Ribonucleotide reductase and SAMHD1: Critical players in nucleotidemetabolism, NRTI efficacy and HIV replication Público

Daly, Michele Brien (2016)

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

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

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