Garcia Diaz, A; (2012) An Investigation of the role of HIV-1 Gag mutation in failure of Protease Inhibitors. Doctoral thesis , UCL (University College London).

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Abstract

Classically, the emergence of resistance to protease inhibitors (PIs) in HIV-1 requires the stepwise accumulation of primary and compensatory mutations in the viral protease (PR). In addition, it was demonstrated that mutations occurring on one of the natural substrates of the PR, Gag, could behave as compensatory mutations in the presence of certain primary PR mutations. Furthermore, mutations on the Gag could account for resistance to PIs when appeared in isolation. So far, most studies assessing the effect of Gag mutations on resistance to PIs have focused on two cleavage sites (CS), namely P7/P1 and P1/P6. However, data on the remaining CS and non-cleavage sites is scarce. In my PhD studies, I developed and optimized an assay for the amplification and sequencing of HIV-1 Gag and PR genes in order to characterize mutations occurring in patients failing PI-based therapy. Initially, I performed a cross-sectional analysis by comparing the Gag and protease sequences from PI-experienced patients and PI-naïve subjects. A number of Gag mutations associated with PI-selective pressure were determined, which were not restricted to P7/P1 and P1/P6 CSs, but present throughout the Gag. Subsequently, I conducted a longitudinal analysis of patients failing a PI-based regimen, which confirmed that under PI-selective pressure the entire Gag evolved along with the PR and that changes were most prominent at P2/P7, P7/P1 and P1/P6 CSs and in the P17 protein outside CSs. Finally, I performed phenotypic characterization of PI susceptibility and replicative capacity studies on patient’s viruses and side-directed mutants. As a result of these investigations I found that the evolution of Gag in patients on unsuccessful PI therapy led to increased levels of PI resistance and improved viral replicative capacity. Specifically, I characterized two novel CS mutations (P17/P24: Y132F, and P2/P7: T375A) that conferred resistance in the context of a wild type backbone.

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