A dip IN the shallow end: SAMHD1 dNTPase specificity and how draining the dNTP pool limits HIV-1 gap repair. Open Access

Goetze, Russell (Fall 2017)

Permanent URL: https://etd.library.emory.edu/concern/etds/kw52j804p?locale=en
Published

Abstract

Lentiviruses, including HIV-1, infect both dividing and nondividing target cells and have adapted strategies to overcome barriers to replication in these types of cells. One such barrier to in nondividing cells is tight regulation of 2’-deoxyribonucleotide (dNTP) synthesis and degradation by ribonucleotide reductase (RNR) and sterile alpha motif and histidine and aspartic acid domain containing protein 1 (SAMHD1), respectively.

SAMHD1 maintains low dNTP concentrations in nondividing cells though its triphosphohydrolase activity and is allosterically regulated by NTPs and dNTPs. Besides canonical dNTPs, SAMHD1 degrades nucleoside analogue drugs that are used to treat cancer and viral disease. However, whether these molecules are able to induce or disrupt activation by binding to either of the SAMHD1 allosteric sites or the catalytic site is unknown. We tested a number of nucleoside analogues to define key characteristics that would enable us to predict whether these molecules would fit into the active site of SAMHD1. We report that while modifications to the base moiety and some modifications to the 2’ position of the sugar moiety are tolerated, modifications to the 3’ and some modifications to the 2’ position of the sugar moiety prevent degradation by SAMHD1. This information is valuable for designing future nucleoside analogue drugs for SAMHD1-expressing cell types.

Lentiviruses require cellular DNA repair pathways to complete integration of the viral genome into a host chromosome. Among the proteins involved, DNA Polymerase b (Pol b) has been proposed as a potential key player in this process. We generated a novel Pol b KO THP-1 cell line to examine HIV DNA repair in dividing and nondividing cells in the absence of Pol b expression. We report that dNTP concentrations, which are suppressed by SAMHD1 in nondividing cells, but not Pol b, control the rate of gap repair. Furthermore, we show that Pol b is dispensable for HIV-1 transduction in both dividing and nondividing THP-1 cells, indicating that other cellular DNA polymerases and/or RT are capable of completing the essential DNA repair step required for integration. Together, the data presented here provide insight into two ways that cellular regulation of dNTPs interfaces with lentivirus replication.

Table of Contents

Table of Contents

Abstract

Acknowledgements

Chapter 1: Introduction………………………………….…………………..………………….1

A.   HIV Biology…………………………………………….…….………………….………..2

1.     AIDS and the Discovery of HIV-1……………………….…………………………...2

2.     The HIV Genome and its Products……………………………………………………3

3.     The HIV Replication Cycle………………………………………………………….10

4.     Treatment of HIV Infection with Direct-acting Antivirals…………………………..15

5.     Factors Controlling HIV Target Cell Specificity…………………………………….16

B.    Cellular Control of Nucleotides………………………………………………………….17

C.    Cellular DNA Damage Sensing and Repair Mechanisms……………………………….22

D.   Genome Editing Techniques……………………………………………………………..26

E.    References………………………………………………………………………………..31

Chapter 2: Substrates and Inhibitors of SAMHD1…………………………………………..53

A.   Abstract…………………………………………………………………………………..54

B.    Background………………………………………………………………………………54

C.    Results……………………………………………………………………………………56

D.   Discussion………………………………………………………………………………..82

E.    Materials and Methods…………………………………………………………………...85

F.    References………………………………………………………………………………..92

Chapter 3: A CRISPR/Cas9 approach reveals that the polymerase activity of DNA Polymerase b is dispensable for HIV-1 infection in dividing and nondividing cells……...100

A.   Abstract…………………………………………………………………………………101

B.    Background……………………………………………………………………………..101

C.    Results…………………………………………………………………………………..104

D.   Discussion..……………………………………………………………………………..122

E.    Materials and Methods...………………………………………………………………..127

F.    References…...………………………………………………………………………….142

Chapter 4: General Discussion……………………………………………………………….141

A.   Collective Results….……………………………………………………………...……142

References.……………………………………………………………………….……..147    

About this Dissertation

Rights statement
  • Permission granted by the author to include this thesis or dissertation in this repository. All rights reserved by the author. Please contact the author for information regarding the reproduction and use of this thesis or dissertation.
School
Department
Degree
Submission
Language
  • English
Research field
Keyword
Committee Chair / Thesis Advisor
Committee Members
Last modified

Primary PDF

Supplemental Files