The Role of the HIV-1 Accessory Proteins Nef and Vpu in Subverting Host Cellular Immunity

Home , Integrase, Nef (protein), Rev (HIV), Viral infectivity factor, Vpu protein

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Electronic Thesis and Dissertation Repository

9-25-2018 2:00 PM

Emily N. Pawlak The University of Western Ontario

Supervisor Dikeakos, Jimmy D. The University of Western Ontario

Graduate Program in Microbiology and Immunology A thesis submitted in partial fulfillment of the equirr ements for the degree in Doctor of Philosophy © Emily N. Pawlak 2018

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Recommended Citation Pawlak, Emily N., "The Role of the HIV-1 Accessory Proteins Nef and Vpu in Subverting Host Cellular Immunity" (2018). Electronic Thesis and Dissertation Repository. 5751. https://ir.lib.uwo.ca/etd/5751

This Dissertation/Thesis is brought to you for free and open access by Scholarship@Western. It has been accepted for inclusion in Electronic Thesis and Dissertation Repository by an authorized administrator of Scholarship@Western. For more information, please contact [email protected]. Abstract

The HIV-1 accessory proteins Nef and Vpu have no known enzymatic activity, yet these viral proteins are critical for HIV-1 viral spread and pathogenicity. To mediate this, these viral proteins bind to and hijack host cellular proteins, including proteins implicated in the membrane trafficking machinery. Key to the effects of these proteins is their ability to alter the subcellular localization of a plethora of cell surface receptors, including the key immune cell receptors CD28 and MHC-I. However, the mechanism utilized by Nef and Vpu to modulate these proteins is not fully understood. Herein, we demonstrated that Nef and Vpu mediate a decrease in both cell surface and total CD28 protein levels in CD4+ T cells. We have implicated the endosomal degradation machinery in this process, as we observed that in the presence of these viral proteins CD28 localizes to LAMP-1 positive compartments in an endosomal acidification-dependent manner. Moreover, in investigating the mechanisms utilized by Nef and Vpu to mediate CD28 downregulation we implicated the Nef LL165 and DD175 and Vpu

S52/56 motifs, which interact with specific cellular membrane trafficking machinery, in CD28 downregulation. Furthermore, upon infection with viruses encoding or lacking Nef and Vpu, we observed differential effects on cellular activation upon stimulation. In seeking to understand the mechanism of MHC-I downregulation by Nef, we examined the roles of membrane trafficking mediators in the sorting nexin family of proteins. Namely, we identified a novel direct Nef binding partner, SNX18, and mapped this interaction with Nef to the SH3 domain of SNX18 and a polyproline PxxP75 motif on Nef. Additionally, using shRNA knock- downs we have implicated SNX18 in Nef-mediated MHC-I downregulation, a key mechanism for HIV-1 viral immune evasion. Overall, this work sheds light on the mechanism of Nef- and Vpu-mediated mis-localization of host cellular proteins, processes key to the pathogenic effects of these viral proteins.

Keywords

Human immunodeficiency virus type 1, Nef, Vpu, immune evasion, viral infection

Co-Authorship Statement

All studies were completed by Emily N. Pawlak, with the exception of the work outlined below. Jimmy Dikeakos contributed to the genesis, design, implementation and interpretation of all experiments, as well as writing and manuscript preparation.

Chapter 2

Sections of Chapter 2 were previously published in:

The HIV-1 accessory proteins Nef and Vpu downregulate total and cell surface CD28 in CD4+ T cells. Pawlak EN, Dirk BS, Jacob RA, Johnson AL and Dikeakos JD. Retrovirology 2018 15:6

Co-authors contributed to the writing of this manuscript as well as experimental work as follows: The microscopy and corresponding analyses presented in Figure 2.9 A, B and Figure 2.10 B, C were completed and analyzed by Brennan S. Dirk. The experiments presented in Figure 2.8 D, E; Figure 2.21 E, F, G; Figure 2.23 were completed and analyzed by Rajesh A. Jacob.

Chapter 3

The work presented in Chapter 3 was completed by Emily N. Pawlak, with the exception of the microscopy experiments presented in Figure 3.4 A, B and Figure 3.6 A, B which were performed and analyzed by Logan R. Van Nynattan.

Acknowledgments

First and foremost, I would like to acknowledge my supervisor Dr. Jimmy Dikeakos, without whom this thesis would not have been possible. I will always be tremendously grateful you took a chance on me as a clueless fourth-year student and gave me the opportunity to work in your lab for all these years. Thank you for your guidance in the lab; from teaching me how to run Western blots, to the use of a French Press, this has served me well. In addition to all that you have taught me about science, I will also take away with me a wealth of knowledge regarding life in general.

Secondly, I would like to recognize my advisory committee members, Drs. Joe Mymryk and Lackshman Gunaratnam, for giving me their time and constructive feedback. This has been critical in helping me produce this thesis.

Finally, to all the Dikeakos lab members, I could not have asked for a better group of people to work with! Additionally, I would like to thank Aaron, Brennan, Logan and Rajesh, who all contributed to the work within this thesis - your respective expertise is what made this possible.

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Table of Contents

Abstract ...... i

Co-Authorship Statement...... ii

Acknowledgments...... iii

Table of Contents ...... iv

List of Tables ...... viii

List of Figures ...... ix

List of Appendices ...... xii

List of Abbreviations ...... xiii

Chapter 1 ...... 1

1 Introduction ...... 1

1.1 General thesis overview ...... 1

1.2 Introduction to lentiviruses ...... 1

1.3 Biology of HIV-1 ...... 4

1.3.1 Clinical course of infection ...... 4

1.3.2 HIV-1 virions and replication cycle ...... 5

1.4 HIV-1 accessory proteins ...... 8

1.4.1 HIV-1 Vpr and Vif ...... 8

1.4.2 HIV-1 Nef and Vpu ...... 9

1.5 Biological effects of HIV-1 Nef and Vpu ...... 9

1.5.1 Evasion of cell-mediated adaptive immune responses ...... 13

1.5.2 Evasion of humoral and innate immune responses ...... 14

1.5.3 Alterations in immune cell homing...... 15

1.5.4 Counteraction of host cell restriction factors ...... 16

1.5.5 Modulation of immune cell activation ...... 17

1.5.6 Modulation of cell death pathways ...... 22

1.5.7 Functional redundancy of HIV-1 Nef and Vpu ...... 23

1.5.8 The importance of Nef and Vpu in vivo...... 25

1.6 How do HIV-1 Nef and Vpu mediate their functions? ...... 25

1.6.1 Nef: host cell protein interactions ...... 33

1.6.2 Nef oligomerization ...... 38

1.6.3 Vpu oligomerization ...... 39

1.6.4 Vpu: host cell protein interactions ...... 39

1.7 Thesis overview ...... 43

1.7.1 Chapter 2 ...... 44

1.7.2 Chapter 3 ...... 44

1.7.3 Significance...... 45

1.8 References ...... 45

Chapter 2 ...... 73

2 The downregulation of CD28 by HIV-1 Nef and Vpu ...... 73

2.1 Introduction ...... 73

2.2 Results ...... 75

2.2.1 The HIV-1 accessory proteins Nef and Vpu downregulate cell surface and total CD28 protein levels ...... 75

2.2.2 Nef and Vpu-mediated effects on CD28 are dependent on the cellular degradation machinery ...... 91

2.2.3 Motifs ascribed to specific Nef: host cell protein interactions are critical for Nef-mediated CD28 downregulation ...... 96

2.2.4 Motifs in Vpu implicated in interactions with specific host cellular proteins are critical for CD28 downregulation ...... 102

2.2.5 Implicating host cell proteins in Nef- and Vpu-mediated CD28 downregulation ...... 107

2.2.6 Patient-derived Vpu proteins mediate CD28 downregulation ...... 114

2.2.7 Nef and Vpu differentially alter responsiveness to CD28-mediated stimulation...... 119

2.3 Discussion ...... 122

2.4 Materials and Methods ...... 130

2.4.1 DNA constructs ...... 130

2.4.2 Cell culture ...... 132

2.4.3 Transfections and Infections ...... 133

2.4.4 Flow cytometry analysis of cell surface receptors ...... 135

2.4.5 Protein analysis ...... 136

2.4.6 Microscopy ...... 137

2.4.7 Cell activation analysis ...... 138

2.4.8 Data and Statistical analysis...... 139

2.5 References ...... 140

Chapter 3 ...... 149

3 Defining the role of the novel interaction between HIV-1 Nef and the membrane trafficking protein SNX18...... 149

3.1 Introduction ...... 149

3.2 Results ...... 151

3.2.1 SNX18 and Nef interact in vitro through a SNX18 SH3 domain: Nef PxxP motif interaction ...... 151

3.2.2 SNX18 and Nef interact in cells ...... 158

3.2.3 SNX18 is required for Nef-dependent reductions in cell surface MHC-I ...... 161

3.2.4 SNX18 is unlikely to be required for Nef-mediated secretion in extracellular vesicles ...... 164

3.3 Discussion ...... 170

3.4 Material and Methods ...... 175

3.4.1 DNA vectors ...... 175

3.4.2 Bacterial protein expression and purification ...... 176

3.4.3 Protein capture assay...... 177

3.4.4 Cell culture ...... 178

3.4.5 Transfections and virus production ...... 178

3.4.6 Western blot analysis ...... 178

3.4.7 Microscopy ...... 179

3.4.8 Extracellular vesicle purification ...... 180

3.4.9 Transient shRNA knock-down experiments ...... 181

3.4.10 Flow cytometry ...... 181

3.4.11 Data analysis ...... 181

3.5 References ...... 183

Chapter 4 ...... 188

4 General Discussion...... 188

4.1 Thesis overview ...... 188

4.1.1 Chapter 2: Findings, limitations and future work ...... 188

4.1.2 Chapter 3: Findings, limitations and future work ...... 196

4.2 Significance...... 200

4.2.1 Implications for HIV-1 treatment ...... 200

4.2.2 Concluding remarks ...... 202

4.3 References ...... 203

Curriculum Vitae ...... 210

vii

List of Tables

Table 1 The HIV-1 genes and their encoded proteins ...... 7

viii

List of Figures

Figure 1.1 HIV-1 genome organization ...... 6

Figure 1.2 Immune cell surface proteins that are targeted by the HIV-1 accessory proteins Nef and Vpu ...... 11

Figure 1.3 Schematic of select molecules found in the immunological synapse ...... 19

Figure 1.4 Structure of HIV-1 Nef and Vpu and motifs that interact with host cell proteins . 28

Figure 1.5 The endosomal trafficking network...... 30

Figure 1.6 General mechanism of vesicle transport...... 31

Figure 2.1 HIV-1 Nef and Vpu downregulate cell surface CD28 protein levels ...... 76

Figure 2.2 Gating of live infected Sup-T1 cells infected with Gag-Pol truncated VSV-G pseudotyped NL4.3 ...... 78

Figure 2.3 Analysis of primary CD4+ T cells infected with replication competent virus ...... 79

Figure 2.4 Schematic of CD28-FLAG construct utilized to overexpress CD28 in cells ...... 81

Figure 2.5 Decreased CD28-FLAG protein levels are detected in the presence of Nef in HeLa cells ...... 82

Figure 2.6 HIV-1 lentiviral vector utilized to express epitope tagged CD28 in the presence or absence of Nef...... 83

Figure 2.7 CD28-FLAG protein levels are reduced in the presence of Nef in CD4+ Sup-T1 T cells ...... 84

Figure 2.8 HIV-1 Nef and Vpu downregulate total CD28 protein levels ...... 86

Figure 2.9 The subcellular localization of CD28 is altered in the presence of Nef or Vpu .... 89

Figure 2.10 Inhibition of the degradation machinery increases CD28 protein levels in infected cells ...... 93

Figure 2.11 Ammonium chloride treatment increases total CD4 levels in infected cells ...... 95

Figure 2.12 Gating of Sup-T1 cells infected with VSV-G pseudotyped NL4.3 encoding various Nef mutants ...... 98

Figure 2.13 Nef: host protein interaction motifs are critical for Nef-mediated CD28 downregulation in the presence of Vpu ...... 99

Figure 2.14 Nef: host cell protein interaction motifs are critical for Nef-mediated CD28 downregulation ...... 101

Figure 2.15 Motifs in Vpu are critical for downregulation of CD28 ...... 105

Figure 2.16 Specific motifs in Vpu are critical for downregulation of CD4 ...... 106

Figure 2.17 Downregulation of cell surface CD28 by Nef and Vpu in the presence of shRNA targeting AP-2 ...... 108

Figure 2.18 Nef-mediated cell surface downregulation of CD28 in the presence of overexpressed wild-type or dominant negative non-functional SNX9 ...... 110

Figure 2.19 HeLa cells with decreased SNX9 expression exhibit reductions in Nef-mediated decreases in total CD28-FLAG ...... 112

Figure 2.20 SNX9-targeting shRNA reduces Nef-mediated decreases in total CD28 in Sup-T1 cells ...... 113

Figure 2.21 Non-NL4.3 Vpu proteins downregulate cell surface CD28 ...... 116

Figure 2.22 Gating of CD4+ peripheral blood mononuclear cells infected with VSV-G pseudotyped NL4.3 ...... 118

Figure 2.23 Infected cell response to CD28-stimulation changes in the absence of Nef or Vpu ...... 120

Figure 2.24 Protein sequence alignment of the Vpu proteins which were tested for their ability to downregulate CD28 ...... 127

Figure 3.1 GST-SNX18, but not GST-SNX9, pulls down His6-Nef ...... 152

Figure 3.2 The direct Nef: SNX18 interaction is dependent on the SH3 domain ...... 154

Figure 3.3 In vitro protein capture of His6-Nef and a Nef mutant, His6-Nef AxxA75, by the SH3 domain of SNX18 ...... 157

Figure 3.4 Mutation of the Nef PxxP75 motif reduces SNX18: Nef BiFC intensity ...... 159

Figure 3.5 Knock-down of SNX18 reduces Nef-mediated MHC-I downregulation ...... 162

Figure 3.6 The SNX18: Nef BiFC complex co-localizes with CD63...... 165

Figure 3.7 Extracellular vesicles purified from Nef-expressing SNX18 knock-down cells . 168

Figure 3.8 Proposed model for the role of SNX18 in Nef-mediated MHC-I trafficking ..... 174

Figure 4.1 Models of Nef- and Vpu-mediated CD28 downregulation ...... 194

Figure 4.2 Model of Nef-mediated evasion of cytotoxic T cell responses ...... 199

List of Appendices

Appendix 1 Copyright permission for published work ...... 209

Appendix 2 Ethics statement ...... 209

xii List of Abbreviations

ADAM A disintegrin and COPI Coatomer protein I metalloprotease CTL Cytotoxic T lymphocyte ADCC Antibody-dependent DCs Dendritic cells cellular cytotoxicity

DMEM Dulbecco’s modified AIDS Acquired immune Eagle’s medium deficiency syndrome DTT Dithiothreitol AP-1 Adaptor protein 1 EDTA Ethylenediaminetetraacetic AP-2 Adaptor protein 2 acid APCs Antigen presenting cells ER Endoplasmic reticulum

ERAD ER-associated degradation APOBEC3 Apolipoprotein B mRNA

editing complex 3 ESCRT Endosomal sorting complexes required for Arf1 ADP-ribosylation factor 1 transport ATCC American Type Culture FBS Fetal bovine serum Collection

GAPDH glyceraldehyde 3- BiFC Bimolecular fluorescence phosphate dehydrogenase complementation

GFP Green fluorescent protein BSA Bovine serum albumin

HAART Highly active antiretroviral BST-2 Bone marrow stromal cell therapy antigen 2

xiii

HIV Human immunodeficiency MHC-II Major histocompatibility virus complex class II

HIV-1 Human immunodeficiency MVBs Multivesicular bodies virus type 1 N-WASP Neural Wiskott-Aldrich HIV-2 Human immunodeficiency syndrome protein virus type 2 NAK Nef-associated kinase HRS Hepatocyte growth factor- NK Natural killer regulated tyrosine kinase

substrate PACS Phosphofurin acidic cluster sorting HTLV Human T-leukemia virus PAK2 p21 activated kinase ICAM-1 Intracellular adhesion molecule 1 PBMCs peripheral blood mononuclear cells IPTG Isopropyl β-D-1-

thiogalactopyranoside PBS Phosphate buffered saline

LAMP-1 lysosomal-associated PHA Phytohemagglutinin membrane protein 1 PI3K Phosphoinositide 3-kinase LAP LC3-associated phagocytosis RPMI Roswell Park Memorial Institute media LAV Lymphadenopathy- associated virus SCF Skp Cullin F-box containing LTRs Long terminal repeats SD Standard deviation MFI Mean fluoresence intensity SE Standard error MHC-I Major histocompatibility complex class I SERINC Serine incorporator

xiv

SFKs Src family kinases v-ATPase vacuolar ATPase

SIV Simian immunodeficiency virus Vif Viral infectivity factor

SNX18 Sorting nexin 18 Vpu Viral protein U

SNX9 Sorting nexin 9 VSV-G Vesicular stomatitis virus envelope glycoprotein TCR T cell receptor β-TrCP β-transducin-repeat TNFα Tumor necrosis factor- containing protein alpha

xv 1

Chapter 1 1 Introduction 1.1 General thesis overview

The Oxford English Dictionary defines a virus as “an infectious, often pathogenic agent or biological entity which is typically smaller than a bacterium, which is able to function only within the living cells of a host animal, plant, or microorganism, and which consists of a nucleic acid molecule (either DNA or RNA) surrounded by a protein coat, often with an outer lipid membrane”[1]. Indeed, viruses require host cells they can infect, as they are obligate intracellular parasites and require eukaryotic or bacterial host cells to replicate. Thus, as will be highlighted in this thesis, viruses have evolved ingenious mechanisms to make use of host cellular machinery to turn their hosts into efficient virus production factories. Moreover, given their generally minute size, many viruses have small genomes with limited coding capacity. Therefore, they must encode proteins which function in numerous different ways to make the host a prime location for replication.

1.2 Introduction to lentiviruses

In the early 1980’s a new illness appeared in North America which baffled medical professionals. This ailment was characterized by an increased incidence of opportunistic infections and rare malignancies that resulted in a rapid death [2]. This illness was later recognized as being caused by a severe immune deficiency, thus it was termed acquired immune deficiency syndrome (AIDS) by the Centers for Disease Control. Originally, AIDS was falsely believed to only affect men who have sex with men, however it was later proven that this illness could be acquired through exposure to contaminated bodily fluids through transfusion of blood products, heterosexual sex, and mother to child transmission during childbirth or breastfeeding [3-7].

In 1983, Dr. Luc Montagnier’s laboratory at the Institut Pasteur in France isolated a virus from the lymph node of a patient exhibiting lymphadenopathy. This patient was considered high risk for developing the above mentioned novel immune deficiency syndrome [8]. Montagnier’s group named the isolated virus lymphadenopathy-associated virus (LAV)

2 and classified it as a retrovirus belonging to the human T-leukemia virus (HTLV) family. These findings were published in Science concurrently with an article by Dr. Robert Gallo and colleagues at the National Cancer Institute in Bethesda, Maryland. In the latter publication, Gallo’s group identified a virus within an AIDS patient they believed to be HTLV, thus they termed this virus HTLV-III [9].

The following year, Gallo and colleagues published a series of articles illustrating that this novel virus was the causative agent of the acute immune deficiency termed AIDS [10-12]. By 1986, it was widely recognized that LAV and HTLV-III were the same virus, hence this newfound consensus in the virology community resulted in the creation of a new virus name: human immunodeficiency virus (HIV) [13]. In the three decades since its initial discovery, this deadly virus has spread to millions of people globally [14], making it a major area of research interest.

Extensive phylogenetic analysis of HIV viral genomes has demonstrated that the transmission of HIV to humans occurred many years prior to the first documented cases of AIDS, most likely around the 1930’s in what is now known as Kinshasa in the Democratic Republic of the Congo [15-17]. Specifically, the epidemic is believed to have arisen from transmission of the primate simian immunodeficiency virus (SIV) from an infected chimpanzee to a human, likely through the act of bush meat hunting [18-20]. Interestingly, phylogenetic studies suggest that there have been multiple transmission events of SIV from infected primates to humans, however a single transmission event led to the group of HIV- 1 viruses that is responsible for the vast majority of infections world-wide. Specifically, the human immunodeficiency virus type 1 (HIV-1) is classified into four phylogenetically related clusters termed groups M, N, O and P [21-23]. Group M is so named as it is the “major” group of HIV-1, having caused the majority of HIV cases in the global epidemic. This group is believed to have arisen through transmission from an SIV infected chimpanzee [24, 25]. Similar to Group M, Group N likely arose from a single transmission event of SIV from an infected chimpanzee [25], while HIV-1 groups O and P are thought to have arisen from two independent transmission events from SIV infected gorillas [23, 26]. In addition to HIV-1, there is also human immunodeficiency virus type 2 (HIV-2), which is distinct from HIV-1 and is derived from transmission of SIV from infected sooty

3 mangabeys [27-29]. Similar to HIV-1, HIV-2 is separated into numerous groups which derived from independent transmission events [19]. However, in contrast to HIV-1, HIV- 2 exhibits decreased transmissibility [30, 31], and only the minority of HIV-2 infected individuals develop immunodeficiency [32-34]. Since HIV-1 Group M is responsible for the vast majority of HIV infections globally, it is the primary focus of most current HIV research and much of what we know about HIV is from experiments using Group M viruses.

As a result of a highly error prone viral replication apparatus, HIV viruses are highly diverse. Indeed, HIV-1 group M itself can be classified into numerous phylogenetically related subtypes or clades [35-37]. However, most HIV research has been performed using HIV-1 group M subtype B viruses as a result of these being the most prominent viruses in the developed world [38]. However, it is becoming increasingly clear that there are functional differences between group M subtypes, specifically in relation to transmission and disease progression [39-42]. Therefore, while the majority of this thesis will describe work primarily completed using HIV-1 Group M subtype B viruses, it must be acknowledged that there may be differences between HIV-1 viruses of different subtypes.

HIV-1, and the related HIV-2 and SIV, are all classified as retroviruses in the genus Lentivirus. The family Retroviridae are enveloped viruses, which carry positive sense RNA in their virions [43]. These viruses are unique in that they encode a viral reverse transcriptase enzyme, which transcribes a double stranded DNA copy of the viral RNA genome [43]. The viral genome encoding DNA, known as pro-viral DNA, becomes permanently integrated into the host cell genome, a characteristic which has driven interest in the use of retroviral vectors in gene therapy [44]. From the integrated pro-viral DNA, viral RNAs and their encoded proteins can be produced to facilitate the production of progeny viruses and all retroviruses encode the structural and enzymatic proteins required to make virions [43]. However, the retroviruses in the genus Lentivirus are generally quite complex, as they have evolved a larger repertoire of proteins to ensure optimal virus replication [45, 46]. Specifically, lentiviruses have evolved regulatory proteins, such as HIV-1 Tat and Rev, which ensure enhanced gene expression [47]. Moreover, select lentiviruses, in particular lentiviruses that infect primates, have evolved multiple accessory

4 proteins which enhance viral spread and persistence within the host [48]. As such, primate lentiviruses are highly successful at hijacking host cells to facilitate viral spread.

1.3 Biology of HIV-1

1.3.1 Clinical course of infection

Unlike in the 1980’s, when the virus was first discovered, infection with HIV-1 no longer presages certain death. Typically, when an individual becomes infected with HIV-1, an immune response is mounted against this foreign invasion. Patients experience flu-like symptoms during the first few weeks of the acute infection, as the virus disseminates, primarily infecting the CD4+ T cell component of the immune system [49, 50]. Unfortunately, unlike for many other viruses, the immune system in unable to clear HIV- 1. If left untreated, the virus continues to replicate and this chronic infection slowly depletes the CD4+ T cells of infected individuals. During this period, which can last up to a decade, infection often lacks visible symptoms [51, 52]. Eventually, infected individuals experience a drastic and catastrophic loss in immune system function, which leads to opportunistic infections and malignancies. Clinically, AIDS is defined in adults as a CD4+ T cell count lower than 200 cells per microliter of blood [53]. However, since the mid- 1990’s, with the implementation of a combination therapy called highly active antiretroviral therapy (HAART) that targets different aspects of the viral life cycle, progression of HIV-1 infection to the immunocompromised state can be prevented [54]. Yet, it is important to note that HIV-1 infected individuals must remain on therapy for the remainder of their lives, as a pool of latently infected cells branded the “latent reservoir” remains present during HAART treatment. This latent reservoir can re-populate the body with virus upon cessation of anti-retroviral treatment [55]. Thus, a significant amount of the current HIV-1 research is focused on finding an HIV-1 cure through targeting this latent reservoir. Finally, it is important to acknowledge that while the implementation of effective HAART means that HIV-1 infection can be treated, access to HAART is not universal and The Joint United Nations Programme on HIV/AIDS estimates that approximately half of all HIV infected individuals world-wide are currently not on treatment [56].

1.3.2 HIV-1 virions and replication cycle

HIV-1 virions are enveloped and contain two copies of the viral genome which is comprised of 9 genes that encode for 15 proteins (Figure 1.1). The HIV-1 proteins are classified as: structural, enzymatic, regulatory or accessory (Table 1; [43, 45]). Within virions, the RNA genome is associated with the viral nucleocapsid protein, which is further surrounded by the capsid protein to form the viral core complex [57]. This core is surrounded by a host cell derived lipid envelope, which associates with the viral matrix protein and contains the membrane-embedded viral glycoproteins gp120 and gp41.

To initiate the viral replication cycle (reviewed in [45]), the HIV-1 virion surface glycoproteins engage the viral entry receptor, CD4, and the co-receptors, CCR5 or CXCR4. These virus-host interactions facilitate fusion of the virion envelope with the host cell envelope, thereby ensuring entry [58]. The strict requirement for CD4 and CCR5 or CXCR4 to enter cells implies that HIV-1 infections are limited to CD4+ cells, namely the following immune cell types: monocytes, dendritic cells (DCs) and CD4+ T cells [59-62]. Post-entry, the viral core dissociates and the HIV-1 RNA genome is reverse transcribed to DNA by the viral reverse transcriptase. The resulting double stranded DNA, or pro-viral DNA, is transported into the nucleus and integrated into the host cell genome via the viral integrase enzyme. Ultimately, the integrated viral DNA will be transcribed and the resulting mRNAs generate HIV-1 proteins that fulfill specific functions within infected cells. Specifically, the HIV-1 regulatory proteins Tat and Rev enhance transcription and transport of RNA encoding viral proteins out of the nucleus for translation [47]. In this manner, the RNA and proteins necessary to form new virions are produced and bud from the host cell plasma membrane, after which the viral protease enzyme mediates proteolytic cleavage of viral protein precursors to form mature, infective virions. Overall, the process of replication is promoted by the HIV-1 accessory proteins Nef, Vif, Vpr and Vpu, which will be discussed in further detail below.

5’ LTR gag pol vpr env 3’ LTR

vif vpu nef

rev

tat

Figure 1.1 HIV-1 genome organization

The 5’ and 3’ ends of the HIV-1 genome encode the long terminal repeats (LTRs), repeating sequences necessary for integration of the viral genome into the host cell genome. The LTRs also contain host cell transcription factor binding regions that enhance viral gene transcription [63]. The gag, pol and env genes encode the viral enzymatic and structural proteins. The rev and tat genes encode regulatory elements that enhance viral gene expression. The accessory proteins are encoded by the vif, vpr, vpu and nef genes.

Table 1 The HIV-1 genes and their encoded proteins Gene Protein Function gag p17/matrix Targets viral proteins to sites of viral assembly at the plasma membrane p24/capsid Constitutes the major structural components of the viral core p7/nucleocapsid Incorporation of viral genomic RNA into virions and coating of viral RNA within the virion core Structural p6 Virion budding and incorporation of Vpr into budding virions env gp120/surface Binding to viral entry receptor/co- receptors gp41/transmembrane Facilitates fusion of the virion envelope with the host cell membrane during viral entry pol Integrase Integration of viral genome into host cell genome Reverse transcriptase Transcription of pro-viral DNA from Enzymatic virion-derived viral RNA Protease Proteolytic cleavage of viral proteins within virions to form mature virus rev Rev Regulates viral mRNA expression via shuttling of viral mRNA from the Regulatory nucleus tat Tat Enhances transcription of viral genome vpu Vpu Mediates immune evasion and increases viral release and spread vpr Vpr Enhances viral replication Accessory vif Vif Prevents viral genome hypermutation nef Nef Mediates immune evasion and increases virion infectivity

1.4 HIV-1 accessory proteins

Unlike other lentiviruses, HIV and the related SIV encode multiple accessory proteins. These proteins were originally termed “accessory” as they were not required for viral replication in vitro. However, the accessory proteins have since been deemed necessary to facilitate viral spread and to mediate evasion of the immune response during infections in vivo [48]. Indeed, the importance of HIV-1 accessory proteins for viral dissemination and pathogenicity has been demonstrated using both mouse and non-human primate infection models [64-68]. Moreover, these proteins fulfill their functions without possessing any known enzymatic activity and therefore function solely by interacting with and hijacking host cell proteins. Specifically, these proteins primarily mediate their effects by co-opting the host cell’s protein degradation or membrane trafficking machinery (reviewed in [48, 69]). In this way, they sequester host cellular proteins within specific locations in the cell or target them for degradation via the lysosome or proteasome. Given their reliance on the host cellular machinery to fulfill their functions, the accessory proteins exemplify viruses’ strict requirement for a host.

1.4.1 HIV-1 Vpr and Vif

HIV-1 Vpr, or viral protein R, is an ~14 kDa protein that is actively incorporated into budding virions through its association with the HIV-1 p6 protein [70-73]. Multiple effects have been attributed to Vpr (reviewed in [74]), but this accessory proteins’ functions still remain poorly defined. Notably, Vpr is critical for efficient viral replication in macrophages in vitro [75], but it remains scantily understood how this is mediated. It has been postulated that this effect in macrophages may relate to Vpr’s ability to facilitate degradation of an as of yet undefined host anti-viral factor which inhibits virion envelope incorporation [76-78]. Moreover, during infection of lymphoid cells, Vpr inhibits G2 to S- phase cell cycle progression by inducing G2 cycle arrest, resulting in cells lagging in G2 phase, during which HIV-1 transcription is highly active [79, 80]. In addition, Vpr has been implicated in modulating cell survival [81]. While the mechanisms that mediate the above described consequences of Vpr expression remain poorly defined, these effects may be related to Vpr’s ability to readily hijack the host cellular ubiquitin ligase machinery to

9 mediate the ubiquitination and subsequent degradation of DNA repair pathway proteins [82-86].

Similar to Vpr, the HIV-1 Viral infectivity factor (Vif) protein contributes to efficient viral replication by targeting host cellular proteins for ubiquitination. Specifically, Vif mediates the poly-ubiquitination of host cell restriction factors, cellular apolipoprotein B mRNA editing complex 3 (APOBEC3) proteins, leading to their degradation [87, 88]. Members of the APOBEC3 family of proteins limit viral replication by inducing viral genome hypermutations during synthesis of cDNA from viral RNA. Specifically, APOBEC3 proteins mediate deamination of viral RNA, such that upon reverse transcription the newly transcribed viral cDNA contains G to A mutations, which impede the viral life cycle [89]. Vif counteracts this by targeting APOBEC3 proteins for degradation, thereby preventing their incorporation into budding virions [88]. Alternatively, degradation-independent mechanisms of APOBEC3 protein counteraction by Vif have also been proposed [90].

1.4.2 HIV-1 Nef and Vpu

In contrast to the relatively small number of host cell proteins currently described as targets for intracellular sequestration or degradation by Vif or Vpr, the accessory proteins Nef and Vpu modulate the expression, localization, and function of an exorbitant number of host cellular proteins. Large scale screens have identified >30 host cell proteins that are modulated by Nef or Vpu [91-93]. Thus, Nef and Vpu have evolved the amazing ability to mediate numerous functions within host cells. Due to the sheer number of host cellular proteins that are altered by Nef and Vpu, we have only begun to understand the functions of Nef and Vpu, how these functions are mediated, and the complex interplay between these two proteins. The accessory proteins Nef and Vpu will be the focus of this thesis, with their functions described in more detail below.

1.5 Biological effects of HIV-1 Nef and Vpu

HIV-1 Nef and Vpu are viral proteins that, despite their lack of enzymatic activity, facilitate the creation of highly efficient virus production factories within their host. These proteins are especially interesting as they have distinct, but sometimes overlapping functions. HIV- 1 Nef, or negative factor, so named due to initial descriptions of it negatively affecting viral

10 replication [94-96], and viral protein u (Vpu), are small membrane associated proteins of ~27 and ~16 kDa, respectively [97-99]. Vpu is a type-I transmembrane protein [100], whereas Nef is post-translationally modified through the addition of a lipid myristoyl group, which mediates Nef’s association with membranes [101]. Due to their membrane association, it is unsurprising that these proteins primarily usurp host cellular membrane trafficking pathways in order to alter the host cell environment [69]. As with the accessory proteins Vpr and Vif, many of these functions are mediated by sequestering host cellular proteins within the cell or targeting host proteins for degradation, with many of the proteins targeted by Nef and Vpu being cell surface associated (Figure 1.2). A critical role for HIV- 1 Nef and Vpu during infection is their contributions to viral immune evasion. Both proteins actively contribute to counteracting the innate and adaptive immune response. Collectively, these Nef and Vpu functions antagonize immune responses to promote viral spread and persistence.

