Bibby, David Frank; (2002) Drug resistance in HIV-1. Doctoral thesis (Ph.D.), University College London (United Kingdom).

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Abstract

Treatment of HIV-1 infection with 3'-azido-2',3'-dideoxythymidine (AZT) frequently results in the evolution of viruses harbouring varying combinations of point mutations within the reverse transcriptase (RT) domain which confer increasing levels of resistance to AZT. It has been demonstrated that two such mutations (M41L and T215Y) reduce the replicative capacity of HIV-1 and are selected against in the absence of AZT. For this thesis, a patient undergoing AZT monotherapy was identified (A3), in which M41L and T215Y emerged only after therapy had been discontinued. It was proposed that rather than universally decreasing fitness, these mutations could increase fitness in the context of different RT genotypes. A system was developed to generate recombinant HIV-1 stocks containing populations of sample-derived RT domains in a constant genetic background. The widely-used recombinant virus assay in which sample-derived sequences are inserted into a virus backbone by homologous recombination was adapted in order to prevent undefined and/or undesired sample-derived sequences being incorporated into recombinant viruses. Phenotypic differences between recombinant viruses could thus be ascribed solely to their differing RT domains. One of two silent point mutation markers was engineered into each recombinant, allowing the relative frequencies of mixed virus populations to be assayed by a highly-reproducible quantitative point mutation assay. Measuring the rate of change of these frequencies during pair-wise competition experiments enabled calculation of relative fitnesses. Recombinant viruses were generated containing RT populations rescued from serial plasma samples taken from patient A3, and their relative fitness calculated from competitions against a reference virus. Significant differences between relative fitness values were observed. The onset of therapy resulted in lowered fitness, but over time, the RT population fitness returned to the pre-treatment level, concomitant with the emergence of M41L and T215Y, together with many other sequence changes observed throughout the duration of therapy.

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