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2015

Insight into -Mediated Signaling via the Discovery of a Novel Isoform With Biologically Distinct Properties

Luis Jose Cocka University of Pennsylvania, [email protected]

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Recommended Citation Cocka, Luis Jose, "Insight into Tetherin-Mediated Signaling via the Discovery of a Novel Isoform With Biologically Distinct Properties" (2015). Publicly Accessible Penn Dissertations. 1661. https://repository.upenn.edu/edissertations/1661

This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/1661 For more information, please contact [email protected]. Insight into Tetherin-Mediated Signaling via the Discovery of a Novel Isoform With Biologically Distinct Properties

Abstract Viruses are obligate, intracellular pathogens that hijack host machinery to replicate. Innate immunity is the first line of defense against such perceived threats through recognition of broadly conserved pathogen signatures. Tetherin (also known as BST2/CD317/HM1.24) is an innate immune factor that senses and restricts egress, the final step of viral replication. Tetherin potently reduces cell-free virus spread by indiscriminately “tethering” particles at the cell surface via direct anchoring to the host membrane. The majority of previous studies on Tetherin focused on elucidating the minimal structural features necessary for tethering viral particles and understanding how viruses counter Tetherin function. While the cytoplasmic tail of Tetherin is dispensable for restricting virus release in the absence of certain viral antagonists, our work, in part, has focused on conserved residues within the tail, hypothesizing these conserved features could provide insight into previously under-studied biology, including a poorly characterized signaling function. In Chapter 2 we show that human Tetherin can exist as two alternatively translated isoforms [long (l-) and short (s-)]. s-Tetherin lacks the first 12 amino acids, a difference that confers functional differences. We found s-Tetherin to be exquisitely resistant to downregulation by the best characterized Tetherin antagonist, HIV-1 Vpu. l-Tetherin, which is sensitive to Vpu, was shown to be a potent activator of NF-κB. A dual-tyrosine motif, unique to l-Tetherin, was identified as an important determinant. In Chapter 3 we describe additional determinants of signaling. Interestingly, we identified compensatory mutations that produce a tyrosine-independent signaling-competent Tetherin called SY. We demonstrate that SY Tetherin utilizes a similar signaling pathway as wt Tetherin. We believe this mutant, at the very least, can be used as a tool to further elucidate how Tetherin engages signaling machinery. Moreover, the identified compensatory changes in the cytoplasmic tail may be useful in exploring what we hypothesize to be a previously uncharacterized regulatory motif. In this dissertation, we explored two novel aspects of Tetherin biology. Our understanding of how Tetherin isoform expression and Tetherin- mediated signaling is regulated could provide mechanisms by which we can target and enhance immune responses during infection by clinically relevant viruses.

Degree Type Dissertation

Degree Name Doctor of Philosophy (PhD)

Graduate Group Cell & Molecular Biology

First Advisor Paul Bates

Keywords BST2, HIV-1, Isoform, NF-kappaB, Signaling, Tetherin

Subject Categories Microbiology | Molecular Biology | Virology

This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/1661 INSIGHT INTO TETHERIN-MEDIATED SIGNALING VIA THE DISCOVERY

OF A NOVEL ISOFORM WITH BIOLOGICALLY DISTINCT PROPERTIES

Luis Jose Cocka

A DISSERTATION

in Cell and Molecular Biology

Presented to the Faculties of the University of Pennsylvania

in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

2015

Supervisor of Dissertation:

______

Paul Bates, Ph.D., Professor of Microbiology

Graduate Group Chairperson:

______

Daniel S. Kessler, Ph.D., Associate Professor of Cell and Developmental Biology

Dissertation Committee:

Ronald G. Collman, MD, Professor of Medicine

Sara Cherry, Ph.D., Associate Professor of Microbiology

Michael S. Marks, Ph.D., Professor of Pathology and Laboratory Medicine

Margaret M. Chou, Ph.D., Associate Professor of Pathology and Laboratory Medicine

INSIGHT INTO TETHERIN-MEDIATED SIGNALING VIA THE DISCOVERY

OF A NOVEL ISOFORM WITH BIOLOGICALLY DISTINCT PROPERTIES

COPYRIGHT

2015

Luis Jose Cocka

This work is licensed under the Creative Commons Attribution-

NonCommercial-ShareAlike 3.0 License

To view a copy of this license, visit http://creativecommons.org/licenses/by-ny-sa/2.0/

I would like to dedicate this dissertation to my biggest supporters, my parents, Gilma &

Jose Cocka, who taught me the value of a good education at an early age, always encouraged me to pursue my interests, believed in and reassured me of my abilities, and most importantly, for

being there, together, every step of the way.

A special dedication to the Oberdorfer family, especially Janyne Oberdorfer, who passed before I

started college; I will never forget the love and support you showed my family. I wish you were

here to share this moment with us.

iii ACKNOWLEDGEMENTS

The following dissertation in Cell and Molecular Biology (CAMB), conducted in the Microbiology

Department at the Perelman School of Medicine at the University of Pennsylvania, could not have

materialized without the support and guidance of many people.

First, I want to thank my mentor, Dr. Paul Bates, for allowing me to undertake this project and make it my own. Paul’s insight was invaluable as the project moved along. His vast knowledge and laid-back demeanor made it easy to discuss any subject, and made coming into lab a real joy everyday. I will always value our chats, both lab-related and especially the entertaining ones about topics outside of science. I especially want to thank Paul, for believing in me during a time when I lost a lot of my confidence and reassuring me things would be all right. I also want to thank all the members of the Bates Lab, past and present who, just like Paul, played a crucial role, supporting me while I completed this work. From feedback during lab meetings to our daily lunch routine, I will always value the camaraderie and friendships I made during my tenure as a grad student. You guys were awesome people to work with. I honestly could not have asked for a better work environment. I am especially thankful for the lab members present during the preparation of this dissertation: MJ Drake, Ben Dyer, Stephen Bart, Nathan Vande Burgt and

Natalia Shalginskikh.

To my dissertation committee: Ron Collman, Sara Cherry, Mickey Marks and Margaret

Chou - I truly wish I would have taken advantage of your expertise on many more occasions, but the advice I received from you all in our handful of meetings was crucial to keep me on track. I want to especially thank Ron, for our numerous discussions and guidance during a turbulent time in my life. To our Joint Lab Meeting group, past and present (Doms Lab, Cherry Lab Ross Lab,

Lopez Lab): having a forum to regularly present my work and hear the work of my peers served in developing my communication skills as a scientist. A special thanks to the Doms lab, present and

iv past, for sharing both reagents and laughs during my entire tenure. You guys were amazing friends in and out of lab. And an extra special thank you goes out to Zahra Parker, who was gracious enough to provide extensive feedback throughout my preparation for my thesis defense.

To the amazing Penn community: Firstly, to all those who contributed discussions/reagents/expertise along the way, whether or not they directly resulted in the work presented in this dissertation, I am grateful. A special shout out goes out to Shilpa Iyer and

Amber Riblett for their feedback during the preparation of my thesis defense presentation. The fostering of collaboration I witnessed here at Penn should serve as a model to other institutions.

To the individuals behind the scenes, whose daily work can, but most definitely should not be taken for granted, To the folks in BGS and CAMB offices, the Microbiology department administrators, the maintenance staff, the folks who run various cores on campus and the guys at the Cell Center- thank you for keeping things running smoothly, allowing us to focus on our science.

To where the dream started, UMBC: I will forever be grateful to the Meyerhoff and MARC

Scholars Programs, where I was introduced to scientific research and was initiated into an extensive, and ever growing, network of brilliant young thinkers who value diversity in STEM disciplines. To my friends from UMBC, many of whom I still remain close with today and have had the honor of knowing they have earned their advanced degrees, you guys have been great inspirations. To where the dream has continued and is culminating, UPenn: Thanks to the student organizations, especially EE Just Biomedical and Fontaine Societies, for serving as an outlet to unwind and meet incredibly smart, motivated and outgoing people to share countless social outings with, as well as many of the values instilled in me at UMBC. To all my Philly friends, thank you for making my time in an amazing city that much more enjoyable! And last but certainly not least, words are not enough to describe the love I have for those who were there before this journey was even a remote possibility: To my closest friends from back home in Maryland, we did it!

And last, but most certainly not least, to my family, los quiero infinitamente!

v TABLE OF CONTENTS

ACKNOWLEDGEMENTS ...... iv

PREFACE ...... ix

LIST OF FIGURES ...... x

ABSTRACT ...... xii

CHAPTER 1: INTRODUCTION TO TETHERIN – AN INTRINSIC RESTRICTION FACTOR AND

IMMUNE SENSOR OF ENVELOPED VIRUS EGRESS ...... 1

General Overview of Innate Immune Responses to Viral Infection ...... 1

Viral sensing by intrinsic immune receptors ...... 1

Overview of NF-κB signaling ...... 2

Overview of IFN signaling ...... 4

General Overview of Enveloped Virus Egress ...... 5

Tetherin: Discovery, Expression, Structure and Topology ...... 9

Tetherin Antiviral Function ...... 11

Identification of Tetherin as a cellular restriction factor ...... 11

Breadth of Tetherin restriction and viral antagonists ...... 12

Structures/domains important for viral particle tethering ...... 14

Tetherin- co-evolution ...... 16

Cellular functions of Tetherin ...... 17

Other immunological functions of Tetherin ...... 18

Tetherin as an Immune Sensor ...... 19

Determinants of Tetherin-mediated activation of NF-κB ...... 19

Tetherin as a novel pattern recognition receptor ...... 21 vi Tetherin-mediated signaling as an acquired function in humans during

speciation ...... 22

Current model for Tetherin-mediated signaling ...... 23

Project Aims ...... 23

FIGURES ...... 26

CHAPTER 2: IDENTIFICATION OF ALTERNATIVELY TRANSLATED TETHERIN ISOFORMS

WITH DIFFERING ANTIVIRAL AND SIGNALING ACTIVITIES ...... 32

ABSTRACT ...... 33

AUTHOR SUMMARY ...... 34

INTRODUCTION ...... 35

RESULTS ...... 37

DISCUSSION ...... 45

MATERIALS AND METHODS ...... 50

ACKNOWLEDGEMENTS ...... 54

FIGURES ...... 55

CHAPTER 3: CHARACTERIZING ADDITIONAL DETERMINANTS OF TETHERIN-MEDIATED

SIGNALING ...... 67

ABSTRACT ...... 68

INTRODUCTION ...... 69

RESULTS ...... 71

DISCUSSION ...... 78

ACKNOWLEDGEMENTS ...... 84

MATERIALS AND METHODS ...... 85

FIGURES ...... 88

vii CHAPTER 4: GENERAL DISCUSSION AND FUTURE PERSPECTIVES ...... 95

Summary and perspectives on Tetherin isoforms ...... 95

Summary and perspectives on Tetherin-mediated activation of NF-κB ...... 101

FIGURES ...... 109

APPENDIX- LIST OF ABBREVIATIONS ...... 111

BIBLIOGRAPHY ...... 112

viii PREFACE

I have always been fascinated with understanding why things exist as they are. Because of this curiosity, my two favorite subjects in high school were history and biology. History taught me about the events of the past and how WE as society have been shaped in the present. War has been at the center of shaping the world, as we know it today. Countless lives sacrificed and an innumerable amount of damage both directed and collateral; long-lasting memories ingrained in the minds of the survivors with the hope that we learn from the past to progress as a society, moving forward toward a better future.

Biology peaked my interest because of my curiosity of how WE, as individuals, function.

War between men has shaped the world we live in, but what I found even more remarkable and fascinating, is that microscopic wars are constantly being waged within men, with many aspects paralleling what we see on actual battlefields. The continual struggle between host and pathogen peaked my interest in infectious disease. The inner workings of the immune system and its molecular “arms race” with pathogens have provided insight into our evolutionary history.

