Mechanisms of HIV-1 Restriction by the Host SAMHD1

Dissertation

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

By Jenna Marie Antonucci Graduate Program in Microbiology

The Ohio State University 2018

Dissertation Committee Li Wu, Ph.D., Advisor Irina Artsimovitch, Ph.D. Jesse Kwiek, Ph.D. Karin Musier-Forsyth, Ph.D.

Copyrighted by

Jenna Marie Antonucci

2018

Abstract

Human immunodeficiency type 1 (HIV-1) is a human that replicates in cells via a well-characterized viral lifecycle. Inhibition at any step in the viral lifecycle results in downstream effects that can impair HIV-1 replication and restrict infection. For decades, researchers have been unable to determine the cause of myeloid-cell specific block in HIV-1 infection. In 2011, the discovery of the first mammalian deoxynucleoside triphosphate (dNTP) triphosphohydrolase

(dNTPase) sterile alpha motif and HD domain containing protein 1 (SAMHD1) answered that question and introduced an entirely novel field of study focused on determining the mechanism and control of SAMHD1-mediated restriction of HIV-1 replication. Since then, the research on SAMHD1 has become a timely and imperative topic of virology. The following body of work includes studies furthering the field by confirming the established model and introducing a novel mechanism of SAMHD1-mediated suppression of HIV-1 replication.

SAMHD1 was originally identified as a dGTP-dependent dNTPase that restricts

HIV-1 infection by hydrolyzing intracellular dNTPs to a level that inhibits efficient reverse transcription of HIV-1 genomic RNA into complementary DNA (cDNA).

Although this model was confirmed by several studies, work published in 2014 suggested that SAMHD1 is a nucleic-acid binding protein that restricts HIV-1

i

replication through its (RNase) activity against the viral RNA genome in non-dividing immune cells. These findings revealed a new mechanism of

SAMHD1-mediated HIV-1 restriction and raised important questions as to the contribution of each activity, dNTPase and RNase, to HIV-1 restriction.

Based on previous studies, we tested our hypothesis that the RNase activity of

SAMHD1 might limit HIV-1 protein production in virus producing cells, as transcribed viral RNA would be subject to cleavage by SAMHD1. Our data suggest that newly transcribed mRNAs of HIV-1, Influenza A virus (IAV), and Sendai virus

(SeV) are not subjected to nucleolytic cleavage by SAMHD1’s RNase activity. We further confirmed SAMHD1 expression does not affect HIV-1 protein production, viral particle release, and infectivity of newly synthesized HIV-1 when the block in reverse transcription is bypassed. While SAMHD1 had no effect on incoming viral genomic RNA levels, we confirmed that SAMHD1 reduces intracellular dNTPs and inhibits efficient production of HIV-1 late reverse transcription products in non- dividing cells. Taken together, our study confirmed a dNTPase-dependent restriction of HIV-1 infection by SAMHD1.

A critical barrier to developing a cure for HIV-1 infection is the long-lived viral reservoir that exists in resting CD4+ T-cells, the main targets of HIV-1. The viral reservoir is maintained through a variety of mechanisms, including regulation of the HIV-1 long terminal repeat (LTR) promoter. Recombinant SAMHD1 binds HIV-

1 DNA or RNA fragments in vitro, but the function of this binding remains unclear. ii

SAMHD1 restricts HIV-1 replication in non-dividing cells and is highly expressed in resting CD4+ T-cells, but its role in HIV-1 latency remains unknown. Here we report a new function of SAMHD1 in regulating HIV-1 latency. We found that

SAMHD1 suppressed HIV-1 LTR promoter-driven expression and reactivation of viral latency in cell lines and primary CD4+ T-cells. Furthermore,

SAMHD1 bound to the HIV-1 LTR in vitro and in a latently infected CD4+ T-cell line, suggesting that the binding may negatively modulate reactivation of HIV-1 latency. Our findings indicate a novel role for SAMHD1 in regulating HIV-1 latency, which enhances our understanding of the mechanisms regulating proviral in CD4+ T-cells.

To further understand the mechanism regulating the activity of SAMHD1, we solved novel crystal structures of full-length mouse SAMHD1 (mSAMHD1) to identify residues essential for the intra- and inter-subunit interaction between the

SAM and HD domains. Interestingly, while the SAM domain of human SAMHD1

(hSAMHD1) is dispensable for HIV-1 restriction, we found that the SAM domain of mSAMHD1 is required for efficient HIV-1 restriction. Further, we determined that destabilization of the SAM-to-HD domain interaction abrogated the HIV-1 restriction activity of mSAMHD1. Interestingly, stabilization of the SAM-to-HD domain interaction in hSAMHD1 resulted in enhanced HIV-1 restriction. These data increase our understanding of the mechanism regulating SAMHD1-mediated

HIV-1 restriction.

iii

The following studies have been critical for defining the mechanism of SAMHD1- mediated HIV-1 restriction. Taken together, our results can help better understand

HIV-1 pathogenesis and how SAMHD1 functions as a retroviral restriction factor.

iv

Dedication

Dedicated to Wendell – for his advice, his patience, and his faith in me. Because he always understood.

v

Acknowledgments

I am overwhelmingly grateful for the patience and guidance of my advisor, Dr. Li

Wu. At times he expected more of me than I thought I had to give; however, I now know that if you make every day better than the last, put your heart fully into everything you do, and never give up, anything is possible. I am indebted to my committee members, Dr. Irina Artsimovitch, Dr. Jesse Kwiek, and Dr. Karin Musier-

Forsyth, for their advice and encouragement throughout my graduate career.

Thank you for emboldening me to feel confident as a student and a scientist, and for always lending an ear when needed.

For those who have never visited the basement of the Veterinary Medicine

Academic Building, you could not imagine a more supportive, encouraging, laughter-filled, and lovely place to work – in spite of the lack of windows. Dr. Sarah

Fritz, Dr. Jacob Al-Saleem, Dr. Amanda Panfil, Dr. Michael Martinez, and my Wu labmates Dr. Feifei Wang, Dr. Suresh de Silva, Dr. Serena Bonifati, Dr. Nagaraja

Tirumuru, Dr. Wuxun Lu, Dr. Shuliang Chen, Zhihua Qin, Victoria Maksimova,

Taiwei Li, and Sunhee Kim: They say it takes a village, and I couldn’t be more grateful I had you to get me through the ups and downs of the past five years.

I don’t think that I could’ve gotten through graduate school without the support of my lab mentor and friend Dr. Corine St Gelais. You make me think better and

vi

harder everyday, and you helped me realize that the best version of myself is the one that is happiest.

I want to especially thank Dr Alice Duchon and Brent Simpson; the best friends anyone could ask for. Brent, your support has never wavered since day one. I am so grateful for all our Sundays watching Game of Thrones, the Pokémon Go hikes, sushi dates, and movie nights. You made me feel calm in times of chaos, and I will be forever thankful for your support. Alice, you made everyday coming to work fun, exciting, and filled with laughter. Thank you for editing virtually everything I wrote in graduate school, for all the advice about science and life, for the mall trips, lunch dates, and yoga classes. Thank you for teaching me to let the bad go and for making the hardest days great again.

Lastly, I want to thank my family. The Johnsons: Amy, Kevin, Frances, Andrew, and my nieces and nephew Beatrice, Tabitha, and Alistair: I could not imagine joining a family more supportive and encouraging of my research career. Thank you for welcoming me into your family, and for always being interested in my work.

No words can describe how grateful I am for my father, Greg, my mother, Jenine, and my siblings, Cristina, Elizabeth, Joseph, and my new brother-in-law Brian. God knew I would need strong people to raise me - to watch over me while I found my way, to forgive me when I stumbled, and to teach me how to be better - which is why he gave me to you! Thank you for cheering me on as I pursued my dreams. vii

Thank you for never letting me give up. Thank you for giving me a home to run away to. Thank you for taking my calls, for checking in, and for reminding me that this is just the beginning. I am so blessed.

Lastly, to the love of my life Wendell. I will never forget the gentle presence you’ve been every minute of this journey. You came to Columbus without any reservations and fiercely encouraged me to achieve my dreams. You are my biggest inspiration and my best friend. I am so excited for this beautiful life ahead of us.

viii

Vita

September 25, 1988…………………………..……………….Born—Champagne, IL

2007………..……………………………………….…..Glenbrook South High School

2013……………………………………………….…. B.S. Biology, Suffolk University

2013 to present……………………………………..…Graduate Research Associate Department of Microbiology The Ohio State University

Fellowships

2013-2014…………………………………….The Ohio State University Fellowship The Ohio State University

2014-2015……..The Howard Hughes Medical Institute Med-into-Grad Fellowship The Ohio State University College of Medicine

2016-2018………………………………………………….The C. Glenn Barber Fund The Ohio State University College of Veterinary Medicine

Awards

2016……………………………..The RNA Center Symposium poster award winner The Ohio State University

2016……The College of Veterinary Medicine Research Day poster award winner The Ohio State University

2017……………………..……The Department of Microbiology travel award winner The Ohio State University

2018…...The American Society of Microbiology Ohio Branch poster award winner

ix

Publications

Antonucci JM, St Gelais C, de Silva S, Yount JS, Tang C, Ji X, Shepard C, Xiong Y, Kim B, Wu L “SAMHD1-mediated HIV-1 restriction in cells does not involve ribonuclease activity.” Nature Medicine 2016. 22, 1072-1074

Antonucci JM, St. Gelais C, Wu L. The dynamic interplay between HIV-1, SAMHD1, and the innate antiviral response. Frontiers in Immunology 2017. 8 (1541)

Buzovetsky O, Tang C, Knecht K, Antonucci JM, Wu L, Ji X, Xiong Y. The SAM domain of mouse SAMHD1 is critical for its activation and regulation. Nature Communications 2018. 9 (411)

Chen S, Bonifati S, Qin Z, St. Gelais C, Kodigepalli MK, Barrett BS, Kim SH, Antonucci JM, Ladner KJ, Buzovetsky O, Knecht KM, Xiong Y, Yount JS, Guttridge D, Santiago ML, and Wu L. SAMHD1 suppresses the innate immune responses to viral infections and inflammatory stimuli. Proceedings of the National Academy of Sciences USA 2018. 115 (16): E3798-E3807

Antonucci JM, Kim SH, St Gelais C, Bonifati S, Buzovetsky O, Knecht K, Duchon AA, Xiong Y, Musier-Forsyth K, and Wu L. SAMHD1 suppresses HIV-1 gene expression and reactivation of viral latency in CD4+ T-cells. In revision, Journal of Virology 2018.

Fields of Study

Major Field: Microbiology

x

Table of Contents

Abstract ...... i

Dedication ...... v

Acknowledgments ...... vi

Vita ...... ix

Table of Contents ...... xi

List of Tables ...... xvi

List of Figures ...... xvii

Chapter 1: Introduction ...... 1

1.1 The Discovery of Acquired Immunodeficiency Syndrome (AIDS) and Human

Immunodeficiency Virus (HIV-1) ...... 1

1.2 Pathogenesis ...... 3

1.3 Treatment of HIV and AIDS...... 4

1.4 The Origins and Types of HIV ...... 5

1.5 The HIV-1 Genome ...... 8

1.5.1 Long Terminal Repeats ...... 8

xi

1.5.2 Gag-Pol ...... 10

1.5.3 Env Protein ...... 11

1.5.4 Regulatory ...... 11

1.5.5 Accessory Proteins ...... 12

1.6 HIV-1 Lifecycle ...... 13

1.6.1 Binding and Entry...... 15

1.6.2 Reverse Transcription...... 15

1.6.3 Nuclear Localization and Integration of HIV-1 cDNA...... 18

1.6.4 Proviral Gene Expression ...... 18

1.6.5 Assembly and Maturation ...... 19

1.6.6 Transmission ...... 20

1.7 Innate Immune Response to HIV-1 ...... 21

1.8 Innate Immune Evasion by HIV-1 ...... 24

1.9 HIV-1 Latency ...... 25

1.10 HIV-1 Restriction Factors ...... 28

1.11 Introduction to SAMHD1 ...... 29

1.12 SAMHD1 Dysfunction and Associated Human Diseases ...... 30

1.12.1 Aicardi-Goutières Syndrome ...... 30

1.12.2 LINE-1 Retrotransposition ...... 30

xii

1.12.3 Cancer ...... 31

1.13 SAMHD1 and dNTP Regulation ...... 32

1.14 SAMHD1 Structure and Function ...... 33

1.14.1 Nuclear Localization Signal ...... 33

1.14.2 SAM Domain ...... 34

1.14.3 Histidine/Aspartic Acid (HD) Domain ...... 35

1.14.4 C-terminus ...... 35

1.15 SAMHD1-Mediated HIV-1 Restriction ...... 36

1.15.1 Introduction ...... 36

1.15.2 SAMHD1 and the Cell-Intrinsic Antiviral Response ...... 37

1.15.3 Model Under Scrutiny ...... 40

1.15.4 Regulation by Post-Translational Modifications ...... 41

1.16 SAMHD1 is a Nucleic Acid Binding Protein ...... 42

1.17 Remaining Questions ...... 44

Chapter 2: SAMHD1-Mediated HIV-1 Restriction in Cells Does Not Involve

Ribonuclease Activity ...... 46

2.1 Abstract ...... 46

2.2 Introduction ...... 47

2.3 Materials and Methods ...... 48

xiii

2.4 Results ...... 52

2.5 Discussion ...... 63

2.6 Acknowledgements ...... 67

2.7 Contributions ...... 67

Chapter 3: SAMHD1 Suppresses LTR-Driven Gene Expression and Reactivation of Viral Latency in CD4+ T-cells ...... 69

3.1 Abstract ...... 69

3.2 Introduction ...... 70

3.3 Materials and Methods ...... 72

3.4 Results ...... 80

3.5 Discussion ...... 101

3.6 Acknowledgements ...... 105

3.7 Contributions ...... 105

Chapter 4: Mutations to mSAMHD1 that Destabilize the SAM-to-HD Domain

Tetramer Interface Abrogate HIV-1 Restriction ...... 107

4.1 Abstract ...... 107

4.2 Introduction ...... 108

4.3 Materials and Methods ...... 110

4.4 Results ...... 111

xiv

4.5 Discussion ...... 119

4.6 Acknowledgements ...... 120

4.7 Contributions ...... 122

Chapter 5: Summary and Future Directions ...... 123

5.1 Summary ...... 123

5.2 Future Directions ...... 128

Bibliography ...... 132

xv

List of Tables

Table 1: PCR primer sequences...... 100

Table 2: Sequences of oligonucleotides used in anisotropy binding assays. ... 101

Table 3: Characterization of human and mouse SAMHD1 mutants and the predicted effect on HIV-1 restriction based on crystal structure data...... 115

xvi

List of Figures

Figure 1: The typical course of HIV infection...... 4

Figure 2: HIV-1 groups and subtypes ...... 7

Figure 3: Schematic of the HIV-1 genome...... 8

Figure 4: The HIV-1 5’ LTR with transcription factor binding sites...... 10

Figure 5: Summary of the HIV-1 replication cycle...... 14

Figure 6: The process of reverse transcription...... 17

Figure 7: Schematic of a mature HIV-1 virion...... 20

Figure 8: Innate immune sensing of HIV-1 DNA and cellular responses...... 23

Figure 9: Tetramerization of SAMHD1 in cells...... 33

Figure 10: Schematic of SAMHD1 and its domains...... 36

Figure 11: SAMHD1 suppresses innate immune sensing of HIV-1 DNA...... 39

Figure 12: Structure-based control of SAMHD1-mediated HIV-1 restriction...... 43

Figure 13: Overexpression of SAMHD1 in HEK293T cells does not reduce HIV-1 Gag protein synthesis and viral particle release...... 54

xvii

Figure 14: Flow cytometry raw data...... 56

Figure 15: SAMHD1 restricts HIV-1, not SeV and IAV, in PMA-differentiated U937 cells...... 57

Figure 16: Both D137N and Q548A mutants of SAMHD1 restrict HIV-1 infection in PMA-differentiated U937 cells by decreasing viral cDNA synthesis, but not viral genomic RNA...... 60

Figure 17: Characterization of SAMHD1 enzymatic activities in vitro...... 62

Figure 18: Expression of SAMHD1 WT and mutants by different constructs in PMA- differentiated U937 cells...... 66

Figure 19: SAMHD1 suppresses HIV-1 LTR-driven luciferase expression...... 82

Figure 20: SAMHD1 suppresses gene expression driven by the LTR from HIV-1 and HTLV-1, but not from MLV...... 85

Figure 21: Nonphosphorylated and dNTPase-inactive SAMHD1 mutants have impaired suppression of HIV-1 LTR activity...... 88

Figure 22: WT SAMHD1 impairs HIV-1 reactivation in latently infected J-Lat cells...... 92

Figure 23: WT SAMHD1 binds to proviral DNA in latently infected J-Lat cells. .. 95

Figure 24: Specific binding of WT SAMHD1 to an HIV-1 LTR fragment in vitro . 97

Figure 25: SAMHD1 impairs HIV-1 reactivation in latently infected primary CD4+ T-cells...... 99

Figure 26: The HD domain of mSAMHD1 is insufficient for dNTPase activity. . 112

xviii

Figure 27: Crystal structure indicating the transparent surface representation of SAM-to-HD interface...... 114

Figure 28: Mutations destabilizing the SAM-HD inter- and intra-subunit interaction result in abrogated HIV-1 restriction by mSAMHD1...... 117

Figure 29: Mutations stabilizing the SAM-HD interaction enhance hSAMHD1 enzyme function...... 118

xix

Chapter 1: Introduction

1.1 The Discovery of Acquired Immunodeficiency Syndrome (AIDS) and

Human Immunodeficiency Virus (HIV-1)

The first official reporting of what would become known as acquired immune deficiency syndrome (AIDS) came in the form of a 1981 U.S. Center for Disease

Control and Prevention (CDC) Morbidity and Mortality Weekly Report describing five cases of rare Pneumocystis carinii pneumonia lung infection in previously healthy gay men in Los Angeles1. The men presented with additional unique and unusual infection, including candidiasis and cytomegalovirus (CMV) retinitis. The prevalence of opportunistic diseases suggested that immune system dysfunction was taking place1. The disease was so catastrophic that two of the five men had died before the report was published. Once published, the CDC began to receive reports of similar cases, including reports of the extremely rare and aggressive

Kaposi’s Sarcoma, in New York and California. By the end of 1981, 45% of the

270 reported cases resulted in death2. The term “gay cancer” had been attributed to this outbreak in opportunistic infections, influencing both the public opinion and medical profession to believe the epidemic was only concerned with gay men3,4.

This misattribution had fatal consequences for all other populations, most especially hemophiliacs5,6.

1

The CDC used the term AIDS for the first time while releasing the first case definition of AIDS7. As funding through the CDC and the National Institutes of

Health began to pour into laboratories in the United States, uncovering the cause of AIDS became the biggest target for research8. Although originally identified as

Lymphadenopathy associated virus (LAV)9 and then Human T-cell Lymphotropic

Virus 3 (HTLV-III)10, the retrovirus soon to be known as Human immunodeficiency virus type 1 (HIV-1) was first isolated from a patient suffering from acquired immune deficiency syndrome (AIDS) and identified as the causative agent of the deadly AIDS epidemic in 19849,10 by Dr. Luc Montagnier at the Pasteur institute in

Paris and Dr. Robert Gallo of the National Cancer Institute in the United States. It took an additional three years for the Food and Drug Administration (FDA) to approve the first antiretroviral therapy (ART) zidovudine (AZT), followed by the first immunoblot (IB) based blood test kit for HIV-1 infection11,12. Today, 35 million people worldwide have died from HIV-1 infection13.

According to the 2016 UNAIDS global AIDS update, there are currently 36.7 million people living with HIV-1 infection worldwide, with over 1.5 million deaths per year being associated with AIDS13. Although the total number of people living with HIV-

1 world-wide has increased since its discovery, the yearly global rates of infection have decreased from 3.2 million in 2000 to 2.1 million in 2016, with 66% of new infections occurring in sub-Saharan Africa13. Additionally, the number of HIV positive patients on ART has increased from less than 1 million in 2000 to 18.2 2

million in 2016, with the number of pills needed per day to treat HIV dropping from

8 to just 113. Yearly AIDS related deaths have also decreased by 400,000 since

2010, with the goal being complete prevention of AIDS-related deaths by 203013.

The difficulty in identifying and treating HIV-1 infection stems from the complex viral lifecycle which allows for sophisticated immune evasion and high rates of mutation14,15. Despite 30 years of research dedicated to HIV/AIDS, there is still no vaccine or cure16.

1.2 Pathogenesis

Without therapeutic intervention, HIV-1 infection leads to progressive depletion of the immune system in three phases17 (Figure 1). Acute infection lasts approximately 3-5 weeks and is characterized by high plasma viremia and a sharp decrease in CD4+ T lymphocytes18. Clinical latency may last up to 10 years and is characterized by a decrease in peripheral viremia coupled with a steady decline in

CD4+ T lymphocytes18. The clinically apparent disease phase can last up to three years and is characterized by a profound reoccurrence of plasma viremia and a complete depletion of CD4+ T lymphocytes18. The depletion of CD4+ T cells results in a rapid decline in immunity19 which allows for the development of opportunistic infections20, a condition known as AIDS. Without proper treatment, patients die during this phase of infection17.

3

Figure 1: The typical course of HIV infection. The red line follows the decline in CD4+ T lymphocyte levels, while the blue line follows the increase in plasma HIV-1 RNA levels as disease progresses. Adapted from Pantaleo et al.199321. Described in detail in 1.2.

1.3 Treatment of HIV and AIDS

To manage HIV/AIDS, treatment options include the use of combinational highly active antiretroviral therapies (HAART) that targets HIV-1 at various steps in the viral lifecycle22-24. Current drug regiments include the use of chemokine receptor antagonists (CCR5 antagonists), fusion inhibitors (FIs), nucleoside analog inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors

(NNRTIs), integrase inhibitors (INSTIs), and protease inhibitors (PIs). HAART

4

lowers the viral load, allowing for proper immune function in HIV infected individuals. HAART also prevents the development of deadly opportunistic infections caused by AIDS-associated immune depletion25. Without a cure, HIV infected individuals must remain on expensive and side-effect laden drug regiments for the rest of their lives26. Although HAART saved an estimated 700,000 lives in 201025, the side effects during the early treatment era, including hepatotoxicity, hyperglycemia, and osteonecrosis, can become so intolerable that drug treatment is terminated, resulting in reactivation of HIV-1 infection27,28.

Recently, novel approaches to therapy development have focused on the host response to viral infection to identify new and less toxic drug targets29-31.

1.4 The Origins and Types of HIV

Although HIV-1 was first identified as the pathogenic virus causing AIDS, the morphologically similar but antigenically distinct HIV-2 was found to cause AIDS in

West African patients32,33. It was determined that HIV-2 was more closely related to an immunodeficiency-causing virus in captive macaques than to HIV-134. This observation led to the discovery of additional simian immunodeficiency

(SIV) in several monkey species from sub-Saharan Africa35. Further identification of SIV relatives of HIV-1 in chimpanzees36 and HIV-2 in sooty mangabees37 provided the first evidence that HIV-1 had co-emerged in humans and non-human primates by cross-contamination of primate lentiviruses35. Eventually it was determined that HIV-1 and HIV-2 were the result of cross-species zoonotic transfers of viral infection in African primates38. 5

HIV-1 is a member of the family Retroviridae. are divided into the subfamilies and , the latter which includes the family of which HIV-1 belongs to39. Retroviruses are all positive-sense single-stranded RNA viruses that utilize reverse transcription to convert their RNA genome into DNA39. Integration of viral DNA into the host genome is an obligatory replication step for all retroviruses. HIV-1 is a Lentivirus, a family of retrovirus that infects many species of animals, including primates, horses, cows, and rabbits, and is characterized by a conical capsid and long incubation periods40.

HIV-1 can be organized into four groups named M, N, O, and P (Figure 2) based on genetic similarity and geographical distribution. Each of the distinct lineages resulted from a single cross-species transmission event, and each can cause CD4+

T-cell depletion and AIDS. The pandemic form of HIV-1, known as the main (M) group, was the first identified and is responsible for the over 65 million known infections41. Cross-species transmission from chimpanzees has been identified as the cause of HIV-1 Group M42. Group M can be subdivided into nine genetically distinct subtypes, each with a specific geographical distribution, with subtypes B and C being the most widespread (Figure 2). While Subtype B is largely restricted to North America, Australia, and Europe, Subtype C infections are located in Africa,

India, and Nepal43.

6

In 1990, the less prevalent Group O (outlier) was discovered in the surrounding region of Cameroon44,45, with its origin associated to Gorillas46. In 1998, the very rare Group N was also identified in Cameroonian patients47. To date, there are less than 20 confirmed cases of infection with HIV-1 Group N48. Lastly, Group P has only been identified in two Cameroonians, with current phylogenetic data suggesting a Gorilla origin49. While the exact mechanism of human acquisition of

HIV-1 groups M, N, O, and P is still unknown, it is widely accepted that the mandatory exposure through cutaneous or mucosal membrane exposure to infected blood or body fluids likely happened during bush meat hunting in Western

Africa50. Interestingly, Subtypes C, D, and G appear to have higher rates of disease progression and AIDS when compared to subtype A51-54.

Figure 2: HIV-1 groups and subtypes The phylogeny is organized based on genetic similarity and geographical associations. Described in detail in 1.4. CRFs, Circulating recombinant forms

7

1.5 The HIV-1 Genome

The HIV-1 genome is an approximately 10 kilobase (Kb) RNA that is reverse transcribed into DNA during replication (Figure 3). The HIV-1 genome contains 9 that encode 15 proteins39. The major gene products, gag, pol, and env, encode the structural and enzymatic proteins needed for HIV-1 to replicate in cells.

The minor gene products, tat and rev, are essential for transcription and translation of viral RNA. The accessory gene products, vif, vpr, vpu, and nef, are nonessential for replication but can alter viral pathogenesis through interaction with cellular factors39 (Figure 3).

tat nef rev vpu 5’ LTR gag vif 3’ LTR

pol vpr env

Kb 1 2 3 4 5 6 7 8 9

Figure 3: Schematic of the HIV-1 genome. The HIV-1 genome is depicted with major gene coding sequences indicated. Kilobase (Kb) are labeled below the genome. Described in detail in 1.5.

1.5.1 Long Terminal Repeats

Long terminal repeats (LTR) are identical DNA sequences, located at both the 5’ and 3’ ends of proviral DNA, that are a formed as a byproduct of reverse

8

transcription18 (Figure 3, Figure 4, Figure 6). The LTR serves as a meeting place for both viral and host transcription factors and machinery55,56 (Figure 4). HIV-1 utilizes LTR-specific integrases to insert the proviral DNA into the host chromosome57,58. Although the integrated proviral LTRs are identical, the 5’ LTR is used as a promoter while the 3’ LTR has a role in the polyadenylation of newly transcribed viral RNAs59. Retroviral LTRs are segmented into U3, R, and U5 regions on both the 5’ and 3’ end60 (Figure 4). Most important for transcriptional control, the 5’ U3R region is broken into the modulatory, enhancer, and core regions. The modulatory region consists of protein binding sites whose activity determine the activation or repression of the enhancer and core regions61. The enhancer region contains a variety of transcription factor binding sites while the core region includes several GC-rich binding sites for Sp transcription factors56,62.

Importantly, the trans-activation response element (TAR) serves as a binding site for the viral protein Tat and allows for transactivation of the LTR promoter62,63.

Research has shown NF-B induced LTR activation occurs at levels comparable to those of Tat, indicating transcription factor binding can greatly affect LTR activation or repression64.

9

Figure 4: The HIV-1 5’ LTR with transcription factor binding sites. The 5’ LTR of the HIV-1 proviral genome is pictured, with several transcription factor binding sites identified by colorful shapes. Basepair (bp) locations are labeled above. Figure adapted from Fisher-Smith and Rappaport, 200665 and Li et al. 201266. Described in detail in 1.5.1.

1.5.2 Gag-Pol

The gag gene allows for translation of a 55 kDa precursor polyprotein p5567. Post translation, p55 is myristoylated at its N-terminus, thereby initiating its association with cytoplasmic cell membranes67,68. The Gag protein is essential for viral assembly, as cytoplasmic Gag polyprotein recruits and assembles two copies of

HIV genomic RNA with other viral proteins, thus triggering the budding of the newly synthesized viral particles from the surface of the infected cell67,69. After budding, p55 is processed by the viral protease into p24 capsid (CA), p17 matrix (MA), p7. nucleocapsid (NC), and p6 proteins67. The gag-pol genomic region encodes the

HIV , including reverse transcriptase (RT), integrase (IN), and protease

(Pro), and is produced by ribosome frameshifting near the 3’ end of gag67,70,71. This frameshift occurs in 5% of transcription, and results in a Gag/Gag-Pol ratio of approximately 20:170. During maturation, the viral protease cleaves the Pol protein

10

off Gag and digests it further into protease p10, RT p50, RNase H p15, and integrase p3167,72,73.

1.5.3 Env Protein

The env genomic region encodes a large precursor glycoprotein gp160, which when processed, produces the external glycoprotein gp120 and the transmembrane glycoprotein gp4174. After processing and during viral assembly, the gp120/gp41 protein complex associate as a trimer on the cell surface, with gp120 containing the primary and secondary receptor binding sites, making it an attractive target for therapeutics75-77.

1.5.4 Regulatory Proteins

The trans-activator of transcription (Tat) protein is the HIV-1 regulatory factor responsible for transactivation of HIV gene expression63,78. Both the major and minor forms of Tat are expressed at low levels in persistently infected cells, with a primarily nuclear localization. Tat binding to the trans-activation response element

(TAR) stem loop activates initiation of transcription and enhances elongation from the LTR promoter63,79. LTR-driven gene expression in the absence of Tat produces short RNA fragments caused by premature termination of transcription.

Additionally, Tat is released and taken up by cells in culture80.

The regulator of expression of virion proteins, or Rev, is a shuttling phosphoprotein essential for nuclear export of viral mRNAs63,79,81. Binding to the rev response 11

element (RRE) allows for the stabilization and export of mRNA by Rev. Removal of Rev or the RRE prevents translation of viral mRNAs and blocks productive infection by HIV79.

1.5.5 Accessory Proteins

Accessory proteins are so named as they are not required for virus replication in some cell types82. Viral infectivity factor (Vif) enhances infectivity by counteracting the cellular restriction factor APOBEC3, a cytidine deaminase which cause G-to-A hypermutations that suppress viral replication83,84.

Viral protein R (Vpr) is essential for efficient HIV-1 replication in CD4+ T-cells and macrophages85. Vpr is incorporated into newly synthesized virions and facilitates the nuclear translocation of pre-integration complexes85,86. Vpr is involved in activating LTR-driven transcription87, and has been identified as a putative player in the production of the HIV-1 latent reservoir in and latently infected

T-cell lines88,89. Additionally, cell cycle disruption and T-cell dysfunction have all been associated with Vpr expression90,91.

Viral protein U (Vpu) is a type I integral membrane protein92,93 which both enhances release of newly synthesized HIV-1 virions from the cell membrane94 and degrades CD4 in the endoplasmic reticulum95,96. Interestingly, Vpu antagonizes the (IFN)--induced glycoprotein bone-marrow stromal cell

12

antigen 2 (BST-2/tetherin) that is responsible for inhibiting the release of enveloped virus, including HIV-1 and Ebola virus (EBOV)97,98.

The negative regulatory factor (Nef) protein is a myristoylated protein that associates with the plasma membrane through its N-terminal myristoylated glycine residue and increases viral infectivity99. Nef is translated early in infection100 and is the most immunogenic of the HIV accessory proteins101,102. Although Nef is not required for HIV-1 replication in cultured cells, it greatly enhances pathogenicity and disease progression in infected hosts103,104. Additionally, Nef downregulates

MHC class I molecules and CD4105,106 and enhances particle infectivity through antagonism of the host restriction factor serine incorporator 3 and 5

(SERINC3/5)107-109. Defective nef genes have been identified in primary HIV-1 isolates from long-term nonprogressors, suggesting Nef expression is essential for disease progression in patients110.

