DISTINCT TRANSCRIPTIONAL PATHWAYS OF TAR-DEPENDENT AND TAR-INDEPENDENT HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 TRANSACTIVATION BY TAT

Tat stimulates HIV-1 gene expression during transcription initiation a nd elongation. Tat functions primarily through specific interactions w ith TAR RNA and several putative cellular cofactors to increase the pr ocessivity of RNA polymerase II complexes during HIV-1 transcription e longation. Although HIV-1 transactivation by Tat in most cell types re quires intact TAR sequences, previous reports demonstrate that Tat tra nsactivates HIV-1 long terminal repeat (LTR)-directed gene expression in several central nervous system-derived astrocytic/glial cell lines in the absence of TAR. Within this study, transient expression assays performed in the astrocytic/glial cell line, U87-MG, confirm that kapp a B elements within the HIV-1 LTR mediate TAR-independent transactivat ion by Tat and demonstrate additionally that distinct amino acid resid ues within the cysteine-rich activation domain of Tat are required for TAR-independent versus TAR-dependent transactivation. Established U87 -MG cell lines expressing a transdominant negative mutant of l kappa B alpha, l kappa B alpha Delta N, fail to support TAR-independent trans activation by Tat, suggesting that binding of NF-kappa B to kappa B en hancer elements within the HIV-1 LTR is necessary for Tat-mediated tra nsactivation in the absence of TAR. Ribonucleic acid protection analys es of promoter-proximal and -distal transcripts derived from TAR-delet ed and TAR-containing HIV-1 LTR reporter constructs in U87-MG cells in dicate that the predominant effect of Tat during TAR-independent trans activation occurs at the level of transcription initiation, whereas a prominent elongation effect of Tat is observed in the presence of TAR. These data suggest an alternative regulatory pathway for Tat transact ivation in specific cells derived from the central nervous system that is independent of TAR and that requires direct or indirect interactio n of Tar with NF-kappa B-binding sites in the HIV-1 LTR. (C) 1997 Acad emic Press.