Lessons learned from this basic biology has been instrumental in developing treatments to some of the most debilitating and deadly maladies, tipping the fulcrum in OUR, the hosts’, favor.

However, pathogens evolve and find a way to evade host responses. There is a lot of evidence of host-pathogen co-evolution, though molecular détente is typically not an immediate option,

The following work delves into understanding an innate immune factor encoded by our cells as part of its arsenal to battle viral infection. This dissertation sought to elucidate previously understudied biology of the antiviral factor, Tetherin. Our work has shed to a previously uncharacterized signaling function, which has bettered our understanding of host-pathogen interactions. I am thankful for being afforded the opportunity to pursue this work and to develop it as my own. Even through the ups and downs, the experience has been extremely enriching and humbling. Many thanks again, to those involved directly and indirectly, for allowing this work to move forward.

ix LIST OF FIGURES

CHAPTER 1: INTRODUCTION TO TETHERIN – AN INTRINSIC RESTRICTION FACTOR AND IMMUNE SENSOR OF ENVELOPED VIRUS EGRESS Figure 1-1: Schematic of the type I IFN signaling pathway Figure 1-2: Schematic of classical NF-κB signaling by plasma membrane receptor Figure 1-3: Schematic of enveloped virus egress Figure 1-4: Cartoon representations of Tetherin domains, topology and function Figure 1-5: Determinants of Tetherin-mediated signaling Figure 1-6: A proposed model for Tetherin-mediated NF-κB signaling

CHAPTER 2: IDENTIFICATION OF ALTERNATIVELY TRANSLATED TETHERIN ISOFORMS WITH DIFFERING ANTIVIRAL AND SIGNALING ACTIVITIES Figure 2-1: Alignment of Tetherin sequences and comparison to a consensus Kozak translation initiation sequence Figure 2-2: Tetherin exists as two isoforms Figure 2-3: Tetherin isoforms have different sensitivities to viral antagonists Figure 2-4: Effect of viral antagonists on surface levels of Tetherin Figure 2-5: Resistance to Vpu antagonism requires mutations in the tyrosine and serine/threonine motifs in Tetherin Figure 2-6: Tetherin isoforms differentially activate NF-κB Figure 2-7: s-Tetherin modulates l-Tetherin-mediated NF-κB induction Figure 2-S1: Comparison of Kozak translation initiation sequences of mammalian Tetherin messages Figure 2-S2: Resolution of heterogeneous Tetherin expression profile by removal of carbohydrate modification with PNGase Figure 2-S3: Endogenous expression of Tetherin isoforms. Figure 2-S4: Expression profiles of rhesus and murine Tetherin. Figure 2-S5: Isoforms produce homo- and heterodimers Figure 2-S6: NF-κB induction varies with Tetherin expression level

CHAPTER 3: CHARACTERIZING ADDITIONAL DETERMINANTS OF TETHERIN-MEDIATED SIGNALING Figure 3-1: Identification of a signaling-competent, tyrosine-independent Tetherin.

x Figure 3-2: SY signaling can be regulated by s-Tetherin Figure 3-3: Identification of additional point mutations important for Tetherin-mediated signaling Figure 3-4: Tyrosine-independent Tetherin mutant can signal through a similar pathway as wt

Tetherin

Figure 3-5: EBOV GP indiscriminately counters SY function and allows incorporation into VLPs Figure 3-6: Minimal changes in STS that confer signaling in tyrosine mutant Tetherin Figure 3-7: Assessing the plasticity of STS region to mutation

CHAPTER 4: GENERAL DISCUSSION AND FUTURE PERSPECTIVES Figure 4-1: Compilation of secondary structure analysis of Tetherin cytoplasmic tail. Figure 4-2: Our proposed models for Tetherin-mediated signaling

xi ABSTRACT

INSIGHT INTO TETHERIN-MEDIATED SIGNALING VIA THE DISCOVERY OF A NOVEL

ISOFORM WITH BIOLOGICALLY DISTINCT PROPERTIES

Luis J. Cocka

Paul Bates

Viruses are obligate, intracellular pathogens that hijack host cell machinery to replicate. Innate immunity is the first line of defense against such perceived threats through recognition of broadly conserved pathogen signatures. Tetherin (also known as BST2/CD317/HM1.24) is an innate immune factor that senses and restricts egress, the final step of viral replication. Tetherin potently reduces cell-free virus spread by indiscriminately “tethering” particles at the cell surface via direct anchoring to the host membrane. The majority of previous studies on Tetherin focused on elucidating the minimal structural features necessary for tethering viral particles and understanding how viruses counter Tetherin function. While the cytoplasmic tail of Tetherin is dispensable for restricting virus release in the absence of certain viral antagonists, our work, in part, has focused on conserved residues within the tail, hypothesizing these conserved features could provide insight into previously under-studied biology, including a poorly characterized signaling function. In Chapter 2 we show that human Tetherin can exist as two alternatively translated isoforms [long (l-) and short (s-)]. s-Tetherin lacks the first 12 amino acids, a difference that confers functional differences. We found s-Tetherin to be exquisitely resistant to downregulation by the best characterized Tetherin antagonist, HIV-1 Vpu. l-Tetherin, which is sensitive to Vpu, was shown to be a potent activator of NF-κB. A dual-tyrosine motif, unique to l-

Tetherin, was identified as an important determinant. In Chapter 3 we describe additional determinants of signaling. Interestingly, we identified compensatory mutations that produce a tyrosine-independent signaling-competent Tetherin called SY. We demonstrate that SY Tetherin

xii utilizes a similar signaling pathway as wt Tetherin. We believe this mutant, at the very least, can be used as a tool to further elucidate how Tetherin engages signaling machinery. Moreover, the identified compensatory changes in the cytoplasmic tail may be useful in exploring what we hypothesize to be a previously uncharacterized regulatory motif. In this dissertation, we explored two novel aspects of Tetherin biology. Our understanding of how Tetherin isoform expression and

Tetherin-mediated signaling is regulated could provide mechanisms by which we can target and enhance immune responses during infection by clinically relevant viruses.

xiii CHAPTER 1: INTRODUCTION TO TETHERIN – AN INTRINSIC RESTRICTION

FACTOR AND IMMUNE SENSOR OF ENVELOPED VIRUS EGRESS

“All delays are dangerous in war.” – John Dryden

General Overview of Innate Immune Responses to Viral Infection

Viral sensing by intrinsic immune receptors

Viruses are highly abundant pathogens in nature that can present a burden to every living organism from unicellular bacteria to multicellular plants and animals. Though not considered technically alive, viruses share some properties with living things to ensure species survival, including maintenance across generations and an ability to adapt to the environment.

Viral replication is contingent on infecting a host cell. However, cells express immune barriers that can block essentially every stage of viral replication. This selective pressure, coupled with error- prone replication machinery, particularly in the case of RNA viruses, drives viral evolution in the form of tolerable mutations to escape recognition by both cell-intrinsic and -extrinsic immune responses (113, 188, 245). The dynamic “arms race” between host and pathogen has resulted in convergent evolution observed in viruses from disparate families that target similar cellular immune sensing pathways. Cells constitutively express both cytosolic and membrane-bound referred to collectively as pattern recognition receptors (PRRs), which bind conserved pathogen signatures. Examples of membrane-bound sensors include the plasma membrane (PM) localized Toll-like receptors (TLRs), TLR2 or 4, which have been traditionally described as sensors of bacteria-derived products such as bacterial lipopolysaccharide (LPS), and C-type lectin receptors (CLRs). Both these families of receptors can also sense viral-encoded structural proteins that contain carbohydrate- and lipid-rich moieties (85, 103). Additionally, intracellular

1 TLRs that localize to endosomal membranes, like TLR3, 7 and 9, sense viral nucleic acids (103).

Additional viral nucleic acid sensors localize to the cytoplasm including the retinoic acid-inducible I (RIG-I)-like receptors (RLRs), which include RIG-I and melanoma differentiating factor 5

(MDA5), both of which specifically recognize viral RNAs (29). More recently, cyclic GMP-AMP synthase (cGAS) was identified as the cytoplasmic sensor for viral DNA (221). PRR sensing of pathogen-derived ligands activates downstream signaling networks that converge and synergize, leading to nuclear translocation of various transcription factors that modulate expression of proteins involved in immune responses (Figure 1-1). These include members of the nuclear factor kappa-light chain-enhancer of activated B cells (NF-κB), regulatory factor (IRF), activator 1 (AP-1) and signal transducer and activator of transcription (STAT) families

(212, 266).

Overview of NF-κB signaling

NF-κB refers to a superfamily of conserved, rapidly responding transcription factors involved in regulating various cell processes including development, cell growth, apoptosis and responses to cellular stresses such as infection (80). In mammals, the family is comprised of 5 members: p65 (RelA), RelB, c-Rel, p105/p50 and p100/p52, which function as either homo- or heterodimers with the other members (81). A highly conserved Rel homology domain regulates function including dimerization, nuclear translocation, and binding to DNA (151). Generally, the

NF-κB family members recognize a consensus 10bp DNA sequence, 5′-GGGRNYYYCC-3′

(R=purine, Y=pyrimidine, N=any base), in the promoter or enhancer regions of target (40).

Though different family members bind to similar sequences, specific responses are directed by cell intrinsic factors in combination with input stimuli. Specificity of the family for particular binding sites allows researchers to assess activation using reporter systems (luciferase, GFP, etc.) as a surrogate for NF-κB activation. Signaling is tightly regulated: sub optimal activation, in the case of infection, may permit pathogen persistence; excessive activation has been associated with numerous immunopathologies including cancer and autoimmune disorders as a result of chronic inflammation (45, 222). 2 There are two known NF-κB activation pathways: classical and alternative. Classical NF-

κB is ubiquitous, and primarily regulates cellular immune responses, cell growth and death (80,

81). The alternative signaling pathway is more specific to lymphoid cells and is crucial for development in cells of that lineage (48). When the cell is at rest, all NF-κB members are sequestered in the cytoplasm and require a proteolytic event for activation and subsequent nuclear translocation. In the alternative pathway one half of the NF-κB heterodimer exists as a precursor protein, p100, which is bound to RelB (p100/RelB). p100 contains inhibitor domains that keeps the dimer cytoplasmic. Nuclear translocation requires proteosomal processing of p100 to p52, generating the activated p52/RelB heterodimer (207, 253). Classical signaling is primarily mediated by the activation of the NF-κB heterodimer p65/p50, which is kept inactive in the cytoplasm by NF-κB inhibitor alpha (IκBα) (Figure 1-2). Upon receptor binding/sensing of a ligand, a series of phosphorylation events mediate recruitment and activation of downstream kinase complexes. Phosphorylation of the Skp, Cullin, F-box containing (SCF) E3 ligase complex mediates proteosomal degradation of IκBα, freeing the sequestered NF-κB heterodimer to translocate and accumulate in the nucleus where it binds κB sites on the allowing regulation of (Figure 1-2, (81)).

NF-κB can be activated by various PRRs, as well as receptors that sense early-secreted cytokines like tumor necrosis factor alpha (TNFα). Effector kinases that regulate NF-κB signaling also feed into other signaling pathways including the mitogen-activated protein kinase (MAPK) pathway to activate additional transcription factors like AP-1, which together with NF-κB regulate transcription of various genes involved in cell homeostasis. NF-κB regulates genes involved in immune responses, including those that encode cytokines, receptors for direct immune recognition and antigen presentation, and factors important for cell transmigration (167). Negative regulators of NF-κB signaling, like IκBα, are also regulated by NF-κB, allowing for negative feedback to dampen prolonged activation (81, 167).

