Ribonucleotide reductase and SAMHD1: Critical players in nucleotide metabolism, NRTI efficacy and HIV replication 公开

Daly, Michele Brien (2016)

Permanent URL: https://etd.library.emory.edu/concern/etds/jq085k242?locale=zh
Published

Abstract

2' Deoxyribonucleotides (dNTPs) are essential for the DNA replication of all organisms and many viruses. Cellular dNTP regulation is an exquisitely complex process, which includes two major enzymes: ribonucleotide reductase (RNR) and sterile alpha motif and histidine-aspartic domain containing protein 1 (SAMHD1). RNR is responsible for the de novo synthesis of dNTPs through the reduction of ribonucleotides. Reciprocally, SAMHD1 is responsible for the hydrolysis of dNTPs to dNs and triphosphates. These two enzymes, working in concert, maintain cellular dNTP concentrations at the proper levels for genomic replication and DNA repair. Even modest disruptions in the specific balance of dNTPs can lead to poor DNA polymerase fidelity and proofreading, resulting in genome instability and mutagenesis.

Human immunodeficiency virus (HIV) is a significant human pathogen with approximately 36.9 million people infected worldwide. Without treatment, chronic HIV infection depletes CD4 lymphocytes, which are necessary for maintaining immunocompetence, leading to acquired immunodeficiency syndrome (AIDS). HIV replication occurs via reverse transcription of the viral RNA genome to DNA by the viral polymerase, reverse transcriptase (RT). This process, which requires cellular dNTPs, has been clinically exploited with nucleoside reverse transcriptase inhibitors (NRTIs). NRTIs are structurally analogous to dNTPs, and due to the low fidelity of RT they are readily incorporated during viral replication. All seven of the FDA-approved NRTIs induce obligate chain termination, because unlike dNTPs they do not have the chemical requirements to make a bond with the next incoming dNTP.

Therapeutic innovation is essential due to the increased prevalence of NRTI resistance, transmission of drug resistant variants and necessity of salvage therapy. Here, we investigate multiple NRTIs, which are varied in their mechanism of action, and the role that RNR and SAMHD1 have in modulating their antiviral activity. First, we investigated the purine analog clofarabine, an FDA approved anticancer compound. Clofarabine works via two mechanisms. It inhibits RNR causing a decrease in cellular dNTPs and its incorporation by RT induces delayed chain termination. RNR inhibition limits competition for the incorporation of clofarabine by RT and its antiviral activity is self-potentiated. Second, we examined the anti-HIV mechanism of 2 FDA-approved lethal mutagens, 5-azacytidine and 5-aza-2'deoxycytidine. Our results show that RNR rapidly reduces 5-azacytidine to 5-aza-2'deoxycytidine, and therefore the active drug is the deoxyribose form. Lastly, we studied how SAMHD1 induced cellular dNTP depletion affects the competitive landscape for NRTIs. As expected, SAMHD1 dNTP depletion increased NRTI efficacy. Importantly SAMHD1 does not effectively degrade the clinically relevant NRTIs. Taken together, our results indicate that novel NRTIs, whether they are an obligate chain terminator, delayed chain terminator, or lethal mutagen should be screened for the following characteristics: 1) Inhibition of RNR, 2) Inhibition of HIV-RT, including NRTI resistant variants and 3) Resistance to degradation by SAMHD1.

Table of Contents

TABLE OF CONTENTS
CHAPTER I: INTRODUCTION
A. HIV/AIDS
a. HIV Origins………………………………………………………………………1
b. Global Impact…………………………………………………………………….2
B. Biology of HIV
a. HIV Replication Cycle…………………………………………………….……...2
i. Structure……………………………………………………………….…2
ii. Binding & Fusion………………………………………………………..3
iii. Reverse Transcription…………………………………………….……...3
iv. Integration…………………………………………………………….….6
v. Replication……………………………………………………………….6
vi. Assembly, Budding & Maturation……………………………………….6
b. HIV Pathogenesis
i. Transmission & Acute Infection………………………………….……...6
ii. Viral Tropism…………………………………………………………….7
iii. Cellular Permissivity…………………………………………………….7
iv. Progression to AIDS……………………………………………………..8
c. HAART……………………………………………………………….…………..9
i. NRTIs……………………………………………………………………9
ii. Viral Mutagenesis & NRTI Resistance………………………...………11
d. Viral Reservoirs & HIV Cure…………………………………………...………12
C. Nucleic Acid Metabolism
a. Nucleotide Biosynthesis………………………………………………….….….13
b. Deoxyribonucleotide Metabolism……………………………….…………..…..13
c. Ribonucleotide Reductase……………………………………….…………..…..14
i. Function
ii. Structure
iii. Expression & Regulation
d. SAMHD1……………………………………………………………..…………15
i. Function
ii. Structure
iii. Expression & Regulation
e. Nucleotide regulation and Disease…………………………………………..….20
i. Cancer
ii. Viral Infections
iii. Aicardi-Goutières Syndrome
CHAPTER II: DUAL ANTI-HIV MECHANISM OF CLOFARABINE…………………...21
Abstract………………………………………………………………………………......22
Background…………………………………………………………………………..…..23
Results & Discussion…………………………………………………………………….25
Methods………………………………………………………………………………......34
References ………………………………………………………………………….…....50
CHAPTER III: 5-AZACYTIDINE ENHANCES THE MUTAGENESIS OF HIV-1 BY THE REDUCTION TO 5-AZA-2'-DEOXYCYTIDINE....................................................................56
Abstract………………………………………………………………………………......57
Background……………………………………………………………………….……...57
Materials and Methods…………………………………………………………….……..60
Results…………………………………………………………………………….……...64
Discussion……………………………………………………………………….…….....68
References……………………………………………………………………….…….....72
CHAPTER IV: SAMHD1 REGULATION OF DNTPS AFFECTS THE EFFICACY OF NRTIs……………………………………………………………………………………….……82
Abstract………………………………………………………………………….……….83
Background……………………………………………………………….………….......83
Experimental Procedures…………………………………………………….…………..85
Results ……………………………………………………………….…………………..89
Discussion………………………………………………………….…………………….95
References…………………………………………………………….………………...104
CHAPTER V: SAMHD1 CONTROLS CELL CYCLE STATUS, APOPTOSIS AND HIV-1 INFECTION IN MONOCYTIC THP-1 CELLS.....................................................................109
Abstract……………………………………………………………………….………...110
Introduction………………………………………………………………….……….....110
Results…………………………………………………………………….………….....112
Discussion……………………………………………………………….……………...117
Materials and Methods…………………………………………………….…………....120
References………………………………………………………………….…………...133
CHAPTER VI: DISCUSSION...................................................................................................141
The HIV Epidemic……………………………………………………………………...141
Nucleotide Regulation, Viruses & Cancer………………………………………….......141
References……………………………………………………………………………....147