Figure 1.2 Immune cell surface proteins that are targeted by the HIV-1 accessory proteins Nef and Vpu

Multiple cell surface associated proteins undergo modifications in their subcellular localization within HIV-1 infected immune cells through the activities of HIV-1 Nef and Vpu. These modifications reduce the abundance of these molecules on the cell surface and can alter immune responses. Molecules can be targeted by Nef (blue), Vpu (purple) or both Nef and Vpu (green). Modifications include: decreases in cell surface MHC-I thereby preventing detection by cytotoxic T cells [102-104], Nef-mediated decreases in the cell surface chemokine receptors CCR5 and CXCR4 [105], Nef and Vpu evasion of NK cell responses by downregulation of ligands that bind to NK cell activating receptors [106-109], and decreases in cell surface CD4 to prevent detection of infected cells by NK cells through inducing exposure of antibody-dependent cellular cytotoxicity (ADCC)-mediated epitopes on the HIV-1 envelope protein [110-112]. Nef and Vpu decrease cell surface levels of the host cell restriction factors serine incorporator (SERINC) 5 and bone marrow stromal cell antigen 2 (BST-2; also called tetherin),

12 respectively, inhibiting incorporation of these anti-viral factors into budding virions [113-116]. Finally, the activation of infected T cells by antigen presenting cells is inhibited by the downregulation of activating cell surface molecules in both infected CD4+ T cells and in infected antigen presenting cells [117-119].

1.5.1 Evasion of cell-mediated adaptive immune responses

During viral infections, the primary immune cell type that target infected cells are CD8+ cytotoxic T lymphocytes (CTLs). Specifically, CTLs recognize and eliminate cells which present virus-derived peptides or antigens on major histocompatibility complex class I (MHC-I) molecules (reviewed in [120]). Mature MHC-I molecules are heterodimers composed of a common β2-microglobulin light chain and a variable heavy chain that binds to 8-10 amino acid peptide antigens [120]. To prevent detection of infected cells by CTLs, Nef and Vpu mediate decreases in cell surface levels of MHC-I. Specifically, Nef mediates the endocytosis of cell surface MHC-I, followed by its sequestration in the cellular paranuclear region [102, 121-123]. In contrast, Vpu disrupts the formation of newly synthesized MHC-I in the endoplasmic reticulum (ER) [103]. Interestingly, Nef preferentially targets HLA-A and HLA-B [124], while Vpu targets HLA-C [125]. Ultimately, the downregulation of MHC-I molecules by HIV-1 has been implicated in reducing CTL killing of infected cells [104].

The second arm of cell-mediated adaptive immune responses is comprised of helper CD4+ T cells, which function during infection to regulate the immune response [126]. CD4+ T cells are activated comparably to CD8+ CTLs, through the presentation of antigens on MHC class II (MHC-II) molecules by antigen presenting cells (APCs), including HIV-1 infected monocytes and dendritic cells. Mature MHC-II molecules are presented on the cell surface subsequent to being loaded with peptide antigen in an endocytic cellular compartment. Prior to this, MHC-II molecules are complexed with the MHC-II invariant chain (CD74; Ii), which is degraded when the MHC-II molecule is loaded with a peptide antigen [127]. Both Nef and Vpu alter the expression of mature MHC-II molecules on the cell surface [119, 128-130], owing to their ability to interact with the MHC-II invariant chain. Briefly, Nef induces accumulation of the invariant chain on the cell surface or in intracellular compartments [129, 131]. Alternatively, Vpu may decrease the levels of cell surface invariant chain, though there are conflicting reports [92, 128]. Taken together, these alterations in invariant chain subcellular localization likely contribute to Nef- and Vpu- mediated decreases in the quantity of peptide-loaded mature MHC-II molecules on the cell surface [119, 128, 130]. This decrease has been associated with a diminished ability of

14 monocytic cells to activate T cells [128]. Moreover, a correlation has been observed between rapid disease progression in HIV infected children and increased Nef-mediated up-regulation of cell surface invariant chain and downregulation of mature MHC-II [132].

1.5.2 Evasion of humoral and innate immune responses

In addition to cell-mediated adaptive immune responses, a large antibody-mediated or humoral response is developed during HIV-1 infection. Of particular importance is the humoral response targeting the HIV-1 surface glycoprotein, or envelope, which is present on the surface of virions and infected cells. Notably, during virion entry the envelope glycoprotein interacts with the viral entry receptor CD4 and undergoes a conformational change to allow for virion envelope fusion with the host cellular membrane [133-135]. We have recently begun to appreciate that the envelope glycoprotein conformational change induced upon CD4 binding exposes epitopes that are targeted by antibodies that can be detected by innate immune cells to facilitate antibody-dependent cellular cytotoxicity (ADCC) [111, 112, 136, 137]. During ADCC, innate immune cells, such as natural killer (NK) cells, detect infected cells that have been bound by specific antibodies and subsequently mediate their lysis. Importantly, Nef and Vpu counteract this effect through their ability to decrease cell surface and total cellular CD4 levels [111, 112, 138], via targeting CD4 for lysosomal or proteasomal degradation [139-141]. This is believed to limit the interactions between the viral envelope and CD4 on the surface of infected cells, thereby reducing the exposure of CD4-induced epitopes that are targeted by ADCC- mediating antibodies. Ultimately, this leads to a reduction in ADCC in the presence of Nef and Vpu [111, 112], with lower levels of cell surface CD4 correlating with a decreased susceptibility to ADCC [138].

The ability of NK cells to target and eliminate HIV-1 infected cells is not solely dependent on ADCC-mediating antibodies binding to infected cells. Indeed, viral infections can alter the cell surface levels of NK cell activating or inhibitory receptors, allowing for NK cells to detect and lyse virally infected cells [142]. However, viruses have evolved the ability to counteract this innate immune mechanism by directly altering these receptors. Briefly, upon infection with HIV-1 lacking a functional nef gene, Jurkat T cells exhibit increases in receptors that are detected by the NK cell activating receptor, NKG2D [106]. However,

15 upon infection with viruses encoding Nef, Nef mediates the downregulation of NKG2D ligands, such that infection does not increase the abundance of these receptors on the cell surface [106]. This activity can prevent NK cell-mediated targeting of infected cells, as a correlation exists between the increased ability of Nef alleles to downregulate NKG2D ligands and decreases in NK cell mediated-ADCC [110]. Nef has also been implicated through an undefined mechanism in the downregulation of NKp44L, the ligand for the NK cell activating receptor NKp44, with Nef expression reducing NKp44L-mediated killing of infected CD4+ T cells [107].

The modulation of NK activating receptors is also attributable to Vpu. Specifically, Vpu mediates decreases in cell surface levels of the NK cell activating receptor NTB-A through altering the processing and glycosylation of newly translated NTB-A [143]. This activity decreases NK cell-mediated effects against virally infected cells [108]. Moreover, reductions in NK cell lysis of infected T cells has also been linked to the ability of Vpu to decrease surface levels of the adhesion molecule intracellular adhesion molecule 1 (ICAM- 1), which limits the formation of adhesions between T cells and NK cells that are necessary for NK cell-mediated killing [144]. Finally, both Nef and Vpu target the NK cell activating receptor PVR (CD155), limiting cell surface levels to decrease susceptibility to NK cell killing [109, 145]. Overall, the presence of a functional Nef and Vpu protein has been shown to reduce NK cell-mediated killing of HIV infected CD4+ T cells [106-109].

1.5.3 Alterations in immune cell homing

Critical to immune responses in vivo is the ability of immune cells to survey an organism for infections and be recruited to sites where immune cell responses are established. Adhesion molecules are essential for homing of immune cells to the secondary lymphoid organs, namely the lymph nodes, where immune responses are developed (reviewed in [146]). Nef and Vpu independently contribute to decreases in cell surface levels of the adhesion molecule L-selectin (CD62L; [147]), which mediates entry of T cells into lymph nodes. This is accomplished by interactions between the viral proteins and L-selectin, which facilitates the inhibition of L-selectin transport to the cell surface via Nef-mediated decreases in total L-selectin protein levels and Vpu-mediated intracellular sequestration of L-selectin [147]. Viral protein induced reductions in cell surface L-selectin results in

16 decreased adhesion of infected CD4+ T cells to the L-selectin ligand fibronectin and reduces L-selectin-mediated immune cell activating signaling [147]. Moreover, recruitment of immune cells to sites of infection is often mediated by migration in response to various stimuli, including chemokines. Nef and Vpu can limit this migration in response to chemokines by decreasing cell surface levels of chemokine receptors, including the HIV- 1 viral entry co-receptors CCR5 and CXCR4, or by altering the ability of cells to migrate in response to chemokines [105, 148-153]. Notably, Nef inhibits migration of infected T cells in response to the CXCR4 ligand SDF1 alpha [152, 153]. Additionally, Vpu reduces cell surface levels of CCR7 on HIV-1 infected cells, reducing migration in response to the CCR7 ligand [154]. The above-mentioned alterations in the migration and homing of HIV- 1 infected immune cells may alter the ability of infected cells to be detected by the immune system and may increase viral dissemination by promoting continued circulation of infected cells throughout the body.

1.5.4 Counteraction of host cell restriction factors

In addition to the aforementioned mechanisms mediated by HIV-1 Nef and Vpu to evade adaptive and innate immune responses, these viral proteins also mediate evasion of intrinsic antiviral proteins known as restriction factors. Restriction factors are key components of innate immunity and function to impede viral replication [155]. Specifically, Vpu’s most recognized function is undoubtedly its ability to counteract BST-2, an interferon induced protein. BST-2 inhibits release of enveloped viruses by incorporating into the budding virions’ envelope to tether these virions to the surface of infected cells [156]. Indeed, in cells expressing endogenous BST-2 the presence of Vpu can increase HIV-1 virion release by 20-fold [113, 115]. Vpu mediates increased virion release through interacting with BST- 2 and preventing its incorporation into budding virions. This is facilitated through numerous proposed mechanisms, including removing BST-2 from sites of viral assembly [157, 158], and decreasing cell surface levels of BST-2 [113]. Decreases in the abundance of cell surface BST-2 are achieved through inhibiting transport of newly synthesized BST- 2 to the cell surface [159], sequestering BST-2 away from the plasma membrane [160], and degrading BST-2 in lysosomes [161-163]. Moreover, it was recently shown that decreases in cell surface BST-2 are important for evasion of ADCC, as the tethering of

17 virions on the cell surface increases the exposure of CD4-induced envelope epitopes to which ADCC-mediating antibodies may bind [164]. Finally, the counteraction of BST-2 has been implicated in facilitating viral dissemination early in infection [165].

Similar to Vpu, Nef also counteracts restriction factors, specifically the newly identified restriction factors belonging to the SERINC family of proteins, SERINC3 and SERINC5 [114, 116]. Nef has long been known for its’ ability to increase virion infectivity [166, 167], but it was not until 2015 that it was discovered that this is primarily due to the ability of Nef to counteract SERINC5 [114, 116]. Specifically, Nef limits the incorporation of SERINC5 into budding virions [114, 116, 168]. This action is facilitated by binding to SERINC5, which results in the redirection of SERINC5 from viral assembly sites at the plasma membrane to the lysosome where it is degraded [168, 169]. How SERINC5 limits infectivity of virions is not yet clear, but it may be related to effects on the virion envelope proteins as some HIV-1 envelopes appear to confer increased resistance, or alternatively sensitivity, to the effects of SERINC5 [170, 171].

1.5.5 Modulation of immune cell activation

CD4+ T cells, the primary cell type infected by HIV-1 [172], are classically activated through signaling provided by APCs, including B cells, dendritic cells and monocytic cells. Specifically, the T cell receptor (TCR) is engaged by binding to antigen presented in the context of MHC-II molecules on the surface of APCs. Subsequent to initial TCR engagement, additional cell surface receptors and intracellular signaling molecules are recruited to the site of the TCR: MHC-II complex. This forms the immunological synapse, the sum of interactions between T cells and APCs that mediate T cell activation (Figure 1.3; reviewed in [173]). When the immunological synapse is formed, intracellular signaling pathways and transcription factors are activated, leading to increased cytokine production. These effects can result in immune cell proliferation, which is essential for amplification of the immune response. Nef and Vpu can influence CD4+ T cell activation, which is particularly important for HIV-1 as viral gene transcription and subsequent virion production can be induced by transcription factors that are upregulated upon T cell activation [174-176]. Therefore, HIV-1 propagation may be optimized by promoting an

18 environment in which activation is sufficient to mediate viral replication and dissemination, whilst also preventing anti-viral immune responses and cell death.

Figure 1.3 Schematic of select molecules found in the immunological synapse

Immunological synapses form between T cells and antigen presenting cells to mediate sustained T cell activating signaling (reviewed in [173]). In CD4+ T cells, these interactions include TCR binding to antigen presented in the context of MHC-II molecules, which is facilitated by the co-receptor CD4. TCR-mediated signaling is also enhanced or inhibited by binding of the co-stimulatory or co-inhibitory receptors CD28 and CTLA-4, respectively, to B7.1/B7.2 on antigen presenting cells. Engagement of the TCR allows for recruitment of downstream signaling molecules, including Zap70 and Lck. Moreover, to ensure sustained interactions between T cells and antigen presenting cells, adhesion molecules, such as ICAM-1, bind to their ligands on the cognate cell.

1.5.5.1 Disruption of the immunological synapse

Alterations in immune cell activation may be facilitated by changes in the cell surface architecture of HIV infected CD4+ T cells, as cell surface associated molecules can influence the formation, maintenance, and downstream signaling of the immunological synapse. Indeed, these processes are thwarted by HIV-1 Nef. Specifically, Nef reduces the formation of immunological synapse-like complexes between T cells and peptide loaded APCs [177]. This is facilitated by changes to immunological synapse-associated molecules that are mediated by HIV-1 Nef and these changes are briefly described below.

Cell surface co-stimulatory or co-inhibitory receptors and their cognate ligands are key components of the immunological synapse. Thus, they are commonly targeted by intracellular parasites to evade immune responses [178]. Specifically, T lymphocytes express cell surface CD28, which functions as a co-stimulatory molecule to initiate a signaling cascade that acts in concert with TCR signaling to promote T cell activation [179]. This is facilitated by the binding of CD28 on T cells to the ligands B7.1/B7.2 (CD80/CD86) on the surface of APCs (Figure 1.3). However, following T cell activation, the co-inhibitory molecule CTLA-4 is also upregulated to prevent over-activation of T cells. Consequentially, CTLA-4 competes with CD28 for binding to B7.1/B7.2 on APCs. However, HIV-1 alters these responses, as Nef reduces T cell surface levels of CTLA-4 and CD28 [118, 180], as well as B7.1/B7.2 on monocytic cells [117]. Specifically, HIV-1 Nef interacts with CD28 and decrease its abundance on the cell surface [118, 181]. In addition, Nef decreases total cellular CTLA-4 protein levels, presumably by mediating CTLA-4 degradation [180]. Finally, Nef also subverts the immunological synapse by sequestering B7.1/B7.2 within an intracellular compartment to limit availability for binding to CD28 or CTLA-4 [117, 182, 183]. Moreover, cellular receptors outside of the immunological synapse can act as co-stimulatory molecules by mediating signaling upon ligand binding. This includes L-selectin, which is downregulated from the cell surface by Nef and Vpu, a function that is associated with decreased T cell proliferation [147].

In addition to altering cell surface receptors that modulate T cell activation, Nef can alter molecules that facilitate signaling downstream of these receptors. Specifically, Nef sequesters Lck, a kinase that plays a key role in signaling downstream of the TCR, in the

21 trans-Golgi network to prevents its recruitment to the immunological synapse [177, 184- 186]. Moreover, Nef impedes the immunological synapse recruitment of phosphoproteins that facilitate downstream signaling. Namely, Nef inhibits the clustering of phosphorylated neural Wiskott-Aldrich syndrome protein (N-WASP), an actin cytoskeleton regulator, near the immunological synapse, which may contribute to Nef’s ability to reduce actin ring formation and spreading subsequent to TCR stimulation [184, 187]. This process has been linked to the ability of Nef to form a Nef:kinase complex, called the Nef-associated kinase (NAK) complex, which contains the Nef-binding serine-threonine kinase p21 activated kinase 2 (PAK2) [188]. These Nef-induced changes to the mediators that potentiate signaling upon TCR engagement likely limit the signaling pathways that classically occur upon TCR activation. Overall, limiting the association of the aforementioned molecules with the immunological synapse may inhibit the formation of fully competent immunological synapses, and thus inhibit the activation of T cells by external stimuli [189].

1.5.5.1 Inhibition of immune cell proliferation

HIV-1 can also modulate immune cell activation independently of its ability to alter immunological synapses or signaling pathways. Namely, HIV-1 can inhibit the key downstream consequence of T cell activation, T cell proliferation, which serves to amplify the immune response. These effects are mediated through HIV-1 altering amino acid metabolism. Specifically, a proteomic screen for host cell plasma membrane proteins that are modulated by Nef and Vpu identified the amino acid transporter SNAT1 as being downregulated in the presence of Vpu [91]. This is mediated by the ability of Vpu to bind to SNAT1 and facilitate its degradation in lysosomes [91]. SNAT1 depletion results in decreased alanine uptake and reduced proliferation upon T cell stimulation [91].

1.5.5.2 Alterations in transcription factor activation

Despite the ability of Nef to limit T cell activation by APCs in a classical TCR-dependent manner, Nef is paradoxically known to facilitate increases in T cell activation. Specifically, Nef lowers the threshold for activation of pathways that lead to stimulation of the activities of transcription factors NF-κB and NFAT [185, 190-193]. This function induces expression of antiviral mediators and cytokines, including IL-2 [185, 190-193]. The exact mechanism

22 by which Nef alters the threshold for activation of these pathways remains enigmatic, but is likely through Nef’s ability to modulate intracellular kinases and actin cytoskeleton regulators [186, 187, 194]. Moreover, it has been proposed that Nef reduces the threshold for activation to stimulate these specific pathways while alternatively limiting the activation of others [195].

In contrast to the effects of Nef, Vpu inhibits the immune cell activating transcription factor NF-κB. Briefly, HIV-1 Vpu counteracts NF-κB through its’ ability to downregulate BST- 2, thereby preventing clustering of virion-bound BST-2 on the cell surface [196]. Otherwise, BST-2 would initiate a signaling cascade activating NF-κB. Moreover, Vpu has BST-2-independent effects on NF-κB, whereby it prevents the nuclear translocation of functional NF-κB in part through inhibiting degradation of the NF-κB inhibitor IκB [197, 198]. It has been proposed that the seemingly opposing effects of Nef and Vpu are due to differences in Nef and Vpu expression patterns during infection. Indeed, Nef is expressed early in infection, while Vpu is expressed at later time points [199-201]. In this way, early in infection Nef can activate NF-κB, which facilitates HIV-1 viral genome transcription, while later in infection Vpu reverses this Nef-mediated effect to mitigate hyper activation [202, 203].

1.5.6 Modulation of cell death pathways

While HIV-1 may increase immune cell activation in order to enhance viral gene transcription, the virus ensures that cellular activation is controlled to guarantee viral immune evasion and to prevent excessive activation which may lead to programmed cell death, or apoptosis [189]. Nef and Vpu can regulate this balance between activation and cell death through their effects on cellular activation, as well as their described pro- and anti-apoptotic functions. Indeed, both Nef and Vpu have ascribed pro- and anti-apoptotic effects, rendering it difficult to conclude if these proteins are pro- versus anti-apoptotic. However these differential effects may be related to differences that occur in cell types and time points post infection [204]. Namely, it was proposed that these viral proteins may act in an anti-apoptotic manner early in infection to allow for viral replication, followed by acting in a pro-apoptotic manner later in infection [204]. Both Nef and Vpu are implicated in influencing the tumour suppressor p53 which activates apoptosis. Specifically, Vpu was

23 shown to increase p53 protein levels [205], while Nef is reported to bind to p53 and prevent p53-induced apoptosis [206]. Nef also inhibits apoptosis through its ability to bind to the kinase PAK2, as this induces PAK-mediated phosphorylation of BAD, inhibiting this pro- apoptotic protein [207]. Moreover, Nef binds to and inhibits the apoptosis signal-regulating kinase 1, a kinase that functions to induce apoptosis [208]. Finally, Nef inhibits apoptosis by binding to the membrane trafficking adaptor protein AP-2 [209]. In contrast to these anti-apoptotic effects, both Nef and Vpu contribute to increases in Fas/Fas ligand mediated apoptosis, whereby Vpu induces increased sensitivity to Fas-mediated apoptosis [210], while Nef increases Fas ligand expression to induce apoptosis in neighbouring cells [211]. Interestingly, AIDS disease progression is correlated with increased Fas expression on T cells [212]. Finally, Nef inhibits the anti-apoptotic proteins Bcl-2 and Bcl-XL to promote apoptosis [213].

The effects of Nef on the secretion of the cytokine transcription necrosis factor-alpha (TNFα) is a prime example of HIV-1 balancing cellular activation with induction of apoptosis. Specifically, through formation of the Nef-associated kinase complex, Nef can induce the secretion of extracellular vesicles containing Nef, Vpu, TNFα and A disintegrin and metalloprotease (ADAM)-17 [214], the metalloproteinase which cleaves pro-TNFα to facilitate TNFα shedding [215, 216]. Surprisingly, vesicles containing these components are detectable in the plasma of HIV-1 positive individuals who are on anti-retroviral therapy [217]. Nef-induced secretion of these vesicles is associated with TNFα-mediated cell activation, which promotes viral transcription and enhances HIV-1 replication [214]. Nef thus induces TNFα production to enhance viral replication, despite TNFα being a pro- apoptotic mediator [218].

1.5.7 Functional redundancy of HIV-1 Nef and Vpu

The relationship between HIV-1 Vpu and Nef is complex and dynamic. Clear examples exist of these viral proteins functioning together to achieve the same effect. Specifically, Nef and Vpu both contribute to decreasing levels of cell surface MHC-I, CD4, MHC-II, PVR and L-selectin [102, 103, 109, 119, 124, 125, 128-130, 139-141, 147]. This redundancy may be due to the sheer importance of these modifications for successful

24 dissemination and persistence of the virus. In this way, Nef and Vpu could compensate for each other should defects in the other protein arise.

Given the ability of Nef and Vpu to perform similar or related functions, there are interesting evolutionary dynamics between them. Specifically, in SIVs closely related to HIV-1, Nef counteracts the effects of the restriction factor BST-2 [219]. However, small differences between the primate and human BST-2 protein prevent chimpanzee SIV Nef proteins from counteracting human BST-2 [219-221]. Therefore, HIV-1 Vpu took over this SIV Nef function by evolving the ability to counteract human BST-2 [219], a function which was likely essential for the zoonotic transmission of SIV from chimpanzees to humans. Moreover, while most HIV-1 Group M viruses encode Vpu proteins that counteract this restriction factor, there have been viruses identified in which Vpu has lost the ability to counteract BST-2 and the Nef protein from these viruses performs this function [222]. Moreover, in the case of HIV-1 Groups O and N, viruses have been identified that encode either Nef or Vpu proteins that counteract BST-2 [219, 223-225]. Thus, the counteraction of BST-2 by Nef and Vpu clearly demonstrate the close relationship between these proteins’ functions and their ability to compensate for each other to facilitate key tasks.

Another example of compensation is through the control of cellular activation. Specifically, SIV viruses encode Nef proteins capable of downregulating cell surface levels of CD3, a protein which functions to stabilize the TCR [226, 227]. The ability of SIV Nef proteins to downregulate CD3 is key to limiting extensive cell activation and may contribute to the decreased pathogenicity associated with SIV infections, relative to HIV-1 infections [202, 228, 229]. Interestingly, Nef-mediated downregulation of CD3 is a function that was often lost when SIV viruses gained a vpu gene [229-231]. With the recent demonstration that HIV-1 Vpu can reduce T cell activation through its ability to suppress NF-κB activity, it was proposed that Vpu may be compensating for the loss of CD3 downregulation ability in HIV-1 Nef proteins to prevent overly pathogenic effects [202, 203]. Ultimately, the control of cellular activation may represent another example of the functional interrelatedness of Nef and Vpu.

1.5.8 The importance of Nef and Vpu in vivo

Overall, the HIV-1 accessory proteins Nef and Vpu mediate a plethora of effects in HIV-1 infected cells. These effects can in turn impact pathogenicity and viral spread in vivo. Evidence for the importance of Nef in HIV-1 pathogenicity first came from a cohort of individuals who were infected with HIV-1 via transfusion of blood products obtained from an HIV-1 positive donor who was infected with a virus lacking a functional nef gene. Interestingly, individuals infected with Nef defective virus exhibited extremely slow disease progression [232]. Moreover, transgenic mice that were developed to express Nef in CD4+ cells in the absence of all other HIV-1 proteins exhibited an AIDS-like phenotype [233], illustrating the pathogenic potential of this protein in the absence of infection. A more recently developed mouse model for studying the effects of HIV-1 was also utilized to demonstrate that nef-deficient viruses replicate less rapidly and induce loss of a lower number of T cells [234]. In addition, the importance of Nef has been illustrated through the use of SIV infections in non-human primate models, as nef genes encoding a premature stop codon readily revert to nef genes that encode a functional Nef protein [235].

In the case of Vpu, multiple groups have illustrated the importance of Vpu for viral replication and dissemination using humanized mouse models [68, 236, 237]. A recent study suggested that this effect is primarily mediated though the ability of Vpu to counteract the restriction factor BST-2 [165]. Moreover, in infections of pig-tailed macaques with chimeric simian-human immunodeficiency viruses, the inhibition of Vpu protein production, the scrambling of the amino acids in the Vpu transmembrane domain, or the mutation of functionally important Vpu residues, all resulted in decreased viral pathogenicity [66, 67, 238]. Thus, despite Nef and Vpu being classified as “accessory” proteins, they are critical for infection and pathogenicity of the virus in vivo.

1.6 How do HIV-1 Nef and Vpu mediate their functions?

HIV-1 Nef and Vpu mediate their multiple effects without possessing their own enzymatic activity. This is amazing given their relatively small size, with the prototypical HIV-1 laboratory strain NL4.3 encoding Nef and Vpu proteins containing 206 and 81 amino acids, respectively. This capacity to facilitate many modifications within host cells is due to their

26 ability to co-opt ubiquitous trafficking pathways. The net result of subverting trafficking pathways is typically the modification of the subcellular localization of many host cell proteins, which in turn alters their function.

Nef and Vpu can co-opt host trafficking pathways due to the presence of a number of motifs that allow them to bind cellular proteins by mimicking signals utilized by host proteins (Figure 1.4). Importantly, despite the high diversity observed within the HIV-1 Group M viruses, these Nef and Vpu motifs are often very well conserved [239-243]. Additionally, both proteins contain regions that lack structure, and this intrinsic disorder may facilitate an increased number of host cell protein interactions.

Figure 1.4 Structure of HIV-1 Nef and Vpu and motifs that interact with host cell proteins

Left: Cartoon illustrations of the Nef (A; blue) and Vpu (B; purple) proteins. Right: schematics illustrating motifs within NL4.3 Nef (A) or Vpu (B). The location of the protein motifs and their binding partners or functions are indicated. (A) NL4.3 Nef is a 206 amino acid cytoplasmic protein which is associated with membranes through myristoylation of G2 [99, 244-246]. Nef associates with the membrane trafficking adaptor proteins AP-1 and AP-2 through interactions mediated by the

W13/V16/M20/EEEE65 and ExxxLL165/DD175 motifs, respectively [247-249]. The vesicular coat protein β-COP interacts with Nef R17/19 [181, 250, 251]. The membrane trafficking regulators phosphofurin acidic cluster sorting (PACS)-1 and -2 bind to the

Nef acidic cluster, EEEE65, and W113/Y120 [122, 123, 252]. The v-ATPase mediates interactions with DD175 [253, 254]. The Nef-targeted cargo protein CD4 interacts with

Nef via WL58 [255]. The kinases PAK2 and SFKs bind to H89/F191 and PxxP75, respectively [256-262]. Nef dimerizes via an interface comprised of

R105/I109/L112/Y115/F121/D123 [257, 263]. (B) The NL4.3 Vpu protein is an 81 amino acid transmembrane protein containing a short luminal domain and two cytoplasmic helixes.

The transmembrane domain amino acids A10/A14/A18/W22 mediate interactions with the

Vpu targeted cargo BST-2 [264, 265]. The RL45 and ExxxLV64 motifs mediate binding to the adaptor proteins AP-1 and AP-2 [266]. The E3 ubiquitin ligase component β-TrCP binds to the Vpu DSGpS56 motif upon serine phosphorylation [267].

The host cell machinery that Nef and Vpu hijack are primarily involved in membrane trafficking. This fundamental cellular process can be defined as the movement of cargo molecules from one location to another in membrane bound vesicles. Nef and Vpu may have a specific preference for these cellular processes as they themselves are membrane associated proteins [246, 268]. Specifically, the host cell proteins to which Nef and Vpu bind are proteins which facilitate trafficking through the endosomal trafficking network (Figure 1.5), or the ubiquitin ligase machinery which modifies cargo proteins to target them for trafficking to specific locations [69]. The movement of vesicle-associated molecules around the cell requires a large number of trafficking proteins which not only facilitate the physical movement of cargo along the cellular cytoskeleton, but also dictate directionality and specificity. These processes unambiguously determine where vesicles from one compartment fuse with vesicles from another compartment (Figure 1.6; [269]). Notably, this process is regulated in part by sequences within proteins or post translational modifications, such as ubiquitination, that serve to direct where cargo go by giving specificity to interactions with cellular trafficking proteins. The importance of regulating vesicular trafficking led to the 2013 Nobel prize in Physiology or Medicine being award to Drs. James Rothman, Randy Schekman and Thomas Südhof, who were recognized for their invaluable contributions to understanding how these membrane trafficking processes occur [270]. Importantly, viruses and viral proteins, including HIV-1 Nef and Vpu, have evolved the ability to mimic or alter these trafficking signals and thereby alter the localization of their target proteins to fulfill their functions [271].

Figure 1.5 The endosomal trafficking network

Proteins destined for the cell surface or compartments in the endocytic network are translated on the rough ER and trafficked through the Golgi complex to the trans-Golgi network wherein proteins are processed. From here, proteins can be shuttled in membrane enclosed vesicles to the plasma membrane and various intracellular compartments, including: early endosomes, recycling endosomes, late endosomes or multivesicular bodies (MVBs), and lysosomes. Arrows indicate the pathways through which vesicle transport can occur.

Figure 1.6 General mechanism of vesicle transport

(1) Signals in the cytoplasmic tail of cargo proteins recruit adaptor proteins to specific membrane regions. This process can be further regulated by changes in membrane phospholipids. Other proteins necessary for subsequent stages of transport may also be recruited, including v-SNAREs. (2) Adaptor proteins contain specific sequences mediating recruitment of vesicular coat proteins which oligomerize at sites of vesicle budding. This oligomerization can be regulated by the ADP-ribosylation factor family of GTPases. (3) Membrane bending ensues and the scission of the vesicle membranes from the donor membrane occurs, often assisted by host cell enzymes that polymerize along the neck of budding vesicles and through regulated actin polymerization. Once a transport vesicle buds, the associated coat and adaptor proteins disassociate and recycle back to the donor membrane to be used in future vesicle budding events. (4/5) At

32 acceptor membranes, various tethering factors bring the vesicle into proximity of the acceptor membrane and proteins known as t-SNAREs interact with vesicle incorporated v-SNAREs to reduce the energy barrier needed to fuse membranes. Tethering factors or protein complexes, SNARE proteins, and enzymes in the Rab GTPase family all function to mediate specificity of transport, such that they ensure vesicle fusion occurs with the correct target membranes. Figure is modified from [269].

1.6.1 Nef: host cell protein interactions

In order for Nef to mediate its effects it associates with host cell membranes. This is facilitated by the post-translational modification of Nef on glycine residue two, which is modified by the addition of a lipid myristoyl group derived from myristic acid and effectively functions as a lipid anchor [99, 244-246]. When HIV-1 Nef myristoylation is inhibited through mutation of glycine residue two to alanine, virtually all of Nef’s functions are inhibited, including downregulation of CD4, MHC-I, mature MHC-II, NK cell activating receptors, and SERINC5 [106, 109, 114, 119]. The association of Nef with membranes presumably brings this protein within close proximity of host cell membrane- associated proteins, to which Nef must bind to facilitate its functions.

1.6.1.1 Vesicular adaptor proteins: AP-1 and AP-2

Key membrane-associated proteins to which Nef binds are the membrane trafficking adaptor proteins adaptor protein 1 (AP-1) and adaptor protein 2 (AP-2). AP-1 and AP-2 are heterotetrameric protein complexes that function as vesicular adaptors to connect cargo to vesicular coat proteins [272]. These proteins are recruited to specific cargo based on sorting signals present in the cytoplasmic tail of cargo proteins. Two prototypical sorting signals recognized by AP-1 and AP-2 are the tyrosine (YxxØ, where x is any amino acid and Ø is any amino acid with a bulky hydrophobic amino acid) and dileucine ([D/E]xxxL[L/I], where x is any amino acid) based sorting signals (reviewed in [272]). These distinct signals are bound by different subunits of the adaptor protein complexes, with the μ subunits typically recognizing tyrosine based motifs and the combination of γ – σ (AP-1) or α– σ (AP-2) subunits recognizing dileucine motifs. Nef has evolved the ability to hijack these signals to forms complexes with specific cargo and adaptor proteins. Namely, Nef can bind the cytoplasmic tail of MHC-I molecules by facilitating recognition of an unconventional tyrosine-based sorting signal (Y320SQA) in the MHC-I cytoplasmic tail by the AP-1 μ subunit [247]. Specifically, a crystal structure containing Nef, the AP-1 μ subunit, and the cytoplasmic tail of MHC-I revealed that these proteins form a ternary complex which allows for AP-1 to bind the MHC-I Y320SQA motif despite it lacking the hydrophobic residue normally found in canonical tyrosine sorting signals [247]. Formation of the ternary complex is facilitated through the creation of a hydrophobic pocket

containing Nef W13, V16, M20 and an acidic cluster, Nef EEEE65, which together mediate interactions with AP-1 [247, 248]. Moreover, mutation of Nef M20 and EEEE65 inhibit MHC-I downregulation [247, 248, 273, 274], indicating that the ability of Nef to form this Nef: AP-1: MHC-I ternary complex is essential for the ability of Nef to downregulate MHC-I.