NF-κB plays a crucial role during infection, in part, by regulating expression of proinflammatory cytokines (114), which are among the earliest danger signals secreted during

3 infection. Acute inflammatory signals are important for recruiting rapidly responding leukocytes

[e.g. neutrophils, professional antigen presenting cells (APCs) like macrophages and dendritic cells (DCs), and natural killer (NK) cells] to the site of damage in order to clear the infectious agent and help prime the adaptive immune system (19). Because acute inflammatory responses are important early in infection, many viruses encode proteins to subvert activation (117), while others have evolved ways to manipulate NF-κB expression to positively regulate replication.

Certain viruses can activate NF-κB via virally encoded proteins to enhance replication (167). NF-

κB is essential for retroviral replication. In fact, NF-κB was first discovered in studies of human immunodeficiency virus 1 (HIV-1) replication (153) where it was identified as a transcription factor. Additionally, proinflammatory responses have been suggested to enhance cell targets for

HIV-1 and other viruses, further promoting infection (142). Conversely, the HIV-1 accessory protein U (Vpu) has been demonstrated to interfere with NF-κB activation by binding and sequestering the SCF E3 ligase complex which stabilizes IκBα (28) suggesting HIV-1 needs to modulate NF-κB activation to efficiently replicate. Just like the host, chronically infecting viruses can modulate the proinflammatory state of a cell to ensure efficient prolonged replication and subsequent transmission. Some viruses that cause acute infections, particularly those that manifest as hemorrhagic fevers, stimulate uncontrolled cytokine production which causes the host to go into shock (166). Elucidating novel NF-κB activating pathways that may directly affect viral replication is paramount to not only further our understanding of host-pathogen dynamics, but also to identify potential new targets for therapeutics.

Overview of IFN signaling

Interferons (IFNs),of which there are three major characterized types (I, II, and III), are a second class of cytokine, and like proinflammatory cytokines are also expressed during early immune responses. Type I IFNs, primarily α and β, robustly activate the host antiviral state in an auto- and paracrine manner through the Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway, downstream of binding to the IFNα/β receptor

(IFNAR) (212). IFN signaling activates the innate immune response through upregulation of 4 genes that code for interferon-stimulated genes (ISGs), as well as negative and positive regulators of the pathway (Figure 1-1). Through a number of these upregulated ISGs, IFN- stimulated cells can more readily undergo apoptosis upon viral infection to halt replication (214).

Moreover, IFNs help prime cells of the adaptive immune system by enhancing T cell effector function and enhancing B cell humoral responses (62, 115).

ISGs present a major hurdle to viral replication across many disparate families (196).

Viruses can dampen the antiviral response by targeting both direct expression of IFNs and IFN- mediated signaling in infected cells (Figure 1-1). Type I IFN expression is regulated, in part, by

IRFs. IRF3, which is ubiquitously and constitutively expressed in all tissue allowing for rapid expression of IFNβ during infection, can be targeted by various viruses (11, 178). HIV-1 and ebolavirus (EBOV) are enveloped viruses, retro- and filoviruses respectively, with distinct replication cycles, express proteins that meditate direct degradation of IRF3 or block IRF3 activation by preventing nucleic acid association with cytoplasmic PRRs, respectively (36, 208).

Additionally, viruses can target the JAK/STAT pathway directly to decrease expression of ISGs

(154). Moreover, viruses can target ISGs directly (187). Viral are typically very compact, encoding proteins with multiple functions. These different strategies exemplify how viruses use a limited number of encoded gene products to counter host defenses and ensure replication. Innate immunity is the first line of host defense and broad recognition redundancy allows for pathogen sensing at all stages of replication from entry to egress.

General Overview of Enveloped Virus Egress

Egress is the final step of viral replication. For both DNA and RNA enveloped viruses, this process involves assembly of genetically encoded structural proteins at host membranes [either plasma membrane (PM) or at intracellular structures that are topologically similar, like the Golgi apparatus or endoplasmic reticulum (ER)]. These viral proteins subsequently facilitate budding and release from the host cell. Viruses express two potential driving forces of membrane vesiculation (i.e. formation of vesicles): the viral “core” proteins (e.g. , matrix and/or 5 nucleocapsid) and the (s). Viral “core” proteins assemble into higher order oligomeric structures prior to or at the site of budding providing the scaffold on which nascent virions are generated (Figure 1-3). “Core” proteins themselves are thought to be the major drivers of membrane curvature. Mechanistically, this can occur through direct shallow penetration into the inner leaflet of the PM, disrupting membrane order to induce curvature (159,

209); this parallels what is observed with numerous cellular molecules (e.g. BAR domain containing proteins, the clathrin adaptor epsin, charged phospholipids) that can sense and/or induce similar morphologic changes at membranes (133). Viral , which stud the surface of the viral particle and mediate entry into host cells, are targeted to the site of budding where some can potentiate membrane coalescence and mediate low levels of vesicle formation

(39, 139, 145, 161, 237), a property that is thought to enhance viral assembly at sites of budding

(132). The budding process is tightly orchestrated and requires, in many cases, the aid of cellular proteins for completion (38).

Though viral structural proteins can initiate vesiculation at sites of budding, many enveloped viruses encode, within structural proteins, short motifs known as late- budding domains (l-domains) that recruit host cell machinery to complete the budding process

(68). The endosomal sorting complexes required for transport (ESCRT) proteins are commonly recruited by viral protein l-domains. Many viruses are dependent on this machinery to complete release from the host cell. Under normal cellular conditions, the ESCRT proteins (ESCRT-0, -I, -II and -III), perform a topologically similar function during biogenesis of multivesicular bodies

(MVBs), which involves formation and sorting of vesicles containing ubiquitinated cargo, a process conserved from yeast to humans (12, 13, 102). Recruitment of ESCRT proteins is a highly conserved mechanism shared by disparate families of enveloped viruses (23). For example, HIV-1 and EBOV both recruit the ESCRT-I protein TSG101 via a P(T/S)AP motif found on either HIV-1 or EBOV “core” protein, Gag (p6 region) and VP40 matrix, respectively (49, 64,

138, 233). Additionally, both viruses can also recruit other proteins to perform similar functions; for instance, the ESCRT related proteins Alix/AIP1 and/or the HECT NEDD4 via

6 additional l-domain motifs encoded within the “core” proteins (216, 248, 257). Engagement of

TSG101 leads to the recruitment of ESCRT-III complex members, like CHMP2 and CHMP4, in the case of HIV-1 (149). Oligomerization of ESCRT-III complex proteins at the budding neck constricts the membrane leading to scission (Figure 1-3A and B). The ATPase Vsp4 mediates dissociation and recycling of ESCRT-III components for subsequent rounds of viral replication recapitulating what is observed in cellular vesicle biogenesis (14). Other viruses, such as and respiratory syncytial virus (RSV), are capable of completing budding independent of

ESCRT machinery (31, 39, 230). The influenza M2 protein is recruited to sites of budding where it inserts into the membrane and in tandem with the drive membrane curvature and release of viral particles (183).

Mutations in “core” protein l-domains or perturbation of the ESCRT pathway (genetically via RNAi, dominant negative mutants, etc. or chemically via small molecule inhibitors, etc.) can block viral budding (57, 76). Mutations typically affect the final scission step of budding.

Phenotypically, budding-defective viruses have incorporated cellular membranes, but particles remain contiguous with the host cell (Figure 1-3C), leading to accumulation of immature virions on the cell surface (68). These observations suggest viral budding is a targetable stage of replication for therapeutic purposes and therefore an important stage of viral replication to study.

Expression of “core” proteins alone, like HIV-1 Gag or EBOV VP40, in cell culture systems is sufficient to produce virus-like particles (VLPs), which are morphologically indistinguishable from infectious virions. This has afforded researchers a tool to study the budding process in isolation, using easier and safer VLP-based systems as surrogates of mature virion production for numerous clinically relevant viruses that would otherwise require higher biosafety level containment.

With or without host machinery, all enveloped viruses acquire a lipid shell derived from host cell membrane upon budding. Many viruses are thought to bud at PM microdomains commonly referred to as lipid rafts. Lipid rafts are small liquid-ordered domains suggested to range between 10-200nm in size (175). These microdomains are heterogeneous in composition,

7 primarily enriched in cholesterol, but also containing various sphingolipids and lipidated proteins typically containing modifications like myristoylation or glycophosphatidylinositol (GPI) anchors.

Viral membrane lipid composition is thought to be important for steps in the viral replication cycle, particularly at steps involving fusion like entry and egress (128, 238). HIV-1 and EBOV membranes have been demonstrated to contain lipid raft-enriched molecules like the ganglioside

GM1 as well as the phospholipid, phosphatidylserine (20, 32, 160). Additionally, numerous signaling platforms have been show to localize to lipid rafts (206). Moreover, there is a dense actin cytoskeletal network underpinning lipid rafts: likely mediated by the interaction between raft- localized signaling molecules with actin cytoskeleton regulatory components, which concentrate upon receptor clustering within these specific microdomains (42). The actin cytoskeletal network plays a role at various stages of viral replication including viral assembly and budding (226).

Enveloped viruses face the task of budding in an environment dense in cellular proteins. Many viruses prevent bulk host protein incorporation into particles; however, certain host molecules present at sites of budding can be preferentially acquired into nascent virions. Acquisition of host molecules, in some instances, can be beneficial for viral replication as observed with enhanced binding and entry of various enveloped viruses that become enriched with host phosphatidylserine (94, 147). As a result of incorporation into virions, host proteins like the tetraspanins have been shown to impair infection by restricting membrane fusion. However, it is believed HIV-1 can modulate tetraspanin expression on the cell surface during late stages of infection to facilitate cell-cell transmission via virological synapses, while reducing production of unfavorable syncytia (227). HIV-1 Vpu has been shown to downregulate a number of host proteins to regulate HIV-1 egress and replication, including tetraspanins (74, 112), CD4 (250,

251) the NK coactivation receptor NTB-A (200) and Tetherin (158, 231).

The interplay between virus and raft-resident molecules at sites of concentrated signaling platforms, suggests the cell could sense perturbation of this network during viral replication. ISGs restrict various early events of viral replication, though there are a few examples of ISGs that can block egress including ISG15, which interferes with ESCRT-viral “core” protein interactions, and

8 viperin, which disrupts raft domains by perturbing lipid composition (155, 163, 164, 244). The focus of this dissertation highlights previously understudied properties of lipid raft resident ISG involved in blocking viral release, Tetherin, which we now know, not only restricts, but can sense viral egress.

Tetherin: Discovery, Expression, Structure and Topology

Originally described as HM1.24, Tetherin was identified as a specific surface marker on terminally differentiated B cells, suggesting a potential role in B cell development (67, 91). As an antigen on immune cells, it was later assigned a cluster of differentiation (CD) nomenclature as

CD317 (267). The cDNA cloned from the associated gene product recognized by a monoclonal antibody against HM1.24 matched a human gene, 540 base pairs in length (180 amino acids), located on annotated as bone marrow stromal cell antigen 2, BST2 (91), consisting of 5 exons, 4 of which code for protein (162). The presence of STAT1/3, IRF1/2 and

IFN-stimulated gene factor 3 (ISGF3) binding sites upstream of the predicted translational start site suggested expression could be immune modulated (162). Tetherin has been confirmed as a bona fide ISG and is upregulated by IFNs, primarily type I, but also type II and type III (2, 157).

