Activation of the HIV-1 promoter by the virally encoded Tat protein is characterized by efficient processive transcription, mediated by host cell factors that are tethered to the promoter with the Tat-TAR RNA complex. Importantly, viral gene activation has been shown to be stimulated in mitogenically induced cells, although the link between cell cycle regulation and viral gene activation is unclear. We reported a Tat-associated CAK/CTD kinase from mitogenically induced primary human T-cells (TTK) (S. Nekhai et al., 1997, J. Virol. 71, 7436-7441). Here, biological activity of the kinase has been studied by direct microinjection at the individual-cell level. The TTK-dependent Tat response is maximal during G1 phase as shown by co-injection with Tat protein in cells synchronized at the various stages of the cell cycle. The cell cycle dependence of the Tat response was confirmed by inhibiting G0 --> G1 progression with the expression of dominant negative mutant Ras(Asn17) or the cyclin-dependent kinase CDK4. The results support a mechanism whereby transactivation of the HIV promoter is regulated by cell growth signal transduction pathways that target the Tat cofactor.

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.

Human immunodeficiency virus type 1 (HIV) infection is often complicated by focal glomerulosclerosis (FGS) and other renal lesions collectively termed HIV associated nephropathy (HIVAN). FGS is characterized by glomerular mesangial expansion and increased synthesis of matrix components. The molecular pathogenic mechanisms associated with the development of HIV associated nephropathy are unknown. Experimental animal models suggest a role for cytokines and growth factors, particularly transforming growth factor beta (TGF-beta), in the pathogenesis of glomerulosclerosis. Patients with AIDS have elevated plasma and tissue levels of TGF-beta. We carried out experiments to determine whether primary human mesangial cells (HMC) in culture can be transfected with HIV-1 genes. HMC were transfected with a chloramphenicol acetyl transferase (CAT) reporter construct containing HIV-1 acetyl transferase (CAT) reporter construct containing HIV-1 LTR sequences. Our results show successful transfection of HMC with HIV-1 LTR gene. HMC transfected with LTR gene are responsive to the HIV-1 regulatory gene product Tat. To study whether TGF-beta can modulate the expression of HIV-1 LTR gene in HMC, HMC transfected with an HIV-1 LTR CAT plasmid were treated with TGF-beta and other growth factors two hours before harvest. TGF-beta specifically increased the expression of the HIV-1 gene in HMC in a dose dependent manner. We further studied whether up-regulation of HIV-1 LTR expression in HMC was mediated by the effect of TGF-beta on the interaction of transcription factors to their binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)