1.6 HIV-1 Lifecycle

HIV-1 is a retrovirus which replicates intracellularly via a well characterized life cycle (Figure 5). Understanding details of the replication cycle of HIV-1 allows researchers to develop strategies for therapeutics. Additionally, an extensive knowledge of how HIV-1 replicates allows for the discovery of novel interactions between HIV-1 and host factors.

13

7. Maturation of HIV-1 newly synthesized 1. Binding of HIV-1 virion to the host cell surface and fusion of viral and cellular 6. Assembly at membranes cellular plasma membrane Y

5. Synthesis of gRNA viral proteins gRNA

mRNA 2. Reverse transcription gRNA of genomic cDNA RNA to cDNA vRNA

dsDNA 3. Nuclear 4. Transcription of mRNA and import and full-length genomic RNA integration

Nucleus Cytoplasm

Figure 5: Summary of the HIV-1 replication cycle. HIV replicates inside host cells via a very distinct and well characterized viral lifecycle. Inhibition at any step in this viral lifecycle leads to downstream affects that result in inefficient replication and restriction of infection. HIV-1 binds to the primary CD4 receptor and a co-receptor, including CCR5 or CXCR4, to mediate fusion between viral and cellular membranes. Once inside the host cell, HIV-1 must reverse transcribe its single-stranded RNA to double stranded cDNA through the incorporation of intracellular dNTP’s and reverse transcriptase. After uncoating and reverse transcription, viral cDNA is integrated into the host chromatin, where it can be transcribed via host polymerases into viral genomic RNA or mRNA. Viral mRNA is then translated into proteins which are assembled with the unspliced viral genomic RNA into the budding virion. Described in detail in 1.6.

14

1.6.1 Binding and Entry

HIV-1 is a retrovirus that infects immune cells, such as CD4+ T-cells, macrophages, and dendritic cells (DCs)18,111. Viral tropism is determined by specific interactions between the HIV-1 surface glycoprotein, gp120, and cellular receptors112. Virions which associate with CXCR4 are T-topic113, while virions that utilize the CCR5 receptor are classified as M-tropic113. The HIV lifecycle begins with the virus binding to the target cell as mediated by the protein and host cell membrane proteins (Figure 5 step 1). Binding of gp120 to the primary CD4 receptor and secondary cellular receptors (either CXCR4 or CCR5) mediates fusion with the cell membrane76,114. Fusion allows the HIV-1 viral genome to enter the cytoplasm where it begins the uncoating process while reverse transcription is initiated39,115.

1.6.2 Reverse Transcription

Following membrane fusion and release of the viral core into the cytoplasm, the single-stranded RNA (ssRNA) genome of HIV-1 is reverse transcribed into double- stranded cDNA115 (Figure 5 step 2, Figure 6). This process is carried out by the

HIV-1 protein RT, which maintains RNA-dependent and DNA-dependent polymerase activities, and which incorporates host deoxynucleoside triphosphates

(dNTPs) into the cDNA product60. RT processivity is directly affected by changes in intracellular dNTP pools. For example, dNTP depletion by hydroxyurea prevents

HIV-1 late RT product synthesis while the addition of exogenous dNTPs increases cDNA synthesis116,117. 15

Reverse transcription is a stepwise process39, as shown in Figure 6, that begins with the binding of the host tRNALys3 to the primer binding site (PBS). This binding initiates the synthesis of of the minus strong-stop DNA by DNA polymerase.

RNAse H then degrades the 5’ viral RNA strand, thereby releasing the newly synthesized DNA fragment which then binds the 3’ complementary R region. This is the first strand transfer. Reverse transcription leads to complete elongation of the first strand of DNA from the RNA template. RNAse H will degrade the template

RNA, leaving only the protected polypurine tracts (PPT). Next, the PP will prime the reverse transcription of the second strand of DNA. This newly synthesized strong-stop DNA is released by further degradation of the PPT and tRNAlys, before binding the complementary PBS on the 3’ end of the first DNA strand. This is the second strand transfer. Both strands elongate into full-length complimentary double-stranded viral cDNA39,118.

16

Figure 6: The process of reverse transcription. Incoming viral genomic RNA (vRNA, darker color) and newly synthesized cDNA (lighter color) are labeled along with the steps of reverse transcription. Described in detail in 1.6.2.

17

1.6.3 Nuclear Localization and Integration of HIV-1 cDNA

The HIV-1 cDNA interacts with several host proteins, forming the pre-integration complex (PIC) which is then imported into the nucleus119 (Figure 5 step 3). The

HIV-1 CA protein is hypothesized to aid in nuclear translocation of the PIC in non- cycling cells120-122. The viral protein integrase allows for the covalent insertion of the proviral cDNA into the host chromosome58, likely into actively transcribed genic regions where RNA polymerase II (RNAPII) can initiate transcription of HIV transcripts123,124.

1.6.4 Proviral Gene Expression

Using the integrated proviral DNA as a template, host proteins transcribe viral messenger (mRNA) and genomic RNAs (gRNA) which will then be exported out of the nucleus for translation of proteins and packaging into newly formed virions, respectively18 (Figure 5 steps 4-6).Transcription of integrated provirus occurs in two phases: Tat-independent and Tat-dependent125-128. Initially, the low availability of Tat proteins allows for low transcriptional activity of proviral DNA.

This initial transcription allows for the translation of mRNA encoding Tat protein and the enhancement of transcription through the Tat-TAR binding and recruitment of the positive elongation factor (P-TEFb) complex, with subsequent enhanced processivity of RNAP II63,64,126,127. Splicing of newly transcribed full-length mRNA allows for the production of all 15 viral proteins. Unspliced or singly spliced mRNA

(env, pol, gag) is exported by Rev into the cytoplasm79. Multiply-spliced mRNA

(nef, rev, tat) leave the nucleus independent of Rev. The Gag/pol polyprotein is 18

translated in the cytoplasm, while Env proteins are produced in the endoplasmic reticulum and are glycosylated39,129.

1.6.5 Assembly and Maturation

The newly synthesized HIV proteins will assemble at the cellular plasma membrane (Figure 5 step 6). Budding of the immature virion occurs and is dependent on the endosomal sorting complex required for transport protein130.

Maturation occurs post proteolytic processing of viral polyproteins (Figure 5 step

7). The HIV-1 protease is an aspartyl protease131 that is essential for cleavage of

Gag and Gag-Pol precursors. Once proteolytic processing is complete, the mature virion is infectious (Figure 7). Knowledge of the structure and function of viral protease has allowed for the development of a class of drugs targeting its function132,133.

19

Figure 7: Schematic of a mature HIV-1 virion. The virion takes on its mature form after proteolytic processing by the HIV-1 aspartyl protease. HIV-1 proteins are labeled, as is the genomic RNA. Described in 1.6.5.

1.6.6 Transmission

A primarily sexually-transmitted disease, HIV-1 also spreads percutaneously and perinatally134. Horizontal transmission occurs through unprotected sex, needle sticks, and blood transfusions135, while vertical transmission occurs through mother-to-child transmission136. HIV-1 primarily infects through exposure at mucosal surfaces137. Subtype characteristics can also affect co-receptor usage and subsequent transmissibility. For example, viruses that enter the cell through binding the co-receptor CCR5 (R5) have a higher rate of transmission than CXCR4 viruses138. Additionally, it has been suggested that subtype C viruses have greater transmission rates, both vertically and horizontally, likely due to the prevalence of

R5 HIV-1139-142.

20

1.7 Innate Immune Response to HIV-1

Innate immunity is the cell-intrinsic defense mechanism that senses incoming pathogens and is characterized by type-I interferon (IFN-I) induction and the release of inflammatory cytokines that upregulate antiviral IFN-stimulated genes

(ISGs)143,144. The activation of the innate response to pathogens is dependent on cellular pattern recognition receptors (PRRs) that detect pathogen associated molecular patterns (PAMPs), including viral structures or nucleic acids.

Recognition of PAMPs results in induction of IFN-I and ISGs to control initial infection and spread, while the concomitant induction of the inflammatory response and cytokines can initiate adaptive immune responses145,146. Modulation of IFN-I activation is essential for viral clearance. However, overstimulation of IFN pathways can lead to inflammatory autoimmune disease147.

HIV-1 offers a variety of PAMPs for immune recognition, including ssRNA with a

5’ ppp, ssDNA produced as an intermediate during RT, and structural proteins148,149. After a PAMP has been identified, the innate immune system triggers mechanisms which activate genes responsible for bringing the defending cell into an anti-viral state150,151. Although HIV-1 can be sensed by the innate immune system, the prevailing theory is that HIV-1 avoids immune surveillance through poor replication in immune cells causing ineffective triggering of innate cytosolic sensors152. Several studies have identified the molecular basis of cytosolic sensors important for targeting viral pathogens (Figure 8). After sensing viral nucleic acids, cyclic guanosine monophosphate-adenosine monophosphate 21

(GMP-AMP) synthase (cGAS) generates the second messenger, cyclic guanosine monophosphate-adenosine monophosphate153-155, that activates the stimulator of

IFN genes (STING)146. STING activation leads to phosphorylation of TANK binding kinase 1 (TBK1) and the subsequent phosphorylation and dimerization of interferon-regulatory transcription factors IRF3 and IRF7. Nuclear translocation of the IRF3/IRF7 homo-or-hetero dimers will activate IFN-I gene expression. This signaling cascade results in an upregulation of IFN-I and ISGs as a defense against viral infection156,157 (Figure 8). Reverse transcribed HIV-1 DNA was identified as the trigger to the cGAS-STING pathway158. Although cGAS is the primary sensor of cytosolic viral DNA, IFI16 can also act as a sensor of HIV-1 single-stranded DNA that induces an IFN-β response in macrophages by a cGAS-

STING-dependent pathway159.

22

HIV-1

Immune activation Induction of antiviral state

Y

gRNA gRNA

mRNA

gRNA cDNA

vRNA IFI16

dsDNA Type I IFN cGAS P IRF3 P IRF7 IRF3 P IRF7

STING TBK1 P

IRF7 IRF3 Nucleus Cytoplasm

Figure 8: Innate immune sensing of HIV-1 DNA and cellular responses. HIV-1 undergoes uncoating through the interaction between viral capsid and host factors160,161. Reverse transcribed HIV-1 DNA, mainly abortive transcripts, activates cytosolic DNA sensors IFI16 and cGAS resulting in TBK1-mediated phosphorylation and nuclear translocation of hetero-or-homo dimers of IRF3 and IRF7 and induction of type-I IFN response. Expression of ISGs allows for immune activation and the induction of an antiviral state of the cell. gRNA, HIV-1 genomic RNA; cDNA, complementary DNA; vRNA, viral RNA; dsDNA, double-stranded DNA; STING, stimulator of IFN genes; the letter P indicates phosphorylation. Adapted from Antonucci, et al.2017162. Described in detail in 1.7.

23

1.8 Innate Immune Evasion by HIV-1

HIV-1 is sensitive to ISGs and the IFN-induced antiviral response; so it is not surprising that HIV-1 is a poor inducer of IFN163. HIV-1 benefits from evading innate immune activation and utilizes a variety of tactics to escape detection110,164.

Professional antigen-presenting cells located in mucosal tissues, including DCs and macrophages, are critical for recognizing HIV-1 at the site of initial exposure.

However, these cells are less permissive to HIV-1 infection compared to activated

CD4+ T-cells, mainly due to host restriction factors that control the establishment or spread of viral infection. Several host proteins can restrict HIV-1 at various points in the viral lifecycle, including APOBEC proteins, tripartite motif-containing protein 5 (TRIM5a), SAMHD1, and tetherin165-168. However, HIV-1 can exploit innate immune cells and their cellular factors to avoid detection and clearance by the host immune system169.

Although cytosolic HIV-1 DNA is abundant in permissive cells, including activated

CD4+ T-cells, a cell-autonomous IFN response is not triggered170. This is due at least in part to host protein TREX1. As a single-stranded DNA , TREX-

1 digests cytoplasmic DNA from retroviral DNA intermediates, thereby preventing the activation of mislocalized DNA by an innate immune sensor170. Cytosolic HIV-

1 DNA is accumulated in HIV-1 infected TREX1-deficient CD4+ T-cells and macrophages, which leads to inhibition of TBK1-dependent IFN-I response170. This suggests a competition between two DNA sensors: cGAS leading to antiviral effects, and TREX1 leading to enhanced viral replication171. 24

Two models suggest that, in MDDCs, HIV-1 attempts to hide its genomic RNA and newly synthesized cDNA from cytosolic sensors by obstructing the nucleic acids using viral capsid. The models differ with respect to the effect of recruitment of cellular cyclophillins and cleavage and polyadenylation-specific factor 6 by capsid.

One model suggests increased cyclophillin A binding to the capsid increases sensitivity to innate sensing172, while another proposes CypA binding coordinates uncoating, reverse transcription, and nuclear import of the pre-integration complex173, all to minimize the exposure of viral nucleic acids to cytosolic sensors.

Future work is needed to clarify the contribution of CypA and other host factors to the negative regulation of the innate immune response in myeloid cells to provide insight into HIV-1 mechanisms of evasion.

1.9 HIV-1 Latency

Alternatively, integrated proviral DNA may become transcriptionally silenced by a variety of mechanisms, including chromatin structure, transcriptional interference, and sequestration of transcription factors, leading to the formation of a latent reservoir that is hidden from the host immune system26,149. Latent proviral DNA may become reactivated at a later time, once again allowing for productive infection27, although these cells eventually die due to cytopathic effects of the virus, clearance by the host immune system, or by a reduced half-life174,175.

Macrophages, follicular dendritic cells, and latently infected memory T cells have been identified as possible reservoirs of latent infection176. 25

In Chapter 3, we will discuss contributions to viral latency in memory CD4+ T-cells, which are produced after infected effector T-cells revert back into resting cells which contain transcriptionally silent proviral DNA that can be reactivated upon stimulation177-179. Naïve T-cells are resting cells that are produced in the bone marrow, educated in the thymus, and circulate throughout the blood stream and lymphatic system awaiting activation by foreign antigens. Naïve T-cells are not efficiently infected by HIV-1180. Naïve T-cells are activated by a stimulation event in three parts: First, an antigen presenting cell’s MHC II molecule binds the T-cell receptor181. Next, the antigen presenting cell’s CD28 co-stimulatory molecule binds CD80/CD86 on the Naïve T-cell. Lastly, the paired stimulation of TCR and

CD80/CD86 activate IL-2 and B-cell lymphoma 2 protein, thereby inducing activation of the effector T-cell and subsequent cellular proliferation181. These effector T-cells will either die or return to a quiescent state, thereby become resting memory T-cells182,183. Activated T-cells are highly susceptible to HIV-1 infection and typically die upon infection due to cytopathic effects or immune clearance184-

186. However, an infected activated T-cell may revert back to a quiescent state, becoming a long-lived resting memory T-cell that contains an integrated provirus26,184. These latently infected memory T-cells can be reactivated with stimulation, and once activated may produce infectious virus27,175,187-190.

Latency is maintained by several mechanisms, including epigenetic modifications of chromatin structure by methylation, acetylation, phosphorylation, and 26

ubiquitination191-195, and transcriptional control of the HIV-1 LTR promoter78,196.

HIV-1 proviral DNA typically integrates into regions of DNA that are actively transcribed, with accessible chromatin being essential for viral gene expression123,124,197-199. Post-translational modifications to chromatin negatively regulate HIV-1 gene expression and can lead to genetic silencing and latency195,200-204. Transcriptional interference occurs in different forms and include disruption of Pol II engagement, occlusion of transcription factors, and even collision due to proviral DNA and host genes existing in opposite polarity174.

HAART is able to substantially decrease peripheral viremia to levels below detection. However, patients treated and then removed from long-term HAART therapy still produce infectious virus, indicating the existence of latently-infected cell reservoirs within the body26,205. It was hypothesized that HAART therapy combined with reactivation of latently infected cells could lead to a purge of viral reservoirs; clearance of latently infected cells with protection from further infection by antiretroviral therapy could effectively cure HIV-1 infection24,206-208. However, clinical success of this “shock and kill” method has not been achieved24,206-210.

Discerning the mechanism of LTR regulation will lead to a greater understanding of HIV-1 latency and will contribute to the development of therapies targeted specifically at latently infected cells211.

27

1.10 HIV-1 Restriction Factors

One category of innate immune regulators includes the HIV-1 restriction factor

(RF)212. The human immune system contains RF proteins that interact with and inhibit viral replication at distinct phases of the viral life cycle212. RFs are cellular proteins that significantly decrease HIV-1 infectivity, therefore HIV-1 often evolves an equally strong mechanism of counter-restriction213. RFs typically express signs of rapid evolution to counteract viral antagonists214. Furthermore, RF expression is induced through innate immune signaling215-218. Several known HIV-1 restriction factors include the APOBEC family proteins APOBEC3F and APOBEC3G219,

TRIM5220,221, tetherin222,223, SERINC5224, and SAMHD1225,226. The APOBEC3F and APOBEC3G DNA cytosine deaminases inhibit HIV-1 infection through converting cytosines to uracils on the retroviral minus-strand cDNA. These high frequency C→U conversions result in lethal G→A hypermutations in the plus- strand219,227. HIV-1 counteracts APOBEC-mediated restriction through Vif, which facilitates proteasomal degradation of APOBEC3 proteins219. The tripartite motif

TRIM5 restricts HIV-1 through interaction with the viral capsid. Recognition of capsid protein motifs and binding to the capsid lattice allows TRIM5 to interfere with the uncoating processes220,221. Tetherin, is a type II single-pass transmembrane protein that “tethers” mature virions to the cell surface, thereby inhibiting viral release222,223. The HIV-1 protein Vpu antagonizes TRIM5- mediated restriction by reducing levels of TRIM5206. The serine incorporator

SERINC5 is included into newly synthesized HIV-1 virions and interferes with viral

28

entry224. HIV-1 protein Nef antagonized this activity by reducing the incorporation of SERINC5 into virions by targeting SERINC5 to the endosome and lysosome system by receptor-mediated endocytosis108,228. This dissertation focuses on

SAMHD1-mediated restriction of HIV-1 infection, thus SAMHD1 will be described in great detail beginning in 1.11.

1.11 Introduction to SAMHD1

SAMHD1 is an IFN-inducible host protein capable of blocking replication of retroviruses and several DNA viruses168,229-232. SAMHD1 is a dNTPase233,234 that converts dNTPs into the constituent deoxynucleoside and inorganic triphosphate upon stimulation by dGTP or GTP233-235 (Figure 9). SAMHD1 is constituently expressed at various levels in all cell types and highly expressed in myeloid lineage and resting CD4+ T-cells168,229,236. IFN-I treatment increases SAMHD1 expression in certain cell types with low endogenous SAMHD1 levels237,238. SAMHD1 has been implicated as a negative regulator of the type I interferon (IFN-I) inflammatory response239-241, however the underlying mechanism is not fully understood. While

HIV-2 encodes the SAMHD1 antagonist , the more pathogenic HIV-1 does not.

It was hypothesized that HIV-1 lacks a countermeasure against SAMHD1 because it may be beneficial for infection. Although there is no evidence concluding

SAMHD1 confers a benefit to HIV-1 infection, we discuss this question further in

1.15.2.

29

1.12 SAMHD1 Dysfunction and Associated Human Diseases

1.12.1 Aicardi-Goutières Syndrome

Mutations in SAMHD1 that affect its enzyme activity are associated with Aicardi-

Goutières syndrome (AGS), an encephalopathic autoimmune disease characterized by symptoms mimicking chronic viral infection239. The accumulation of intracellular dNTPs caused by mutations in the genes encoding proteins involved in nucleic acid metabolism, including SAMHD1 and TREX1242, are sensed by PRRs, resulting in aberrant production of IFN-I243. AGS patients present with increased production of IFN-ɑ, the chemokine most characteristic of congenital virus infection. AGS patients with SAMHD1 mutations can present with signs of erythematosus, with many symptoms mimicking those of HIV-1 infection239,244. Furthermore, cells isolated from AGS patients with homozygous

SAMHD1 mutations revealed that SAMHD1-deficient monocytes supported productive infection by HIV-1237, suggesting a link between SAMHD1 function in both autoimmunity and HIV-1 restriction.

1.12.2 LINE-1 Retrotransposition

Long interspersed element 1 (LINE-1) is the only autonomous and active human retroelement capable of producing new genomic insertions through its endogenous and reverse transcriptase activities245,246. A study on

AGS-related SAMHD1 mutations indicate that all disease-related mutations reduced LINE-1 inhibition in dividing cells247. Recent work suggests that SAMHD1 potently blocks LINE-1 transposition in cycling cells by triggering the sequestration 30

of LINE-1 ORF1p into stress granules248. Impaired inhibition of LINE-1 retrotransposition may lead to triggering of the autoimmune response by stimulating toll-like receptors (TLR)249, although this has not been confirmed.

Impaired dNTPase activity and LINE-1 suppression by mutant SAMHD1 could explain the chronic inflammatory response characteristics of AGS disease. These studies outlining the pathogenic effect of SAMHD1 deficiency on autoimmune disease implicate SAMHD1 as a negative regulator of the innate immune system.

1.12.3 Cancer

The level of intracellular dNTPs changes during the cell cycle, with high levels present during S phase and low levels occurring during the G1/G0 phase.

Predictably, SAMHD1 is expressed highly during the G1/G0 phase, with low expression observed during S phase250,251. Further, the dNTPase activity of

SAMHD1 is modulated through phosphorylation by interacting with cyclin- dependent CDK1, CDK2, and cyclin A2252-254. The SAMHD1-mediated modulation of dNTP pools is tightly associated with cell cycle progression255,256. The intracellular accumulation of dNTPs is associated with insertion errors by DNA polymerases and misaligned intermediates resulting from impaired DNA repair, all of which may lead to tumor development257. Therefore, the suppression of dNTP pools by SAMHD1 was hypothesized to prevent spontaneous mutagenesis and tumor development by enhancing genomic stability251. In support of that hypothesis, SAMHD1 knockout (KO) in THP-1 cells results in enhanced cell proliferation and reduced apoptosis255. 31

Somatic mutations in the SAMHD1 gene have been identified in several cancer types, including lung adenocarcinoma258,259, colon carcinoma260, chronic lymphocytic leukemia (CLL)261,262, and recently in T-cell prolymphocytic leukemia263. Downregulation of SAMHD1 is observed in CLL patients261 and is involved in the DNA damage response261,264,265. Recent work suggests SAMHD1 promotes DNA end resection to facilitate double-strand break (DSB) repair by homologous recombination (HR), as Vpx-mediated degradation of SAMHD1 causes cells to be hypersensitive to chemicals that promote DSB266. This function of SAMHD1 is independent of its dNTPase activity and requires recruitment of the

DNA-end resection factor C-terminal binding protein interacting protein to damaged DNA. Taken together, these studies provide evidence that dysfunctional

SAMHD1 is associated with cancers and plays a role in genome stability251.

1.13 SAMHD1 and dNTP Regulation

SAMHD1 is a dNTPase233,234 that converts dNTPs into the constituent deoxynucleoside and inorganic triphosphate upon stimulation by dGTP or GTP233-

235 (Figure 9). SAMHD1 and ribonuclease reductase, the enzyme responsible for de novo dNTP synthesis through the conversion of ribonucleotide diphosphates to deoxyribonucleotides267, are allosterically regulated to achieve balanced

268 intracellular dNTP levels in a cell-cycle dependent manner . During G1 to S- phase transition in actively proliferating cells, ribonuclease reductase expression increases, leading to expansion of the dNTP pool to facilitate DNA synthesis 256,269. 32

The dNTPase activity of SAMHD1 is activated by high dNTP levels, and degradation of nucleic acids in the absence of DNA replication protects the cell from innate immune activation and cancer development251,270. Structural studies strengthened a model of nucleotide-dependent tetramer assembly of SAMHD1271-

273, where GTP binds to guanine-specific allosteric sites and dNTP binds to non- specific activator sites, initiating the formation of enzymatically active tetramers with the catalytic core of the HD domain233,234,268,274.

Figure 9: Tetramerization of SAMHD1 in cells. SAMHD1 monomers dimerize after allosteric stimulation by GTP or dGTP. Further allosteric stimulation through binding of a dNTP allows for tetramerization and activation of SAMHD1’s dNTPase activity. Described in 1.13 and 1.14.

1.14 SAMHD1 Structure and Function

1.14.1 Nuclear Localization Signal

The nuclear localization signal (NLS) of SAMHD1 is located at residues 11KRPR14 on the N-terminus of SAMHD1275 (Figure 10). Removal of the NLS does not effect

33

the ability of SAMHD1 to hydrolyze dNTPs, although NLS-deficient cytoplasmic

SAMHD1 are resistant to Vpx-mediated degradation275.

1.14.2 SAM Domain

The SAM domain of SAMHD1 is located between residues 44-108 (Figure 10).

This domain is not required for the dNTPase activity of SAMHD1276. In fact, the function of the SAM domain of SAMHD1 has yet to be described272,276. We can gain insight into the potential physiological function of the SAM domain of SAMHD1 by exploring evolutionarily conserved functions of SAM domains. SAM domains are found in a wide variety of eukaryotic proteins and are implicated in regulation of numerous developmental processes277. SAM domains can hetero-oligomerize, homo-oligomerize, and bind RNA277. Through protein-protein and protein-RNA interactions, SAM domains have been shown to mediate transcriptional and translational activation278, kinase activity279, signal transduction278, apoptosis280, and protein localization within the cell281. Tumor suppressor proteins , its homologue p73, and p61 all contain a C-terminal SAM domain that mediate interactions with cellular factors, indicating these domains may be important for protein-protein interactions278,280. SAM-domain containing protein 1 is a human nuclear-localized protein that negatively regulates B-cell activation and down- regulates cell proliferation in vitro282. Taken together, these studies highlight the importance of SAM domains in regulation of transcription factors and indicate a potential role for SAM domain-mediated regulation of tumor suppressors in cancer development. Experimental characterization of the SAM domain of SAMHD1 may 34

uncover a novel function that could be applied to various signal transduction pathways that involve SAM-domain containing proteins278.

1.14.3 Histidine/Aspartic Acid (HD) Domain

The HD domain of SAMHD1 contains the core enzymatic region and is located between residues 115 and 562276 (Figure 10). The HD domain is critical for dGTP- dependent phosphohydrolase activity233,234,272. The enzymatic core of SAMHD1 is located at residues H206 and D207, and mutations to these residues results in abrogated dNTPase activity.

1.14.4 C-terminus

Located between residues 562 and 626, the C-terminal region of SAMHD1 stabilizes the catalytically active tetramer276,283 (Figure 10). This region also contains the phosphorylation residue T592 and a cyclin binding motif252-254,284 at residues 451-453. Certain mutations to the cyclin binding motif disrupts SAMHD1 tetramer formation and abrogates its dNTPase activity both in vitro and in cells284.

The C-terminus also contains the Vpx-interacting domain, where the HIV-2 accessory protein Vpx targets SAMHD1 for degradation285. The interaction between Vpx and SAMHD1 occurs in the nucleus275.

35

Figure 10: Schematic of SAMHD1 and its domains. Human SAMHD1 is a 626 amino acid-long protein containing an N-terminal nuclear localization signal followed by a sterile-alpha motif (SAM) and histidine/aspartic acid (HD) domain. locations are labeled on the gray bar below the protein schematic. Important residues are labeled above schematic. The phosphorylation site T592 is identified in green. The structure and function of SAMHD1 is further described in 1.13, 1.14, and 1.15.4.

1.15 SAMHD1-Mediated HIV-1 Restriction

1.15.1 Introduction

HIV-1 replicates inefficiently in non-diving cells, such as quiescent CD4+ T-cells,

DCs, and monocytes. HIV-1 infection can be enhanced in these cells by Vpx, an accessory protein encoded by HIV-2 and certain lineages of simian immunodeficiency viruses (SIV)286,287. This hinted at the existence of a cellular restriction factor counteracted by Vpx 286. SAMHD1 was identified as the mystery

HIV-1 restriction factor by a mass spectrometry analysis of cellular proteins immunoprecipitated from cells expressing Vpx168,229. Vpx interacts with the C- terminal domain of SAMHD1, thereby initiating proteasomal degradation by an E3 ubiquitin ligase complex, and relieving SAMHD1-mediated lentiviral restriction168,229,288,289.

36

1.15.2 SAMHD1 and the Cell-Intrinsic Antiviral Response

SAMHD1 cDNA was originally identified as an orthologue of the mouse IFN-γ- induced gene Mg11 in human DCs290. A link to the innate immune response was strengthened by the discovery that cytokines, including toll-like agonists and IFNs, can induce SAMHD1 expression291,292. Cell lines treated with IFN-I238,293 and human primary monocytes treated with IFN-ɑ and IFN-γ237,291,294 show enhanced expression of SAMHD1. While SAMHD1 is highly expressed in monocyte-derived macrophages (MDMs), monocyte-derived dendritic cells (MDDCs), and primary

CD4+ T-cells, IFN treatment does not increase SAMHD1 protein levels further238,295-297. However, treatment of MDMs and MDDCs with IFN-I results in reduced phosphorylation of SAMHD1 at residue T592252, indicating a shift from catalytically inactive to active SAMHD1. Interestingly, the SAMHD1 promoter is a direct target of IRF3. The overexpression and activation of IRF3 enhances

SAMHD1 promoter activity in HeLa cells293.

HIV-1 does not trigger a sterilizing immune response298 and is a poor activator of inflammatory pathways163, resulting in an impaired response to HIV-1 and the development of persistent infection. The DC response to HIV-1 infection contributes to this dysfunctional immune response299. Myeloid cells constantly sample the cellular environment to identify pathogens and send out danger signals in the form of IFN-I. DCs are essential for activating the adaptive immune response to infection, as maturation leads to T-cell responses through antigen priming298,300.

Interestingly, HIV-1 infects DCs without activating an effective antiviral response. 37

As SAMHD1 limits HIV-1 cDNA synthesis in myeloid cells168,301, it was hypothesized that degradation of SAMHD1 by Vpx in DCs would result in productive HIV-1 infection and the synthesis of viral proteins that would directly enter antigen presentation, thereby strengthening the T-cell response to infection172. This could be why the vpx gene was lost from the ancestor of HIV-1 during the coevolution of primate SAMHD1 and lentiviruses302. Vpx-mediated degradation of SAMHD1 in DCs leads to enhanced HIV-1 infection, and studies in primary MDMs and MDDCs indicate that Vpx-mediated SAMHD1 degradation results in cGAS stimulation and IRF3 activation158 (Figure 11).

Discovering the mechanisms used by HIV-1 to avoid innate immune sensors is critical for the design of new therapies to eradicate HIV-1 infection. Therapeutic strategies aiming to inhibit host factors that promote HIV-1 replication, and to stimulate the immune response could diminish viral infection and transmission.

Current work aims to determine whether a role exists for drugs targeting SAMHD1.

Expression of SAMHD1 can increase the susceptibility of HIV-1 to nucleoside reverse transcriptase inhibitors by reducing the levels of competitive dNTPs117,303-

305, suggesting modulation of SAMHD1 function may be a means to enhance drug effectiveness. Conversely, as SAMHD1 expression enables immune evasion by

HIV-1169, it is tempting to hypothesize that SAMHD1 could be used as a drug target to enhance the innate immune response to viral infection. However, research is just beginning to uncover mechanisms to modify the dNTPase activity of

SAMHD1306,307. Importantly, as an ISG and a negative regulator of the innate 38

immune system, SAMHD1 may be involved in an unknown negative feedback loop aimed at modulating the complex and delicate system of inflammatory pathways.

HIV-1

Myeloid cell or Y CD4+ T-cell

gRNA No immune activation No Induction of antiviral state dNTPs Low dNTPs

gRNA cDNA

Low IFI16 cDNA yield dNTPs dN+PPP dsDNA cGAS SAMHD1 SAMHD1 SAMHD1 SAMHD1 SAMHD1 SAMHD1

dNTP

GTP dGTP STING TBK1

SAMHD1 IRF7 IRF3

Nucleus Cytoplasm

Figure 11: SAMHD1 suppresses innate immune sensing of HIV-1 DNA. SAMHD1 blocks HIV-1 infection through intracellular dNTP depletion, thus preventing the accumulation of viral DNA accessible to sensing by IFI16 and cGAS and the activation of the IFN-I response. Adapted from Antonucci, et al.2017162. Described in 1.15.2.

