Transcription elongation factor P-TEFb mediates Tat activation of HIV-1 transcription at multiple stages

EMBO J. 1998 Jul 1;17(13):3681-91. doi: 10.1093/emboj/17.13.3681.

Abstract

Tat stimulates human immunodeficiency virus type 1 (HIV-1) transcription elongation through recognition of the transactivation response (TAR) RNA stem-loop structure at the 5' end of nascent viral transcripts. Recently, a human transcription elongation factor P-TEFb, consisting of CDK9 kinase, cyclin T and other associated factors, has been shown to interact with Tat to restore Tat activation in HeLa nuclear extract depleted of P-TEFb. Here, we report the purification of a P-TEFb complex fraction containing epitope-tagged wild-type CDK9 or kinase-inactive CDK9 and five tightly associated polypeptides. Only wild-type P-TEFb complex with an active CDK9 kinase was able to hyperphosphorylate the C-terminal domain of RNA polymerase II and mediate Tat transactivation in P-TEFb-depleted HeLa nuclear extract. Tat also stimulated transcription elongation by recruitment of the P-TEFb complex to the HIV-1 promoter through a Tat-TAR interaction. A possible mechanism for P-TEFb to become associated with polymerase elongation complexes and function as a general elongation factor was demonstrated by an interaction of P-TEFb with double-stranded RNA molecules through an 87 kDa subunit. Finally, P-TEFb was found to interact with and phosphorylate Tat-SF1, a Tat cofactor required for Tat transactivation. Our data indicate that the various subunits of the human P-TEFb complex may play distinct roles at multiple stages to mediate Tat activation of HIV-1 transcription elongation.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Base Sequence
  • Cell Line, Transformed
  • Cyclins / metabolism
  • Gene Products, tat / genetics*
  • HIV Long Terminal Repeat
  • HIV-1 / genetics*
  • HeLa Cells
  • Humans
  • Molecular Sequence Data
  • Nucleic Acid Conformation
  • Peptide Chain Elongation, Translational
  • Phosphorylation
  • Positive Transcriptional Elongation Factor B
  • Protein Kinases / metabolism
  • Protein Serine-Threonine Kinases / metabolism*
  • RNA, Double-Stranded / metabolism
  • RNA, Viral / chemistry
  • RNA, Viral / metabolism
  • Trans-Activators / metabolism
  • Transcriptional Activation*
  • tat Gene Products, Human Immunodeficiency Virus

Substances

  • Cyclins
  • Gene Products, tat
  • HTATSF1 protein, human
  • RNA, Double-Stranded
  • RNA, Viral
  • Trans-Activators
  • tat Gene Products, Human Immunodeficiency Virus
  • Protein Kinases
  • Positive Transcriptional Elongation Factor B
  • Protein Serine-Threonine Kinases