Phosphorylation of bovine leukemia virus Tax protein is required for in vitro transformation but not for transactivation

Abstract

The Tax proteins of the oncovirinae viruses are phosphorylated transcriptional activators that exhibit oncogenic potential. The role of phosphorylation 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 leukemogenesis in vivo. Here, we show that the BLV Tax protein is phosphorylated on serine residues 106 and 293 both in insect and in mammalian cells. These sites can also be eciently phosphorylated by the cdc2 and MAP kinases in vitro. Mutation of these residues does not aect the capacity of the Tax protein to function as a transactivator. Indeed, the Tax proteins mutated at one or both serines increase LTR-directed viral transcription at levels similar to those obtained with wild-type Tax in cell culture. Moreover, inhibition of Tax phosphorylation by W7, a calmodulin antagonist, does not alter its transactivation activity. Thus, phosphorylation on serines 106 and 293 is not required for transactivation by Tax. However, simultaneous substitution of both serines into alanine residues destroys the capacity of Tax to cooperate with the Haras oncogene to transform primary rat embryo ®bro-blasts and induce tumors in nude mice. When the serines were replaced with aspartic acid residues, the oncogenic potential of Tax was maintained indicating 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 mutants were constructed and injected into sheep. It appeared that the mutated proviruses replicate at levels similar to the wild-type virus in vivo. We conclude that Tax phosphorylation is dispensable for transactivation and viral replication in vivo but is required for its oncogenic potential in vitro.

Increased cell proliferation, but not reduced cell death, induces lymphocytosis in bovine leukemia virus-infected sheep

“…Three sheep (nos. 8, 105, and 293) were persistently infected with wild-type pBLVIX (29), whereas animals 104, 2658, 2667, and 2668 harbored provirus pBLVTax106 ϩ 293, pLTR-NF1, pLTR-NF2, and pLTR-Ebox, respectively (31,32). Importantly, all proviruses behaved as wild type during pathogenesis, despite the presence of some mutations.…”

The Homeobox Protein MSX2 Interacts with Tax Oncoproteins and Represses Their Transactivation Activity

“…Indeed mutations in the zinc finger structure abrogate the capacity of Tax to activate viral expression without concomitant loss of immortalization (14). On the other hand, phosphoserines 106 and 293 of BLV Tax are required for in vitro transformation but not for transactivation (16). Dissociation of these two activities of Tax might be a major process which allows cell immortalization in the absence of viral structural gene expression.…”

“…It thus appears that MSX2 represses Tax-dependent transactivation but still permits cell transformation indicating that both functions of Tax are differently modulated by MSX2. It should be mentioned here that separable domains within the BLV Tax protein mediate transactivation and transformation (16). Indeed mutations in the zinc finger structure abrogate the capacity of Tax to activate viral expression without concomitant loss of immortalization (14).…”

“…Plasmids pSGCREB and pPKA express, respectively, the bovine CREB2 protein and the protein kinase A (28). Plasmids pBDTax, pBDTax(106 ϩ 293), p53, and pLaminC express the DNA binding domain (BD) of the yeast transactivator Gal4 fused to the BLV Tax protein, the Tax mutant in which serine residues 106 and 293 were replaced by alanine sequences (16), the murine p53 (amino acids 72-390), and the human Lamin C (amino acids 67-230), respectively. Plasmids pADMSX2 and pSV40 code for the Gal4-activating domain (AD) fused to the human MSX2 protein or the amino acids 84 -708 of the SV40 large T-antigen, respectively.…”

“…Both transactivation and immortalizing functions of Tax can be dissociated by mutations in specific regions of the protein. For example, mutation of the phosphorylation sites at serines 106 and 293 of BLV Tax abrogates immortalization potential in vitro but maintains transcriptional activity and viral oncogenicity in vivo (16,17). Conversely, the transactivation of the LTR promoter is not required for Tax to transform primary cells in vitro (14).…”