39

1.15.3 Model Under Scrutiny

The mechanism and modulation of SAMHD1-mediated HIV-1 restriction is an area of intense scrutiny. Overexpression of SAMHD1 in phorbol 12-myristolate 13- acetate (PMA)-treated monocytic U937 cells results in a depletion of dNTP levels301. PMA is used to differentiate monocytic cells into -like cells, and enhances exogenous expression of CMV-driven SAMHD1 expression in transduced U937 cells. It was later confirmed that SAMHD1 restricts the replication of retroviruses and several DNA viruses by depleting the concentration of intracellular dNTPs to levels insufficient to support viral DNA synthesis168,229-

232,308,309. Although the accepted consensus is that SAMHD1 restricts HIV-1 infection through the depletion of intracellular dNTPs, several studies suggested the existence of an additional yet-undiscovered mechanism of SAMHD1-mediated retroviral restriction. This undefined antiviral activity appears to be dependent on phosphorylation252,310,311 and is not fully dependent on low dNTP levels312.

SAMHD1 acts as a single-stranded nucleic acid binding protein that degrades ssDNA and RNA via a metal-dependent 3’-5’ exonuclease activity in vitro313-315. It has been suggested that SAMHD1 utilizes its nucleic acid binding potential to exert a ribonuclease activity against incoming HIV-1 genomic RNA (gRNA) in a phosphorylation-dependent manner316. SAMHD1 was shown to restrict retroviruses though degradation of HIV-1 RNA in human monocyte-derived macrophages (MDM), monocytes, and CD4+ T-cells316,317. It was proposed that

SAMHD1 degrades incoming HIV-1 genomic RNA, thereby restricting infection and preventing innate immune sensing of viral nucleic acids. However, recent 40

studies have been unable to confirm the controversial findings308,318-320. As a nuclear-localized protein275, incoming viral genomic RNA would be inaccessible by

SAMHD1 for hydrolysis. Additional studies showed that dNTPase inactive

SAMHD1 mutant retained exonuclease activities in vitro, indicating the exonuclease activity could not be attributed to the known dNTP-binding active site318. Seamon et al.318 suggested that the activity attributed to SAMHD1 was due to contamination during purification. Cell-based assays also failed to recapitulate the findings, thereby confirming the lack of SAMHD1 RNase activity to restrict HIV-1 in infected cells308,319. Ryoo et al.suggested that the differences in experimental conditions are responsible for the conflicting results, including a shorter infection time and the use of RNase H-defective reverse transcriptase321.

They further identified SAMHD1 as a phosphorolytic not hydrolytic ribonuclease322, although these findings have not been independently confirmed.

1.15.4 Regulation by Post-Translational Modifications

As SAMHD1 is also highly expressed in activated CD4+ T-cells that support productive infection by HIV-1, several studies demonstrated post-translational modification as a means of mechanistic regulation of SAMHD1 function in restricting HIV-1. SAMHD1 is phosphorylated at several residues, including S278,

S33, T25, T21, and S18, however phosphorylation of threonine 592 was identified as essential for the negative modulation of its HIV-1 restriction activity252,253,310,311,323 and tetramer formation324,325. SAMHD1 is phosphorylated by cyclin-dependent kinase 1 (CDK1) and CDK2 in complex with cell cycle regulatory 41

proteins cyclin A252-254. This regulation of SAMHD1 function is associated with the cell cycle, as CDK1 and cyclin A are highly expressed in dividing cells.

Furthermore, S-phase requires elevated dNTP levels, indicating modulation of the dNTPase activity of SAMHD1 during the cell cycle323. SAMHD1 protein levels may be altered during various stages of the cell cycle depending on cell type250,326.

Interestingly, proliferation-induced oxidation of SAMHD1 by hydrogen peroxide reversibly inhibits its dNTPase activity through the formation of tetramer-inhibiting disulfide bonds327, suggesting a dynamic structure-based regulatory mechanism of SAMHD1’s dNTPase activity that is influenced by the cell cycle (Figure 12).

1.16 SAMHD1 is a Nucleic Acid Binding Protein

SAMHD1 is a single-stranded nucleic acid (ssNA) binding protein313-315, although the function of this binding activity in cells remains unknown. It has been reported that SAMHD1 preferentially binds to ssDNA over dsDNA, and that an increase in

NA secondary structure results in a stronger binding313. The SAM domain is required for maximal nucleic acid binding activity, as the purified SAM domain binds to both ssDNA and ssRNA stronger than the purified isolated HD domain313.

Additionally, mutations to either H206 or D207 abrogated ssDNA binding in vitro

313. The binding of ssNA occurs at the dimer-dimer interface on free monomers and dimers of SAMHD1. This interaction prevents the formation of catalytically active tetramers328 (Figure 12). Furthermore, the addition of dGTP to a binding reaction consisting of ssRNA and SAMHD1 results in diminished NA binding313.

These studies suggest a dynamic mechanism where SAMHD1 may regulate its 42

potent dNTPase activity through NA binding. However, the effect of NA binding on the HIV-1 restriction or other functions of SAMHD1 remains to be explored.

HIV-1

HIV-1 replication is restricted

Y

dNTPs dN+PPP

gRNA SAMHD1 SAMHD1 SAMHD1 SAMHD1 Low SAMHD1 SAMHD1 dNTPs dNTP

gRNA GTP cDNA dGTP

Low SAMHD1 SAMHD1 SAMHD1 SAMHD1 dNTPs

P S S dsDNA Phosphorylation Oxidation ssNA binding

Nucleus

Cytoplasm

Figure 12: Structure-based control of SAMHD1-mediated HIV-1 restriction. The enzymatic activity of SAMHD1 is dependent on its tetramerization. Regulation of tetramer formation thereby leads to regulation of SAMHD1’s potent dNTPase activity. Described in 1.15.4 and 1.16.

43

1.17 Remaining Questions

The existence of a yet-undiscovered mechanism of HIV-1 restriction that is dependent on phosphorylation of SAMHD1 can not be overlooked311. A study examining the effect of SAMHD1 on HIV-1 2-LTR circle formation determined that pretreatment with Vpx enhanced 2-LTR circle numbers in MDMs329, thereby confirming that the decrease in reverse transcription kinetics conferred by

SAMHD1-mediated modulation of dNTP levels negatively regulates the rate of proviral DNA synthesis and integration in non-dividing cells. When transcription is silenced, integrated proviral DNA can lead to latency330. Although SAMHD1 is highly expressed in cells purported to harbor latent provirus26,236 and the HIV-1 proviral promoter is activated by transcription factors331, the effect of SAMHD1 expression on latency development or reversal has not been explored. It is possible that SAMHD1 utilizes its nucleic acid binding ability to restrict HIV-1 infection post-integration, although a study confirmed SAMHD1 does not effect

HIV-1 Gag synthesis, viral particle release, and virus infectivity in human embryonic kidney 293T cells transfected with proviral DNA308. 293T cells are robust virus-producing cells that maintain high dNTP levels and undetectable

SAMHD1 levels. SAMHD1 may exert a direct effect on proviral DNA through binding, as purified recombinant SAMHD1 was shown to bind in vitro transcribed fragments of gag and tat cDNA313, or indirect effects may occur due to SAMHD1 modulation of inflammatory pathways.

44

The identification of SAMHD1 as a dNTPase, a DNA binding protein, and a regulator of the innate immune response to viral infection has led to the development of an exciting field of research. The structural and functional studies of SAMHD1 connect the cellular antiviral response to the physiology of HIV-1 infection. Further work will aid in the development of stratagems to enhance the treatment of HIV infection.

45

Chapter 2: SAMHD1-Mediated HIV-1 Restriction in Cells Does Not Involve

Ribonuclease Activity

2.1 Abstract

Since the discovery of the first mammalian dNTPase SAMHD1 as a novel anti-

HIV-1 protein in 2011, the research on SAMHD1 has become a timely and imperative topic of virology. Ryoo et al. 316 reported that SAMHD1 restricts HIV-1 replication through its RNase activity, which results in the nucleolytic cleavage of the viral RNA genome in non-dividing cells. These findings revealed a new mechanism of SAMHD1-mediated HIV-1 restriction and raised important questions as to whether SAMHD1’s RNase activity inhibits HIV-1 protein synthesis in virus producing cells. Based on Ryoo et al.’s findings, we tested our hypothesis that

SAMHD1’s RNase activity might limit HIV-1 protein production in virus producing cells, as the transcribed viral RNA genome and mRNAs would be subject to cleavage by SAMHD1. We also tested whether SAMHD1’s RNase activity could restrict the replication of other RNA viruses, including Sendai virus and influenza

A virus. Overall, our data suggest that newly transcribed mRNAs of HIV-1, Sendai virus (SeV), and influenza A virus (IAV) are not subjected to nucleolytic cleavage by SAMHD1’s RNase activity. Additionally, our data confirmed that SAMHD1

46

reduced dNTP levels and inhibited efficient cDNA synthesis in non-dividing cells.

Further, SAMHD1 expression does not affect HIV-1 protein production, viral particle release, and infectivity of newly synthesized virions when the block in reverse transcription is removed, indicating SAMHD1 does not degrade newly synthesized viral mRNA or genomic RNA. Our results obtained from new approaches can help better understand how SAMHD1 functions as a novel viral restriction factor.

2.2 Introduction

The dNTPase SAMHD1 regulates intracellular dNTP pools and restricts the replication of a variety of retroviruses and certain DNA viruses332. SAMHD1 restricts HIV-1 in myeloid cells and resting CD4+ T-cells by degrading dNTPs and limiting viral reverse transcription229,236,309,333,334. Purified recombinant SAMHD1 possesses an exonuclease activity when synthetic nucleic acids or in vitro transcripts of HIV-1 RNAs were used as substrates313. Ryoo et al. suggest that

SAMHD1 restricts HIV-1 infection through its RNase activity by cleaving the viral

RNA genome335. Using SAMHD1 mutants purported to specifically retain dNTPase

(Q548A) or RNase (D137N) activities, Ryoo et al. proposed that the RNase activity of SAMHD1, but not its dNTPase activity, is essential for HIV-1 restriction in non- dividing cells317,335. They also suggested that SAMHD1 phosphorylation at threonine 592 (T592) negatively regulated its RNase activity335. In this study, we

47

aimed to to confirm whether SAMHD1 exercises its newly-characterized RNase activity to inhibit HIV-1 infection, thereby replacing the established model of

SAMHD1-mediated restriction of HIV-1 infection.

2.3 Materials and Methods

Immunoblotting and . Cells were harvested at 24 hr after transfection or as specifically indicated, and lysed with cell lysis buffer (Cell Signaling) containing protease inhibitor cocktail (Sigma-Aldrich). Cell lysates were prepared for immunoblotting253 and the blots were detected using antibodies specific against

HA (Covance, Ha.11 clone 16B12), glyceraldehyde-3-phosphate dehydrogenase

(GAPDH) (AbD serotec). Monoclonal to HIV-1 p24 Gag (clone #24-2) was obtained from the NIH AIDS Reagent Program. Rabbit antibodies specific for phospho-SAMHD1 were generated by ProSci Inc. using phosphorylated hSAMHD1 peptide336: FTKPQDGDVIAPLI(Tp)PQKKE. Immunoblotting images were captured and analyzed using the Luminescent Image analyzer (LAS 4000) as described253. Image J software was used to calculate the densitometry of cellular Pr55 Gag, virion p24 CA and GAPDH bands in three independent experiments.

HIV-1, SeV or IAV infection assays. U937 cells were transduced with pLenti-puro based lentiviral vectors that do not express (vector control) or express WT or

48

mutant SAMHD1 (D137N, Q548A, or T592A) to generate puromycin-resistant stable cell lines as described253. Each cell line was differentiated with PMA (100 ng/mL) for 24 hr and subsequently infected with HIV-1-GFP (GFP reporter virus) or HIV-1-Luc (luciferase reporter virus) pseudotyped with vesicular stomatitis virus

G protein (VSV-G)253, SeV-GFP337 (a gift from Dominique Garcin, University of

Geneva), or IAV338 at the indicated MOI. For HIV-1 infection, cells were pre-treated with DMSO control or neviraprine (NVP, 10 µM) for 1 hr prior to HIV-1 infection.

DMSO or NVP was maintained in the culture medium for the duration of the infection assay. The number of HIV-1-GFP infected cells was determined by flow cytometry at 48 hr post-infection by the percentage of GFP-positive cells compared to an uninfected sample. At 24 hr post-infection, the number of SeV and IAV infected cells was determined by the percentage of GFP-positive cells and cells positive for IAV nucleocapsid protein338, respectively.

Intracellular dNTP measurement. For dNTP analysis and quantification, U937 cells were lysed and extracted samples were processed for the single nucleotide incorporation assay as described339.

Quantitative real-time PCR (qPCR) for HIV-1 genomic RNA and cDNA measurement. U937 cells expressing WT SAMHD1 and mutants and the vector control cells were differentiated with 30 ng/mL PMA for 20 hr and then infected

49

with HIV-1-Luc/VSV-G at a MOI of 1 or as indicated. All virus stocks were treated with DNase I (40 U/ml; Ambion) prior to infections to avoid plasmid DNA contamination. Total RNA was isolated from the cells at 1, 3, and 6 hours post- infection (hpi) using an RNeasy kit (Qiagen), and cDNA synthesized using the

SuperScript III First-Strand Synthesis System(ThermoFisher Scientific). The levels of HIV-1 genomic RNA from infected U937 cells were measured by SYBR-green based pPCR using the method and HIV-1 gag-specific primers described by Ryoo et al.335. The levels of HIV-1 late reverse transcription products in infected U937 cells were quantified by SYBR-green based pPCR analysis using primers (5’-

GACATAGCAGGAACTACTAGTACCC-3’ and 5’-GGTCCT

TGTCTTATGTCCAGAATGC-3’) and methods previously described238,340. Briefly, genomic DNA (50 ng) from HIV-1 infected U937 cells were used as input for the detection of late reverse transcription products. GAPDH was used for additional input control. Genomic DNA from HIV-1 infected cells at 12 and 24 hpi was isolated using a DNeasy Blood and Tissue kit (Qiagen). NVP treatment was used as a negative control of HIV-1 infection.

Protein expression and purification. WT full-length SAMHD1 was cloned into a pET28a expression vector with a 6×His-tag at the N terminus and expressed in

Escherichia coli. SAMHD1 proteins were purified using a nickel-nitrilotriacetic acid

(Ni-NTA) affinity column. The eluted peak fractions were collected and dialyzed

50

into the assay buffer as previously described 272 and then further purified with size- exclusion chromatography.

Analytical size-exclusion chromatography. Purified samples of SAMHD1 constructs (2 mg/mL, 200 μl) were loaded onto an analytical Superdex 200 10/300

GL column (GE Healthcare), which is pre-equilibrated in assay buffer. The elution profiles (UV absorbance at 280 nm) were recorded.

SAMHD1 dNTPase activity assays. SAMHD1 dNTPase activity assay was performed with 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM MgCl2, 0.5 mM TCEP and 1 mM substrate dGTP in a 500 μl reaction volume. Reactions were initiated at

37°C by the addition of purified SAMHD1 samples to a final concentration of 500 nM. Aliquots of reactions were collected at various times points and terminated by

5-fold dilution into ice cold buffer containing 10 mM EDTA, followed by spinning through an Amicon Ultra 0.5 ml 10 kDa filter (Millipore) at 16,000 ×g for 20 min.

Deproteinized samples were analyzed by HPLC using a Synergi C18 Column

150×4.6 mm (Phenomenex), pre-equilibrated in 20mM ammonium acetate, pH 4.5

(buffer A). Samples were eluted with a methanol (buffer B) gradient over 14 min at a flow rate of 1 mL/min and the elution profiles (UV absorption at 260nm) were recorded.

51

SAMHD1 nuclease activity assays. The exonuclease activity assays were performed in 100 μl reaction mixtures containing 1 μM synthesized 5’ fluorescence labeled 30-nt ssDNA (5’- /56-FAM/ TAGAAAGGGA GATTCTAAGA

GGAAAGGTGA -3’) or 20-nt ssRNA (5'- /56-FAM/ rCrUrCrCrArUrCrArCrC rCrUrCrCrArUrCrArCrC -3') substrates (Integrated DNA Technologies), and 1 μM purified recombinant proteins in the assay buffer at 37°C. 25 μl aliquots of the reactions were terminated after 1 hr by the addition of 7 μl of 6× DNA loading Dye

(New England Biolabs). The products were separated in 15% polyacrylamide gels containing 8 M urea and buffered with 0.5× Tris-borate-EDTA and then pictured by

ChemiDoc MP gel imaging system (BIO-RAD).

Statistical analysis. The data were analyzed by one-way ANOVA, non- parametric T-test using Graphpad 5.0. The statistical significance was defined as p <0.05.

2.4 Results

Overexpression of SAMHD1 in HEK293T cells does not reduce HIV-1 Gag protein synthesis and viral particle release. In an effort to extend the findings of Ryoo et al.335, we first measured HIV-1 protein synthesis and virion production in the presence of SAMHD1 when the requirement for intracellular dNTP-

52

dependent HIV-1 reverse transcription was bypassed. To achieve this goal, we co- transfected an HIV-1 proviral DNA plasmid (pNL4-3) with a plasmid expressing wild-type (WT) SAMHD1 or a phospho-ablative but dNTPase-active mutant of

SAMHD1 (T592A)253,311,341 into HEK293T cells and assessed intracellular HIV-1

Gag protein synthesis and viral particle release in the cell supernatants (Figure

13) This transfection-based HIV-1 production is independent of the reverse transcription step that requires intracellular dNTPs as precursors of viral DNA synthesis, but is dependent on HIV-1 mRNA-mediated gene expression342.

Intracellular HIV-1 Gag protein levels (Figure 13A-B), p24 capsid levels in released HIV-1 virions (Figure 13C), and viral infectivity (Figure 13D) were not reduced by the overexpression of WT SAMHD1 or the constitutively active T592A mutant. Despite the purported RNase activity of SAMHD1 against incoming HIV-1 genomic RNA335, our results demonstrate that, regardless of its phosphorylation at

T592, SAMHD1 cannot inhibit HIV-1 production when the requirement for dNTPs is relieved in proviral DNA transfected cells. These data are also consistent with our previous studies demonstrating that overexpression of WT SAMHD1 or the

T592A mutant in dividing HEK293T or HeLa cells does not restrict HIV-1 infection238,253. Our results do not rule out a specific nucleolytic interaction between

SAMHD1 and incoming viral genomic RNA, but indicate that SAMHD1 does not have broad nuclease activity.

53

Aa Co-transfected with HIV-1 pNL4-3 (1 µg) Vector WT T592A SAMHD1 1.5 0.5 1.0 1.5 0.5 1.0 1.5 µg kDa 72 SAMHD1

72 Phospho-T592

55 Pr55 Gag Cells 41

24 p24 CA

36 GAPDH

24 p24 CA Virions

Bb Cc Dd

y t

4.0 1.5 i 2.0

s

v

l

i

s

l

t

e

e

v

c

v

e

e

l f

e 1.5

3.0 l

n

g

I A

a 1.0

1

C

G

-

4 V

5 2.0 1.0

I

2

5

r

p

H

P e

0.5 e

v

e

v i

i 0.5

1.0 t

v

t

i

t

a

l

a

l

a

l

e

e

e

R R R 0.0 0.0 0.0 Vector WT T592A Vector WT T592A Vector WT T592A SAMHD1 SAMHD1 SAMHD1

Figure 13: Overexpression of SAMHD1 in HEK293T cells does not reduce HIV-1 Gag protein synthesis and viral particle release. Transfected HEK293T cells were lysed and the expression levels of SAMHD1, HIV-1 Gag, and p24 CA were determined by immunoblotting. T592- phosphorylated SAMHD1 (Phospho-T592) was determined using specific antibodies. GAPDH was used as a loading control. (B and C) Semi-quantitative analysis of the relative levels of Pr55 Gag protein in cells (B) and p24 CA in virions (C) in the absence or presence of SAMHD1 WT and T592A protein expression. For normalization, the densitometry of the intracellular Pr55 Gag band in each lane was normalized to the densitometry of the corresponding GAPDH band. The normalized densitometry of the Pr55 Gag band and the p24 CA band in the vector 54

control sample was set as 1 in B and C, respectively. Error bars represent the standard deviation of triplicate experiments. (D) The relative infectivity of HIV-1 virions generated from transfected HEK293T cells in the presence of SAMHD1 was determined using a reporter cell line and normalized to the vector control cells (set as 1). There is no statistical difference compared among vector control, WT SAMHD1, and the T592 mutant (p ≥0.08) in B-D. Statistical analysis was performed using a Student’s t-test.

SAMHD1 restricts HIV-1, but not SeV and IAV, in PMA-differentiated U937 cells. We next tested whether RNase activity of SAMHD1 could restrict the replication of other RNA viruses, such as Sendai virus (SeV) and influenza A virus

(IAV)338, which replicate their genomes in the cytoplasm and nucleus of infected cells, respectively, and do not utilize dNTPs for viral replication. We found that these RNA viruses were not inhibited by WT or T592A mutant SAMHD1 in non- dividing U937 cells differentiated with phorbol 12-myristate 13-acetate (PMA). HIV-

1 infection was efficiently restricted by WT or T592A mutant SAMHD1 as previously reported253,311,341 (Figure 14, Figure 15). Together with the observation that SAMHD1 did not reduce HIV-1 protein production and virion release from virus producing cells (Figure 13), our results suggest that newly transcribed mRNAs of

HIV-1, SeV and IAV are not subjected to nucleolytic cleavage by an RNase activity of SAMHD1. Our data are consistent with a recent report that SAMHD1 has no effect on the replication of non-retro RNA viruses317. Although it is possible that

SAMHD1 may associate with and degrade retrovirus genomic RNA, these data

55

suggest that this process does not occur with other RNA viral genomes, and a mechanism for this ostensible specificity is currently unknown.

Mock Infected Vector WT T592A

0 .42 9 30 3.8 1 4.26 HIV-1 (MOI=0.6)

r 0.068 44 48 47.1

e

t

t

a c

s SeV

e (MOI=5)

d

i S

0.441 25.2 24.5 24.5

IAV (MOI=5)

Virus-positive cells (%)

Figure 14: Flow cytometry raw data. Representative samples of viral infections (higher MOIs) showing the flow cytometric analysis and gating strategy. The numbers in the plots indicate the percentages of virus-infected U937 cells. HIV-1 and IAV reporter virus infections were measured by GFP rexpression. SeV infection was measured by nucleocapsid staining.

56

A

B

Figure 15: SAMHD1 restricts HIV-1, not SeV and IAV, in PMA-differentiated U937 cells. U937 cells were transduced with pLenti-puro based lentiviral vectors that do not express (vector control) or express wild-type SAMHD1 (WT) or the phospho- ablative mutant (T592A) to generate stable cell lines. (A) Each cell line was differentiated with PMA and subsequently infected with HIV-1-GFP/VSV-G, SeV- GFP, or IAV at the indicated multiplicity of infection (MOI). The number of HIV-1- GFP infected cells was determined by flow cytometry at 48 hr post-infection by the percentage of GFP-positive cells. At 24 hr post-infection, the number of SeV and IAV infected cells was determined by the percentage of GFP-positive cells and cells positive for IAV nucleocapsid protein, respectively. Three independent experiments were carried out and a representative experiment is presented. Error bars represent the standard deviation of triplicate samples and statistical analysis was performed using a Student’s t-test where * denotes p <0.05. (B) The expression levels of SAMHD1 WT and T592A and T592-phosphorylated SAMHD1 (Phospho-T592) in PMA-differentiated U937 cells were determined by immunoblotting. GAPDH was used as a loading control.

57

Both D137N and Q548A mutants of SAMHD1 restrict HIV-1 infection in PMA- differentiated U937 cells by decreasing viral cDNA synthesis, but not viral genomic RNA. Given the preponderance of previous data implicating the dNTPase activity of SAMHD1 as its primary antiviral mechanism, and our inability to identify a broad nucleolytic activity for SAMHD1, we sought to directly reproduce the experiments of Ryoo et al335. The conclusion that SAMHD1 restricts HIV-1 through RNase activity was based upon the differential activity of SAMHD1 mutants, D137N and Q548A335. We thus independently generated these two mutant constructs and confirmed the expected mutations by DNA sequencing to ensure there were no other disabling mutations in the constructs and further verified the mutations by sequencing cDNA derived from our stable cell lines made with these constructs. We then examined the anti-HIV-1 activity and intracellular dNTP regulation of these mutants in comparison to WT SAMHD1 following the protocol of Ryoo et al335. WT SAMHD1 was expressed at higher levels as compared with D137N and Q548A mutants, but still lower than primary monocyte- derived macrophages (MDM) (Figure 16A), and each cell line differentiated similarly into macrophage-like cells, as measured by CD11b surface marker staining compared to MDM (Figure 16B-C). Each cell line restricted HIV-1 infection and significantly decreased dNTP levels in PMA-differentiated U937 cells

(Figure 16D-F). Thus, in contrast to what was previously reported by Ryoo et al335,

58

these mutants do not have differing antiviral activities and neither lacks the ability to lower cellular dNTP levels.

Specifically addressing the ability of SAMHD1 to degrade HIV-1 genomic RNA reported by Ryoo et al335, we found that expression of WT SAMHD1 and D137N and Q548A mutants did not affect HIV-1 genomic RNA levels at 1, 3 and 6 hr post- infection in PMA-differentiated U937 cells as compared with the vector control cells

(Figure 16G). Importantly, although we used the same qRT-PCR assay described by Ryoo et al335, we could not observe decreased levels of HIV-1 genomic RNA in

PMA-differentiated U937 cells expressing WT SAMHD1 and D137N mutant at 3 and 6 hr post-infection as reported by Ryoo et al335. Thus, our data do not support a role for SAMHD1 in degrading viral genomic RNA during the early stage of the

HIV-1 life cycle. To further support our findings, we measured the levels of HIV-1 late reverse transcription products in PMA-differentiated U937 cells at 12 and 24 hr post-infection, which represent viral cDNA synthesis dependent on the intracellular dNTP pool. Our results show that expression of WT SAMHD1 and

D137N and Q548A mutants significantly reduced HIV-1 late reverse transcription products at both 12 and 24 hours post infection (Figure 16H), which correlate well with reduced intracellular dNTP pool (Figure 16E). Together, these results suggest that the dNTPase and RNase functions of SAMHD1 cannot be effectively

59

distinguished by these two mutants, and that dNTP depletion accounts for

SAMHD1-mediated HIV-1 restriction.

A B C 100

s 50

M s

D Vector l

80 l

M

e

c 40 WT

or 7N 8A e e M t 3 4 v

c i D e T 1 5 v D137N

kDa t 60 i

M V W D Q i

t i

s 30

s Q548A o

anti-SAMHD1 o

72- p p Isotype

40 b

b 20 1

Phospho-T592 1 1

72- 1 D

D 20 10

C

C

36- GAPDH

% % 0 0 Isotype CD11b Media PMA

D E F 150 15000 Vector

) 300

% WT ( ***

n D137N

o

i

t

) ) c 10000 Q548A

100 M 200

e

M f

*** n

n

n

(

i

(

1 ***

P

P

-

T

T

V

I

N C

H 5000 d 100

50 d

e v

i ** t

a ***

l e R 0 0 0 dATP dCTP dGTP dTTP r T N A NVP (-) NVP (+) to 7 8 c W 3 4 e 1 5 V D Q

G H 1.5 Vector

A 1.5 1.5

N WT 12 hpi Vector 24 hpi Vector

R

r

r

c

e i

D137N WT e WT

b

b

m

o m

1.0 Q548A D137N m D137N

n u

1.0 u 1.0

e

n

n

g Q548A Q548A

y

y

p 1

p *

-

o

o V

c **

c

I

H **

e e

0.5

v

v

i

i

e t

0.5 t 0.5

v

a a

i ***

l l

t ***

e e

a ***

l

R

R e

R 0.0 1 3 6 0.0 0.0 Hours post infection NVP (-) NVP (+) NVP (-) NVP (+)

Figure 16: Both D137N and Q548A mutants of SAMHD1 restrict HIV-1 infection in PMA-differentiated U937 cells by decreasing viral cDNA synthesis, but not viral genomic RNA. (A) lysates from PMA-differentiated cells were collected for immunoblotting to confirm SAMHD1 expression and T592-phosphorylated SAMHD1 (Phospho- T592). GAPDH was used as a loading control. Expression of SAMHD1 in U937 cell lines was compared to endogenous expression in MDMs (Lane 1). (B-C) PMA-

60

differentiated cells were tested for differentiation through CD11b surface marker staining. The CD11b positive cell population (%) of MDMs (B) and the SAMHD1- expressing U937 cells (C) were compared to isotype or media control, respectively. (D) PMA-differentiated cells were infected with single-cycle HIV-1-Luc/VSV-G at a MOI of 1. At 24 hours post-infection, luciferase assay was used to measure HIV-1 infectivity. Graph depicts relative percentage of luciferase per 10 µg protein, with the vector being set as 100%. Data are presented as mean ± SEM (n=6). (E-F) Decreased dNTP levels of PMA-differentiated U937 cell lines expressing WT or mutant SAMHD1. Data are presented as mean ± SEM (n=4). Panel (E) shows relative dNTP concentrations (dATP of the vector control cells was set as 1) and panel (F) shows dCTP concentrations in different cell lines. (G) The levels of HIV- 1 genomic RNA were measured at 1, 3, and 6 hr post-infection as described at an MOI of 1 (N=6). Data are presented as mean ±SEM (n=6) and depicted as relative HIV-1 genomic RNA, with the vector being set as 1. RNA samples without reverse transcription were used as negative controls. (H) HIV-1 late reverse transcription products were measured by qPCR. The reverse transcriptase inhibitor nevirapine (NVP) was used as a control. Data are presented as mean ±SEM (n=4 for 12 hpi and n=6 for 24 hpi). Statistical analysis was performed using the one way ANOVA with Dunnett’s correction, *** denotes p <0.001, ** denotes p <0.01, * denotes p <0.05.

Characterization of SAMHD1 enzymatic activities in vitro. Seamon et al. reported that trace exonuclease activities of recombinant SAMHD1 arise from contaminants during protein purification318. To clarify whether SAMHD1 has a nuclease activity, we tested its ability to degrade ssRNA and ssDNA using 5’- fluorescence labeled 30-nucleotide (nt) ssDNA and 20-nt ssRNA and highly pure full-length (FL) SAMHD1. In our in vitro activity assays, purified SAMHD1 (Figure

17A) exhibited a weak nuclease activity on both ssDNA and ssRNA in some protein preparations (Figure 17B, sample 1), while only background level of nuclease activity was observed for other protein preparations (Figure 17B,

61

sample 2). These SAMHD1 samples were prepared by the same expression and

purification procedure. In all preparations, SAMHD1 had robust dNTPase activity

as expected (Figure 17C). The observed activity on ssDNA and ssRNA resulted

in products with distinct cleavage patterns, which are similar to previous

observations313,318,335. However, the inconsistency of the nuclease activity of

SAMHD1 between different batches of protein preparations suggests that the

nuclease activity is likely associated with contaminant proteins, consistent with the

findings reported by Seamon et a318

A B

C

Figure 17: Characterization of SAMHD1 enzymatic activities in vitro. 62

(A) The purity of recombinant wild-type SAMHD1 from different batches of preparations (sample 1 and sample 2) demonstrated by analytical size exclusion chromatography and SDS-PAGE. SAMHD1 (150 μl) at 2 mg/mL were applied to a Superdex 200 10/300 GL column. Fraction numbers on the SDS-PAGE indicate elution volumes. (B) Moderate exonuclease activities on both ssDNA and ssRNA were observed for sample 1, only background-level activities were detected for sample 2. Ctrl indicates a control with no protein. (C) The dNTPase activity of SAMHD1 proteins (0.5 μM) was assayed with 1 mM dGTP from 5-15 min. The amount of dN products generated in the reactions was quantified by HPLC. Error bars represent standard error of the mean from triplicate experiments.

