV, has neither typical oncogene (2) nor preferential integration site in the host chromosome with the subsequent activation of its flanking cellular sequences (4, 5, 8). The integrated BLV provirus is quiescent in vivo (1, 4). Furthermore, no overexpression of most known oncogenes can be detected in the leukemic cells (6). Thus, the mechanism of the cellular transformation by BLV still remains enigmatic. However, the presence of a BLV provirus in all the leukemic cells (1) and the ability of BLV to transform cells in vitro (9, 10) suggest that the BLV genome possesses a possible transforming gene. Recent molecular biological (16) and biochemical (14) studies suggest that the BLV genome harbors the.unique XBL gene encoding a non-structural 38 kda protein, corresponding to the X gene of HTLV (16, 17). Analysis of the defective proviruses of HTLV in ATL tumor cells showed that deletion occurred in the 5'LTR, gag, pol, and env regions, and that the X and 3'LTR regions were retained (20). In contrast to HTLV, little is known about the genomic 1009

2 1010 Y. OGAWA ET AL structures of defective proviruses of BLV. A previous analysis of abnormal restriction patterns of BLV proviruses suggested that deletion was liable to occur in the 7.7 kb fragment located at the 5'side of the proviral genome (4). However, the remaining and deleted regions of the single defective provirus of BLV have been unclear. Here, to know these regions, we examined the organization of a single defective provirus of BLV by Southern blotting analysis with molecularly cloned and well-defined BLV DNA as probes. MATERIALS AND METHODS Tissues and cells. Bovine tumor cells were obtained from leukemic (EBL) cattle. BLSC-KU1, a bovine B cell line established from EBL tumor 89, was maintained in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum (7). Extraction of genomic DNA. Genomic DNA was extracted as previously described (8). Hybridization probes. Various 32P-labeled probes (1-3 x 108 cpm/,ug) were prepared with [cc-32p]dctp (3,000 Ci/mmol; Amersham) by nick translation (11) from Figures 1B and 2A show the regions of each probe: the major part of the long terminal repeat (LTR) and a 5'portion of the gag gene (probe LG), portions of the gag and protease genes (probe G), the pol and env genes (probe PE), and the major part of the XBL gene (probe XBL), corresponding to the X gene of HTLV (16, 17) of BLV proviral DNA. Southern blotting hybridization. Ten itg each of DNA were digested with restriction enzymes by procedures recommended by the supplier (Takara-Shuzo, Kyoto, Japan), electrophoresed on an agarose gel, transferred to a nitrocellulose filter, and hybridized with various 32P-labeled probes (19). RESULTS DNAs obtained from nine independent EBL tumors, each carrying a single BLV proviral copy per cell, were digested with Sstl and then hybridized with probe XBL. The very small Sst1 fragments (of 0.1 kb) were not detected because of the difficulty in transferring them to a nitrocellulose filter. In most leukemic cells tested (74, 77, 79, 80, and 81), two proviral fragments of 1.3 and 6.8 kb were detected as expected from a representative BLV provirus (Fig. 1A). Three other tumors tested (13, 70, and 72) showed the same bands of 1.3 and 6.8 kb (data not shown). But, in only one tumor (89) and its deriving lymphoid cell line (BLSC-KU1) (lanes 89 and BL in panel XBL/SstI of Fig. 1A, respectively), a hybridization fragment of 5.2 kb, instead of 6.8 kb, could be commonly detected. This clearly indicates that it was a single copy of provirus that underwent deletion. Tumor 89 and BLSC-KU1 cells showed the same hybridization patterns when other enzymes and probes were used (Fig. 1A). These results revealed that the BLV provirus in

