Sink or Swim: Mechanisms of dNTP Pool Elevation by Lentiviruses and Cancer Público
Bowen, Nicole (Spring 2023)
Abstract
The sole utility of deoxynucleoside triphosphates (dNTPs) is to serve as substrates for DNA polymerase during DNA synthesis. Correspondingly, dNTP concentrations elevate at the G1/S phase transition to accommodate novel DNA synthesis. In contrast, non-dividing quiescent cells that lack chromosomal replication harbor consistently low dNTP pools. Appropriate intracellular dNTP levels are maintained by a delicate balance of de novo and salvage biosynthetic pathways and hydrolysis by dNTPase, SAMHD1. Importantly, the regulation of these pathways is tied closely to the cell cycle. Proper maintenance of dNTP levels is essential to the health of a host as abnormal dNTP levels can contribute to decreased polymerase fidelity and mutation synthesis. Lentiviruses infecting non-diving cells and fast-replicating cancer cells are faced with the barrier of insufficient dNTP pools for copying their genomes. To address this, lentiviruses such as HIV-2 and some SIVs code for Viral protein X (Vpx), which targets host SAMHD1 for proteasomal degradation and elevates intracellular dNTP levels. Similarly, cancer cells elevate dNTP levels 6-11-fold and this alteration in metabolism has been suggested as a hallmark of cancer. Therefore, SAMHD1 counteracting lentiviruses and cancer cells both elevate intracellular dNTP pools during the course of their pathogenesis. In this work, I first explore necessary determinants for Vpx-mediated dNTP elevation in non-diving cells. Here, I uncover active dNTP biosynthesis in primary non-dividing macrophages. I then find that Vpx-mediated dNTP elevation and rescue of infection in non-dividing cells after SAMHD1 depletion is dependent on this ongoing dNTP biosynthesis. In subsequent work, I identify cancer associated SAMHD1 mutants that have similar expression and stability profiles to wild type. I then used these mutants as tools to probe which functions of SAMHD1 may contribute to cancer phenotypes. Here, I find only dNTPase activity has been altered by these mutations, suggesting cancer-associated mutations in SAMHD1 can contribute to the elevated dNTP level characteristic of cancer cells. Together, this work provides mechanistic insights into the shared phenomenon of dNTP elevation during viral infection and oncogenesis.
Table of Contents
Chapter 1: Introduction. 1
1.1 Intracellular dNTP pool maintenance. 2
1.2 Retroviruses. 3
1.3 Regulation of cellular dNTP pools is a major determinant of HIV-1 replication kinetics in target cells. 4
1.4 Regulation of SAMHD1. 5
1.5 HIV-1 restriction by human SAMHD1. 8
1.6 Viral Protein X (Vpx) counteracts restriction by SAMHD1. 9
1.7 Host and viral evolution due to the host-pathogen arms-race. 10
1.8 SAMHD1 and the restriction of other retroviruses. 11
1.9 SAMHD1 modulates host innate immunity. 12
1.10 The role of SAMHD1 in DNA damage and cell cycle. 13
1.11 SAMHD1 in cancer. 14
1.12 Framework and overview of the dissertation. 16
Chapter 2: Vpx requires active dNTP biosynthesis to effectively counteract the anti-lentivirus activity of SAMHD1 in macrophages. 19
2.1 Abstract 20
2.2 Introduction 21
2.3 Results 23
2.3.1 Ribonucleotide reductase is expressed in monocyte-derived macrophages. 24
2.3.2 Macrophages, but not monocytes, express dNTP biosynthesis enzymes from the de novo and salvage pathways. 25
2.3.3 SAMHD1 in monocytes predominantly remains active/dephosphorylated, but SAMHD1 phosphorylation increases during differentiation to
macrophages. 26
2.3.4 Monocytes enter a G1/S phase-like state during differentiation to macrophages. 27
2.3.5 Monocytes have extremely low dNTP levels that cannot be raised by Vpx. 29
