Mechanism of Activation of the Integrated Promoter of the Human Immunodeficiency Virus Type1

A thorough understanding of the mechanisms regulating the activation and the rate of HIV-1 gene expression is of basic importance in the race towards effective treatment of AIDS. In this respect, the results reported in this thesis cover several critical aspects of the regulation of HIV-1 transcription. Chapter 1 provides a general introduction on HIV-1 classification and structural and genomic organization. Here are described the structure of the viral promoter and the factors involved in transcription regulation of the virus after integration in the genome of the infected cells. This first chapter is also intended as a broad overview on the issues raised and dealt with in depth in the ensuing chapters. In Chapter 2 are reported the results of the study on the role of transcription factor USF in the modulation of L TR-driven transcription. They demonstrate that USF is a positive modulator of HIV-1 transcription and that this activity results from the specific binding of the protein to a cis-acting element present in the negative regulatory element of the L TR. Strikingly, binding of USF results in marked promoter bending. This observations is in agreement with previous reports that other L TR-binding factors are able to induce DNAbending upon binding. A novel function of the viral transactivator Tat is described in Chapter 3. It is demonstrated that Tat binds p300 and CBP, two proteins with histone-specific acetyltransferase activity able to remodel the chromatin scaffold on the L TR. These proteins are specifically directed by Tat to the HIV-1 promoter for viral transactivation. These data provide a molecular explanation for the long-standing observation that Tat increases the rate of transcription initiation. Moreover, they represent the first evidence that an RNAtargeted transactivating factor such as Tat is able to recruit histone acetyltransferases. Chapter 4 describes a quantitative system for monitoring HIV-1 nucleic acids in infected samples. The system combines the sensitivity of PCR with the precision deriving from the use of a single competitor for viral DNA and RNA quantitation and normalization to cellular standards. The system was used to monitor HIV-1 activation in two different in vitro models of infection. In the Appendix to this chapter the application of this competitive PCR procedure is described for the study the correlation between the proliferative state of infected lymphocytes and HIV-1 activation. Following repeated cycles of mitogen stimulation, the level of HIV-1 gene expression is shown to follow closely that of cellular activation. Furthermore, viral transcription is rapidly shut off after the cells return to a non-proliferative state but can be rapidly re-activated upon a second cell stimulation. In Chapter 5 conclusions are drawn based on the results described in the previous chapters.