The Interplay Between Reverse Transcription and SAMHD1 Degradation: Mechanistic Differences Between Reverse Transcriptases from HIV-1, HIV-2 and SIV Lentiviruses Able or Unable to Degrade SAMHD1 Público

Abstract

Two distinct zoonoses, one from a SIV strain infecting chimpanzees and the other from a SIV strain infecting sooty mangabeys, have led to HIV-1 and HIV-2, respectively. Despite the difference in origin, both lentiviruses HIV-1 and HIV-2 are able to infect dividing CD4+ T cells and nondividing myeloid cells including macrophages and microglia. However, HIV-1 and HIV-2 display very distinct replication kinetics in nondividing myeloid cells such as macrophages. Nondividing cells remain in resting phase of the cell cycle and do not need to replicate their genome for upcoming divisions. An overexpressed enzyme, SAM domain and HD domain-containing protein 1 (SAMHD1), maintains low levels of dNTPs by hydrolyzing them into dNs in nondividing cells. Thus, one major difference between HIV-1 and HIV-2 is that HIV-2 is able to degrade SAMHD1 through its viral protein X (Vpx) and replicate under high cellular dNTP concentrations in nondividing macrophages. HIV-1 lacks Vpx and thus replicates under very limited dNTP conditions found in nondividing macrophages. Previous research has shown that cellular environment may affect viral replication kinetics. Gammaretroviruses infect dividing cells with high dNTP concentrations but are unable to replicate in nondividing cells like HIV-1. Comparing these viral polymerases, the binding affinity for nucleotides is much tighter for HIV-1 reverse transcriptase (RT) versus gammaretroviral RT. This suggests that viral polymerase kinetics interplay with cell tropism. Indeed, this work furthers that hypothesis by showing that RTs of multiple subtypes of HIV-1 reach maximum velocity at lower concentrations of dNTPs which enables them to remain highly active even in a low dNTP environment found in macrophages, compared to RTs of many Vpx coding lentiviruses. Mechanistically, RTs from Vpx encoding lentiviruses display similar binding affinities but lower incorporation rates particularly at pause sites, compared to RTs of Vpx noncoding HIV-1, supporting that faster rates of incorporation contribute to why the Vpx noncoding viral RTs show more efficient DNA synthesis at low dNTP concentration. We hypothesize that the RTs of the Vpx noncoding viruses have evolved to have faster rates of dNTP incorporation in order to overcome the SAMHD1-mediated dNTP dearth found in nondividing myeloid cells.

Table of Contents

Abstract

Acknowledgements

Chapter 1- Introduction

1.1 HIV-1 is the Causative Agent of AIDS

1.2 The HIV Pandemic

1.3 The Origins of HIV-1, HIV-2, and SIVs

1.4 Retroviral Genome Organization

A. Gag

B. Pol

C. Env

D. Vif

E. Vpr

F. Vpx

G. Vpu

H. Nef

I. Tat

J. Rev

1.5 The Retroviral Replication Cycle

A. Viral Entry

B. Uncoating

C. Reverse Transcription

D. Integration

E. Transcription and Translation

F. Virion Assembly, Budding, and Maturation

1.6 HIV-1/ HIV-2 Pathogenesis

A. Transmission and Progression Towards AIDS

B. Target Cells

i. Tropisms and Co-receptors

ii. Nucleotide Pools and SAMHD1

C. Viral Latency

1.7 HIV-1 Reverse Transcriptase

A. Discovery of HIV-1 RT

B. Functions of RT

i. Viral Replication

ii. Source of Mutagenesis

C. RT as a Drug Target

i. NRTIs

ii. NNRTIs

iii. Resistance

D. Structural Features of RT

E. Polymerization Reaction Pathway

i. Steady-state

ii. Pre-steady-state

F. Viral Polymerase Comparison

1.8 Thesis Hypothesis

Chapter 2- Kinetic Variations Between Reverse Transcriptases of Viral Protein X coding and Non-coding Lentiviruses

2.1 Summary

2.2 Introduction

2.3 Experimental Procedures

2.4 Results

2.5 Discussion

Chapter 3- Mechanistic and Kinetic Differences Between Reverse Transcriptases of Vpx Coding and Non-coding Lentiviruses

3.1 Summary

3.2 Introduction

3.3 Experimental Procedures

3.4 Results

3.5 Discussion

Chapter 4- General Discussion

4.1 Collective Results

4.2 Implications of Findings

4.3 Limitations and Future Directions

4.4 Conclusion

References

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Molecular & Systems Pharmacology
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Chemistry, Biochemistry Health Sciences, Pharmacology Biology, Virology
Palavra-chave	Kinetics HIV-1 Lentivirus
Committee Chair / Thesis Advisor	Kim, Baek, Emory University
Committee Members	Schinazi, Raymond F, Emory University Hepler, John R, Emory University Doering, Christopher, Emory University

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