Evidence for phosphorylation oftrans-activator p40^x of human T-cell leukemia virus type I produced in insect cells with a baculovirus expression vector

Human T-cell leukemia virus type I (HTLV-I) encodes a trans-activator protein p40^x which positively regulates transcription of the viral RNA as well as interleukin-2 and its receptor genes. We placed a cDNA coding for p40^x in baculovirusBombyx mori nuclear polyhedrosis virus (BmNPV) expression vectors. Infection of BmN cells derived from an insect,B. mori (silkworm), with a recombinant virus led to the production of soluble p40^x. The biological activity of the recombinant p40^x was demonstrated by introducing the protein into intact NIH 3T3 cells that had been selected for genomic integration of HTLV-I LTR connected with the CAT gene. Immunocytochemical and cell fractionation analyses showed the localization of p40^x in both the cytoplasmic and nuclear fractions of BmN cells. Analyses of³²P-labeled proteins of BmN cells by cell fractionation and subsequent immunoprecipitation revealed that the p40^x present in each subcellular fraction was phosphorylated. The post-translational modification was inhibited by the addition of a protein kinase inhibitor K252a during the metabolic labeling of BmN cells. Phosphoamino acid analysis indicated that the phosphorylation occurred on serine residues of p40^x.