Caswell, Sarah Jane; (2019) Biochemical analysis of the catalytic activity of HIV-1 restriction factor SAMHD1. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

SAMHD1 is a cellular enzyme that has deoxynucleotide triphosphate triphosphohydrolase activity and is vital for maintaining the homeostatic balance of cellular dNTPs. SAMHD1 is also an important effector of innate immunity and cell cycle regulation, and mutations to SAMHD1 are frequently found in relation to both the autoimmune disease Aicardi-Goutières syndrome and in many types of cancer. In 2011, it was demonstrated that SAMHD1 restricts HIV-1 replication in terminally differentiated myeloid-lineage cells and resting CD4+ T cells. However, competing models have been published regarding how SAMHD1 activity results in HIV-1 restriction. These are divided between dNTP triphosphohydrolase activity, which is proposed to deplete cellular dNTPs to levels that no longer support productive reverse transcription, and a nuclease activity, suggested to cleave viral nucleic acid prior to integration. In addition, SAMHD1 is proposed to bind to nucleic acids, although the exact nature of this activity is unclear. The first part of this thesis addresses the question of “which activity of SAMHD1 is responsible for HIV-1 restriction?” Cell-based virological assays demonstrate that the HIV-1 genome remains intact in the presence of SAMHD1, which supports the notion that SAMHD1 triphosphohydrolase activity rather than any nuclease activity is responsible for restriction. These observations are reinforced by in vitro biochemical and enzymological assays that demonstrate a robust SAMHD1 triphosphohydrolase activity but only a weak nuclease activity that can be removed with increased stringency of protein purification. Studies of SAMHD1 nucleic acid binding revealed that SAMHD1 is a nucleic acid binding protein with preference for RNA over DNA. However, binding of nucleic acids appears to have little impact on SAMHD1 oligomerisation and triphosphohydrolase activity. In the second part of this thesis, in vitro assays employing both single and multi-dNTP substrates as well as dNTP analogues were used to characterise SAMHD1 oligomerisation and triphosphohydrolase activity. The analogue studies probed the SAMHD1 catalytic mechanism and provided a rationale for SAMHD1 stereo-selectivity and inhibition. Mutational studies showed how changes in SAMHD1 at residues that either mediate dNTP hydrolysis, direct allosteric activation or promote SAMHD1 tetramerisation all diminish dNTP triphosphohydrolase activity. Moreover, complementary cell-based, dNTP quantification and HIV-1 restriction assays reveal that the same mutations result in increased cellular dNTP levels and abolish SAMHD1 mediated restriction of HIV-1.

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