INVIVO INFECTION OF SHEEP BY BOVINE LEUKEMIA-VIRUS MUTANTS

Direct inoculation of a cloned bovine leukemia virus (BLV) provirus in to 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 express ion in cell culture. The proviruses were then inoculated into sheep in order to assess viral infectivity in vivo. Of three variants cloned f rom BLV-induced tumors (344, 395, and 1345), one (344) was found infec tious in vivo. This particular provirus was used to engineer 12 BLV mu tants. A hybrid between the 5' region of the complete but noninfectiou s provirus 395 and the 3' end of mutant 344 was infectious in vivo, su ggesting that the tax/rex sequences were altered in virus 395. As expe cted, several regions of the BLV genome appeared to be essential for v iral infection: the protease, pol, and env genes. Even discrete modifi cations in the fusion peptide located at the NH, end of the transmembr ane gp30 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 v ivo. This region of unknown function could thus be used to introduce f oreign sequences. A BLV recombinant carrying a ribozyme directed again st the tax/rex sequences was still infectious in vivo. Cotransfection of two noninfectious mutants carrying deletions led to infection in tw o of four independent injections, the infectious virus being then a re combinant between the two deletants. The experimental approach describ ed here should help to gain insight into essential mechanisms such as in vivo viral replication, cooperation between deletants for viral inf ectivity, and viral superinfections. The gene products in the X3 and X 4 region which are dispensable for in vivo infection could be involved in leukemogenesis, and thus proviruses deleted in these sequences cou ld constitute the basis for a live attenuated vaccine.