IFNβ- and γ-induced unphosphorylated STAT1 can stabilize Tetherin mRNA 48h after induction allowing for potentially prolonged expression (41). Additionally, expression has also been shown to be regulated by alternative mechanisms including a novel, uncharacterized IFN-independent,

IRF7-dependent pathway (22), the cytokine interleukin 27 (IL-27) (73) and regulated by a chromosome proximal, IFN-stimulated long non-coding RNA (lncRNA) (101). Tetherin can be highly upregulated in various IFN-responsive cultured cell lines though in primary cells and tissue upregulation is thought to be more modest (54). Endogenously expressed Tetherin is present in specialized cells across a variety of tissues, as well as cells of the immune system including monocytes, macrophages, dendritic cells, NK cells, and B and T cells, (primarily those of Th1 lineage) (54, 129, 234), and preferentially expressed in certain transformed and invasive cell types compared to non-transformed counterparts (33, 194, 239, 240, 242, 243, 252). To date,

Tetherin orthologues have only been identified in mammals, with the exception of an annotated 9 protein that is Tetherin-like in sequence in Chinese alligator (accession #s: XP_006017475,

XP006017476), though it has not been tested for functionality. Additionally, in ruminants, gene duplication of Tetherin allow for expression of functionally distinct isoforms (9, 152, 225).

Tetherin is a type II integral membrane glycoprotein of about 30-36kDa in size consisting of a short N-terminal cytoplasmic tail, a transmembrane (TM) domain, long coiled-coil ectodomain and a C-terminal GPI anchor (Figure 1-4A). It exists as a dimer under physiological conditions

(91, 109, 162). Dimerization is mediated by bond formation via three conserved cysteine residues localized in the ectodomain. Monomeric forms of Tetherin dimerize in a parallel orientation (160). Structurally, Tetherin is unusual, anchored at both the N- and C- termini, by a transmembrane domain and GPI anchor respectively (53, 109). To date, the only other mammalian protein identified with a similar structure is an alternative form of the prion protein

(215). Though it is universally accepted that Tetherin is GPI-anchored, at least one line of evidence suggests that human Tetherin may express a second transmembrane domain in lieu of the processed GPI-anchor signal sequence (5), presumably still allowing to be anchored at both termini. The ectodomain, which is extracellular (or lumenal), is primarily a long coiled-coil (~165-

170 Angstroms (Å) relaxed) that can form higher ordered oligomeric species (83, 197, 223, 256).

Key hydrophobic residues in the ectodomain important for multimerization have been mapped via structural data. Tetherin is proposed to also exist as a multimer of anti-parallel dimer of dimers

(~275Å) interacting via residues near the N-terminus (83, 197, 256). The ectodomain structure has been described as “irregular”, with a N-terminal proximal kink observed via small angle x-ray scattering approximately about a third of the way into the ectodomain. This feature is believed to provide flexibility to the protein (197, 223). Though the cytoplasmic tail and GPI anchor are primarily thought to mediate surface Tetherin partitioning, the ectodomain has also been suggested to have some contribution to PM distribution (75). The Tetherin ectodomain has two N- linked glycosylation sites that add ~14-17kDa of complex carbohydrate mass as observed by diffuse immunoblot expression profiles. Tight protein resolution can be achieved using PNGase to

10 remove N-linked glycans (Full-length, deglycosylated Tetherin is predicted to be ~19kDa in size; confirmed by Western blot analysis after PNGase treatment) (6, 109).

Tetherin has a short N-terminal cytoplasmic tail, 21 amino acids in length (Figure 1-4A).

Residues in the tail are important for trafficking and localization. Tetherin localizes primarily to detergent-resistant microdomains at the PM, though it is also present in intracellular compartments like the trans-Golgi network (TGN) and endosomes (109). Trafficking between these compartments is mediated by a tyrosine-based sequence in the cytoplasmic tail that resembles overlapping putative recycling motifs (Figure 1-4A). In both rat and human Tetherin, the dual-tyrosine motif engages the clathrin-associated adaptor proteins (AP) complexes 1 and 2.

The AP2 complex mediates Tetherin endocytosis from the PM to endosomes where AP1 engages and redirects cargo to the TGN where presumably Tetherin is transported back to the

PM (140). Mutations at both tail tyrosines (Y6, Y8) in the motif are required to fully disrupt

Tetherin intracellular trafficking, leading to PM stabilization (140, 181). Individual tyrosine mutations have no discernable localization defect, though each residue has been proposed to interact differently with trafficking machinery by yeast two-hydrid screening (Y6 with AP2 and Y8 with AP1), suggesting the individual tyrosine residues are perhaps not performing redundant trafficking functions (140).

Tetherin Antiviral Function

Identification of Tetherin as a cellular restriction factor

The accessory protein Vpu has been described as essential for HIV-1 replication in certain refractory cell lines (156, 184). HIV-1 lacking Vpu (delU HIV-1) was shown to accumulate at the PM and within intracellular compartments (104). These observations suggested the presence of a cell intrinsic factor involved in blocking HIV-1 replication that could be countered by

Vpu. Additionally, type I IFN stimulation enhanced the budding restriction phenotype (157), further evidence that implicated the presence of an uncharacterized immune molecule. The factor in

11 question would remain unknown for almost two decades from the time of the initial observations.

Neil et al., employing targeted screening of candidate proteins that differed in expression between refractory and permissive cell lines, identified an IFN-inducible physiological “tether” that prevented release of cell-free delU HIV-1. The gene associated with this phenotype was BST2, previously described as HM1.24 and CD317, and thereafter referred to as Tetherin (158) due to its “tethering” function of viral particles at the cell surface.

Breadth of Tetherin restriction and viral antagonists

In cell culture, Tetherin has been demonstrated to indiscriminately restrict a broad, and continually expanding, list of enveloped viral families including alpha- (98, 165), arena- (177,

185), filo- (99, 100, 157), flavi- (168), rhabdo- (246), alphaherpes- (27, 263), gammaherpes-

(136), hepadna- (131, 255), orthomyxo- (134), paramyxo- (16), and representative viruses from all families of mammalian (including endogenous retroviruses (ERVs)) tested to date

(65, 69, 99, 158). Mouse knockout and knockdown models have suggested Tetherin contributes to murine retrovirus restriction in vivo (97, 124, 224). Significant restriction in the murine knockout models is believed to require additional IFN stimulation of endogenous Tetherin expression (124).

Additionally, Tetherin has been demonstrated to enhance cell-mediated immune responses during infection (122).

With few exceptions, most Tetherin-sensitive virus families described to date are believed to primarily bud from the PM. There is evidence that Tetherin in macrophages can restrict HIV-1 in intracellular MVB-like compartments called “virus-containing compartments” (VCCs) (43).

Hepatitis C virus (HCV), which buds into intracellular membranes, has been suggested to be

Tetherin-sensitive (46, 169), though work in an inducible system suggests only high Tetherin expression levels have any significant effect (258). Recently, Tetherin was shown to also restrict (HBV), which like HCV buds into intracellular membranes. The HBV work suggests interactions in MVBs as Tetherin mutants that have aberrant trafficking and potentially accumulate in intracellular compartments have potent function against HBV, but not against a PM budding virus like HIV-1 (131). Additionally, 1 and human coronavirus 229E 12 (HCoV-229E), both of which also bud into intracellular membranes, have been shown to be

Tetherin-sensitive (27, 241).

Many Tetherin-sensitive viruses encode proteins that counteract tethering function.

Mechanistically, most virally encoded Tetherin antagonists act by downregulating Tetherin (i.e. removing from the sites of budding). Downregulated Tetherin can either be actively degraded or sequestered intracellularly. Both the Kaposi Sarcoma-associated Herpesvirus (KSHV) K5 protein and HIV-1 Vpu target Tetherin for degradation. KSHV K5 is an E3 ubiquitin ligase that mediates

Tetherin polyubiquitination, targeting it to the endolysosomal pathway for degradation (18, 136).

HIV-1 Vpu recruits the SCF-βTrCP E3 ubiquitin ligase complex via a cytoplasmic di-serine motif to mediate ESCRT-dependent endolysosomal degradation of Tetherin (51, 92, 93, 148, 231). Vpu binds Tetherin in the TGN to also prevent trafficking back to the PM (7, 52, 79, 195). Intracellular sequestration is believed to contribute to maintaining low PM expression. The importance of Vpu has been highlighted in humanized mouse models of HIV-1 infection, where Vpu was shown to be important in early dissemination of the virus (47, 190). Both HIV-1 Vpu and KSHV K5 are thought to interact with Tetherin intracellularly and target lysine residues in the Tetherin cytoplasmic tail to mediate downregulation. A serine-threonine-serine region in the tail was shown to be required for efficient Vpu targeting of Tetherin (44, 229), though the mechanism is unclear

(72).

Unlike HIV-1, most simian immunodeficiency viruses (SIVs) utilize the accessory protein negative regulatory factor () to counter Tetherin. SIV Nef specifically engages simian Tetherin through residues in the cytoplasmic tail (see below) and enhances recruitment of the AP-2 complex, which downregulates surface Tetherin. Contrasting Vpu or K5, Nef activity does not result in loss of cellular Tetherin (264) suggesting a sequestration model. Though most SIVs utilize Nef to counter Tetherin function, at least one SIV from the tantalus monkey (SIVtan) uses its envelope glycoprotein () (70). Inhibition by a retroviral Envs has also been observed in feline immunodeficiency virus (FIV), various ERVs and HIV-2 (70, 118, 121, 150). A sequestration model has been suggested for primate lentiviral Env-mediated antagonism of Tetherin and at

13 least in HIV-2, occurs by enhancing Tetherin endocytosis via AP-2 (79). HIV-2 Env is thought to mediate this interaction via its ectodomain, recognizing an N-terminal region of the Tetherin ectodomain (55). Recently, the Chikungunya (CHIKV) accessory protein non-structural protein 1

(nsP1) (98) and 1 (HSV1) M (27) protein have been shown to downregulate

Tetherin though the mechanisms remain unclear

Other Tetherin antagonists have been described that utilize less conventional mechanisms to counter Tetherin function. Filoviruses also utilize their glycoprotein, GP (100).

However, unlike SIV and HIV-2 Envs, filovirus GPs are thought to counter Tetherin without actively removing it from sites of budding or degrading the protein (100, 126). Recent evidence suggests that EBOV GP may facilitate incorporation of Tetherin in budding particles to overcome restriction (71). FIV also does not actively downregulate Tetherin and is thought to require incorporation into budding particles to function. However, unlike filoviral GPs, which can rescue budding of and incorporate into different viral particles in the presence of Tetherin, FIV Env has been shown to only rescue release of its cognate FIV “core” protein, p24, suggesting specificity between FIV Env and FIV Gag (150).

Structures/domains important for viral particle tethering

Tetherin-restricted particles are distinct from viral particles still associated with host membrane due to either mutations in l-domains or mutations in key ESCRT proteins. Particles restricted by Tetherin have completed membrane scission and are considered mature (compare

Figure 1-3 and 4C). This tethering mechanism is direct and proteinaceous in nature as trapped particles can be released from the PM by proteinase treatment (158). Tetherin requires anchoring at both the N- and C-terminus to function. Rendering Tetherin a single membrane anchor protein by either deleting the GPI anchor or TM domain allows surface expression, but prevents particle retention (158, 171). These non-functional, single-anchored Tetherin mutants have been observed to incorporate into budded virions (171). Restoring anchoring at either terminus by engineering chimeric Tetherins partially restores tethering function. An artificial Tetherin (art-

Tetherin) comprised entirely of domains (TM, coiled-coil ectodomain and GPI-anchor) from 14 heterologous proteins has been demonstrated to be sufficient to tether HIV-1 particles, suggesting that the cellular restriction function of Tetherin is inherent and primarily mediated by structure (171). However, the potency of this art-Tetherin is greatly reduced compared to wt

Tetherin suggesting some degree of functional specificity to the native structure of the protein.

