Genetic tests to reveal TAT homodimer formation and select TAT homodimer inhibitor.
The HIV Tat protein is essential for productive infection and is a potent activator of viral gene expression. By constructing a genetic fusion between the amino-terminal DNA-binding domain of the lambda repressor (as a reporter for dimerization) and Tat, we show that Tat forms dimers in vivo. By deletion analysis and site-directed mutagenesis, we show that (i) the peptide encoded by exon-1 of Tat is sufficient to promote dimerization and (ii) cys37 is essential for homo-dimerization of Tat protein. Furthermore, by using a new E. coli strain in which the expression of beta-galactosidase is under the negative control of the cl::Tat repressor, we select a protein (CD10/Nep) expressed by human Jurkat T-cells which inhibits Tat dimerization.
Battaglia
P A
PA
Dipartimento di Biopatologia Umana, Università La Sapienza, Policlinico Umberto I, Roma, Italy.
Longo
F
F
Ciotta
C
C
Del Grosso
M F
MF
Ambrosini
E
E
Gigliani
F
F
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D004268:DNA-Binding Proteins / Q000096:biosynthesis*; D004926:Escherichia coli; D005091:Exons; D015696:Gene Products, tat / Q000032:analysis / Q000096:biosynthesis*; D005821:Genetic Techniques; D006678:HIV / Q000378:metabolism*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D017354:Point Mutation; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000096:biosynthesis; D012097:Repressor Proteins / Q000032:analysis / Q000096:biosynthesis*; D015183:Restriction Mapping; D017384:Sequence Deletion; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus
D004268:DNA-Binding Proteins; D015696:Gene Products, tat; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins; D054322:tat Gene Products, Human Immunodeficiency Virus