Structural and functional characterization of human immunodeficiency virus tat protein.
Site-directed mutagenesis was used to identify functional domains present within the human immunodeficiency virus (HIV) tat protein. Transient cotransfection experiments showed that derivatives of tat protein with amino acid substitutions either at the amino-terminal end or at cysteine residue 22, 37, 27, or 25 were no longer able to transactivate HIV long terminal repeat-directed gene expression. Incubation of Tat expressed in Escherichia coli with zinc demonstrated that both authentic Tat and cysteine mutation derivatives could form metal-protein complexes. The tat proteins that contained alterations within the cluster of positively charged amino acid residues retained their ability to transactivate gene expression, albeit at markedly reduced levels. Indirect immunofluorescence showed that the authentic tat protein and the amino-terminal and cysteine substitution mutants all localized in the nucleus, with accumulation being most evident in the nucleolus. In contrast, nuclear accumulation was greatly reduced with the basic-substitution mutations. Consistent with this result, a fusion protein that contained amino acids GRKKR, derived from the basic region, fused to the amino-terminal end of beta-galactosidase also accumulated within the nucleus. These results demonstrate that the 14-kilodalton tat protein contains at least three distinct functional domains affecting localization and transactivation.
Ruben
S
S
Department of Molecular Oncology, Roche Institute of Molecular Biology, Nutley, New Jersey.
Perkins
A
A
Purcell
R
R
Joung
K
K
Sia
R
R
Burghoff
R
R
Haseltine
W A
WA
Rosen
C A
CA
D000595:Amino Acid Sequence; D002460:Cell Line; D002466:Cell Nucleolus / Q000032:analysis; D002467:Cell Nucleus / Q000032:analysis; D003001:Cloning, Molecular; D003545:Cysteine / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D005786:Gene Expression Regulation; D015696:Gene Products, tat; D006678:HIV / Q000032:analysis / Q000235:genetics* / Q000648:ultrastructure; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D014157:Transcription Factors / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D014162:Transfection; D015032:Zinc / Q000378:metabolism; D054322:tat Gene Products, Human Immunodeficiency Virus
D015696:Gene Products, tat; D014157:Transcription Factors; D054322:tat Gene Products, Human Immunodeficiency Virus; D015032:Zinc; D003545:Cysteine