Conservative mutations in the putative metal-binding region of human immunodeficiency virus tat disrupt virus replication.
The tat trans-activator proteins of the primate immunodeficiency viruses contain a highly conserved cysteine-rich domain. In human immunodeficiency virus type 1 tat there are seven cysteines located between residues 22 and 37 that are thought to form a metal-nucleic acid-binding structure. Most of the previous mutagenesis studies had demonstrated that these residues are essential for tat activity and virus expression. Here we show that potentially conserved cysteine-histidine substitutions within the proposed tetrahedral structure still eliminate tat activity and virus expression. Consistent with previous studies, one cysteine-to-histidine mutation (amino acid 31) had little effect on trans-activation. We have studied the functional properties, stability and subcellular localization of several tat protein mutants. Most of the mutants are stable and properly localized to the nucleus and/or nucleolus. However, cysteine-to-glycine at position 34 affected tat stability. Our studies with the histidine mutants suggest that tat does not assume the prototype "zinc finger" structure for metal binding.
Sadaie
M R
MR
Laboratory of Tumor Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892.
Mukhopadhyaya
R
R
Benaissa
Z N
ZN
Pavlakis
G N
GN
Wong-Staal
F
F
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D003545:Cysteine / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D015967:Gene Expression Regulation, Viral; D015696:Gene Products, tat / Q000235:genetics* / Q000378:metabolism; D015756:Genes, tat; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D008670:Metals / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011233:Precipitin Tests; D015533:Transcriptional Activation; D014779:Virus Replication / Q000235:genetics*; D054322:tat Gene Products, Human Immunodeficiency Virus
D015696:Gene Products, tat; D008670:Metals; D054322:tat Gene Products, Human Immunodeficiency Virus; D003545:Cysteine