In addition to hijacking AP-1 complexes, Nef hijacks AP-2 in a similar manner. Specifically, Nef forms a ternary complex with AP-2 and the cytoplasmic tail of the host cell receptor CD4 [275]. Unlike the Nef: AP-1: MHC-I ternary complex, the Nef: AP-2: CD4 ternary complex can be formed through the recognition of a dileucine based motif in

Nef (ExxxLL165) by the AP-2 α and σ subunits, as defined by a crystal structure of Nef and AP-2 α and σ [249]. The Nef: AP-2 structure also revealed a surface wherein CD4 could bind to form a Nef: CD4: AP-2 ternary complex [249]. This predicted CD4 binding site contained Nef WL58 [249], which has been previously implicated in the interaction between Nef and the cytoplasmic tail of CD4 [255]. The recruitment of AP-2 to the CD4 cytoplasmic tail by Nef is believed to facilitate the recruitment of the vesicular coat protein clathrin to mediate endocytosis of CD4-containing vesicles from the cell surface, thereby decreasing cell surface levels of CD4. Indeed, mutation of the Nef AP-2 binding motif

(LL165) or CD4 binding motif (WL58) abolishes the ability of Nef to downregulate cell surface CD4 [276, 277].

The Nef: AP-2 α and σ structure also suggested that the Nef diacidic motif (DD175), which takes the form of aspartic or glutamic acid residues depending on the Nef protein, functions to stabilize the interaction between Nef and AP-2 [249]. This may explain, to some extent, the requirement of this motif in the downregulation of CD4 [278]. However, the Nef diacidic motif has also been implicated in the binding of Nef to the vacuolar ATPase (v- ATPase) [253, 254], a proton pump which acidifies endosomes [279]. It has been proposed that since v-ATPase binds to both Nef and the μ subunit of AP-2 it connects Nef to AP-2 to mediate CD4 downregulation [253]. Interestingly, the mechanisms utilized by Nef to hijack AP-2-mediated trafficking to downregulate CD4 may also be co-opted to downregulate other cell surface receptors. Indeed, mutation of the Nef dileucine motif

(LL165) and the Nef diacidic motif (DD175) have been shown to abolish the ability of Nef

35 to alter the subcellular localization of other cell surface proteins including: the MHC-II invariant chain [131, 280], CD28 [118, 280], CTLA-4 [180], and the restriction factor SERINC5 [114, 281]. Overall, the ability of Nef to co-opt classical membrane trafficking adaptors, namely AP-1 and AP-2, are key to Nef’s functions.

1.6.1.2 Vesicular coat proteins and their regulators: Arf1 and β-COP

The oligomerization of adaptor protein-containing vesicular coats is a highly regulated process. Indeed, the GTPase ADP-ribosylation factor 1 (Arf1) regulates the formation of AP-1 containing coats by interacting with AP-1 and promoting formation of an “open complex” wherein the cargo-binding regions are exposed [282]. Interestingly, a functional Arf1 protein is necessary for Nef-mediated MHC-I downregulation in T cells [283]. Moreover, the expression of a constitutively active Arf1 increased the ability of MHC-I to co-immunoprecipitate with AP-1 in the presence of Nef [283]. A more recent cryo-electron microscopy study revealed that Arf1 may be contributing to Nef-mediated MHC-I downregulation [284]. This study illustrated that a Nef: MHC-I cytoplasmic tail fusion protein could form a trimeric complex with Arf1 and AP-1, implicating Arf1 in promoting the formation of AP-1 coated vesicles [284]. Arf1 is also known to regulate the formation of vesicular coats containing the coat protein coatomer protein I (COPI) [285, 286], which Nef has also been implicated in hijacking. Indeed, it was demonstrated that immunoprecipitation of Nef can pull down the COPI subunit β-COP from CHO cell lysate [287]. While this Nef: β-COP interaction was initially mapped to the Nef diacidic motif

[287], this was later challenged and the interaction mapped to Nef R17/19 [181, 250, 251]. Through the use of short hairpin RNAs to deplete β-COP and the overexpression of Nef

R17/19 mutants, β-COP was implicated in Nef-mediated degradation of MHC-I and CD4, presumably in lysosomes [181, 251]. Generally, Nef may utilize components of the COPI trafficking pathway, including Arf1 and β-COP to mediate its distinct functions.

1.6.1.3 Vesicular trafficking regulators: PACS-1 and PACS-2

There are many proteins that regulate membrane trafficking by promoting or inhibiting the association of specific cargo with trafficking adaptors or coat proteins. A prime example of trafficking regulators are PACS-1 and PACS-2, which link acidic cluster containing

36 cargo molecules with membrane trafficking machinery [288]. Specifically, PACS-1 and PACS-2 link cargo to the vesicular adaptor proteins AP-1 and COPI, respectively [289,

290]. Interestingly, Nef contains an acidic cluster, EEEE65, which in conjunction with residues W113 and Y120 facilitates binding to PACS-1 and PACS-2 [122, 123, 252]. Given that PACS-1 forms ternary complexes with AP-1 and cargo proteins [289], and that PACS- 1 binds to Nef and AP-1 [123, 289], PACS-1 may facilitate the formation of multi-protein complexes that include Nef, AP-1 and cargo molecules [291]. Indeed, the PACS proteins have been implicated in the ability of Nef to downregulate cell surface MHC-I, with mutation of the PACS-1/2 binding acidic cluster (EEEE64) or overexpression of dominant negative forms of PACS-1/2, inhibiting Nef-mediated MHC-I downregulation [122, 252]. Finally, the interaction between the PACS proteins and Nef may be important for mediating additional Nef functions, as mutation of the Nef EEEE65 acidic cluster also inhibits the ability of Nef to downregulate cell surface MHC-II [119], CD28 [280], and a variety of chemokine receptors [105].

1.6.1.4 Vesicular trafficking sorting signals: ubiquitin

In addition to mediating recruitment of the membrane trafficking machinery through specific protein motifs, Nef can alter the localization of target proteins through pathways that are regulated by post-translational modifications. One key protein modification is the covalent addition of 8 kDa ubiquitin peptides in a process known as ubiquitination. Ubiquitination is classically thought of as functioning to mark proteins destined for degradation in the cytosolic proteasome, however ubiquitination also plays a role during membrane trafficking (reviewed in [292]). Specifically, ubiquitination functions as a signal for endocytosis from the cell surface by interacting with vesicular adaptor proteins. Moreover, endocytosed ubiquitinated proteins present in early endosomes are detected by components of the endosomal sorting complexes required for transport (ESCRT) machinery and are subsequently sorted into intraluminal vesicles that are degraded in the lysosome. HIV-1 Nef can hijack ubiquitin-dependent pathways in order to downregulate various cell surface receptors. Specifically, Nef hijacks the ESCRT associated protein ALIX, which binds to ubiquitinated cargo [293], by binding directly to ALIX to link un- ubiquitinated CD4 to the ESCRT machinery [294]. This interaction mediates CD4’s sorting

37 into intraluminal vesicles and subsequent lysosomal degradation [294, 295]. Nef also mediates the accelerated trafficking of ubiquitinated SERINC5 to lysosomes where this restriction factor is degraded [169]. Additionally, Nef induces ubiquitination of the chemokine receptor CXCR4 to promote its downregulation from the cell surface [148].

Nef also enhances the ubiquitination and degradation of Giα2 which functions in signaling downstream of chemokine receptors [296]. Finally, the Nef protein itself can be ubiquitinated at K144 and mutation of this residue inhibits the ability of Nef to downregulate both CD4 and MHC-I [297, 298]. However, it is unknown if the ubiquitination of Nef is required for CD4 and MHC-I downregulation.

1.6.1.5 Kinases: SFKs and PAK2

Membrane trafficking pathways and proteins may be altered by signaling molecules, such as intracellular kinases. Nef may therefore modulate trafficking though its ability to bind to specific kinases, namely members of the Src family kinases (SFKs) and PAK2. Specifically, Nef binds to and mediates activation of the tyrosine kinases Hck, Lyn, and c- Src, members of the SFKs [299]. The details of this interaction have been elucidated using mutational analyses, crystal structures, and solution NMR structures. These studies indicate that the RT loop of the SH3 domain in SFKs interacts with the Nef type II polyproline helix, specifically the P69xRPxxPxRP78 encoding region [256-260]. Similar SH3: polyproline helix interaction interfaces are commonly used by host cellular proteins

[300]. Moreover, the Nef PxxP75 motif is critical for this interaction, as a Nef AxxA75 mutant is unable to activate SFKs [299]. The Nef: SFK interaction has been implicated in the formation of a multi-kinase signaling complex which activates a phosphoinositide 3- kinase (PI3K)-signaling pathway that induces the endocytosis of cell surface MHC-I [121,

122, 301]. Therefore, mutation of the Nef PxxP75 motif or inhibition of Nef: SFK binding using small molecule inhibitors can block Nef-mediated downregulation of cell surface MHC-I [121, 301]. Interestingly, inhibition of the Nef: SFK interaction has also been shown to negatively affect the ability of Nef to increase virion infectivity and alter cellular activation [302, 303]. In addition, mutation of the Nef PxxP75 motif can inhibit Nef- mediated downregulation of MHC-II [119, 304], NK cell activating receptors [109, 305], and chemokine receptors [105, 149-151]. While these effects may be due to the inability

of Nef to bind SFKs, the Nef PxxP75 motif is also implicated in facilitating the binding of Nef to the serine-threonine kinase PAK2 [306, 307].

When Nef-associated serine-threonine kinase activity was first observed in T cells, the identity of the Nef-associated kinase was unknown. Thus, this complex was initially termed the Nef-associated kinase (NAK; [307, 308]). It was later recognized that the Nef- associated kinase is PAK2 [309-311]. Unlike the Nef-binding SFKs, PAK2 lacks a SH3 domain to interact with the Nef PxxP75 motif. Rather, mutational analysis and protein modeling suggests that PAK2 interacts with a hydrophobic patch on Nef containing H89 and F191 [261, 262]. Thus, the requirement of the Nef PxxP75 motif for PAK2 binding has been suggested to be due to PAK2 forming a complex with Nef concomitantly with other associated proteins, including the SH3-domain containing protein Vav, which may act to stabilize the interaction between Nef and PAK2 [312]. The association of Nef with PAK2 may facilitate PAK2 activation in cells [311]. The Nef: PAK2 interaction is primarily associated with the ability of Nef to alter T cell activation [187, 188, 194, 313], modulate T cell survival [207], and to mediate the secretion of extracellular vesicles [314]. Overall, Nef-mediated interactions with cellular kinases, including SFKs and PAK2, are key to many alterations in the cellular environment facilitated by Nef.

1.6.2 Nef oligomerization

In order for Nef to mediate its functions, including activating cellular kinases, protein dimerization may be required. Indeed, Nef dimers and trimers were observed in vitro [263, 315]. Based on crystal structures and mutational analyses, a dimer interface was proposed which consists of a hydrophobic patch made up of Nef residues I109, L112, Y115 and F121 flanked by electrostatic interactions between R105 on one monomer and D123 on the other monomer [257, 263]. Studies suggest that in cells Nef can oligomerize and that Nef dimerization requires the Nef I109/L112/Y115/F121 hydrophobic patch [316-318]. Moreover, the significance of this dimerization interface was supported by findings that mutation of

D123 or the hydrophobic patch inhibits various Nef functions, including CD4 downregulation, MHC-I downregulation and enhancement of virion infectivity [316, 319]. However, more recent work suggests that the Nef dimer interface may be altered when Nef interacts with various binding partners [260, 320], and the importance of Nef D123 for

39 dimerization has been questioned [247, 260, 321]. Nonetheless, it is believed that Nef’s ability to dimerize is critical for its function and the creation of small molecule inhibitors to block Nef’s effects via targeting dimerization have been proposed [318, 322].

1.6.3 Vpu oligomerization

The HIV-1 Vpu protein, like Nef, is membrane associated given that it contains a transmembrane domain. Specifically, Vpu is a type I integral membrane protein with a single alpha helical transmembrane domain [268]. Interestingly, Vpu oligomerizes and is classified as a class IA viroporin, similar to the unrelated influenza A’s matrix 2 protein [323]. Computer modeling and NMR spectroscopy suggest Vpu forms pentameric oligomers [268, 324-326], thereby forming a pore within membranes which can mediate selective transport of cations [327, 328]. The functional significance of ion channel formation by Vpu remains enigmatic, though pore formation has been correlated with the ability of Vpu to downregulate CD4 and enhance virion release [328, 329]. In contrast, it has been suggested that the active form of Vpu for CD4 downregulation is monomeric, as mutation of Vpu residue W22 increases Vpu oligomerization and decreases the ability of Vpu to downregulate CD4, without inhibiting Vpu: CD4 interactions [330]. Moreover, a

Vpu transmembrane domain mutation (S23A) which inhibits Vpu ion channel activity did not inhibit the ability of Vpu to counteract BST-2 [331]. Finally, the effects of Vpu ion channel formation may be restricted to specific subcellular locations given that live cell imaging indicates that Vpu does not oligomerize in the ER [326].

1.6.4 Vpu: host cell protein interactions

1.6.4.1 Target protein binding

In addition to mediating oligomerization, the transmembrane domain of Vpu is implicated in forming key interactions with host cellular proteins that Vpu targets for downregulation. Indeed, Vpu counteracts BST-2 by binding to BST-2 via an interaction interface that is mediated by the transmembrane domains of these proteins. This interface has been mapped using mutational analysis and NMR spectroscopy to Vpu residues A10, A14, A18 and W22, mutation of which inhibit Vpu: BST-2 interactions and the ability of Vpu to antagonize BST-2 [264, 265]. Moreover, structural modeling of Vpu suggests that the transmembrane

domain forms a helix with A10, A14, A18 and W22 located on same face of the helix [264, 325]. This model implies that these residues could constitute an interaction interface. The transmembrane domain of Vpu has also been implicated in Vpu’s ability to downregulate cell surface CD4. Specifically, while a number of studies suggest that Vpu and CD4 interact via their cytoplasmic domains [332-336], mutation of transmembrane domain residues or replacement of the Vpu transmembrane domain with that of other proteins have been shown to inhibit the ability of Vpu to degrade or bind CD4 [330, 337-339]. Additionally, combinations of mutations within the Vpu transmembrane domain residues

A19, A14, A18 and W22 or scrambling of the Vpu transmembrane domain sequence inhibit the ability Vpu to downregulate NK cell activating receptors [108, 145, 305] and the chemokine receptor CCR7 [154]. These findings demonstrate that the Vpu transmembrane domain is critical for Vpu to mediate its function, likely through facilitating binding to target proteins.

1.6.4.2 Vesicular adaptor proteins: AP-1 and AP-2

The interactions between Vpu and host cell proteins enable Vpu to alter the subcellular localization of target proteins by linking them to cellular machinery, similar to the functions of Nef. Recently, it was demonstrated that Vpu can hijack membrane trafficking adaptor proteins. Specifically, the cytoplasmic tail of Vpu contains a dileucine-like motif

(ExxxLV64; Figure 1.4), similar to the conventional adaptor protein-binding dileucine motif ([D/E]xxxL[L/I/V]). Using chromatography this motif in Vpu was shown to mediate a direct interaction with AP-1 and AP-2 [340]. Moreover, the formation of a fusion protein containing the cytoplasmic tails of BST-2 and Vpu exhibited enhanced AP-1 binding, compared to BST-2 or Vpu alone [340]. The latter BST-2: Vpu fusion was successfully co- crystalized with AP-1 [340]. This structure indicated that Vpu forms a ternary complex with BST-2 and AP-1 whereby an atypical tyrosine binding motif, YxYxxØ (where Ø, where x is any amino acid and Ø is any amino acid with a bulky hydrophobic amino acid), in BST-2 binds to AP-1 μ. This structure also demonstrated that the Vpu dileucine-like motif binds to the AP-1 γ and σ subunits, while Vpu residues RL45 interact with AP-1 at the γ-σ interface [340]. The formation of a Vpu: BST-2: AP-1 complex may link BST-2 to the endocytic trafficking network to facilitate Vpu-mediated exclusion of BST-2 from viral

41 assembly sites and BST-2 lysosomal degradation. This possibility is supported by mutation of the Vpu dileucine-like (ExxxLV64) or RL45 motifs, which inhibits both Vpu-mediated decreases in cell surface BST-2 and increases in virion release [340-342]. Moreover, AP- 2 may form a ternary complex with Vpu and BST-2 similar to AP-1, as AP-2 was co- immunoprecipitated with Vpu in BST-2 expressing HEK293T cells in a manner dependent on the Vpu ExxxLV64 motif [266]. Interestingly, the recruitment of AP-1 or AP-2 to BST- 2 to form a ternary complex may be regulated by phosphorylation of Vpu, specifically at serine residues S52/56 [266], which are phosphorylated by casein kinase II [343].

1.6.4.3 LC3-associated phagocytosis mediators: LC3C

In addition to mediating interactions with host cellular adaptor proteins, the Vpu ExxxLV64 motif mediates an interaction with the protein LC3C [344]. LC3C is a member of the Atg8 family of ubiquitin-like proteins that are conjugated to phospholipids. This conjugation leads to membrane association of cargo and facilitates selective transport of LC3 conjugated membranes to either autophagosomes or a non-canonical phagocytic pathway termed LC3-associated phagocytosis (LAP) [345]. Interestingly, depletion of autophagy or LAP proteins revealed that proteins specifically facilitating LAP are required for Vpu to enhance virion release in a BST-2 dependent manner [344]. Therefore, formation of a Vpu: LC3C complex may contribute to the downregulation of BST-2 by connecting BST-2 to LAP machinery [344].

1.6.4.4 Ubiquitin ligase machinery: β-TrCP

Vpu mediates some of its effects by hijacking the cellular ubiquitin ligase machinery. Specifically, Vpu links target host cell proteins CD4 and BST-2 to β-transducin-repeat containing protein (β-TrCP) [162, 267, 346], a component of the Skp Cullin F-box containing (SCF) E3 ubiquitin ligase complex. Beta-TrCP functions as an adaptor molecule that binds phosphorylated cargo to connect it to the ubiquitin conjugating machinery [347]. Vpu usurps this machinery through its casein kinase phosphorylated serine residues which are part of the DSGNES56 motif in NL4.3 Vpu (Figure 1.4). This sequence partially matches the canonical β-TrCP recognition motif, DpSGøxpS, where x is any amino acid, ø is a hydrophobic residue, and the serine residues are phosphorylated [347]. Binding of Vpu

42 to β-TrCP facilitates formation of a ternary complex between Vpu, β-TrCP, and CD4 or BST-2 [162, 267, 346]. In the case of CD4, the formation of Vpu: CD4: β-TrCP complexes in the ER allows for β-TrCP to recruit additional components of the SCF E3 ubiquitin ligase machinery which mediates polyubiquitination of the CD4 cytoplasmic tail [139, 267, 348]. Subsequently, components of the cellular ER associated degradation (ERAD) pathway mediate the translocation of ubiquitinated CD4 from the ER to the cytoplasm where CD4 is degraded by the proteasome [140, 337, 348]. Key to this process is the initial

Vpu: β-TrCP interaction, as mutation of the DSGNES56 motif serine residues to non- phosphorylatable residues inhibits β-TrCP binding to Vpu and Vpu-mediated CD4 degradation [267, 335, 349, 350].

In contrast to CD4 downregulation, Vpu binds to BST-2 on endosomes or at the trans- Golgi network [161, 351]. Formation of Vpu: BST-2: β-TrCP ternary complexes can subsequently mediate lysosomal degradation of BST-2 [161, 351]. Specifically, binding of β-TrCP to Vpu that is bound to BST-2 may facilitate recruitment of E3 ubiquitin ligases which enhance the ubiquitination of BST-2 [351]. The ubiquitination of BST-2 serves as a signal to sort BST-2 into degradative lysosomes through detection by components of the ESCRT machinery. Namely, this activity is regulated by the ESCRT-0 protein hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) which binds ubiquitinated cargo and links them to ESCRT machinery that mediates trafficking to lysosomes [292]. Indeed, HRS co-immunoprecipitated with BST-2 and Vpu while siRNA knock-down of HRS inhibited the ability of Vpu to downregulate cell surface and total BST-2 protein levels [352]. Moreover, inhibition of endosomal acidification or the use of lysosomal inhibitors reduced Vpu-mediated BST-2 downregulation [161, 163, 353]. Furthermore, siRNA- or shRNA-mediated knock-downs implicated β-TrCP in Vpu-mediated decreases of cell surface and total BST-2 protein levels [161, 353, 354]. However, mutation of the β-TrCP binding site does not completely abolish the ability of Vpu to enhance virion release [161, 351, 353]. Thus, β-TrCP binding may only constitute one element of the mechanism through which Vpu counteracts BST-2.

In addition to mediating the downregulation of CD4 and BST-2, the binding of β-TrCP to Vpu may have additional effects within infected cells. Specifically, the Vpu: β-TrCP

43 interaction may inhibit the ability of β-TrCP to fulfill its normal cellular functions. Briefly, when the NF-κB signaling pathway is initiated, the NF-κB inhibitor IκB is phosphorylated and subsequently bound by β-TrCP (reviewed in [355]). This interaction facilitates IκB ubiquitination and proteasomal degradation to allow for NF-κB to translocate into the nucleus and function as a transcription factor. However, binding of Vpu to β-TrCP may sequester β-TrCP away from this canonical target and prevent IκB degradation. This dissociation may contribute to Vpu’s ability to suppress NF-κB activation [197, 198, 356]. Similar to IκB, the pro-apoptotic protein p53 is also phosphorylated and subsequently degraded by β-TrCP [357]. Accordingly, expression of Vpu was shown to inhibit the degradation of p53 leading to increased p53-induced apoptosis [205]. Overall, Vpu mediates hijacking of the ubiquitin ligase machinery by connecting it to specific host proteins and may prevent β-TrCP from facilitating its normal functions.

1.7 Thesis overview

It is established that HIV-1 Nef and Vpu are essential for viral spread and contribute to AIDS pathogenesis. This is due to the ability of these viral proteins to alter and evade immune responses. Many specific functions of Nef and Vpu have been described that contribute to creating an optimal host cell environment for HIV. This is facilitated by the hijacking of host cell membrane trafficking machinery to alter the sub-cellular localization of target proteins. However, our understanding of how HIV-1 Nef and Vpu create the optimal host cell environment is not yet complete.

Our general hypothesis is that these undefined pathways are mediated by Nef and Vpu commandeering host cell proteins that are implicated in membrane trafficking events. The work within this thesis involves investigating the mechanisms by which HIV-1 Nef and Vpu alter the host cellular environment, which may facilitate evasion of the immune system and support viral spread. Specifically, we examine the mechanisms by which HIV-1 Nef and Vpu reduce cell surface levels of the co-stimulatory molecule CD28 (Chapter 2) and the mechanism by which Nef reduces cell surface levels of MHC-I (Chapter 3).

1.7.1 Chapter 2

Previous studies illustrated that the HIV-1 Nef protein can decrease cell surface levels of the T cell co-stimulatory molecule CD28 [118, 181]. However, these studies did not define the mechanistic details of CD28 downregulation during an HIV-1 infection. Therefore, our objectives were to:

• Determine the subcellular fate of CD28 upon HIV-1 infection

• Describe the HIV-1 viral proteins and their host cell binding motifs implicated in CD28 downregulation

• Examine the functional consequences of CD28 downregulation

In undertaking our investigation into the mechanism by which HIV-1 Nef reduces endogenous levels of cell surface CD28 on CD4+ T cells, we also observed downregulation of cell surface CD28 mediated by HIV-1 Vpu. Therefore, we examined the mechanism of CD28 downregulation mediated by both HIV-1 Nef and Vpu. We found that both Nef and Vpu decrease total CD28 protein levels and facilitate transport of CD28 to LAMP-1 positive lysosomal compartments. Moreover, we identified viral protein motifs that were essential for this function, thereby demonstrating that specific membrane trafficking proteins known to interact with these motifs may be important for downregulating CD28. Finally, we postulate that the downregulation of CD28 is playing a role in the broad mechanisms through which Nef and Vpu modulate T cell activation.

1.7.2 Chapter 3

It is known that HIV-1 Nef reduces cell surface levels of MHC-I through its ability to facilitate trafficking of MHC-I to an intracellular compartment wherein this receptor is sequestered. A number of Nef-interacting host cell membrane trafficking proteins have been implicated in MHC-I downregulation, however given the sheer complexity of moving cargo within the cell, there are undoubtedly many additional host cell proteins that take part in this process. We hypothesized that the sorting nexin proteins SNX9 and SNX18 were potential trafficking mediators that could facilitate this Nef function. The rationale for this being that SNX18 was previously shown to form a complex with Nef that localizes to AP-1 positive compartments, subcellular locales through which MHC-I traffics during

Nef-mediated downregulation [358]. Moreover, both SNX18 and SNX9 interact with Nef- binding proteins, namely AP-1 and AP-2, respectively. Our objectives in this study were to:

• Determine if Nef directly binds SNX18 and SNX9, and map this interaction

• Determine if the Nef: SNX18 complex plays a role during MHC-I downregulation

In this study, we showed that SNX18, but not SNX9, is a Nef-binding protein. We mapped this interaction to the SH3 domain of SNX18 and the Nef PxxP75 motif. Moreover, we demonstrated that reducing SNX18 protein levels reduces the ability of Nef to downregulate cell surface levels of SNX18. Overall, we have identified a novel Nef: host protein interaction which mediates MHC-I downregulation, a Nef function that is key to HIV-1 pathogenesis [359, 360].

1.7.3 Significance

The work presented in this thesis details mechanisms through which the HIV-1 viral proteins Nef and Vpu subvert host cellular machinery to alter the sub-cellular localization of the immune cell receptors CD28 and MHC-I. The ability of viruses, like HIV-1, to hijack host cell machinery is key to their ability to replicate, as viruses cannot produce progeny virions without the host cell. Therefore, viruses have evolved ingenious ways to usurp host cellular pathways. Understanding how viruses take control of cellular pathways and the cellular machinery not only informs us about how viruses mediate their effects, but also about normal cellular function.

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Chapter 2 2 The downregulation of CD28 by HIV-1 Nef and Vpu 2.1 Introduction

Adaptive immune responses are primarily driven by B and T lymphocytes, which facilitate humoral and cell-mediated immunity (reviewed in [1]). Although a simplification, thymus derived lymphocytes, or T cells, can be classified as CD8+ CTLs or CD4+ T helper cells, and these cells play key roles during anti-viral responses. Naïve T cells require signaling from APCs to become competent effector cells. According to the two-signal model of T cell activation, APCs provide two signals to enable these cells to become productive effector cells. The first signal is established during MHC-restricted binding of the TCR: antigen complex, while signal 2 consists of co-stimulatory signaling (reviewed in [2, 3]). A lack of co-stimulatory signaling results in cells becoming unresponsive or anergic. Key co-stimulatory signaling is provided by binding of CD28, a transmembrane receptor expressed on the surface of T cells, to B7.1/B7.2 on the surface of APCs. Indeed, CD28 receptor ligation initiates downstream signaling, which in conjunction with TCR signaling, results in cell activation and proliferation essential for mounting an immune response [4, 5].

The importance of the TCR and co-stimulatory signaling to the development of an effective immune response makes them prime targets of intracellular pathogens. Indeed, many viruses have evolved the capabilities to intricately modulate these T cell activation cues to optimize their replication and persistence. For instance, lymphotropic viruses, including HIV-1, HTLV, and Epstein-Barr Virus inhibit T cell activation by downregulating components of the TCR and the TCR-associated kinases essential for TCR signaling [6- 11]. Moreover, viruses inhibit signaling downstream of co-stimulatory receptors, or alter the levels of cell surface co-stimulatory or inhibitory molecules [12-15]. Ultimately, virus- induced changes in T cell activation can result in immune evasion, enhanced viral replication, and increased viral persistence. The importance of viral alterations to immune cell activation are evidenced by the specific HIV-1 proteins that play key roles in T cell activation.

HIV-1 encodes four accessory proteins that lack any known enzymatic or structural functions: Vif, Vpr, Nef and Vpu (reviewed in [16-18]). Collectively, these viral proteins play critical roles in increasing infectivity, persistence and pathogenesis. While Nef and Vpu are arguably the most extensively studied accessory proteins, their roles in T cell activation are not fully understood. The ability of Nef and Vpu to downregulate various host cell receptors is a key function mediating their effects. Indeed, both Nef and Vpu downregulated multiple receptors in a screen to identify cell surface proteins that are altered by these viral proteins [19]. Specifically, Nef and Vpu facilitate degradation or intracellular sequestration of host receptors, including restriction factors and immune cell receptors, to thwart their cell surface expression and ultimately, their function [16]. By hijacking membrane trafficking proteins, such as the adaptor proteins AP-1 or AP-2, Nef and Vpu connect multiple cellular receptors to additional cellular trafficking machinery which alters the receptors’ subcellular localization. For example, Nef hijacks AP-1 to facilitate the endocytosis and sequestration of MHC-I molecules [17, 20-22], limiting recognition of infected cells by the immune system [23]. In parallel, the ability of Nef to hijack AP-2 results in the endocytosis and degradation of CD4, which limits superinfection and ADCC [24-31]. Similarly, Vpu hijacks BST-2 trafficking by associating with adaptor proteins to facilitate its sequestration and degradation [32-36]. Vpu also enables the degradation of CD4 by targeting newly synthesized CD4 to the ERAD pathway [37]. Interestingly, Nef expression leads to endocytosis of CD28 from the cell surface, in a manner dependent on both AP-1 and AP-2 [38, 39]. However, the fate of CD28 after Nef- mediated endocytosis remains poorly understood and the effects of Vpu on CD28 are unexplored.

In this chapter, we report that Nef and Vpu decrease cell surface and total cellular CD28 levels in infected CD4+ T cells. Moreover, these decreases in total CD28 are blocked by inhibiting the cellular degradation machinery, consistent with Nef or Vpu facilitating trafficking of CD28 to an acidic lysosomal compartment. Additionally, a mutant Nef protein associated with impaired binding to the vacuolar ATPase was compromised in its ability to reduce cell surface and total CD28 levels, while a non-phosphorylatable Vpu protein unable to recruit protein degradation machinery was impaired in its ability to reduce cell surface and total CD28 levels. Moreover, we sought to determine if knock-downs of

75 specific host cell trafficking machinery impaired the observed downregulation of CD28. Finally, we found that the ability of Vpu to downregulate CD28 is not limited to the lab adapted HIV-1 strain NL4.3 Vpu, and infection of cells with viruses encoding Nef or Vpu have differential effects on activation upon stimulation of CD3 and CD28.

2.2 Results

2.2.1 The HIV-1 accessory proteins Nef and Vpu downregulate cell surface and total CD28 protein levels

To investigate the effects of the HIV-1 accessory proteins Nef and Vpu on endogenous CD28 levels, CD4+ Sup-T1 T cells were infected with Gag-Pol truncated, vesicular stomatitis virus envelope glycoprotein (VSV-G) pseudotyped and eGFP expressing HIV- 1 NL4.3 viruses encoding both Nef and Vpu (NL4.3), Vpu alone (dNef), Nef alone (dVpu), or neither accessory protein (dVpu dNef). Infected Sup-T1 cells were analyzed by flow cytometry after staining for cell surface CD28 (Figure 2.1). Upon gating on live (Zombie RedTM-), infected (GFP+) single cells (Figure 2.2), significantly greater cell surface levels of CD28 were present on cells infected with viruses lacking Nef (dNef), Vpu (dVpu) or both Nef and Vpu (dVpu dNef), compared to cells infected with virus encoding both Nef and Vpu (NL4.3; Figure 2.1A, B, C). Accordingly, Western blot analysis confirmed the presence or absence of Nef and Vpu in infected Sup-T1 cells (Figure 2.1D). Moreover, we examined the ability of Nef and Vpu expressed from replication competent NL4.3 to downregulate cell surface CD28 in infected CD4+ T cells purified from peripheral blood mononuclear cells (PBMCs) (Figure 2.1E, F; Figure 2.3). In line with our observations in Sup-T1 cells, in the absence of Nef (dNef), Vpu (dVpu), or both Nef and Vpu (dVpu dNef) significantly greater cell surface levels of CD28 were present when compared to cells infected with NL4.3 (Figure 2.1E), indicating that Nef and Vpu independently downregulate cell surface CD28 in both primary CD4+ T cells and the immortal CD4+ T cell line, Sup-T1 cells.

Figure 2.1 HIV-1 Nef and Vpu downregulate cell surface CD28 protein levels

CD4+ Sup-T1 and primary CD4+ T cells were infected with VSV-G pseudotyped or replication competent NL4.3. Viruses encoded Nef and Vpu (NL4.3, red), lacked Nef (dNef,

77 blue) or Vpu (dVpu, orange), or lacked both Nef and Vpu (dVpu dNef, green). Infected cells were stained for CD28 and analyzed by flow cytometry. Live infected Sup-T1 cells were analyzed by gating on Zombie RedTM- and GFP+ cells and infected primary CD4+ T cells were analyzed by gating on p24+ cells. (A) Representative dot plots illustrating cell surface CD28 (APC) and infection (GFP+) of live (Zombie RedTM-) Sup-T1 cells. (B) Representative histograms illustrating cell surface levels of CD28 or the appropriate isotype control on Sup-T1 cells after gating on live (Zombie RedTM-) and infected (GFP+) cells. CD28 (APC) geometric mean fluorescence intensities (MFI) are indicated. (C) Summary of the relative mean (+/-SE) cell surface CD28 levels on infected (GFP+) Sup-T1 cells based on MFIs (n=17). (D) Western blot illustrating expression of Nef and Vpu in infected Sup- T1 cells. (E) Representative histograms illustrating cell surface levels of CD28 or the appropriate isotype control on uninfected (UI) or infected (p24+) CD4+ PBMCs. MFIs are indicated. (F) Summary of the relative mean (+/-SE) cell surface CD28 levels on infected CD4+ T cells based on MFIs obtained from infection of CD4+ T cells from two healthy donors (n ≥3). (UI: uninfected; SE: standard error; Mr: molecular weight; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; GFP: green fluorescent protein; SE: standard error; *p≤ 0.05; **p≤ 0.01; ****p≤ 0.0001).