List of Figures
CHAPTER I
Figure 1: HIV Reverse Transcription……………………………………………………..4
Figure 2: SAMHD1 and RNR regulate cellular dNTP pool together…………………...19
CHAPTER II
Figure 1: Anti-HIV-1 activity of clofarabine in primary human activated CD4+ T cells and monocyte derived macrophages……………………………………………………..41
Figure 2: Clofarabine induced depletion of cellular dNTPs and inhibition of reverse transcription. Effect of clofarabine on cellular dNTP levels in primary activated CD4+
T cells…………………………………………………………………………………….42
Figure 3: Biochemical examination of the dual mechanism of clofarabine. a Direct clofarabine-TP incorporation by HIV-1 RT………………………………………...…...44
Figure 4: Model for the anti-HIV-1 dual action mechanisms of clofarabine in macrophages……………………………………………………………………………..46
Figure S1: Infectivity and toxicity in T cells and macrophages……...............................47
Figure S2: Clofarabine induced depletion of cellular dNTPs in MAGI cells……….......49
CHAPTER III
Figure 1: 5-Azacytidine and 5-aza-2′-deoxycytidine induce similar levels of G-to-C and C-to-G transversion mutations during HIV-1 replication………………………………..77
Figure 2: 5-Azacytidine and 5-aza-2′-deoxycytidine induce similar patterns of mutation during HIV-1 replication………………………………………………………………....78
Figure 3: 5-Aza-dCTP levels are comparable in cells treated with 5-aza-C or
5-aza-dC………………………………………………………………………………….79
Figure 4: HIV-1 RT incorporates 5-aza-CTP much less efficiently than 5-aza-dCTP in vitro………………………………………………………………………………………80
Figure 5: Model of 5-azacytidine- and 5-aza-2′-deoxycytidine-mediated HIV-1 mutagenesis………………………………………………………………………………81
CHAPTER IV
Figure 1: Knockdown of SAMHD1 decreases NRTI efficacy in THP1 cells………......98
Figure 2: Vpx-mediated degradation of SAMHD1 decreases NRTI efficacy in macrophages…………………………………………………………………………......99
Figure 3: Vpx-mediated degradation of SAMHD1 decreases the efficacy of combination NRTI treatment………………………………………………………………………....100
Figure 4: Vpx-mediated degradation of SAMHD1 decreases NRTI efficacy in activated T cells…………………………………………………………………………………...101
Figure 5: SAMHD1 enzymatic activity toward ddNTPs and allosteric activation with ddGTP…………………………………………………………………………………..102
Supplemental Figure 1: Degradation of SAMHD1 in activated CD4+ T cells increases cellular dNTPs……………………………………………………………….…………103
CHAPTER IV
Figure 1: SAMHD1 knockout in THP-1 cells by CRISPR/Cas9………………….…..126
Figure 2: THP-1 SAMHD1 knockout cells have increased cell proliferation and altered cell cycle status…………………………………………………………………….…...127
Figure 3: Reduced apoptosis in SAMHD1 knockout THP-1 cells compared to control cells……………………………………………………………………………………..128
Figure 4: Knockout of SAMHD1 increases HIV-1 infection of non-differentiated and differentiated THP-1 cells………………………………………………………………129
Supplementary Figure 1: Effects of SAMHD1 silencing on dNTP levels, cell proliferation and cell cycle progression of THP-1 cells……………………………...131
Supplementary Figure 1: SAMHD1 induces spontaneous apoptosis………………132

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