While art-Tetherin was expressed at the cell surface, the contributions of the individual heterologous domains toward PM localization was not assessed.

Tetherin incorporation into particles favors GPI-insertion, while the N-terminal transmembrane domain anchors the protein to the host cell (232). The cytoplasmic tail of Tetherin has been shown to be excluded from raft domains (24), which presents a model where Tetherin remains anchored outside the site of budding upon viral particle “tethering” at lipid rafts (Figure 1-

4B and C). Tethered particles are internalized in a Rab5 dependent manner and trafficked to endolysosomal compartments where they are degraded. Trafficking to endosomal compartments exposes viral protein and nucleic acid to endosomal sensors that may further enhance immune responses to infection. Multiple Tetherin dimers are thought to remain present at the assembly site. Super-resolution microcopy suggests between 4-7 dimers of Tetherin within single microdomains associate with a budding HIV-1 particle, though at least one report using quantitative immunoblotting methods suggests this may be an underestimate based on certain limitations of the microscopy technique (120, 232).

The length and post-translational modifications of the ectodomain of Tetherin appear to be important for its function. The ectodomain is able to tolerate many triple alanine substitutions, though there are specific regions and point mutations that disrupt function, correlating with disruption of ectodomain structure (4, 75, 171). Tetherin glycosylation has been shown to be dispensable for restriction of certain viruses like Lassa, Marburg and HBV, but not HIV-1 (131,

171, 185), though surface localization of the double-glycosylation mutant is greatly impaired

(171). Additionally, Tetherin dimerization appears to be important for tethering HIV-1, but not

Lassa virus (186). The cytoplasmic tail of Tetherin has been shown to be unnecessary for tethering VLPs for various viruses. However, the tail is important in the context of viruses that

15 encode antagonists that bind and/o target the tail with modifications that counter Tetherin- mediated particle restriction.

Tetherin-retrovirus co-evolution

Intrinsic immune factors, including but not limited to TRIM5α, APOBEC3G and SAMHD1, have all been demonstrated to potently restrict retroviral replication, limiting cell- and species- specific tropism (205, 218). This relationship has been well studied in primate , which encode proteins that can antagonize the aforementioned cellular restriction factors (217). TRIM5α is believed to block cross-species transmission of primate lentiviruses (77), while SAMHD1 appears to restrict cellular tropism of specific viruses like HIV-1 from infecting cells of myeloid lineage, which express inhibitory levels of SAMHD1 endogenously (86, 111). APOBEC3G presents a block to HIV-1 in target CD4 T cells, however, HIV-1 encodes the accessory protein, viral infectivity factor (Vif), which targets APOBEC for degradation (137, 202). Tetherin has been proposed to contribute to limiting primate lentiviral cross-species transmission (192). While

Tetherin indiscriminately restricts enveloped viruses, including representative members of all retroviral families tested to date, primate lentiviruses specifically counteract Tetherin expressed in cognate cell types. HIV-1 Vpu specifically antagonizes human Tetherin through unique recognition via the TM domain (105, 143, 235). Sequence variations present in the TM domains of various non-human primate Tetherins prevents recognition by HIV-1 Vpu. To date, only three other SIVs [greater spot nose monkey (SIVgsn), mona monkey (SIVmon) and mustached monkey

(SIVmus)] have been identified that use Vpu to downregulate their cognate Tetherins through TM recognition (192). Vpu also serves to downregulate surface CD4, a function that contributes to efficient viral egress. Though all Vpus from SIVs and HIV-1 subgroup (with the exception of group

N) can efficiently downregulate CD4, only a subset these have been demonstrated to have anti-

Tetherin function suggesting that the ability of certain Vpus to counteract Tetherin was an acquired function through species-specific selective pressure (192).

HIV-1 arose from independent cross-species transmission events of either SIVcpz or SIVgor

(201). However, both of these viruses, like most other SIVs, use the accessory protein Nef. 1 6 Tetherin recognition by Nef requires a five amino acid motif within the cytoplasmic domain found on non-human primate Tetherins, but has been lost in human Tetherin. Consequently SIV Nef does not counteract human Tetherin. Human Tetherin can be engineered to be Nef-sensitive by the introduction of this motif (95, 265). Evidence of SIV adaptability to Tetherin has been observed in SIV virus lacking the Nef protein (SIV delNef) passaged in vivo. Viral mutants with compensatory changes in Env arise that confer anti-Tetherin function (198). This observed adaption has demonstrated how lentiviral Env recognition of Tetherin could have evolved in various SIVs that were thought to have given rise to the less prevalent HIV-2. SIVcpz is thought to have arisen from transmission of SIVs from two different monkey lineages: one that utilized Vpu and one that used Nef. Through selection, Nef ultimately become the dominant Tetherin antagonist. This is also reflected in the potency of HIV-1 Vpus form the three different phylogenetic groups (M, N, O). HIV-1 from group M have both strong anti-Tetherin and anti-CD4 function, in part, contributing to group M viruses becoming the pandemic strain. Based upon these observations Tetherin has been implicated as a major barrier to primate speciation. Whether additional biological functions of Tetherin are also impacted by adaptation to lentivirus infection is an open question and will be further discussed in Chapters 3 and 4.

Cellular functions of Tetherin

Tetherin is hypothesized to act as an organizer of lipid rafts, influencing diffusion and distribution of proteins to and between microdomain clusters (25). This function is coupled to its interaction with the actin cytoskeleton through its interaction with the protein RICH2 (25, 180).

Interfering with this interaction leads to disruption of apical actin network in polarized cells (180).

Regulation of monocyte adhesion to Tetherin-expressing endothelial cells has also been observed (260). Additionally, the hamster Tetherin orthologue, Golgi-resident GPI-anchored protein (GREG) is primarily Golgi-localized and involved in maintaining Golgi-apparatus integrity

(123). Of note, hamster Tetherin as well all other mammalian Tetherins contain long coiled-coil domains that are reminiscent of golgin family proteins involved in regulating vesicular cargo movement within the Golgi-apparatus. Though not topologically similar during function, golgin 17 ectodomain homology potentially highlights the importance of Tetherin structure in its ability to restrict viral particles (see Structure/Domains Important for Tetherin Viral Particles).

Recently, Tetherin was suggested to be involved in regulating autophagy and mitophagy by sequestering a suppressor protein LRPPRC, preventing it from inhibiting Beclin 1 and Bcl-2 function (268).

Other immunological functions of Tetherin

Apart from being a cellular restriction factor, Tetherin has been shown to have additional immune functions. Tetherin has been implicated in dampening type I IFN secretion in both murine and human cells. Tetherin-expressing murine IFN-producing cells cross-linked by an immobilized

Tetherin mAb demonstrated reduced IFNα secretion after TLR stimulation with single-stranded

DNA (26). In human plasmacytoid dendritic cells (pDCs), Tetherin has been shown to interact with the pDC receptor ILT7, leading to dampening of IRF3 dependent IFNα secretion in activated pDCs (35). Recently, it was suggested that during particle restriction at the cell surface, Tetherin epitopes that mediate interaction with ILT7 are occluded, which would allow pDCs to secrete IFN

(21). However, in the presence of Vpu, Tetherin molecules that were not downregulated were observed to be relocated from budding sites, leading to pDC dysfunction (i.e. blocking IFN secretion) (21) Additionally, Tetherin retention of budding particles on the cell surface enhances natural killer (NK) cell mediated antibody-dependent cellular cytotoxicity (ADCC) (1, 8). Enhanced presentation of HIV-1 specific epitopes on whole virions suggests Tetherin can serve as a link between the innate and adaptive responses. Recently, Tetherin has also been suggested to restrict long interspersed nucleotide element-1 (LINE-1) retrotransposition (66). Tetherin was identified as a potent activator of the transcription factor NF-κB in a screen attempting to identify novel and uncharacterized activators (191). NF-κB is important for many cell functions (see below). The mechanism, including determinants, by which Tetherin activates NF-κB were unknown prior to the start my thesis work. Additionally, it still remains unclear whether described binding factors (e.g. ILT7) can affect NF-κB signaling. Additionally, the immune consequences of

18 Tetherin-mediated activation of NF-κB, during or independent of, infection still remain to be elucidated.

Tetherin as an Immune Sensor

Determinants of Tetherin-mediated activation of NF-κB

BST2 (aka Tetherin) was identified in a large cDNA screen for novel activators of the NF-

κB and MAPK pathways (141). Based upon this initial observation several groups, including ours

(see Chapters 2 and 3), have over the past 3 years identified determinants of, and requirements for, Tetherin-mediated NF-κB activation (25, 44, 59, 60, 228). Tetherin appears to specifically activate NF-κB, but not AP-1 or NF-AT (44, 59, 228). Tetherin-induced NF-κB signaling can be dampened by a dominant-negative IKKβ suggesting signaling is activated via the classical pathway (44, 59, 228). Tetherin isoforms [long (l-) and short (s-)] highlighted in Chapter 2 have different signaling functions. s-Tetherin acts as a dominant-negative regulator of l-Tetherin- mediated signaling (44). Additional signaling-null Tetherin mutants, including s-Tetherin, have been shown to also act as a dominant negative, suggesting dimerization and/or multimerization of the cytoplasmic tails may be important (see below). Tetherin-mediated signaling, like its viral restriction function, is dependent on specific structural domains, as well as sequence motifs.

The cytoplasmic tail is critical and encodes several determinants of Tetherin-mediated signaling. Loss of the dual-tyrosine motif significantly reduces signaling (44, 59, 228). Individual tyrosine mutations have varying effects on signaling. Y6 within the dual-tyrosine motif has been demonstrated as being crucial residue in what is believed to be a tyrosine-based signaling motif similar to an ITAM (60). Tyrosine-based trafficking motifs (YxxΦ, where Φ=bulky hydrophobic) resemble tyrosine-based signaling motifs observed in immunoreceptor tyrosine-based activation motifs (ITAMs), which have individual tyrosine based motifs space by a series of non-specific amino acids (YxxL/IX(6-8)YxxL/I). ITAMs are phosphorylated at the tyrosine residues, providing a docking site for src-homology 2 (SH2) domain containing proteins, which typically are intracellular kinases that amplify signaling pathways. Tetherin can be phosphorylated at either or both

19 cytoplasmic tyrosine, Y6 or Y8 (60, 211). However, the Tetherin dual-tyrosine motif does not have the proper spacing between tyrosine residues observed in classical ITAMs. hemITAMs, however, represent a class of signal activating motifs with single tyrosine residues. This motif has been observed on the dimeric immune sensor C-type lectin 2 (CLEC-2) and meditates recruitment of

Syk kinase. Tetherin dimerization is important for signaling suggesting that Tetherin phosphorylation could parallel what is seen with receptors like CLEC-2 (87).

Triple alanine scanning mutagenesis has been performed on the cytoplasmic tail identifying additional sets of amino acids that are required for signaling. Mutations at both an arginine and valine residue believed to be under positive selection at positions 10 and 11 (RV) abrogate signaling (59). Additionally, a region of basic residues that is proximal to the inner leaflet of the PM was identified to be important. The arginine (R19) at the center of the set of triple mutants in the basic patch has been identified as a rare polymorphism in humans (R19H).

Mutation at this site alone abrogates signaling (60, 191). Across all the point mutants tested, ability to restrict particles was never compromised suggesting residues are specific for signaling function.