Figure 2.2 Gating of live infected Sup-T1 cells infected with Gag-Pol truncated VSV-G pseudotyped NL4.3

To examine live infected cells, dead cells were excluded by gating on Zombie RedTM- cells, doublets were excluded and subsequently infected (GFP+) cells were gated on. In a representative experiment, 80.3% of cells were live (Zombie RedTM-) and 82.1% of live single cells were infected (GFP+). Gates were set based on FMO (fluorescence minus one) controls stained for all fluorophores except that which is being gated on.

Figure 2.3 Analysis of primary CD4+ T cells infected with replication competent virus

Primary CD4+ T cells were purified and infected with replication competent NL4.3 viruses and stained for cell surface CD28 and intracellular p24. (A) To examine the cell surface CD28 levels on infected cells, single cells were gated on followed by gating on the p24 (PE) and CD28 (APC) high populations. In a representative experiment, 5.22% of cells were p24 high. (B) Representative dot plots illustrating p24 (PE) and CD28 (APC) on cells that were either uninfected and unstained or stained with the appropriate APC isotype control or infected and stained with the appropriate PE isotype control or both anti-CD28 (APC) and anti-p24 (PE).

Given that HIV-1 Nef and Vpu are known to decrease cell surface levels of various receptors by mediating their degradation, we next sought to determine if these viral proteins alter total CD28 protein levels. To examine total CD28 protein levels and due to the unavailability of an antibody capable of detecting endogenous CD28 on Western blots, we engineered an expression vector to facilitate overexpression of CD28 in cells. This vector was designed to optimize protein expression by fusing the CD8 α signal peptide to the CD28 protein coding sequence [40], as has been done previously to overexpress other cell receptors [41, 42]. Moreover, this vector contains an epitope (FLAG) tag to enable detection (Figure 2.4). We subsequently examined the effects of Nef on total CD28-FLAG protein levels, as Nef had the largest effect on cell surface CD28, as measured by flow cytometry (Figure 2.1C). Thus, HeLa cells were co-transfected with expression vectors encoding CD28-FLAG and Nef-eGFP or eGFP alone and total protein was examined by Western blot. A decrease in CD28-FLAG levels in the presence of Nef was observed (Figure 2.5). To further investigate Nef-mediated decreases in total CD28 protein levels in T cells, CD4+ Sup-T1 cells were transduced with VSV-G pseudotyped HIV-1 NL4.3 engineered to encode epitope tagged CD28 and Nef (F2A CD28-FLAG Nef) or no Nef (F2A CD28-FLAG dNef; Figure 2.6). Forty-eight hours post transduction cells were lysed and total cellular CD28-FLAG was visualized by Western blot (Figure 2.7). As in transfected HeLa cells, decreases in CD28-FLAG in the presence of Nef were observed. Notably, Nef-mediated decreases in CD28-FLAG were observed inconsistently, which we hypothesize may be due to the overexpression of CD28-FLAG above level of endogenous CD28 protein, thus limiting our ability to detect viral protein mediated decreases.

Figure 2.4 Schematic of CD28-FLAG construct utilized to overexpress CD28 in cells

The CD8α signal peptide (blue) was cloned in frame with the coding sequence for the CD28 protein (residues 19-220 of CD28 protein), including the CD28 extracellular domain (orange; residues 153-179 of CD28 protein), transmembrane domain (red; residues 153-179 of CD28 protein) and cytoplasmic tail (green; residues 180-220 of CD28 protein) and was epitope (purple; DYKDDDDK) tagged on the C-terminus. Numbers denote residues in the CD28-FLAG construct.

Figure 2.5 Decreased CD28-FLAG protein levels are detected in the presence of Nef in HeLa cells

HeLa cells were transfected with plasmids encoding CD28-FLAG and Nef-eGFP or eGFP. Forty-eight hours post transfection, cells were lysed and lysate analyzed for the presence of CD28-FLAG (top), Nef-eGFP or eGFP (middle) and a loading control (actin, bottom) via Western blot. Indicated are relative intensities of the band corresponding to CD28-FLAG as determined using LI-COR Image Studio software (top). (eGFP: enhanced green fluorescent protein; Mr: molecular weight; NT: non-transfected)

Figure 2.6 HIV-1 lentiviral vector utilized to express epitope tagged CD28 in the presence or absence of Nef

Illustration of a linear map of the lentiviral vector utilized to overexpress epitope tagged CD28 in T cells. The vector was modified from previously described lentiviral vectors produced from modification of the HIV-1 laboratory strain NL4.3 [43]. The HIV-1 gag and pol genes are truncated to encode the foot-and-mouth disease virus 2A peptide (F2A) and the CD8α signal peptide fused to a FLAG-tagged CD28-encoding gene. The remaining pUC vector backbone is not shown.

Figure 2.7 CD28-FLAG protein levels are reduced in the presence of Nef in CD4+ Sup-T1 T cells

Sup-T1 T cells were transduced with a VSV-G pseudotyped lentivirus encoding CD28- FLAG and Nef (F2A CD28-FLAG Nef) or CD28-FLAG and no Nef (F2A CD28-FLAG dNef). Forty-eight hours post transduction cells were lysed and proteins were analyzed by Western blot. Shown is CD28-FLAG (top), Nef (middle) and actin (bottom). (Mr: molecular weight)

Due to the inconsistent nature of our results obtained when using overexpressed epitope tagged CD28, we next sought to examine the effects of Nef and Vpu on endogenous CD28 total protein levels within CD4+ T cells, a more biologically relevant system. Briefly, Sup- T1 cells or whole peripheral blood mononuclear cells (PBMCs) were infected with VSV- G pseudotyped HIV-1 NL4.3 viruses, as in Figure 2.1. Infected cells were stained for total CD28 levels by permeabilizing the cells prior to staining for CD28 and cells were subsequently analyzed by flow cytometry. Levels of CD28 were analyzed in Sup-T1 cells after gating on single live (Zombie NIRTM-) infected (GFP+) cells (Figure 2.2), while PBMCs were gated on CD4+ and infected (GFP+) cells. As with cell surface CD28, total CD28 levels were significantly higher in cells infected with viruses lacking Nef (dNef) or Vpu (dVpu), relative to cells infected with viruses encoding both viral proteins (NL4.3), in both Sup-T1 and primary CD4+ cells (Figure 2.8). Moreover, the highest total CD28 levels were observed in Sup-T1 cells infected with virus lacking functional nef and vpu genes (dVpu dNef; Figure 2.8C).

Figure 2.8 HIV-1 Nef and Vpu downregulate total CD28 protein levels

CD4+ Sup-T1 cells or peripheral blood mononuclear cells were infected with VSV-G pseudotyped NL4.3. Viruses encoded Nef and Vpu (NL4.3, red), lacked Nef (dNef, blue), lacked Vpu (dVpu, orange) or lacked both Nef and Vpu (dNef dVpu, green). Forty-eight hours post-infection, cells were permeabilized, stained for CD28 and analyzed by flow cytometry. (A) Representative dot plots illustrating total CD28 (APC)

87 and infection (GFP) levels of live (Zombie RedTM-) Sup-T1 cells. (B) Representative histograms illustrating total levels of CD28 (APC) after gating on live (Zombie RedTM-) infected (GFP+) cells. Geometric mean fluorescence intensities (MFI) are indicated. (C) Summary of mean (+/-SE) relative total CD28 based on MFI (n=12). (D) Representative dot plots illustrating purified CD4+ T cells infection (GFP) and total CD28 (APC) levels. (E) Summary of mean (+/-SE) total CD28 levels on infected (GFP+) CD4+ T cells (n=5). Data were obtained from infection of cells obtained from two healthy donors. (UI: uninfected; SE: standard error; *p≤ 0.05; **p≤ 0.01; ***p≤ 0.001; ****p≤ 0.0001; GFP: green fluorescent protein).

To alternatively confirm that the HIV-1 accessory proteins Nef and Vpu alter endogenous CD28, we infected Sup-T1 cells with VSV-G pseudotyped NL4.3 viruses encoding or lacking the viral proteins and examined CD28 localization using widefield microscopy (Figure 2.9). Specifically, due to the observed decreases in total CD28 protein levels (Figure 2.5; Figure 2.7; Figure 2.8), we stained cells for endogenous CD28 and lysosomal- associated membrane protein 1 (LAMP-1), a marker of the degradative lysosomal compartment. Interestingly, we observed that in the presence of Nef and Vpu, CD28 localized away from the cell surface (NL4.3; Figure 2.9A), inconsistent with the plasma membrane localization observed in uninfected cells (UI; Figure 2.9A). Moreover, upon elimination of the viral proteins we observed a rescue in CD28’s localization to the cell surface (dVpu dNef; Figure 2.9A). Upon quantification of CD28: LAMP-1 co-localization, we found that in the presence of both viral proteins (NL4.3) the co-localization of CD28 with LAMP-1 was significantly greater than in the absence of either (dNef, dVpu) or both (dVpu dNef) viral proteins (Figure 2.9B). This microscopy analysis confirms that Nef and Vpu downregulate endogenous CD28 and suggests this downregulation is mediated by transport of CD28 to the degradative lysosome.

Figure 2.9 The subcellular localization of CD28 is altered in the presence of Nef or Vpu

CD4+ Sup-T1 cells were infected with VSV-G pseudotyped NL4.3 viruses encoding or lacking Nef and/or Vpu. Infected cells were stained for CD28 and the lysosomal marker LAMP-1 and visualized by widefield microscopy. (A) Shown are representative infected (GFP+) cells (left) and a graphical representation of CD28 (blue) and LAMP-1 (red) fluorescence intensity relative to the maximum along the illustrated line (right). The scale bar indicates 10 m and the vertical lines labelled PM indicate where the illustrated line meets the plasma membrane (PM). Insets in the merged panel illustrate the GFP channel. (B) The percentage overlap between CD28 and LAMP-1 is calculated based on the mean (+/- SD) Manders’ overlap coefficients from at least 30 cells and 3 independent experiments. (UI: uninfected; LAMP-1: Lysosomal-associated membrane protein 1; SD: standard deviation; ****p≤ 0.0001).

2.2.2 Nef and Vpu-mediated effects on CD28 are dependent on the cellular degradation machinery

Cellular protein levels can be reduced via degradation by acidic hydrolases within acidified endosomal compartments [44]. Since we observed the co-localization of CD28 and LAMP- 1 in infected cells (Figure 2.9), we sought to determine if Nef- and Vpu-mediated reductions in total CD28 could be blocked by inhibition of endosomal acidification. To test this, we treated infected Sup-T1 cells with the base ammonium chloride to increase intra- vesicular pH [45]. We specifically measured total CD28 within Sup-T1 cells infected with pseudotyped NL4.3 and treated with ammonium chloride prior to staining. Interestingly, upon ammonium chloride treatment, total CD28 detected by flow cytometry increased relative to untreated cells (Figure 2.10). Moreover, the fold increase, or recovery, of total CD28 protein levels upon ammonium chloride treatment was significantly lower when cells were infected with viruses lacking either (dNef, dVpu) or both Nef and Vpu (dVpu dNef), compared to Nef- and Vpu-encoding virus (NL4.3; Figure 2.10A). As a control, we measured total CD4 within infected cells treated or not with ammonium chloride. Similar to CD28, the recovery of CD4 levels upon ammonium chloride treatment was significantly less in cells infected with virus lacking one or both of the accessory proteins (dNef, dVpu, dVpu dNef; Figure 2.11). Therefore, in the presence of the viral accessory proteins Nef and Vpu, a decrease in total CD28 occurs and this effect is most likely mitigated by inhibition of endosomal acidification.

To further confirm these findings, we used widefield microscopy to visualize CD28 within Sup-T1 cells infected with VSV-G pseudotyped NL4.3. Specifically, we examined the subcellular localization of CD28 within infected cells treated with the general endosome acidification inhibitor ammonium chloride or treated with Bafilomycin A1 or Concanamycin A, specific inhibitors of the endosome acidifying proton pump, the vacuolar ATPase [46, 47]. We observed that upon treatment with ammonium chloride, Bafilomycin A1 or Concanamycin A, the distribution of CD28 within VSV-G pseudotyped NL4.3 infected cells was altered relative to vehicle (DMSO) treated cells (Figure 2.10). Indeed, inhibitor treatment resulted in the increased localization of CD28 to the cell surface when compared to cells treated with vehicle. Moreover, quantification of the co-localization of CD28 and LAMP-1 in inhibitor treated cells indicated that upon inhibitor treatment CD28

92 co-localizes significantly less with the lysosomal marker LAMP-1 (Figure 2.10C). These results suggest that the ability of Nef and Vpu to downregulate CD28 is dependent on lysosomal acidification.

Figure 2.10 Inhibition of the degradation machinery increases CD28 protein levels in infected cells

CD4+ Sup-T1 cells were infected with VSV-G pseudotyped NL4.3 encoding or lacking Nef and/or Vpu. Infected cells were analyzed 48 hours post-infection after treatment for 24 hours with 40 mM ammonium chloride or treatment for five hours with 100 nM Bafilomycin A1 or 100 nM Concanamycin. Cells were stained for CD28 and the lysosomal marker LAMP-1 and analyzed by widefield microscopy or stained for CD28 and analyzed by flow cytometry. (A) Representative histograms illustrating total CD28 levels of infected cells treated with complete RPMI with (blue) or without (red) 40 mM ammonium chloride. Geometric mean fluorescence intensities (MFIs) are indicated. MFI of infected cells were determined after gating on live (Zombie RedTM-), infected (GFP+) cells and the relative fold increase (+/-SE) in total CD28 upon ammonium chloride (n≥5) treatment is illustrated (right). (B) Representative infected (GFP+) Sup-T1 cells subjected to treatment with vehicle (DMSO), ammonium chloride (NH4Cl), Concanamycin A (ConA) or Bafilomycin A1 (BafA) and visualized by widefield microscopy (left). The scale bar indicates 10 m and insets in merged panel illustrate GFP channel. A graphical representation of CD28 and LAMP-1 fluorescence intensity relative to max along the illustrated line is shown (right). The vertical lines labelled PM indicate where the illustrated line meets the plasma membrane (PM). (C) The percentage of overlap between CD28 and LAMP-1 is based on the mean (+/-SD) Manders’ overlap coefficient measured on at least 30 cells from 3 independent experiments (UI: uninfected; SD: standard deviation; LAMP-1: Lysosomal-associated membrane protein; ****p≤ 0.0001).

Figure 2.11 Ammonium chloride treatment increases total CD4 levels in infected cells

CD4+ Sup-T1 cells were infected with Gag-Pol truncated VSV-G pseudotyped NL4.3 encoding or lacking Nef and/or Vpu. Infected cells were treated with 40 mM ammonium chloride for twenty-four hours prior to staining for CD4 and analyzed by flow cytometry. (A) Representative histograms illustrating CD4 (APC) levels on live infected cells. Mean geometric fluorescence intensities (MFIs) are indicated. (B) MFIs of infected cells were determined after gating on live, infected (Zombie RedTM- and GFP+) cells and the relative fold increase (+/-SE) in total CD4 (n≥5) upon ammonium chloride treatment is illustrated. (SE: standard error; ****p≤ 0.0001).

2.2.3 Motifs ascribed to specific Nef: host cell protein interactions are critical for Nef-mediated CD28 downregulation

To further elucidate the mechanisms used by Nef to downregulate CD28, we sought to determine if this function is dependent on motifs within Nef that have been previously implicated in interacting with membrane trafficking regulators that act to downregulate other cell surface receptors. Accordingly, Sup-T1 cells were infected with isogenic Gag- Pol truncated VSV-G pseudotyped NL4.3 viruses that only differed at the following Nef motifs: the myristoylation site (G2; [48]), the methionine residue critical for Nef: AP-1:

MHC-I complex formation (M20; [21]), the dileucine motif implicated in adaptor protein- complex formation (LL165; [49]) and the diacidic motif implicated in vacuolar ATPase binding (DD175; [50]). Specifically, Sup-T1 cells were infected with pseudotyped NL4.3 encoding a non-functional Vpu protein and the various Nef mutants. Subsequently, live and infected single cells were analyzed (Figure 2.2). In addition, cells were infected with pseudotyped NL4.3 encoding a functional Vpu protein and the various Nef mutants (Figure

2.12; Figure 2.13). As expected, cells infected with virus encoding Nef G2A [48], which disrupts most known Nef functions, exhibited significantly higher cell surface levels of CD28 compared to cells infected with virus encoding wild-type Nef (Figure 2.13; Figure

2.14A, B). Interestingly, cells infected with viruses encoding the LL165AA and DD175GA Nef mutations also showed significantly higher cell surface levels of CD28, suggesting that the LL165 and DD175 motifs are critical for cell surface CD28 downregulation (Figure

2.13A; Figure 2.14A, B). Conversely, mutation of M20 (M20A), a residue essential for the downregulation of MHC-I via the interaction with AP-1 [21, 51], did not affect CD28 cell surface downregulation (Figure 2.13A; Figure 2.14A, B). Importantly, the dileucine

(LL165AA) and diacidic (DD175GA) motif mutations did not inhibit other Nef functions, as these mutated Nef proteins retained the ability to downregulate cell surface MHC-I (Figure 2.13C), demonstrating that the dileucine and diacidic motifs function in downregulating CD28.

In parallel, Sup-T1 cells were infected with the isogenic Gag-Pol truncated VSV-G pseudotyped NL4.3 viruses that only differed at various Nef motifs and total CD28 levels were measured by flow cytometry. The observed decrease in total CD28 in the presence of

Nef (Figure 2.14C) was abolished by mutation of the myristolation site (G2A) and the

diacidic motif (DD175GA), as cells infected with viruses encoding these mutated Nef proteins exhibited significantly higher total levels of CD28 compared to those infected with virus encoding wild-type Nef (Figure 2.13; Figure 2.14C). However, unlike cell surface CD28, the total CD28 levels did not differ significantly between cells infected with virus encoding wild-type Nef versus Nef LL165AA in cells infected with viruses also encoding Vpu (Figure 2.13B). Contrastingly, in cells infected with virus lacking Vpu and encoding the Nef LL165AA mutation, we observed significantly greater levels of cell surface CD28 (Figure 2.14C). Finally, expression of the mutated Nef proteins was confirmed by Western blot (Figure 2.14). Taken together, these findings implicate the Nef diacidic motif (DD175) in the downregulation of total and cell surface CD28.

Figure 2.12 Gating of Sup-T1 cells infected with VSV-G pseudotyped NL4.3 encoding various Nef mutants

To examine the population of interest, cells were gated on, followed by gating on infected (GFP+) cells. In a representative experiment 97.9% of cells were infected (GFP+).

Figure 2.13 Nef: host protein interaction motifs are critical for Nef-mediated CD28 downregulation in the presence of Vpu

Infected CD4+ Sup-T1 cells were stained for CD28 or MHC-I and analyzed by flow cytometry. Cells infected with VSV-G pseudotyped wild-type NL4.3 (NL4.3, red) or NL4.3 lacking Nef (dNef, blue) were used as controls. (A) Mean (+/- SE) relative cell surface CD28 of cells infected with NL4.3 encoding various mutations in the nef gene

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(n ≥5). (B) Mean (+/-SE) relative cell surface MHC-I on cells infected with NL4.3 encoding various nef mutations (n≥4). (C) Relative mean (+/- SE) total CD28 within live cells infected with NL4.3 encoding various nef mutations (n≥6). (SE: standard error; *p≤ 0.05; **p≤ 0.01; ****p≤ 0.0001)

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Figure 2.14 Nef: host cell protein interaction motifs are critical for Nef-mediated CD28 downregulation

Infected CD4+ Sup-T1 cells were stained for CD28 and analyzed by flow cytometry. Cells infected with VSV-G pseudotyped NL4.3 lacking Vpu (dVpu, orange) or Vpu and Nef (dVpu dNef, green) were used as controls. (A) Representative dot plots illustrating CD28 (APC) and infection (GFP) in live (Zombie RedTM-) cells. (B) Mean (+/-SE) relative cell surface CD28 MFIs of cells infected with NL4.3 lacking Vpu and encoding the indicated mutations in nef (n ≥4). (C) Relative mean (+/-SE) total CD28 within live cells infected with NL4.3 encoding various Nef mutations (n≥5). (D) Western blot illustrating expression of the mutated Nef proteins. (Mr: molecular weight; UI: uninfected; SE: standard error; *p≤ 0.05; ***p≤ 0.001; ****p≤ 0.0001)

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2.2.4 Motifs in Vpu implicated in interactions with specific host cellular proteins are critical for CD28 downregulation

Next, we sought to gain insight into the mechanism used by Vpu to downregulate CD28 by examining the ability of mutant Vpu proteins to modulate cell surface and total CD28 levels. Mutated motifs within the NL4.3 vpu gene which have been previously implicated in the ability of Vpu to downregulate CD4 [52-54] or the restriction factor BST-2 [55-57] were inserted into a NL4.3 viral backbone encoding eGFP, but lacking a functional nef gene (pNL4.3 dG/P eGFP dNef). Sup-T1 cells were infected with these VSV-G pseudotyped isogenic NL4.3 viruses, which only differ in the specific vpu gene mutations. Relative to cells infected with virus encoding wild-type Vpu (dNef), significantly greater levels of cell surface CD28 were present on cells infected with viruses encoding Vpu mutations at the adaptor protein interaction interface (LV64AA Vpu dNef) and serine residues (S52/56N Vpu dNef) which are phosphorylated by casein kinase II to facilitate recruitment of E3 ubiquitin ligase complex components (Figure 2.15; [37]). In contrast, relative to cells infected with virus encoding wild-type Vpu (dNef), cell surface CD28 levels were not significantly different on cells infected with virus encoding a mutation within a Vpu transmembrane domain residue implicated in downregulation of CD4 and

BST-2 (W22L Vpu dNef [53, 56]; Figure 2.15A, B). The functionality of the mutant Vpu proteins was tested by examining the effects of the mutations on Vpu-mediated CD4 downregulation in Sup-T1 cells (Figure 2.16). Indeed, upon infection with viruses encoding the W22L, S52/56N or LV64AA mutations, significantly greater cell surface levels of CD4 were present relative to cells infected with virus encoding wild-type Vpu (dNef;

Figure 2.16A), suggesting that the W22 motif is critical for CD4, but not cell surface CD28 downregulation.

In parallel, viruses encoding mutated Vpu proteins were also tested for their effects on the downregulation of total CD28. Total CD28 levels were not significantly different when comparing cells infected with virus encoding wild-type Vpu or the W22L or LV64AA mutations (Figure 2.15C). In contrast, there was significantly greater total CD28 in the absence of Vpu (dVpu dNef) and in the presence of Vpu encoding the S52/56N mutation

(S52/56N dNef) relative to virus encoding wild-type Vpu (dNef; Figure 2.15C). As an additional control, we demonstrated that cells infected with virus encoding the W22L,

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S52/56N or LV64AA mutations exhibited significantly greater total levels of CD4 relative to cells infected with virus encoding wild-type Vpu (Figure 2.16B). Expression of all mutated Vpu proteins was confirmed by Western blot (Figure 2.15D). As with our findings for cell surface CD28 downregulation, this suggests that the molecular determinants of CD28 downregulation by Vpu are distinct from those utilized to downregulate CD4.

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Figure 2.15 Motifs in Vpu are critical for downregulation of CD28

Infected CD4+ Sup-T1 cells were stained for CD28 and analyzed by flow cytometry. Mean geometric fluorescence intensities (MFI) of cells were determined after gating on live and infected (Zombie RedTM- and GFP+) cells. Cells infected with VSV-G pseudotyped NL4.3 lacking Nef (dNef, blue) and both Nef and Vpu (dNef dVpu, green) were used as controls. (A) Representative histograms illustrating cell surface CD28 on live (Zombie RedTM-) infected (GFP+) cells. MFIs are indicated. (B) Mean (+/-SE) relative cell surface CD28 on cells infected with viruses encoding mutations in vpu (n ≥9). (C) Relative mean (+/-SE) total CD28 within cells infected with viruses encoding the indicated vpu mutations (n≥7). (D) Western blot illustrating expression of mutated Vpu proteins. (Mr: molecular weight; UI: uninfected; SE: standard error; *p≤ 0.05; **p≤ 0.01; ****p≤ 0.0001)

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Figure 2.16 Specific motifs in Vpu are critical for downregulation of CD4

Infected CD4+ Sup-T1 cells were stained for CD4 and analyzed by flow cytometry. Mean geometric fluorescence intensities of cells (MFI) were determined after gating on live and infected (Zombie RedTM- and GFP+) cells. Cells infected with VSV-G pseudotyped NL4.3 lacking Nef (dNef, blue) and both Nef and Vpu (dNef dVpu, green) were used as controls. (A) Mean (+/-SE) relative cell surface CD4 on cells infected with NL4.3 encoding various mutations in vpu (n≥4). (B) Relative mean (+/- SE) total CD4 within cells infected with NL4.3 encoding various Vpu mutations (n≥5). (SE: standard error; *p≤ 0.05; **p≤ 0.01; ***p≤ 0.001; ****p≤ 0.0001)

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2.2.5 Implicating host cell proteins in Nef- and Vpu-mediated CD28 downregulation

We next sought to utilize the information gained through our mutational analysis to test for the requirements of putative host cell proteins in Nef- or Vpu-mediated downregulation of

CD28. Specifically, the Nef LL165 and DD175 motifs, which were critical for CD28 downregulation (Figure 2.13; Figure 2.14), and the Vpu LV64 motif, which was critical for cell surface CD28 downregulation (Figure 2.15), have been implicated in binding to the membrane trafficking adaptor protein AP-2 [24, 50, 57]. Thus, we sought to test for the requirement of AP-2 in CD28 downregulation. To test this, CD4+ Sup-T1 T cells were infected with lentiviral vectors encoding eGFP and shRNAs targeting the AP-2 α and μ subunits. The next day, these cells were infected with VSV-G pseudotyped NL4.3 encoding mStrawberry and Nef, Vpu, or Nef and Vpu. Forty-eight hours later cell surface CD28 was measured by flow cytometry. Live (Zombie NIRTM-) cells transduced with both the shRNA encoding viruses (eGFP+) and viral protein encoding viruses (mStrawberry+) were analyzed for cell surface CD28 levels by gating on double positive cells (eGFP+ mStrawberry+), and the relative downregulation mediated by Nef or Vpu was determined. Compared to cells transduced with virus encoding a non-specific (NS) shRNA, no significant effect of AP-2 α and μ knock-down on Nef or Vpu mediated CD28 downregulation was observed (Figure 2.17A). Knock-down of the AP-2 subunits was confirmed using Western blotting (Figure 2.17 B, C). These findings suggest that Nef- and Vpu-mediated decreases in cell surface levels of CD28 may be AP-2 independent, or alternatively may demonstrate that cells can compensate for the loss of the AP-2 α and μ subunits.

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Figure 2.17 Downregulation of cell surface CD28 by Nef and Vpu in the presence of shRNA targeting AP-2

Sup-T1 T cells were infected with lentiviruses encoding eGFP and non-specific (NS) or AP-2 α and μ subunit targeting shRNA. Twenty-four hours post transduction cells were infected with VSV-G pseudotyped NL4.3 encoding mStrawberry and encoding or lacking Nef and Vpu. Forty-eight hours later, cells were stained for cell surface CD28 and analyzed by flow cytometry. Cell surface CD28 levels on live (Zombie NIRTM-), dual infected (eGFP+ mStrawberry+) cells was determined. (A) Mean (+/-SD) relative levels of CD28 downregulation mediated by the various viral proteins in the presence or absence of Nef and/or Vpu (n=8). Downregulation was calculated by comparing cell surface levels of CD28 on cells expressing vs. lacking the viral protein of interest, and downregulation in the presence of the AP-2 α- and μ-targeting shRNAs was normalized to downregulation in the presence of non-specific (NS) control shRNA (set to 1). (B) Western blot illustrating AP-2 μ protein levels in cells transduced with virus encoding the knock-down (KD) shRNA or non-specific shRNA (NS). (C) Western blot illustrating AP-2 α protein levels in cells transduced with virus encoding the knock-down (KD) shRNA or non-specific shRNA (NS). (SD: standard deviation; AP-2: adaptor protein 2; NS: non-specific; KD: knock-down; GFP: green fluorescent protein; GAPDH: glyceraldehyde 3-phosphate dehydrogenase)

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In order to further confirm that knock-down of AP-2 does not affect Nef-mediated CD28 downregulation, we tested for the requirement of the membrane trafficking regulator sorting nexin 9 (SNX9), which functions in membrane trafficking by binding to AP-2 [58]. Moreover, in uninfected cells CD28 is removed from the cell surface in a SNX9 dependent manner [59]. To test for the requirement of SNX9 in Nef-mediated cell surface CD28 downregulation, CD4+ Sup-T1 cells were transduced with VSV-G pseudotyped NL4.3 which was modified to express a previously described non-functional dominant negative SNX9 protein lacking the PX and BAR domains [58], as well as Nef-eGFP or a non- functional mutant Nef G2A-eGFP. In parallel, cells were infected with viruses encoding wild-type SNX9 and Nef-eGFP or Nef G2A-eGFP. These vectors also lacked a functional vpu gene to ensure only Nef-mediated CD28 downregulation could occur. Forty-eight hours post transduction, cells were analyzed for cell surface CD28 levels using flow cytometry. The downregulation of CD28 in live, transduced (Zombie RedTM- and GFP+) cells was calculated based on mean fluorescence intensity of cells expressing Nef-eGFP versus Nef G2A-eGFP. Subsequently, cell surface CD28 downregulation was compared between cells transduced with virus encoding wild-type SNX9 (F2A SNX9 (WT)) or dominant negative SNX9 (F2A SNX9 (DN); Figure 2.18A). Expression of the SNX9 and Nef proteins of interest within transduced cells was confirmed by Western blotting (Figure 2.18B). No difference in Nef-mediated downregulation of cell surface CD28 was seen between cells expressing wild-type versus dominant negative SNX9 (Figure 2.18A), indicating that SNX9 may be dispensable for cell surface downregulation of CD28 by Nef. Alternatively, cells may compensate for the disruption in SNX9-mediated trafficking by utilizing alternate trafficking pathways.

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Figure 2.18 Nef-mediated cell surface downregulation of CD28 in the presence of overexpressed wild-type or dominant negative non-functional SNX9

Sup-T1 T cells were infected with VSV-G pseudotyped lentiviruses encoding wild-type or dominant negative SNX9 and Nef-eGFP or a non-functional Nef mutant (Nef G2A- eGFP). Forty-eight hours later, cells were stained for cell surface CD28 and analyzed by flow cytometry. (A) Mean (+/-SD) relative levels of CD28 downregulation mediated by Nef in live (Zombie RedTM-) and infected (eGFP+) cells. (n=8) (B) Western blot illustrating expression of wild type or dominant negative SNX9-FLAG and Nef-eGFP or Nef G2A-eGFP within transduced cells. (SD: standard deviation; SNX9: sorting nexin 9; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; eGFP: enhanced green fluorescent protein; WT: wild-type; DN: dominant negative; UI: uninfected)

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In addition to testing the requirement of SNX9 for the downregulation of cell surface CD28, we also sought to determine if SNX9 is required for Nef-mediated decreases in total CD28 protein levels. To test this, HeLa cell lines stably expressing SNX9-targeting or non- specific shRNA were generated. These cells were subsequently transfected with expression vectors encoding epitope tagged CD28 and Nef, and total protein was examined using Western blotting (Figure 2.19). Decreased CD28-FLAG was observed in cells expressing Nef and a non-specific shRNA, while this Nef-mediated decrease in CD28-FLAG was absent in cells expressing shRNA targeting SNX9 (Figure 2.19), indicating SNX9 may be required for Nef-mediated reductions in total CD28 protein levels. To test this in T cells, CD4+ Sup-T1 cells were infected with VSV-G pseudotyped lentiviruses encoding SNX9- targeting or non-specific shRNA. The next day, infected cells were transduced with a modified NL4.3 virus, as in Figure 2.7, that encodes CD28-FLAG and Nef (F2A CD28- FLAG Nef) or no Nef (F2A CD28-FLAG dNef; Figure 2.6). Forty-eight hours post transduction, cells were lysed and total CD28-FLAG was visualized by Western blot (Figure 2.20). In cells transduced with virus expressing a non-specific shRNA (NS), there was a reduction in CD28-FLAG detected in cells expressing Nef, compared to those lacking Nef. However, in cells transduced with lentivirus encoding a SNX9-targeting shRNA there was no difference in CD28-FLAG levels in the presence or absence of Nef (Figure 2.20). Overall, these findings suggest that the AP-2-binding membrane trafficking regulator SNX9 may be required for the ability of Nef to decrease total CD28 protein levels.

