Orchard, Kim Harold; (1999) Cellular factors that interact with the negative regulatory element of the 5'-long terminal repeat of human immunodeficiency virus type 1. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Transcriptional regulation of HlV-1 gene expression has been shown to be regulated by a combination of viral and cellular proteins which bind to regulatory elements in the viral 5' long terminal repeat (5'LTR) Functionally the LTR can be divided into three regulatory regions: the TAR region, extending from nucleotides 1 to 60 relative to the start site of transcription, contains sequences with which the viral trans-activator Tat interacts. The adjacent region from nucleotides -1 to -78 contains the core promoter with elements crucial for both basal and Tat-induced expression. The third region, extending from -79 to -454, contains numerous elements with which a variety of cellular factors may interact, resulting in either positive or negative modulation of LTR-driven transcription. The work contained within this thesis describes the discovery and delineation of two new transcription factor binding sites, designated as site A and site B, within the 5'-LTR of HIV-1. The majority of the work focused on site B itself, involving the characterisation of cellular proteins that specifically interacted with the nucleotide sequences in this site. Site B was found to contain a palindromic sequence TGACC involved in protein-DNA contact separated by a 9 base-pair spacer sequence that was not important for protein binding. This palindrome resembles the consensus binding site for members of the nuclear hormone receptor super-family of transcription factors. Although several members of this super-family of transcription factors were shown to interact in vitro with site B, the predominant protein present in T-lymphocyte nuclear extracts did not correspond to any of those previously characterised. The T-cell protein was shown to have a relative molecular size of 100-110 kD for the monomeric polypeptide and bound to site B as a dimer. Maximal binding to site B required both halves of the palindrome. Functionally site B was shown to act as a repressor element of both basal transcription and of transcription activated by phorbol ester in T- lymphocytes. Site B was also shown to function as a retinoic acid response element (RARE) in a heterologous promoter. The ability to function as either a positive or negative regulatory element is a recognised characteristic of nuclear hormone response elements and in part is a function of the relative abundance of factors able to interact with the site or to form complexes with one another. The overall effect of site B upon LTR-directed transcription may similarly depend upon the complex interaction of multiple factors which themselves depend on the cell type, cell activation state and degree of differentiation.

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