2.5 Discussion

Previous studies have used the D137A mutant to explore the effects of the dGTP binding site (D137) on the dNTPase activity, tetramer formation, and HIV-1 restriction of SAMHD1233,343,344. The D137A mutant has no detectable in vitro dNTPase activity or HIV-1 restriction due to its inability to form a stable tetramer343,344. It is possible that the D137N mutant may be stabilized in cells to remain in a tetrameric form, thus maintaining its ability to reduce intracellular dNTP levels and restrict HIV-1 infection. In contrast to the results of Ryoo et al.335, the

D137N and Q548A mutants in our experiments do not have differing anti-HIV-1 activities and neither lacks the ability to lower cellular dNTP levels.

Ryoo et al. reported an approximately 2-fold decrease in HIV-1 gRNA levels in

PMA-differentiated U937 cells expressing WT SAMHD1 or D137N, but not Q548A mutant, compared to the control cells at 3 and 6 h post-infection (hpi), suggesting

63

SAMHD1-mediated HIV-1 gRNA degradation335. In contrast, we detected comparable levels of HIV-1 gRNA in PMA-differentiated U937 cells expressing

SAMHD1 (WT, D137N and Q548A mutants) and the vector control cells at 1, 3 and 6 hpi, respectively (Figure 16D), showing that in our hands SAMHD1 cannot degrade HIV-1 gRNA during early infection. To further support our findings, we measured the levels of HIV-1 late reverse transcription products in infected cells at 12 and 24 hpi, which represent viral cDNA synthesis dependent on the intracellular dNTP pool. We found that expression of WT SAMHD1 and D137N and

Q548A mutants significantly reduced HIV-1 late reverse transcription products

(Figure 16E), correlating well with reduced intracellular dNTP pool (Figure 16C).

Thus, in our hands these two mutants of SAMHD1 cannot distinguish its dNTPase and RNase functions, and dNTP depletion accounts for SAMHD1-mediated HIV-1 restriction.

Purified SAMHD1 (Figure 17A) exhibited a weak in vitro nuclease activity on both ssDNA and ssRNA in some protein preparations (Figure 17B, sample 1), while only background level of nuclease activity was observed for other independent protein preparations under identical conditions (Figure 17B, sample 2). In all preparations, SAMHD1 had robust dNTPase activity as expected (Figure 17C).

The inconsistency of the RNase activity of SAMHD1 between different protein

64

preparations suggests that the RNase activity is likely associated with contaminants, consistent with the results by Seamon et al.318

Moreover, SAMHD1 acts as a dNTPase in cells because the addition of exogenous deoxynucleosides rescues HIV-1 infectivity in SAMHD1-expressing cells309,345.

Hofmann et al. reported that the anti-HIV activity of SAMHD1 is reversible345, further suggesting that dNTP depletion is the primary mechanism of SAMHD1- mediated HIV-1 restriction and arguing against a nucleolytic mechanism. Our data and recent two studies346,347 do not support the conclusion that the RNase activity of SAMHD1 is involved in its anti-HIV-1 activity335, but are in agreement with its dNTPase activity in HIV-1 restriction.

The discrepancy between our results and Ryoo et al335 may be due to different experimental approaches, although we repeated HIV-1 infection experiments and qRT-PCR experiments using the similar methods described in Ryoo et al335. One notable difference was our use of the pLenti-puro vector to stably express high levels of SAMHD1 in PMA-differentiated U937 cells to perform HIV-1 restriction assays253, while Ryoo et al. used a pMSCV-puro vector to express SAMHD1 in the same cell line335. We were not able to express high levels of SAMHD1 proteins in

U937 cells using the pMSCV-puro vector provided by Ryoo et al335 (Figure 18).

This might be due to lower expression driven by the MSCV LTR promoter, which

65

may be influenced by the context of genome insertion, as compared to the CMV immediate early promoter driving higher SAMHD1 expression from the pLenti vector.

SAMHD1 (HA tagged)

GAPDH

1 2 3 4 5 6 7 8 9

• Lanes #1-7: pMSCV-puro vector and it derived SAMHD1 constructs;

• Lanes #1-2: pMSCV-puro vector and WT SAMHD1 construct from the Ahn lab.

• Lanes #3-7: SAMHD1 constructs generated by the Xiong lab

• Lanes # 8-9: pLenti-puro vector and it derived construct (from the Landau lab)

• FL: full-length

Figure 18: Expression of SAMHD1 WT and mutants by different constructs in PMA-differentiated U937 cells. U937 cells were transduced by pLenti-puro of pMSCV-puro vectors expressing WT or mutant SAMHD1. Two separated clones of pMSCV expressing WT SAMHD1 were tested (from Dr. Ahn (Ryoo et al.) or Dr. Xiong’s labs, respectively). The result

66

shows a long exposure and there were multiple bands for pLenti-expressed WT SAMHD1. Cells were differentiated with PMA and lysed and the expression levels of SAMHD1 were determined by immunoblotting with anti-HA antibodies. GAPDH was used as a loading control. FL, full-length.

Indeed, previous studies showed that the efficiency of SAMHD1-mediated HIV-1 restriction in PMA-differentiated U937 cells is dependent on the levels of SAMHD1 expression268,343. It is also plausible that different HIV-1 reporter vectors or other reagents used in these studies may account for the discrepancy of the results despite our best efforts to recapitulate the findings of Ryoo et al335. Nevertheless, here we provide independent evidence suggesting that intracellular dNTP reduction by SAMHD1 is the primary mechanism of its anti-HIV-1 activity in non- dividing cells.

2.6 Acknowledgements

I would like to thank Nathaniel Landau and Jacek Skowronski for reagents and

Jocelyn Hach for technical assistance. The antibody to HIV-1 p24 Gag (clone #24-

2) and nevirapine were obtained from the NIH AIDS Reagent Program.

2.7 Contributions

The purification of recombinant SAMHD1 protein and the in vitro characterization of SAMHD1’s nuclease activity (Figure 17) were performed by members of the 67

Yong Xiong lab. The SeV and IAV infections (Figure 15) were performed by members of the Jacob Yount lab. The immunoblot comparing the expression levels of SAMHD1 in the pMSCV or pLenti backbones (Figure 18) was performed by Dr.

Suresh de Silva. All other experiments were performed by Jenna Antonucci.

68

Chapter 3: SAMHD1 Suppresses LTR-Driven Gene Expression and

Reactivation of Viral Latency in CD4+ T-cells

3.1 Abstract

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) restricts human immunodeficiency virus type 1 (HIV-1) replication in non-dividing cells by degrading intracellular deoxynucleoside triphosphates (dNTPs). SAMHD1 is highly expressed in resting CD4+ T-cells that are important for the HIV-1 reservoir and viral latency; however, whether SAMHD1 affects HIV-1 latency is unknown.

Recombinant SAMHD1 binds HIV-1 DNA or RNA fragments in vitro, but the function of this binding remains unclear. Here we investigate the effect of SAMHD1 on HIV-1 gene expression and reactivation of viral latency. We found that endogenous SAMHD1 impaired HIV-1 LTR activity in monocytic THP-1 cells and

HIV-1 reactivation in latently infected primary CD4+ T-cells. Overexpression of WT

SAMHD1 suppressed HIV-1 long terminal repeat (LTR)-driven gene expression at the level of transcription. SAMHD1 overexpression also suppressed LTR activity from human T-cell leukemia virus type 1 (HTLV-1), but not from (MLV), suggesting specific suppression of retroviral LTR-driven gene expression. WT SAMHD1 bound to proviral DNA and impaired reactivation of HIV- 69

1 gene expression in latently infected J-Lat cells. In contrast, a nonphosphorylated mutant (T592A) and a dNTP triphosphohydrolase (dNTPase) inactive mutant

(H206D/R207N, or HD/RN) of SAMHD1 failed to efficiently suppress HIV-1 LTR- driven gene expression and reactivation of latent virus. Purified recombinant WT

SAMHD1, but not T592A and HD/RN mutants bound to fragments of the HIV-1

LTR in vitro. These findings suggest that SAMHD1-mediated suppression of HIV-

1 LTR-driven gene expression contributes to regulation of viral latency in CD4+ T- cells.

3.2 Introduction

SAMHD1 is the only identified mammalian dNTPase233,234 with a well- characterized role in downregulation of intracellular dNTP levels250, a mechanism by which SAMHD1 acts as a restriction factor against the infection of retroviruses

301,348 and several DNA viruses 231,232,349-351 in non-dividing myeloid cells168,229 and quiescent CD4+ T-cells236,334. Additionally, in vitro studies indicate that SAMHD1 is a single-stranded nucleic acid (NA) binding protein313-315,328, although the function of this binding activity in cells remains unknown. One report suggested that

SAMHD1 uses its RNA binding potential to exert a ribonuclease activity against

HIV-1 genomic RNA316; however, recent studies do not support this observation162,308,318,320.

70

SAMHD1 less efficiently restricts retroviral replication in dividing cells due to phosphorylation of SAMHD1 at Thr 592 (T592)253,255,311,336,341,352. The dNTPase activity of SAMHD1 requires the catalytic H206 and D207 residues of the HD domain268,276. While mutations to either H206 or D207 abrogated ssDNA binding in vitro313, the effect of nonphosphorylated T592 on ssDNA binding has not been described. The binding of ssNA occurs at the dimer-dimer interface on free monomers and dimers of SAMHD1. This interaction prevents the formation of catalytically active tetramers328, suggesting a dynamic mechanism where

SAMHD1 may regulate its potent dNTPase activity through NA binding. However, the effect of SAMHD1 and NA binding on HIV-1 infection or viral gene expression is unknown.

HIV-1 latency occurs post-integration when a proviral reservoir is formed within a population of resting memory CD4+ T-cells330. By forming a stable reservoir and preventing immune clearance of infection, HIV-1 is able to persist in the host despite effective treatment with antiretroviral therapy205. Although HIV-1 proviral

DNA is transcriptionally silent in latently infected CD4+ T-cells, reactivation of intact provirus can result in the production of infectious virions353,354. There are several mechanisms that contribute to HIV-1 latency, including sequestration of host transcription factors in the cytoplasm and transcriptional repression330,354. The 5´

LTR promoter of HIV-1 proviral DNA contains several cellular transcription factor-

71

binding sites, with transcription factors activated by external stimuli to enhance

HIV-1 gene expression355. Known cellular reservoirs of latent HIV-1 proviral DNA include quiescent CD4+ T-cells and macrophages356-358. Although HIV-1 does not productively replicate in resting CD4+ T-cells, a stable state of latent infection does exist in these cells359,360. SAMHD1 blocks reverse transcription leading to HIV-1 restriction in resting CD4+ T-cells236,334; however, whether SAMHD1 affects reactivation of HIV-1 proviral DNA in latently infected CD4+ T-cells remains unknown.

In this study, we demonstrate that SAMHD1 suppresses HIV-1 LTR-driven gene expression and binds to the LTR promoter in a latently infected cell line model.

Furthermore, endogenous SAMHD1 suppresses HIV-1 LTR-driven gene expression in a monocytic THP-1 cells and viral reactivation in latently infected primary CD4+ T-cells. Our findings suggest that SAMHD1-mediated suppression of HIV-1 gene expression contributes to regulation of viral latency in primary CD4+

T-cells, thereby identifying a novel role of SAMHD1 in modulating HIV-1 infection.

3.3 Materials and Methods

Cell culture. Human embryonic kidney 293T (HEK293T) cells were obtained from the American Type Culture Collection (ATCC) and maintained as described 253.

Jurkat cell-derived J-Lat cells (clone 9.2) were obtained from the NIH AIDS reagent

72

program and maintained as described331. THP-1 control cells and derived

SAMHD1 KO cells were maintained as described255. All cell lines tested negative for mycoplasma contamination using a PCR-based universal mycoplasma detection kit (ATCC, #30-101-2k). Healthy human donors’ peripheral blood mononuclear cells (PBMCs) were isolated from the buffy coat as previously described361. Naive CD4+ T-cells were isolated from PBMCs by MACS microbread- negative sorting and the naive CD4+ T-cell isolation kit (Miltenyi Biotec). Primary

CD4+ T-cells were cultured in complete RPMI-1640 media in the presence of 30

IU/mL of recombinant interleukin 2 (rIL-2) (Obtained from the NIH AIDS Research and Reference Reagent Program, catalog number 136)253.

Plasmids. The pLenti-puro vectors encoding hemagglutinin (HA)-tagged WT

SAMHD1 (driven by the CMV immediate-early promoter) and the empty vector were described301 and provided by Nathaniel Landau (New York University). The pLenti-puro vector expressing HA-tagged T592A and HD/RN SAMHD1 mutant constructs were generated using a Quikchange mutagenesis kit (Agilent

Technologies)253. The HTLV-1-LTR luciferase reporter plasmid and pcTax were provided by Patrick Green (The Ohio State University)362. The HIV-1 FF-luc (pGL3-

LTR-luc) was provided by Jian-Hua Wang (Pasteur Institute of Shanghai) xfnaf1

363. The pRenilla-TK plasmid was provided by Kathleen Boris-Lawrie (University of

Minnesota). The pTat plasmid is a pcDNA3-based HIV-1 Tat expression construct

73

342 provided by Vineet KewalRamani (National Cancer Institute). The MLV-LTR reporter (pFB-Luc) contains the MLV 5’ LTR, truncated gag, 3’ LTR, and firefly luciferase, which was provided by Vineet KewalRamani (National Cancer

Institute).

Transfection assays. HEK293T cells (5.0 × 104 in experiments of Figure 19 and

1.0 × 105 in experiments of Figure 20-21) were co-transfected with a viral LTR- driven luciferase construct (HIV-1, HTLV-1, or MLV), TK-driven Renilla luciferase construct, and increasing amounts of SAMHD1 WT, T592A or HD/RN mutant- expressing plasmid using calcium phosphate as described253. The total amount of

DNA transfected was maintained through addition of empty vector. Transfection media was replaced with fresh media at 16 h after transfection. Nucleofection of control and SAMHD1 KO THP-1 cells with HIV-1-LTR-Luc and TK-Renilla was performed using the Amaxa Cell Line Nucleofector Kit V (Lonza).

Immunoblotting and antibodies. Cells were harvested 24 h after transfection or as specifically indicated, washed with phosphate-buffered saline (PBS) and lysed with cell lysis buffer (Cell Signaling, #9803) containing protease inhibitor cocktail

(Sigma-Aldrich P8340). Cell lysates were prepared for immunoblotting as described253. HA-tagged SAMHD1 and endogenous glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were detected using antibodies specific to HA

74

(Covance, Ha.11 clone 16B12) at a 1:1,000 dilution, and GAPDH (BioRad,

AHP1628) at a 1:3,000 dilution, respectively. Polyclonal SAMHD1-specific antibodies (Abcam, ab67820) were used at a 1:1,000 dilution for immunoblotting, as described 238. Immunoblots were imaged and analyzed using the Amersham imager 600 (GE Healthcare). Validation for all antibodies is provided on the manufacturers’ websites.

Densitometry quantification of immunoblots. Densitometry analysis was performed on unaltered low-exposure images using the ImageJ software.

Densitometry values were normalized to GAPDH.

Protein expression and purification. Full-length cDNA of WT, T592A, and

HD/RN SAMHD1 were cloned into a pET28a expression vector with a 6 × His-tag at the N- terminus and expressed in E. coli. SAMHD1 proteins were purified using a nickel-nitrilotriacetic acid affinity column as described 272. The eluted peak fractions were collected and dialyzed into the assay buffer, and then further purified with size-exclusion chromatography as described 308. SAMHD1 protein was stored in buffer containing 50 mM Tris-HCL (pH 8.0), 150 mM NaCl, 5 mM MgCl2, 0.5 mM

Tris-(2-carboxyethyl) phosphine at -80 oC.

75

Synthetic DNA oligonucleotides. Oligonucleotides used in FA binding assays, and as primers for qPCR, were synthesized by Integrated DNA Technologies.

Sequences of oligonucleotides and primers are shown in Tables 1 and 2. A 90- mer 6-FAM-labeled DNA oligonucleotide derived from the scrambled sequence of the 3’ U5 region of the HIV-1 LTR was obtained using the Sequence Manipulation

Suite (Bioinformatics.org).

FA binding assays. The assays were performed as described 364,365 using 5'-6-

FAM-labeled DNA sequences shown in Table 2. Briefly, proteins were incubated with 10 nM DNA at room temperature for 30 min in 20 mM Tris-HCl, pH 8, 1 mM

MgCl2, 0.25 mM HEPES, 50 M 2-mercaptoethanol, and 50, 100, or 150 mM monovalent ions (25, 50, or 75 mM of each NaCl and KCl). Each measurement was performed in triplicate over a range of WT or mutant SAMHD1 (5-8,300 nM).

Binding affinities were calculated by fitting the data to a 1:1 binding model, as described 366. Fluorescence measurements were obtained using a SpectraMax M5 plate reader (Molecular Devices, Sunnyvale, CA).

Generation of SAMHD1-expressing J-Lat cell lines. HEK293T cells were transfected with pLenti-puro vector or HA-tagged SAMHD1 (WT, T592A, and

HD/RN) expressing plasmids, pMDL packaging construct, pVSV-G, and pRSV-rev to produce lentiviral stocks for spinoculation at 2,000 × g for 2 h at room

76

temperature. Lentiviral stocks were harvested, filtered, and concentrated through a sucrose cushion at 48 h post transfection. Concentrated lentivirus stock was resuspended in RPMI-1640 media and applied to J-Lat cells (clone 9.2) with polybrene (8 µg/mL) prior to spinoculation. Afterwards, cells were cultured in complete RPMI media for 72 h before undergoing selection with 0.8 µg/mL puromycin.

HIV-1 reactivation assay in J-Lat cells. J-Lat cells (clone 9.2) stably expressing

WT, mutant T592A or HD/RN SAMHD1 were generated as described above. Cells were treated with 10 ng/mL TNF, or 32 nM PMA with 1 µM ionomycin (2× PMA+i) unless otherwise described in figure legends. At 24 h post-treatment, media was removed and cells were washed and placed in untreated complete RPMI-1640 media for an additional 12 h. Cells were collected, washed twice with 1× PBS, and suspended in 2% fetal bovine serum in PBS. Cells were evaluated by flow cytometry using Guava EasyCyte Mini Flow Cytometer (Millipore), with data analyzed by FlowJo software.

IP of SAMHD1 in J-Lat cells. J-Lat cells (clone 9.2) expressing WT or mutant

SAMHD1 and the vector control cells were differentiated using either 64 nM PMA

(WT) or 128 nM PMA (vector, T592A, and HD/RN) for 24 h. At 36 h post-treatment, cells were treated with 1% paraformaldehyde for 10 min before the reaction was

77

quenched with 0.125 M glycine. Cells were lysed in non-SDS containing radioimmunoprecipitation assay buffer and sonicated to shear cellular chromatin.

Monoclonal anti-HA-agarose beads were incubated with 250 µg of cell lysate from

SAMHD1-expressing (WT, T592A, HD/RN) or vector control cells at 4°C for 2 h.

Beads were washed 3 times with PBS containing 0.1% Tween. To confirm IP efficiency, bound proteins were eluted from beads by boiling in 1× SDS-sample buffer, and the supernatants were analyzed by immunoblot as described 253. Total

DNA was isolated from proteinase-K treated sonicated input lysate and IP products using a DNeasy kit (Qiagen).

qPCR assay. For quantification of Renilla or firefly luciferase mRNA in transfected

HEK293T cells, total cellular RNA was extracted using the RNeasy mini kit

(Qiagen). Equal amounts of total RNA from each sample were used as a template for first-strand cDNA synthesis using Superscript III first-strand synthesis system and oligo (dT) primers (Thermo Fisher Scientific). SYBR green-based PCR analysis was performed using specific primers detailed in Table 1 and methods described 238. Quantification of spliced GAPDH mRNA was used for normalization as described 238. Calculation of relative gene expression was performed using the

2-∆∆CT method as described 367. The levels of SAMHD1-bound HIV-1 genomic

DNA from PMA-treated latently infected J-Lat cells were measured by SYBR- green-based qPCR using primers detailed in Table 1 and methods as described

78

238,340. DNA samples without primer templates were used as negative controls.

Genomic DNA (50 ng) from PMA-treated SAMHD1-expressing or vector control J-

Lat cells after IP was used as input for the detection of HIV-1 genes. Data was normalized to vector background levels and presented as percent of total input

DNA.

Generation of shRNA vectors. HEK293T cells were transfected with pLKO.1- puro empty vector (GE DHarmacon) and SAMHD1-specific shRNA expressing plasmids (GE DHarmacon, clone ID: TRCN0000145408), psPAX2 packaging construct, and vesicular stomatitis virus G-protein-expressing construct (pVSV-G) to produce lentiviral stocks for spinoculation at 2,000 × g for 2 h at room temperature. Lentiviral stocks were harvested, filtered, and concentrated through a sucrose cushion at 48 h post transfection. Concentrated lentivirus stock was resuspended in RPMI-1640 media and applied to isolated naïve CD4+ T-cells with polybrene (8 µg/mL) prior to spinoculation. Afterwards, cells were cultured in complete RPMI-1640 media with 30 IU/mL of IL-2.

HIV-1 latency reactivation assay in primary TCM cells. We utilized the primary

TCM model of latency as described 190,363. In brief, naïve CD4+ T-cells cells were stimulated for 72 h with anti-CD3/CD28-antibody coated magnetic beads

(Dynabeads). After an additional 4 days of culture, cells were infected with VSV-

79

G-pseudotyped HIV-1-GFP 359 and cultured for 7 days to produce latently infected

TCM. Next, cells were transduced with lentiviral vectors containing vector control or SAMHD1 shRNA for 3 days before activation with or without anti-CD3/CD28 antibody-coated magnetic beads for 3 days. HIV-1 reactivation was measured by flow cytometry.

3.4 Results

Exogenous SAMHD1 expression suppresses HIV-1 LTR-driven gene expression in HEK293T cells. Transcriptional activation of the HIV-1 provirus is regulated by interactions between the LTR promoter and several host and viral proteins 355. However, the effect of SAMHD1 expression on HIV-1 LTR-driven gene expression is unknown. To address this question, we performed an HIV-1 LTR- driven firefly luciferase (FF-luc) reporter assay using HEK293T cells. To examine transfection efficiency, a Renilla luciferase (Ren-luc) reporter driven by the herpes simplex virus (HSV) thymidine kinase (TK) promoter was used as a control 368.

Expression of increasing levels of exogenous SAMHD1 did not change Ren-luc protein or mRNA expression (Figure 19A-C), indicating comparable transfection efficiency among different samples, and that SAMHD1 overexpression did not affect TK-promoter driven gene expression. In contrast, when normalized with the

Ren-luc control and compared to an empty vector, SAMHD1 expression resulted in 70-85% suppression of FF-luc activity (Figure 19D) and FF-luc mRNA levels

80

(Figure 19E) in a dose-dependent manner. These data suggest that exogenous

SAMHD1 expression suppresses HIV-1 LTR-driven gene expression at the level of gene transcription.

SAMHD1 silencing in THP-1 cells enhances HIV-1 LTR-driven gene expression. To determine whether endogenous SAMHD1 can suppress LTR- driven gene expression in cells, we performed the HIV-1 LTR reporter assay using human monocytic THP-1 cells expressing a high level of endogenous SAMHD1

(Ctrl) and SAMHD1 knockout (KO)255. THP-1 control or KO cells were nucleofected with plasmids expressing FF-luc and Ren-luc. The lack of SAMHD1 expression in

THP-1 KO cells was confirmed by immunoblotting (Figure 19F). Consistent with the results from HEK293T cells, the Ren-luc activity was unchanged in THP-1 control and KO cells, confirming comparable transfection (Figure 19G). When normalized with Ren-luc activity, KO cells showed a 4.5-fold increase of FF-Luc activity compared to control cells (Figure 19H), indicating that endogenous

SAMHD1 impairs HIV-1 LTR-driven gene expression in THP-1 cells.

81

A pSAMHD1 (ng) V 250 500 1000 kDa 72- HA-SAMHD1

36- GAPDH

0.0 0.3 0.6 1.0 Relative SAMHD1 levels

B C

1.5 1.5

y

t

A

i

v

N

i t

1.0 R 1.0

c

m

a

c c

u

u

l

l

- - n

0.5 n 0.5

e

e

R R

0.0 0.0 V 250 500 1000 V 250 500 1000 pSAMHD1 (ng) pSAMHD1 (ng) D E

1.2 1.2

y

t A

i 1.0 1.0

v

N

i

t R

c 0.8 0.8

m

a

c

c u

u **** l

l 0.6 0.6

-

- n

n **** e

e 0.4 0.4

R

R

/

/ F

F 0.2 F F 0.2 0.0 0.0 V 250 500 1000 V 250 500 1000 pSAMHD1 (ng) pSAMHD1 (ng) F G H

1.2 6

y t

i **

y

t

v i

THP-1 cell 1.0 i 5

t

v

i

c

t a

c 0.8 4

Ctrl KO

a c

kDa

u c

0.6 l 3

-

u l

72- n

SAMHD1 -

0.4 e 2

n

e

R

/ R 36- 0.2 F 1

GAPDH F 0.0 0 Ctrl KO Ctrl KO

Figure 19: SAMHD1 suppresses HIV-1 LTR-driven luciferase expression. (A-E) An HIV-1 LTR-driven firefly luciferase (FF-Luc) construct was co-transfected with an empty vector (V) or increasing amounts of a plasmid encoding HA-tagged SAMHD1 (pSAMHD1) into HEK293T cells. Co-transfection of a construct encoding 82

HSV TK-driven Renilla luciferase (Ren-Luc) was used as a control of transfection efficiency. (A) Overexpression of SAMHD1 was confirmed by immunoblotting. GAPDH was used as a loading control. Quantification of relative SAMHD1 expression levels by densitometry was normalized to GAPDH, with the level of 1000 ng pSAMHD1 sample set as 1. Ren-luc activity (B) and mRNA (C), and FF- luc activity (D) and mRNA (E) were measured at 24 h post-transfection. (B) Ren- luc activity was normalized to total protein concentration. (D and E) FF-luc activity and mRNA levels were normalized to Ren-luc activity and mRNA levels, with vector levels set as 1. (B-E) Error bars show standard deviation (SD) of at least three independent experiments as analyzed by one-way ANOVA with Dunnett’s multiple comparison post test. ****, p≤0.0001 compared to vector control cells. (F- H) FF-luc and Ren-luc constructs were expressed by nucleofection in THP-1 control (Ctrl) cells or SAMHD1 knockout (KO) cells. SAMHD1 KO was confirmed by immunoblotting, with GAPDH used as a loading control (F). Luciferase activity was measured at 48 h post-transduction, and raw Ren-luc values were normalized to total protein (G), and FF-luc activity normalized to Ren-luc activity (H). **, p≤ 0.01 compared to control cells.

SAMHD1 suppresses gene expression driven by the LTR from HIV-1 and

HTLV-1, but not from MLV. To examine the specificity of SAMHD1-mediated suppression of LTR-driven gene expression, we tested luciferase reporters driven by LTR promoters derived from HTLV-1 or MLV LTR in addition to the HIV-1 LTR

FF-luc reporter. The TK promoter-driven Ren-luc reporter was used as a transfection control. As HIV-1 and HTLV-1 utilize viral proteins Tat 63 and Tax 369 respectively, to enhance viral transcription via transactivation, we compared the ability of SAMHD1 to suppress HIV-1 and HTLV-1 LTR-driven gene expression with or without transactivation. Increased levels of exogenous SAMHD1 expression in transfected HEK293T cells were confirmed by immunoblotting

83

(Figure 20A, C, E). Although SAMHD1 suppressed HIV-1 LTR-driven gene expression in the absence of Tat (Figure 20B, black bars), Tat expression led to a 20 to 28-fold enhancement of HIV-1 LTR activity that was not suppressed by

SAMHD1 (Figure 20B, gray bars). Conversely, HTLV-1-LTR activity was potently suppressed by SAMHD1 expression, with up to 75% reduction in luciferase activity at the highest level of SAMHD1 expression in the presence or absence of Tax

(Figure 20D). SAMHD1 expression had no effect on MLV-LTR activity (Figure

20F). These data suggest that SAMHD1 selectively suppresses retroviral LTR- driven gene expression.

84

A B

50

y t

i - Tat

v 40 i

t + Tat

- Tat + Tat c 30

a

c 20

u l V 25 75 225 V 25 75 225 pSAMHD1 (ng) - 10

kDa R

T 1.5

72- L

HA-SAMHD1

1 1.0 *

- ** V 36- GAPDH I 0.5 H 0.0 V 25 75 225 pSAMHD1 (ng)

C D

y **** t

i 1.3

v i t 1.0

+ Tax - Tax c a

0.7

c

u l V 25 75 225 V 225 pSAMHD1 (ng) - 0.4

kDa R

T 0.1

72- L

HA-SAMHD1 0.02 1

- **** V

L 0.01

36- GAPDH T H 0.00 V 25 75 225 V 225 pSAMHD1 (ng) + Tax - Tax

E F y

t 1.2

i

v i

t 1.0

c a

V 25 75 225 pSAMHD1 (ng) 0.8 c

kDa u l

- 0.6

72- HA-SAMHD1 R

T 0.4

L

36- GAPDH V 0.2 L

M 0.0 V 25 75 225 pSAMHD1 (ng)

Figure 20: SAMHD1 suppresses gene expression driven by the LTR from HIV-1 and HTLV-1, but not from MLV. (A-F) HEK293T cells were transfected with an empty vector (V) or increasing amounts of constructs expressing HA-tagged SAMHD1 and either an HIV-1 LTR- driven FF-luc construct with or without HIV-1 Tat-expressing plasmid (A-B), HTLV- 1 LTR-driven FF-luc construct with or without HTLV-1 Tax-encoding plasmid (C- D), or MLV LTR-driven FF-luc construct (E-F). Overexpression of SAMHD1 was analyzed by immunoblotting (A, C, E) with GAPDH as a loading control. Co- transfection of Ren-luc was used as a control of transfection efficiency, with LTR- driven FF-luc activity normalized to Ren-luc activity. (B, D, F). Luciferase activity was determined 24 h post transfection. Error bars show standard error mean 85

(SEM) of three (HIV-luc +/- tat, HTLV-luc +tat) or two (HTLV-luc -tat, MLV-luc) independent experiments. Statistical analysis was performed by one-way ANOVA with Dunnett’s multiple comparison post test. *, p≤ 0.05, **, p≤ 0.01, and ****, p≤0.0001, compared to vector (V) control cells.

Nonphosphorylated and dNTPase-inactive SAMHD1 mutants have impaired suppression of HIV-1 LTR activity. SAMHD1 is predominantly phosphorylated in HEK293T cells253,341. To assess the effect of dNTPase activity and T592 phosphorylation of SAMHD1 on suppression of LTR-driven gene expression, we tested a catalytically inactive SAMHD1 mutant HD/RN276 and a nonphosphorylated

T592A mutant311. HEK293T cells were transfected with increasing amounts of plasmids encoding WT, T592A, or HD/RN mutant SAMHD1, along with the HIV-1

LTR-driven FF-luc reporter. Comparable WT and mutant SAMHD1 expression was confirmed by immunoblotting (Figure 21A). Undetectable phosphorylation of the

T592A mutant was confirmed in our previous studies308. The TK promoter-driven

Ren-luc reporter showed similar activity across all samples (Figure 21B), confirming comparable transfection efficiency. Compared to the vector control and normalized with Ren-luc activity, WT SAMHD1 suppressed HIV-1 LTR-driven FF- luc expression up to 60% in a dose-dependent manner (Figure 21C). In contrast to WT SAMHD1, low amounts of HD/RN or T592A mutants (125 and 250 ng plasmid input) did not significantly inhibit HIV-1 LTR activity. However, a modest inhibition of LTR-driven FF-luc activity (between 25-31%) was observed at the

86

highest levels of mutant SAMHD1 expression (500 ng plasmid input) (Figure 21C).

These results indicated that T592A and HD/RN mutants have a diminished ability to suppress HIV-1 LTR-driven gene expression compared to WT SAMHD1, suggesting that SAMHD1-mediated suppression of HIV-1 gene expression is partially dependent on its dNTPase activity and T592 phosphorylation.