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Figure 2.19 HeLa cells with decreased SNX9 expression exhibit reductions in Nef- mediated decreases in total CD28-FLAG

HeLa cell lines were established that stably express shRNA targeting SNX9 (clone D1) or non-specific shRNA (clone NS1). These cells were transfected with vectors encoding CD28-FLAG and Nef-eGFP or eGFP alone. Forty-eight hours post transfection cells were lysed and proteins analyzed by Western blot. (A) Western blot illustrating CD28- FLAG and Nef-eGFP or eGFP expression in cells expressing SNX9-targeting (anti- SNX9; left) or non-specific (right) shRNA. (B) Western blot illustrating SNX9 protein levels in the stable cell lines utilized. Clone D1 was transduced with a virus encoding SNX9-targeting shRNA, clone NS1 was transduced with virus encoding a non-specific shRNA. (eGFP: enhanced green fluorescent protein; Mr: molecular weight; SNX9: sorting nexin 9)

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Figure 2.20 SNX9-targeting shRNA reduces Nef-mediated decreases in total CD28 in Sup-T1 cells

CD4+ Sup-T1 T cells were transduced with lentiviruses encoding shRNA that targets SNX9 or non-specific shRNA. The next day, cells were transduced with lentiviruses encoding CD28-FLAG and Nef (F2A CD28-FLAG Nef) or no Nef (F2A CD28-FLAG dNef). Forty-eight hours post infection cells were lysed and analyzed by Western blot. Shown are Western blots illustrating CD28-FLAG (top) protein levels in cells transduced with viruses encoding SNX9-targeting (left) or non-specific-targeting (right) shRNAs and Nef (middle) and actin (bottom) protein levels in these cells. (Mr: molecular weight)

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2.2.6 Patient-derived Vpu proteins mediate CD28 downregulation

Our findings suggest that HIV-1 NL4.3 can downregulate CD28 by both Nef and Vpu. However, as HIV-1 is genetically diverse, functions observed in the laboratory strain NL4.3 may not necessarily play a prominent role in the epidemic at large. We therefore wanted to determine if Vpu-mediated CD28 downregulation is a conserved function. Thus, we tested the ability of multiple group M Vpu proteins to downregulate cell surface and total CD28. To test this, the NL4.3 vpu gene from a gag-pol truncated NL4.3 virus lacking Nef (NL4.3 dG/P eGFP dNef) was replaced with vpu genes from a HIV-1 subtype B reference strain (B.US.86.JRFL), a subtype C reference strain (C.BR.92.92BR025; [60]), or a gene encoding a subtype C consensus protein (CC). Sup-T1 cells were infected with these VSV-G pseudotyped isogenic viruses which differed only in the encoded Vpu proteins, and cell surface and total CD28 levels were measured by flow cytometry. Cells infected with virus encoding a subtype C consensus protein or a subtype B reference strain (B.US.86.JRFL) Vpu protein did not significantly differ in cell surface levels of CD28 relative to cells infected with virus encoding NL4.3 Vpu (Figure 2.21A, B), suggesting that these additional Vpu proteins were capable of downregulating cell surface CD28. In contrast, cells infected with virus encoding a subtype C reference strain (C.BR.92.92BR025) vpu gene had significantly greater cell surface levels of CD28, as with virus lacking a functional vpu gene (dVpu dNef; Figure 2.21A, B). Moreover, staining for total cellular CD28 revealed that cells infected with virus encoding the B.US.86.JRFL and consensuses C vpu genes did not differ in total cellular CD28 levels relative to cells infected with virus encoding NL4.3 vpu, while the C.BR.92.92BR025 Vpu protein did not downregulate total CD28 (Figure 2.21C). To confirm that these findings were not unique to the Sup-T1 cell line, PBMCs were infected with the same pseudotyped NL4.3 viruses (Figure 2.21A) and cell surface CD28 levels on infected (GFP+) CD4+ cells was measured (Figure 2.1E, F, G; Figure 2.22). A similar trend to that observed in Sup-T1 cells was seen whereby the levels of CD28 were greater on cells infected with virus lacking the vpu gene than in cells infected with virus encoding the B.US.86.JRFL vpu gene. However, unlike in Sup-T1 cells, significantly more cell surface CD28 is present on cells infected with virus encoding CC Vpu than NL4.3 Vpu. Moreover, expression of these proteins in a heterologous system revealed equivalent protein levels for all Vpus except for Vpu from

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C.BR.92.92BR025, which is weakly expressed (Figure 2.21D). Overall, this suggests that Vpu-mediated cell surface and total CD28 downregulation is not limited to the laboratory adapted HIV-1 strain NL4.3.

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Figure 2.21 Non-NL4.3 Vpu proteins downregulate cell surface CD28

CD4+ Sup-T1 and peripheral blood mononuclear cells (PBMCs) were infected with VSV-G pseudotyped NL4.3 viruses lacking Nef and encoding wild-type Vpu (dNef,

117 blue), no Vpu (dNef dVpu, green) or three non-NL4.3 Vpu proteins (grey). Cells were analyzed by flow cytometry and mean geometric fluorescence intensities (MFI) of infected (GFP+) cells were determined. (A) Representative histograms illustrating cell surface CD28 on live infected Sup-T1 cells. MFIs are indicated. (B) Mean (+/-SE) relative cell surface CD28 on Sup-T1 cells infected with NL4.3 lacking a functional Nef protein (dNef), but encoding various Vpu proteins (n=5). (C) Mean (+/-SE) relative total cellular CD28 of Sup-T1 cells infected with NL4.3 encoding various Vpu proteins (n≥8). (D) Western blot illustrating expression of eGFP-tagged versions of the analyzed Vpu proteins from transfected HEK293T cells. (E) Representative dot plots illustrating GFP and cell surface CD28 (APC) on CD4+ cells. (F) Representative histograms illustrating cell surface CD28 on either uninfected (isotype control, uninfected) or CD4+ infected (GFP+) cells. MFIs are indicated. (G) Mean (+/-SE) relative cell surface CD28 on CD4+ cells infected with viruses encoding various vpu genes (n=8). Data were obtained from infection of PBMCs from two healthy donors. (NT: non-transfected; Mr: molecular weight; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; GFP: green fluorescent protein; SE: standard error; UI: uninfected; *p≤ 0.05; **p≤0.01; ****p≤ 0.0001).

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Figure 2.22 Gating of CD4+ peripheral blood mononuclear cells infected with VSV-G pseudotyped NL4.3

To examine the population of interest, lymphocytes were gated on, followed by gating on CD4+ (APC-Cy7) and infected (GFP+) positive cells. In a representative experiment 35.9% of single lymphocytes were CD4+ and 1.3% of these were infected (GFP+). Gates were set based on isotype stained (APC-Cy7) and uninfected controls.

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2.2.7 Nef and Vpu differentially alter responsiveness to CD28-mediated stimulation

Given that HIV-1 decreases cell surface levels of CD28 (Figure 2.1), a key immune cell stimulatory receptor, we postulated that decreased cell surface levels of CD28 on infected cells leads to a decreased ability of cells to become activated by CD28/CD3 stimulation. Thus, we wished to test what effects Nef and Vpu may have on activation in cells stimulated by CD28/CD3. We therefore infected primary CD4+ T cells with VSV-G pseudotyped NL4.3 encoding both Nef and Vpu (NL4.3), Nef alone (dVpu), Vpu alone (dNef) or lacking both Nef and Vpu (dVpu dNef). Infected cells were stimulated with soluble anti-CD28 and plate bound anti-CD3, and activation was determined by measuring intracytoplasmic production of IL-2, an event that occurs downstream from CD28 activation (Figure 2.23; [61]). Upon infection with virus encoding Vpu alone (dNef), the percentage of infected cells that produced IL-2 after stimulation was significantly lower than in cells infected with virus encoding Nef and Vpu (NL4.3; Figure 2.23B), suggesting that the presence of Nef leads to increased activation, consistent with Nef’s previously reported role in T-cell activation upon anti-CD3 and anti-CD28 treatment of Jurkat cells [62]. In contrast, in primary cells infected with a virus lacking Vpu (dVpu), the proportion of IL-2 positive infected cells were significantly increased, suggesting that Vpu decreases the anti-CD3/CD28 dependent cell activation. Overall, this suggests that the Nef and Vpu, which influence cell surface and total CD28 levels, differentially regulate the responsiveness to CD28 stimulation.

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Figure 2.23 Infected cell response to CD28-stimulation changes in the absence of Nef or Vpu

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Purified CD4+ T cells were infected with VSV-G pseudotyped NL4.3 encoding Nef and Vpu (NL4.3, red), lacking Nef (dNef, blue) or Vpu (dVpu, orange), or lacking both Nef and Vpu (dVpu dNef, green). Twenty-four hours post-infection cells were activated with anti-CD3/anti-CD28 for 24 hours. Cells were then incubated with Brefeldin A for 12 hours and stained for intracellular IL-2 prior to analysis by flow cytometry. (A) Representative dot plots illustrating intracellular IL-2 (APC) and infection (eGFP) levels. Quadrants were selected based on the IL-2 isotype and uninfected controls. The percentage of infected cells that are IL-2 positive is indicated (red). The percentage of cells making up the uninfected and IL-2 negative population (Q4) are as follows: uninfected unstimulated: 99.8%, isotype control: 99.7%, uninfected stimulated: 92.8%, NL4.3: 91.3, dNef: 95.5%, dVpu: 90.1, dVpu dNef: 89.3%. (B) Mean (+/-SE) percentage of infected cells that are IL-2 positive (n≥6). The means were obtained by analysis of infected cells from two healthy donors. (SE: standard error; *p≤ 0.05)

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2.3 Discussion

In this chapter, we describe the ability of both HIV-1 Nef and Vpu to decrease cell surface and total levels of CD28 in CD4+ T cells. Specifically, we have shown cell surface CD28 downregulation with VSV-G pseudotyped NL4.3 in Sup-T1 cells and primary cells, as well as downregulation in primary CD4+ T cells infected with replication competent NL4.3 (Figure 2.1; Figure 2.21). These results are consistent with observations made in cell lines transiently transfected with Nef and/or Vpu expression vectors. Downregulation of endogenous CD28 was previously illustrated in Jurkat cells transiently expressing Nef [39]. Moreover, Haller et al. completed a large screen for receptors downregulated by Nef and Vpu through transient expression of the viral proteins in A3.01 T lymphocytes and observed downregulation of CD28 by both HIV-1 Nef and Vpu [19]. Interestingly, the latter study demonstrated varying degrees of downregulation by HIV-1 Nef and Vpu, as well as SIVmac239 Nef, with respect to the multiple tested receptors [19]. Accordingly, we observed that the effects of Nef and Vpu on CD28 are subtler than the effects of Nef and Vpu on other receptors, namely MHC-I and CD4 (Figure 2.13; Figure 2.16), but nevertheless in the presence of Nef and Vpu this receptor traffics to a degradative lysosomal compartment and decreased total CD28 protein levels are observed (Figure 2.8; Figure 2.9; Figure 2.10). Moreover, it has been shown that there are varying degrees of CD28 downregulation by various Nef proteins, with HIV-2 and certain SIV derived Nef proteins exhibiting more efficient downregulation than HIV-1 Nef proteins [19, 63]. However, this does not limit the potential physiological relevance of CD28 downregulation by HIV-1 and our current findings demonstrate mechanistic details of Nef and Vpu- mediated CD28 downregulation.

Our results demonstrate that slight increases in cell surface CD28 levels can be observed in cells infected with viruses lacking Nef and Vpu (dVpu dNef) in comparison to uninfected cells (Figure 2.1B, E). This suggests that HIV-1 proteins other than Nef and Vpu may also be contributing to changes in cell surface CD28 levels upon infection. The latter effects may be due to events independent of membrane trafficking and could be the consequence of changes at the level of CD28 transcription. Notably, CD28 expression

123 levels may be affected at the transcriptional level via the HIV-1 tat protein, as it interacts with the Sp1 transcription factor which positively affects CD28 transcription [64, 65].

Given that Nef and Vpu collaborate to mediate the downregulation and degradation of cellular receptors, specifically CD4 [27, 66-68], we hypothesized that the observed decreases in total CD28 levels in the presence of Nef and Vpu were due to CD28 degradation. This is supported by our observation that CD28 co-localizes with the lysosome marker LAMP-1 in the presence of the viral proteins (Figure 2.9). Moreover, we found that we could limit Nef- and Vpu-mediated decreases in total CD28 and CD4 protein levels upon treatment with ammonium chloride (Figure 2.10; Figure 2.11). The effects of ammonium chloride on CD4 downregulation are of interest given the previously reported Vpu-dependent proteasomal degradation of CD4 [69]. In our experiments, ammonium chloride may be playing a non-specific role by impacting cellular physiology by affecting general cell acidification. Importantly, we have used the specific endosomal acidification inhibitors Bafilomycin A1 and Concanamycin A to provide a more unambiguous mechanistic explanation of Nef- and Vpu-dependent effects on CD28 (Figure 2.10), thereby bypassing the more general effects of using ammonium chloride. The specific inhibition of the vacuolar ATPase with these molecules limits the co-localization of CD28 with LAMP-1 in Nef and Vpu-expressing cells (Figure 2.10).

We also report how CD28 depletion involves the distinct ability of Nef and Vpu to interact with host cellular proteins. Indeed, cell surface CD28 downregulation was inhibited by mutation of Nef LL165 and DD175, while maintaining the ability to downregulate MHC-I

(Figure 2.13; Figure 2.14). The findings that Nef LL165 and DD175 are critical for cell surface CD28 downregulation is in accordance with previously published reports [39, 70], and suggests that the Nef LL165 and DD175 motif binding proteins AP-2 [49] and the vacuolar ATPase [50] may be critical for cell surface downregulation by Nef. Interestingly, mutation of the G2, LL165 and DD175 motifs abolished Nef-dependent decreases in total levels of cellular CD28 in the absence of Vpu (Figure 2.14), while mutation of the LL165 motif did not significantly alter total CD28 levels in the presence of Vpu (Figure 2.13). This may be due to Vpu’s ability to compensate for specific Nef mutations. Our observed increases in total cellular CD28 when the ability of Nef to interact with the vacuolar

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ATPase is inhibited further supports the idea that Nef mediates degradation of CD28

(DD175GA; Fig. 5C). The Nef DD175 motif is also critical for Nef-mediated lysosomal degradation of the co-inhibitory receptor CTLA-4 [71], which interestingly, competes with CD28 for binding the same receptors, B7.1/B7.2 [72]. Overall, this suggests that as with cell surface downregulation, the downregulation of total CD28 levels is dependent on specific Nef: host cell protein interaction motifs.

We have also provided mechanistic details on how Vpu modulates CD28 expression and localization within HIV-1 infected cells. Intriguingly, the mutations exhibited different effects on CD28 and CD4 downregulation (Figure 2.15; Figure 2.16), indicating that CD28 downregulation by Vpu may have evolved independently. Specifically, mutation of the

Vpu W22, S52/56 and LV64 motifs inhibited the ability of Vpu to downregulate cell surface CD4 (Figure 2.16), in agreement with previous reports [52-54]. In contrast, mutation of the highly conserved W22 did not inhibit cell surface downregulation of CD28 (Figure 2.15A, B), suggesting that this residue is dispensable for CD28 downregulation despite being implicated in the downregulation of CD4 and BST-2 [53, 56, 73, 74].

Similarly to CD4 downregulation, mutation of Vpu S52/S56 and LV64 inhibited cell surface downregulation of CD28 (Figure 2.15A, B). Interestingly, these Vpu motifs play distinct roles with AP-1 and AP-2, in addition to contributing to mechanisms that promote the degradation of cellular proteins. Indeed, the non-phosphorylatable mutants of Vpu (Vpu

S52/56N) do not recruit AP-1 and AP-2 and subsequently fail to downregulate BST-2 [36], whereas Vpu LV64 promotes the degradation of BST-2 via phagocytosis [75]. Thus, the ability of Vpu to downregulate CD28 from the cell surface may require AP-1 or AP-2 binding to the LV64 motif in a Vpu S52/S56 dependent manner to exclude CD28 from the cell surface post-endocytosis or prior to anterograde transport.

Interestingly, only the S52/56N mutations in Vpu inhibited Vpu-mediated decreases in total

CD28, whereas the W22, S52/56 and LV64 motifs were all necessary to decreasing total CD4

(Figure 2.15C; Figure 2.16). The adaptor protein-binding motif in Vpu (LV64) was critical for cell surface downregulation, but dispensable for decreasing total CD28 (Figure 2.15B, C). This motif may be unnecessary for reducing total CD28 levels as additional host cell

125 binding factors, either known or currently unidentified, may compensate for mutation of the LV64 motif. Overall, the mechanism of Vpu-mediated CD28 cell surface downregulation is discrete from that which mediates total decreases in cellular CD28, and this mechanism is distinct from Vpu-mediated downregulation of total CD4.

In addition to utilizing mutations in host cell binding motifs to shed light on the mechanisms of Nef- and Vpu-mediated CD28 downregulation, we employed shRNA knock-downs to potentially implicate specific host cellular proteins in CD28 downregulation. Interestingly, despite the fact that mutation of the Nef and Vpu AP-2 binding sites abolished the ability of these proteins to downregulate cell surface CD28 (Figure 2.13; Figure 2.14; Figure 2.15), shRNA depletion of the AP-2 α and μ subunits in CD4+ Sup-T1 cells did not alter the ability of Nef or Vpu to downregulate surface CD28 relative to cells expressing a non-specific control shRNA (Figure 2.17). The incomplete depletion of these AP-2 subunits may be insufficient to completely abolish the ability of AP-2 to mediate trafficking. Alternatively, the dileucine binding motifs in Nef and Vpu may be facilitating hijacking of AP-1, rather than AP-2, to downregulate CD28. The dispensability of AP-2 for cell surface CD28 downregulation is supported by our finding that a dominant negative SNX9 protein, which inhibits AP-2 dependent trafficking events [58], did not inhibit Nef-mediated cell surface CD28 downregulation (Figure 2.18). However, despite our findings that suggest AP-2 and SNX9 may be dispensable for cell surface CD28 downregulation, it is possible that upon AP-2 depletion or inhibition of SNX9 trafficking pathways additional membrane trafficking proteins compensate for these losses to facilitate CD28 downregulation. This is supported by the redundant functions observed for SNX9 and the related SNX18 protein [76, 77]. Moreover, shRNA-mediated knock-down of SNX9 inhibited Nef-mediated decreases in total protein levels of overexpressed epitope tagged CD28 in HeLa and Sup-T1 cells (Figure 2.19; Figure 2.20). This discrepancy between the effects of SNX9 on Nef-mediated cell surface and total protein downregulation may be due to differences that occur as a result of overexpressing CD28 versus examining endogenous CD28. Indeed, our observations made using overexpressed CD28 were often much less reproducible than our findings with endogenous CD28. Alternatively, SNX9, and perhaps AP-2, may be dispensable for mediating

126 decreases in cell surface CD28, but are required for degradation of CD28, as CD28 degradation may occur independently of cell surface downregulation.

We demonstrated that the ability of Vpu to downregulate cell surface and total CD28 is not specific to the laboratory adapted NL4.3 strain Vpu protein. Specifically, a subtype C consensus Vpu protein and a subtype B reference strain (B.US.86.JRFL) protein downregulated cell surface and total CD28 in infected Sup-T1 cells (Figure 2.21). Contrastingly, the Vpu protein derived from a subtype C reference strain (C.BR.92.92BR025) did not downregulate CD28 (Figure 2.21), which may be attributable in part to decreased expression (Figure 2.21D). Interestingly, in a separate study we found that this subtype C reference strain encodes a non-functional Nef protein and has previously been shown to have lower fitness when compared to other well described subtype reference strains [78, 79]. Moreover, we found that in infected CD4+ PBMCs both the NL4.3 and B.US.86.JRFL derived Vpu proteins were capable of downregulating cell surface CD28 (Figure 2.21E, F, G). However, unlike in Sup-T1 cells, PBMCs infected with virus encoding the consensus C Vpu protein did not exhibit downregulation (Figure 2.21G). The observed difference between Sup-T1 cells and CD4+ PBMCs may be due to intrinsic differences between Sup-T1 and primary cells, as well as potential differences in the expression pattern of the various Vpu proteins in different cell types. Interestingly, upon examination of the amino acid sequences of these various Vpu proteins, we observed that they exhibit conservation at the S52/56 motif we demonstrated to be critical for NL4.3 Vpu- mediated CD28 downregulation (Figure 2.15; Figure 2.24). However, the B.JFRL, CC and

C.BR92025 Vpu proteins all lack the LV64 motif found in the NL4.3 Vpu protein, which was critical for NL4.3 Vpu cell surface CD28 downregulation (Figure 2.15B; Figure 2.24). Therefore, perhaps non-NL4.3 Vpu proteins contain unique residues which influence CD28 downregulation ability. Nonetheless, the ability of distinct Vpu proteins to downregulate cell surface and total CD28 suggests that this function is conserved to some extent, despite high HIV-1 diversity [80].

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Figure 2.24 Protein sequence alignment of the Vpu proteins which were tested for their ability to downregulate CD28

The protein sequences of the NL4.3, B.JFRL, CC and C.BR92025 Vpu proteins were aligned using Clustal Omega [81]. The residues in NL4.3 Vpu that were mutated and tested for their effects on CD28 downregulation are indicated.

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HIV-1 has evolved multiple means of downregulating CD28, which are in part genetically separable from CD4 and MHC-I downregulation, implying that CD28 downregulation is critical during infection. Moreover, we hypothesize that the function of CD28 downregulation is relevant during infection and that CD28 downregulation by Nef and Vpu may alter cell activation through CD28 receptor stimulation. Indeed, we observed that in primary CD4+ T cells infected with pseudotyped virus encoding Vpu alone (dNef), a significantly smaller proportion of infected cells secreted IL-2 upon CD3/CD28 stimulation, relative to cells infected with virus encoding Nef and Vpu (NL4.3; Figure 2.23B). In contrast, cells infected with virus lacking Vpu (dVpu) displayed an increase in cell activation (Figure 2.23), indicating that Nef alone increases CD28 stimulation responsiveness.

The observed differences in cell activation in the presence or absence of Nef and Vpu may be partially attributable to other reported functions of these viral proteins. Namely, Nef alters the subcellular localization of the T cell receptor and immunological synapse associated kinase Lck, leading to a physical separation of the T cell receptor from the immunological synapse [6, 82]. Nef also binds and activates the serine/threonine kinase PAK2, which induces T cell activation [62]. Nef thus alters the activation status of cells, perhaps inducing the correct balance between activating and inhibitory signaling, thereby enabling optimal viral replication without inducing anergy or activation induced cell death [83]. However, when compared to Nef, little is known regarding how Vpu alters T cell activation. Nonetheless, Vpu may interfere with T cell activation and IL-2 production through its ability to inhibit NF-κB [84]. Ultimately, the evolution of CD28 downregulation may be one component of the multitude of alterations within infected cells that act to attain optimal levels of cell activation.

The role or function of CD28 downregulation in vivo remains elusive, however we speculate it may play a role in cell activation. CD28 is critical for T cell activation, providing co-stimulatory signaling necessary for activation upon binding to CD80/CD86 [4, 5] and in the absence of CD28, T cell receptor ligation can lead to an unresponsive, anergic state [85]. The importance of CD28 in vivo has been demonstrated through the use of anti-CD28 therapies to induce immunosuppression following transplantation, in patients

129 with autoimmune diseases (reviewed in [86]) and during cancer immunotherapies via CD28 activation [87]. In addition, reductions in IL-2 levels, as observed in the presence of Vpu (Figure 2.23), may lead to impairments in T cell survival, proliferation and function (reviewed in [88]). Interestingly, decreases in IL-2 levels are observed in HIV infected individuals [89, 90], and clinical trials have examined the benefit of administering recombinant IL-2 in combination with anti-retroviral therapy [91]. IL-2 has also been associated with reactivation of latently infected cells [92, 93]. Therefore, it is conceivable that CD28 downregulation alters responsiveness and activation of infected T cells.

Alterations in the activation status of infected cells, which may in part be achieved through CD28 downregulation, could have effects in vivo during HIV-1 infection. Upon HIV-1 infection, a transcriptionally silent latent reservoir of cells is formed, which currently present the largest obstacle for achieving a HIV-1 cure [94]. A decrease in cell activation as a result of alterations in cell surface CD28 levels may allow an infected cell to enter a transcriptionally silent, latent state. Indeed, CD28 activation is capable of inducing HIV-1 transcription and replication, even in the absence of TCR activation [95]. Moreover, viral microRNA-mediated silencing of genes that contribute to cellular activation, which may include CD28 [96], have been suggested to be important for the development of latency [97]. Furthermore, CD28 activation is utilized to reactivate latent cells, as mTOR, a kinase activated downstream of CD28 signaling [98], was identified as a critical controller of HIV-1 latency [99].

Overall, we have demonstrated that the HIV-1 Nef and Vpu accessory proteins downregulate CD28 from the cell surface and at the cellular level. This effect is observed with more than just the laboratory adapted NL4.3 strain, indicating this function is conserved. We propose that the decreases in total cellular CD28 may be potentiated by lysosomal degradation of CD28. Moreover, our analysis of Nef and Vpu mutants suggest that the Nef: vacuolar ATPase interaction and phosphorylation of Vpu S52/56 are both critical for downregulation of cell surface and total protein levels of this key co-stimulatory molecule. Finally, the presence or absence of Nef and Vpu modulates the ability of cells to respond to CD28-mediated stimulation.

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2.4 Materials and Methods

2.4.1 DNA constructs

2.4.1.1 Expression vectors

To facilitate efficient overexpression of epitope tagged CD28, the human CD28 gene was amplified out of the MegaMan Human Transcriptome Library (Agilent Technologies, Santa Clara, CA) and subsequently amplified with a forward primer containing the CD8α signal peptide and a reverse primer encoding the FLAG epitope tag. The CD8α signal peptide-CD28-FLAG encoding DNA was subsequently sub-cloned into the peGFP-N1 expression vector (Clontech, Mountain View, CA) in which eGFP was removed by restriction digestion. For expression of Nef-eGFP and eGFP, the NL4.3 nef gene was subcloned into the peGFP-N1 expression vector or this vector was left unmodified. To test the expression of the non-NL4.3 Vpu proteins the Vpu encoding fragments were PCR amplified and sub-cloned into the peGFP-N1 expression vector (Clontech).

2.4.1.2 Lentiviral vectors

Viral infection plasmids used for VSV-G pseudotyped lentivirus production were engineered from the previously described pNL4.3 dG/P eGFP or pNL4.3 dG/P eGFP dNef backbones [100, 101]. To make viral constructs lacking Vpu, HIV-1NL4-3 vpu/nef/UD Deletion Mutant (p230-11; NIH-AIDS reagents catalog number 2535) was digested with EcoRI and BamHI and the fragment encoding the vpu gene was sub-cloned into pNL4.3 dG/P eGFP or pNL4.3 dG/P eGFP dNef. NL4.3 based lentiviral vectors encoding mStrawberry, CD28-FLAG and SNX9 were produced through insertion into a previously described vector encoding truncated gag and pol genes and the foot and mouth disease 2A (F2A) site [43]. Specifically, to make a viral vector for overexpression of CD28-FLAG, CD8α-CD28-FLAG from the aforementioned expression vector was PCR amplified and sub-cloned into the F2A-containing lentiviral vectors of interest. For the expression of wild-type or dominant negative SNX9, the full SNX9 gene or only residues 2 through 182, respectively, were PCR amplified from a SNX9/pGEX-KG vector obtained from R. Prekeris (University of Colorado, Aurora, CO) with a forward primer encoding the FLAG epitope.

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Mutations in the NL4.3 nef gene were produced by site directed mutagenesis within a pN1 expression vector encoding NL4.3 Nef. Mutagenesis primers were created with the Agilent Technologies QuickChange Primer Design program (Agilent Technologies). The nef genes containing mutations were subsequently PCR amplified with primers encoding XmaI and NotI cut sites, and were inserted into a previously described vector encoding XmaI and NotI cut sites flanking the nef gene [43]. Mutations in the NL4.3 vpu gene were produced by site directed mutagenesis in a pN1 Vpu-FLAG expression vector. Vpu genes encoding mutations were subsequently PCR amplified and inserted into pRECnfl HIV-1 dVpu/URA3 (obtained from Eric Arts, University of Western Ontario), using a previously described yeast recombination system [102]. The vpu-encoding fragment was then sub- cloned into a NL4.3 backbone lacking a functional nef gene (pNL4.3 dG/P eGFP dNef) using the EcoRI and BamHI restriction sites. Vpu genes derived from a subtype C reference strain (C.BR.92.92BR025), subtype B reference strain (B.US.86.JRFL) or a consensus C protein synthesized using Invitrogen GeneArt Synthesis (Thermo Fisher Scientific, Mississauga, ON) were inserted into the pRECnfl HIV-1 dVpu/URA3 vector followed by sub-cloning into pNL4.3 dG/P eGFP dNef, as described above.

Viral infection plasmids utilized to make replication competent NL4.3 were engineered from the previously described pNL4.3 vector [101]. A Nef deficient plasmid (pNL4.3 dNef) was obtained from Gary Thomas (University of Pittsburgh) and Vpu (dVpu) and Nef and Vpu (dVpu dNef) deficient plasmids were produced by subcloning the vpu encoding portion of HIV-1NL4-3 vpu/nef/UD Deletion Mutant into pNL4.3 or pNL4.3 dNef, as described above.

2.4.1.3 shRNA encoding vectors

The shRNA encoding lentiviral vectors encoding a non-specific (catalog number TR30021) or SNX9 (catalog number TL307006D) targeting shRNA were obtained from OriGene (OriGene Technologies, Inc., Rockville, MD). The lentiviral vectors encoding AP-2 targeting shRNAs were produced as previously described [83].

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2.4.2 Cell culture

HEK293T (American Type Culture Collection (ATCC)), HeLa (ATCC) and U87 CD4+ CXCR4+ (National Institutes of Health-AIDS Research and Reference Reagent program; catalog number 4036) cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 4 mM L-glutamine, 4500 mg/L glucose and sodium pyruvate (HyClone, Logan, UT) and supplemented with 1% Penicillin-Streptomycin (Hyclone) and 10% fetal bovine serum (FBS; Wisent, St-Bruno, QC). Sup-T1 cells were maintained in Roswell Park Memorial Institute media (RPMI) 1640 media with L-glutamine supplemented with 100 g/ml penicillin-streptomycin, 1% sodium pyruvate, 1% non- essential amino acids, 2 mM L-glutamine (Hyclone) and 10% FBS. All cell lines were grown at 37°C in the presence of 5% CO2 and sub-cultured in accordance with supplier’s recommendations.

Primary peripheral blood mononuclear cells were isolated from four healthy donors by density centrifugation using Histopaque (Sigma-Aldrich, Oakville, ON) and cryopreserved. Upon revival, cells were maintained in RPMI with L-glutamine supplemented with 100 μg/ml penicillin-streptomycin, 1% sodium pyruvate, 1% non- essential amino acids, 2 mM L-glutamine (Hyclone) and 10% FBS at 37°C and 5% CO2 in the presence or absence of IL-2 and phytohemagglutinin (PHA) stimulation, as indicated. CD4+ T cells were purified from PBMCs using the MojoSortTM human CD4 T cell isolation kit (BioLegend, San Diego, CA) and Magnetic-activated cell sorting cell separation columns (Miltenyi Biotec, Auburn, CA), according to the manufacturer’s protocol.

For ammonium chloride treatment, cells were pelleted and culture medium was replaced with complete RPMI containing or lacking 40 mM ammonium chloride (Sigma-Aldrich) in phosphate buffered saline (PBS) twenty-four hours prior to analysis. For vacuolar ATPase inhibitor treatment, cells were pelleted and culture medium was replaced with complete RPMI containing vehicle, 100 nM Concanamycin A (Santa Cruz Biotechnology, Dallas, TX) or 100 nM Bafilomycin A1 (Sigma-Aldrich) in DMSO 5 hours prior to analysis.

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HeLa cells stably expressing shRNA with a non-specific target or targeting SNX9 were produced by transduction with shRNA-encoding lentiviral vectors. Briefly, HeLa cells were transduced with VSV-G pseudotyped lentivirus produced as described below with pGFP-C-shLenti plasmid encoding a scrambled shRNA (TR30021, Origene) or a SNX9 targeting shRNA (TL307006D, Origene). Transduced cells were selected for using 0.8 ug/mL puromycin and clones were selected. Cells were subsequently maintained in DMEM containing 0.4 ug/mL puromycin.

2.4.3 Transfections and Infections

2.4.3.1 Transfections

To examining CD28 expression levels in HeLa cells in the presence or absence of Nef, HeLa cells were transfected with the appropriate plasmids using PolyJet (FroggaBio, North York, ON), as per the manufacturer’s protocol.

2.4.3.2 Lentivirus production

For VSV-G pseudotyped lentivirus production, HEK293T cells were triply transfected with the lentiviral vector of interest, the VSV-G envelope-encoding pMD2.G plasmid (Addgene; catalog number 12259) and pCMV-DR8.2 (Addgene; catalog number 12263) using PolyJet (FroggaBio) as per the manufacturer’s protocol. Forty-eight hours post transfection, lentivirus was harvested via cell culture supernatant clarification by spinning at 1500 x g, followed by 20 m filtration. For production of NL4.3 provirus, viral vectors were transfected into HEK293T cells using PolyJet. Forty-eight hours post-transfection, cell supernatant was applied to U87 CD4+ CXCR4+ cells to propagate the virus. Cell supernatant was harvested four to six days post infection as described above. Viruses were stored in 20% FBS at -80C.