Though the cytoplasmic tail plays an important role in signaling, additional residues and domain have also been identified (Figure 1-5). Residues in the transmembrane domain have been suggested (see below). The GPI anchor appears to be important, as addition of a premature stop codon before the GPI-anchor signal sequence abrogates signaling (59). However, merely deleting the C-terminal serine residues that could potentially act as GPI anchor sites still allows

Tetherin to signal (228). It remains unclear whether this deletion mutant has actually lost the addition of the GPI-anchor. Replacing the GPI anchor with TMs from heterologous proteins, including ones that localize to rafts, do not restore signaling to wt Tetherin levels though it is suggestive that a second anchor is important (25). Furthermore, the chimeric proteins used in the study did not restore wt Tetherin microdomain localization suggesting raft localization plays an important role for signaling (25). Additionally, loss of both N-linked glycosylation dampens signaling most likely due to mislocalization as this mutant has been previously observed to

20 reduce PM expression (171). Based on the body of literature available, Tetherin-mediated signaling is believed to be enhanced by clustering of Tetherin multimers at the PM. Mutations at residues that promote multimerization (L70D, L123P) abrogate signaling (59, 228) and do not promote enhanced activation during viral egress. Tetherin has been suggested to organize raft microdomains (25). Changes in Tetherin structure, through viral particle sensing, presumably enhances coalescing of raft microdomain and subsequent migration and recruitment of raft resident signaling platforms.

Tetherin as a novel pattern recognition receptor

Tetherin restriction of a broad range of enveloped viruses coupled with this previously uncharacterized signaling function suggested a role in sensing viral egress. In Tetherin expressing cells NF-κB activation, as measured by reporter assay, was enhanced compared to

Tetherin-null or knockdown cells, using both VLP-based systems and replication competent viruses (MLV and delU HIV-1) in stable cell lines. This correlated with enhanced cytokine mRNA expression in primary CD4 T cells (59). Tetherin-mediated signaling leads to activation of various cytokines including the proinflammatory cytokines IL-6 and CXCL10. When HIV-1 expresses Vpu, reduced NF-κB activation is observed. Though a Vpu mutant (A14L) that has lost its ability to interact with Tetherin allowed activation of NF-κB (59, 235), it is unclear whether this Vpu mutant affects signaling independent of Tetherin as Vpu has been shown to dampen NF-κB by stabilizing

IκB (28). Additionally, enhanced IFNβ mRNA expression was observed in the presence of replication competent virus. Tethered particles are thought to be endocytosed and degraded in endolysosomal compartments. The current theory suggests that enhanced IFNβ is the result of viral nucleic acid being sensed by an endosomal receptor. Sensing and subsequent signaling is dependent on efficient assembly and budding of viral particles. L-domain mutant MLV fails to robustly activate NF-κB. Replacing the mutant l-domain with the p6 region of HIV-1 restores activation (59). Additionally, blocking endocytosis enhances signaling suggesting virion accumulation plays an important role. Clustering of particles leads to aggregation of Tetherin at the PM, a model that is also supported by the observation that Ab crosslinking of Tetherin at the 21 PM also enhances signaling (59). Sensing of viral particles correlates with phosphorylation of the dual tyrosine motif. Genetic and chemical perturbations suggest activation by the putative hemITAM is Src and Syk kinase dependent. The interaction between Tetherin and the actin cytoskeleton through RICH2 is crucial as knockdown of RICH2 or disruption of the actin polymerization by cytochalasin D reduces the hypothesized Tetherin-mediated Syk activation

(60).

Tetherin-mediated signaling as an acquired function in humans during primate speciation

Regions in the cytoplasmic tail are thought to have undergone positive selection in . Signaling by human Tetherin appears to be an acquired function from these adaptations. This function, in part, has been suggested to have contributed to the selection of the current HIV-1 pandemic strain, M; subtypes of which make up about 90% of the circulating cases worldwide (82). Human Tetherin is the only primate Tetherin with signaling function identified to date. Tetherins from chimpanzee, gorilla, African green monkey and rhesus macaques do not signal, nor does Tetherin from mouse and various felids; a caveat of these findings being that signaling was tested in human cells (59). It is unclear whether non-human Tetherin proteins signal in the appropriate cognate cell, though NF-κB signaling is fairly well conserved among mammals. Recently, two equine Tetherins were demonstrated to activate NF-κB in human cells independent of a tyrosine-based motif (259). It is currently unclear whether they activate similarly to what has been described for human Tetherin.

Tetherin antagonism by primate lentiviruses is specific. Important residues that have evolved in primates contribute to hTetehrin signaling (Figure 1-5) including the five amino acid

Nef recognition motif, which impairs chimp Tetherin, the closest related Tetherin to humans.

Deletion of this Nef recognition sequence allows chimp Tetherin to signal; conversely, introduction of this sequence into human Tetherin abrogates signaling (59). The RV region described above is under positive selection in primates. The previously described RV tail sequence and a two amino acid insertion into the TM only present in human and chimp Tetherin are also important. Additionally, the STS region was previously identified to be important for Vpu 22 mediated downregulation of human Tetherin (229). Introduction of the rhesus sequence (PIL), but not the chimp sequence which is point mutation S5L, also abrogates signaling (60). This region is explored in Chapter 3 of this dissertation.

Current model for Tetherin-mediated signaling

To summarize the current model from Galao et al of Tetherin-mediated activation of NF-

κB: Tetherin in a resting cell is believed to interact with RICH2 via its cytoplasmic domain. In this model, the actin cytoskeleton, in part mediates sensing of viral particles. Tethered-particles clustered at the cell surface enhance phosphorylation of the dual-tyrosine motif in the Tetherin cytoplasmic tail by a currently unknown Src kinase. This phosphorylated tyrosine motif is believed to act as a hemITAM, which recruits Syk kinase. Syk subsequently activates a series of effectors including TRAF2/6 and the TAK1 complex leading to downstream liberation of NF-κB. NF-κB induced genes are upregulated (Figure 1-4, (60)), though their role downstream of Tetherin, in vivo, is unknown.

Project Aims

Tetherin restricts a wide range of clinically relevant enveloped viruses, including HIV-1. A large body of work has elucidate how Tetherin directly “tethers” budding viral particles. Functional studies have identified the structural features required for viral particle restriction. The Tetherin cytoplasmic tail has been shown to be dispensable for tethering particles. However, we know aspects of Tetherin biology, like trafficking, are regulated by motifs in the tail. Additionally, virally encoded Tetherin antagonists can bind/target sequences in the tail to mediate PM downregulation. The human Tetherin cytoplasmic tail has few conserved features across other orthologues apart from the previously described dual-tyrosine motif. We sought to identify additional features of the cytoplasmic tail that could potentially regulate poorly characterized aspects of Tetherin biology.

In Chapter 2, we describe an internal methionine in the cytoplasmic tail of human

Tetherin, which is highly conserved in Tetherin orthologues. We hypothesized this codon could

23 act as a downstream in-frame translation initiation site (TIS) based on analysis the Kozak sequences flanking the putative upstream start codon and the conserved downstream AUG.

Because this would yield a truncated isoform lacking, at minimum, the dual-tyrosine motif, we hypothesized that the full-length and truncated isoforms could be functionally distinct. There was precedent for this hypothesis. Blasius et al. identified Tetherin cDNA from murine cells, which only encoded a downstream methionine compared to the full-length annotated sequence (26).

Upon cloning the full-length form, Blasius et al. observed reduced expression off the upstream start codon leading to the hypothesis that the second AUG codon could be the primary start site and that the full-length form could have “instability or altered cellular localization” (26).

Functionally we assessed the ability of the Tetherin isoforms to restrict particle release in the presence and absence of the Tetherin antagonist Vpu, and to activate NF-κB. Because NF-κB plays an important role in priming early responses to viral infection and Tetherin acts to restrict virus, we sought to elucidate this understudied Tetherin function in an attempt to develop a more comprehensive picture of Tetherin as an innate immune factor.

Observations described in Chapter 2, in combination with concurrent work out of the Neil and Guatelli groups implicated the dual-tyrosine trafficking motif for Tetherin-mediated signaling.

We initially hypothesized that differences in signaling between l- and s-Tetherin isoforms would help focus on the most important determinants for signaling. It has since been suggested that the tyrosine motif can act as a hemITAM (60). However, numerous determinants have been identified beyond the dual tyrosine motif within and outside of the cytoplasmic tail (59, 60, 191, 228), which suggests a more complex signaling model. Syk has been suggested to interact with Tetherin via the hemITAM, however a direct interaction has not been definitively shown. The studies described in Chapter 3 were started before a hemITAM was implicated in the current model of

Tetherin signaling. During the process of identifying determinants of signaling, we discovered that our data, from a series of single and multiple point mutations, does not appear to align with the current model described above, thereby questioning the mechanism by which Tetherin is activating NF-κB. In Chapter 3 we assess the importance of the tyrosine residues within the

24 hypothesized hemITAM, as well as suggest the role of an amino terminal motif that we hypothesize regulates Tetherin-mediated signaling.

25 z z z z

FIGURES

(TFs)

s

, which leads to β IFN ISGs ISGs APOBECs TRIMs IFITMs Viperin ISG15 TETHERIN , which propagates signal JAK STAT2 STAT2 IFNAR Cells Cells that encounter virus sense

) P P IFN IRF9 POS. REGs POS. IRF7 PKR OAS REGs NEG. SOCS

KSHV membrane membrane bound receptors (e.g. TRAM, [

EBOV EBOV , as well as other transcription factor , as as well transcription other TYK2 STAT1 STAT1 IRF7 EBV MARV MARV at the surface or upon entry in the cytoplasm or

IRF3 s) responsive sequence element (IRSE) on target DNA - PRR P P ISGF3 ISGF3 Follow from left to right ( adaptor molecule

. α IFN signaling and ISGs. Viruses that target IFN expression and and IFN expression target that and Viruses ISGs. IFN signaling IFN F3 binds IFN via via binding motifs or modified residues

TLRs

HCV P

VACV P

β P P IFN events transcription factors like

slocate slocate the nucleus and bind cognate TF binding sites

TLRs, CLRsTLRs, . P

s (MAVS, STING)] IRF3 P or autocrine manner to activate JAK/STAT pathway. STAT1/STAT2 phosphorylation leads to RLRs cGAS

- dimerize, tran phosphorylation

) MAVS/ MAVS/ STING 2 - rane rane receptors recognize proteins on the surface of the particle (e.g. glycoprotein). Intracellular both positive and negative regulators of toplasmic toplasmic sensor Figure 1 ze ze viral nucleic acid. Activated receptors recruit signaling highlighted in red in highlighted signaling Schematic Schematic of the type I IFN signaling pathway (see (see

, Through a series of

1. - s. κ B - mediated mediated - igure igure 1 F ( receptors recognition pattern by ligands associated pathogen conserved endosome. Plasma memb receptors recogni TRIFF, MyD88), cy cascade like NF expression. IFN β signals in para dimerization and formation of complex with IRF9 (ISGF3). ISG leading to expression of IFN

26

Rest Activated PRR

Adaptor

P SH2 containing kinase P TAK complex

IKK complex P IκB

NF-κB P Proteosome

Other transcription factor

P

Proinflammatory cytokines

Figure 1-2. Schematic of classical NF-κB signaling through a surface receptor. When the cell is rest, NF-κB dimers remains cytoplasmic, sequestered by IκB. Ligand binding by a receptor leads to receptor or adaptor phosphorylation at signaling motifs like ITAMs, which recruits SH2 containing kinases, like Src- or Syk-family kinases. Central to activation of NF-κB via stimuli, like

TNFα and bacterial LPS. is the TAK1 complex. Phosphorylation leads to activation the IκB kinase

(IKK) complex, which is comprised of 3 subunits, IKKα, IKKβ and NEMO. The IKK complex mediates ubiquitination of IκB and subsequent proteosomal degradation, which frees NF-κB for nuclear translocation. NF-κB regulates expression of various genes involved in immunity, including proinflammatory cytokines.

