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Complex oncoviruses contain, in addition to the classical retroviral genes (gag, pol, and env), a region (X) located between the envelope sequences and the 3' long terminal repeat. The X region contains two genes, tax and rex, whose protein products are involved in transcriptional and posttranscriptional regulation of viral expression. In addition to these activators, the bovine leukemia virus (BLV) and the human T-cell leukemia virus (HTLV) contain alternative open reading frames (R3 and G4 for BLV; p30, p13, and p12 for HTLV). As a virus/animal model for HTLV-induced leukemogenesis, BLV provirus can be injected intradermally into sheep, where it induced B-lymphocyte transformation. Deletion of the R3 and G4 sequences from an infectious and tumorigenic BLV provirus greatly impaired the in vivo propagation of the viruses as demonstrated by DNA polymerase chain reaction, RNA blots, structural-protein ELISA, and immunofluorescence analysis. Our results show that the alternative open reading frames are required for maintaining high virus loads during the course of persistent infection in vivo. Thus, R3 and G4 are candidates for antiviral drug development. Furthermore, viruses with a deletion in these sequences should be tested as live attenuated vaccines.

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 METHODSBovine 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) Liquid hybridization was performed as described (2). RESULTS Persistent lymphocytosisAs shown in Fig. 1A, BLV [32P]cDNA hybridized on a large number of EcoRI fragments from leukocyte DNA of animals in PL. Molecular weights of the fragments cover a wide range, varying between 5-6 X 106 and 10-12 X 106. In some samples, hybridization bands emerged over the continuous background, an indication that some integration sites were preferred. No fragment with Mr lower than 5 X 106 was apparent. The control, calf thymus DNA digest, showed a weak band at Mr 5 X 106, corresponding to ribosomal DNA (13,14). The integrated BLV proviruses examined so far (13) showed none or at the most one EcoRI restriction site.BLV [32P]cDNA also hybridized on two BamHI fragments, Mr 2.0 X 106 and 1.3 X 106, from the above DNAs. Weak hybridization bands were also observed in some cas...

Direct inoculation of a cloned bovine leukemia virus (BLV) provirus into sheep has allowed study of the viral infectivity of genetic mutants in vivo. Three BLV variants cloned from BLV-induced tumors and 12 in vitro-modified proviruses were isolated and analyzed for viral expression in cell culture. The proviruses were then inoculated into sheep in order to assess viral infectivity in vivo. Of three variants cloned from BLV-induced tumors (344, 395, and 1345), one (344) was found infectious in vivo. This particular provirus was used to engineer 12 BLV mutants. A hybrid between the 5' region of the complete but noninfectious provirus 395 and the 3' end of mutant 344 was infectious in vivo, suggesting that the tax/rex sequences were altered in virus 395. As expected, several regions of the BLV genome appeared to be essential for viral infection: the protease, pol, and env genes. Even discrete modifications in the fusion peptide located at the NH2 end of the transmembrane gp3O glycoprotein destroyed the infectious potential. In contrast, mutations and deletions in the X3 region present between the env gene and the 3' tax/rex region did not interfere with viral infection in vivo. This region of unknown function could thus be used to introduce foreign sequences. A BLV recombinant carrying a ribozyme directed against the tax/rex sequences was still infectious in vivo. Cotransfection of two noninfectious mutants carrying deletions led to infection in two of four independent injections, the infectious virus being then a recombinant between the two deletants. The experimental approach described here should help to gain insight into essential mechanisms such as in vivo viral replication, cooperation between deletants for viral infectivity, and viral superinfections. The gene products in the X3 and X4 region which are dispensable for in vivo infection could be involved in leukemogenesis, and thus proviruses deleted in these sequences could constitute the basis for a live attenuated vaccine.