2.4.3.3 Lentivirus transduction

For infection of Sup-T1 cells with VSV-G pseudotyped NL4.3 viruses to examine effects on endogenous CD28, 8 x 105 Sup-T1 cells were pelleted and re-suspended in the appropriate volume of pseudovirus in 20% FBS, 8 g/mL polybrene and brought to one mL with 10% complete RPMI. Forty-eight hours post infection, cells were analyzed via

134 flow cytometry, microscopy or Western blotting. For infection of peripheral blood mononuclear cells with VSV-G pseudotyped viruses, cells were cultured for three days in 10 ng/mL IL-2 (PeproTech, Rocky Hill, NJ) and 5 μg/mL PHA (Sigma-Aldrich). Cells were then pelleted and re-suspended in the appropriate volume of pseudovirus containing 8 g/mL polybrene. Cells were subsequently spinoculated for four hours at 2880 x g at room temperature, re-suspended in fresh RPMI and incubated for two days prior to analysis. For infection of primary CD4+ T cells with replication competent NL4.3 viruses, cells were cultured for three days in 10 ng/mL IL-2 and 5 g/mL PHA-L. CD4+ T cells were then purified as described above and re-suspended in the appropriate volume of virus in 20% FBS and 8 g/mL polybrene. Subsequently, cells were spinoculated for four hours at 2880 x g at room temperature, re-suspended in fresh complete RPMI containing 5 ng/mL IL-2 and 5 g/mL PHA-L. Two days post-infection, cells were pelleted and re-suspended in fresh media containing PHA and IL-2 and four days post-infection cells were fixed and stained. For infection of purified CD4+ T cells with VSV-G pseudotyped virus, cells were cultured for three days in 10 ng/mL IL-2 and 5 g/mL PHA-L. CD4+ T cells were then purified as described above and re-suspended in the appropriate volume of virus in 20% FBS and 8 g/mL polybrene. Subsequently, cells were spinoculated for four hours at 2880 x g at room temperature, re-suspended in fresh complete 10% RPMI and analyzed forty- eight hours post-infection.

For examination of the effects of AP-2 μ or α knock-down on endogenous CD28 downregulation, 1 x 106 Sup-T1 cells were suspended in 8 g/mL polybrene and virus preparations in 20% FBS produced as described above from pGFP-C-shLenti vectors encoding different shRNAs. Cells were then spinoculated for four hours at 2880 x g at room temperature and re-suspended in fresh RPMI. The next day, cells were pelleted and re-suspended in 10% RPMI (mock infection) or pNL4.3 dG/P F2A-mStrawberry Nef/dNef pseudovirions in 20% FBS and 8 g/mL polybrene. Forty-eight hours later, cells were analyzed via flow cytometry or Western blotting. For examination of the effects of SNX9 knock-down on CD28-FLAG, cells were infected as above with pGFP-C-shLenti vectors encoding NS or SNX9 targeting shRNAs, infected the next day with viruses encoding CD28-FLAG and Nef or no Nef, and forty-eight hours later analyzed by Western blot.

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2.4.4 Flow cytometry analysis of cell surface receptors

Cells were analyzed using a BD Biosciences FACSCanto SORP (BD Biosciences, San Jose, CA). Data analysis was performed using FlowJo software (version 9.6.4, FlowJo LLC, Ashland, OR).

2.4.4.1 Sup-T1 cells

For cell surface staining of Sup-T1 cells, forty-eight hours post infection, cells were washed twice with PBS, followed by staining for 20 minutes at room temperature with 1:6000 Zombie RedTM (BioLegend) or 1:6000 Zombie NIRTM (BioLegend) in PBS, where appropriate. Cells were then washed in FACS buffer (1% FBS, 50 mM ethylenediaminetetraacetic acid (EDTA) in PBS) and fixed in 1% paraformaldehyde (PFA) for 20 minutes at room temperature. Cells were subsequently washed twice with FACS buffer and stained with the appropriate antibodies by rocking for 40 minutes at room temperature. Cells were then washed twice with FACS buffer and re-suspended in PBS. For staining of CD28 and CD4, 1:25 APC-conjugated mouse-anti-CD28 (clone CD28.2, BioLegend) and 1:25 APC-conjugated mouse-anti-CD4 (clone OKT4, BioLegend) were utilized, respectively. For cell surface MHC-I staining, cells were stained with W6/32 (anti- MHC-I, pan-selective for HLA-A, B and C, provided by D. Johnson, Oregon Health and Science University), washed twice with FACS buffer, incubated with an APC-conjugated species specific secondary antibody, followed by washing with FACS buffer and re- suspending in PBS. For analysis of total CD28 or CD4 levels, cells were prepared as above, but were permeabilized and blocked prior to staining. Specifically, after fixation, cells were permeabilized by incubation with 0.5% saponin for 15 minutes at room temperature. Cells were then washed with 1% FBS, 0.1% saponin, 5 mM EDTA and blocked for thirty minutes with blocking buffer (10% FBS, 0.1% saponin, 5 mM EDTA in PBS). Cells were incubated with primary antibody diluted in blocking buffer followed by washing with 1% FBS, 0.1% saponin, 5 mM EDTA and re-suspending in PBS.

2.4.4.2 Primary cells

For cell surface staining of peripheral blood mononuclear cells infected with VSV-G pseudotyped NL4.3 encoding eGFP, cells were fixed in 2% PFA for 20 minutes at room

136 temperature, followed by washing twice with cell staining buffer (BioLegend). Cells were then incubated for 40 minutes at room temperature with the appropriate fluorescently labeled primary antibodies (1:50 APC-Cy7 conjugated anti-CD4 (clone OKT4, BioLegend), 1:25 APC conjugated anti-CD28 (clone CD28.2, BioLegend)). Cells were then washed twice with cell staining buffer and re-suspended in PBS prior to analysis.

For analysis of total CD28 in primary CD4+ T cells infected with VSV-G pseudotyped NL4.3 provirus, cells were washed twice with cell staining buffer (BioLegend) and fixed and permeabilized in BD Cytofix/Cytoperm solution (BD Biosciences). Cells were subsequently washed twice with Perm/Wash buffer (BD Biosciences) and stained for 30 minutes at 4°C for CD28 level analysis (1:25 APC conjugated anti-CD28 (clone CD28.2, BioLegend)). Cells were then washed, re-suspended in PBS and analyzed.

For analysis of cell surface CD28 on primary CD4+ T cells infected with replication competent NL4.3 provirus, cells were washed twice with cell staining buffer (BioLegend) and incubated for 30 minutes at 4°C with fluorophore conjugated primary antibodies against the appropriate cell surface antigen (1:25 APC conjugated anti-CD28 (clone CD28.2, BioLegend)). Cells were then washed twice with cell staining buffer and then fixed and permeabilized in BD Cytofix/Cytoperm solution (BD Biosciences, San Jose, CA). Cells were subsequently washed twice with Perm/Wash buffer (BD Biosciences) and stained for 30 minutes at 4°C for p24 level analysis (1:50 anti-p24; clone KC57, Beckman Coulter). Cells were then washed, re-suspended in PBS and analyzed. The following isotype control antibodies were used in lieu of primary antibody as required: APC-Cy7 conjugated mouse IgG2b, κ isotype (clone MOPC-21, BioLegend), APC mouse IgG1 κ isotype control (clone MOPC-21, BioLegend), PE mouse IgG1κ (clone: MOPC-21, BioLegend).

2.4.5 Protein analysis

Infected Sup-T1 cells and HeLa or HEK293T cells transfected as described above were lysed forty-eight hours post transduction or transfection for protein expression analysis. Briefly, cells were pelleted and lysed by rocking in lysis buffer (0.5 M HEPES, 1.25 M

NaCl, 1 M MgCl2, 0.25M EDTA, 0.1% Triton X-100 and 1X complete Protease Inhibitor

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Tablets (Roche, Indianapolis, IN)) for one hour at 4C. The supernatant was then clarified by spinning at 16100 x g for 30 minutes at 4C, mixed with SDS-PAGE sample buffer (0.312 M Tris pH 6.8, 2.6 M 2-Mercaptoethanol, 50% glycerol, 10% SDS) and boiled at 95C for 10 minutes. Samples were run on 12 or 14% SDS-PAGE gels followed by transferring to nitrocellulose. Membranes were blocked with 5% milk in TBST (50 mM Tris, 150 mM NaCl, 0.1% Tween 20) for one hour at room temperature, followed by incubation overnight at 4C with the appropriate primary antibody in 5% milk in TBST. The following primary antibodies were used: 1:1000 rabbit anti-Nef polyclonal antibody (NIH-AIDS Research and Reference Reagent program, catalog number 2949), 1:2000 rabbit anti-GFP polyclonal antibody (Clontech), 1:5000 rabbit anti-Vpu polyclonal antibody (NIH-AIDS Research and Reference Reagent program, catalog number 969), 1:2000 mouse anti-Actin monoclonal antibody (Thermo Fisher Scientific, 1:1000 rabbit anti-GAPDH polyclonal antibody (Thermo Fisher Scientific), 1:500 rabbit anti-AP2 μ (BD Biosciences), 1:1000 rabbit anti-AP-2 α (Sigma-Aldrich), 1:800 rabbit anti-SNX9 (Sigma- Aldrich), 1:1000 rat anti-DYKDDDK (BioLegend). The next day, membranes were washed three times with TBST and incubated for two hours at room temperature with the appropriate species specific HRP-conjugated secondary antibodies (1:2000, Thermo Fisher Scientific) in 5% milk in TBST. Blots were subsequently washed and developed using ECL substrates (Millipore, Etobicoke, ON) and a C-DiGit chemiluminescence Western blot scanner (LI-COR Biosciences, Lincoln, NE). Relative intensities of protein bands were determined using LI-COR Image Studio software.

2.4.6 Microscopy

For microscopy experiments, cells were infected and treated with inhibitors as described above. Cells were then adhered to poly-L-lysine (Sigma-Aldrich) coated coverslips and fixed in 2% PFA for 10 minutes at room temperature. Cells were subsequently washed twice with PBS and permeabilized with methanol for 20 minutes. Cells were then washed twice with PBS and blocked in 2% bovine serum albumin (BSA) in PBS for 1 hour at room temperature. Cells were subsequently stained for two hours with mouse anti-CD28 (Thermo Fisher Scientific) and rabbit anti-LAMP-1 (Developmental Studies Hybridoma Bank, University of Iowa) diluted at 1:200 in 0.2% BSA in PBS, washed twice with 0.2%

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BSA in PBS and incubated for one and a half hours with the appropriate fluorophore conjugated secondary antibodies (Alexa-Fluor-647 conjugated anti-mouse and Cy3- conjugated anti-rabbit; Jackson ImmunoResearch, West Grove, PA) at 1:400 in 0.2% BSA in PBS. Finally, cells were washed twice with PBS and mounted on coverslips with DAPI Fluoromount-G (SouthernBiotech, Birmingham, AL). Cells were imaged on a Leica DMI6000 B at 63X or 100X magnification using the FITC, Cy3, Cy5 and DAPI filter settings and imaged with a Hamamatsu Photometrics Delta Evolve camera. Images were subsequently deconvolved using the Advanced Fluorescence Deconvolution application (Lecia, Wetzlar, Germany) on the Leica Application Suite software. Co-localization analysis was conducted using Mander’s Coefficient from the ImageJ plugin JACoP, as described previously [103].

2.4.7 Cell activation analysis

To examine activation in infected cells, cryopreserved PBMCs were revived and rested for + 6 hours in complete RPMI media (without PHA/IL-2) at 37º C and 5% CO2. CD4 T cells were then purified as described above and spinoculated with the appropriate VSV-G pseudotyped NL4.3 virus encoding both Nef and Vpu (NL4.3 dG/P eGFP), encoding Vpu alone (NL4.3 dG/P eGFP dNef), encoding Nef alone (NL4.3 dG/P eGFP dNef), or lacking both Nef and Vpu (NL4.3 dG/P eGFP dVpu dNef). After spinoculation, cells were incubated in complete 10% RPMI for 24 hours prior to incubating with anti-CD3 (10 μg/ml, clone OKT3, BioLegend) immobilized on a plate and soluble anti-CD28 (5μg/ml, clone CD28.2, BioLegend). After 24 hours of activation, cells were treated with Brefeldin A (1:1000 dilution, BD Biosciences) for 12 hours before proceeding for intracellular IL-2 staining.

For intracellular staining of IL-2, infected CD4+ T cells were fixed using Cytofix/Cytoperm solution (BD Biosciences) for 20 minutes at 4ºC, washed twice with Perm/Wash buffer (BD Biosciences) and stained for intracellular IL-2 with an APC-conjugated anti-IL-2 antibody (clone MQ1-17H12, BD Biosciences), or the appropriate isotype control (APC Rat IgG2a, κ isotype control, clone R35-95, BD Biosciences), for 1 hour at 4ºC (1:20 dilution in Perm/Wash buffer). Cells were then washed twice using Perm/Wash buffer prior to analysis by flow cytometry. In order to determine the percentage of the infected cells

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+ + that were IL-2 positive, the percentage of GFP IL-2 cells (Q2, Figure 2.23) was divided + + + - by the total percentage of infected cells (sum of GFP IL-2 (Q2, Fig. 8) and GFP IL-2

(Q3, Figure 2.23) and multiplied by 100.

2.4.8 Data and Statistical analysis

2.4.8.1 Flow cytometry data analysis

For analyses of flow cytometry data obtained for Sup-T1 cells infected with VSV-G pseudotyped NL4.3 encoding Vpu and various Nef mutations, relative levels of receptors were determined by normalizing geometric mean fluorescence intensity after gating on infected (GFP+) cells. For all other analysis of cell surface receptors on Sup-T1 cells infected with a single VSV-G pseudotyped virus, relative levels of receptors were determined by normalizing geometric mean fluorescence intensity after gating on single, live (Zombie RedTM-), infected (GFP+) cells. Relative levels of CD28 on primary CD4+ T cells infected with replication competent NL4.3 were determined by normalizing geometric mean fluorescence intensity after gating on single, p24 high and CD28+ cells. Relative levels of CD28 on VSV-G pseudotyped NL4.3 infected PBMCs, were determined by normalizing geometric mean fluorescence intensity after gating on CD4+ and infected (GFP+) lymphocytes. For all cell surface or total receptor analysis by flow cytometry, the geometric mean fluorescence intensity of the control in each experiment (left-most sample on each graph) was set to 1 and the other sample MFIs were calculated relative to the control having an MFI of 1.

To calculate the relative increase in intracellular staining upon ammonium chloride treatment, the geometric mean fluorescence intensity of the live and infected cells treated with ammonium chloride was divided by the geometric mean fluorescence intensity of cells that were untreated. This ratio was then normalized such that the fold increase in MFI for cells infected with NL4.3 was equal to one.

For analysis of cell surface levels of CD28 on Sup-T1 cells infected with VSV-G pseudotyped viruses encoding shRNAs and eGFP and mStrawberry and Nef and/or Vpu, geometric mean fluorescence intensity of CD28 was determined after gating on single, live (Zombie NIRTM-), doubly infected (mStrawberry+ GFP+) cells. The relative Nef or Vpu-

140 mediated downregulation of cell surface CD28 in the presence of the shRNA vectors was then determined based on the MFI of cells infected with virus encoding versus lacking the accessory protein of interest. The downregulation of CD28 was then normalized to the downregulation mediated by the same viral protein in the presence of the non-specific shRNA.

To analyze the effects of overexpression of wild-type SNX9 or dominant negative SNX9, levels of CD28 downregulation were calculated by determining geometric mean fluorescence intensity after gating on single, live (Zombie RedTM-), infected (GFP+) cells and normalizing the levels on cells expressing Nef-eGFP to those expressing Nef G2A- eGFP. Downregulation levels were then compared between cells transduced with virus encoding wild-type SNX9 or dominant negative SNX9.

2.4.8.2 Statistical analyses

All statistics for analysis of receptor levels were conducted using a one-way analysis of variance with Bonferroni’s multiple comparison test. For analysis of CD28: LAMP-1 co- localization, the mean Manders’ overlap coefficients were compared using a one-way analysis of variance with Bonferroni’s multiple comparison test to compare wild-type infected cells to cells infected with viruses encoding or lacking Nef and/or Vpu (Fig. 3) or wild-type infected cells treated with vehicle to cells treated with various inhibitors (Fig. 4). Alternatively, for analysis of the percentage of IL-2 positive infected cells, a paired two- tailed t-test was used. All statistical tests were completed using Graph Pad Prism (Graph Pad Software Inc., La Jolla, CA).

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Chapter 3 3 Defining the role of the novel interaction between HIV-1 Nef and the membrane trafficking protein SNX18

3.1 Introduction

In order for viruses to persist within their hosts, they must evade host anti-viral immune responses. This is particularly critical for viruses that cause chronic infections, such as HIV-1, which fails to be eliminated by the immune response upon establishment of infection. Key to the ability of HIV-1 to evade the immune response are the actions of the accessory protein Nef. Nef mediates multiple functions to facilitate the evasion of both innate and adaptive immune responses (reviewed in [1]). A central mechanism by which Nef enables evasion of immune responses is via the reduction of cell surface levels of MHC-I molecules [2]. Briefly, cell surface MHC-I molecules present antigens to the adaptive immune cells CD8+ cytotoxic T cells, which secrete cytotoxic enzymes to induce the death of cells presenting viral antigens on MHC-I molecules [3]. Therefore, as a result of the Nef-dependent reduction of cell surface MHC-I molecules, cells infected with HIV- 1 encoding a functional nef gene are less effectively killed by CD8+ cytotoxic T cells [4]. Moreover, increased downregulation of MHC-I by Nef correlates with faster disease progression and higher viral loads in rhesus macaques infected with the related simian immunodeficiency virus [5-8].

Nef mediates its functions, including decreasing surface MHC-I molecules, by interacting with host cell proteins and subverting their functions. Specifically, many Nef-dependent functions require Nef to alter the subcellular localization of various host cell proteins by hijacking host proteins implicated in cellular membrane trafficking. A well characterized example of this is the hijacking of the membrane trafficking adaptor proteins AP-1 and AP-2. Indeed, Nef binds to both AP-1 and AP-2 to connect host cell cargo to the trafficking machinery, whereby these Nef: adaptor protein: cargo complexes mediate the transport of cellular cargo to specific subcellular locations [9-12]. However, in order for cargo to be trafficked to different locations within the cell a multitude of host cellular machinery are necessary, as vesicular trafficking is complex and generally requires many factors [13],

150 leading to the possibility that Nef recruits one or more additional factors to facilitate cargo trafficking.

An additional protein that may facilitate Nef-dependent cargo trafficking is SNX18, which was shown to form a complex with Nef in infected HeLa cells [14]. SNX18 is a member of a subgroup of sorting nexin proteins that contain an SH3 domain, which also includes the proteins SNX9 and SNX33 (SNX30) [15]. Together, these sorting nexin proteins facilitate membrane trafficking by mediating the formation of tubulated membranes [15, 16], which may ultimately form vesicles. In addition, SNX9, SNX18 and SNX33 have been reported to interact with and recruit additional membrane trafficking proteins, thereby making these key regulators of membrane trafficking events. Notably, the SH3 domains of SNX18, SNX9 and SNX33 interact with the GTPase dynamin-II [15, 17, 18], which mediates the scission of membranes to form vesicles during membrane trafficking [19]. Interestingly, SNX9 and SNX18 also interact with the Nef-binding vesicular adaptor proteins. Specifically, SNX9 immunoprecipitated with AP-2 and SNX18 with AP-1 from K562 and HeLa cells, respectively [15, 20]. Moreover, endogenous SNX18 co-localizes with the Nef and AP-1 binding protein PACS-1 in HeLa cells [15]. Given that the classical trafficking organelles, clathrin coated vesicles, contain upwards of 200 discrete proteins which can contribute to their trafficking within cells [21], the relatively small number of host cell proteins currently characterized as essential for Nef-mediated trafficking of host cell cargo are undoubtedly not sufficient to facilitate membrane trafficking. Therefore, we hypothesize that additional host cell factors are required during Nef-mediated trafficking events and have focused on SNX18 and SNX9 as potential candidates.

Herein, we investigate the SNX18: Nef interaction further by mapping the protein domains implicated in this complex using in vitro protein binding assays. We demonstrate that Nef interacts directly with SNX18, but not the related protein SNX9, and map this interaction to the SH3 domain of SNX18 and the PxxP75 motif of Nef. Moreover, we investigate the role of the Nef: SNX18 interaction in cells, specifically by examining the requirement for this interaction for Nef-mediated MHC-I downregulation. Our findings suggest that SNX18 may be important for Nef to mediate decreases in cell surface MHC-I in T cells.

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3.2 Results

3.2.1 SNX18 and Nef interact in vitro through a SNX18 SH3 domain: Nef PxxP motif interaction

We first sought to map the interaction between Nef and members of the sorting nexin family of proteins, in addition to determining if this interaction is direct. Interestingly, the

HIV-1 Nef protein contains a motif (PxxP75) implicated in direct interactions with SH3- domain containing proteins [22-25], and these same SH3 domains are present in the sorting nexin proteins SNX18 and SNX9. We first tested if Nef and SNX18 or SNX9 interact directly. To monitor this, in vitro capture assays were performed using bacterially produced and subsequently purified proteins. Specifically, purified His6-Nef (SF2) was incubated with glutathione conjugated beads and increasing concentrations of GST-tagged SNX18 and SNX9 or GST alone. In the presence of GST-SNX18, His6-Nef was detected via Western blot in the pulled-down proteins, unlike in the presence of SNX9 (Figure 3.1A). Relative to our control, GST, significantly greater SNX18, but not SNX9, pulled down

Nef, and there was greater His6-Nef detected when an increased amount of SNX18 bait protein was utilized (Figure 3.1B). Overall, this suggests that SNX18 interacts with Nef in vitro, thus demonstrating a direct interaction, while the related protein SNX9 does not interact with Nef.

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Figure 3.1 GST-SNX18, but not GST-SNX9, pulls down His6-Nef

Various GST-tagged proteins and His6-Nef protein were bacterially produced and subsequently purified. His6-Nef was then pulled down by sepharose beads bound with the indicated amounts of GST, GST-SNX18 or GST-SNX9 and detected by Western blot. (A) Anti-His6 Western blot of 5% of input (top); Anti-His6 Western blot of protein remaining associated with glutathione sepharose after washing with 500 mM sodium chloride (middle); Ponceau S stain of membrane from pulled down proteins (bottom).

(B) Mean (+/-SD) band intensity of relative His6-Nef pulled down by GST, GST-SNX18 or GST-SNX9. Mean His6-Nef binding was compared using a one-way Anova and the Bonferroni’s multiple comparisons test was utilized to compare binding to GST. Assays were performed in quadruplicate. (SD: standard deviation; * p≤ 0.05; **** p≤ 0.0001; WB: Western blot)

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Next, we mapped the SNX18: Nef interaction interface. To determine if the SH3 domain of SNX18 mediates the Nef: SNX18 interaction, as it does with the SFK interaction with Nef [26], a GST-tagged variant of the SH3 domain of SNX18 was bacterially produced and subsequently purified, along with a GST-tagged version of SNX18 that is devoid of its SH3 domain (SH3; Figure 3.2A). Upon performing in vitro pull-down assays, we observed that His6-Nef was indeed pulled down by the SH3 domain of SNX18, but not by the SH3 protein (Figure 3.2B, C). This result suggests that the SH3 domain is necessary and sufficient for interacting with Nef in vitro. Moreover, to further test the specificity of the SNX18 SH3 domain in mediating a direct interaction with Nef, we designed a GST- tagged chimeric protein in which the SH3 domain of the SNX18 protein was replaced with the SH3 domain of SNX9 (Figure 3.2A), which fails to bind Nef in vitro (Figure 3.1), and tested for its ability to pull down His6-Nef. Interestingly, the SNX9 SH3/SNX18 chimera failed to pull down His6-Nef in an in vitro binding assay, in a similar manner to the GST protein negative control (Figure 3.2D, E), confirming the specificity and importance of the SNX18 SH3 domain for binding to Nef in vitro.

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Figure 3.2 The direct Nef: SNX18 interaction is dependent on the SH3 domain

GST-tagged bait proteins and His6-Nef were bacterially produced and subsequently purified. His6-Nef was then pulled-down by glutathione-sepharose beads coated with the indicated GST-tagged proteins and detected by Western blot. (A) Schematics indicating the GST-tagged constructs utilized as bait proteins. (B) His6-Nef was captured by glutathione sepharose bound GST, GST-tagged SH3 domain of SNX18 or GST-tagged

ΔSH3 (SNX18 lacking the SH3 domain) and detected by Western blotting. Anti-His6

Western blot of 5% of input (top); Anti-His6 Western blot of protein remaining associated with glutathione sepharose after washing with 500 mM sodium chloride (middle); Ponceau S stain of membrane from pulled down proteins (bottom). (C) Mean band intensity of relative His6-Nef pulled down by GST, GST-SH3 or GST-ΔSH3. Assays were performed multiple times (n=7) and the results are presented as the mean

(+/-SD). Mean His6-Nef binding was compared using a one-way Anova and the

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Bonferroni’s multiple comparisons test was utilized to compare binding in all conditions versus GST. (E) Anti-His6 Western blot of 5% of input (top); Anti-His6 Western blot of protein remaining associated with glutathione sepharose after washing with 500 mM sodium chloride (bottom). (F) Mean band intensity of relative His6-Nef pulled down by GST, GST-SNX18, GST-SNX9 or a GST-SH3 domain chimera comprised of the SH3 domain of SNX9 fused to the entire SNX18 protein except for the SH3 domain. Assays were performed in triplicate and results are presented as the mean (+/-SD). Mean His6- Nef binding was compared using a one-way Anova and the Bonferroni’s multiple comparisons test was utilized to compare binding in all conditions versus GST. (SD: standard deviation; * p≤ 0.05; ** p≤ 0.01; *** p≤ 0.001; **** p≤ 0.0001; WB: Western blot)

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It has been previously demonstrated that Nef interactions that are dependent on SH3 domains are mediated by the Nef PxxP75 motif [23, 26], which has defined roles in AIDS pathogenesis and immune evasion [26-30]. To test for the requirement of the Nef PxxP75 motif for interacting with the SNX18 SH3 domain and thereby establishing a Nef motif implicated in the Nef: SNX18 interaction, we repeated our in vitro protein pull-down assay using purified His6-Nef or His6-Nef AxxA75, a Nef mutant that fails to bind SFKs ([26];

Figure 3.3). We observed that significantly less His6-Nef AxxA75 was pulled down by the

GST-tagged SNX18 SH3 domain when compared to wild-type His6-Nef (Figure 3.3A, B). Overall, this data demonstrates that SNX18 and Nef interact directly in vitro, unlike the related SNX9 protein which fails to bind Nef. Moreover, mapping of this interaction revealed its dependence on the SH3 domain of SNX18 and the Nef PxxP75 motif.

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Figure 3.3 In vitro protein capture of His6-Nef and a Nef mutant, His6-Nef AxxA75, by the SH3 domain of SNX18

Bacterially produced and subsequently purified GST-tagged SH3 domain of SNX18 was bound to sepharose beads and tested for its ability to pull down wild-type or AxxA75 mutant His6-tagged Nef. (A) His6-Nef or His6-Nef AxxA75 was captured by glutathione- sepharose bound GST or GST tagged SH3 domain of SNX18 and detected by Western blotting. Top: Anti-His6 Western blot of 5% of input; bottom: Anti-His6 Western blot of protein remaining associated with glutathione sepharose after washing with 500 mM sodium chloride. (B) Mean (+/-SD) band intensity of hexa-histidine tagged protein pulled down by GST-tagged SNX18 SH3 domain, relative to protein pulled down by GST. Assays were performed in triplicate and the relative mean band intensities were compared using a Student’s t-test. (SD: standard deviation; **= p≤ 0.01; WB: Western blot)

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3.2.2 SNX18 and Nef interact in cells

We next sought to define the molecular determinants implicated in the Nef: SNX18 interaction in cells. Our group previously described the detection of a close association between SNX18 and Nef within cells using the technique of Bimolecular Fluorescence Complementation (BiFC) [14], which reconstitutes a split fluorophore to demonstrate a protein-protein interaction. Therefore, with the molecular details of the Nef: SNX18 interaction interface acquired in vitro (Figure 3.3), we wished to validate the previously observed Nef: SNX18 BiFC signal. To test this, we expressed vectors encoding wild-type or the PxxP75 mutant Nef (NL4.3) protein fused to a C-terminal fragment of the Venus fluorophore (Nef-VC or Nef AxxA75-VC) and SNX18 fused to a N-terminal fragment of

Venus (SNX18-VN) in HeLa cells and examined the reconstitution of the fluorophore using wide field microscopy (Figure 3.4). We observed that the mean fluorescence intensity of the Nef: SNX18 BiFC signal was significantly reduced with the mutant Nef (Nef AxxA75-

VC), compared to wild type Nef (Figure 3.4B). Moreover, expression of the Nef-VC and

Nef AxxA75-VC proteins was confirmed by Western blotting (Figure 3.4C). These findings suggest that the Nef: SNX18 interaction occurs within cells and is mediated at least in part by the Nef PxxP75 motif.

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Figure 3.4 Mutation of the Nef PxxP75 motif reduces SNX18: Nef BiFC intensity

HeLa cells were co-transfected with vectors encoding SNX18-VN-FLAG and either Nef-

VC or Nef AxxA75-VC. Twenty-four hours post transfection, cells were incubated in the dark for 1 hour to mature the fluorophore, followed by fixation in 4% PFA and subsequent visualization by wide field microscopy. (A) Representative images of

160 transfected cells illustrating Nef: SNX18 BiFC signal (left), anti-FLAG staining (middle) and merge (right). (B) Mean (+/-SD) fluorescence intensity of non-transfected (NT),

SNX18-VN-FLAG and Nef-VC transfected, or SNX18-VN-FLAG and Nef AxxA75-VC transfected cells (n=30). The mean fluorescence intensities were compared using a one- way ANOVA followed by Tukey’s multiple comparisons test. (C) Western blots demonstrating expression of Nef-VC and Nef AxxA75-VC. (*** p≤ 0.001; NT: non- transfected; Mr: molecular weight)

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3.2.3 SNX18 is required for Nef-dependent reductions in cell surface MHC-I

We next sought to identify if the Nef: SNX18 interaction is required for Nef-mediated decreases in cell surface MHC-I, a key mechanism utilized by HIV-1 to evade immune surveillance. Indeed, it has been previously demonstrated that the Nef PxxP75 motif which mediates SNX18 binding also binds SFKs to trigger an intracellular signaling cascade which downregulates cell surface MHC-I [30, 31]. To test this, CD4+ Sup-T1 T cells were transduced with lentiviral vectors encoding both GFP and shRNA sequences targeting SNX18 or an irrelevant non-specific shRNA. In addition, these same Sup-T1 cells were transduced with a modified HIV-1 NL4.3 vector encoding the fluorescent protein mStrawberry in addition to encoding (Nef) or lacking Nef (dNef). Cell surface MHC-I levels on dual infected (GFP+ mStrawberry+) cells were quantified by flow cytometry (Figure 3.5A). We observed a small, but significant, increase in the amount of cell surface MHC-I in the presence of Nef in cells infected with virus decreasing SNX18 expression, compared to cells expressing the non-specific shRNA (Figure 3.5B, C), suggesting that Nef-mediated decreases in cell surface MHC-I are dependent on SNX18 expression. Moreover, efficient knock-down of SNX18 in Sup-T1 cells was confirmed using Western blot analyses of infected cells (Figure 3.5D). Indeed, we observed a significant decrease in cellular SNX18 levels in cells transduced with SNX18 targeting shRNAs, compared to non-specific shRNA (Figure 3.5E). Overall, these findings suggest that the presence of SNX18 is important for the ability of Nef to decrease cell surface MHC-I levels.

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Figure 3.5 Knock-down of SNX18 reduces Nef-mediated MHC-I downregulation

CD4+ Sup-T1 T cells were infected with viruses encoding both GFP and non-specific or SNX18 targeting shRNAs in addition to a virus encoding mStrawberry and encoding or lacking Nef. Infected cells were stained for cell surface MHC-I (HLA-A, B, C) and analyzed by flow cytometry. (A) Illustration of gating strategy. Dual infected (GFP+ mStrawberry+) cells were gated on and cell surface MHC-I was quantified on dual infected cells. (B) Representative histograms illustrating cell surface MHC-I levels on dual infected cells infected with non-specific (NS) shRNA or SNX18 targeting (anti- SNX18) shRNA and with virus encoding (Nef; blue) or lacking (dNef; orange) Nef. (red: isotype stained cells) (C) Relative mean (+/-SD) cell surface MHC-I in cells infected

163 with virus encoding Nef and a non-specific (NS) or SNX18 (anti-SNX18) targeting shRNA. (n=16) Relative means were compared using a paired Student’s t-test. (D) Western blot illustrating endogenous levels of SNX18 in uninfected Sup-T1 cells (UI) or cells infected with viruses encoding a non-specific (NS) or SNX18 (anti-SNX18) targeting shRNA. (E) Quantification of relative mean (+/-SD) SNX18 levels in uninfected Sup-T1 cells (UI) or cells infected with viruses encoding a non-specific (NS) or SNX18 (anti-SNX18) targeting shRNA detected by Western blots (n=8). Mean SNX18 levels were compared using a one-way Anova and Dunnett’s multiple comparisons test. (GFP: green fluorescent protein; MHC-I: major histocompatibility complex type I; Mr: molecular weight; * p≤ 0.05; ** p≤ 0.01; SD: standard deviation; UI: uninfected; SNX18: sorting nexin 18)

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3.2.4 SNX18 is unlikely to be required for Nef-mediated secretion in extracellular vesicles

In order to decrease cell surface levels of MHC-I, Nef facilitates the endocytosis and intracellular sequestration of cell surface MHC-I [32]. However, it has also been reported that Nef can facilitate the secretion of extracellular vesicles containing MHC-I via exocytosis [33, 34]. To perform this function, Nef activates host cell signaling proteins to form the Nef-associated kinase complex [35], which facilitates the complex’s interaction with the scaffold protein Paxillin [34]. Interestingly, Paxillin has been previously shown to interact with the SNX18-binding metalloproteinases ADAM-10 and ADAM-17 to promote the inclusion of Nef, ADAM-17 and ADAM-10 into extracellular vesicles [34, 36]. Therefore, we hypothesized that SNX18 may contribute to Nef-mediated decreases in cell surface MHC-I by facilitating the secretion of extracellular vesicles. Thus, we examined if the SNX18: Nef BiFC complex localizes to the late endosome or mutivesicular body (MVB) compartment, which contains vesicles that can serve as precursors for extracellular vesicles [37]. Specifically, to label the MVB compartment we utilized an antibody that recognizes the tetraspanin protein CD63. We observed that the Nef: SNX18 BiFC signal co-localized with CD63 in HeLa cells (Figure 3.6). Interestingly, the SNX18: Nef BiFC signal co-localized with CD63 to a greater extent than GPF-tagged SNX18 or the related protein SNX9, which does not bind Nef (Figure 3.1), in the absence of Nef (Figure 3.6). Therefore, given that neither SNX9 or SNX18 localize to CD63 positive compartments in the absence of Nef, this suggests that at least in the case of SNX18, this membrane trafficking protein is being diverted to MVBs in the presence of Nef.