87

A SAMHD1

WT T592A HD/RN

kDa V 125 250 500 125 250 500 125 250 500 pSAMHD1(ng) 72- HA-SAMHD1 36- GAPDH 0.0 0.1 0.6 1.0 0.1 0.3 0.8 0.4 0.7 0.9

Relative SAMHD1 levels B

1.50

y

t

i v

i 1.25

t

c

a

c 1.00

u

l -

n 0.75

e

R

e 0.50

v

i

t a

l 0.25

e R 0.00 V 125 250 500 125 250 500 125 250 500 WT T592A HD/RN pSAMHD1 (ng)

C y

t 1.50

i

v

i t

c 1.25

a

c u

l 1.00 -

n **** ** *

e 0.75 R

/ **** ****

F F

0.50

e

v

i t

a 0.25

l e

R 0.00 V 125 250 500 125 250 500 125 250 500 WT T592A HD/RN pSAMHD1 (ng)

Figure 21: Nonphosphorylated and dNTPase-inactive SAMHD1 mutants have impaired suppression of HIV-1 LTR activity. (A-C) An HIV-1 LTR-driven FF-luc construct was co-transfected with increasing amounts of plasmids encoding HA-tagged WT, nonphosphorylated T592A, or dNTPase-inactive HD/RN mutant SAMHD1 into HEK293T cells. Co-transfection of Ren-luc was used as a control of transfection efficiency. (A) SAMHD1 expression was confirmed by immunoblotting. GAPDH was used as a loading control. Quantification of relative SAMHD1 expression levels by densitometry was 88

normalized to GAPDH. Relative Ren-luc units were normalized to total protein concentration (B), and relative FF-luc units were normalized to Ren-luc levels (C). Vector cell luciferase activity set as 1. Statistical analysis was performed by one- way ANOVA with Dunnett’s multiple comparison post test. Error bars show SD of at least three independent experiments. *, p≤ 0.05, **, p≤0.01, and ****, p≤0.0001 compared to vector control cells.

WT SAMHD1 impairs HIV-1 reactivation in latently infected J-Lat cells. To investigate whether SAMHD1 suppresses HIV-1 gene expression in CD4+ T-cells, we used Jurkat CD4+ T cell line-derived J-Lat cells331. J-Lat cells have been used as an HIV-1 latency model, as they contain a full-length HIV-1 provirus with a green fluorescent protein (gfp) gene inserted in the nef region331. Treatment of J-Lat cells with latency reversing agents (LRAs) results in activation of LTR-driven gene expression, indicated by an increase in GFP expression189,331. As J-Lat cells do not express detectable endogenous SAMHD1 protein, likely due to gene promoter methylation as reported in Jurkat cells370, we stably expressed WT SAMHD1 in J-

Lat cells by lentiviral transduction. Empty vector-transduced cells were used as a control. Previous studies showed that efficient SAMHD1 expression driven by the cytomegalovirus (CMV) immediate-early promoter of stably integrated lentiviral vector in monocytic cell lines is dependent on treatment of cells with phorbol 12- myristate 13-acetate (PMA)253,301, which is a protein C kinase agonist that activates the NF-B signaling pathway371,372.

89

To activate HIV-1 gene expression in J-Lat cells we applied two LRAs, tumor necrosis factor alpha (TNF) which induces HIV-1 gene expression by activating the NF-B pathway189,373,374, and PMA in conjunction with ionomycin (PMA+i) that has been shown to be the strongest activator of HIV-1 gene expression in several

J-Lat cell clones189. Treatment of J-Lat cells with TNF (Figure 22A) resulted in

GFP expression in 38% of vector control cells (Figure 22B), consistent with published data189,331. In contrast, expression of WT SAMHD1 reduced TNFα- induced GFP expression to 27% (Figure 22B), suggesting that SAMHD1 impairs

TNF-induced HIV-1 reactivation. Treatment of WT SAMHD1-expressing J-Lat cells with PMA+i resulted in a significant increase in SAMHD1 expression (Figure

22A) and a significant decrease in GFP-expression by 20% compared to vector control cells (Figure 22B). While PMA+i treatment resulted in a 1.5-fold reduction of GFP mean fluorescence intensity (MFI) of SAMHD1-expressing cells compared to vector, the MFI of TNF-treated cells was not significantly reduced by SAMHD1 expression (Figure 22B).

To examine whether increased SAMHD1 expression in J-Lat cells could more efficiently suppress HIV-1 reactivation, we compared J-Lat cells treated with two

PMA+i concentrations with a 8-fold difference (Figure 22C and 22D). To examine whether the dNTPase activity or T592 phosphorylation of SAMHD1 affects its suppression of HIV-1 reactivation in J-Lat cells, we performed the analysis in J-Lat

90

cells stably expressing WT SAMHD1, T592A, or HD/RN mutant by lentiviral transduction. Similar expression levels of WT SAMHD1 and the mutants were observed in 1× PMA+i-treated cells, while 8× PMA+i treatment highly increased the expression levels of WT SAMHD1, and mutant SAMHD1 to a lesser degree

(Figure 22C). WT SAMHD1-expressing cells had a 15% lower GFP-positive cell population compared to vector control cells at 1× PMA+i; however, this was not further enhanced with increased WT SAMHD1 expression at 8× PMA+i (Figure

22D). While WT SAMHD1 suppressed HIV-1 reactivation at both 1× and 8× PMA+i treatment, neither T592A nor HD/RN mutant had a suppression effect (Figure

22D), suggesting that T592 phosphorylation and dNTPase activity of SAMHD1 are likely involved in reactivation of HIV-1 latency.

91

A B 60 1400 **** Vector

) 1200 **** WT SAMHD1

% 50 **

(

s

Vector WT SAMHD1 l

l

s l

e **

l 1000

c e

40

TNFɑ PMA+i TNFɑ PMA+i c

+

P

kDa e 800

v

F i

t 30 i

72- SAMHD1 G

f

s 600

o

o

I

p 20 -

36- GAPDH F 400

P

M F

0.0 0.0 1.0 3.3 G 10 200 Relative SAMHD1 levels 0 0 α α TNF TNF PMA+i PMA+i C D 60 1x PMA+i 8x PMA+i 1x PMA+i ) 8x PMA+i

% 50 ( A N A N 2 R 2 R s / / l 9 9 l T 5 D T 5 D

V W T H V W T H e 40 **

c

e

kDa v i

t 30

i ***

72- HA-SAMHD1 s o

p 20

- P 36- GAPDH F G 10 0.0 0.4 0.4 0.4 0.0 1.0 0.8 0.7 0

Relative SAMHD1 levels WT WT Vector T592A HD/RN Vector T592A HD/RN

Figure 22: WT SAMHD1 impairs HIV-1 reactivation in latently infected J-Lat cells. (A, C) HA-tagged SAMHD1 WT or mutants, or an empty vector were stably expressed in J-Lat cells by lentiviral transduction. Quantification of relative SAMHD1 expression levels by densitometry was normalized to GAPDH. (A-B) The cells were treated with either TNF, or PMA+i. At 24 h post-treatment, the expression of SAMHD1 was detected by IB. The percentage of GFP-positive cells and the relative GFP mean fluorescence intensity (MFI) were determined by flow cytometry (B). J-Lat cells expressing T592A and HD/RN mutants were treated with 1× or 8× PMA+i (1× corresponds to 16 nM PMA and 0.5 µM ionomycin), with expression of SAMHD1 measured and quantified by immunoblotting (C). Latency reversal, as measured by percentage of GFP-positive cell population and MFI of GFP-positive cells, was determined by flow cytometry (D). Error bars in (B and D) represent SD from at least three independent experiments analyzed by two-way ANOVA and Dunnett’s multiple comparisons test. **, p≤0.01, ***, p≤0.001, and ****, p≤0.0001 (compared to vector cells in panels B and D).

92

WT SAMHD1 binds to HIV-1 LTR of proviral DNA in J-Lat cells. One common mechanism by which host proteins modulate HIV-1 LTR activity is transcriptional repression by directly binding to the promoter174. SAMHD1 is a DNA binding protein318 capable of interacting with in vitro transcribed HIV-1 gag DNA fragments313. However, the interaction between SAMHD1 and integrated HIV-1 proviral DNA in cells has not been reported. To address this question, we performed a chromatin immunoprecipitation coupled with quantitative real-time

PCR (ChIP-qPCR) experiment in J-Lat cells expressing WT, T592A, or HD/RN

SAMHD1. To induce high levels of SAMHD1 for efficient immunoprecipitation (IP), we treated the cells with increased PMA+i concentrations. Treatment with 8×

PMA+i allowed for maximum SAMHD1 expression without cell death (data not shown). However, WT SAMHD1 expressed 20-30% greater than mutants under this condition (Figure 22C). We treated the WT SAMHD1-expressing cells with

50% less PMA+i compared to that used for mutant-expressing cells, and obtained comparable levels of SAMHD1 (Figure 23A). HIV-1 reactivation in all cell lines was measured by GFP expression (Figure 23B). The WT SAMHD1-expressing

J-Lat cells had a 17% lower GFP-positive population compared to vector control,

T592A, and HD/RN-expressing cells, which was reflected in a 1.6-fold lower MFI

(Figure 23B).

93

After IP of WT or mutant SAMHD1 from cells treated with PMA+i (Figure 23A), total bound DNA was eluted and quantified by qPCR. We used PCR primers specific for different regions in the HIV-1 genome, including the LTR, gag, vpr, and rev genes, to characterize the regions of interaction between SAMHD1 and proviral

DNA (Figure 23C, Table 1). We also included gfp-specific PCR primers as an additional control, as gfp is a non-viral gene inserted in the nef gene of HIV-1 in J-

Lat cells 331. We observed that only DNA fragments derived from the LTR (12% of input) bound to WT SAMHD1 (Figure 23D). WT SAMHD1 did not bind other HIV-

1 genes tested or the gfp gene. These data suggest that the SAMHD1-DNA interaction occurs in the LTR promoter region of HIV-1 provirus. Interestingly, analysis of the DNA eluted from IP products of T592A and HD/RN SAMHD1 revealed that neither mutant bound to tested HIV-1 DNA sequences or gfp cDNA

(Figure 23D). Taken together, these data indicate that mutant SAMHD1 cannot suppress latency reactivation or bind to proviral DNA, suggesting that direct binding to the HIV-1 LTR is partially responsible for the mechanism of SAMHD1- mediated suppression of LTR-driven gene expression.

94

A B

50 1000

)

%

(

s

l l

Input IP:HA s 40 800

l

e

l

c

e

c

r r +

o A N o A N 30 *** 600 e t 2 R t 2 R P

c 9 / c 9 / *** v

T T F e 5 D e 5 D i V W T H t

V W T H i G

kDa s

20 f 400 o HA-SAMHD1 o

72-

I

p

- F

0.0 0.8 0.6 0.7 0.0 1.0 1.0 1.0 P 10

M 200 F Relative SAMHD1 levels G 0 0

WT WT Vector T592AHD/RN Vector T592AHD/RN C gfp tat nef rev vpu 5’ LTR gag vif 3’ LTR

pol vpr env

ltr gag vpr rev gfp Amplicon 1 2 3 4 5 6 7 8 9

HIV-1 genome (Kb) D 15 WT

) T592A

% ( HD/RN

A 10

N

D

t

u 5

p

n I 0 ltr gag vpr rev gfp

Figure 23: WT SAMHD1 binds to proviral DNA in latently infected J-Lat cells. (A-D) J-Lat cells were seeded in the presence of PMA+i for 24 h. SAMHD1 expression in input and IP lysates was analyzed by immunoblotting and densitometry analysis (A). Latency reversal, as measured by percentage of GFP- positive cell population and MFI of GFP-positive cells (B). Three independent experiments were analyzed by two-way ANOVA and Dunnett’s multiple comparisons test, with error bars in (B) representing SEM. ***, p≤0.001 compared to vector cells. (C) Diagram of the location of the qPCR amplicons. Quantitative PCR data were normalized to spliced GAPDH levels and presented as percent of input in SAMHD1-expressing cells over vector cells (D). Error bars in (D) represent SEM from two independent experiments.

95

Purified recombinant WT SAMHD1 binds to HIV-1 LTR fragments in vitro. To investigate the underlying mechanism of SAMHD1-mediated suppression of HIV-

1 LTR activity and viral reactivation in cells, we determined whether this suppression effect correlated with SAMHD1 binding to HIV-1 LTR in vitro.

Fluorescence anisotropy (FA)365 was used to measure the binding of WT

SAMHD1, T592A or HD/RN mutant to a 90-mer 5'-6-carboxyfluorescein (6-FAM)- labeled DNA oligonucleotide derived from the HIV-1 LTR (Table 2). Binding was measured over a range of SAMHD1 concentrations and three monovalent ion concentrations (50, 100, and 150 mM) to determine whether the interaction is mediated by electrostatic interactions. While WT SAMHD1 binding to the HIV-1

LTR fragment was detected at all three salt concentrations tested, higher salt reduced the observed binding (Figure 24A-C), which suggests that the interaction is mediated, at least in part, by electrostatic contacts. In contrast, no significant binding was observed for the T592A and HD/RN mutants even at the highest protein concentration (8,300 nM) (Figure 24A-C). For WT SAMHD1, saturated or near-saturated binding was observed at 50 and 100 mM monovalent ions (Figure

24A-B) with calculated apparent Kd values of 93 ± 8 and 242 ± 51 nM, respectively.

Importantly, none of the SAMHD1 proteins bound to a 90-mer 6-FAM-labeled DNA oligonucleotide derived from a scrambled sequence of the HIV-1 LTR (Table 2), even at low (50 mM) monovalent ion concentration (Figure 24D). These data

96

indicate that WT SAMHD1 binds specifically to HIV-1 LTR-derived fragments in a salt sensitive manner.

A B Monovalent ions: 50 mM Monovalent ions: 100 mM WT DNA: HIV-1 LTR DNA: HIV-1 LTR 0.25 0.25 T592A SAMHD1

y HD/RN y 0.19

0.19 p

p

o

o

r

r

t t

o 0.13

o 0.13

s

s

i

i

n n

A 0.07

A 0.07

0.01 0.01 1 100 10000 1 100 10000 SAMHD1 (nM) SAMHD1 (nM) C D Monovalent ions: 150 mM Monovalent ions: 50 mM DNA: HIV-1 LTR DNA: Scramble

0.25 0.25 y

y 0.19 0.19

p

p

o

o

r

r

t

t o

0.13 o 0.13

s

s

i

i

n

n A 0.07 A 0.07

0.01 0.01 1 100 10000 1 100 10000 SAMHD1 (nM) SAMHD1 (nM)

Figure 24: Specific binding of WT SAMHD1 to an HIV-1 LTR fragment in vitro Results of FA binding assays for WT, T592A or HD/RN mutant SAMHD1 binding to a 90-mer fragment of the HIV-1 LTR in 50 mM, 100 mM, or 150 mM monovalent ions (25, 50, or 75 mM of each NaCl and KCl) (A-C, respectively). Binding to a 90- mer scrambled DNA oligonucleotide was also tested at 50 mM monovalent ions (25 mM of each NaCl and KCl) (D). Error bars indicate the SD from three independent experiments.

SAMHD1 knockdown promotes HIV-1 reactivation in latently infected primary CD4+ T-cells. To examine the effect of endogenous SAMHD1 on HIV-1 97

reactivation in primary CD4+ T-cells, we utilized the established central memory T-

190,359 cell (TCM) model of HIV-1 latency as a depicted in the protocol in Figure 25A.

Using a SAMHD1-specific shRNA and established method190,363, we knocked down 40-50% of endogenous SAMHD1 expression in latently infected TCM derived from naïve CD4+ T-cells isolated from three healthy donors (Figure 25B). Next, we activated latently infected GFP-reporter HIV-1 in TCM by CD3/CD28 antibody treatment and measured GFP expression as a readout of latency reactivation. As a negative control, TCM treated with media did not express GFP (1% background).

Upon activation of latently infected TCM, partial knockdown of SAMHD1 enhanced

HIV-1 reactivation by 1.6-fold compared to cells transduced with an empty shRNA vector, as measured by % GFP-positive cell population and MFI of GFP-positive cells (Figure 25B-C). These results confirm that endogenous SAMHD1 acts as a negative regulator of HIV-1 reactivation in latently infected primary CD4+ T-cells.

98

A

PBMC Remove Transduce Transduce Anti-CD3/ cells with cells with +/- Anti-CD3/ Measure GFP CD28 HIV-GFP shRNA CD28 expression Naïve CD4+ T-cell

Day -7 -4 0 7 10 13

Anti-CD3/CD28 IL-2

Naïve Activated Memory Anti-CD3/CD28

Reactivated B C

2.5 * 80 * Donor

s

l l

e 1

s c

l 2.0

Donor 1 Donor 2 Donor 3 l + 60

e 2

P

c

F

1 1 1 +

D D D G 3

r r r P 1.5

o H o H o H f

t t t F

c M c M c M o

G

e A e A e A I 40

shRNA

kDa V S V S V S

F

e

v M i 1.0

72-

SAMHD1 t

e

a

l

v

i e

t 20

R a

36- GAPDH 0.5 l e

1.0 0.5 1.0 0.6 1.0 0.5 R 0.0 0 Relative SAMHD1 levels

Vector Vector sh SAMHD1 sh SAMHD1

Figure 25: SAMHD1 impairs HIV-1 reactivation in latently infected primary CD4+ T-cells. (A) Protocol summary. Naïve CD4+ T-cells were isolated from PBMCs from three different healthy donors, activated by incubation with anti-CD3/CD28 antibody- coated beads, and transduced with a single-cycle HIV-1 containing a GFP reporter (HIV-GFP) to produce a primary TCM cell model of latency. After infection and culture to produce latently infected quiescent CD4+ T-cells, transduction with either empty vector or lentiviral vectors containing SAMHD1-specific shRNA to knockdown of SAMHD1. SAMHD1 expression was measured by immunoblotting and GAPDH was a loading control (B). After stimulation of the cells with anti- CD3/CD28, HIV-1 reactivation was measured by flow cytometry. Relative changes in the GFP-positive cell population and MFI of GFP-positive cells were quantified (C), with each line indicating the result from one donor. *, p≤ 0.05.

99

Table 1: PCR primer sequences.

PCR primers DNA sequence (5' -3') ltr F 1 CGAACAGGGACTTGAAAGC ltr R 1 CATCTCTCTCCTTCTAGCCTC gag F CTAGAACGATTCGCAGTTAATCCT gag R CTATCCTTTGATGCACACAATAGAG vpr F GCCGCTCTAGAACCATGGAACAAGCCCCAGAAG ACCAA vpr R GCCGCCGGTACCGGATCTACTGGCTCCATTTCTT GCT rev F CGGCGACTGCCTTAGGCATC rev R CTCGGGATTGGGAGGTGGGTC gapdh F GATGGCATGGACTGTGGTCATG gapdh R TGGATATTGCCATCAATGACC gfp F ACGTAAACGGCCACAAGTTC gfp R AAGTCGTGCTGCTTCATGTG Ren-luc 2 F GAGCATCAAGATAAGATCAAAGCA Ren-luc 2 R CTTCACCTTTCTCTTTGAATGGTT FF-luc 3 F GGTTGGCAGAAGCTATGAAAC FF-luc 3 R CATTATAAATGTCGTTCGCGGG

1F, forward; R, reverse. 2Ren-luc, Renilla luciferase; 3FF-Luc, firefly luciferase;

100

Table 2: Sequences of oligonucleotides used in anisotropy binding assays.

Oligonucleotides (90-mer) DNA sequence (5' -3'), 5'-6-FAM-labeled

HIV-1 LTR AGCAGTGGCGCCCGAACAGGGACTTGAAA GCGAAAGTAAAGCCAGAGGAGATCTCTCGA CGCAGGACTCGGCTTGCTGAAGCGCGCAC GG

Scrambled DNA ACTAGCTCAAGCGGGGGACACCCAGGTGT CTCCAACAGTCCGTAGATCGGACGGAGAAG AGGGGACCCCGCTAATGAGCGTGAGCAGA GA

3.5 Discussion

One of the hallmarks of HIV-1 persistence is the maintenance of a long-lived stable proviral reservoir that is formed after infection in resting CD4+ T-cells26,330.

Although the integrated provirus is transcriptionally silent, it is capable of full reactivation and production of infectious virus upon discontinuation of therapy or treatment with LRAs26,330,360. In this study, we tested the hypothesis that SAMHD1 plays a role in negatively regulating HIV-1 reactivation and viral latency by suppressing HIV-1 LTR-driven gene expression.

We demonstrated that WT SAMHD1 suppresses HIV-1 LTR-driven gene expression, in the absence of Tat, in HEK293T and THP-1 cells. Previous work 101

confirmed that SAMHD1 does not degrade HIV-1 genomic RNA or mRNA308,318, thereby excluding the possibility that mRNA degradation causes the suppression.

We observed that SAMHD1 potently suppressed the HTLV-1 LTR independently of Tax expression but had no effect on the MLV LTR or HIV-1 LTR in the presence of Tat. These differences in suppression could be the result of variations in transcriptional control of each LTR. Tat transactivation of the HIV-1 LTR occurs through direct binding of Tat to the HIV-1 transactivation-responsive region63,375.

Tat transactivation may saturate LTR activity and mask a SAMHD1-mediated suppressive effect. The Tat-TAR binding affinity is particularly tight, with a Kd of 1-

3 nM, making effective competition by SAMHD1 very unlikely63,79. Moreover, we previously observed that SAMHD1 expression does not affect HIV-1 Gag expression from transfected HIV-1 proviral DNA where Tat is present308.

Conversely, Tax transactivation occurs through the mediation of interactions with host factors, specifically the protein complex consisting of cAMP response element binding protein (CREB), the CREB binding protein, and p300369,376,377. Whether

SAMHD1 interacts with host proteins to further suppress HTLV-1 LTR activity through disruption of Tax activity is unknown. As a simple retrovirus, MLV does not encode transactivation accessory proteins; however, its LTR has several binding sites for transcription factors, including nuclear factor 1378. Our data suggests that

SAMHD1-mediated suppression of LTR activity may be specific for complex

102

human retroviruses and could be influenced by certain transcription factors that bind to each respective LTR.

SAMHD1 enzyme activity can be regulated by mutations to its catalytic core or by post-translational modifications379. Thus, we used two SAMHD1 mutants to determine the contribution of phosphorylation and dNTPase activity to the suppression of LTR-driven gene expression. The nonphosphorylated T592A mutant had reduced ability to suppress HIV-1 LTR-driven gene expression.

Additionally, the dNTPase-inactive HD/RN mutant did not efficiently suppress HIV-

1 LTR-driven gene expression. It is unlikely that a reduction in dNTP levels is required for the effect on LTR-driven gene expression as dNTP levels are high in

HEK293T cells despite SAMHD1 overexpression238. Interestingly, whereas WT

SAMHD1 was observed to bind specifically to the HIV-1 LTR both in ChIP-qPCR and in vitro binding experiments, the SAMHD1 mutants failed to bind to the HIV-1

DNA regions tested. It is possible that SAMHD1 oligomerization may play a role in the ability of SAMHD1 to bind DNA. Dimeric SAMHD1 binds ssNA; however, previous reports have shown that tetramerization of SAMHD1 inhibits NA binding318,328. Phosphorylation of SAMHD1 at residue T592 destabilizes tetramer formation and impairs the dNTPase activity of SAMHD1324,325, with binding of phosphomimetic T592E to ssRNA and ssDNA being identical to WT SAMHD1318.

In vitro, the HD/RN mutant tetramerizes to a greater extent than WT SAMHD1343,

103

and mutations of either H206 or D207 residues result in loss of ssDNA binding313.

It is possible that the T592A and HD/RN mutants form more stable tetramers and, as a consequence, lose the ability to bind the LTR and suppress activation.

However, experiments to determine the oligomeric states of WT, T592A, and

HD/RN SAMHD1 in the presence of fragments of the HIV-1 LTR can help to further test this possibility. Future studies are required to map the region of interaction between SAMHD1 and the HIV-1 LTR and to examine the contribution of binding to the suppression of LTR-driven gene expression. Together, our data suggests a mechanism for suppression of LTR activity in which WT SAMHD1 is able to bind directly to the LTR and possibly occlude transcription factors required for LTR activity.

As suppression of the HIV-1 LTR is a common mechanism contributing to latency174, we aimed to determine whether SAMHD1 affects latency reversal in cells. We utilized two HIV-1 latency cell models, the J-Lat cell line and primary

TCM cells190,331,359,363,380. In both models, SAMHD1 expression resulted in a suppression of latency reactivation. The modest effect observed could be due to saturation of the NF-B pathway by PMA and TNF (Figure 22) or anti-CD3/CD28

(Figure 25), as significant activation of the LTR may mask the suppressive effect of SAMHD1189,373,381,382.

104

In summary, our data indicate a correlation between SAMHD1 binding to the HIV-

1 LTR and SAMHD1-mediated suppression of viral gene expression and reactivation of HIV-1 latency, suggesting that SAMHD1 is among the host proteins involved in the transcriptional regulation of proviral DNA. Our results further implicate that the T592 and H206/D207 residues of SAMHD1 are important for

LTR binding and suppression of HIV-1 gene expression. Future studies using latently infected cells from HIV-1 patients and primary HIV-1 isolates will further inform the function and mechanisms of SAMHD1 as a novel modulator of HIV-1 latency.

3.6 Acknowledgements

I would like to thank Kathleen Boris-Lawrie, Patrick Green, Vineet KewalRamani,

Baek Kim, Nathaniel Landau, Vicente Planelles, and Jian-Hua Wang for sharing reagents. The following reagents were obtained through the NIH AIDS Reagent

Program, Division of AIDS, NIAID, NIH: J-Lat Full Length GFP Cells (clone 9.2) from Dr. Eric Verdin, and human rIL-2 from Dr. Maurice Gately, Hoffmann - La

Roche Inc.

3.7 Contributions

The purified recombinant SAMHD1 protein was produced by members of the Yong

Xiong lab. The IB showing expression levels of SAMHD1 in ctrl and KO THP-1

105

cells was performed by Serena Bonifati (Figure 19F). The HTLV-1-LTR and MLV-

1-LTR driven reporter assays and corresponding immunoblots were performed by

Sun Hee Kim and Corine St. Gelais (Figure 20C-F). All other experiments were performed by Jenna Antonucci.

106

Chapter 4: Mutations to mSAMHD1 that Destabilize the SAM-to-HD Domain

Tetramer Interface Abrogate HIV-1 Restriction

4.1 Abstract

Human SAMHD1 (hSAMHD1) is a retroviral restriction factor that blocks HIV-1 infection by depleting the cellular required for viral reverse transcription. SAMHD1 is allosterically activated by nucleotides that induce assembly of the active tetramer. Although the catalytic core of hSAMHD1 has been studied extensively, previous structures have not captured the regulatory

SAM domain. Although mouse SAMHD1 (mSAMHD1) and hSAMHD1 are highly similar in sequence and function, the flexible nature of hSAMHD1 has proved difficult to crystallize. In this collaborative study, we report the crystal structure of FL SAMHD1 by capturing mSAMHD1 structures. Based on these crystal structures, residues essential for the inter- and intra-subunit interaction between the SAM and HD domains were identified. Cell-based HIV-1 infection assays confirmed the importance of the identified residues and the inter- and intra- subunit interactions between the SAM and HD domain. Our results provide insights into the regulation of SAMHD1 activity, thereby facilitating the

107

improvement of mouse models and the development of new therapies for certain cancers, autoimmune diseases, and HIV-1 infection.

4.2 Introduction

The work presented in this chapter is part of a contribution to a larger published study (Buzovetsky et al, The SAM domain of mouse SAMHD1 is critical for its activation and regulation, Nature Communications 2018)383.

hSAMHD1 is a retroviral restriction factor that blocks HIV-1 infection by depleting the cellular dNTPs required for viral reverse transcription229,233,236,309,384-386. hSAMHD1 tightly controls the cellular dNTP levels through its deoxyribonucleoside triphosphate triphosphohydrolase (dNTPase) activity. Cellular dNTPs bind the two allosteric sites of each subunit of SAMHD1 to initiate tetramerization268,272,344 (as described in section 1.13 and Figure 9). The SAM domain of hSAMHD1 was shown to be dispensable for the tetramerization and dNTPase activity of hSAMHD1. In fact, removal of the SAM domain led to an enhanced enzymatic and antiviral activity in hSAMHD1 compared to FL protein268. Taken together, this data suggest that the SAM domain of hSAMHD1 may regulate the dNTPase activity of

SAMHD1: however, neither the function of the SAM domain nor the FL SAMHD1 crystal structure has been solved.

108

SAMHD1 is a protein that is conserved amongst mammalian species. Two isoforms of mSAMHD1, which share 72-74% protein sequence identities with hSAMHD1352, also restrict HIV-1 by their dNTPase activity271,352. As dysfunction of

SAMHD1 expression in humans results in disease, such as AGS and cancer

(detailed in 1.12), it is tempting to hypothesize that SAMHD1 null mouse models would be a great tool to study SAMHD1-related disease. However, the use of

SAMHD1 null mouse models remains controversial. Both of the two lineages of

SAMHD1 knockout mice generated by LoxP-FRT trap and Cre-Lox240 system387,388 did not exhibit disease, although they did display an increased spontaneous type I

IFN induction240,332,387. Although the activation and mechanism of restriction of hSAMHD1 has been well-studied, the understanding of the activation and regulation of mSAMHD1 remains incomplete.

The comparison between the molecular properties of human and mouse SAMHD1 using extensive biochemical, enzymatic, and structural studies led to the first FL crystal structure of mSAMHD1 tetramers383. These structures shed light on the structural basis for the SAMHD1 allosteric activation process and highlight the importance of the SAM domain in stabilizing the tetramer, including interactions that have not yet been observed in hSAMHD1 structures. Additionally, these results present a more complicated nucleotide-induced oligomer process for mSAMHD1 compared to hSAMHD1383.

109

Importantly, these biochemical, enzymatic, and structural studies identified several residues in human and mouse SAMHD1 that either stabilize or destabilize the

SAM-to-HD domain interaction. In this collaborative study, we utilize the identified mutants in functional cell-based viral restriction assays to determine the contribution of the SAM-to-HD interaction to HIV-1 restriction. The results provide new insights into the mechanisms of HIV-1 restriction by SAMHD1, the regulation of its antiviral activity, and its effect on dNTP homeostasis, thereby allowing for the improvement of mouse models for the study of HIV-1 and the development of novel therapy for certain cancers and autoimmune diseases.

4.3 Materials and Methods

HIV-1 restriction assay: Stable U937 cells expressing FL or mutant SAMHD1 were generated and cultured under puromycin selection as described 308. U937 cells were differentiated using phorbol 12-myristate 13-acetate (PMA) (100 ng/mL) for 24 h, PMA was removed and cells were cultured for a further 24 h. All cell lines were tested negative for mycoplasma contamination using a universal mycoplasma detection kit (ATCC, #30-101-2K). To quantitate HIV-1 infection in

PMA-differentiated SAMHD1 expressing stable U937 cell lines, cells were infected with single-cycle, vesicular stomatitis virus protein G (VSV-G) pseudotyped HIV-1

(HIV-Luc/VSV-G) at a multiplicity of infection of 0.5 for 2 h before removal of virus.

110

Cells were then cultured for 48 h prior to collection of cell lysates for luciferase assay (Promega) as described308.

4.4 Results

The HD domain of mSAMHD1 is insufficient for dNTPase activity. Several in vitro assays, including high performance liquid chromatography of highly purified recombinant mSAMHD1 with dGTP and malachite green (MG) colorimetric assays, determined that both isoforms of FL mSAMHD1 have similar rates of dNTP hydrolysis as hSAMHD1383. Removal of the SAM domain of mouse SAMHD1

(mHD), however, results in severely reduced catalytic activity. This is in contrast to the hHD which maintains higher dNTPase activity than FL hSAMHD1383. It was further confirmed that mHD is deficient in tetramer formation, thereby suggesting the SAM domain contributes to the allosteric activation of mSAMHD1383.

