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
C. Sune et al., 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, VIROLOGY, 274(2), 2000, pp. 356-366
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.