Evidence for phosphorylation of trans-activator p40x 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 p40x which positively regulates transcription of the viral RNA as well as interleukin-2 and its receptor genes. We placed a cDNA coding for p40x in baculovirus Bombyx 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 p40x. The biological activity of the recombinant p40x 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 p40x in both the cytoplasmic and nuclear fractions of BmN cells. Analyses of 32P-labeled proteins of BmN cells by cell fractionation and subsequent immunoprecipitation revealed that the p40x 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 p40x.
Nyunoya
H
H
Virology Division, National Cancer Center Research Institute, Tokyo, Japan.
Akagi
T
T
Ogura
T
T
Maeda
S
S
Shimotohno
K
K
D000818:Animals; D001681:Biological Assay; D012831:Bombyx; D002451:Cell Compartmentation; D002478:Cells, Cultured; D005786:Gene Expression Regulation; D015694:Gene Products, rev; D005822:Genetic Vectors; D006678:HIV / Q000502:physiology*; D007304:Insect Viruses; D010750:Phosphoproteins / Q000235:genetics* / Q000502:physiology; D010766:Phosphorylation; D010768:Phosphoserine / Q000032:analysis; D011233:Precipitin Tests; D012191:Retroviridae Proteins / Q000235:genetics* / Q000502:physiology; D014157:Transcription Factors / Q000378:metabolism*; D054321:rev Gene Products, Human Immunodeficiency Virus
D015694:Gene Products, rev; D010750:Phosphoproteins; D012191:Retroviridae Proteins; D014157:Transcription Factors; D054321:rev Gene Products, Human Immunodeficiency Virus; D010768:Phosphoserine