Functional interaction between cyclin T1/cdk9 and Purα determines the level of TNFα promoter activation by Tat in glial cells

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Abstract

In addition to its stimulatory effect on transcription of the HIV-1 LTR, the early protein of HIV-1, Tat, exhibits detrimental effects on the CNS by deregulating the expression of several cytokines and immunomodulators including TNFα. Activation of the viral promoter by Tat requires several cellular proteins including cyclin T1 and its partner, cdk9, which upon association with the TAR sequence of the LTR, forms a complex that enhances the activity of RNA polymerase II. Here, we examined the involvement of cyclin T1/cdk9 in Tat-mediated transcriptional activation of the TNFα promoter which has no TAR sequence. Results from transfection of human astrocytic cells revealed that both cyclin T1 and cdk9 stimulate the basal promoter activity of TNFα, although the level of such activation is decreased in the presence of Tat. Ectopic expression of Purα, a brain-derived regulatory protein which binds to Tat, enhanced the basal level of TNFα transcription, yet exerted a negative effect on the level of Tat activation of the TNFα promoter. The antagonistic effect of Purα and Tat upon the TNFα promoter was diminished in the presence of cyclin T1 and cdk9, suggesting cooperativity of Purα with cyclin T1 and cdk9 in Tat activation of the TNFα promoter. Results from protein–protein binding studies showed the interaction of Purα with both cyclin T1 and cdk9 through distinct domains of Purα which are in juxtaposition with each other. Interestingly, the site for cyclin T1 binding within Purα is adjacent to the region which is important for Tat/Purα association. In light of these observations, we propose a model which ascribes a bridging role for Purα in assembling Tat, cyclin T1, and cdk9 around the promoter region of TAR-negative genes such as TNFα, which is responsive to Tat activation.

Introduction

HIV-1 infects the central nervous system (CNS) and frequently causes dementia and neurologic disorders in AIDS patients. Although several studies have suggested the potential role of the viral envelope in neurologic HIV-1 disease (Dreyer et al., 1990), the extent of clinical and histopathological abnormalities is clearly disproportionate to the amount of viral particles produced, suggesting the involvement of other factors and secondary mechanisms of pathogenesis in the CNS. Since astrocytes, like microglia, are infected and apparently not killed by viral infection (Dewhurst et al., 1987), dysregulation of host cellular gene expression by viral regulatory proteins may lead to abnormal glial cell function, potentially contributing to neuronal degeneration. In this respect, much attention has been paid to Tat. The HIV-1 Tat protein is a potent transactivator of HIV-1 transcription (Biswas et al., 1995). Tat mediates transactivation of the HIV-1 LTR promoter through its interaction with the Tat activation response element, TAR, a stable stem loop structure at the 5′ end of all HIV-1 mRNAs. Further, Tat binds to TAR and recognizes the UCU bulge region Luo et al., 1993, Taylor et al., 1992. The specificity for Tat interaction with TAR by itself is not impressive and presumably Tat recognizes TAR RNA in vivo through interaction with cellular factors. Since the CUGGG motif in the TAR loop is required for transactivation, but not Tat binding in vitro, it has been proposed that this region of TAR interacts with critical cellular factor(s) involved in Tat-mediated transactivation. A cellular Tat-associated kinase (TAK) has been identified with affinity for lentiviral Tat proteins, which has the ability to phosphorylate the carboxy-terminal domain (CTD) of the large subunit of RNA polymerase II (Herrmann and Rice, 1995). TAK is a component of the multi-subunit elongation complex p-TEFb, required for Tat transactivation of the HIV-1 LTR (Garcia-Martinez et al., 1997). The catalytic subunit of TAK, cdk9, is a member of the cyclin-dependent kinase family and has been identified as the cdc2-related kinase, PITALRE, which has elevated activity upon activation of peripheral blood lymphocytes and monocytes (Gold et al., 1998). Cdk9 is a component of the elongation factor p-TEFb (Zhu et al., 1997) and its activity in elongation appears to involve phosphorylation of the carboxy-terminal domain (CTD) of RNA polymerase II Herrmann and Rice, 1995, Zauli et al., 1992. Cyclin T1, an 87-kDa subunit of the Tat-associated kinase and partner for cyclin dependent kinase 9 (cdk9), forms a complex with Tat, which then recognizes TAR loop sequences and increases its affinity for TAR RNA Garcia-Martinez et al., 1997, Jones, 1997, Yang et al., 1997. Many other studies underline the critical role of the TAR complex in the Tat transactivation pathway. For example, ectopic expression of human cyclin T1 permits transcriptional activation of the HIV-1 LTR via TAR in murine cells, a species which is not permissive for Tat-mediated activation through TAR RNA (Wei et al., 1998).

