Transcriptional activation in vitro by the human immunodeficiency virus type 1 Tat protein: evidence for specific interaction with a coactivator(s).
The Tat protein encoded by human immunodeficiency virus type 1 is a strong transcriptional activator of gene expression from the viral long terminal repeat and is essential for virus replication. We have investigated the molecular mechanism of Tat trans-activation by using a cell-free transcription system. We find that the trans-activation domain of Tat, amino acid residues 1-48 [Tat-(1-48)], can inhibit specifically--i.e., "squelch," transcriptional activation by full-length Tat [Tat-(1-86)]. Squelching depends upon the functional integrity of the Tat trans-activation domain because the mutant [Ala41]Tat-(1-48), which is defective in Tat trans-activation in vivo and in vitro, does not squelch in vitro Tat trans-activation. Inhibition is selective because Tat-activated transcription, but not Tat-independent transcription, is squelched. Preincubation experiments with Tat or Tat-(1-48) and nuclear extracts show that the trans-activation region of Tat can interact with cellular coactivator(s) required for Tat trans-activation and that this interaction can occur in the absence of the human immunodeficiency virus long terminal repeat promoter. Furthermore, the putative coactivator(s) mediating trans-activation by Tat differ from those mediating trans-activation by the acidic activator VP16, as shown by reciprocal squelching experiments in vitro. Our results suggest that specific cellular coactivator(s) are required for mediating activated transcription by human immunodeficiency virus type 1 Tat.
Song
C Z
CZ
Institute for Molecular Virology, Saint Louis University Health Sciences Center, MO 63110.
Loewenstein
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PM
Green
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M
D000595:Amino Acid Sequence; D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis; D017931:DNA Primers; D015696:Gene Products, tat / Q000378:metabolism*; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000378:metabolism* / Q000502:physiology; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping; D015534:Trans-Activators / Q000378:metabolism*; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014779:Virus Replication; D054322:tat Gene Products, Human Immunodeficiency Virus
D017931:DNA Primers; D015696:Gene Products, tat; D011994:Recombinant Proteins; D015534:Trans-Activators; D054322:tat Gene Products, Human Immunodeficiency Virus; D015500:Chloramphenicol O-Acetyltransferase