Abstract
Human immunodeficiency virus type 1 (HIV-1) is the causative agent of a progressive failure in the host immune system, leading to acquired immunodeficiency syndrome (AIDS). Antiretroviral therapies effectively treat the disease but fail to eradicate the virus from the patient, presenting the need for further knowledge of virus-host interactions to reach the ultimate goal of a vaccine or cure. Previous work has either taken binary approaches, involving the knockdown or overexpression of specific genes, to finding host-encoded dependency and restriction factors that interact with the virus and modulate replication, or have sought associated genetic polymorphisms (e.g. HLA-B and CCR5) that correlate with outcomes of natural infection.This project utilises the natural genetic variation between patients to identify cellular HIV-1 regulatory factors by conducting a permissivity screen in characterised induced pluripotent stem cells (iPSCs) reprogrammed from samples of peripheral blood mononuclear cells (PBMCs) from the multicentre AIDS cohort study (MACS) – a prospective study of men at high risk of HIV-1 infection whose clinical histories have been described in great detail. The consistency in generation and culture of the iPSCs means that differences between iPSC lines are predominantly attributable to the genetic variation of the patient from which they were derived. This, along with their ability to self-renew, makes iPSCs an excellent resource to utilise for a permissivity screen and has the potential to result in more reproducible permissivity phenotypes when compared to the use of other cell types such as PBMCs from a patient’s peripheral blood. Clinical data describing disease outcomes of patients in the MACS can then be evaluated in the context of in vitro iPSC permissivity phenotypes, genome sequence and transcriptomic profiles.
An optimised reprogramming pipeline was established and enabled the successful generation of 50 MACS-derived iPSC lines from 18 MACS participants who demonstrated extreme HIV-1 disease progression clinical phenotypes. The in vitro permissivity phenotypes of the iPSCs were then identified and comparative transcriptomic analysis of those exhibiting extreme opposite phenotypes allowed for the identification of cellular factors, pathways and networks that possibly influence HIV-1 infection and pathogenesis. Numerous candidate genes were identified and require validation of their influence on HIV-1 infection as either potential restriction or dependency factors. As well as suggesting novel host factors, the work in this thesis demonstrates a functioning pipeline that utilises host natural variation to identify host factors involved in HIV-1 infection that can serve as a foundation for future work. Defining new viral regulatory networks will build on our understanding of HIV-1-host interactions whilst providing clues to potential therapeutic targets.
| Date of Award | 1 Jun 2024 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Michael Malim (Supervisor) & Davide Danovi (Supervisor) |