3 DEFECTIVE PROVIRUS OF BLV 1011 A B Fig. 1. Integration of BLV proviruses in the genomes of six independent EBL tumors and a cell line. DNAs of EBL tumors (74, 77, 79, 80, 81, and 89) and a cell line BLSC-KU1 (BL) were cleaved with Sstl or EcoRI, electrophoresed on 0.8% agarose gels and transferred to nitrocellulose filters. The filters were hybridized with 32P-labeled probes LG and XBL (A). HindIII-digested ADNA and HaeIII-digested OX174 DNA markers were used as reference (in kb). Location in the BLV proviral genome of restriction sites and probes used here is shown (B). Pv, Pvull; RI, EcoRI; S 1, Sall; St, Sstl. BLSC-KU 1 (BLV/BLSC-KU 1) was identical to that in the EBL tumor 89. Therefore, BLV/BLSC-KU1 was chosen for further studies. To examine the deletion of BLV/BLSC-KU1, SallTvull restriction patterns of BLV/BLSC-KU1 obtained with four probes (LG, G, PE, and XBL in Fig. 2B) were compared with those of a representative BLV provirus in tumor 81 (BLV/ Tumor 81) (Fig. 2A). This double digestion gave rise to several common proviral fragments in both the DNAs with three probes: 1.1 and 3.1 kb with probe LG, 2.0 and 3.1 kb with probe G, and 1.5 kb with probe XBL. The fragment of 3.1 kb probably resulted from the partial digestion of the Sall site within BLV proviral DNA, because Sail enzyme does not cleave its recognition site (5'GTCGAC3') if the internal cytosine residue is methylated. Probe LG detected one more additional fragment which is most likely to be composed of 3'LTR and its flanking cellular sequences, because each fragment was a different length (5.2 kb in lane 81, and 0.8 kb in lane BL of probe LG; Fig. 2A). These results revealed that no major deletion was in the LTRs, gag and XBL regions of BLV/BLSC-KU 1. In contrast, a strikingly different pattern was observed in both the DNAs with probe PE; the fragment

4 1012 V. OGAWA ET AL A B Fig. 2. Hybridization analysis of deletion of BLV/BLSC-KU I. DNAs from 17,131, tumor 81 (81) and BLSC-KU1 (BL) cells were doubly digested with Sall and Pvull, electrophoresed on 1.2% agarose gels, and then hybridized with four 32P-labeled probes (LG, G, PE, and XBD. The hybridization patterns obtained were compared between 131 _,V/Tumor 81 and BLV/BLSC-KU1 (A). Only the restriction sites of interest and location of four hybridization probes are shown here (B). The broken line indicates the region in which deletion of BLV/ BLSC-KU1 occurred in a representative BLV proviral DNA. PT, protease; Bg, Bgl II ; 13m, BamHI; RI, EcoRl; Pv, Pvull; of the incomplete provirus was 1.9 kb (lane BL of probe PE in Fig. 2A), which was smaller than that of the complete provirus (lane 81 of probe PE in Fig. 2A). This finding exhibited that its pot and/or env regions apparently underwent deletion. To examine the deletion accurately, the Pvu11-digested DNAs of BLSC-KU I and tumor 81 cells were further digested with various enzymes, and then probed with PE (Fig. 3). In BLSC-KU1 cells, the Pvulll BamH1 restriction pattern (a fragment of 1.9 kb) was quite similar to that of the PvuII digestion, indicating that BLV/BLSC-KU1 had no BamHI site (Fig. 3). Moreover, it most likely lacked the HindIII site; in HindIII/HincII-digested preparation, although a fragment of 1.1 kb was commonly detected in both the DNAs, one fragment of 0.8 kb could be detected in BLV/BLSC-KU 1 instead of twofragments of 1.0 and 1.4 kb in the complete BLV/Tumor 81. A summary of the results obtained in these experiments shows that the region from the 5' Xhol site (at 0.35 kb from the 5'PvuII site) to the Hindi site probably underwent deletion of 1.6 kb, and that the HindIII-BamHI fragment of a representative BLV provirus was likely deleted in BLV/BLSC-KU 1; a 3'portion of the pol gene and a 5/portion of the env gene was deleted (Fig. 3, D and E).

