As part of their larger effort to understand unique aspect of HIV biology that might be exploited in novel therapeutic approaches, we focus in this proposal on a newly defined function of the Tat protein of HIV. Tat is a potent transcriptional activator of the HIV promoter and exists in infected cells under two forms: 72 (Tat72) and 101 amino acids (Tat101). The transactivating activity of Tat suggested that Tat might also affect the expression of cellular genes during infection. The P.I. has obtained evidence that Tat101 expressed alone or in the context of HIV viral infection, increases the response to CD28 signaling, a crucial costimulatory signal involved in T cell activation. This hypersensitivity to CD28 signaling results in a 5-10 fold increase in IL-2 and IL-8 secretion in response to T cell activation. Tat mediates this effect at the transcriptional level by upregulating DNA-binding complex bound to the CD28 responsive element in the IL-2 and IL-8 promoters. The P.I. has also observed that Tat101 expression in T cells renders them resistant to several apoptotic stimuli. It is now proposed to further define the modulating role of Tat on T cell functions such as cytokine secretion and the control of apoptosis. New T cell lines will be generated containing inducible Tat vectors. These cell lines will be used both to identify genes that are differentially regulated by Tat expression and to define the role of Tat expression on the control of apoptosis. To confirm these observations in the context of HIV infection, he will generated a tagged-HIV expressing the green fluorescent protein. This marker will allow to follow infected cells individually using flow cytometry and to correlate the process of HIV infection with changes in cytokine secretion and apoptosis. He will analyze in molecular details the mechanism of Tat action by examining the composition and the regulation of the CD28 responsive complex. Point mutations will be introduced into the Tat open reading frame to delineate the amino acids critical for this activity. To identify cellular partners necessary for Tat activity, he will use the last 29 amino acids of Tat101 as a bait in the two-hybrid system and clone Tat-interacting factors. These studies will further define the role of Tat as modulator of T cell differentiated functions and establish its role in HIV pathogenesis.
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| Dorr, Alexander; Kiermer, Veronique; Pedal, Angelika et al. (2002) Transcriptional synergy between Tat and PCAF is dependent on the binding of acetylated Tat to the PCAF bromodomain. EMBO J 21:2715-23 |
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