An in vitro transcription system that recapitulates equine infectious anemia virus Tat-mediated inhibition of human immunodeficiency virus type 1 Tatactivity demonstrates a role for positive transcription elongation factor b and associated proteins in the mechanism of Tat activation

Equine infectious anemia virus (EIAV) activates transcription via a Tat pro tein, a TAR element, and the equine elongation factor positive transcriptio n elongation factor b (P-TEFb). In human cells, EIAV Tat (eTat) can inhibit the ability of human immunodeficiency virus type 1 (HIV-1) Tat (hTat) to a ctivate transcription from the HIV-1 long terminal repeat, demonstrating th at EIAV Tat can interact nonproductively with human P-TEFb. To study the me chanism of EIAV Tat and HIV-1 Tat activation, we developed an in vitro elon gation assay that recapitulates EIAV Tat-mediated inhibition of HIV-1 Tat t rans-activation. We found that eTat specifically inhibits activation of elo ngation by HIV-1 Tat while having no effect on basal transcription elongati on. The competitive inhibition of hTat activation was reversed by an activi ty present in HeLa cell nuclear extracts, most likely a form of P-TEFb. Rec ombinant P-TEFb (cyclin T1 and CDK9) overcame the inhibition of transcripti on by eTat but in a nonspecific manner. EIAV Tar affinity chromatography wa s used to purify the activity present in nuclear extract that was capable o f reversing eTat inhibition. We characterized the protein components of thi s activity, which include cyclin T1, CDK9, Tat-SF1, and at least three unid entified proteins, These data suggest that additional factors are involved in the mechanism of Tat activation. (C) 2000 Academic Press.