Hinks, J.A.; (2005) Studies of retroviral proteases. Doctoral thesis , University of London.

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Abstract

This work is primarily concerned with the expression, purification, and characterisation of aspartic proteases from three retroviruses of the lentivirus subgroup, specifically the Human Immunodeficiency Viruses types 1 and 2, and the Simian Immunodeficiency Virus isolated from the African Green Monkey (HIV-1 PR, HIV-2 PR, SIVagm PR respectively). These viruses cause immunodeficiency syndromes within their respective hosts, and understanding their molecular biology would facilitate development of Acquired Immunodeficiency Syndrome (AIDS) treatments in man. The proteases are essential to viral maturation and infection and are of great interest with respect to the development of new antivirals. This thesis describes attempts to develop improved methods for the over-expression and purification of these cytotoxic proteins for use in structural and biochemical studies. The development of a system for expression and purification of active, crystallisable HIV-1 PR is described, followed by a preliminary analysis of two compounds intended to act as irreversible "suicide inhibitors" of HIV-1 PR. The expression, purification, crystallisation, and preliminary crystallographic data for the native HIV-2 PR using the same expression system are also reported. Mutagenesis of the HIV-2 protease is described, whereby the conserved active site aspartic acids of the native homodimer were changed to histidine and cysteine, with the intention of modifying the enzyme's mechanism, whilst maintaining its native substrate specificity and overall structure. Finally the production of an insoluble, non-toxic, histidine tagged fusion of mutant E.coli Uracil DNA Glycosylase (UDG) and the SIVagm PR is reported. This was intended to produce high levels of a non-toxic fusion protein, allow one-step affinity purification, and provide native soluble protease following autocatalytic cleavage from the fusion protein. The effects of protease toxicity and codon usage on yields are discussed in light of the results presented.

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