2.3.6 dNTP concentrations found in monocytes block efficient reverse transcription. 30
2.3.7 Vpx requires ongoing dNTP biosynthesis to accelerate reverse transcription and rescue HIV-1 transduction. 31
2.4 Discussion 32
2.5 Experimental Procedures 34
2.5.1 Cell culture 34
2.5.2 Vectors 35
2.5.3 RNR inhibitor treatment 37
2.5.4 Western Blot 37
2.5.5 dNTP extraction 38
2.5.6 RT-based cellular dNTP measurement 38
2.5.7 EdU assay 39
2.5.8 Mitochondrial DNA copy number qPCR 40
2.5.9 LC-MS/MS-based dNTP measurement 40
2.5.10 VLP Vpx treatment of monocytes and macrophages 41
2.5.11 Cell volume of monocytes and macrophages 41
2.5.12 Monocyte and macrophage dNTP concentration 42
2.5.13 Primer extension assay 42
2.5.14 HIV-vector and VLP treatment of monocytes and macrophages 43
2.5.15 Statistical analyses 44
2.6 Data Availability 44
2.7 Funding 44
2.8 Author Contributions 45
2.9 Conflict of Interest Statement 45
2.10 Acknowledgements 45
Chapter 3: Elevation of intracellular dNTP Levels: A mechanistic role of SAMHD1 cancer mutations. 60
3.1 Abstract 61
3.2 Introduction 61
3.3 Results 64
3.3.1 R366C/H mutants are cancer-associated mutants with wild type protein expression level. 64
3.3.2 Biochemical analyses of protein stability and structural integrity of R366C/H mutants. 65
3.3.3 Cancer-associated SAMHD1 mutants have significantly reduced dNTPase activity. 66
3.3.4 X-ray crystal structures of R366C/H mutants. 68
3.3.5 Impact of the R366C/H mutation on SAMHD1 restriction of HIV-1. 69
3.3.6 R366C/H mutants have unaltered interactions with CtIP for dsDNA break repair and Cyclin A2. 69
3.3.7 R366C/H mutants suppress HIV-1 LTR activation and innate immune activation. 71
3.3.8 R366C/H mutants showed reduced nucleic acid binding activity. 71
3.3.9 SAMHD1 reduction in human primary normal dividing cells further elevates intracellular dNTP levels. 72
3.4 Discussion 73
3.5 Experimental Procedures 74
3.5.1 Cell culture 74
3.5.2 Structural model with location of mutant residues 75
3.5.3 Mutant cellular expression 75
3.5.4 Immunoblots 76
3.5.5 SAMHD1 protein expression and purification 76
3.5.6 Thermal Shift Assay 77
3.5.7 Cross-linking based tetramerization assay 78
3.5.8 SAMHD1 degradation assay 79
3.5.9 Thin-layer chromatography based dNTPase assay 79
3.5.10 Crystallization and data collection 80
3.5.11 Structure and refinement 80
3.5.12 Generation of U937 cells expressing SAMHD1 mutations 81
3.5.13 HIV-1 vector transduction 81
3.5.14 Cellular dNTP measurement 82
3.5.15 Immunoprecipitation 82
3.5.16 DSB reporter assay 83
3.5.17 LTR and ISRE luciferase assays 83
3.5.18 Oligonucleotide binding by fluorescence polarization 84
3.5.19 Virus-like particle transduction of CD4+ T-cells 85
3.5.20 Statistical analyses 85
3.6 Data Availability 86
3.7 Funding 86
3.8 Author Contributions 87
3.9 Conflict of Interest Statement 87
Chapter 4: Concluding remarks. 108
4.1 Abstract 109
4.2 Macrophage tropism as a driver of lentiviral evolution. 109
4.2.1 Lentiviruses have acquired adaptations to infect macrophages over evolutionary time. 109
4.2.2 Herpesviruses have evolved adaptations distinct from lentiviruses for macrophage infection. 112
4.2.3 The inhibitory “cost” of individual adaptations to infect macrophages depends on viral background. 113
4.2.4 The search for the significance of macrophage infection to lentiviruses is ongoing. 114
4.3 Mechanisms of cancer cell dNTP pool elevation and therapeutic implications. 118
4.3.1 Cancer cells utilize diverse mechanisms to elevate intracellular dNTP pools. 118
4.3.2 Cancer therapies target dNTP metabolism. 120
4.3.3 Therapeutic implications of SAMHD1. 121
4.3.4 Refining the role of SAMHD1 in cancer continues. 123
4.4 Summary 125
References 126
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