Functional analysis of interactions between Tat and the trans-activation response element of human immunodeficiency virus type 1 in cells.
Transcriptional trans-activation of the human immunodeficiency virus type 1 long terminal repeat requires that the virally encoded Tat effector interacts with its target trans-activation response element (TAR) RNA stem-loop. Although the arginine-rich region of Tat from amino acids 49 to 59 is sufficient to bind to TAR RNA in vitro, the RNA-binding domain of Tat has not been defined in vivo. Human immunodeficiency virus type 1 also encodes the Rev protein, which acts through an RNA stem-loop called the Rev-response element to transport unspliced and singly spliced viral RNA species from the nucleus to the cytoplasm. To map the RNA-binding domain of Tat, we performed assays that relied on Rev function using the heterologous RNA-tethering mechanism of Tat and the TAR. By examining the effects of selected targeted mutations of Tat on the abilities of hybrid Tat/Rev proteins to rescue the expression of unspliced mRNA via the TAR, we demonstrated that residues throughout the N-terminal 59 amino acids of Tat are required for binding of Tat and TAR RNA in vivo.
Luo
Y
Y
Howard Hughes Medical Institute, University of California, San Francisco 94143.
Madore
S J
SJ
Parslow
T G
TG
Cullen
B R
BR
Peterlin
B M
BM
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis; D015870:Gene Expression; D015694:Gene Products, rev / Q000378:metabolism*; D015696:Gene Products, tat / Q000235:genetics / Q000378:metabolism*; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010957:Plasmids; D011485:Protein Binding; D012333:RNA, Messenger / Q000096:biosynthesis*; D012367:RNA, Viral / Q000096:biosynthesis / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection; D054321:rev Gene Products, Human Immunodeficiency Virus; D054322:tat Gene Products, Human Immunodeficiency Virus
D015694:Gene Products, rev; D015696:Gene Products, tat; D012333:RNA, Messenger; D012367:RNA, Viral; D011994:Recombinant Proteins; D054321:rev Gene Products, Human Immunodeficiency Virus; D054322:tat Gene Products, Human Immunodeficiency Virus; D015500:Chloramphenicol O-Acetyltransferase