PHOSPHORYLATION OF BOVINE LEUKEMIA-VIRUS TAX PROTEIN IS REQUIRED FOR IN-VITRO TRANSFORMATION BUT NOT FOR TRANSACTIVATION

The Tax proteins of the oncovirinae viruses are phosphorylated transcr iptional activators that exhibit oncogenic potential. The role of phos phorylation in their functional activities remains unknown. As a model for the Human T-cell leukemia virus type I (HTLV-I), Bovine Leukemia Virus (BLV) permits the characterization of viral replication and leuk emogenesis in vitro. Here, we show that the BLV Tax protein is phospho rylated on serine residues 106 and 293 both in insect and in mammalian cells. These sites can also be efficiently phosphorylated by the cdc2 and MAP kinases in vitro. Mutation of these residues does not affect the capacity of the Tax protein to function as a transactivator. Indee d, the Tax proteins mutated at one or both serines increase LTR-direct ed viral transcription at levels similar to those obtained with wild-t ype Tax in cell culture, Moreover, inhibition of Tax phosphorylation b y W7, a calmodulin antagonist, does not alter its transactivation acti vity. Thus, phosphorylation on serines 106 and 293 is not required for transactivation by Tax, However, simultaneous substitution of both se rines into alanine residues destroys the capacity of Tax to cooperate with the Haras oncogene to transform primary rat embryo fibroblasts an d induce tumors in nude mice, When the serines were replaced with aspa rtic acid residues, the oncogenic potential of Tax was maintained indi cating that the negative charge rather than the phosphate group itself was required for Tax oncogenicity, Finally, to assess the role of the serine residues in vivo, recombinant viruses which express the Tax mu tants were constructed and injected into sheep. It appeared that the m utated proviruses replicate at levels similar to the wild-type virus i n vivo. We conclude that Tax phosphorylation is dispensable for transa ctivation and viral replication in vivo but is required for its oncoge nic potential in vitro.