27

A B C

Viral core protein ESCRT proteins Cholesterol Actin Viral glycoprotein Cellular protein Raft-enriched phospholipid

Figure 1-3. Schematic representation of viral egress. (A) Oligomerized or monomeric subunits are targeted to sites of budding. Aggregation of viral proteins at the membrane induces curvature, initiating the budding process. Viral “core” proteins recruit ESCRT proteins like TST101 (green circle) to facilitate budding and scission. TSG101 recruitment leads to ESCRTIII complex (green ring) constricting budding neck, leading to scission (B) Fully budded enveloped virion completed scission, acquiring host membrane and specific host molecules in the process. (C) l-domain mutant viruses and/or mutations in ESCRT proteins prevents viral budding.

Particles remain “tethered” to plasma membrane via contiguous host membrane.

28

A C

* * * Extracellular CT TM Ectodomain GPI

Tetherin- MASTSYDYCRVPMEDGDKRCK mediated endocytosis of YxYΦxΦ viral particle Cytoplasmic B

Side View

Top View

Figure 1-4. Cartoon representations of Tetherin domains, topology and function. (A) Linear cartoons highlighting important functional domains. Top: Graphical depiction of domains and modifications with * denoting cysteine residues involved in disulfide bond formation. Two sites of

N-linked glycosylation are labeled. Bottom: Linear domain model. Residues of the cytoplasmic tail of human Tetherin labeled. Dual-tyrosine motif underlined and aligned with putative consensus for trafficking motif YxxΦ, where Φ =hydrophobic residue. CT=cytoplasmic tail,

TM=transmembrane domain (B) Tetherin topology. Top left: Topological cartoon representation of a parallel Tetherin dimers (black monomer in front, grey monomer in back) anchored at the plasma membrane. Raft domain represented by yellow shapes. Top right: Cartoon depiction of

Tether tetramers interacting via N-terminal hydrophobic faces on ectodomain Bottom: Looking down on cell surface, Tetherin molecules depicted spanning lipid rafts (yellow clusters) as dimers and tetramers (C) Tetherin localization at sites of budding allows incorporation of one anchored end into particle (preferably the GPI anchor), while other end remains membrane associated with host membrane. Tethered particles are subsequently endocytosed and degraded. 29 L70D L123P GPI?

* * *

Human MASTSYDYCRVPM-----EDGDKRCKLLLGIGI Chimpanzee MASTLYDYCRVPMDDIWKKDGDKRCKLLLGIGI Gorilla MASTLYDYCRVPMDAILKKDGGKRCKLLLGIGI AGM MAPILYDYCKMPMDDICKEDRDKCCKLAV--GI Rhesus MAPILYDYCKMPMDDIWKEDGDKRCKLVI--GI Mouse MAPSFHYLPVPMDEMGGKQGWGSHRQWLGAAIL Cat ------MVPGRSLGWQRWLGG--- Horse ------MGDHRLLRW-LLLVV-V Donkey ------MGDHRLLRW-LLL----

Cytoplasmic tail TM

Figure 1-5. Determinants of Tetherin-mediated signaling. Cartoon model of linear Tetherin structure described in Figure 1-4A. Red highlighted sequences on cartoon structure abrogate signaling when mutated in human Tetherin *= triple cysteines substitution. Alignment of cytoplasmic tail and partial sequence of tail proximal TM sequence expanded. Cytoplasmic tail residues highlighted in red are single point mutations identified in Chapter 4 that drastically impact signaling. Residues highlighted in blue previously identified as important for signaling in combination via triple alanine scanning mutagenesis. Residues in gold when introduced into the human Tetherin background, abrogates signaling. Tetherins observed to signal highlighted in green.

30

Rest Activated

RICH2 P P P P P P P P Syk

TAK1 TRAF2/6? TAB1/2 AP-1

IL-6 CXCL10

Figure 1-6. A proposed model for Tetherin-mediated NF-κB signaling. When cell is at rest,

Tetherin associates with actin cytoskeleton via RICH2. Activation through Tetherin clustering by sensing assembling/budding viral particle (or Ab crosslinking) leads to actin-dependent phosphorylation of cytoplasmic tyrosine motif by an unknown src-kinase. Syk recruitment to this hemITAM activates downstream complexes consisting of TRAF2/6, TAK1, and TAB1/2, which leads to IKK complex activation. IKK mediates ubiquitination of IκB, liberating NF-κB for nuclear translocation where it upregulates genes like IL-6 and CXCL10. Unlike other traditional TAK1 mediated signaling pathways, Tetherin does not activate AP-1.

31 CHAPTER 2: IDENTIFICATION OF ALTERNATIVELY TRANSLATED

TETHERIN ISOFORMS WITH DIFFERING ANTIVIRAL AND SIGNALING

ACTIVITIES

Luis J. Cocka and Paul Bates#

Department of Microbiology, Perelman School of

Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104

#To whom correspondence should be addressed: [email protected]

Originally published in PLoS Pathog 8(9): e1002931. doi:10.1371/journal.ppat.1002931

September 27, 2012

32 ABSTRACT

Tetherin (BST-2/CD317/HM1.24) is an IFN induced transmembrane protein that restricts release of a broad range of enveloped viruses. Important features required for Tetherin activity and regulation reside within the cytoplasmic domain. Here we demonstrate that two isoforms, derived by alternative translation initiation from highly conserved methionine residues in the cytoplasmic domain, are produced in both cultured human cell lines and primary cells. These two isoforms have distinct biological properties. The short isoform (s-Tetherin), which lacks 12 residues present in the long isoform (l-Tetherin), is significantly more resistant to HIV-1 Vpu- mediated downregulation and consequently more effectively restricts HIV-1 viral budding in the presence of Vpu. s-Tetherin Vpu resistance can be accounted for by the loss of serine-threonine and tyrosine motifs present in the long isoform. By contrast, the l-Tetherin isoform was found to be an activator of nuclear factor-kappa B (NF-κB) signaling whereas s-Tetherin does not activate

NF-κB. Activation of NF-κB requires a tyrosine-based motif found within the cytoplasmic tail of the longer species and may entail formation of l-Tetherin homodimers since coexpression of s-

Tetherin impairs the ability of the longer isoform to activate NF-κB. These results demonstrate a novel mechanism for control of Tetherin antiviral and signaling function and provide insight into

Tetherin function both in the presence and absence of infection.

33 AUTHOR SUMMARY

Regulation of innate immunity is critical to maintain a balance between control of a perceived threat and immunopathology. The interferon induced cellular factor Tetherin has been shown to restrict budding of a broad range of enveloped viruses including the human immunodeficiency virus. Though Tetherin appears to be a bona fide viral restriction factor, additional cellular functions have been observed including an involvement in actin cytoskeleton organization in polarized cells, regulating interferon secretion and signaling through nuclear factor-kappa B (NF-κB). Our studies present a mechanism by which Tetherin function is regulated at the translational level through the production of alternatively translated isoforms. The short isoform of Tetherin was observed to be significantly more resistant to HIV-1 Vpu. In contrast, the longer isoform can induce NF-κB activity, a function lacking in the short isoform. Critical NF-κB signaling residues include a dual tyrosine motif, which is only present in the long isoform.

Identification of these isoforms helps to illuminate how Tetherin functions, not only as a restriction factor, but also as a signaling molecule. These data highlight a previously unappreciated level of regulation and furthers our understanding of additional Tetherin functions.

34 INTRODUCTION

Tetherin (BST-2/CD317/HM1.24) is an interferon (IFN) induced, type II transmembrane glycoprotein which has been shown to function as an intrinsic antiviral factor that restricts release of a broad range of enveloped viruses, including members of the arenavirus (177, 185), filovirus

(100, 185), gammaherpesvirus (136, 170), paramyxovirus (177), retrovirus (9, 69, 99) and rhabdovirus (189, 246) families. Selective pressure from Tetherin on the aforementioned families of viruses is evident because many of these Tetherin sensitive viruses encode proteins that counter Tetherin function. To date, several virally encoded Tetherin antagonists have been identified, a few of which function through different mechanisms. These include HIV-1 Vpu (158,

231), KSHV K5 (136), SIV Nef (95), HIV-2 and SIV Env (70, 118), and ebolavirus GP (100).

Tetherin has an unusual topology that has been shown to be critical for function as a restriction factor. Tetherin is a lipid raft resident protein anchored to the cellular membrane by a

N-terminal transmembrane domain and a C-terminal GPI anchor (109). Localization to sites of enveloped virus budding and anchoring at both ends is needed for Tetherin function as a cellular restriction factor through a direct “tethering” mechanism which bridges the viral and host membranes (171). The N-terminus also includes a cytoplasmic tail, which contains a tyrosine- based motif that mediates trafficking between lipid rafts at the plasma membrane and intracellular compartments, including the trans-Golgi Network (TGN), via adaptor complexes involved in clathrin-mediated endocytosis (140, 181). Additionally, the Tetherin cytoplasmic tail has a serine- threonine-serine stretch and lysine residues that were identified as targets for HIV-1 Vpu- mediated tetherin downregulation through the recruitment of cellular ubiquitin ligase complexes

(51, 148, 229).

A number of studies have examined the ability of Tetherin to function as a cellular restriction factor; however, there is evidence that Tetherin has additional roles that may be important both in the presence and absence of viral infection. Tetherin was originally described as a marker on differentiated B cells and was suggested to be involved in B cell development (67,

35 91). It is apparent now that Tetherin is constitutively expressed on various other cells of the immune system including macrophages, plasmacytoid dendritic cells (pDCs) and T lymphocytes

(26, 234). Tetherin is also preferentially expressed on the surface of various transformed and metastatic cell types (33, 162), though whether Tetherin is actively contributing to the transformed state of the cell is unknown. Additionally, Tetherin has been proposed to act as a scaffold protein in polarized cells (180) and contribute to monocyte adhesion to endothelial cells (260). Apart from directly restricting viral egress, Tetherin has been shown to have immunomodulatory properties.

Tetherin regulates cytokine secretion in murine (26) and human pDCs. In human pDCs, Tetherin acts as a ligand for immunoglobulin-like transcript 7, which results in modulation of Toll-like receptor-mediated IFN and proinflammatory cytokine secretion (35). Additionally, a cDNA screen identified BST-2 (aka Tetherin) as one of several uncharacterized cellular factors that could activate NF-κB (191). However, this potential role for Tetherin in signaling has not been further explored.

Here we identify a previously undescribed short isoform of human Tetherin generated by alternative translation initiation at an in-frame codon 33 nucleotides downstream of the canonical translation start site which results in the production of two Tetherin species. The long (l-) and short (s-) Tetherin isoforms are generated in both cultured and primary human cells. Importantly, the two isoforms have distinct biological properties. s-Tetherin, which lacks 12 residues present in the long isoform, is significantly more resistant to HIV-1 Vpu-mediated downregulation and consequently more effectively restricts HIV-1 release in the presence of Vpu than l-Tetherin. In contrast, the ebolavirus glycoprotein (GP) effectively counters both Tetherin isoforms. We also find that Tetherin is a significant activator of NF-κB. However, the Tetherin isoforms have dramatically different signaling potential, with NF-κB activation requiring a tyrosine motif found in the l-Tetherin isoform but absent in s-Tetherin. These observations provide insight into how the expression of alternatively translated Tetherin isoforms regulates multiple cellular functions and begins to shed more light on a poorly characterized signaling property.

36 RESULTS

Alternative translation initiation produces two isoforms of Tetherin

Sequence analysis of the cytoplasmic tail of Tetherin revealed minimal amino acid conservation across species. Residues that were highly conserved included two cytoplasmic methionine residues and a dual tyrosine motif (Figure 2-1A), previously found to be involved in

Tetherin trafficking between the plasma membrane and intracellular compartments (140, 181).

