title: Second-site long terminal repeat (LTR) revertants of replication-defective human immunodeficiency virus: effects of revertant TATA box motifs on virus infectivity, LTR-directed expression, in vitro RNA synthesis, and binding of basal transcription factors TFIID and TFIIA.
abstract: Second-site revertants from replication-incompetent molecular clones of human immunodeficiency virus (HIV) contain base substitutions adjacent to the TATA motif. The altered TATA box motifs were analyzed for their effect(s) on virus infectivity, long terminal repeat (LTR)-directed expression in transient transfection assays, in vitro RNA synthesis, and assembly of the TFIID-TFIIA preinitiation complex. The revertant TATA boxes accelerated the kinetics of HIV replication when present in the context of an LTR containing a Sp1 mutation (deletion or site specific); no effect was observed on the infectivity of wild-type HIV. In chloramphenicol acetyltransferase assays and in vitro transcription systems, the altered TATA box motifs led to elevated basal levels of RNA synthesis from NF-kappa B- and Sp1-mutagenized and wild-type templates, respectively, but did not increase responsiveness to Tat transactivation. The revertant TATA boxes accelerated the binding of TFIID and TFIIA to the LTR and stabilized their association with the promoter. The revertants did not assemble a more-processive elongation complex. These results suggest that in the context of an impaired enhancer/promoter (viz., three mutated Sp1 elements), a series of HIV revertants emerge which contain LTR alterations that significantly augment basal RNA synthesis. The TATA motif revertants are capable of rescuing the enhancer/promoter defect and sustain virus infectivity.
authors.affiliation: Laboratory of Molecular Virology, National Cancer Institute, Bethesda, Maryland 20892.
authors.forename: F
authors.initials: F
authors.lastname: Kashanchi
authors.forename: R
authors.initials: R
authors.lastname: Shibata
authors.forename: E K
authors.initials: EK
authors.lastname: Ross
authors.forename: J N
authors.initials: JN
authors.lastname: Brady
authors.forename: M A
authors.initials: MA
authors.lastname: Martin
mesh_terms: D001483:Base Sequence; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis; D015696:Gene Products, tat / Q000378:metabolism; D016325:HIV Long Terminal Repeat / Q000235:genetics*; D015497:HIV-1 / Q000235:genetics* / Q000254:growth & development*; D006801:Humans; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009154:Mutation; D016328:NF-kappa B / Q000378:metabolism; D011485:Protein Binding; D012367:RNA, Viral / Q000096:biosynthesis*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D016329:Sp1 Transcription Factor / Q000378:metabolism; D016385:TATA Box / Q000235:genetics*; D035561:Transcription Factor TFIIA; D035362:Transcription Factor TFIID; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection; D014779:Virus Replication; D054322:tat Gene Products, Human Immunodeficiency Virus
chemical_list: D015696:Gene Products, tat; D046911:Macromolecular Substances; D016328:NF-kappa B; D012367:RNA, Viral; D011993:Recombinant Fusion Proteins; D016329:Sp1 Transcription Factor; D035561:Transcription Factor TFIIA; D035362:Transcription Factor TFIID; D014157:Transcription Factors; D054322:tat Gene Products, Human Immunodeficiency Virus; D015500:Chloramphenicol O-Acetyltransferase