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Title: Investigating SAMHD1 restriction of HIV-1 in stem cell-derived macrophages
Author: Warner, Damien
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2020
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SAMHD1 is potent in restriction of lentiviruses in primary bone marrow-derived monocytes, macrophages, and dendritic cells and is variably neutralised in these cells by the SIV accessory protein Vpx. However, the dynamics of SAMHD1 restriction of lentiviruses in yolk sac derived tissue-resident macrophages has not been formally demonstrated. Here I use a human stem cell differentiation protocol, competent in the production of macrophages that are developmentally related to, and thus a good model for, tissue-resident macrophages such as microglia, Langerhans cells, and alveolar macrophages. This system produced stem cell origin Yolk-sac-Derived-like Macrophage Precursor cells (scYDMPs) and Macrophages (scYDMs) over extended periods, which were subsequently used as a tool to investigate SAMHD1 restriction of HIV-1 and antagonisation by Vpx. Using a loss-of-function approach, I showed that the phenotype of SAMHD1 expression, hydrolysis of dNTPs, restriction of HIV-1, and antagonisation by Vpx in scYDMs is very similar to that reported in blood MDMs. I also showed that Vpx-mediated rescue of HIV-1 infection was more potent than the shRNA knockdown of SAMHD1. I, therefore, hypothesised that either Vpx facilitates HIV-1 infection via a SAMHD1 independent mechanism or the SAMHD1 protein remaining after knockdown was of sufficient potency to facilitate HIV-1 restriction and dNTP hydrolysis. I used a CRISPR-Cas9 knockout strategy to show that Vpx did not rescue HIV-1 infection by a SAMHD1 independent mechanism in scYDMs. However, the application of Vpx to SAMHD1 knockout cells was concomitant with an increase in dNTPs. This showed that in scYDMs, Vpx functions to increase dNTPs by a mechanism independent of SAMHD1. Using a dNTPase defective SAMHD1 mutant scYDM cell line, I was able to show for the first time that the dNTPase function of SAMHD1 is dispensable for HIV-1 infection in scYDMs. I also showed for the first time that Vpr of SIVmac degrades SAMHD1 and rescues HIV-1 infection in scYDMs. Moreover, and in contrast to Vpx-mediated rescue of HIV-1 infection at the steady-state in scYDMs, Vpx (and Vpr) rescued HIV-1 infection from the antiviral state established by type I interferons and LPS in scYDMs. This result implies that in scYDMs, Vpx and Vpr may interact with additional components of the innate immune system that are independent of SAMHD1. Taken together, these results highlight the experimental flexibility and integrity of data generated using a stem cell to macrophage differentiation strategy combined with genetic modification, and show that these cells represent a good model for investigating SAMHD1 restriction of HIV-1.
Supervisor: James, William ; Moore, Michael Sponsor: Biotechnology and Biological Sciences Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Virology