While these observations may assist in unraveling the mechanism of Tat transactivation of HIV-1 via the TAR region, the pathway by which Tat modulates transcriptional activity of several cellular and other viral genes, which lack the TAR sequence remains unknown. This becomes a particularly important question in the central nervous system, as several studies have indicated that while it is clear that HIV-1 has a causative role in CNS disease, the broad range pathology associated with HIV-1 may be a consequence of infiltration of inflammatory cells and cytokine dysregulation in the CNS rather than the amount of virus in the brain Brown, 1999, Taylor et al., 1992. In this respect, Tat activates transcription of a variety of cytokine genes including TNFα Glass et al., 1995, Nuovo and Alfieri, 1996, Sawaya et al., 1998, TGFβ-1 Cupp et al., 1993, Glass et al., 1995, Nottet et al., 1996, Sawaya et al., 1998, Zauli et al., 1992, IL-1, IL-6, and chemokines such as MCP-1 (Conant et al., 1998). However, the mechanism by which Tat enhances transcription of the TAR-negative cellular promoters remains unknown. Previously, we have identified a novel DNA-binding protein from brain nuclear extract, named Purα, which has the ability to specifically form a complex with GA/GC DNA or RNA motif and affect transcription of the genes containing this motif. Interestingly, our recent studies have shown that Purα also interacts with HIV-1 Tat and its association is dependent on RNA molecules with GA/GC sequences which are detected in CNS cells (Gallia et al., 1999). In light of these observations and our previous results demonstrating the ability of Purα to stimulate transcription of the Tat-responsive genes via the region which is important for Tat to exert its regulatory effect Chepenik et al., 1998, Gallia et al., 1999, we launched a series of transfection studies to examine functional interplay of Tat with three cellular proteins, i.e. Purα, cyclin T1, and cdk9, upon transcription of the TNFα promoter. Our results provide compelling evidence for the functional and physical interaction of Purα with both cyclin T1 and cdk9, and the modulation of Tat-induced transactivation of the TNFα promoter by these cellular proteins in human astrocytic cells.

Section snippets

Plasmids

The GST–cdk9 and GST–cyclin T1 constructs have been described previously (Sawaya et al., 2000). pCMV–cyclin T1-HA was kindly provided by Dr. K. Jones (Salk Institute, La Jolla, CA) (Wei et al., 1998). pFlag-CMV2–cdk9 was kindly provided by Dr. A. Rice (Baylor College of Medicine, TX) (Yang et al., 1997). pTNFα–Luc was described in Sawaya et al. (1998). The Purα constructs, pCMV–Purα, GST–Purα, and its mutant variants have been described previously (Gallia et al., 1998).

Cell culture and transfection

Human astrocytic cells,

Results and discussion

In the first series of experiments, we examined the transcriptional activity of the TNFα promoter in the absence and presence of cyclin T1 and cdk9, alone or in combination, in human astrocytic glial cells, U-87MG. Toward this end, cells were transfected with the reporter plasmid containing the TNFα promoter fused to a Luciferase reporter gene and the level of Luciferase activity, which is indicative of the TNFα promoter activity, was determined after 36 h. As shown in Fig. 1, Panel A, in the

Acknowledgements

We wish to thank past and present members of the Center for Neurovirology and Cancer Biology for their insightful discussion, and sharing of reagents and ideas. We wish to thank C. Schriver for editorial assistance. This work was made possible by grants awarded by NIH to BES and SA.

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