Intracellular analysis of in vitro modified HIV Tat protein

• Published in: The Journal of biological chemistry 1994-03, Vol.269 (11), p.8366-8375
• Main Authors: KOKEN, S. E. C, GREIJER, A. E, VERHOEF, K, VAN WAMEL, J, BURKRINSKAYA, A. G, BERKHOUT, B
• Format: Article
• Language: English
• Subjects: AIDS/HIV; Base Sequence; Biological and medical sciences; Cadmium - metabolism; Cadmium - pharmacology; Cell Line; Chloramphenicol O-Acetyltransferase - metabolism; Dithionitrobenzoic Acid; Dithiothreitol - pharmacology; DNA Primers; Electroporation; Escherichia coli; Fundamental and applied biological sciences. Psychology; Gene Products, tat - biosynthesis; Gene Products, tat - isolation & purification; Gene Products, tat - metabolism; Glutathione Transferase - biosynthesis; Glutathione Transferase - isolation & purification; Glutathione Transferase - metabolism; HIV-1 - metabolism; HIV-2 - metabolism; human immunodeficiency virus 1; human immunodeficiency virus 2; Humans; Kinetics; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; Oxidation-Reduction; Polymerase Chain Reaction; Recombinant Fusion Proteins - biosynthesis; Recombinant Fusion Proteins - isolation & purification; Recombinant Fusion Proteins - metabolism; Replication; RNA-Binding Proteins - metabolism; Sulfhydryl Compounds - pharmacology; T-Lymphocytes; tat Gene Products, Human Immunodeficiency Virus; Transfection; Zinc - metabolism; Zinc - pharmacology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(17)37203-4

Human immunodeficiency viruses HIV-1 and HIV-2 encode a Tat protein that specifically activates transcription from the viral long terminal repeat. To characterize the properties of the Tat proteins, we have expressed them in Escherichia coli. The purified Tat protein was biochemically analyzed and tested for activity upon electroporation into human cell lines. This protein electroporation was used for the intracellular analysis of in vitro modified Tat protein. Our results indicate that the transcriptionally active form of the Tat protein is a monomer. Furthermore, we found that Tat activity is dramatically inhibited by preincubation of the protein with strongly reducing agents. In contrast, no inhibitory effect was measured upon incubation with metal-chelating reagents. These results suggest that the cysteine residues of Tat are involved in the formation of intramolecular disulfide bonds.