Genomic integration of bovine leukemia provirus: Comparison

Integration of bovine leukemia proviral DNA in the genome of infected cells was investigated in cattle affected by either the persistent lymphocytosis or the lymph node tumor form of enzootic bovine leukosis. In persistent lymphocytosis, proviral DNA was found to be integrated at a large number of genomic sites in one-fourth to one-third of circulating leukocytes. In the lymph node tumor form, in contrast, proviral DNA was found to be integrated at one or very few sites in the genomes of a larger fraction of both circulating leukocytes and lymph node tumor cells. Enzootic bovine leukosis (EBL) is a lymphoproliferative disease of cattle induced by an exogenous retrovirus, the bovine leukemia virus (BLV) (1-3), whose target cell is the B lymphocyte (4). Two forms of the disease are known: persistent lymphocytosis (PL), characterized by a permanent large number of peripheral lymphocytes (5-7), and a tumoral form, characterized by lymph node tumors. These two forms generally represent the early and the late stage of the disease, respectively. In some cases, however, these two pathological conditions do not affect the same cattle herds, suggesting that they may represent separate responses to BLV infection, both of them being under the control of genetic factors (8-10). In general, the fate of BLV-infected animals is variable and depends upon several factors, including age, genetic make-up, environmental factors, and immunological surveillance (see ref. 11 for a review). In the present work, we have compared proviral integration into the genomes of host cells of naturally infected animals that were in either the PL or the tumor stage of the disease. The main result obtained in these investigations was that proviral copies were found to be integrated at several sites in the genomes of one-fourth to one-third of the leukocytes of animals in PL, but at only one or very few sites in the genomes of a larger fraction of leukocytes or tumor cells of animals in the tumor stage of the disease. MATERIALS AND METHODS Bovine tissues and cells were collected from seven animals with EBL. Four animals (nos. 928, 641, 2586, and 4) were in PL and three (nos. 15, 950, and 82) in the tumor stage of the disease. Leukocytes (W928, W641, W2586, W4, W15, W950, and W82), lymph node tumors (T15 and T950), and the thymus of a normal calf were used as sources of DNA. Preparation of virus and viral DNA, cDNA synthesis, DNA The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 2577 extraction, digestion with restriction endonucleases, and gel electrophoresis were done as described (12, 13). Gel transfer and filter hybridization were carried out as described (13, 14), except that BLV [32P]cDNA was prehybridized for 16 hr with 3 mg of sonicated, denatured calf thymus DNA per ml; this largely prevented spurious hybridization of the probe with satellite and ribosomal DNA (13). Filters were exposed at -70°C to preflashed Kodak RP-Royal X-Omat film in the presence of Siemens "special" intensifying screens for 2-5 days. Liquid hybridization was performed as described (2).