Host dNTPase SAMHD1, Lentiviral Accessory Protein Vpx/Vpr, and the Evolutionarily Honed Reverse Transcriptases of SAMHD1 Non-Counteracting Lentiviruses Público

Abstract

Since its devastating appearance in 1981, human immunodeficiency virus (HIV) has remained a major global health concern. With approximately 37.9 million people infected worldwide, the need to understand and adequately treat HIV infections is ever-present. HIV-1 and HIV-2 are the result of two independent cross-species transmission events, with the originating viruses being simian immunodeficiency virus (SIV) from chimpanzees and sooty mangabeys respectively. During the course of viral pathogenesis, HIV/SIV infects dividing (i.e. CD4⁺ T cells) and nondividing (i.e. macrophages and microglia) CD4⁺ cells within the host immune system. With no necessity to support DNA replication, nondividing myeloid cells express high levels of SAM domain- and HD domain-containing protein 1 (SAMHD1), an enzyme that hydrolyzes dNTPs into 2’-deoxynucleoside (dNs) and actively depletes intracellular dNTP pools in nondividing cells. While sharing a target cell tropism, HIV-1 and HIV-2 display distinct replication kinetics in nondividing macrophages: contrary to HIV-1 infection, which is restricted in macrophages, HIV-2 and some SIVs readily replicate in this target cell type. This is because HIV-2 and some SIVs target host SAMHD1 for proteasomal degradation using their viral protein R (Vpr) or viral protein X (Vpx) proteins. Virus-induced degradation of SAMHD1 elevates intracellular dNTP concentrations and enables efficient viral replication in macrophages. Unlike HIV-2, HIV-1 cannot counteract SAMHD1 and thus replicates under low dNTP conditions in nondividing myeloid cells.

Previous studies have shown that reverse transcriptase (RT) proteins from lentiviruses without the ability to counteract SAMHD1 (i.e. HIV-1) reach maximum velocity at lower dNTP concentrations and are able to incorporate dNTP substrates faster than RTs from SAMHD1 counteracting lentiviruses (i.e. SIVmac239). The enhanced kinetics of HIV-1 RT enable complete proviral DNA synthesis in restrictive dNTP concentrations. This dissertation builds upon previous knowledge by showing that RTs from SAMHD1 non-counteracting lentiviruses circumvent SAMHD1 restriction by executing a faster polymerase conformational change during dNTP incorporation. Further, RTs from SIVmac239 infections devoid of Vpx display enhanced enzyme kinetics when compared to RTs from wild type infections—suggesting that intracellular dNTP environments and the lentiviral ability to counteract host SAMHD1 can influence RT kinetics and evolution during the infection of a single host.

Table of Contents

Chapter 1: Introduction 1

1.1 HIV/AIDS 1

A. Global Impact 1

B. Origins 2

1.2 Viral Genome and Replication Cycle 6

A. Viral Genome Organization 6

B. Viral Components 8

C. Viral Replication Cycle 15

D. Disease Progression to AIDS 25

E. HIV/SIV Target Cells 29

i. Viral Tropism 29

ii. Intracellular dNTP Pools and SAMHD1 31

1.3 Reverse Transcriptase 33

A. Structural Features and Drug-Induced Viral Mutagenesis 36

B. DNA Polymerase Reaction Pathway and Kinetics 39

C. Differences Between RTs from SAMHD1 Counteracting and Non-Counteracting Lentiviruses 40

1.4 Dissertation Direction 43

Chapter 2: Evolutionarily Improved Pre-Catalytic Conformational Change of SAMHD1 Non-Counteracting Lentiviral Reverse Transcriptase Proteins 44

2.1 Abstract 45

2.2 Introduction 46

2.3 Experimental Procedures 49

2.4 Results and Discussion 53

2.5 References 68

Chapter 3: Enhanced Enzyme Kinetics of Reverse Transcriptase Variants Cloned from Animals Infected with SIVmac239 Lacking Viral Protein X 75

3.1 Abstract 76

3.2 Introduction 77

3.3 Experimental Procedures 80

3.4 Results 84

3.5 Discussion 98

3.6 References 103

Chapter 4: General Discussion 109

References 117

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Biochemistry, Cell & Developmental Biology
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Chemistry, Biochemistry Biology, Virology
Palabra Clave	Kinetics HIV Evolution SIV Lentivirus SAMHD1 Reverse Transcriptase
Committee Chair / Thesis Advisor	Baek Kim, Emory University
Committee Members	Daniel Reines, Emory University David Lynn, Emory University Graeme Conn, Emory University Gregory Melikian, Emory University

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