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Title: Investigation of the genetic and molecular basis of HIV-1 cellular tropism in vivo
Author: Marsden, Matthew David
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2003
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The aim of this project was to further characterise the evolutionary relationships between HIV-1 variants in the brain and lymphoid system, and in particular to search for evidence of recombination between variants in the two compartments by analysing sequence segments both within and outside the major genetic determinants of HIV-1 cellular tropism. The evolutionary relationships manifest in the p17gag and V3 genomic regions were assessed between HIV-1 variants derived from the brain and lymphoid system of four infected individuals with evidence of HIVE. P17gag distances between brain and lymph node variants were calculated, allowing approximate divergence times to be inferred. In two cases this indicated that p17gag sequences present in the brain and lymphoid compartments had diverged prior to the individual progressed to AIDS, possibly indicating an autonomous pre-AIDS infection of the brain in these two study subjects. A correlation was also noted between the severity of HIVE and both the V3 sequence divergence within brain, and the extent to which variants in brain differed from those in the lymphoid system. In addition, p17gag and V3 phylogenetic tree topologies generated with sequences obtained from the same tissues exhibited discordant phylogenetic relationships in each study subject. The most obvious explanation for these complex evolutionary relationships is the occurrence of recombination among HIV-1 variants present within the brain, and also between brain and lymphoid derived HIV-1 variants. Limiting dilution PCR was used to obtain entire HIV-1 env sequences for genetic analysis. Inter-tissue recombination was apparent in several env sequences, providing further evidence for the occurrence of this mutational mechanism in the context of a single HIV-1 infection. Close relationships between particular brain and lymphoid tissue derived HIV-1 variants were also noted in certain genomic regions, despite apparent tissue specificity in others. A novel technique was developed for the addition of promoter and polyadenylation sequences directly to PCR products, allowing expression of open reading frames in mammalian cells without the need for prior molecular cloning. The efficiency of this technique was verified by using two reporter genes and HIV-1 env and nef sequences derived from autopsy tissue. As envelope genes were amplified at limiting dilution, use of this method ensured that sequence characteristics and genetic linkages present in vivo were maintained, and not affected by the various in vitro artefacts that may significantly alter retrieved sequences. Together these results indicate that recombination between HIV-1 variants have been diverged during a single infection is an extremely common event. The identification of recombination between brain and lymphoid derived virus also suggests that physical separation or differences in viral turnover rates are not the source of the commonly observed genetic differences between vi8ral variants within these two compartments. Therefore it may be speculated that those regions of the viral genome that do show tissue specificity are the result of an adaptive process.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available