To test the ability of mHD to restrict HIV-1 infection, we stably expressed FL and

HD-domain only mSAMHD1 isoform 2 alongside the FL mouse isoform 1 protein in U937 cells (Figure 26A). After differentiation upon PMA treatment, the cells were infected with an HIV-1 luciferase reporter virus. While the FL mSAMHD1 restricted HIV-1 infection, the mHD domain alone could not. Interestingly, there was a 1.6-fold enhancement of infection in mHD-expressing cells compared to vector control cells (Figure 26B). Taken together, these data suggest that while

111

the HD domain of hSAMHD1 maintains strong antiviral activities, the HD domain of mSAMHD1 is not sufficient for HIV-1 restriction. This important observation allows for comparative studies to discern the contribution of the SAM domain to hSAMHD1.

A B

200 iso1 iso2

iso1 iso2 ) 175 ***

% ( Vector FL FL HD 150

kDa n

o i

76- t 125

HA-SAMHD1 c e

60- f 100

n

i

36- GAPDH e 75

v i

t 50

a l e 25 R *** *** 0 Vector FL FL HD

Figure 26: The HD domain of mSAMHD1 is insufficient for dNTPase activity. (A) Immunoblotting confirmed HA-tagged mSAMHD1 expression. GAPDH was used as a loading control. (B) HIV-1 restriction was measured in mSAMHD1- expressing U937 cells after PMA differentiation. Single-cycle HIV-1 infection of vector control cells was set as 100%. FL, full length. HD, HD domain only. Each experiment was performed in triplicate. Error bars, s.d. ***p ≤ 0.0001.

The crystal structure of full-length mSAMHD1 iso1 in complex with GTP and dGTP captures the relationship between the SAM and HD domains. Although the FL crystal structure of hSAMHD1 would allow for a more complete 112

understanding of the functional state of the protein, previous efforts have only resulted in crystallization of the HD domain233,268,344, as the disordered SAM region could not be visualized324. That being said, the SAM domain is not critical for hSAMHD1 activity and crystallization of the HD domain has given incredible insight into the mechanism of dNTP binding and hydrolysis. However, as the mSAM domain is required for dNTP hydrolysis and likely regulates mSAMHD1 activity, It was essential to solve the structure of both the SAM and HD domains of mSAMHD1.

It was found that the overall folds of both the SAM and HD domains of mSAMHD1 are conserved with hSAMHD1383. Interestingly, the most significant deviation between mSAMHD1 and hSAMHD1 subunit structures occurs at the mSAM-to- mHD domain linker region383. The interaction between the SAM and HD domain in both intra-subunit (within the same subunit) and inter-subunit (from adjacent subunits) interactions help position the SAM domain in the correct orientation to cap the allosteric sites383. This stabilization of the tetramer through SAM-to-HD domain interactions may not be essential in hSAMHD1, as the SAM domain has been shown to be dispensable for hSAMHD1 enzyme activity. To test this hypothesis, several residues essential to the SAM-to-HD domain interaction in mSAMHD1 were identified. Modeling the corresponding residues in hSAMHD1

113

into the SAM-to-HD interfaces of mSAMHD1 predicts disruption of the observed

tetramer-stabilizing interactions383 (Figure 27 and Table 3).

A B Inter-subunit interface Intra-subunit interface

S566T S142 S566 S142I R143H E225 R143 E225

F112 N567 F112C N567A Q137 Q137 H161 H161 Mutant Model F109 F109L Y289 Y289 A B Inter-subunit interface Intra-subunit interface Mutant Model S566T S142 S566 S142I R143H E225 R143 E225

F112 N567 F112C N567A Q137 Q137 H161 H161 Mutant Model F109 F109L Y289 Y289

Mutant Model

Figure 27: Crystal structure indicating the transparent surface representation of SAM-to-HD interface.

114

The important residues shown in sticks. In both panel A and B: Left, original configuration and residues in WT mSAMHD1 protein. Right, model of SAM-to- HD interface after mSAMHD1 residues are mutated to the corresponding hSAMHD1 residues. The red cross in A indicates a steric clash.

Table 3: Characterization of human and mouse SAMHD1 mutants and the predicted effect on HIV-1 restriction based on crystal structure data.

SAMHD1 variant Location of Tetramer dNTPase HIV-1 mutation formation activity restriction

mIso 1/2 Full-length N/A Yes Yes Yes (mFL)

mIso 2 SAM Less than No No HD only domain hHD (predicted) (mHD)

mIso 2 Tetramer Inter- No No S142I/S566T/N567A interface subunit (predicted) (ITA) defective

mIso 1 SAM Intra- No No F109L/F112C/R143H domain subunit (Predicted) (LCH) defective

hSAMHD1 N/A Yes Yes Yes Full-length (hFL)

hSAMHD1 SAM More Hyperactive Enhanced L77F/C80F/H111R domain stable (Predicted) (FFR) than FL

Predicted = by structure modeling in Buzovetsky, et al. 2018383

115

The SAM-HD inter-subunit and intra-subunit interactions are important for mSAMHD1 function in vivo. To assess the effect of disruption of the inter-subunit

SAM-to-HD interaction, the mSAMHD1 residues S142, N566, and N567 were swapped for their hSAMHD1 counterparts (Table 3), which was predicted to cause steric clash and disruption of stable tetramerization (Figure 27A). In in vitro assays, the triple mutant S142I/S566T/N567A (ITA) affecting the inter-subunit interaction resulted in a defective tetramerization and substantial reduction in dNTPase activity383. The defect in tetramerization and dNTPase activity resulted in abrogated restriction of HIV-1 infection compared to FL mSAMHD1 in PMA- treated U937 cells (Figure 28), confirming the importance of inter-subunit SAM-to-

HD domain interactions in stabilizing the catalytically active mSAMHD1 tetramer.

To assess the importance of the SAM-to-HD domain intra-subunit interactions to tetramerization and enzyme function, mutations of mSAMHD1 residues to hSAMHD1 residues were again made. This time, modeling of the interactions between the SAM domain residues R143, F109, and F112 indicated that R143 forms a salt bridge with the HD domain residue E225, with F109 and F112 forming stacking interactions with HD domain residues H161 and Y289, thereby stabilizing the intra-subunit interaction383 (Figure 27B). Swapping of the mSAMHD1 residues with hSAMHD1 counterparts produced the triple mutant F109L/F112C/R143H

(LCH) (Table 3). Modeling of this interaction indicated a disruption in the

116

electrostatic and stacking interactions that position the SAM domain to stabilize the tetramer (Figure 27B). It was confirmed that the LCH mutant mSAMHD1 does not form tetramers or exhibit dNTPase activity in vitro383. Importantly, infection of

PMA-treated U937 cells expressing LCH show abrogated HIV-1 restriction compared to FL mSAMHD1 (Figure 28). Taken together, these data demonstrate that the SAM-to-HD domain inter-subunit and intra-subunit interactions are essential for mSAMHD1 function in cells, as they stabilize the catalytically active tetramer.

A B

125 mSAMHD1 iso1

iso1 ) %

( 100 Vector FL ITA LCH

kDa n

o i

76- t

HA-SAMHD1 75

c

e f

36- n

GAPDH i

50

e

v

i t

a ***

l 25

e R 0 Vector FL ITA LCH

Figure 28: Mutations destabilizing the SAM-HD inter- and intra-subunit interaction result in abrogated HIV-1 restriction by mSAMHD1. (A) mSAMHD1 Iso1 FL and mutants were stably expressed in U937 cells. (B) HIV-1 restriction by mSAMHD1 was examined after treatment with PMA. Immunoblotting confirmed HA-tagged mSAMHD1 or hSAMHD1 expression. GAPDH was used as a loading control. Single-cycle HIV-1 infection of vector control cells was set as 100% (B). Each experiment was performed in triplicate. Error bars, s.d. ***p ≤ 0.0001.

117

Mutations stabilizing the SAM-to-HD domain interaction enhance hSAMHD1 enzyme function. To further demonstrate the effect of tetramer-stabilizing residues at the SAM-to-HD interface, we performed complimentary mutation studies in hSAMHD1 with corresponding SAM-to-HD interface residues from mSAMHD1383. We hypothesized that stabilization of the SAM-to-HD interface would result in enhanced tetramerization and HIV-1 restriction. (Table 3). This

L77F/C80F/H111R (FFR) triple mutant displayed higher dNTPase activity and formed a more stable tetramer than WT hSAMHD1 in vitro383. Correspondingly, we observed a 2-fold increase in HIV-1 restriction by the FFR mutant compared to FL

WT hSAMHD1 (Figure 29). These data suggest that enhancing interactions between the SAM and HD domains of hSAMHD1 lead to a more stable tetramer with higher enzymatic and HIV-1 restriction activities.

A B

125

hSAMHD1 ) %

( 100

Vector FL FFR n

kDa o i t 75

72- HA-SAMHD1 c

e

f

n i 50

36- GAPDH e hSAMHD1

v

i

t a

l 25 *** e

R *** 0 Vector FL FFR

Figure 29: Mutations stabilizing the SAM-HD interaction enhance hSAMHD1 enzyme function. 118

(A) HIV-1 restriction by FL hSAMHD1 (WT and mutants) stably expressed in U937 cells after PMA differentiation. (B) Immunoblotting confirmed HA-tagged mSAMHD1 or hSAMHD1 expression. GAPDH was used as a loading control. Single-cycle HIV-1 infection of vector control cells was set as 100%. Each experiment was performed in triplicate. Error bars, s.d. ***p ≤ 0.0001.

4.5 Discussion

Crystallization of the FL hSAMHD1 protein has been challenging, in part due to the disorganized and flexible nature of the hSAMHD1 SAM domain. Conversely, the

SAM domain of mSAMHD1 is much more stable. This inherent stability allows for easier crystallization of FL mSAMHD1 protein. A rigorous biochemical analysis and crystal structures of FL mSAMHD1 allowed Buzovetsky et al383 to capture the interactions between the SAM and HD domains in FL mSAMHD1383. Mutations that destabilized the SAM-to-HD domain interface of mSAMHD1 resulted in diminished ability to restrict HIV-1 infection. In conjunction, mutations stabilizing the SAM-to-HD domain interaction of hSAMHD1 led to an enhanced ability to restrict HIV-1 infection. Taken together, these data confirm the in vitro findings383 that the SAM-to-HD domain interface stabilizes SAMHD1 tetramers.

The SAM domains of hSAMHD1 and mSAMHD1 may play different roles in the regulation of enzymatic and other cellular functions. In contrast to mSAMHD1, hSAMHD1 does not require the SAM domain for stable formation of the tetrameric

119

catalytic core. In fact, the hHD domain is more active than the FL protein, so it is possible that the SAM domain imposes a negative regulatory effect on the catalytic activity of hSAMHD1. These opposing effects of SAM domain on mSAMHD1 and hSAMHD1 activities might reflect differences in the sensitivity to nucleotides. It has been shown that the dNTP levels in human cells are lower than those in mouse cells387. As a result of being exposed to higher concentrations of dNTPs, mHD may have evolved lower affinities for nucleotide activators whose binding are enhanced by the SAM domain. Conversely, the hHD itself may have developed a higher affinity for nucleotides at relatively lower dNTP concentrations.

These differences in oligomerization and enzymatic activity may help to explain the differences between human and mouse SAMHD1 and inform the improvement of a mouse model for the study of HIV-1, and novel therapy for certain cancers and autoimmune diseases.

4.6 Acknowledgements

The work presented in this chapter is part of a larger published study (Buzovetsky et al., The SAM Domain of mouse SAMHD1 is critical for its activation and regulation, Nature Communications 2018)383 that I contributed to (Figures 1I, 3C, and 3F).

120

The full authorship is as follows383:

Olga Buzovetsky1,4, Chenxiang Tang1, 4, Kirsten Knecht1, Jenna M. Antonucci2,

Li Wu2, Xiaoyun Ji3 and Yong Xiong1

1Department of Molecular Biophysics and Biochemistry, Yale University, New

Haven, CT 06520, USA. 2 Center of Retrovirus Research, Department of

Veterinary Biosciences; The Ohio State University, Columbus, OH, 43210, USA 3

The State Key Laboratory of Pharmaceutical Biotechnology, School of Life

Sciences, Nanjing University, Nanjing, Jiangsu 210023, People’s Republic of

China.4These authors contributed equally to this work.

The acknowledgements for the full study are as follows383:

We thank B. Slater, J. Wang, T.C. Cheng, S.S. Smaga, T. Cheng, and C.S.

Gelais for technical assistance, and K. Digianantonio and W. Wang for assistance and discussion. We also thank the staff at the Advanced Photon

Source beamline 24-ID C and E. This work was supported in part by the NIH grants AI102778 (Y.X.), AI120845 (X.J.), AI104483 (L.W.). O.B. was supported by the predoctoral program in Cellular and Molecular Biology T32 GM007223 and by the National Science Foundation Graduate Research Fellowship. K.M.K. was supported by the NIH T32 grant GM008283. J.M.A. was supported by C.

121

Glenn Barber funds from the College of Veterinary Medicine at The Ohio State

University

4.7 Contributions

In this chapter, the crystal structure data (Figure 27) was produced by members of the Yong Xiong lab. The HIV-1 restriction assays (Figures 26, 28, 29) were performed by Jenna Antonucci.

122

Chapter 5: Summary and Future Directions

5.1 Summary

This dissertation has one major focus: to better understand the molecular mechanisms contributing to the SAMHD1-mediated restriction of HIV-1 infection in non-dividing cells.

In Chapter 2, we aimed to investigate findings that introduced a controversial new model of HIV-1 restriction by SAMHD1. In 2011, SAMHD1 was identified as a dGTP-dependent dNTPase that restricted HIV-1 infection in non-cycling cells by reducing the intracellular dNTP pool to a level that inhibited efficient reverse transcription of viral cDNA168,229,233,234. This model was confirmed using in vitro dNTPase assays and through the use of dNTPase-deficient mutants of

SAMHD1233,272,276. Interestingly, Beloglazova et al313 published a study identifying

SAMHD1 as a nucleic acid binding protein that maintains a novel nuclease activity.

Using end-labeled oligonucleotide fragments and in vitro transcribed fragments of

HIV-1 gag, Beloglazova et al. showed that SAMHD1 was capable of degrading

HIV-1 RNA transcripts in a 3’ to 5’ direction313. This data introduced the hypothesis that the nuclease activity of SAMHD1 may contribute to its HIV-1 restriction. 123

Subsequent work further determined that SAMHD1 uses a ribonuclease activity to degrade HIV-1 RNA by nucleolytic cleavage316. The study used two mutants of

SAMHD1 which were identified as dNTPase defective (D137N) or RNAse defective (Q548A). In developing these variants of SAMHD1, Ryoo et al. aimed to determine the contribution of each enzymatic activity to the restriction of HIV-1 infection316. They concluded that SAMHD1 primarily restricts HIV-1 infection through nucleolytic cleavage of incoming HIV-1 genomic RNA. They postulated that the standard model of restriction by SAMHD1 was incomplete and offered their work as a replacement.

We felt that it was important to clarify the work done by Ryoo el al. to better understand how SAMHD1 blocks HIV-1 infection. Our study recreated several key experiments shown in Ryoo et al. alongside additional assays, all to determine whether SAMHD1 degrades HIV-1 RNA308. Utilizing monocytic U937 cells that stably expressed WT, D137N, and Q548A SAMHD1 upon PMA differentiation, we confirmed that SAMHD1 does not degrade incoming HIV-1 gRNA but does restrict

HIV-1 infection and reduce dNTP levels significantly. Interestingly, both of our mutants, D137N and Q548A, behaved like WT SAMHD1 in all of our assays, suggesting that these residues do not delineate the dNTPase or RNAse activity in cells. Our work further confirmed that SAMHD1 does not affect HIV-1 protein

124

synthesis, viral particle release, or infectivity of newly synthesized virions in a proviral DNA transfection-based system, suggesting that SAMHD1 does not degrade HIV-1 mRNA or newly synthesized gRNA. Additionally, we presented data indicating that SAMHD1 does not restrict RNA viruses, such as SeV and IAV, which do not have DNA intermediates. Using purified recombinant SAMHD1, we were able to show that the SAMHD1-associated nuclease activity seen in vitro is likely due to bacterial contamination during the purification process. This observation was confirmed in additional studies that categorically determined that

SAMHD1 has no active-site associated nuclease activity318. However, while the nucleic acid binding potential was confirmed in subsequent studies, the purpose of this binding remains unknown313,318,328.

In Chapter 3, we investigated the possibility that SAMHD1 uses its DNA binding potential to suppress HIV-1 LTR-driven gene expression. SAMHD1 is highly expressed in quiescent CD4+ T-cells, which are reticent to HIV-1 infection.

However, activated T-cells are readily infected by HIV-1 and, once reverted back to a quiescent state, these central memory T-cells have been shown to harbor latent proviral DNA and contribute to the viral reservoir389. Our study aimed to determine whether SAMHD1 expression affects transcription of HIV-1 proviral

DNA and the reactivation of viral latency.

125

To do so, we utilized several cell lines and primary cells. By performing gene reporter assays, we found that SAMHD1 potently suppressed HIV-1 LTR-driven gene expression at the level of transcription, with luciferase protein activity and luciferase mRNA levels reduced in a dose-dependent manner with increased

SAMHD1 expression. Additionally, SAMHD1 suppressed HTLV-1 LTR activity greater than the HIV-1 LTR, although further studies are required to determine the cause of this effect. As SAMHD1 suppressed HIV-1 LTR-driven gene expression, we aimed to determine whether SAMHD1 could suppress reactivation of proviral

DNA in a relevant model of latency. We stably expressed SAMHD1 in J-lat cells before treatment with LRAs. We found that SAMHD1-expressing J-lat cells displayed lower levels of latency reactivation relative to vector control cells lacking

SAMHD1 expression. Further, our data indicated that SAMHD1 binds the HIV-1

LTR promoter in cells, suggesting that SAMHD1 may act as a transcription factor that negatively regulates LTR activity. We tested this hypothesis in a primary CD4+

T-cell model of latency in which we achieved 40-50% knockdown of SAMHD1.

Upon stimulation with PMA and ionomycin, the SAMHD1 knockdown cells exhibited 1.6-fold higher levels of latency reactivation compared to SAMHD1- expressing control cells. These data are the first to identify SAMHD1 as a contributor to HIV-1 latency.

126

To discern the mechanism of SAMHD1-mediated suppression of HIV-1 LTR-driven gene expression and latency reactivation, we aimed to determine if the phosphorylation or enzymatic core of SAMHD1 were required for the observed effects. Both phosphoablative (T592A) and dNTPase impaired (HD/RN) SAMHD1 mutants could not suppress LTR-driven gene expression or reactivation of viral latency, and both failed to bind integrated proviral DNA in cells. Our subsequent in vitro binding assays confirmed that while WT SAMHD1 can bind fragments of the

HIV-1 LTR, the T592A and HD/RN mutants can not. Further studies are needed to fully characterize the mechanism by which WT SAMHD1 can bind to the LTR. As the binding-deficient mutants were also unable to suppress LTR-driven gene expression and the reversal of latency in cells, we concluded that the mechanism of SAMHD1-mediated suppression of HIV-1 gene expression likely requires the binding of SAMHD1 to the HIV-1 LTR promoter.

In Chapter 4 we examined how destabilization of the SAM-to-HD domain interaction in mSAMHD1 abrogates the ability of mSAMHD1 to restrict HIV-1 infection. Utilizing the first full-length crystal structure of mSAMHD1, we identified residues essential for both intra-subunit and inter-subunit interactions between the

SAM and HD domains of SAMHD1. We confirmed that destabilizing these interactions in mSAMHD1 by swapping essential residues for the comparable hSAMHD1 residues resulted in abrogated HIV-1 restriction. Interestingly,

127

stabilizing tetramer formation of hSAMHD1 by mutating inter-subunit residues to comparable mSAMHD1 residues led to an enhancement of HIV-1 restriction.

Taken together, these data indicate a role of the SAM domain of mSAMHD1 in regulating its dNTPase activity, and lays the framework for further study of hSAMHD1.

5.2 Future Directions

In Chapter 2 we confirmed that SAMHD1 primarily restricts HIV-1 infection through depletion of the intracellular dNTP pool and that the observed nuclease activity of

SAMHD1 is likely due to bacterial contamination during the protein purification process. However, it has been confirmed that SAMHD1 is a DNA and RNA binding protein capable of binding HIV-1 nucleic acids. Thus, determining the contribution of HIV-1 DNA binding to the suppression of HIV-1 infection will be an important area of future research. Additionally, the current literature has described a model of complex structure-based regulation of SAMHD1’s dNTPase activity. However, several questions still exist, including the effect of phosphorylation, even at sites other than T592, on HIV-1 restriction. Further, the FL human SAMHD1 crystal structure has not been identified. Although cell-based and in vitro crosslinking assays allow us to determine oligomerization of SAMHD1, crystallization of

SAMHD1 with post-translational modifications or with nucleic acids will allow us to clearly study the structural and biochemical changes to SAMHD1 under various

128

conditions. Additionally, conflicting reports could be due to the use of different cell types, and the differential use of clinical HIV-1 isolates, replication-competent lab strains, and single-cycle HIV-1. It is essential to confirm experimental findings with primary cells that accurately recapitulate in vivo mucosal infection sites. Further understanding of SAMHD1-mediated restriction of early HIV-1 infection will allow for a better understanding of HIV-1 pathology.

In Chapter 3, we utilized an established primary TCM model of latency in cells from healthy donors. We used shRNA to achieve a 40-50% knockdown of SAMHD1 in these cells. Enhancement of SAMHD1 knockdown by Vpx-VLP transduction will allow for a clearer result to confirm the effect of SAMHD1 on latency in T-

238,253 cells . Treatment of SAMHD1 KD TCM with different LRAs, such as the histone deacetylase inhibitor vorinostat189 or TNF-190, will allow us to examine whether histone modifications or different stimulatory pathways enhance or abrogate the

SAMHD1-mediated suppression of latency reversal. Validation of the TCM results with additional CD4+ T-cell based model of HIV-1 latency189 will strengthen our conclusion that SAMHD1 expression results in suppressed reactivation of viral latency in CD4+ T-cells.

To validate our in vitro results, we propose an ex vivo study using resting CD4+ T- cells from HIV-1 infected individuals undergoing HAART. Isolation of resting CD4+

129

T-cells from HIV-1-infected individuals on HAART380,390,391 coupled with knockdown of SAMHD1 and reversal of latency will allow us to investigate the contribution of SAMHD1 to maintaining latency in a truly physiologically relevant system.

Mapping the specific sites of interaction between SAMHD1 and the HIV-1 LTR sequence will allow us to investigate the mechanisms underlying the SAMHD1- mediated suppression of LTR-driven gene expression. Mapping can be accomplished using the extensively used hydroxyl-radical footprinting assay392.

The hydroxyl radical abstracts hydrogen atoms from the deoxyribose sugar backbone, resulting in subsequent cleavage of DNA in a sequence and base- independent manner393 and allowing for single-nucleotide resolution mapping of protein-DNA interactions394,395. After identification of the SAMHD1 footprint, mutagenesis of putative binding sites on HIV-1 LTR DNA will confirm the sequence specificity of SAMHD1 binding.

In Chapter 4 we performed cell-based HIV-1 restriction assays to confirm biochemical assays based on novel full-length mSAMHD1 crystal structures. In doing so, we discovered the importance of SAM-to-HD domain interactions in the stabilization of catalytically active mSAMHD1 tetramers and how tetramerization is required for HIV-1 restriction. Further cell-based functional assays will be

130

performed to further refine and confirm essential intra-subunit and inter-subunit interactions in both human and mouse SAMHD1. In doing do, we will further understand the structure-based regulatory mechanism behind SAMHD1’s dNTPase activity, which will further our work in SAMHD1-mediated restriction of retroviral infection.

131

Bibliography

1. Centers for Disease Control and Prevention (CDC), Pneumocystis pneumonia--Los Angeles. MMWR Morb Mortal Wkly Rep 30, 250-252 (1981).

2. Haverkos, H.W. & Curran, J.W. The current outbreak of kaposi's sarcoma and opportunistic infections. Vol. 32 (John Wiley & Sons, Ltd, CA: A Cancer Journal for Clinicians, 1982).

3. Picard, A. How the advent of AIDS advanced gay rights. (The Globe and Mail, 2014).

4. Wright, J. Remembering the early days of "Gay Cancer". in All Things Considered (ed. Block, M.) (NPR.org, 2006).

5. Bierhoff, E., et al. Autopsy findings in patients with human immunodeficiency virus infection with emphasis on the risk factor of hemophilia. Gen Diagn Pathol 141, 295-304 (1996).

6. Lowe, G.D. Haemophilia, blood products and HIV infection. Scott Med J 32, 109-111 (1987).

7. Centers for Disease Control and Prevention (CDC), Current Trends Update on Acquired Immune Deficiency Syndrome (AIDS) --United States. (1982).

8. Kellerman, S., Wortley, P. & Fleming, P. The Changing Epidemic of HIV. Curr Infect Dis Rep 2, 457-465 (2000).

9. Barré-Sinoussi, F., et al. Isolation of a T-lymphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome (AIDS). Science 220, 868-871 (1983).

132

10. Popovic, M., Sarngadharan, M.G., Read, E. & Gallo, R.C. Detection, isolation, and continuous production of cytopathic retroviruses (HTLV-III) from patients with AIDS and pre-AIDS. Science 224, 497-500 (1984).

11. Broder, S. Clinical applications of 3'-azido-2',3'-dideoxythymidine (AZT) and related dideoxynucleosides. Med Res Rev 10, 419-439 (1990).

12. Mitsuya, H., et al. 3'-Azido-3'-deoxythymidine (BW A509U): an antiviral agent that inhibits the infectivity and cytopathic effect of human T- lymphotropic virus type III/lymphadenopathy-associated virus in vitro. Proc Natl Acad Sci U S A 82, 7096-7100 (1985).

13. UNAIDS. AIDS by the numbers: 2016. (Geneva, Switzerland, 2016).

14. Weiss, R.A. How does HIV cause AIDS? Science 260, 1273-1279 (1993).

15. Lu, L., Yu, F., DU, L.Y., Xu, W. & Jiang, S.B. Tactics used by HIV-1 to evade host innate, adaptive, and intrinsic immunities. Chin Med J (Engl) 126, 2374-2379 (2013).

16. Liu, C., Ma, X., Liu, B., Chen, C. & Zhang, H. HIV-1 functional cure: will the dream come true? BMC Med 13, 284 (2015).

17. CDC. About HIV/AIDS. Vol. 2015 (2015).

18. Fields, B., Knipe, D. & Howley, P. Fields' Viro0logy, 5th Edition, (Wolters Kluwar Health/ Lippincott Williams & Wilkins, Philedelphia, 2007).

19. Doitsh, G., et al. Cell death by pyroptosis drives CD4 T-cell depletion in HIV- 1 infection. Nature 505, 509-514 (2014).

20. Holmes, C.B., Losina, E., Walensky, R.P., Yazdanpanah, Y. & Freedberg, K.A. Review of human immunodeficiency virus type 1-related opportunistic infections in sub-Saharan Africa. Clin Infect Dis 36, 652-662 (2003).

133

21. Pantaleo, G., Graziosi, C. & Fauci, A.S. New concepts in the immunopathogenesis of human immunodeficiency virus infection. N Engl J Med 328, 327-335 (1993).

22. Burns, D.N., et al. Toward an endgame: finding and engaging people unaware of their HIV-1 infection in treatment and prevention. AIDS Res Hum Retroviruses 30, 217-224 (2014).

23. Hammer, S.M., et al. A controlled trial of two nucleoside analogues plus indinavir in persons with human immunodeficiency virus infection and CD4 cell counts of 200 per cubic millimeter or less. AIDS Clinical Trials Group 320 Study Team. N Engl J Med 337, 725-733 (1997).

24. Gulick, R.M., et al. Treatment with indinavir, zidovudine, and lamivudine in adults with human immunodeficiency virus infection and prior antiretroviral therapy. N Engl J Med 337, 734-739 (1997).

25. Fauci, A.S. & Folkers, G.K. Toward an AIDS-free generation. JAMA 308, 343-344 (2012).

26. Finzi, D., et al. Latent infection of CD4+ T cells provides a mechanism for lifelong persistence of HIV-1, even in patients on effective combination therapy. Nat Med 5, 512-517 (1999).

27. Shan, L. & Siliciano, R.F. From reactivation of latent HIV-1 to elimination of the latent reservoir: the presence of multiple barriers to viral eradication. Bioessays 35, 544-552 (2013).

28. Nsagha, D.S., et al. Risk Factors of Cardiovascular Diseases in HIV/AIDS Patients on HAART. Open AIDS J 9, 51-59 (2015).

29. Kumar, A., Coquard, L. & Herbein, G. Targeting TNF-alpha in HIV-1 infection. Curr Drug Targets 17, 15-22 (2015).

30. Badia, R., et al. Zinc finger endonuclease targeting PSIP1 inhibits HIV-1 integration. Antimicrob Agents Chemother 58, 4318-4327 (2014). 134

31. Chan, E., Towers, G.J. & Qasim, W. Gene therapy strategies to exploit TRIM derived restriction factors against HIV-1. Viruses 6, 243-263 (2014).

32. Clavel, F., et al. Isolation of a new human retrovirus from West African patients with AIDS. Science 233, 343-346 (1986).

33. Clavel, F., et al. Human immunodeficiency virus type 2 infection associated with AIDS in West Africa. N Engl J Med 316, 1180-1185 (1987).

34. Guyader, M., et al. Genome organization and transactivation of the human immunodeficiency virus type 2. Nature 326, 662-669 (1987).

35. Sharp, P.M., et al. The origins of acquired immune deficiency syndrome viruses: where and when? Philos Trans R Soc Lond B Biol Sci 356, 867- 876 (2001).

36. Huet, T., Cheynier, R., Meyerhans, A., Roelants, G. & Wain-Hobson, S. Genetic organization of a chimpanzee lentivirus related to HIV-1. Nature 345, 356-359 (1990).

37. Hirsch, V.M., Olmsted, R.A., Murphey-Corb, M., Purcell, R.H. & Johnson, P.R. An African primate lentivirus (SIVsm) closely related to HIV-2. Nature 339, 389-392 (1989).

38. Hahn, B.H., Shaw, G.M., De Cock, K.M. & Sharp, P.M. AIDS as a zoonosis: scientific and public health implications. Science 287, 607-614 (2000).

39. Fields, B., Knipe, D. & Howley, P. Fields' Virology, 5th Edition, (Wolters Kluwar Health/ Lippincott Williams & Wilkins, Philedelphia, 2007).

40. Freed, E. & Martin, M. Fields Virology. (eds. Knipe, D. & Howley, P.) 1971- 2041 (Lippincott, Williams & Wilkins, Philedelphia, PA, 2001).

41. Merson, M.H., O'Malley, J., Serwadda, D. & Apisuk, C. The history and challenge of HIV prevention. Lancet 372, 475-488 (2008). 135

42. Keele, B.F., et al. Chimpanzee reservoirs of pandemic and nonpandemic HIV-1. Science 313, 523-526 (2006).

43. Taylor, B.S. & Hammer, S.M. The challenge of HIV-1 subtype diversity. N Engl J Med 359, 1965-1966 (2008).

44. De Leys, R., et al. Isolation and partial characterization of an unusual human immunodeficiency retrovirus from two persons of west-central African origin. J Virol 64, 1207-1216 (1990).

45. Peeters, M., et al. Geographical distribution of HIV-1 group O viruses in Africa. AIDS 11, 493-498 (1997).

46. D'arc, M., et al. Origin of the HIV-1 group O epidemic in western lowland gorillas. Proc Natl Acad Sci U S A 112, E1343-1352 (2015).

47. Simon, F., et al. Identification of a new human immunodeficiency virus type 1 distinct from group M and group O. Nat Med 4, 1032-1037 (1998).

48. Delaugerre, C., De Oliveira, F., Lascoux-Combe, C., Plantier, J.C. & Simon, F. HIV-1 group N: travelling beyond Cameroon. Lancet 378, 1894 (2011).

