Shiu, Carlum; (2004) Functional analyses of HIV-1 glycoproteins: Fusogenic potential in relation to viral pathogenesis. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

HIV-1 entry into host cells is mediated by the viral surface glycoprotein (gp160 or Env) that is processed into gp120 and gp41, carrying receptor-binding and membrane-fusion activities respectively. Interplay between these two activities determines the overall fusogenic potential of processed Env. Comparison of full-length env-genes recovered directly from a patient cohort has revealed amino acid differences across Env between long-term non-progressor and rapid progressor patient groups that could affect the efficiency of membrane-fusion. To assess this the aim of my project was to develop a cell-cell fusion assay for HIV-1 Env. The ability of the assay described in this thesis to distinguish non-functional and functional Envs was determined with HIV-1NL43 Env constructs either mutated in the gp160 KAKRRVVQREKR processing site, or progressively truncated from the C-terminus. A novel attenuating single residue amino acid substitution in the KAKRR motif has been identified and the results have been corroborated by virus infectivity assays following insertion of the env-genes into C2, an env-gene cosseting infectious molecular clone. Other mutations in the VVQ motif highlight qualitative differences in the function of Env when expressed either on a cell surface or on a virus surface, suggesting additional roles of the KAKRR motif in Env function. The study of Env truncation mutants, in the context of recent revised models of the gp41 cytoplasmic domain, provided functional evidence for regions adjacent to and involving the gp41 Kennedy domain playing a role in membrane fusion. The application of the cell-cell fusion assay to longitudinal samples of env-genes derived from four patients who are progressing to AIDS after 12 or more years of asymptomatic HIV-1-infection, as determined by declining CD4+ cell numbers and increasing viral load, indicated a lack of Envs with high fusogenic capacities. The C2 system indicated that many of these Envs were unable to support chimeric virus infection of PBMCs. The assay developed could thus provide a rapid screen for functional Envs to study in the C2 infectious molecular clone. The developed cell-cell fusion assay is complementary to existing methods of analyses in our laboratory, and could be adapted in the future for assessing other aspects related to clinical treatment of patients, such as surveillance for the emergence of Envs that are resistant to entry inhibitors.

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