5 DEFECTIVE PROVIRUS OF BLV 1013 A B C E Fig. 3. Comparison of BLV/BLSC-KU1 with BLV/Tumor 81. DNAs from EBL tumor 81 and BLSC-KU1 cells were digested with PvuII and further digested with BamHI, BglII, HindIII/HincII, XbaI, or XhoI, followed by hybridization with probe PE. The size of hybridization fragments were compared between the complete (Tumor 81) and incomplete (BLSC-KU1) BLV proviruses (A). Partial restriction maps (C and D) and genomic organizations (B and E) of these proviruses were also compared. The hybridization fragments that were commonly detected are indicated by asterisks. Restriction sites indicated by open circles are absent from BLV/BLSC-KU 1. Partially deleted genes are indicated by prime. Based on Southern blotting analysis of BLV/BLSC-KU1, the areas in which deletion occurred are indicated by closed box. PT, protease; Bg, BglII; Bm, BamHI ; Hc, Hinc- II ; Hd, HindIII ; Pv, PvulI; Xb, XbaI; Xh, XhoI. DISCUSSION In the present experiment, we examined the structure of a single defective provirus of BLV to know its remaining and deleted regions. We found a single defective provirus of BLV integrated into the genomes of EBL tumor 89 and BLSC-KU 1 cells, a bovine B cell line established from this tumor (7). Using various portions of a moleculary cloned and well-defined BLV DNA as probes, Southern blotting analysis of this defective provirus revealed that the regions encompassing the gag, protease, and XBL genes and both the LTRs underwent no major deletion, and that the region spanning the pol and env genes was partially deleted. The observed deletion, which might occur accidentally after the neoplastic transformation, supports the result that BLSC-KU1 cells produce no BLV (7). However, a possibility that the deleted pol and env genes might encode altered proteins could not be ruled out.

6 1014 Y. OGAWA ET AL Considering the transcriptional activator proteins encoded by the X (18) and XBL (12) genes and the conservation of the X region in the tumors of ATL (20), our observation may raise the possibility that the XBL region plays a role in development of EBL. The biological significance of the observed deletion, however, remains unknown since this occurred in only one case. On the other hand, in HTLV, it was reported that the 5'side of the proviral genome was prone to deletion irrespective of leukemogenesis (3). To understand the relationship of the XBL region to development of EBL, analysis of many single defective BLV proviruses in EBL tumors each carrying one proviral copy per cell would be necessary. This work was supported by Grants-in-Aid for Cancer Research from the Ministry of Education, Science and Culture and for Comprehensive 10-year Strategy of Cancer Control from the Ministry of Health and Welfare, Japan. REFERENCES 1) Burny, A., Bruck, C., Chantrenne, H., Cleuter, Y., Dekegel, D., Ghysdeal, J., Kettmann, R., Leclercq, M., Leunen, J., Mammerickx, M., and Portetelle, D Bovine leukemia virus: molecular biology and epidemiology, p In Klein, G. (ed), Viral oncology, Raven Press, New York. 2) Deshamps, J., Kettmann, R., and Burny, A Experiments with cloned complete tumorderived bovine leukemia virus information prove that the virus is totally exogenous to its target animal species. J. Virol. 40: ) Hiramatsu, K., and Yoshikura, H Frequent partial deletion of human adult T-cell leukemia virus type I proviruses in experimental transmission: pattern and possible implication. J. Virol. 58: ) Kettmann, R., Deshamps, J., Cleuter, Y., Couez, D., Burny, A., and Marbaix, G 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., Deshamps, J., Couez, D., Claustriaux, J.-J., Palm, R., and Burny, A Chromosome integration domain for bovine leukemia provirus in tumors. J. Virol. 47: ) Kettmann, R., Westin, F.H., Marbaix, G., Deshamps, J., Wong-Staal, F., Gallo, R.C., and Burny, A Lack of expression of cellular homologues of retroviral onc genes in bovine tumors, p In Neth, R., Gallo, R.C., Greaves, M., Moore, M., and Winkler, S. (eds), Haematology and blood transfusion, Vol. 28, Spring-Verlag, Berlin. 7) Onuma, M., Koyama, H., Aida, Y., Okada, K., Ogawa, Y., Kirisawa, R., and Kawakami, Y Establishment of B cell line from tumors of enzootic bovine leukosis. Leukemia Res. 10: ) Onuma, M., Sagata, N., Okada, K., Ogawa, Y., Ikawa, Y., and Oshima, K Integration of bovine leukemia virus DNA in the genomes of bovine lymphosarcoma cells. Microbiol. Immunol. 26: ) Onuma, M., Watarai, S., Suneya, M., Mikami, T., and Izawa, H Induction of transformed phenotypes in sheep fibroblasts by culture fluids from cells persistently infected with bovine leukemia virus. Microbiol. Immunol. 25: ) Rhim, J.S., Kraus, M., and Arnstein, P Neoplastic transformation of fetal lamb kidney cells by bovine leukemia virus. Int. J. Cancer 31: ) Rigby, P.W., Dieckmann, M., Rhodes, C., and Berg, P Labelling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J. Mol. Biol. 113:

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