Transcriptional and Non Transcriptional Properties of the TAT Protein of Human Immunodeficiency Virus Type 1

Human immunodeficiency virus type 1 (HIV-1) is the causative agent of AIDS. In addition to the Gag, Pol and Env proteins, which are common to all retroviruses, the HIV-1 genome also encodes six regulatory proteins, among which Tat plays an essential role in the viral life cycle. This protein is a very powerful transcriptional activator of HIV-1 with unusual molecular properties, including the capacity of being released by infected cells. The results reported in this thesis have been obtained by addressing the study of some transcriptional and non transcriptional features of Tat. In Chapter I, a brief molecular overview of HIV-1 replication is presented, with an emphasis on the regulation of viral transcription by the Tat protein. This small protein directly activates transcription from the viral Long Terminal Repeat (L TR) promoter by interacting with the TAR RNA sequence present at the 5' end of the primary viral transcript. Tat exerts its functions by promoting the recruitment of more processive RNA polymerase 11 complexes to the L TR and by increasing the rate of transcriptional initiation. In Chapter 11, the molecular properties of over 16 different mutants of Tat are described, all of which studied as recombinant proteins fused to GST. In particular, it is shown that amino acid substitutions of the cysteines in the cystein-rich domain, of the arginins in the basic domain, deletion of the N-terminal 21 amino acids and a single point mutations at histidine 13 strongly impair protein function. In the same chapter it is also shown that, through its basic domain, Tat specifically interacts with the CBP/p300 histone acetyltransferases (HAT) proteins, and that it recruits these proteins to the viral promoter to induce remodeling of the chromatin scaffold. These data provide a molecular explanation for the long-standing observation that Tat increases the rate of transcription initiation. Moreover, this is the first evidence suggesting that an RNA targeted transcription factor could act by recruiting a HAT activity to a promoter. In Chapter Ill, I address the study of the molecular mechanisms involved in Tat intercellular trafficking. The results obtained provide biochemical and genetic evidence that Tat entry into the cells is mediated by cell surface proteoglycan molecules containing heparan sulfate (HS) as the glucosaminoglycan moiety. Cell lines genetically deficient in HS proteoglycans biosynthesis were unable to support the Tat entry. Consistently, the enzymatic removal of HS residues from the cell surface, or cell treatment with soluble heparin, specifically impaired Tat internalization. Interestingly, HS proteoglycans are not required for the release of Tat outside of the expressing cells. Chapter IV, focuses on the possible role of Tat phosphorylation. It is demonstrated that Tat is an in vitro substrate for the nuclear DNA-dependent protein kinase (DNA-PK) which phosphorylates Tat at two specific serine residues. Amino acid substitution at these sites results in reduced transactivation activity of the mutated Tat protein.