49. Vallari, A., et al. Four new HIV-1 group N isolates from Cameroon: Prevalence continues to be low. AIDS Res Hum Retroviruses 26, 109-115 (2010).

50. Peeters, M., et al. Risk to human health from a plethora of simian immunodeficiency viruses in primate bushmeat. Emerg Infect Dis 8, 451- 457 (2002).

51. Baeten, J.M., et al. HIV-1 subtype D infection is associated with faster disease progression than subtype A in spite of similar plasma HIV-1 loads. J Infect Dis 195, 1177-1180 (2007).

136

52. Touloumi, G., et al. Impact of HIV-1 subtype on CD4 count at HIV seroconversion, rate of decline, and viral load set point in European seroconverter cohorts. Clin Infect Dis 56, 888-897 (2013).

53. Kanki, P.J., et al. Human immunodeficiency virus type 1 subtypes differ in disease progression. J Infect Dis 179, 68-73 (1999).

54. Kiwanuka, N., et al. Effect of human immunodeficiency virus Type 1 (HIV- 1) subtype on disease progression in persons from Rakai, Uganda, with incident HIV-1 infection. J Infect Dis 197, 707-713 (2008).

55. Pereira, L.A., Bentley, K., Peeters, A., Churchill, M.J. & Deacon, N.J. A compilation of cellular transcription factor interactions with the HIV-1 LTR promoter. Nucleic Acids Res 28, 663-668 (2000).

56. Harrich, D., et al. Role of SP1-binding domains in in vivo transcriptional regulation of the human immunodeficiency virus type 1 long terminal repeat. J Virol 63, 2585-2591 (1989).

57. Engelman, A. & Cherepanov, P. The structural biology of HIV-1: mechanistic and therapeutic insights. Nat Rev Microbiol 10, 279-290 (2012).

58. Busschots, K., et al. The interaction of LEDGF/p75 with integrase is lentivirus-specific and promotes DNA binding. J Biol Chem 280, 17841- 17847 (2005).

59. Kilareski, E.M., Shah, S., Nonnemacher, M.R. & Wigdahl, B. Regulation of HIV-1 transcription in cells of the monocyte-macrophage lineage. Retrovirology 6, 118 (2009).

60. Flint, S.J., Enquist, L.W., Racaniello, V.R. & Skalka, A.M. Principles of Virology, (ASM).

61. Hogan, T.H., Krebs, F.C. & Wigdahl, B. Regulation of human immunodeficiency virus type 1 gene expression and pathogenesis by 137

CCAAT/enhancer binding proteins in cells of the monocyte/macrophage lineage. J Neurovirol 8 Suppl 2, 21-26 (2002).

62. Garcia, J.A., et al. Human immunodeficiency virus type 1 LTR TATA and TAR region sequences required for transcriptional regulation. EMBO J 8, 765-778 (1989).

63. Rosen, C.A. & Pavlakis, G.N. Tat and Rev: positive regulators of HIV gene expression. AIDS 4, A51 (1990).

64. Nabel, G. & Baltimore, D. An inducible transcription factor activates expression of human immunodeficiency virus in T cells. Nature 326, 711- 713 (1987).

65. Fischer-Smith, T. & Rappaport, J. Evolving paradigms in the pathogenesis of HIV-1-associated dementia. Expert Rev Mol Med 7, 1-26 (2005).

66. Li, L., et al. Impact of Tat Genetic Variation on HIV-1 Disease. Adv Virol 2012, 123605 (2012).

67. Bell, N.M. & Lever, A.M. HIV Gag polyprotein: processing and early viral particle assembly. Trends Microbiol 21, 136-144 (2013).

68. Bryant, M. & Ratner, L. Myristoylation-dependent replication and assembly of human immunodeficiency virus 1. Proc Natl Acad Sci U S A 87, 523-527 (1990).

69. Göttlinger, H.G., Sodroski, J.G. & Haseltine, W.A. Role of capsid precursor processing and myristoylation in morphogenesis and infectivity of human immunodeficiency virus type 1. Proc Natl Acad Sci U S A 86, 5781-5785 (1989).

70. Jacks, T., et al. Characterization of ribosomal frameshifting in HIV-1 gag- pol expression. Nature 331, 280-283 (1988).

138

71. Dulude, D., Baril, M. & Brakier-Gingras, L. Characterization of the frameshift stimulatory signal controlling a programmed -1 ribosomal frameshift in the human immunodeficiency virus type 1. Nucleic Acids Res 30, 5094-5102 (2002).

72. Kohl, N.E., et al. Active human immunodeficiency virus protease is required for viral infectivity. Proc Natl Acad Sci U S A 85, 4686-4690 (1988).

73. Schubert, U., et al. Proteasome inhibition interferes with gag polyprotein processing, release, and maturation of HIV-1 and HIV-2. Proc Natl Acad Sci U S A 97, 13057-13062 (2000).

74. Kwong, P.D., et al. Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody. Nature 393, 648-659 (1998).

75. Maerz, A.L., Drummer, H.E., Wilson, K.A. & Poumbourios, P. Functional analysis of the disulfide-bonded loop/chain reversal region of human immunodeficiency virus type 1 gp41 reveals a critical role in gp120-gp41 association. J Virol 75, 6635-6644 (2001).

76. Wyatt, R. & Sodroski, J. The HIV-1 envelope glycoproteins: fusogens, antigens, and immunogens. Science 280, 1884-1888 (1998).

77. Wang, H., et al. Asymmetric recognition of HIV-1 Envelope trimer by V1V2 loop-targeting antibodies. Elife 6(2017).

78. Karn, J. & Stoltzfus, C.M. Transcriptional and posttranscriptional regulation of HIV-1 gene expression. Cold Spring Harb Perspect Med 2, a006916 (2012).

79. Karn, J., Dingwall, C., Finch, J.T., Heaphy, S. & Gait, M.J. RNA binding by the tat and rev proteins of HIV-1. Biochimie 73, 9-16 (1991).

80. Rice, A.P. The HIV-1 Tat protein: mechanism of action and target for HIV-1 cure strategies. Curr Pharm Des 23, 4098-4102 (2017). 139

81. O'Carroll, I.P., et al. Contributions of individual domains to function of the HIV-1 Rev response element. J Virol 91, 00746-00717 (2017).

82. Emerman, M. & Malim, M.H. HIV-1 regulatory/accessory genes: keys to unraveling viral and host cell biology. Science 280, 1880-1884 (1998).

83. Malim, M.H. APOBEC proteins and intrinsic resistance to HIV-1 infection. Philos Trans R Soc Lond B Biol Sci 364, 675-687 (2009).

84. Franca, R., Spadari, S. & Maga, G. APOBEC deaminases as cellular antiviral factors: a novel natural host defense mechanism. Med Sci Monit 12, RA92-98 (2006).

85. Kogan, M. & Rappaport, J. HIV-1 accessory protein Vpr: relevance in the pathogenesis of HIV and potential for therapeutic intervention. Retrovirology 8, 25 (2011).

86. Yuan, X., Matsuda, Z., Matsuda, M., Essex, M. & Lee, T.H. Human immunodeficiency virus vpr gene encodes a virion-associated protein. AIDS Res Hum Retroviruses 6, 1265-1271 (1990).

87. Kino, T., et al. Human immunodeficiency virus type 1 (HIV-1) accessory protein Vpr induces transcription of the HIV-1 and glucocorticoid-responsive promoters by binding directly to p300/CBP coactivators. J Virol 76, 9724- 9734 (2002).

88. Romani, B., et al. HIV-1 Vpr reactivates latent HIV-1 provirus by inducing depletion of class I HDACs on chromatin. Sci Rep 6, 31924 (2016).

89. Romani, B., Baygloo, N.S., Hamidi-Fard, M., Aghasadeghi, M.R. & Allahbakhshi, E. HIV-1 Vpr Protein Induces Proteasomal Degradation of Chromatin-associated Class I HDACs to Overcome Latent Infection of Macrophages. J Biol Chem 291, 2696-2711 (2016).

90. Planelles, V., et al. Vpr-induced cell cycle arrest is conserved among primate . J Virol 70, 2516-2524 (1996). 140

91. Shostak, L.D., et al. Roles of p53 and caspases in the induction of cell cycle arrest and apoptosis by HIV-1 vpr. Exp Cell Res 251, 156-165 (1999).

92. Cohen, E.A., Terwilliger, E.F., Sodroski, J.G. & Haseltine, W.A. Identification of a protein encoded by the vpu gene of HIV-1. Nature 334, 532-534 (1988).

93. Strebel, K., Klimkait, T. & Martin, M.A. A novel gene of HIV-1, vpu, and its 16-kilodalton product. Science 241, 1221-1223 (1988).

94. Strebel, K., Klimkait, T., Maldarelli, F. & Martin, M.A. Molecular and biochemical analyses of human immunodeficiency virus type 1 vpu protein. J Virol 63, 3784-3791 (1989).

95. Willey, R.L., Maldarelli, F., Martin, M.A. & Strebel, K. Human immunodeficiency virus type 1 Vpu protein induces rapid degradation of CD4. J Virol 66, 7193-7200 (1992).

96. Chen, M.Y., Maldarelli, F., Karczewski, M.K., Willey, R.L. & Strebel, K. Human immunodeficiency virus type 1 Vpu protein induces degradation of CD4 in vitro: the cytoplasmic domain of CD4 contributes to Vpu sensitivity. J Virol 67, 3877-3884 (1993).

97. Neil, S.J., Sandrin, V., Sundquist, W.I. & Bieniasz, P.D. An interferon-alpha- induced tethering mechanism inhibits HIV-1 and Ebola virus particle release but is counteracted by the HIV-1 Vpu protein. Cell Host Microbe 2, 193-203 (2007).

98. Perez-Caballero, D., et al. Tetherin inhibits HIV-1 release by directly tethering virions to cells. Cell 139, 499-511 (2009).

99. Foster, J.L., Denial, S.J., Temple, B.R. & Garcia, J.V. Mechanisms of HIV- 1 Nef function and intracellular signaling. J Neuroimmune Pharmacol 6, 230-246 (2011).

141

100. Schwartz, S., Felber, B.K., Benko, D.M., Fenyö, E.M. & Pavlakis, G.N. Cloning and functional analysis of multiply spliced mRNA species of human immunodeficiency virus type 1. J Virol 64, 2519-2529 (1990).

101. Yang, O.O., et al. Short conserved sequences of HIV-1 are highly immunogenic and shift immunodominance. J Virol 89, 1195-1204 (2015).

102. Manzourolajdad, A., Gonzalez, M. & Spouge, J.L. Changes in the Plasticity of HIV-1 Nef RNA during the Evolution of the North American Epidemic. PLoS One 11, e0163688 (2016).

103. Kestler, H.W., et al. Importance of the nef gene for maintenance of high virus loads and for development of AIDS. Cell 65, 651-662 (1991).

104. Deacon, N.J., et al. Genomic structure of an attenuated quasi species of HIV-1 from a blood transfusion donor and recipients. Science 270, 988-991 (1995).

105. Wildum, S., Schindler, M., Münch, J. & Kirchhoff, F. Contribution of Vpu, Env, and Nef to CD4 down-modulation and resistance of human immunodeficiency virus type 1-infected T cells to superinfection. J Virol 80, 8047-8059 (2006).

106. Basmaciogullari, S. & Pizzato, M. The activity of Nef on HIV-1 infectivity. Front Microbiol 5, 232 (2014).

107. Trautz, B., et al. The Antagonism of HIV-1 Nef to SERINC5 Particle Infectivity Restriction Involves the Counteraction of Virion-Associated Pools of the Restriction Factor. J Virol 90, 10915-10927 (2016).

108. Rosa, A., et al. HIV-1 Nef promotes infection by excluding SERINC5 from virion incorporation. Nature 526, 212-217 (2015).

109. Trautz, B., et al. The host-cell restriction factor SERINC5 restricts HIV-1 infectivity without altering the lipid composition and organization of viral particles. J Biol Chem 292, 13702-13713 (2017). 142

110. Zaunders, J. & van Bockel, D. Innate and Adaptive Immunity in Long-Term Non-Progression in HIV Disease. Front Immunol 4, 95 (2013).

111. Aggarwal, A., McAllery, S. & Turville, S.G. Revising the Role of Myeloid cells in HIV Pathogenesis. Curr HIV/AIDS Rep 10, 3-11 (2013).

112. Berger, E.A. HIV entry and tropism. When one receptor is not enough. Adv Exp Med Biol 452, 151-157 (1998).

113. Wilen, C.B., Tilton, J.C. & Doms, R.W. HIV: cell binding and entry. Cold Spring Harb Perspect Med 2(2012).

114. Chan, D.C. & Kim, P.S. HIV entry and its inhibition. Cell 93, 681-684 (1998).

115. Arhel, N. Revisiting HIV-1 uncoating. Retrovirology 7, 96 (2010).

116. Gao, W.Y., Cara, A., Gallo, R.C. & Lori, F. Low levels of deoxynucleotides in peripheral blood lymphocytes: a strategy to inhibit human immunodeficiency virus type 1 replication. Proc Natl Acad Sci U S A 90, 8925-8928 (1993).

117. Amie, S.M., Noble, E. & Kim, B. Intracellular nucleotide levels and the control of retroviral infections. Virology 436, 247-254 (2013).

118. Gomez, C. & Hope, T.J. The ins and outs of HIV replication. Cell Microbiol 7, 621-626 (2005).

119. Finzi, D., Dieffenbach, C.W. & Basavappa, R. Defining and solving the essential protein-protein interactions in HIV infection. J Struct Biol 158, 148- 155 (2007).

120. Yamashita, M. & Emerman, M. Capsid is a dominant determinant of retrovirus infectivity in nondividing cells. J Virol 78, 5670-5678 (2004).

143

121. Lee, K., et al. Flexible use of nuclear import pathways by HIV-1. Cell Host Microbe 7, 221-233 (2010).

122. Shah, V.B. & Aiken, C. HIV Nuclear Entry: Clearing the Fog. Viruses 2, 1190-1194 (2010).

123. Brady, T., et al. HIV integration site distributions in resting and activated CD4+ T cells infected in culture. AIDS 23, 1461-1471 (2009).

124. Han, Y., et al. Resting CD4+ T cells from human immunodeficiency virus type 1 (HIV-1)-infected individuals carry integrated HIV-1 genomes within actively transcribed host genes. J Virol 78, 6122-6133 (2004).

125. Bourgeois, C.F., Kim, Y.K., Churcher, M.J., West, M.J. & Karn, J. Spt5 cooperates with human immunodeficiency virus type 1 Tat by preventing premature RNA release at terminator sequences. Mol Cell Biol 22, 1079- 1093 (2002).

126. Fujinaga, K., et al. Dynamics of human immunodeficiency virus transcription: P-TEFb phosphorylates RD and dissociates negative effectors from the transactivation response element. Mol Cell Biol 24, 787- 795 (2004).

127. Kim, Y.K., Bourgeois, C.F., Isel, C., Churcher, M.J. & Karn, J. Phosphorylation of the RNA polymerase II carboxyl-terminal domain by CDK9 is directly responsible for human immunodeficiency virus type 1 Tat- activated transcriptional elongation. Mol Cell Biol 22, 4622-4637 (2002).

128. Parada, C.A. & Roeder, R.G. Enhanced processivity of RNA polymerase II triggered by Tat-induced phosphorylation of its carboxy-terminal domain. Nature 384, 375-378 (1996).

129. Rathore, U., et al. Glycosylation of the core of the HIV-1 envelope subunit protein gp120 is not required for native trimer formation or viral infectivity. J Biol Chem 292, 10197-10219 (2017).

144

130. Scourfield, E.J. & Martin-Serrano, J. Growing functions of the ESCRT machinery in cell biology and viral replication. Biochem Soc Trans 45, 613- 634 (2017).

131. Ashorn, P., et al. An inhibitor of the protease blocks maturation of human and simian immunodeficiency viruses and spread of infection. Proc Natl Acad Sci U S A 87, 7472-7476 (1990).

132. Miller, M., Jaskólski, M., Rao, J.K., Leis, J. & Wlodawer, A. Crystal structure of a retroviral protease proves relationship to aspartic protease family. Nature 337, 576-579 (1989).

133. Navia, M.A., et al. Three-dimensional structure of aspartyl protease from human immunodeficiency virus HIV-1. Nature 337, 615-620 (1989).

134. Hladik, F. & McElrath, M.J. Setting the stage: host invasion by HIV. Nat Rev Immunol 8, 447-457 (2008).

135. Royce, R.A., Seña, A., Cates, W. & Cohen, M.S. Sexual transmission of HIV. N Engl J Med 336, 1072-1078 (1997).

136. Cohen, M.S. & Gay, C.L. Treatment to prevent transmission of HIV-1. Clin Infect Dis 50 Suppl 3, S85-95 (2010).

137. Cohen, M.S., Shaw, G.M., McMichael, A.J. & Haynes, B.F. Acute HIV-1 Infection. N Engl J Med 364, 1943-1954 (2011).

138. Moore, J.P., Kitchen, S.G., Pugach, P. & Zack, J.A. The CCR5 and CXCR4 coreceptors--central to understanding the transmission and pathogenesis of human immunodeficiency virus type 1 infection. AIDS Res Hum Retroviruses 20, 111-126 (2004).

139. Renjifo, B., et al. Preferential in-utero transmission of HIV-1 subtype C as compared to HIV-1 subtype A or D. AIDS 18, 1629-1636 (2004).

145

140. Blackard, J.T., et al. HIV-1 LTR subtype and perinatal transmission. Virology 287, 261-265 (2001).

141. Björndal, A., et al. Coreceptor usage of primary human immunodeficiency virus type 1 isolates varies according to biological phenotype. J Virol 71, 7478-7487 (1997).

142. Cilliers, T., et al. The CCR5 and CXCR4 coreceptors are both used by human immunodeficiency virus type 1 primary isolates from subtype C. J Virol 77, 4449-4456 (2003).

143. McNab, F., Mayer-Barber, K., Sher, A., Wack, A. & O'Garra, A. Type I in infectious disease. Nat Rev Immunol 15, 87-103 (2015).

144. Chan, Y.K. & Gack, M.U. Viral evasion of intracellular DNA and RNA sensing. Nat Rev Microbiol 14, 360-373 (2016).

145. Takeuchi, O. & Akira, S. Pattern recognition receptors and inflammation. Cell 140, 805-820 (2010).

146. Chen, Q., Sun, L. & Chen, Z.J. Regulation and function of the cGAS-STING pathway of cytosolic DNA sensing. Nat Immunol 17, 1142-1149 (2016).

147. Trinchieri, G. Type I interferon: friend or foe? J Exp Med 207, 2053-2063 (2010).

148. Stetson, D.B. & Medzhitov, R. Recognition of cytosolic DNA activates an IRF3-dependent innate immune response. Immunity 24, 93-103 (2006).

149. Manel, N. & Littman, D.R. Hiding in plain sight: how HIV evades innate immune responses. Cell 147, 271-274 (2011).

150. Neil, S. & Bieniasz, P. Human immunodeficiency virus, restriction factors, and interferon. J Interferon Cytokine Res 29, 569-580 (2009).

146

151. Prinz, M. & Knobeloch, K.P. Type I interferons as ambiguous modulators of chronic inflammation in the central nervous system. Front Immunol 3, 67 (2012).

152. Rustagi, A. & Gale, M. Innate antiviral immune signaling, viral evasion and modulation by HIV-1. J Mol Biol 426, 1161-1177 (2014).

153. Ablasser, A., et al. cGAS produces a 2'-5'-linked cyclic dinucleotide second messenger that activates STING. Nature 498, 380-384 (2013).

154. Sun, L., Wu, J., Du, F., Chen, X. & Chen, Z.J. Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway. Science 339, 786-791 (2013).

155. Wu, J., et al. Cyclic GMP-AMP is an endogenous second messenger in innate immune signaling by cytosolic DNA. Science 339, 826-830 (2013).

156. Ablasser, A. & Gulen, M.F. The role of cGAS in innate immunity and beyond. J Mol Med (Berl) 94, 1085-1093 (2016).

157. Ma, Z. & Damania, B. The cGAS-STING Defense Pathway and Its Counteraction by Viruses. Cell Host Microbe 19, 150-158 (2016).

158. Gao, D., et al. Cyclic GMP-AMP synthase is an innate immune sensor of HIV and other retroviruses. Science 341, 903-906 (2013).

159. Jakobsen, M.R., et al. IFI16 senses DNA forms of the lentiviral replication cycle and controls HIV-1 replication. Proc Natl Acad Sci U S A 110, E4571- 4580 (2013).

160. Campbell, E.M. & Hope, T.J. HIV-1 capsid: the multifaceted key player in HIV-1 infection. Nat Rev Microbiol 13, 471-483 (2015).

161. Yamashita, M. & Engelman, A.N. Capsid-Dependent Host Factors in HIV-1 Infection. Trends Microbiol 25, 741-755 (2017). 147

162. Antonucci, J.M., St Gelais, C. & Wu, L. The Dynamic Interplay between HIV- 1, SAMHD1, and the Innate Antiviral Response. Front Immunol 8, 1541 (2017).

163. Doyle, T., Goujon, C. & Malim, M.H. HIV-1 and interferons: who's interfering with whom? Nat Rev Microbiol 13, 403-413 (2015).

164. Arcia, D., Acevedo-Sáenz, L., Rugeles, M.T. & Velilla, P.A. Role of CD8(+) T Cells in the Selection of HIV-1 Immune Escape Mutations. Viral Immunol 30, 3-12 (2017).

165. Yan, N. & Chen, Z.J. Intrinsic antiviral immunity. Nat Immunol 13, 214-222 (2012).

166. Douville, R.N. & Hiscott, J. The interface between the innate interferon response and expression of host retroviral restriction factors. Cytokine 52, 108-115 (2010).

167. Schott, K., Riess, M. & König, R. Role of Innate Genes in HIV Replication, (Springer, Berlin, Heidelberg, 2017).

168. Laguette, N., et al. SAMHD1 is the dendritic- and myeloid-cell-specific HIV- 1 restriction factor counteracted by Vpx. Nature 474, 654-657 (2011).

169. Schott, K., Riess, M. & König, R. Role of Innate Genes in HIV Replication. Curr Top Microbiol Immunol (2017).

170. Yan, N., Regalado-Magdos, A.D., Stiggelbout, B., Lee-Kirsch, M.A. & Lieberman, J. The cytosolic exonuclease TREX1 inhibits the innate immune response to human immunodeficiency virus type 1. Nat Immunol 11, 1005- 1013 (2010).

171. Hasan, M. & Yan, N. Safeguard against DNA sensing: the role of TREX1 in HIV-1 infection and autoimmune diseases. Front Microbiol 5, 193 (2014).

148

172. Manel, N., et al. A cryptic sensor for HIV-1 activates antiviral innate immunity in dendritic cells. Nature 467, 214-217 (2010).

173. Rasaiyaah, J., et al. HIV-1 evades innate immune recognition through specific cofactor recruitment. Nature 503, 402-405 (2013).

174. Siliciano, R.F. & Greene, W.C. HIV latency. Cold Spring Harb Perspect Med 1, a007096 (2011).

175. Shan, L., et al. Stimulation of HIV-1-specific cytolytic T lymphocytes facilitates elimination of latent viral reservoir after virus reactivation. Immunity 36, 491-501 (2012).

176. Katlama, C., et al. Barriers to a cure for HIV: new ways to target and eradicate HIV-1 reservoirs. Lancet 381, 2109-2117 (2013).

177. Ho, D.D., et al. Rapid turnover of plasma virions and CD4 lymphocytes in HIV-1 infection. Nature 373, 123-126 (1995).

178. Bukrinsky, M.I., Stanwick, T.L., Dempsey, M.P. & Stevenson, M. Quiescent T lymphocytes as an inducible virus reservoir in HIV-1 infection. Science 254, 423-427 (1991).

179. Zack, J.A., et al. HIV-1 entry into quiescent primary lymphocytes: molecular analysis reveals a labile, latent viral structure. Cell 61, 213-222 (1990).

180. Meyerhans, A., et al. Restriction and enhancement of human immunodeficiency virus type 1 replication by modulation of intracellular deoxynucleoside triphosphate pools. J Virol 68, 535-540 (1994).

181. Abbas, A., Lichtman, A.H. & Pillai, S. Cellular and Molecular Immunology, (Elsevier, 2017).

149

182. Wightman, F., et al. Both CD31(+) and CD31⁻ naive CD4(+) T cells are persistent HIV type 1-infected reservoirs in individuals receiving antiretroviral therapy. J Infect Dis 202, 1738-1748 (2010).

183. Yuan, J., Crittenden, R.B. & Bender, T.P. c-Myb promotes the survival of CD4+CD8+ double-positive thymocytes through upregulation of Bcl-xL. J Immunol 184, 2793-2804 (2010).

184. Chun, T.W., et al. In vivo fate of HIV-1-infected T cells: quantitative analysis of the transition to stable latency. Nat Med 1, 1284-1290 (1995).

185. Chun, T.W., et al. Early establishment of a pool of latently infected, resting CD4(+) T cells during primary HIV-1 infection. Proc Natl Acad Sci U S A 95, 8869-8873 (1998).

186. Zhou, Y., Zhang, H., Siliciano, J.D. & Siliciano, R.F. Kinetics of human immunodeficiency virus type 1 decay following entry into resting CD4+ T cells. J Virol 79, 2199-2210 (2005).

187. Donahue, D.A. & Wainberg, M.A. Cellular and molecular mechanisms involved in the establishment of HIV-1 latency. Retrovirology 10, 11 (2013).

188. Albert, B.J., et al. Combinations of isoform-targeted histone deacetylase inhibitors and bryostatin analogues display remarkable potency to activate latent HIV without global T-cell activation. Sci Rep 7, 7456 (2017).

189. Spina, C.A., et al. An in-depth comparison of latent HIV-1 reactivation in multiple cell model systems and resting CD4+ T cells from aviremic patients. PLoS Pathog 9, e1003834 (2013).

190. Bosque, A. & Planelles, V. Studies of HIV-1 latency in an ex vivo model that uses primary central memory T cells. Methods 53, 54-61 (2011).

191. Hakre, S., Chavez, L., Shirakawa, K. & Verdin, E. Epigenetic regulation of HIV latency. Curr Opin HIV AIDS 6, 19-24 (2011).

150

192. Van Lint, C., Emiliani, S., Ott, M. & Verdin, E. Transcriptional activation and chromatin remodeling of the HIV-1 promoter in response to histone acetylation. EMBO J 15, 1112-1120 (1996).

193. Boehm, D., et al. SMYD2-Mediated Histone Methylation Contributes to HIV- 1 Latency. Cell Host Microbe 21, 569-579.e566 (2017).

194. Kim, K.C., et al. Identification of novel genes associated with HIV-1 latency by analysis of histone modifications. Hum Genomics 11, 9 (2017).

195. Sheridan, P.L., Mayall, T.P., Verdin, E. & Jones, K.A. Histone acetyltransferases regulate HIV-1 enhancer activity in vitro. Genes Dev 11, 3327-3340 (1997).

196. Akpamagbo, Y.A., et al. HIV-1 Transcription Inhibitors Increase the Synthesis of Viral Non-Coding RNA that Contribute to Latency. Curr Pharm Des 23, 4133-4144 (2017).

197. Lewinski, M.K., et al. Genome-wide analysis of chromosomal features repressing human immunodeficiency virus transcription. J Virol 79, 6610- 6619 (2005).

198. Meehan, A.M., et al. LEDGF/p75 proteins with alternative chromatin tethers are functional HIV-1 cofactors. PLoS Pathog 5, e1000522 (2009).

199. Schröder, A.R., et al. HIV-1 integration in the favors active genes and local hotspots. Cell 110, 521-529 (2002).

200. Felsenfeld, G. & Groudine, M. Controlling the double helix. Nature 421, 448- 453 (2003).

201. Tyagi, M. & Bukrinsky, M. Human immunodeficiency virus (HIV) latency: the major hurdle in HIV eradication. Mol Med 18, 1096-1108 (2012).

151

202. Wolffe, A.P. Nucleosome positioning and modification: chromatin structures that potentiate transcription. Trends Biochem Sci 19, 240-244 (1994).

203. Coiras, M., López-Huertas, M.R., Pérez-Olmeda, M. & Alcamí, J. Understanding HIV-1 latency provides clues for the eradication of long-term reservoirs. Nat Rev Microbiol 7, 798-812 (2009).

204. Verdin, E., Paras, P. & Van Lint, C. Chromatin disruption in the promoter of human immunodeficiency virus type 1 during transcriptional activation. EMBO J 12, 3249-3259 (1993).

205. Richman, D.D., et al. The challenge of finding a cure for HIV infection. Science 323, 1304-1307 (2009).

206. Bowman, M.C., Archin, N.M. & Margolis, D.M. Pharmaceutical approaches to eradication of persistent HIV infection. Expert Rev Mol Med 11, e6 (2009).

207. Savarino, A., et al. "Shock and kill" effects of class I-selective histone deacetylase inhibitors in combination with the glutathione synthesis inhibitor buthionine sulfoximine in cell line models for HIV-1 quiescence. Retrovirology 6, 52 (2009).

208. Chun, T.W., Davey, R.T., Engel, D., Lane, H.C. & Fauci, A.S. Re- emergence of HIV after stopping therapy. Nature 401, 874-875 (1999).

209. Perelson, A.S., et al. Decay characteristics of HIV-1-infected compartments during combination therapy. Nature 387, 188-191 (1997).

210. Wei, X., et al. Viral dynamics in human immunodeficiency virus type 1 infection. Nature 373, 117-122 (1995).

211. Burnett, J.C., et al. Combinatorial latency reactivation for HIV-1 subtypes and variants. J Virol 84, 5958-5974 (2010).

152

212. Bieniasz, P.D. Restriction factors: a defense against retroviral infection. Trends Microbiol 11, 286-291 (2003).

213. Zheng, Y.H., Jeang, K.T. & Tokunaga, K. Host restriction factors in retroviral infection: promises in virus-host interaction. Retrovirology 9, 112 (2012).

214. Jia, X., Zhao, Q. & Xiong, Y. HIV suppression by host restriction factors and viral immune evasion. Curr Opin Struct Biol 31, 106-114 (2015).

215. Goujon, C., et al. Human MX2 is an interferon-induced post-entry inhibitor of HIV-1 infection. Nature 502, 559-562 (2013).

216. Hotter, D., Sauter, D. & Kirchhoff, F. Emerging role of the host restriction factor tetherin in viral immune sensing. J Mol Biol 425, 4956-4964 (2013).

217. Luban, J. Innate immune sensing of HIV-1 by dendritic cells. Cell Host Microbe 12, 408-418 (2012).

218. Sze, A., Olagnier, D., Lin, R., van Grevenynghe, J. & Hiscott, J. SAMHD1 host restriction factor: a link with innate immune sensing of retrovirus infection. J Mol Biol 425, 4981-4994 (2013).

219. Harris, R.S. & Liddament, M.T. Retroviral restriction by APOBEC proteins. Nat Rev Immunol 4, 868-877 (2004).

220. Wagner, J.M., et al. General Model for Retroviral Capsid Pattern Recognition by TRIM5 Proteins. J Virol 92(2018).

221. Morger, D., et al. The Three-Fold Axis of the HIV-1 Capsid Lattice Is the Species-Specific Binding Interface for TRIM5α. J Virol 92(2018).

222. Varthakavi, V., Smith, R.M., Bour, S.P., Strebel, K. & Spearman, P. Viral protein U counteracts a human host cell restriction that inhibits HIV-1 particle production. Proc Natl Acad Sci U S A 100, 15154-15159 (2003).

153

223. Neil, S.J., Zang, T. & Bieniasz, P.D. Tetherin inhibits retrovirus release and is antagonized by HIV-1 Vpu. Nature 451, 425-430 (2008).

224. Fackler, O.T. Spotlight on HIV-1 Nef: SERINC3 and SERINC5 Identified as Restriction Factors Antagonized by the Pathogenesis Factor. Viruses 7, 6730-6738 (2015).

225. Malim, M.H. & Bieniasz, P.D. HIV Restriction Factors and Mechanisms of Evasion. Cold Spring Harb Perspect Med 2, a006940 (2012).

