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Title: Cellular and viral factors affecting HIV-1 silencing and reactivation
Author: Norton, Nicholas James
ISNI:       0000 0004 7651 397X
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2019
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Despite advances in the treatment of HIV-1 a cure remains elusive. A significant barrier to the eradication of the virus from an infected individual is a pool of cells infected with transcriptionally silent proviruses. A key pillar of the strategy to eradicate latent viruses has been called 'kick and kill', whereby the latent virus is stimulated to transcribe rendering the host cell vulnerable to eradication by cytotoxic T cells. Optimising the reactivation signal is therefore critical to this approach. Here the established model system of latency 'J-lat' is used to probe optimum reactivation signals. Single clones are observed to respond to maximal stimulation with a single agent with a fixed proportion of cells. Here it is shown that this proportion can be overcome by dosing with two agents in combination and critically that maximum synergies between agents occur at concentrations of agents close to those achieved in vivo. The role of SETDB1 recruitment by the recently described HUSH complex is examined using shRNA knockdowns of these proteins. Knockdown does not increase expression from the majority of J-lat clones tested. Viral factors which influence silencing and reactivation from latency have not been explored to the same extent. Here mutations affecting the binding of splicing factors to HIV-1 mRNA were cloned into laboratory viruses. A reduction in splice factor binding is seen to change the use of splice junctions required for the production of Tat mRNA; in turn this alters the rate at which proviruses are silenced. In addition the threshold for transcription in response to stimulation is increased in mutants with reduced splice factor binding.
Supervisor: Lever, Andrew Michael Lindsay Sponsor: British Infection Association ; Medical Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: HIV ; transcription ; latency ; virus ; viral latency ; provirus