Structural and functional characterization of mutants of the bovine leukemia virus transactivator protein p34

Mutants of the bovine leukemia virus (BLV) transactivator protein (tat, tax, p34, the XLOR gene product) were constructed by site-directed deletions, in-phase linker insertions, or fragment replacements (swapping) among BLV variants. The mutant constructs were transfected into cos cells and transiently expressed. Western blot analysis using a mixture of monoclonal antibodies to wild-type p34 revealed the presence of mutated XLOR gene products in all the mutants tested. The transactivating activity of 11 tax mutants containing site-directed deletions and in-phase linker insertions was completely abolished. Only the swapping mutant tested, a hybrid between two BLV variants, transactivated LTR-directed gene expression at wild-type levels. These data illustrate the narrow range of structural variations that allow full activity of the BLV tax product and suggest that the present molecular structure of the transactivator protein results from heavy evolutionary constraints.