tat regulates binding of the human immunodeficiency virus trans-activating region RNA loop-binding protein TRP-185.
The TAR element extending from -17 to +80 in the human immunodeficiency virus long terminal repeat (HIV LTR) is required for activation of gene expression by the tat trans-activator protein. TAR RNA forms a stable stem-loop structure, and mutagenesis studies indicate that the stem structure, the primary sequence of the loop, and the bulge element are the major determinants for tat activation. RNA gel retardation analysis demonstrates that both tat and cellular proteins bind to TAR RNA, but the mechanism by which these proteins increase HIV gene expression is unknown. We have fractionated HeLa cell nuclear extracts in an attempt to identify cellular proteins that bind to TAR RNA and are involved in regulating HIV gene expression. RNA gel retardation and UV cross-linking reveal that a cellular protein of 185 kD, which we designate TAR RNA-binding protein 185 (TRP-185), binds with both high affinity and marked specificity to TAR RNA. RNA gel retardation and competition analyses indicate that TRP-185 binding is strongly dependent on the TAR RNA loop sequences. The binding of TRP-185 is modulated by both a set of cellular cofactors and the tat protein. Highly purified preparations of TRP-185 are capable of activating in vitro transcription of wild-type, but not mutated, HIV LTR chloramphenicol acetyltransferase (CAT) constructs. These results characterize a positively acting cellular RNA-binding factor, TRP-185, which is involved in the regulation of HIV gene expression.
Wu
F
F
University of Texas Southwestern Medical School, Dallas 75235.
Garcia
J
J
Sigman
D
D
Gaynor
R
R
D000595:Amino Acid Sequence; D001483:Base Sequence; D002458:Cell Fractionation; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D015967:Gene Expression Regulation, Viral / Q000235:genetics*; D015696:Gene Products, tat / Q000235:genetics* / Q000378:metabolism; D016325:HIV Long Terminal Repeat / Q000235:genetics*; D015497:HIV-1 / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009687:Nuclear Proteins; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D054322:tat Gene Products, Human Immunodeficiency Virus; D000099253:Trans-Activation Responsive RNA-Binding Protein
D015500:Chloramphenicol O-Acetyltransferase; D015696:Gene Products, tat; D009687:Nuclear Proteins; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus; C071598:TARBP1 protein, human; D000099253:Trans-Activation Responsive RNA-Binding Protein