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

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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.