Intracellular analysis of in vitro modified HIV Tat protein.
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.
Koken
S E
SE
Department of Virology, University of Amsterdam, The Netherlands.
Greijer
A E
AE
Verhoef
K
K
van Wamel
J
J
Bukrinskaya
A G
AG
Berkhout
B
B
D001483:Base Sequence; D002104:Cadmium / Q000378:metabolism / Q000494:pharmacology; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D017931:DNA Primers; D004228:Dithionitrobenzoic Acid; D004229:Dithiothreitol / Q000494:pharmacology; D018274:Electroporation; D015696:Gene Products, tat / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D005982:Glutathione Transferase / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D015497:HIV-1 / Q000378:metabolism*; D015498:HIV-2 / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D016133:Polymerase Chain Reaction; D016601:RNA-Binding Proteins / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000494:pharmacology; D013601:T-Lymphocytes; D014162:Transfection; D015032:Zinc / Q000378:metabolism / Q000494:pharmacology; D054322:tat Gene Products, Human Immunodeficiency Virus
D017931:DNA Primers; D015696:Gene Products, tat; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D013438:Sulfhydryl Compounds; D054322:tat Gene Products, Human Immunodeficiency Virus; D002104:Cadmium; D004228:Dithionitrobenzoic Acid; D015500:Chloramphenicol O-Acetyltransferase; D005982:Glutathione Transferase; D015032:Zinc; D004229:Dithiothreitol