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 TNFalpha. 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 TNFalpha 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 TNFalpha, although the level of such activation is decreased in the presence of Tat. Ectopic expression of Puralpha, a brain-derived regulatory protein which binds to Tat, enhanced the basal level of TNFalpha transcription, yet exerted a negative effect on the level of Tat activation of the TNFalpha promoter. The antagonistic effect of Puralpha and Tat upon the TNFalpha promoter was diminished in the presence of cyclin T1 and cdk9, suggesting cooperativity of Puralpha with cyclin T1 and cdk9 in Tat activation of the TNFalpha promoter. Results from protein-protein binding studies showed the interaction of Puralpha with both cyclin T1 and cdk9 through distinct domains of Puralpha which are in juxtaposition with each other. Interestingly, the site for cyclin T1 binding within Puralpha is adjacent to the region which is important for Tat/Puralpha association. In light of these observations, we propose a model which ascribes a bridging role for Puralpha in assembling Tat, cyclin T1, and cdk9 around the promoter region of TAR-negative genes such as TNFalpha, which is responsive to Tat activation.