2ND-SITE LONG TERMINAL REPEAT (LTR) REVERTANTS OF REPLICATION-DEFECTIVE HUMAN-IMMUNODEFICIENCY-VIRUS - EFFECTS OF REVERTANT TATA BOX MOTIFSON VIRUS INFECTIVITY, LTR-DIRECTED EXPRESSION, IN-VITRO RNA-SYNTHESIS, AND BINDING OF BASAL TRANSCRIPTION FACTORS TFIID AND TFIIA

Second-site revertants from replication-incompetent molecular clones o f human immunodeficiency virus (HIV) contain base substitutions adjace nt to the TATA motif. The altered TATA box motifs were analyzed for th eir effect(s) on virus infectivity, long terminal repeat (LTR)-directe d 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 spe cific); no effect was observed on the infectivity of wild-type HIV. In chloramphenicol acetyltransferase assays and in vitro transcription s ystems, the altered TATA box motifs led to elevated basal levels of RN A synthesis from NP-kB- and Sp1-mutagenized and wild-type templates, r espectively, but did not increase responsiveness to Tat transactivatio n. The revertant TATA boxes accelerated the binding of TFIID and TFIIA to the LTR and stabilized their association with the promoter. The re vertants 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 w hich contain LTR alterations that significantly augment basal RNA synt hesis. The TATA motif revertants are capable of rescuing the enhancer/ promoter defect and sustain virus infectivity.