Tat stimulates human immunodeficiency virus, type 1 (HIV-1), transcription elongation by recruitment of the human transcription elongation factor P-TEFb, consisting of CDK9 and cyclin T1, to the TAR RNA structure. It has been demonstrated further that CDK9 phosphorylation is required for high affinity binding of Tat/P-TEFb to the TAR RNA structure and that the state of P-TEFb phosphorylation may regulate Tat transactivation. We now demonstrate that CDK9 phosphorylation is uniquely regulated in the HIV-1 preinitiation and elongation complexes. The presence of TFIIH in the HIV-1 preinitiation complex inhibits CDK9 phosphorylation. As TFIIH is released from the elongation complex between ؉14 and ؉36, CDK9 phosphorylation is observed. In contrast to the activity in the "soluble" complex, phosphorylation of CDK9 is increased by the presence of Tat in the transcription complexes. Consistent with these observations, we have demonstrated that purified TFIIH directly inhibits CDK9 autophosphorylation. By using recombinant TFIIH subcomplexes, our results suggest that the XPB subunit of TFIIH is responsible for this inhibition of CDK9 phosphorylation. Interestingly, our results further suggest that the phosphorylated form of CDK9 is the active kinase for RNA polymerase II carboxyl-terminal domain phosphorylation.

HIV-1 Tat protein regulates viral gene expression by modulating the activity and association of cellular transcription factors with RNA polymerase II (RNAPII). Possible mechanisms include Tat-associated protein kinase(s) and phosphatase(s) that regulate phosphorylation of the C-terminal domain (CTD) of the large subunit of RNAPII. Hypophosphorylated RNAPII (RNAPIIa) is recruited to promoters during formation of a preinitiation complex, whereas hyperphosphorylated RNAPII (RNAPIIo) is associated with the elongation complex. The role of phosphatases in maintaining the equilibrium between the two phosphorylated states of RNAPII, which is required for sustained transcriptional activation from the HIV-1 LTR, is not clear. In this study, we discuss the properties of a Tat-associated CTD phosphatase fractionated from Jurkat T cells. The Tat-associated protein phosphatase (TAPP) is related to the serine/threonine, type 1, protein phosphatase (PP1) family. TAPP dephosphorylates the hyperphosphorylated form of recombinant CTD specifically on serine 2, and augments Tat-mediated transcriptional transactivation of HIV-1 LTR in an in vitro transcription reaction. TAPP is associated with the transcription complex during the early initiation steps, and its release from the HIV-1 promoter coincides with the Tat-specific activation of CDK9. The results suggest a unique role of the Tat-associated phosphatase which regulates viral transcription by target-specific dephosphorylation of RNAPII during the early stages of elongation.