Sink or Swim: Mechanisms of dNTP Pool Elevation by Lentiviruses and Cancer Open Access

Bowen, Nicole (Spring 2023)

Permanent URL: https://etd.library.emory.edu/concern/etds/j3860833m?locale=en
Published

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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