226. Wu, L. Cellular and Biochemical Mechanisms of the Retroviral Restriction Factor SAMHD1. ISRN Biochem (2013).

227. Bishop, K.N., et al. Cytidine deamination of retroviral DNA by diverse APOBEC proteins. Curr Biol 14, 1392-1396 (2004).

228. Shi, J., et al. HIV-1 Nef antagonizes SERINC5 restriction by downregulation of SERINC5 via the endosome/lysosome system. J Virol (2018).

229. Hrecka, K., et al. Vpx relieves inhibition of HIV-1 infection of macrophages mediated by the SAMHD1 protein. Nature 474, 658-661 (2011).

230. Gramberg, T., et al. Restriction of diverse retroviruses by SAMHD1. Retrovirology 10, 26 (2013).

231. Hollenbaugh, J.A., et al. Host factor SAMHD1 restricts DNA viruses in non- dividing myeloid cells. PLoS Pathog 9, e1003481 (2013).

232. Chen, Z., et al. Inhibition of replication by SAMHD1. Biochem Biophys Res Commun 450, 1462-1468 (2014).

233. Goldstone, D.C., et al. HIV-1 restriction factor SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase. Nature 480, 379-382 (2011).

154

234. Powell, R.D., Holland, P.J., Hollis, T. & Perrino, F.W. Aicardi-Goutieres syndrome gene and HIV-1 restriction factor SAMHD1 is a dGTP-regulated deoxynucleotide triphosphohydrolase. J Biol Chem 286, 43596-43600 (2011).

235. Amie, S.M., Bambara, R.A. & Kim, B. GTP is the primary activator of the anti-HIV restriction factor SAMHD1. J Biol Chem 288, 25001-25006 (2013).

236. Descours, B., et al. SAMHD1 restricts HIV-1 reverse transcription in quiescent CD4(+) T-cells. Retrovirology 9, 87 (2012).

237. Berger, A., et al. SAMHD1-deficient CD14+ cells from individuals with Aicardi-Goutières syndrome are highly susceptible to HIV-1 infection. PLoS Pathog 7, e1002425 (2011).

238. St Gelais, C., et al. SAMHD1 restricts HIV-1 infection in dendritic cells (DCs) by dNTP depletion, but its expression in DCs and primary CD4+ T- lymphocytes cannot be upregulated by interferons. Retrovirology 9, 105 (2012).

239. Rice, G.I., et al. Mutations involved in Aicardi-Goutières syndrome implicate SAMHD1 as regulator of the innate immune response. Nat Genet 41, 829- 832 (2009).

240. Behrendt, R., et al. Mouse SAMHD1 has antiretroviral activity and suppresses a spontaneous cell-intrinsic antiviral response. Cell Rep 4, 689- 696 (2013).

241. Maelfait, J., Bridgeman, A., Benlahrech, A., Cursi, C. & Rehwinkel, J. Restriction by SAMHD1 Limits cGAS/STING-Dependent Innate and Adaptive Immune Responses to HIV-1. Cell Rep 16, 1492-1501 (2016).

242. Crow, Y.J., et al. Characterization of human disease phenotypes associated with mutations in TREX1, RNASEH2A, RNASEH2B, RNASEH2C, SAMHD1, ADAR, and IFIH1. Am J Med Genet A 167A, 296-312 (2015).

155

243. Crow, Y.J. Aicardi-Goutières syndrome. Handb Clin Neurol 113, 1629-1635 (2013).

244. Kaliyadan, F. Hiv and lupus erythematosus: a diagnostic dilemma. Indian J Dermatol 53, 80-82 (2008).

245. Feng, Q., Moran, J.V., Kazazian, H.H. & Boeke, J.D. Human L1 retrotransposon encodes a conserved endonuclease required for retrotransposition. Cell 87, 905-916 (1996).

246. Mathias, S.L., Scott, A.F., Kazazian, H.H., Boeke, J.D. & Gabriel, A. Reverse transcriptase encoded by a human transposable element. Science 254, 1808-1810 (1991).

247. Zhao, K., et al. Modulation of LINE-1 and Alu/SVA retrotransposition by Aicardi-Goutières syndrome-related SAMHD1. Cell Rep 4, 1108-1115 (2013).

248. Hu, S., et al. SAMHD1 Inhibits LINE-1 Retrotransposition by Promoting Stress Granule Formation. PLoS Genet 11, e1005367 (2015).

249. Crow, M.K. Long interspersed nuclear elements (LINE-1): potential triggers of systemic autoimmune disease. Autoimmunity 43, 7-16 (2010).

250. Franzolin, E., et al. The deoxynucleotide triphosphohydrolase SAMHD1 is a major regulator of DNA precursor pools in mammalian cells. Proc Natl Acad Sci U S A 110, 14272-14277 (2013).

251. Kretschmer, S., et al. SAMHD1 prevents autoimmunity by maintaining genome stability. Ann Rheum Dis 74, e17 (2015).

252. Cribier, A., Descours, B., Valadão, A.L., Laguette, N. & Benkirane, M. Phosphorylation of SAMHD1 by cyclin A2/CDK1 regulates its restriction activity toward HIV-1. Cell Rep 3, 1036-1043 (2013).

156

253. St Gelais, C., et al. Identification of cellular proteins interacting with the retroviral restriction factor SAMHD1. J Virol 88, 5834-5844 (2014).

254. St Gelais, C., et al. A Putative Cyclin-binding Motif in Human SAMHD1 Contributes to Protein Phosphorylation, Localization, and Stability. J Biol Chem 291, 26332-26342 (2016).

255. Bonifati, S., et al. SAMHD1 controls cell cycle status, apoptosis and HIV-1 infection in monocytic THP-1 cells. Virology 495, 92-100 (2016).

256. Kohnken, R., Kodigepalli, K.M. & Wu, L. Regulation of deoxynucleotide metabolism in cancer: novel mechanisms and therapeutic implications. Mol Cancer 14, 176 (2015).

257. Mathews, C.K. Deoxyribonucleotide metabolism, mutagenesis and cancer. Nat Rev Cancer 15, 528-539 (2015).

258. Imielinski, M., et al. Mapping the hallmarks of lung adenocarcinoma with massively parallel sequencing. Cell 150, 1107-1120 (2012).

259. Liu, J., et al. Genome and transcriptome sequencing of lung cancers reveal diverse mutational and splicing events. Genome Res 22, 2315-2327 (2012).

260. Network, C.G.A. Comprehensive molecular characterization of human colon and rectal cancer. Nature 487, 330-337 (2012).

261. Clifford, R., et al. SAMHD1 is mutated recurrently in chronic lymphocytic leukemia and is involved in response to DNA damage. Blood 123, 1021- 1031 (2014).

262. Landau, D.A., et al. Evolution and impact of subclonal mutations in chronic lymphocytic leukemia. Cell 152, 714-726 (2013).

263. Johansson, P., et al. SAMHD1 is recurrently mutated in T-cell prolymphocytic leukemia. Blood Cancer J 8, 11 (2018). 157

264. Rossi, D. SAMHD1: a new gene for CLL. Blood 123, 951-952 (2014).

265. Cabello-Lobato, M.J., Wang, S. & Schmidt, C.K. SAMHD1 Sheds Moonlight on DNA Double-Strand Break Repair. Trends Genet 33, 895-897 (2017).

266. Daddacha, W., et al. SAMHD1 Promotes DNA End Resection to Facilitate DNA Repair by Homologous Recombination. Cell Rep 20, 1921-1935 (2017).

267. Aird, K.M. & Zhang, R. Nucleotide metabolism, oncogene-induced senescence and cancer. Cancer Lett 356, 204-210 (2015).

268. Ji, X., et al. Mechanism of allosteric activation of SAMHD1 by dGTP. Nature structural & molecular biology 20, 1304-1309 (2013).

269. Rampazzo, C., et al. Regulation by degradation, a cellular defense against deoxyribonucleotide pool imbalances. Mutat Res 703, 2-10 (2010).

270. Bester, A.C., et al. Nucleotide deficiency promotes genomic instability in early stages of cancer development. Cell 145, 435-446 (2011).

271. Miazzi, C., et al. Allosteric regulation of the human and mouse deoxyribonucleotide triphosphohydrolase sterile α-motif/histidine-aspartate domain-containing protein 1 (SAMHD1). J Biol Chem 289, 18339-18346 (2014).

272. Ji, X., Tang, C., Zhao, Q., Wang, W. & Xiong, Y. Structural basis of cellular dNTP regulation by SAMHD1. Proc Natl Acad Sci U S A 111, E4305-4314 (2014).

273. Li, Y., et al. Structural Insights into the High-efficiency Catalytic Mechanism of the Sterile α-Motif/Histidine-Aspartate Domain-containing Protein. J Biol Chem 290, 29428-29437 (2015).

158

274. Hansen, E.C., Seamon, K.J., Cravens, S.L. & Stivers, J.T. GTP activator and dNTP substrates of HIV-1 restriction factor SAMHD1 generate a long- lived activated state. Proc Natl Acad Sci U S A 111, E1843-1851 (2014).

275. Brandariz-Nuñez, A., et al. Role of SAMHD1 nuclear localization in restriction of HIV-1 and SIVmac. Retrovirology 9, 49 (2012).

276. White, T.E., et al. Contribution of SAM and HD domains to retroviral restriction mediated by human SAMHD1. Virology 436, 81-90 (2013).

277. Qiao, F. & Bowie, J.U. The many faces of SAM. Sci STKE 2005, re7 (2005).

278. Schultz, J., Ponting, C.P., Hofmann, K. & Bork, P. SAM as a protein interaction domain involved in developmental regulation. Protein Sci 6, 249- 253 (1997).

279. Grimshaw, S.J., et al. Structure of the sterile alpha motif (SAM) domain of the Saccharomyces cerevisiae mitogen-activated protein kinase pathway- modulating protein STE50 and analysis of its interaction with the STE11 SAM. J Biol Chem 279, 2192-2201 (2004).

280. Chi, S.W., Ayed, A. & Arrowsmith, C.H. Solution structure of a conserved C-terminal domain of p73 with structural homology to the SAM domain. EMBO J 18, 4438-4445 (1999).

281. Harada, B.T., et al. Regulation of enzyme localization by polymerization: polymer formation by the SAM domain of diacylglycerol kinase delta1. Structure 16, 380-387 (2008).

282. Zhu, Y.X., et al. The SH3-SAM adaptor HACS1 is up-regulated in B cell activation signaling cascades. J Exp Med 200, 737-747 (2004).

283. Brandariz-Nuñez, A., et al. Contribution of oligomerization to the anti-HIV-1 properties of SAMHD1. Retrovirology 10, 131 (2013).

159

284. St Gelais, C., et al. A Cyclin-binding Motif in Human SAMHD1 Is Required for Its HIV-1 Restriction, dNTPase Activity, Tetramer Formation, and Efficient Phosphorylation. J Virol 96, 01787-01717 (2018).

285. Hofmann, H., et al. The Vpx lentiviral accessory protein targets SAMHD1 for degradation in the nucleus. J Virol 86, 12552-12560 (2012).

286. Srivastava, S., et al. Lentiviral Vpx accessory factor targets VprBP/DCAF1 substrate adaptor for cullin 4 E3 ubiquitin ligase to enable macrophage infection. PLoS Pathog 4, e1000059 (2008).

287. Goujon, C., et al. Characterization of simian immunodeficiency virus SIVSM/human immunodeficiency virus type 2 Vpx function in human myeloid cells. J Virol 82, 12335-12345 (2008).

288. Ahn, J., et al. HIV/simian immunodeficiency virus (SIV) accessory virulence factor Vpx loads the host cell restriction factor SAMHD1 onto the E3 ubiquitin ligase complex CRL4DCAF1. J Biol Chem 287, 12550-12558 (2012).

289. Wei, W., et al. A novel DCAF1-binding motif required for Vpx-mediated degradation of nuclear SAMHD1 and Vpr-induced G2 arrest. Cell Microbiol 14, 1745-1756 (2012).

290. Li, N., Zhang, W. & Cao, X. Identification of human homologue of mouse IFN-gamma induced protein from human dendritic cells. Immunol Lett 74, 221-224 (2000).

291. Pauls, E., et al. Restriction of HIV-1 replication in primary macrophages by IL-12 and IL-18 through the upregulation of SAMHD1. J Immunol 190, 4736- 4741 (2013).

292. Chen, Z., Zhang, L. & Ying, S. SAMHD1: a novel antiviral factor in intrinsic immunity. Future Microbiol 7, 1117-1126 (2012).

160

293. Yang, S., et al. Interferon regulatory factor 3 is a key regulation factor for inducing the expression of SAMHD1 in antiviral innate immunity. Sci Rep 6, 29665 (2016).

294. Riess, M., et al. Interferons Induce Expression of SAMHD1 in Monocytes through Down-regulation of miR-181a and miR-30a. J Biol Chem 292, 264- 277 (2017).

295. Goujon, C., et al. Evidence for IFNα-induced, SAMHD1-independent inhibitors of early HIV-1 infection. Retrovirology 10, 23 (2013).

296. Schmidt, S., et al. SAMHD1's protein expression profile in humans. J Leukoc Biol 98, 5-14 (2015).

297. Baldauf, H.M., et al. SAMHD1 restricts HIV-1 infection in resting CD4(+) T cells. Nat Med 18, 1682-1687 (2012).

298. Che, K.F., et al. HIV-1 impairs in vitro priming of naïve T cells and gives rise to contact-dependent suppressor T cells. Eur J Immunol 40, 2248-2258 (2010).

299. Miller, E. & Bhardwaj, N. dysregulation during HIV-1 infection. Immunol Rev 254, 170-189 (2013).

300. Banchereau, J. & Steinman, R.M. Dendritic cells and the control of immunity. Nature 392, 245-252 (1998).

301. Lahouassa, H., et al. SAMHD1 restricts the replication of human immunodeficiency virus type 1 by depleting the intracellular pool of deoxynucleoside triphosphates. Nat Immunol 13, 223-228 (2012).

302. Zhang, C., de Silva, S., Wang, J.H. & Wu, L. Co-evolution of primate SAMHD1 and lentivirus Vpx leads to the loss of the vpx gene in HIV-1 ancestor. PLoS One 7, e37477 (2012).

161

303. Huber, A.D., et al. SAMHD1 has differential impact on the efficacies of HIV nucleoside reverse transcriptase inhibitors. Antimicrob Agents Chemother 58, 4915-4919 (2014).

304. Ballana, E., et al. SAMHD1 specifically affects the antiviral potency of thymidine analog HIV reverse transcriptase inhibitors. Antimicrob Agents Chemother 58, 4804-4813 (2014).

305. Ordonez, P., et al. SAMHD1 enhances nucleoside-analogue efficacy against HIV-1 in myeloid cells. Sci Rep 7, 42824 (2017).

306. Hollenbaugh, J.A., et al. Substrates and Inhibitors of SAMHD1. PLoS One 12, e0169052 (2017).

307. Seamon, K.J., et al. Small molecule inhibition of SAMHD1 dNTPase by tetramer destabilization. J Am Chem Soc 136, 9822-9825 (2014).

308. Antonucci, J.M., et al. SAMHD1-mediated HIV-1 restriction in cells does not involve ribonuclease activity. Nat Med 22, 1072-1074 (2016).

309. Lahouassa, H., et al. SAMHD1 restricts the replication of human immunodeficiency virus type 1 by depleting the intracellular pool of deoxynucleoside triphosphates. Nat Immunol 13, 223-228 (2012).

310. White, T.E., et al. The retroviral restriction ability of SAMHD1, but not its deoxynucleotide triphosphohydrolase activity, is regulated by phosphorylation. Cell Host Microbe 13, 441-451 (2013).

311. Welbourn, S., Dutta, S.M., Semmes, O.J. & Strebel, K. Restriction of virus infection but not catalytic dNTPase activity is regulated by phosphorylation of SAMHD1. J Virol 87, 11516-11524 (2013).

312. Welbourn, S. & Strebel, K. Low dNTP levels are necessary but may not be sufficient for lentiviral restriction by SAMHD1. Virology 488, 271-277 (2016).

162

313. Beloglazova, N., et al. Nuclease activity of the human SAMHD1 protein implicated in the Aicardi-Goutieres syndrome and HIV-1 restriction. J Biol Chem 288, 8101-8110 (2013).

314. Tüngler, V., et al. Single-stranded nucleic acids promote SAMHD1 complex formation. J Mol Med (Berl) 91, 759-770 (2013).

315. Goncalves, A., et al. SAMHD1 is a nucleic-acid binding protein that is mislocalized due to aicardi-goutières syndrome-associated mutations. Hum Mutat 33, 1116-1122 (2012).

316. Ryoo, J., et al. The ribonuclease activity of SAMHD1 is required for HIV-1 restriction. Nat Med 20, 936-941 (2014).

317. Choi, J., Ryoo, J., Oh, C., Hwang, S. & Ahn, K. SAMHD1 specifically restricts retroviruses through its RNase activity. Retrovirology 12, 46 (2015).

318. Seamon, K.J., Sun, Z., Shlyakhtenko, L.S., Lyubchenko, Y.L. & Stivers, J.T. SAMHD1 is a single-stranded nucleic acid binding protein with no active site-associated nuclease activity. Nucleic Acids Res 43, 6486-6499 (2015).

319. Bloch, N., et al. A Highly Active Isoform of Lentivirus Restriction Factor SAMHD1 in Mouse. J Biol Chem 292, 1068-1080 (2017).

320. Wittmann, S., et al. Phosphorylation of murine SAMHD1 regulates its antiretroviral activity. Retrovirology 12, 103 (2015).

321. Ryoo, J., Hwang, S.Y., Choi, J., Oh, C. & Ahn, K. Reply to SAMHD1- mediated HIV-1 restriction in cells does not involve ribonuclease activity. Nat Med 22, 1074-1075 (2016).

322. Ryoo, J., Hwang, S.Y., Choi, J., Oh, C. & Ahn, K. SAMHD1, the Aicardi- Goutières syndrome gene and retroviral restriction factor, is a phosphorolytic ribonuclease rather than a hydrolytic ribonuclease. Biochem Biophys Res Commun 477, 977-981 (2016).

163

323. Pauls, E., et al. Cell cycle control and HIV-1 susceptibility are linked by CDK6-dependent CDK2 phosphorylation of SAMHD1 in myeloid and lymphoid cells. J Immunol 193, 1988-1997 (2014).

324. Arnold, L.H., et al. Phospho-dependent Regulation of SAMHD1 Oligomerisation Couples Catalysis and Restriction. PLoS Pathog 11, e1005194 (2015).

325. Tang, C., Ji, X., Wu, L. & Xiong, Y. Impaired dNTPase activity of SAMHD1 by phosphomimetic mutation of Thr-592. J Biol Chem 290, 26352-26359 (2015).

326. Yan, J., et al. CyclinA2-Cyclin-dependent Kinase Regulates SAMHD1 Protein Phosphohydrolase Domain. J Biol Chem 290, 13279-13292 (2015).

327. Mauney, C.H., et al. The SAMHD1 dNTP Triphosphohydrolase Is Controlled by a Redox Switch. Antioxid Redox Signal 27, 1317-1331 (2017).

328. Seamon, K.J., Bumpus, N.N. & Stivers, J.T. Single-Stranded Nucleic Acids Bind to the Tetramer Interface of SAMHD1 and Prevent Formation of the Catalytic Homotetramer. Biochemistry 55, 6087-6099 (2016).

329. Kim, B., Nguyen, L.A., Daddacha, W. & Hollenbaugh, J.A. Tight interplay among SAMHD1 protein level, cellular dNTP levels, and HIV-1 proviral DNA synthesis kinetics in human primary monocyte-derived macrophages. J Biol Chem 287, 21570-21574 (2012).

330. Ruelas, D.S. & Greene, W.C. An integrated overview of HIV-1 latency. Cell 155, 519-529 (2013).

331. Jordan, A., Bisgrove, D. & Verdin, E. HIV reproducibly establishes a latent infection after acute infection of T cells in vitro. EMBO J 22, 1868-1877 (2003).

332. Wu, L. SAMHD1 knockout mice: modeling retrovirus restriction in vivo. Retrovirology 10, 142 (2013). 164

333. Laguette, N., et al. SAMHD1 is the dendritic- and myeloid-cell-specific HIV- 1 restriction factor counteracted by Vpx. Nature 474, 654-657 (2011).

334. Baldauf, H.M., et al. SAMHD1 restricts HIV-1 infection in resting CD4(+) T cells. Nat Med 18, 1682-1687 (2012).

335. Ryoo, J., et al. The ribonuclease activity of SAMHD1 is required for HIV-1 restriction. Nat Med 20, 936-941 (2014).

336. Cribier, A., Descours, B., Valadao, A.L., Laguette, N. & Benkirane, M. Phosphorylation of SAMHD1 by cyclin A2/CDK1 regulates its restriction activity toward HIV-1. Cell Rep 3, 1036-1043 (2013).

337. Strahle, L., et al. Activation of the beta interferon promoter by unnatural Sendai virus infection requires RIG-I and is inhibited by viral C proteins. J Virol 81, 12227-12237 (2007).

338. Hach, J.C., McMichael, T., Chesarino, N.M. & Yount, J.S. Palmitoylation on conserved and nonconserved cysteines of murine IFITM1 regulates its stability and anti-influenza A virus activity. J Virol 87, 9923-9927 (2013).

339. Diamond, T.L., et al. Macrophage tropism of HIV-1 depends on efficient cellular dNTP utilization by reverse transcriptase. J Biol Chem 279, 51545- 51553 (2004).

340. St Gelais, C., Roger, J. & Wu, L. Non-POU Domain-Containing Octamer- Binding Protein Negatively Regulates HIV-1 Infection in CD4(+) T Cells. AIDS Res Hum Retroviruses 31, 806-816 (2015).

341. White, T.E., et al. The retroviral restriction ability of SAMHD1, but not its deoxynucleotide triphosphohydrolase activity, is regulated by phosphorylation. Cell Host Microbe 13, 441-451 (2013).

342. Dong, C., Kwas, C. & Wu, L. Transcriptional restriction of human immunodeficiency virus type 1 gene expression in undifferentiated primary monocytes. J Virol 83, 3518-3527 (2009). 165

343. Yan, J., et al. Tetramerization of SAMHD1 is required for biological activity and inhibition of HIV infection. J Biol Chem 288, 10406-10417 (2013).

344. Zhu, C., et al. Structural insight into dGTP-dependent activation of tetrameric SAMHD1 deoxynucleoside triphosphate triphosphohydrolase. Nat Commun 4, 2722 (2013).

345. Hofmann, H., et al. Inhibition of CUL4A Neddylation causes a reversible block to SAMHD1-mediated restriction of HIV-1. J Virol 87, 11741-11750 (2013).

346. Welbourn, S. & Strebel, K. Low dNTP levels are necessary but may not be sufficient for lentiviral restriction by SAMHD1. Virology 488, 271-277 (2015).

347. Wittmann, S., et al. Phosphorylation of murine SAMHD1 regulates its antiretroviral activity. Retrovirology 12, 103 (2015).

348. Gramberg, T., et al. Restriction of diverse retroviruses by SAMHD1. Retrovirology 10, 26 (2013).

349. Kim, E.T., White, T.E., Brandariz-Núñez, A., Diaz-Griffero, F. & Weitzman, M.D. SAMHD1 restricts herpes simplex virus 1 in macrophages by limiting DNA replication. J Virol 87, 12949-12956 (2013).

350. Jeong, G.U., Park, I.H., Ahn, K. & Ahn, B.Y. Inhibition of hepatitis B virus replication by a dNTPase-dependent function of the host restriction factor SAMHD1. Virology 495, 71-78 (2016).

351. Sommer, A.F., et al. Restrictive influence of SAMHD1 on Hepatitis B Virus life cycle. Sci Rep 6, 26616 (2016).

352. Wang, F., et al. Phosphorylation of mouse SAMHD1 regulates its restriction of human immunodeficiency virus type 1 infection, but not murine leukemia virus infection. Virology 487, 273-284 (2016).

166

353. Shan, L., et al. Influence of host gene transcription level and orientation on HIV-1 latency in a primary-cell model. J Virol 85, 5384-5393 (2011).

354. Margolis, D.M., Garcia, J.V., Hazuda, D.J. & Haynes, B.F. Latency reversal and viral clearance to cure HIV-1. Science 353, aaf6517 (2016).

355. Van Lint, C., Bouchat, S. & Marcello, A. HIV-1 transcription and latency: an update. Retrovirology 10, 67 (2013).

356. Araínga, M., et al. A mature macrophage is a principal HIV-1 cellular reservoir in humanized mice after treatment with long acting antiretroviral therapy. Retrovirology 14, 17 (2017).

357. Abbas, W., Tariq, M., Iqbal, M., Kumar, A. & Herbein, G. Eradication of HIV- 1 from the macrophage reservoir: an uncertain goal? Viruses 7, 1578-1598 (2015).

358. Kumar, A., Abbas, W. & Herbein, G. HIV-1 latency in monocytes/macrophages. Viruses 6, 1837-1860 (2014).

359. Bosque, A. & Planelles, V. Induction of HIV-1 latency and reactivation in primary memory CD4+ T cells. Blood 113, 58-65 (2009).

360. Finzi, D., et al. Identification of a reservoir for HIV-1 in patients on highly active antiretroviral therapy. Science 278, 1295-1300 (1997).

361. Wang, J.H., Janas, A.M., Olson, W.J., KewalRamani, V.N. & Wu, L. CD4 coexpression regulates DC-SIGN-mediated transmission of human immunodeficiency virus type 1. J Virol 81, 2497-2507 (2007).

362. Panfil, A.R., et al. PRMT5 Is Upregulated in HTLV-1-Mediated T-Cell Transformation and Selective Inhibition Alters Viral Gene Expression and Infected Cell Survival. Viruses 8(2015).

167

363. Li, C., et al. Naf1 Regulates HIV-1 Latency by Suppressing Viral Promoter- Driven Gene Expression in Primary CD4+ T Cells. J Virol 91, 01830-01816 (2017).

364. Jones, C.P., Datta, S.A., Rein, A., Rouzina, I. & Musier-Forsyth, K. Matrix domain modulates HIV-1 Gag's nucleic acid chaperone activity via inositol phosphate binding. J Virol 85, 1594-1603 (2011).

365. Rye-McCurdy, T., Rouzina, I. & Musier-Forsyth, K. Fluorescence anisotropy-based salt-titration approach to characterize protein-nucleic acid interactions. Methods Mol Biol 1259, 385-402 (2015).

366. Stewart-Maynard, K.M., et al. Retroviral nucleocapsid proteins display nonequivalent levels of nucleic acid chaperone activity. J Virol 82, 10129- 10142 (2008).

367. Livak, K.J. & Schmittgen, T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25, 402-408 (2001).

368. Shifera, A.S. & Hardin, J.A. Factors modulating expression of Renilla luciferase from control plasmids used in luciferase reporter gene assays. Anal Biochem 396, 167-172 (2010).

369. Andresen, V., et al. Suppression of HTLV-1 replication by Tax-mediated rerouting of the p13 viral protein to nuclear speckles. Blood 118, 1549-1559 (2011).

370. de Silva, S., et al. Promoter methylation regulates SAMHD1 gene expression in human CD4+ T cells. J Biol Chem 288, 9284-9292 (2013).

371. Wolf, M., LeVine, H., May, W.S., Cuatrecasas, P. & Sahyoun, N. A model for intracellular translocation of protein kinase C involving synergism between Ca2+ and phorbol esters. Nature 317, 546-549 (1985).

168

372. Blumberg, P.M. Protein kinase C as the receptor for the phorbol ester tumor promoters: sixth Rhoads memorial award lecture. Cancer Res 48, 1-8 (1988).

373. Folks, T.M., et al. Tumor necrosis factor alpha induces expression of human immunodeficiency virus in a chronically infected T-cell clone. Proc Natl Acad Sci U S A 86, 2365-2368 (1989).

374. Duh, E.J., Maury, W.J., Folks, T.M., Fauci, A.S. & Rabson, A.B. Tumor necrosis factor alpha activates human immunodeficiency virus type 1 through induction of nuclear factor binding to the NF-kappa B sites in the long terminal repeat. Proc Natl Acad Sci U S A 86, 5974-5978 (1989).

375. Taube, R., Fujinaga, K., Wimmer, J., Barboric, M. & Peterlin, B.M. Tat transactivation: a model for the regulation of eukaryotic transcriptional elongation. Virology 264, 245-253 (1999).

376. Gaudray, G., et al. The complementary strand of the human T-cell leukemia virus type 1 RNA genome encodes a bZIP transcription factor that down- regulates viral transcription. J Virol 76, 12813-12822 (2002).

377. Zhang, W., et al. Human T-lymphotropic virus type 1 p30(II) regulates gene transcription by binding CREB binding protein/p300. J Virol 75, 9885-9895 (2001).

378. Sørensen, K.D., et al. Distinct roles of enhancer nuclear factor 1 (NF1) sites in plasmacytoma and osteopetrosis induction by Akv1-99 murine leukemia virus. Virology 334, 234-244 (2005).

379. Wu, L. Cellular and Biochemical Mechanisms of the Retroviral Restriction Factor SAMHD1. ISRN Biochem 2013(2013).

380. Li, C., et al. Naf1 regulates HIV-1 latency by suppressing viral promoter- driven gene expression in primary CD4+ T cells. J Virol 91(2017).

169

381. Thaker, Y.R., Schneider, H. & Rudd, C.E. TCR and CD28 activate the transcription factor NF-κB in T-cells via distinct adaptor signaling complexes. Immunol Lett 163, 113-119 (2015).

382. Schütze, S., Wiegmann, K., Machleidt, T. & Krönke, M. TNF-induced activation of NF-kappa B. Immunobiology 193, 193-203 (1995).

383. Buzovetsky, O., et al. The SAM domain of mouse SAMHD1 is critical for its activation and regulation. Nat Commun 9, 411 (2018).

384. Bermejo, M., et al. Dasatinib inhibits HIV-1 replication through the interference of SAMHD1 phosphorylation in CD4+ T cells. Biochem Pharmacol 106, 30-45 (2016).

385. Laguette, N., et al. SAMHD1 is the dendritic- and myeloid-cell-specific HIV- 1 restriction factor counteracted by Vpx. Nature 474, 654-657 (2011).

386. Berger, A., et al. SAMHD1-deficient CD14+ cells from individuals with Aicardi-Goutieres syndrome are highly susceptible to HIV-1 infection. PLoS Pathog 7, e1002425 (2011).

387. Rehwinkel, J., et al. SAMHD1-dependent retroviral control and escape in mice. EMBO J 32, 2454-2462 (2013).

388. Rehwinkel, J. Mouse knockout models for HIV-1 restriction factors. Cell Mol Life Sci 71, 3749-3766 (2014).

389. Dunay, G.A., et al. Assessment of the HIV-1 reservoir in CD4+ regulatory T cells by a Droplet Digital PCR based approach. Virus Res 240, 107-111 (2017).

390. Han, Y., Wind-Rotolo, M., Yang, H.C., Siliciano, J.D. & Siliciano, R.F. Experimental approaches to the study of HIV-1 latency. Nat Rev Microbiol 5, 95-106 (2007).

170

391. Kim, M., et al. A primary CD4(+) T cell model of HIV-1 latency established after activation through the T cell receptor and subsequent return to quiescence. Nat Protoc 9, 2755-2770 (2014).

392. Jain, S.S. & Tullius, T.D. Footprinting protein-DNA complexes using the hydroxyl radical. Nat Protoc 3, 1092-1100 (2008).

393. Pogozelski, W.K. & Tullius, T.D. Oxidative Strand Scission of Nucleic Acids: Routes Initiated by Hydrogen Abstraction from the Sugar Moiety. Chem Rev 98, 1089-1108 (1998).

394. Tullius, T.D. & Dombroski, B.A. Hydroxyl radical "footprinting": high- resolution information about DNA-protein contacts and application to lambda repressor and Cro protein. Proc Natl Acad Sci U S A 83, 5469-5473 (1986).

395. Tullius, T.D., Dombroski, B.A., Churchill, M.E. & Kam, L. Hydroxyl radical footprinting: a high-resolution method for mapping protein-DNA contacts. Methods Enzymol 155, 537-558 (1987).

171