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Figure 3.6 The SNX18: Nef BiFC complex co-localizes with CD63

HeLa cells were transfected with vectors encoding eGFP, SNX9-eGFP, SNX18-eGFP or

SNX18-VN and Nef-VC. Twenty-four hours post transfection, cells were incubated in the dark to mature the fluorophore, stained for endogenous CD63 and visualized using wide field microscopy. (A) Representative images of transfected cells. Puncta where SNX18: Nef BiFC and CD63 overlap are denoted by arrows. Scale bar = 10 μm. (B) Mean (+/-SD) Pearson correlation coefficient between signal in the FITC (eGFP/BiFC) and Cy3 (CD63) channels (n≥7). Means were compared using one way ANOVA followed by Tukey’s

166 multiple comparisons test. (*** p≤ 0.001; eGFP: enhanced green fluorescent protein; SD: standard deviation)

167

Next, we wanted to determine if SNX18 is required for Nef-mediated secretion of extracellular vesicles. To investigate this, stable HEK293T cell lines expressing shRNA targeting SNX18 (293TαSNX18) or a non-specific target control (293TNS) were produced. These cells were then transfected with the pNL4.3 dG/P eGFP lentiviral vector. The extracellular vesicles from this transfected cell culture supernatant were purified and probed for their content (Figure 3.7). Specifically, the cell lysate and purified extracellular vesicles were probed for the viral proteins Nef and Vpu, which were previously shown to be secreted into extracellular vesicles, and specific markers for intracellular proteins (Calnexin, Actin) and extracellular vesicles (TSG-101). In addition, we probed for the metalloprotease ADAM-17, which has been previous implicated in the sorting of Nef into extracellular vesicles [34]. Through five independent experiments, a significant difference in the amount of Nef secreted from 293TαSNX18 or 293TNS cells was not observed (Figure 3.7B). However, upon Western blotting for Nef in the extracellular vesicle preparations, an additional protein band was consistently observed. Moreover, Nef exhibited an altered migration pattern, as was seen in the extracellular fraction for the HIV-1 protein Vpu and for ADAM-17.

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Figure 3.7 Extracellular vesicles purified from Nef-expressing SNX18 knock-down cells

HEK293T cells stably expressing non-targeting (NS) or SNX18 targeting (anti-SNX18) shRNAs were transfected with a vector encoding all HIV-1 viral accessory proteins (NL4.3 dG/P eGFP). Forty-eight hours post transfection, extracellular vesicles in cell

169 culture supernatants were purified using a commercial purification reagent (exoquick) and the associated proteins analyzed by Western blotting. (A) Representative Western blots illustrating cellular and extracellular vesicle proteins. (B) Mean relative extracellular Nef (+/-SD) from five independent experiments. Mean relative extracellular Nef was not significantly different, as determined using a Student’s t-test. (Mr: molecular weight, SD: standard deviation)

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3.3 Discussion

In the present work, we identify and investigate the underlying mechanism of a novel Nef: host protein interaction, the interaction between Nef and SNX18. Using in vitro protein capture assays, we have shown that Nef binds directly to SNX18 (Figure 3.1). This interaction was dependent on the SH3 domain of SNX18 and was specific to SNX18, as the SH3 domain from a related family member, SNX9, could not mediate binding to Nef (Figure 3.2). Moreover, the Nef: SNX18 interaction was reduced upon mutation of the Nef

PxxP75 motif both in vitro and in mammalian cells (Figure 3.3; Figure 3.4). Notably, while mutation of the Nef PxxP75 motif inhibited complex formation with full length SNX18 within cells (Figure 3.4), the examination of the effects of this Nef mutation on binding in vitro was limited to the interaction with the SH3 domain of SNX18 (Figure 3.3). Therefore, future experiments to examine Nef AxxA75 binding to full length SNX18 protein in vitro may be of value. The finding that this interaction required the SH3 domain of SNX18 and the PxxP75 motif of Nef is consistent with the previously defined role of SH3 domains in binding to the Nef polyproline motif [26], an interaction critical for the immunoevasive effects of Nef [30]. Indeed, the Nef PxxP75 motif is critical for interacting with the SH3 domains of multiple SFKs to mediate the downregulation of cell surface MHC-I [30, 31].

Thus, the use of the Nef PxxP75 motif to hijack two different host proteins, SFKs and SNX18, by recognizing a common SH3 domain, exemplifies the amazing ability of viral proteins to efficiently mediate their functions by expanding their repertoire of binding partners. Moreover, given that proteins containing SH3 domains classically interact with other proteins via polyproline rich interaction interfaces [38], this is a prime example of viruses evolving the ability to mimic host cellular interactions while maintaining a degree of binding specificity, as SH3 domain binding was specific to SNX18 (Figure 3.2). Given that the affinity of previously identified Nef polyproline helix: SH3 domain interactions have been shown to be goverened by a specific region of the SH3 domain, known as the RT-loop [24, 39], future studies to investigate the residues within the SNX18 RT-loop that faciliate Nef binding may be of interest. The specificity of Nef: host protein interactions that required the Nef polyproline motif may also be mediated by differences in subcellular localization. Specifically, upon the localization of Nef to the TGN, wherein SFKs are enriched, Nef can interact with and activate a signaling cascade that initiates MHC-I

171 downregulation [30]. Concomitantly, Nef may interact with SNX18 at peripheral endocytic compartments. This multi-functionality of a viral protein interaction motif is not unlike that seen with host cellular proteins, including SNX18, whereby, in uninfected cells the SH3 domain of SNX18 is implicated in binding to the polyproline rich domains of the proteins N-WASP and dynamin-II [15, 40].

In order to investigate the functional consequence of Nef: SNX18 interactions in cells, we utilized shRNA to silence SNX18 expression in T cells. We observed a reduction in Nef’s ability to downregulate cell surface MHC-I in the absence of SNX18 (Figure 3.5). Notably, the effect of our SNX18 knock-down on MHC-I downregulation was small. These studies may be limited by the functional redundancy between SNX18 and the related protein SNX9 [40, 41], whereby SNX9 may compensate for the knock-down of SNX18 despite the lack of a direct Nef: SNX9 interaction. Specifically, a recent study illustrated that knock-down of both SNX18 and SNX9 was required to inhibit the proper trafficking of the metalloproteinase ADAM9, an effect not observed upon knock-down of the proteins independently [41]. Thus, future studies examining MHC-I downregulation upon reduction of both SNX18 and SNX9 may be of value. Additionally, the knock-down of SNX18 observed via Western blot was variable and limited (Figure 3.5E), with some experiments exhibiting reductions in uninfected (UI) cell SNX18 protein levels relative to cells transduced with lentiviral vectors encoding non-specific (NS) shRNA. These observed decreases in SNX18 protein levels within uninfected cells may be a result of global changes in cells resulting from lentiviral vector infection. These experiments could be improved through creation of SNX18 knock-out cell lines utilizing CRISPR/Cas9 technology [42].

The observed inhibition of Nef-mediated MHC-I downregulation upon knock-down of SNX18 is not likely due to SNX18 facilitating Nef-mediated extracellular vesicle release, as SNX18 was dispensable for Nef-induced extracellular vesicle release in HEK293T cells (Figure 3.7B). However, we did note molecular weight shifts in multiple proteins when comparing migration patterns in the cellular fraction vis-a-vis the extracellular fraction (Figure 3.7A), as has been seen previously observed with Nef in extracellular vesicle preparations [43, 44]. These differences in Nef, Vpu and ADAM-17 migration patterns may reflect post-translation modifications which act as signals to mediate the incorporation

172 of these proteins into intraluminal vesicles destined to be secreted from the cell [45]. However, we have not examined the physical properties or biogenesis pathway of our extracellular vesicle preparations, thus we cannot conclude that we are examining extracellular vesicles derived from the secretion of intraluminal vesicles.

As an alternative to mediating decreases in cell surface MHC-I through secretion of extracellular vesicles, the Nef: SNX18 interaction may be facilitating the endocytosis of MHC-I which is transiting from the cell surface to the TGN for intracellular sequestration, a virus induced activity which limits antigen presentation [32, 46, 47]. It has been previously established that to downregulate MHC-I, Nef and AP-1 form a ternary complex with the cytoplasmic tail of MHC-I [11, 12]. Additionally, it is proposed that PACS-1, which can bind both Nef and AP-1 and co-localizes with SNX18 [15, 32, 48, 49], facilitates this interaction and mediates proper trafficking of the Nef: AP-1: MHC-I ternary complex [32, 47]. Since SNX18 binds AP-1 and Nef, via the SH3 domain and low complexity domain, respectively ([15]; Figure 3.2), it is plausible that SNX18 may be recruited to and interact with the Nef: AP-1: MHC-I ternary complex. Notably, due to the ability of SNX18 to dimerize [15, 40], it is conceivable that despite the potential for competition between Nef and other polyproline rich proteins for SH3 domain binding, a single SNX18 dimer could bind to both Nef and an additional interaction partner simultaneously (Figure 3.8).

There are numerous mechanisms by which Nef-bound SNX18 could facilitate the endocytic trafficking of MHC-I. Firstly, SNX18 contains a BAR domain, which can dimerize with other BAR domains into crescent shaped structures which forms a concave “bent” surface capable of interacting with membranes, thereby contributing to overall membrane bending [50]. Membrane bending is a hallmark state of lipid bilayers that facilitates the scission of membranes, thereby ensuring the formation of small vesicles that bud from one membrane enclosed structure to transport cargo from one location to another [50]. Moreover, SNX18 can promote the scission of membranes by interacting with dynamin-II [15], which uses the energy from GTP hydrolysis to enable membrane scission [19]. Furthermore, SNX18 interacts with N-WASP [40], the recruitment of which may facilitate changes in actin dynamics to promote vesicular trafficking [51]. Ultimately, we propose that the interaction between Nef and SNX18 may be facilitating the vesicular

173 trafficking of MHC-I during Nef-mediated sequestration of MHC-I away from the cells surface.

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Figure 3.8 Proposed model for the role of SNX18 in Nef-mediated MHC-I trafficking

SNX18 can dimerize via its BAR domain and interact with curved membranes. The SNX18 low-complexity domain interacts with AP-1, which also forms a ternary complex with Nef and the cytoplasmic tail of MHC-I. Moreover, the Nef PxxP75 motif interacts with the SH3 domain of SNX18. Nef and AP-1 also interact with PACS-1, which can co-localize with SNX18, to facilitate MHC-I trafficking. While one SNX18 monomer in a single heterodimer interacts with Nef, the other can interact with additional host cellular trafficking proteins through its SH3 domain, including dynamin-II or N-WASP, to further promote membrane trafficking.

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In conclusion, our results identify a novel Nef: host protein interaction, specifically between Nef and SNX18, that may contribute to the ability of Nef to evade the immune system via MHC-I downregulation. This interaction is mediated by a highly conserved polyproline motif in Nef and therefore may be conserved across the diverse HIV-1 clades. The finding that additional Nef: host protein interactions may mediate immune evasion, highlights the complexity of the mechanism evolved by the HIV-1 virus to persist within the host and contribute to pathogenesis.

3.4 Material and Methods

3.4.1 DNA vectors

Plasmids encoding GST-tagged SNX18 and SNX9 in pGEX-6P1 and pGEX-KG backbones, respectively, were kindly provided by R. Prekeris (University of Colorado, Aurora, CO). GST-SNX18 SH3 domain and GST-SNX18 ΔSH3 expression vectors were produced by PCR amplification of the SNX18 SH3 domain (residues: 1-61) or low complexity through BAR domains (residues: 62-624) with primers encoding EcoRI and NotI restriction sites, followed by insertion into pGEX-4T1. A vector encoding the GST- tagged SNX9 SH3 domain- SNX18 chimeric protein was produced by insertion of a HindIII restriction site immediately downstream of the SH3 domain (residue 63) of SNX18 using site directed mutagenesis with mutagenesis primers created with the Agilent Technologies QuikChange Primer Design program (Agilent Technologies, Santa Clara, CA). The SNX18 SH3 domain was subsequently replaced with PCR amplified SNX9 SH3 domain (residues: 1-61) using the inserted HindIII restriction site. The bacterial expression vector for production of His6-Nef encoded hexa-histidine tagged Nef from the HIV-1 SF2 strain and was provide by T. Smithgall (University of Pittsburgh, Pittsburgh, PA). A His6-

Nef AxxA75 mutant was produced by site directed mutagenesis, as above.

The SNX18 gene was sub-cloned into a peGFP-N1 backbone, in which the eGFP fluorophore was replaced with an N-terminal portion of Venus (VN 1-173) FLAG tagged on the N-terminus. The Nef-VC expression vector was previously described [46] and a Nef

AxxA75-VC expression vector was produced using site directed mutagenesis, as described

176 above. The SNX18 and SNX9 genes were sub-cloned into peGFP-N1 to produce SNX18- eGFP and SNX9-eGFP expression vectors.

Viral infection plasmids were engineered via insertion of the mStrawberry encoding region of the previously described pNL4.3 F2A-mStrawberry vector into pNL4.3 dG/P eGFP or pNL4.3 dG/P eGFP dNef backbones [14, 52, 53]. Lentiviral vectors encoding shRNAs were obtained from OriGene (OriGene Technologies, Inc., Rockville, MD) and are described below.

3.4.2 Bacterial protein expression and purification

E. coli BL21(DE3) were transformed with expression vectors encoding GST or hexa- histidine tagged proteins of interest and cultured in LB media containing the appropriate antibiotic to an optical density at 600 nanometers (OD600) between 0.5-0.7. GST tagged sorting nexin proteins or domains of interest were subsequently induced with 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) (Thermo Fisher Scientific, Mississauga,

ON) overnight at 30° C, while GST and His6-Nef were induced for four hours with 1.0 mM IPTG at 37° C and 1.5 mM IPTG at 30° C, respectively. Induced E. coli BL21 (DE3) transformed with GST-tagged protein expression vectors were pelleted at 3,840 x g, re- suspended in lysis buffer [0.1 M sodium phosphate (pH 7.2), 1 M sodium chloride, 10 mM benzamidine, 0.5 mM ethylenediaminetetraacetic acid (EDTA), 1 mM dithiothreitol (DTT)], and lysed using a French press. The lysate was ultra-centrifuged at 256,630 x g to separate the soluble and insoluble lysate components. The supernatant was incubated with glutathione sepharose 4B (GE Healthcare, Baie-D’Urfé, QC), with rocking for one hour at 4° C. The resin was then pelleted, the supernatant removed, and the resin washed to remove unbound or non-specifically bound proteins. Briefly, the resin was washed sequentially as follows: two washes with 12 resin volumes of wash buffer 1 [0.1 M sodium phosphate buffer (pH 7.2), 1 M sodium chloride, 0.5 mM EDTA, 1 mM DTT], three washes with 12 resin volumes of wash buffer 2 [0.1 M sodium phosphate buffer (pH 7.2), 150 mM sodium chloride, 1 mM DTT], and two washes with one resin volume of elution buffer [50 mM Tris buffer (pH 8.0), 15 mM reduced glutathione]. The elution buffer washes were pooled and proteins were further purified using a size exclusion column and fast protein liquid

177 chromatography such that eluted proteins were in 20 mM Tris buffer (pH 8.0), 150 mM sodium chloride, 1 mM DTT, and 0.5 mM EDTA.

The induced E. coli BL21 (DE3) transformed with His6-Nef expression vectors were pelleted at 3,840 x g, re-suspended in lysis buffer [50 mM sodium phosphate (pH 8.0), 200 mM sodium chloride, 10 mM imidazole], lysed using a French press, and the lysate ultra- centrifuged at 256,630 x g. The supernatant was then incubated with nickel-nitriloacetic acid (Ni2+-NTA) agarose (5 PRIME, Mississauga, ON) for one hour at 4°C with rocking. The elution of proteins bound to the Ni2+-NTA agarose resin was performed using buffer containing imidazole. Specifically, the resin was pelleted and washed sequentially as follows: two washes with 12 resin volumes of wash buffer 1 [50 mM sodium phosphate buffer (pH 8.0), 200 mM sodium chloride, 20 mM imidazole], three washes with 12 resin volumes of wash buffer 2 [50 mM sodium phosphate buffer (pH 8.0), 200 mM sodium chloride, 100 mM imidazole], and one wash with 6 resin volumes of wash buffer 3 [50 mM sodium phosphate buffer (pH 8.0), 200 mM sodium chloride, 250 mM imidazole]. The protein eluted in the 100 and 250 mM imidazole washes was concentrated using 10,000 Da cut-off centricon centrifugal filters (EMD Millipore, Billerica, MA) and stored at -80ºC.

3.4.3 Protein capture assay

In vitro captures of His6-Nef were performed using the various GST-tagged bait proteins. Variable amounts of bait protein (GST-tagged proteins), as quantified by Lowry assay, were incubated with rocking for 45 minutes at room temperature with glutathione sepharose in binding buffer [20 mM Tris (pH 8.0), 150 mM sodium chloride, 0.5 mM

EDTA, 0.1% NP-40 alternative]. Subsequently, 8 µg His6-Nef was then combined with each bait protein, at which time a loading control sample was obtained. After incubating for one hour at room temperature with rocking the resin was pelleted at 1,200 x g and washed three times with 25 resin volumes of wash buffer [20 mM Tris (pH 8.0), 500 mM sodium chloride, 0.1 mM EDTA, 0.1% NP-40 alternative], and re-suspended in SDS- PAGE sample buffer for Western blot analysis.

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3.4.4 Cell culture

HEK293T (ATCC) cells and HeLa cells (ATCC) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 4 mM L-glutamine, 4500 mg/L glucose and sodium pyruvate (HyClone, Logan, UT) and supplemented with 1% Penicillin-Streptomycin (Hyclone) and 10% fetal bovine serum (FBS; Wisent, St-Bruno, QC). Sup-T1 cells were maintained in Roswell Park Memorial Institute media (RPMI) 1640 media with L- glutamine supplemented with 100 g/ml penicillin-streptomycin, 1% sodium pyruvate, 1% non-essential amino acids, 2 mM L-glutamine (Hyclone) and 10% FBS. All cell lines were grown at 37°C in the presence of 5% CO2 and sub-cultured in accordance with supplier’s recommendations.

HEK293T cells stably expressing shRNAs against a non-specific target or SNX18 were produced by transduction with shRNA-encoding lentiviral vectors. Briefly, HEK293T cells were transduced with VSV-G pseudotyped lentivirus produced as described below with pGFP-C-shLenti plasmid encoding a scrambled shRNA (TR30021, Origene) or a SNX18 targeting shRNA (TL307432D, Origene). Transduced cells were selected for using 0.8 ug/mL puromycin and clones were selected. Cells were subsequently maintained in DMEM containing 0.4 ug/mL puromycin.

3.4.5 Transfections and virus production

Transfection of HeLa cells for microscopy and Western blotting was performed using PolyJet (FroggaBio, North York, ON) as per the manufacturer’s protocol. For VSV-G pseudotyped lentivirus production, HEK293T cells were triply transfected with the NL4.3 or shRNA encoding vector of interest, the VSV-G envelope-encoding pMD2.G plasmid (Addgene; catalog number 12259) and pCMV-DR8.2 (Addgene; catalog number 12263) using PolyJet (FroggaBio) as per the manufacturer’s protocol. Seventy-two hours post transfection, lentivirus was harvested via cell culture supernatant clarification by spinning at 1500 x g, followed by 20 m filtration. Viruses were stored in 20% FBS at -80C.

3.4.6 Western blot analysis

For Western blotting of whole cell lysates, cells were pelleted and lysed by rocking in lysis buffer [0.5 M HEPES, 1.25 M NaCl, 1 M MgCl2, 0.25M EDTA, 0.1% Triton X-100 and

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1X complete Protease Inhibitor Tablets (Roche, Indianapolis, IN)] for one hour at 4C. The supernatant was then clarified by spinning at 16,100 x g for 30 minutes at 4C, combined with SDS-PAGE sample buffer (0.312 M Tris pH 6.8, 3.6 M 2-Mercaptoethanol, 50% glycerol, 10% SDS) and boiled at 95C for 10 minutes.

All Western blots were completed by running samples on 10 or 12% SDS-PAGE gels, followed by transferring to a nitrocellulose membrane. Membranes were blocked with 5% milk in TBST (50 mM Tris, 150 mM NaCl, 0.1% Triton-X) for one hour at room temperature, followed by incubation overnight at 4C with the appropriate primary antibody in 5% milk in TBST. The next day membranes were washed three times with TBST and incubated for two hours at room temperature with the appropriate species specific HRP-conjugated secondary antibodies (1:2000, Thermo Fisher Scientific) in 5% milk in TBST. Blots were subsequently washed and developed using ECL substrates (Millipore, Etobicoke, ON) and a C-DiGit chemiluminescence Western blot scanner (LI- COR Biosciences, Lincoln, NE). The following primary antibodies were utilized: 1:100 THETM His antibody (GenScript, Piscataway, NJ), 1:1000 rabbit anti-Nef polyclonal antibody (NIH-AIDS Research and Reference Reagent program; catalog number 2949), 1:5000 rabbit anti-Vpu polyclonal antibody (NIH-AIDS Research and Reference Reagent program; catalog number 969), 1:2000 mouse anti-Actin monoclonal antibody (Thermo Fisher Scientific), 1:2000 rabbit anti-Calnexin (abcam, Cambridge, MA; catalog number ab22595), 1:1000 rabbit anti-ADAM17 (abcam; catalog number ab2051), 1:1000 mouse anti-TSG-101 (abcam; catalog number ab83), 1:1000 rabbit anti-SNX18 (Sigma-Aldrich, Oakville Ontario; catalog number HPA037800).

3.4.7 Microscopy

For microscopy experiments, cells were plated on cover slips and transfected with the expression vectors of interest using PolyJet, as described above. Twenty-four hours post transfection cells were incubated at room temperature for 2 hours to mature the fluorophore and fixed with 4% paraformaldehyde. Cells were subsequently washed twice with PBS and permeabilized and blocked in 2% bovine serum albumin (BSA) and 0.1% Triton-X in PBS for 2 hour at room temperature. Cells were subsequently stained for two hours with anti-

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DYKDDDK (BioLegend, San Diego, CA) or anti-CD63 (Developmental Studies Hybridoma Bank, The University of Iowa) diluted down to in 2% BSA in PBS, washed twice with 0.2% BSA in PBS and incubated for one and a half hours with the appropriate fluorophore conjugated secondary antibodies (Alexa-Fluor-647 conjugated anti-mouse or anti-Rat; Jackson ImmunoResearch, West Grove, PA) in 0.2% BSA in PBS. Finally, cells were washed twice with PBS and mounted on coverslips with DAPI Fluoromount-G (SouthernBiotech, Birmingham, AL). Cells were imaged on a Leica DMI6000 B at 63X or 100X magnification using the FITC, Cy5 and DAPI filter settings and imaged with a Hamamatsu Photometrics Delta Evolve camera. Images were subsequently deconvolved using the Advanced Fluorescence Deconvolution application on the Leica Application Suite software (Lecia, Wetzlar, Germany). The mean fluorescence intensity of the fluorescence signal was determined using image J [54] and co-localization analysis was conducted using Pearson correlation coefficient between signal in the FITC (eGFP/BiFC) and Cy3 (CD63) channel using the ImageJ plugin JACoP, as described previously [55].

3.4.8 Extracellular vesicle purification

HEK293T cells stably expressing a non-specific or SNX18 targeting shRNA were transfected with the pNL4.3 dG/P eGFP vector using PolyJet, as described above. Forty- eight hours post transfection the cell culture medium was removed and processed to purify extracellular vesicles, while the cells were washed with 1 x PBS followed by lysis and preparation for Western blotting, as described above. To purify extracellular vesicles, culture supernatant was pelleted at 3000 x g for 15 minutes and the supernatant was run through a 2 m filter. Culture supernatant was then concentrated using a 10,000 Da cut-off PeirceTM protein concentrator (Thermo Fisher Scientific). The extracellular vesicles were then precipitated from concentrated supernatants using ExoQuick exosome precipitation solution (FroggaBio), as per the manufactures protocol. Briefly, the exosome precipitation solution was mixed with the culture supernatant and incubate at 4ºC overnight prior to pelleting extracellular vesicles and removing supernatant via subsequent spins at 1500 x g for 30 mins and 5 mins. Pelleted vesicles were then re-suspended in RIPA-Doc buffer

(140 mM NaCl, 10 mM NaH2PO4, 1% NP-40, 0.05% SDS, 0.5% sodium deoxycholate)

181 and mixed with SDS-PAGE sample buffer (0.312 M Tris pH 6.8, 3.6 M 2- Mercaptoethanol, 50% glycerol, 10% SDS) and boiled at 95C for 10 mins.

3.4.9 Transient shRNA knock-down experiments

For the dual infection of Sup-T1 cells with VSV-G pseudotyped viruses, 1 x 106 Sup-T1 cells were suspended in 8 g/mL polybrene and virus preparations in 20% FBS produced as described above from pGFP-C-shLenti vectors encoding different shRNAs. Cells were then spinoculated for four hours at 2880 x g at room temperature and re-suspended in fresh RPMI. The next day, cells were pelleted and re-suspended in 10% RPMI (mock infection) or pNL4.3 dG/P F2A-mStrawberry Nef/dNef pseudovirions in 20% FBS and 8 g/mL polybrene. Forty-eight hours later, cells were analyzed via flow cytometry or Western blotting.

3.4.10 Flow cytometry

For cell surface staining of MHC-I on transduced Sup-T1 cells, cells were washed twice with PBS, followed by fixing in 1% paraformaldehyde for 20 minutes at room temperature. Cells were subsequently washed twice with FACS buffer (1% FBS, 50 mM ethylenediaminetetraacetic acid (EDTA) in PBS) and stained with the appropriate antibody by rocking for 40 minutes at room temperature. Cells were then washed twice with FACS buffer and re-suspended in PBS. For staining of MHC-I 1:25 APC-Cy7-conjugated mouse- anti-HLA-A, B, C (clone W6/32, BioLegend) or an isotype control antibody (APC-Cy7 conjugated mouse IgG2b, κ isotype, clone MOPC-21, BioLegend) was utilized. Cells were analyzed using a BD Biosciences FACSCanto SORP (BD Biosciences). Data analysis was performed using FlowJo software (version 9.6.4, FlowJo LLC, Ashland, OR).

3.4.11 Data analysis

3.4.11.1 In vitro protein capture assays

For in vitro pull-down assays, Western blots were acquired using a C-DiGit chemiluminescent blot scanner (LI-COR) and band intensities were quantified using Image Studio (LI-COR). The band intensities were normalized to the highest intensity in each assay and the mean normalized intensities were analyzed by one-way analysis of variance

182 followed by Bonferroni’s multiple comparison test (Figures 3.1B, 3.2C, E) or a Student’s t-test (Figure 3.3B).

3.4.11.2 Microscopy

For analysis of Nef: SNX18 BiFC and CD63 overlap, the mean (+/-SD) Pearson correlation coefficient between signals in the FITC (eGFP/BiFC) and Cy3 (CD63) were determined using the ImageJ plugin JACoP, as described previously [55]. Means were compared using an Ordinary one-way analysis of variance followed by Tukey’s multiple comparison test. The mean fluorescence intensity of Nef-VC: SNX18-VN and Nef

AxxA75-VC: SNX18-VN BiFC signal was compared using an unpaired Student’s t-test.

3.4.11.3 Transient shRNA knock-down experiments

For analyses of flow cytometry data obtained for dual infected Sup-T1 cells, double positive (GFP+ mStrawberry+) cells we gated on based on fluorescence minus one controls. The relative levels of MHC-I on double positive cells were determined by normalizing MFI levels on cells infected with virus encoding Nef to those infected with virus lacking Nef (dNef), where cell surface levels of MHC-I in cells infected with dNef virus was set to 100. The relative levels of MHC-I on cells infected with virus encoding Nef was then compared between cells infected with virus encoding a non-specific and SNX18-targeting shRNA using a paired t-test.

To quantify the amount of SNX18 in Sup-T1 cells transduced with virus encoding a non- specific and SNX18-targeting shRNA, SNX18 and actin band intensities obtained by Western blotting, as described above, were quantified using Image Studio (LI-COR). The intensity of the SNX18 protein bands were normalized to actin levels from the same sample and normalized to the SNX18 intensity in cells transduced with virus encoding a non- specific shRNA. The mean relative SNX18 levels were then analyzed by one-way analysis of variance followed by Dunnett’s multiple comparison test.

3.4.11.4 Extracellular vesicle purifications

To quantify the amount of Nef in the extracellular vesicle purifications from transfected cells, the Western blot band intensities were quantified using Image Studio (LI-COR). The

183 intensity of Nef from the extracellular fraction was normalized to the intracellular levels of Nef. The relative levels of extracellular Nef in 293TαSNX18 knock-down cells was than normalized to those in 293TNS cells from the same experiment. Mean levels of extracellular Nef purified from 293TαSNX18 and 293TNS cell supernatants was then compared using a Student’s t-test.

3.4.11.5 Statistical analysis

All statistical tests were completed using Graph Pad Prism (Graph Pad Software Inc., La Jolla, CA).

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35. Witte V, Laffert B, Rosorius O, Lischka P, Blume K, Galler G, et al. HIV-1 Nef mimics an integrin receptor signal that recruits the polycomb group protein Eed to the plasma membrane. Mol Cell. 2004;13(2):179-90. 36. Ebsen H, Lettau M, Kabelitz D, Janssen O. Identification of SH3 domain proteins interacting with the cytoplasmic tail of the a disintegrin and metalloprotease 10 (ADAM10). PLoS One. 2014;9(7):e102899. 37. Simons M, Raposo G. Exosomes - vesicular carriers for intercellular communication. Curr Opin Cell Biol. 2009;21(4):575-81. 38. Saksela K, Permi P. SH3 domain ligand binding: What's the consensus and where's the specificity? FEBS Lett. 2012;586(17):2609-14. 39. Jung J, Byeon IJ, Ahn J, Gronenborn AM. Structure, dynamics, and Hck interaction of full-length HIV-1 Nef. Proteins. 2011;79(5):1609-22. 40. Park J, Kim Y, Lee S, Park JJ, Park ZY, Sun W, et al. SNX18 shares a redundant role with SNX9 and modulates endocytic trafficking at the plasma membrane. J Cell Sci. 2010;123(10):1742-50. 41. Mygind KJ, Storiko T, Freiberg ML, Samsoe-Petersen J, Schwarz J, Andersen OM, et al. Sorting nexin 9 (SNX9) regulates levels of the transmembrane disintegrin and metalloproteinase (ADAM)-9 at the cell surface. J Biol Chem. 2018. 42. Shalem O, Sanjana NE, Hartenian E, Shi X, Scott DA, Mikkelson T, et al. Genome- scale CRISPR-Cas9 knockout screening in human cells. Science. 2014;343(6166):84-7. 43. Sami Saribas A, Cicalese S, Ahooyi TM, Khalili K, Amini S, Sariyer IK. HIV-1 Nef is released in extracellular vesicles derived from astrocytes: evidence for Nef- mediated neurotoxicity. Cell Death Dis. 2017;8(1):e2542. 44. Lee JH, Ostalecki C, Zhao Z, Kesti T, Bruns H, Simon B, et al. HIV Activates the Tyrosine Kinase Hck to Secrete ADAM Protease-Containing Extracellular Vesicles. EBioMedicine. 2018;28:151-61. 45. Moreno-Gonzalo O, Villarroya-Beltri C, Sanchez-Madrid F. Post-translational modifications of exosomal proteins. Front Immunol. 2014;5. 46. Dirk BS, Pawlak EN, Johnson AL, Van Nynatten LR, Jacob RA, Heit B, et al. HIV- 1 Nef sequesters MHC-I intracellularly by targeting early stages of endocytosis and recycling. Sci Rep. 2016;6:37021. 47. Dikeakos JD, Atkins KM, Thomas L, Emert-Sedlak L, Byeon IJL, Jung JW, et al. Small Molecule Inhibition of HIV-1-Induced MHC-I Down-Regulation Identifies a Temporally Regulated Switch in Nef Action. Mol Bio Cell. 2010;21(19):3279- 92. 48. Piguet V, Wan L, Borel C, Mangasarian A, Demaurex N, Thomas G, et al. HIV-1 Nef protein binds to the cellular protein PACS-1 to downregulate class I major histocompatibility complexes. Nat Cell Biol. 2000;2(3):163-7.

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Chapter 4 4 General Discussion 4.1 Thesis overview

The work presented in this thesis aimed to provide novel insights into the mechanisms by which the HIV-1 accessory proteins Nef and Vpu alter the host cellular environment. We posited that the functions of Nef and Vpu are mediated through the ability of these viral proteins to hijack host cellular proteins, specifically those involved in the trafficking of cargo to different cellular locales. In Chapter 2, we observed Nef- and Vpu-mediated downregulation of the immune cell co-stimulatory molecule CD28 and also implicated host cell protein binding motifs located within the viral proteins in this process. In Chapter 3, we identified a novel host cell binding partner of Nef, the membrane trafficking regulator SNX18, which we propose is hijacked by Nef via a host cell protein binding motif to mediate dysregulation of the immune cell receptor MHC-I.

