TRANSCRIPTION ELONGATION-FACTOR P-TEFB MEDIATES TAT ACTIVATION OF HIV-1 TRANSCRIPTION AT MULTIPLE STAGES

Tat stimulates human immunodeficiency virus type 1 (HIV-1) transcripti on 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 o f CDK9 kinase, cyclin T and other associated factors, has been shown t o interact with Tat to restore Tat activation in HeLa nuclear extract depleted of P-TEFb, Here, we report the purification of a P-TEFb compl ex fraction containing epitope-tagged wild-type CDK9 or kinase-inactiv e 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 LI and mediate Tat transactivatio n in P-TEFb-depleted HeLa nuclear extract. Tat also stimulated transcr iption elongation by recruitment of the P-TEFb complex to the HIV-1 pr omoter 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. F inally, 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 ro les at multiple stages to mediate Tat activation of HIV-1 transcriptio n elongation.