4.1.1 Chapter 2: Findings, limitations and future work

In Chapter 2, we set out to examine how the HIV-1 accessory proteins alter the co- stimulatory molecule CD28. Prior studies suggested that Nef downregulates cell surface levels of CD28 [1, 2], however it was unknown how Nef was mediating this process. Moreover, the cell surface downregulation of CD28 by the accessory protein Vpu had only been previously documented in a large-scale screen of receptors that are modulated by Nef and Vpu [3]. Given that Nef and Vpu alter the subcellular localization of other cell surface receptors by hijacking cellular trafficking machinery, we hypothesized that this is how Nef and Vpu mediate their effects on CD28. Overall, the work in this chapter has given us an unprecedented view of the mechanism of Nef- and Vpu-mediated downregulation of endogenous CD28 in CD4+ T cells. Moreover, this is the first examination of Vpu-mediated CD28 downregulation.

4.1.1.1 The subcellular fate of CD28 upon HIV-1 infection

We observed that in infected cells HIV-1 Nef and Vpu mediate decreases in cell surface levels of CD28, as well as decreases in total CD28 protein levels (Figure 2.1; Figure 2.8).

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These decreases in CD28 were accompanied by increased CD28 localization to lysosomal compartments (Figure 2.9). Moreover, our results support a mechanism whereby total CD28 protein levels are decreased by the viral proteins in an endosomal acidification- dependent manner (Figure 2.10). Thus, we concluded that the mechanism by which Nef and Vpu decrease cell surface CD28 is mediated, at least in part, by decreasing total protein levels via mis-localizing CD28 to acidified degradative compartments. Similar mechanisms are utilized by Nef and Vpu to downregulate cell surface CD4 and BST-2, respectively [4-9].

4.1.1.2 Nef and Vpu motifs in CD28 downregulation

We hypothesized that Nef and Vpu mediate their effects within the cell by hijacking cellular trafficking proteins. Therefore, we examined the consequences of introducing mutations in motifs that were previously implicated in mediating interactions between the viral proteins and host cellular proteins. We observed that mutations in specific virus: host protein interaction motifs inhibited downregulation of CD28 at both the cell surface and total protein levels, namely mutation of the Nef LL165 and DD175 motifs (Figure 2.13;

Figure 2.14) and the Vpu S52/56 motif (Figure 2.15). Given that these motifs mediate interactions with host cell proteins to downregulate other cellular receptors [10-14], these findings support our hypothesis that CD28 downregulation is facilitated by Nef and Vpu hijacking host cellular trafficking proteins. To more directly test this hypothesis, we sought to knock-down or inhibit specific cellular trafficking proteins and examined the effects on CD28 downregulation. We did not observe any impairment in Nef-mediated cell surface CD28 downregulation upon knock-down of the membrane trafficking adaptor AP-2 α and μ subunits or overexpression of a dominant negative form of the membrane trafficking regulator SNX9 (Figure 2.17; Figure 2.18). As described in section 2.3, these experiments may be limited by the fact that host cellular trafficking proteins and pathways can be redundant or capable of compensating for the loss of specific trafficking proteins. Indeed, previous work suggests that upon knock-out of adaptor protein subunits, namely AP-1µ, cells may compensate for this loss by upregulating other membrane trafficking proteins [15]. Further studies would benefit from simultaneous knock-down or inhibition of multiple membrane trafficking proteins, though this may be technically difficult.

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Moreover, the effects of knock-downs may be increasingly apparent if more efficient decreases in protein levels are obtained, which could be achieved through the use of gene editing technologies to knock-out genes of interest [16, 17]. Nonetheless, it would be useful to validate our AP-2 knock-downs or SNX9 dominant negative protein by determining if they impede the Nef-mediated downregulation of receptors believed to be downregulated in an AP-2-dependent manner. In addition to the trafficking mediators explored herein, the importance of the Nef LL165 and DD175 motifs and the Vpu S52/56 motif also suggest potential involvement of AP-1, LC3C and β-TrCP in Vpu-mediated downregulation [13, 18, 19], and the v-ATPase in Nef-mediated CD28 downregulation [20, 21]. Thus, these host proteins represent strong candidates for further exploration.

4.1.1.3 Conservation of Vpu-mediated CD28 downregulation

In addition to exploring the mechanism of Nef- and Vpu-mediated CD28 downregulation, we sought to determine if the ability of Vpu to downregulate CD28 was a feature unique to the HIV-1 lab adapted strain NL4.3, or if Vpu proteins from other HIV-1 viruses can downregulate CD28. This is important as the HIV-1 epidemic is comprised of a large array of diverse viruses [22-24]. Thus, if Vpu-mediated CD28 downregulation is observed for multiple distinct viruses, our findings may be more applicable to the epidemic as a whole. We observed that the downregulation of CD28 in CD4+ Sup-T1 cells can be mediated by Vpu proteins derived from the HIV-1 strain B.JFRL and a consensus C Vpu protein (Figure 2.21). However, our analysis was limited to a small number of Vpu proteins. Furthermore, we examined the ability of these Vpu proteins to downregulate CD28 in the context of a viral backbone derived from NL4.3. Therefore, it would be worthwhile for future studies to examine Vpu-mediated downregulation of CD28 by a variety of viruses using full length molecular clones. Additionally, the dileucine-like motif of NL4.3 Vpu was required for cell surface CD28 downregulation (Figure 2.15). However, this motif is absent in the B.JRFL Vpu protein which also mediated efficient CD28 downregulation (Figure 2.21; Figure 2.24). Thus, it will be important to examine if the host cell proteins required to mediate CD28 downregulation by these different Vpu proteins are equivalent. Nonetheless, the finding that non-NL4.3 Vpu proteins can downregulate CD28 validates that Vpu-

191 mediated downregulation of CD28 could be a universal feature across the epidemic, supporting the importance of future research.

4.1.1.4 The function of CD28 downregulation

Given the central role of CD28 in signaling to mediating T cell activation, we proposed that CD28 downregulation by the viral proteins may impact the activation of T cells. We observed that in stimulated cells Nef promoted increased activation, while Vpu inhibited activation (Figure 2.23). These effects could be due to changes in the cell surface levels of CD28, but may also be attributable to the numerous other effects of the viral proteins on immune cell activation, as described in section 1.5.5. Future experiments are necessary to more conclusively link downregulation of CD28 to cell activation; however, this is challenging given the fact that there are no known mutations in the viral proteins that can be made to specifically negate CD28 downregulation without impeding other functions. Thus, it is difficult to inhibit CD28 downregulation without hindering other viral protein functions which could impact cellular activation. Nevertheless, it would be interesting for future experiments to determine if the amount of phosphorylated, and thus signaling competent, CD28 differs in stimulated CD4+ T cells infected with virus encoding or lacking Nef and Vpu. Overall, we postulate that changes in cell surface CD28 mediated by the viral proteins contribute to the overall dysregulation of cellular activation by limiting detection of extrinsic stimuli.

4.1.1.5 Redundancy in CD28 downregulation ability

The ability of two HIV-1 proteins to counteract the same cell surface molecules underscores the importance of this function. In this manner, HIV-1 has a back-up plan to ensure that CD28 is downregulated to some extent. Future experiments may be useful to determine if there are differences in CD28 downregulation at different times during infection. In our experiments in Sup-T1 cells, we examined downregulation at forty-eight hours post infection. However, Nef is expressed early during infection, even from unintegrated HIV-1 DNA [25], while Vpu is a “late gene”, as its translation is dependent on prior expression of the viral Rev protein [26]. Therefore, it would be interesting to examine whether the effects of Vpu are more pronounced if the infection proceeds for an

192 increased amount of time, or if downregulation by Nef and Vpu differ over the course of a number of days. Moreover, given that vpu and nef genes can compensate for each other, we could examine whether there are viruses encoding Nef proteins that poorly downregulate CD28, but encode Vpu proteins that compensate for this defect by downregulating CD28 more efficiently, or vice-versa. Overall, the ability of multiple viral proteins to alter cell surface expression of CD28 suggests that this function is significant.

4.1.1.6 Summary and future directions

In taking together the data presented in Chapter 2 and previous findings, we propose a model of how Nef and Vpu modulate CD28 within infected CD4+ T cells (Figure 4.1). Nef decreases both cell surface and total cellular protein CD28 levels (Figure 2.1; Figure 2.8). It remains unknown if Nef targets CD28 at the cell surface, though it has been proposed that Nef accelerates the internalization of cell surface resident CD28 [1]. Moreover, it remains unclear if Nef mediated downregulation of CD28 occurs through binding to the cytoplasmic tail of CD28, similar to how Nef downregulates MHC-I and CD4 [27-29]. We observed that mutation of the Nef LL165 or DD175 motifs inhibits the ability of Nef to downregulate CD28 (Figure 2.14), suggesting that downregulation of CD28 by Nef may require host cellular proteins known to bind to Nef via these motifs, such as AP-2 or v- ATPase [20, 30]. However, future studies are required to confirm if these proteins are necessary for downregulation of CD28. Finally, we observed that in the absence of Nef there is less CD28 trafficked to LAMP-1 positive compartments (Figure 2.9), suggesting that Nef mediates trafficking to, and potentially degradation of CD28 within, lysosomes. Future studies to better map the trafficking pathway and proteins required for CD28 downregulation by Nef are required. In the case of Vpu, our findings indicate that the S52/56 motif, which mediates binding to β-TrCP [13], is critical for decreasing cell surface and total cellular CD28 protein levels (Figure 2.15). Thus, the mechanism of Vpu-mediated CD28 downregulation may require β-TrCP recruitment. However, it remains unknown if Vpu is hijacking CD28 at the endoplasmic reticulum, as it does CD4 [13, 31], or at the cell surface or intracellular compartments, as it does BST-2 [4, 32]. Moreover, future work is needed to determine if Vpu interacts with CD28, to identify additional host cellular proteins necessary for Vpu-mediated CD28 downregulation, and to further understand how Vpu

193 contributes to increased lysosomal localization of CD28 (Figure 2.9). Given the importance of CD28 in cell activation [33, 34], Nef- and Vpu-mediated decreases in cellular CD28 levels may play a significant role in altering the activation status of the cell. We observed opposite effects of Nef and Vpu on cellular activation (Figure 2.23), which may be a result of changes in cellular CD28 levels in addition to the other effects on cellular activation that are mediated by the accessory proteins (Section 1.5.5). Overall, changes in cellular activation are important, as they can influence antiviral responses and HIV-1 viral gene transcription [35-37].

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Figure 4.1 Models of Nef- and Vpu-mediated CD28 downregulation

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Proposed models of CD28 downregulation by (a) Nef and (b) Vpu. (A) Newly translated CD28 is trafficked to the cell surface via the TGN. In the presence of Nef, CD28 may be targeted at the cell surface to increase endocytosis [1], contributing to decreases in cell surface levels of CD28. The LL165 and DD175 motifs, which facilitate binding of Nef to AP-2 and v-ATPase [20, 30], are critical for CD28 downregulation. In the presence of Nef, CD28 localizes to lysosomes. (B) Vpu mediates decreases in cell surface and total CD28, but it remains unknown if CD28 is targeted by Vpu shortly after translation in the ER, at the cell surface, or at intracellular compartments. The β-TrCP-binding motif of

Vpu, S52/56, is critical for Vpu-mediated downregulation of CD28. In the presence of Vpu, CD28 localizes to lysosomes.

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4.1.2 Chapter 3: Findings, limitations and future work

We propose that HIV-1 Nef and Vpu mediate their functions by interacting with host cellular membrane trafficking proteins and in Chapter 3 we characterize a novel Nef: host protein interaction and examine its importance in Nef-mediated MHC-I downregulation. Given the sheer complexity of membrane trafficking, we hypothesized that numerous host cell proteins are critical for Nef to downregulate MHC-I, including proteins not yet implicated in this process. We were specifically interested in the sorting nexin proteins, which stems from our previously published work that illustrates a SNX18: Nef complex is formed that localizes to compartments containing AP-1 [38], a trafficking adaptor that Nef hijacks to downregulate MHC-I [27].

4.1.2.1 The Nef: SNX18 interaction interface

In this chapter, we illustrated for the first time that SNX18 is a direct Nef binding partner (Figure 3.1). We have mapped the interaction between SNX18 and Nef to the SH3 domain of SNX18 (Figure 3.2). Moreover, the in vitro direct binding, as well as the interaction between SNX18 and Nef in cells, was dependent on the Nef PxxP75 motif (Figure 3.3; Figure 3.4), which is unsurprising given its documented role in interacting with other SH3 domain containing proteins [39, 40]. Notably, the interaction between SNX18 and Nef in cells, as quantified by BiFC fluorescence intensity, was not completely abolished by mutation of the Nef PxxP75 motif (Figure 3.4). This indicates that there may be residues within Nef outside of this motif that contribute to this interaction, or that additional host cell proteins may facilitate this interaction in cells. A candidate host cell protein that could facilitate SNX18 and Nef binding is AP-1, which binds to both SNX18 and Nef [27, 41, 42], and was previously found to co-localize with the Nef: SNX18 complex in cells [38]. Therefore, it would be of interest for future studies to determine if mutation of the Nef

EEEE65 motif, which mediates AP-1 interactions [27, 43], alters the intensity of the Nef: SNX18 BiFC signal. Moreover, it would be interesting to determine if Nef inhibitors such as 2c, which targets the Nef PxxP75: SFK SH3 domain interaction interface [44], influence the Nef: SNX18 interaction, which could contribute to the ability of these inhibitors to block Nef-mediated MHC-I downregulation [44]. In summary, we have identified a novel Nef-binding host protein which we posit may facilitate Nef-mediated functions.

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4.1.2.2 The Nef: SNX18 interaction in MHC-I downregulation

Overall, we propose that the Nef: SNX18 interaction functions to facilitate Nef-mediated MHC-I downregulation. This is supported by our findings that the shRNA knock-down of SNX18 reduced Nef-mediated MHC-I downregulation (Figure 3.5). While the inhibition of downregulation was consistent, the change was small. However, as discussed in section 3.3, given that SNX18 and SNX9 exhibit some redundant functions we cannot exclude the possibility that despite the lack of a direct SNX9: Nef interaction SNX9 may compensate for the loss of SNX18, thereby reducing the effect of SNX18 knock-down on Nef-mediated MHC-I downregulation. Future experiments could examine if knock-down of both SNX18 and SNX9 leads to a greater deficit in Nef-mediated MHC-I downregulation. Moreover, while we would like to illustrate that a physical interaction between SNX18 and Nef is explicitly required for Nef-mediated MHC-I downregulation it is technically challenging given the fact that mutations in the interaction interface also inhibit other protein interactions. Indeed, mutation of the Nef PxxP75 motif has already been implicated in inhibiting MHC-I downregulation through its abolition of the Nef: SFK interaction [45, 46]. Nonetheless, our data suggest SNX18 plays a role in decreasing cell surface levels of MHC-I in the presence of Nef.

Given our interest in the role of host cell proteins in the mechanisms of Nef function, we sought to define SNX18’s specific role during MHC-I downregulation. Our findings indicated that Nef-containing extracellular vesicles are secreted despite reductions in SNX18 protein levels (Figure 3.7). Thus, we do not believe that SNX18 is promoting decreased cell surface levels of MHC-I by facilitating Nef-mediated secretion of vesicles containing MHC-I. However, as described in section 3.3, future experiments to define the components within our extracellular vesicle purifications would be beneficial to determine the purity of our preparations, as we have not fully defined the characteristics of our purified content, nor have we defined the biogenesis pathway from which these vesicles originated. This could be completed by electron microscopy or nanoscale flow cytometry to determine the size and physical properties of the purified content. In addition, we observed that the SNX18: Nef BiFC complex was present at CD63 positive compartments (Figure 3.6). Co-localization of the SNX18: Nef BiFC signal with the CD63 marker could

198 suggest that this interaction is occurring at locations where vesicles destined for secretion accumulate, however late endosomes marked by CD63 can also contain cargo destined for other intracellular compartments. Therefore, the SNX18: Nef interaction may simply be occurring at late endosomes, a compartment through which Nef and MHC-I traffic during MHC-I downregulation [47]. In this way, SNX18 may function to mediate trafficking of MHC-I from the cell surface to the TGN, where MHC-I is sequestered in the presence of Nef [44, 47, 48]. Future experiments are necessary to further map the cellular location of the SNX18: Nef interaction and examine if Nef-mediated sequestration of MHC-I in the TGN is altered in the absence of SNX18. Ultimately, we propose that the binding of Nef to SNX18 facilitates Nef-mediated mis-localization of MHC-I within infected cells.

4.1.2.3 Summary and future directions

The data presented in Chapter 3 sheds light on the mechanism of how Nef contributes to decreases in cell surface levels of MHC-I, which prevents CD8+ cytotoxic T cell detection of virally infected CD4+ T cells (Figure 4.2; [49, 50]). Nef promotes endocytosis of cell surface MHC-I through an Arf1- or Arf6-dependent mechanism [51, 52]. Trafficking of MHC-I away from the cell surface is dependent on Nef’s ability to bind AP-1 and PACS- 1 [53, 54]. This process may be further facilitated by the membrane trafficking mediator SNX18, which we have identified as a novel Nef-binding partner (Figure 3.1). This interaction is mediated by the PxxP75 motif of Nef binding to the SNX18 SH3 domain (Figure 3.2; Figure 3.3). SNX18, like Nef, binds to AP-2 [30]. Upon reductions in SNX18 protein levels, Nef-mediated MHC-I downregulation is reduced (Figure 3.5). However, future studies are needed to confirm the importance of SNX18 in MHC-I downregulation.

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Figure 4.2 Model of Nef-mediated evasion of cytotoxic T cell responses

Nef mediates decreases in CD4+ T cell surface levels of MHC-I, preventing detection by CD8+ cytotoxic T cells (left; [49, 50]). MHC-I downregulation is facilitated by Arf1- or Arf6-mediated endocytosis of cell surface MHC-I (right; [51, 52]). MHC-I is then trafficked to the trans-Golgi network in a manner dependent on Nef, PACS-1 and AP-1 [53, 54]. This trafficking step may be facilitated by SNX18 binding to Nef via a

PxxP75:SH3 domain interaction.

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4.2 Significance

Overall, this thesis provides novel insight into how host cellular receptors are mis-localized by the HIV-1 accessory proteins Nef and Vpu. These alterations in the location of host cell proteins undoubtedly alter how cells function during HIV-1 infection. In the case of the receptors of interest to this work, MHC-I and CD28, the decreases in cell surface levels of these immune cell receptors may alter immune responses, despite the effects on these receptors being modest. Indeed, it is well established that HIV-1-mediated decreases in cell surface levels of MHC-I inhibit detection and killing of virally infected cells [50]. Specifically, cells infected with HIV-1 encoding a functional nef gene are more effeciently killed by CTLs than by cells infected with HIV-1 enconding Nef unable to downregulate MHC-I [50, 55]. Moreover, clinical evidence indicates that MHC-I downregulation ability may correlate with HIV-1 disease progression [56, 57]. In contrast, decreases in cell surface CD28 likely inhibit CD28 receptor activation. This may contribute to inhibiting the responsiveness of cells to external stimuli, in conjunction with the countless other Nef- and Vpu-mediated modifications described in section 1.5.5 that influence immune cell activation. Changes in cellular activation are important during HIV-1 infection, given that HIV-1 transcription is driven by transcription factors that are activated during cell activation, thus influencing viral replication (reviewed in [58]). The importance of altering the subcellular localization of these receptors during a HIV-1 infection is supported by the functional redundancy in Nef and Vpu, whereby both viral proteins contribute to decreases in cell surface MHC-I and CD28. In this way, the virus has a contingency plan to ensure these functions are fulfilled.

4.2.1 Implications for HIV-1 treatment

The functional redundancy of Nef and Vpu emphasizes their significance during infection and implies they could be effective therapeutic targets. However, the development of HIV- 1 therapeutics against the accessory proteins Nef and Vpu has been limited. This is likely owing to the fact that therapeutics inhibiting Nef and Vpu would need to be utilized in combination with other anti-retroviral therapies, as HIV-1 can still replicate in the absence of these proteins. Moreover, given that Nef and Vpu are not enzymes, they cannot be counteracted by targeting catalytic sites or inhibiting enzymatic activity, as has been done

201 for most of the previously developed anti-retrovirals [59]. However, Nef and Vpu functions, including MHC-I and CD28 downregulation, can be targeted based on our knowledge of their interactions with host cell proteins. Specifically, as druggable targets we can utilize the interaction interfaces between the virus and host proteins that are crucial for accessory protein functions. By preventing these binding events through the use of small molecule inhibitors, we can prevent Nef- and Vpu-mediated functions. Importantly, the Nef and Vpu host cell protein binding motifs are good targets as they are generally made up of conserved residues [60-64]. This conservation would allow for targeting of these accessory proteins in a manner applicable across the HIV-1 epidemic.

In addition to being potential targets for new HIV-1 therapeutics, the presence of these proteins needs to be accounted for when developing HIV-1 cure strategies, as the functions of Nef and Vpu alter the ability of the immune system to detect and eliminate HIV-1 infection. Obtaining a cure for HIV-1 for those that are currently infected is of importance due to the large cost of remaining on life-long anti-retroviral treatment and the co- morbidities associated with HIV-1 infection [65, 66]. Currently, the greatest challenge to curing HIV-1 infections is the presence of latently infected cells which remain “hidden” from both the immune system and anti-retroviral therapy by remaining in a resting state where HIV-1 gene expression does not occur [67]. Nevertheless, these latently infected cells can be reactivated to begin producing virus. Thus, these cells must be eradicated in order to eliminate HIV-1 infection. A major approach being used to eliminate latently infected cells is reactivating or “shocking” latently infected cells, such that they begin expressing viral proteins. This process is followed by elimination or “killing” of these reactivated cells via the immune system or viral cytopathic effects [68]. HIV-1 cure approaches such as this shock-and-kill method that rely on the immune system to eliminate the virus will need to address the fact that if the viral accessory proteins are present, they will continue to inhibit the ability of the immune system to eliminate the virus. In this way, the use of inhibitors to block the immune evasion capabilities of the accessory proteins has been suggested to be critical for successful elimination of latently infected cells [69].

It has been proposed that molecules such as the Nef: SFK interaction inhibitor 2c, which inhibits Nef-mediated MHC-I downregulation [44], are good candidates to consider for

202 preventing immune evasion during attempts to eliminate latently infected cells [69]. However, we must also consider the fact that the virus has a back-up plan, with Vpu also contributing to MHC-I downregulation [70, 71]. In addition to inhibiting Nef-mediated MHC-I downregulation, the targeting of Nef and Vpu’s ability to hide from the immune system has also been facilitated through the use of small molecules known as CD4 mimetics. Specifically, Nef- and Vpu-mediated CD4 downregulation prevents the exposure of CD4-induced epitopes on cell surface envelope to ADCC-mediating antibodies which serves as a signal for NK cell-mediated lysis of infected cells [72, 73]. CD4 mimetics can overcome this process by mimicking the presence of cell surface CD4. Specifically, they bind to cell surface envelope and stimulate exposure of CD4-induced epitopes, thereby increases NK cell-mediated killing of infected cells [74].

Ultimately, if we are to successfully cure HIV-1 by eliminating the latent reservoir, we need to consider how we can overcome the incredibly effective and redundant abilities of the HIV-1 accessory proteins Nef and Vpu to evade the immune system.

4.2.2 Concluding remarks

The ability of HIV-1 Nef and Vpu to exhibit functional redundancy is through their shared ability to usurp broad cellular mechanisms that determine protein localization within the cell. Both Nef and Vpu bind to and hijack host cellular proteins to link their target proteins, often cell surface molecules, with the machinery that alter their primary location in the cell. This frequently results in the sequestration of target proteins intracellularly or their trafficking to sites of protein degradation. The viral proteins facilitate this by mimicking canonical host cell protein signals that determine protein localization, such as those that mediate binding of membrane trafficking adaptors or the ubiquitin ligase machinery. Ultimately, functions similar to those exhibited by HIV-1 Nef and Vpu allow for viruses to commandeer host cell machinery to make cells optimally efficient viral production factories.

The study of viruses has broad implications. Notably, researching the fundamental mechanisms of clinically relevant viruses is critical to improving prevention, detection and treatment of pathogenic viruses. Additionally, since viruses require host cells to replicate,

203 these basic studies also enhance our understanding of fundamental host cell functions. Therefore, virology research inevitably leads to an increased understanding of cellular biology and biochemistry. In this way, viruses and viral proteins can also serve as research tools, effectively expanding our understanding of how cellular processes are mediated in the absence of viral infection.

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Appendix 1 Copyright permission for published work

Appendix 2 Ethics statement

Informed consent was obtained from all subjects according to an ethics protocol approved by The University of Western Ontario Research Ethics Board for Health Sciences Research Involving Human Subjects (HSREB; project ID: 103782; project title: Nef is an HIV-1 protein at the forefront of pathogenesis).

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Curriculum Vitae

Emily N. Pawlak

Education

Doctorate of Philosophy May/2015 – current Supervisor: Dr. JD Dikeakos Department of Microbiology and Immunology Schulich School of Medicine and Dentistry The University of Western Ontario, London, Ontario, Canada

Master’s of Science September/2013 – April/2015 Supervisor: Dr. JD. Dikeakos Department of Microbiology and Immunology Schulich School of Medicine and Dentistry The University of Western Ontario, London, Ontario, Canada Completed PhD candidacy examination with distinction

Bachelor of Medical Sciences (Honors) September/2009 – April/2013 Honors Specialization in Biochemistry of Infection and Immunity Schulich School of Medicine and Dentistry The University of Western Ontario, London, Ontario, Canada Graduation with Distinction Western Scholars Designation Western University Gold Medal Recipient Dean’s honor list – 2010, 2011, 2012, 2013

Peer-Reviewed Publications

Van Nynatten L.R., Johnson A.L., Dirk B.S., Pawlak E.N., Jacob R.A., Haeryfar S.M. & Dikeakos J.D. 2018. Identification of Novel Subcellular Localization and Trafficking of HIV-1 Nef Variants from Reference Strains G (F1.93.HH8793) and H (BE.93.VI997). Viruses 10(9)

Pawlak E.N., Dirk B.S., Jacob R.A., Johnson A.L., & Dikeakos J.D. 2018. The HIV-1 accessory proteins Nef and Vpu downregulate total and cell surface CD28 in CD4+ T cells. Retrovirology 15:6

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Jacob R.A., Johnson A.L., Pawlak E.N., Dirk B.S., Van Nynatten L.R., Haeryfar S.M. & Dikeakos J.D. 2017. The interaction between HIV-1 Nef and adaptor protein-2 reduces Nef-mediated CD4+ T cell apoptosis. Virology 509:1-10

Dirk B.S., Pawlak E.N., Johnson AL, Van Nynatten LR, Jacob RA, Heit B & Dikeakos JD. 2016. HIV-1 Nef sequesters MHC-I intracellularly by targeting early stages of endocytosis and recycling. Scientific Reports 6:37021

Dirk B.S., Jacob R.A., Johnson A.L., Pawlak E.N., Cavanagh P.C., Van Nynatten L., Haeryfar S.M. & Dikeakos J.D. 2015. Viral bimolecular fluorescence complementation: a novel tool to study intracellular vesicular trafficking pathways. PLoS One 10(4):e0125619

Pawlak E.N. & Dikeakos J.D. 2015. HIV-1 Nef: A Master Manipulator of the Membrane Trafficking Machinery. Biochimica et Biophysica Acta General Subjects 1850: 733-741.

Conference Presentations

Pawlak E.N., Dirk B.S., Jacob R.A., Johnson A.L., & Dikeakos J.D. The immune cell surface receptor CD28 is hijacked by the HIV-1 accessory proteins Nef and Vpu. (10 May 2018) London Health Research Day, London ON (poster presentation)

Pawlak E.N., Dirk B.S., Jacob R.A., Johnson A.L., & Dikeakos J.D. The HIV-1 accessory proteins Nef and Vpu downregulate the immune cell surface receptor CD28 in CD4+ T cells. (26-29 April 2018) Canadian Conference on HIV/AIDS Research, Vancouver BC (selected oral presentation)

Pawlak E.N. & Dikeakos J.D. (24-28 June 2017) The HIV-1 accessory proteins Nef and Vpu downregulate the cell surface receptor CD28. American Society for Virology Annual Meeting, Madison WI (selected oral presentation)

Pawlak E.N. & Dikeakos J.D. (6-9 June 2017) The HIV-1 Accessory Proteins Nef and Vpu Hijack Membrane Trafficking to Downregulate the Immune Cell Surface Receptor CD28. Canadian Student Health Research Forum, Winnipeg MB (poster presentation, nominated for participation by Schulich School of Medicine and Dentistry)

Pawlak E.N., Van Nynatten L.R. & Dikeakos J.D. (6-9 April 2017) Investigating the Role of Membrane Trafficking Regulator Sorting Nexin Proteins in HIV-1 Nef-mediated Extracellular Vesicle Release. Annual Canadian Conference on HIV/AIDS Research, Montreal QC (poster presentation)

Pawlak E.N. & Dikeakos J.D. (28 March 2017) The HIV-1 accessory proteins Nef and Vpu hijack membrane trafficking to downregulate the immune cell surface receptor CD28. London Health Research Day, London ON (selected oral presentation, *awarded prize)

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Pawlak E.N. & Dikeakos J.D. (23 September 2016) The HIV-1 accessory protein Nef hijacks cell surface receptor trafficking. Infection and Immunity Research Forum, London ON (poster presentation)

Pawlak E.N. & Dikeakos J.D. (29 March 2016) The HIV-1 Nef protein hijacks cell surface receptor trafficking. London Health Research Day, London ON (poster presentation)

Pawlak E.N. & Dikeakos J.D. (12-16 December 2015) The role of membrane trafficking regulator sorting nexin proteins in cell surface receptor downregulation mediated by the HIV-1 accessory protein Nef. American Society for Cell Biology Annual Meeting, San Diego CA (poster presentation)

Pawlak E.N. & Dikeakos J.D. (6 November 2015) Investigating the role of membrane trafficking regulator sorting nexin proteins in HIV-1 Nef-mediated cell surface receptor downregulation. Infection and Immunity Research Forum, London ON (poster presentation *awarded prize)

Pawlak E.N. & Dikeakos J.D. (11-15 July 2015) Investigating the role of membrane trafficking regulator sorting nexin proteins in HIV-1 Nef-mediated cell surface receptor downregulation. American Society for Virology Annual Meeting, London ON (selected oral presentation)

Pawlak E.N. & Dikeakos J.D. (3 May 2015) Exploring the Role of Membrane Trafficking Regulator Sorting Nexin Proteins in HIV-1 Nef MHC-I Downregulation. Annual Canadian Conference on HIV/AIDS Research, Toronto ON (poster presentation)

Pawlak E.N. & Dikeakos J.D. (1 April 2015) A novel protein interaction between the membrane trafficking regulator SNX18 and the HIV-1 accessory protein Nef. London Health Research Day, London ON (selected oral presentation)

Pawlak E.N. & Dikeakos J.D. (6 November 2014) Exploring a novel protein interaction between HIV-1 Nef and the membrane trafficking regulator SNX18 and HIV-1 Nef. Infection and Immunity Research Forum, London, ON (poster presentation)

Pawlak E.N. & Dikeakos J.D. (18 March 2014) Examining the interaction between the membrane trafficking regulator SNX18 and HIV-1 Nef. London Health Research Day, London, ON (poster presentation)

Pawlak E.N. & Dikeakos J.D. (1 November 2013) Molecular dissection of the interaction between HIV-1 Nef and the membrane trafficking regulator SNX18, Infection and Immunity Research Forum, London, ON (poster presentation)

Teaching Experience

Microbiology and Immunology 4300A – Clinical Immunology September – December / 2014; 2015; 2016; 2017

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The University of Western Ontario, London, Ontario Canada *nominated for a graduate teaching award, 2016 & 2017

Scholarships and Awards

➢ Academic scholarship, Canadian Conference on HIV/AIDS Research, April 2018 ➢ Travel award, American Society for Virology, June 2017 ($500) ➢ Oral Presentation Award, London Health Research Day, April 2017 ($600) ➢ Doctoral Excellence Research Award, The University of Western Ontario - $10,000/year, September/2016- current ➢ Alexander Graham Bell Canada Graduate Scholarship, NSERC - $35,000/year for three years, May/2016- current ➢ Queen Elizabeth II Scholarship, Ministry of Training, Colleges and Universities – May/2016 – April/2017 ($15,000)- Declined ➢ Poster prize, Infection and Immunity Research Forum, graduate student over 6 months category, 2015 $100 ➢ 2015 Dr. Frederick W. Luney Graduate Entrance Fellowship, $3000 (September 2015) ➢ 2014 Dr. John Robinson Graduate Scholarship, $1300 (September 2015) ➢ Institute Community Support (Spring 2015) Travel Award – Canadian Institutes of Health Research Institute Community Support (Spring 2015) ($2500) ➢ Ontario Graduate Scholarship (OGS), Ministry of Training, Colleges and Universities – May/2015 – April/2016 ($15000) ➢ Microbiology and Immunology Graduate Travel Award, Department of Microbiology and Immunology, Western University – May/2015 ($1000) ➢ Ontario Graduate Scholarship (OGS), Ministry of Training, Colleges and Universities – May/2014 – April/2015 ($15000 Declined) ➢ Frederick Banting and Charles Best Canada Graduate Scholarship, Canadian Institutes of Health Research Masters Award – May/2014 – April/2015 ($17,500) ➢ Dr. Frederick W. Luney Graduate Entrance Fellowship, Department of Microbiology and Immunology graduate entrance scholarship - 2013 ($2000)