Methionine codons can function as translation start sites when they are flanked by a Kozak consensus sequence, which influences ribosomal binding at the site of translation initiation in eukaryotic cells. Analysis of the human tetherin mRNA sequence revealed that the Kozak sequences flanking the first AUG (M1) appears to be a potentially “leaky” signal due to a non- consensus pyrimidine at the -3 position (Figure 2-1B) (107). The downstream methionine (M13) appears to be a stronger translation start site with compensatory cytosine residues at positions -1 and -2, which could enhance the strength in the absence of the purine in the -3 position (106).

Although neither of these methionine residues can be defined as strong Kozak consensus start sites, both sequences display conservation at the important +1 through +4 positions. Analysis of the mRNA sequences of Tetherin from other species revealed a similar potentially “leaky” Kozak signal for the upstream methionine (Figure 2-S1), suggesting that production of multiple isoforms may be a conserved Tetherin characteristic.

To address whether alternatively initiated Tetherin isoforms could be produced, mutations that individually abrogated each of the AUG codons were generated in the wt cDNA (Figure 2-

2A). N-linked glycosylation of Tetherin produces a heterogeneous population of proteins that is not well resolved by electrophoresis [(6) and Figure 2-S2], complicating unambiguous identification of the observed Tetherin bands. Therefore, to better differentiate the potential isoforms that are predicted to differ by 12 residues (~1.5 kDa), lysates from transiently expressing cells were treated with Peptide: N-Glycosidase F (PNGase) to remove N-linked glycosylation before analysis by Western blot. Analysis of deglycosylated lysates from HT1080 cells

37 expressing the wt Tetherin cDNA reveals two major bands of between 15-20kDa (Figs. 2-S2 and

2-2B); the predicted sizes of proteins initiated at the M1 and M13 translation initiation sites is approximately 18 and 16.5kDa respectively. Transient expression of the wt tetherin cDNA produces more of the larger form compared to the smaller species. Expression of the M13I mutant in HT1080 cells produces a single major product that aligns with the larger protein species seen in wt cDNA transfected cells (Figure 2-2B). The M1A mutant, that abolishes the upstream potential initiation site, generates a protein that migrates similarly to the smaller species produced by wt cDNA. Co-transfection of plasmids expressing the M1 and M13 mutants recapitulates the pattern seen with wt Tetherin (Figure 2-2B). Overall, these data demonstrate that transient expression of the wt cDNA produces two alternatively initiated species that we refer to as the l-

Tetherin (long) and s-Tetherin (short) isoforms.

To address whether expression of the s-Tetherin isoform was due to translational leakage, an optimized translational initiation sequence was generated by converting the sequence at the -3 position of the upstream M1 ATG in the wt Tetherin cDNA from a pyrimidine

(T) to a purine (A). Upon introduction into HT1080 cells of a vector with a strong Kozak sequence at the M1 AUG, expression of l-Tetherin was greatly enhanced while expression of s-Tetherin was almost undetectable (Figure 2-2B). This mutational analysis, coupled with the data presented above, demonstrates that Tetherin utilizes a non-canonical Kozak sequence to produce alternatively initiated proteins with different cytoplasmic tails.

To determine whether both isoforms of Tetherin are endogenously expressed, non- stimulated and interferon (IFN)-stimulated 293T, HT1080, HeLa, primary human CD4 T-cell lysates were analyzed as described above and compared to 293T cells transiently expressing the

M13I and M1A mutants. As predicted from previous results, no Tetherin was seen upon analysis of deglycosylated proteins from unstimulated 293T and HT1080 cells. Interferon treatment of

293T and HT1080 cells induces Tetherin expression and upon PNGase treatment, collapses a diffuse banding pattern to two bands that migrate similarly to the two isoforms produced by transfection of l-Tetherin and s-Tetherin encoding plasmids (Figure 2-2C, 1st panel and Figure 2-

38 S3). This demonstrates that IFN stimulation produces both alternatively initiated forms of

Tetherin. Analysis of lysates from HeLa cells, which constitutively express relatively high levels of

Tetherin, revealed the presence of the doublet in the absence of IFN (Figure 2-2C, 2nd panel).

Both bands appear to be enhanced after IFN stimulation (Figure 2-S3). Similarly to HeLa cells, analysis of untreated primary CD4 T-cells revealed two Tetherin species that were enhanced upon IFN stimulation (Figure 2-2C, 3rd panel). In all cell types tested for endogenous expression of Tetherin, roughly equal expression of both isoforms was observed after 48 h of IFN stimulation.

Expression of the M13I and M1A mutants in 293T cells produced the l- and s-isoforms seen above in HT1080 cells, however in this case smaller molecular mass species are seen (denoted by an asterisk on the 4th panel of Figure 2-2C and throughout the manuscript). These species are only observed in transiently expressing 293T, and not in transfected HT1080 or interferon- induced cells. Though the derivation of these species is unclear, they may represent a precursor or proteolyzed product. Finally, analysis of cells transiently expressing rhesus or murine Tetherin cDNA or IFN treated murine J774 cells demonstrated a doublet indicative of production of two isoforms (Figure 2-S4A and B) indicating that production of long and short isoforms of Tetherin is a highly conserved feature. In contrast, the rhesus cell line FRhK-4 appears to produce only a single Tetherin isoform (Figure 2-S4B). Taken as a whole, the presence of these isoforms in numerous cell types, under various conditions and in multiple species is suggestive of important biological roles for the newly identified l-and s-Tetherin isoforms.

Tetherin isoforms assemble into homo- and heterodimers

Tetherin exists as a disulfide-linked dimer that is believed to represent the functional form necessary for viral restriction. Structural studies on the ectodomain suggest that Tetherin dimers may also form higher order aggregates, which may be functionally important (83, 197). To address whether the isoforms interact to form heterodimers, lysates from HT1080 cells expressing wt, l- or s-Tetherin were separated under non-reducing conditions and analyzed by

Western blot. Expression of either the l- or s-Tetherin isoform produced single bands of a

39 molecular mass indicative of homodimer formation (Figure 2-S5A). In comparison, the wt cDNA produced a broader expression profile that overlapped with the l- and s-profiles suggesting both homo- and heterodimer formation (Figure 2-S5A). To directly assess the interaction of the l- and s- isoforms, a C-terminally FLAG-tagged l-Tetherin and N-terminally AU1 tagged s-Tetherin were co-expressed and immunoprecipitated using antibodies to the epitope tags (Text 2-S1). As anticipated for efficient heterodimer formation, immunoprecipitation with either tag effectively co- precipitated the other isoform (Figure 2-S5B). As discussed below, the ability to form homo- and heterodimers may have significant effects upon the biological activities of Tetherin.

Both Tetherin isoforms restrict particle release, but differ in their sensitivities to viral antagonists

The cytoplasmic tail of Tetherin is dispensable for function as a viral restriction factor

(171). However, residues in the cytoplasmic tail have been shown to confer sensitivity to viral antagonists. For example, a serine-threonine-serine sequence, which is unique to l-Tetherin, has been found to be important for efficient Vpu antagonism (229). Using a transient HIV-1 virus-like particle (VLP) budding assay, the l- and s- isoforms were analyzed for their ability to restrict viral release. Both isoforms were effective viral restriction factors and displayed strong inhibition of

HIV-1 VLP release (Figure 2-3A, top panel). The antiviral activity of wt Tetherin, which expresses both Tetherin isoforms, is antagonized by co-expression of HIV-1 Vpu with VLP release correlating with the level of Vpu expression. In contrast, antiviral activity of the l-Tetherin isoform is exquisitely sensitive to Vpu with significant rescue of viral budding seen at the lowest levels of

Vpu expression analyzed (Figure 2-3A, top panel). Additionally, a decrease in l-Tetherin protein levels was readily observed at higher levels of Vpu expression (Figure 2-3A, third panel).

Conversely, the s- isoform appears to be resistant to antagonism by Vpu, with very little VLP budding rescue observed at the highest levels of Vpu tested (Figure 2-3A, top panel). Moreover, turnover of the s- isoform also appears to be resistant to Vpu as there was no obvious decrease in s-Tetherin expression even at the highest levels of Vpu (Figure 2-3A, third panel).

40 To determine whether the differential sensitivity to viral antagonists was a common property of the Tetherin isoforms, sensitivity to a different viral factor, the ebolavirus glycoprotein,

GP, was analyzed. GP is hypothesized to rescue virus budding in Tetherin-expressing cells through a non-cytoplasmic tail-associated mechanism (127) and might therefore be predicted to interact similarly with both Tetherin isoforms. In contrast to the results with Vpu, within the range of GP levels tested, GP-mediated rescue of budding was similar for wt and the two Tetherin isoforms (Figure 2-3B, top panel). Additionally, total cellular Tetherin protein levels appeared unchanged for wt Tetherin and each individual isoform even at the highest levels of ebolavirus

GP co-expression (Figure 2-3B, third panel).

To determine whether the differential particle release by the isoforms in response to Vpu was due to altered Tetherin cell surface levels, flow cytometric analysis of live cells stained for

Tetherin was performed. In Vpu expressing cells, surface wt Tetherin expression followed the same trend as the total cell Tetherin protein levels (compare Figure 2-3A third panel and Figure

2-4). Surface expression of l-Tetherin was downregulated by Vpu to a greater extent than the s-

Tetherin isoform, while wt Tetherin appeared to have an intermediate level of downregulation

(Figure 2-4). Analysis of lysates from the cells prepared in parallel with those used for flow cytometry confirmed the enhanced degradation of l-Tetherin compared to s-Tetherin observed above in both the reduced monomers and stable dimers. Additionally, preferential degradation of the l-isoform was seen in lysates of cells expressing wt Tetherin and high levels of Vpu (Figure 2-

4; arrow). In comparison, there is no difference in the effect of ebolavirus GP on surface or total levels for the l- and s- isoforms though a decrease in surface staining was observed across all

Tetherin constructs tested. The minimal 2-fold decrease in the mean fluorescence intensity of

Tetherin surface staining observed in ebolavirus GP expressing cells is consistent with steric shielding of epitopes previously documented for this glycoprotein (56, 179) and appears similar to the slight decrease noted recently by Lopez and colleagues (127). Overall, these observations suggest that the Tetherin isoforms can have different sensitivities to viral antagonists, which may have important consequences during natural infection.

41

Resistance to Vpu antagonism requires mutations in the tyrosine and serine/threonine motifs in Tetherin

To further explore the basis for the relative Vpu resistance of s-Tetherin compared to l-

Tetherin, a series of mutations were engineered into the 12 residue region unique to l-Tetherin

(Figure 2-5A). Two motifs within this region have been proposed to play a role in Vpu mediated antagonism; a dual tyrosine motif (amino acids 6 and 8, Figure 2-5A) involved in endocytic cycling of Tetherin (195) and a stretch of serine and threonine residues (amino acids 3 to 5, Figure 2-5A) that can be ubiquitinated (72, 229). Analysis of HIV-1 VLP release from 293T cells transiently expressing a tyrosine mutant AxA produced in l-tetherin expressing cDNA shows decreased release in response to Vpu as compared to parental l-Tetherin (lane l-AxA, Figure 2-5B).

However, the observed decreased Vpu antagonism of AxA is not as pronounced as that seen with s-Tetherin. Altering the serine/threonine residues also impairs Vpu mediated particle release

(lane l-STS, top panel Figure 2-5B). However, as with the tyrosine mutations the effect seen is intermediate between l- and s-Tetherin. A mutant that combines the tyrosine and serine/threonine changes appears to recapitulate the high level of resistance to Vpu antagonism observed with s-

Tetherin (lane l-SY, top panel Figure 2-5B). Thus it appears that both these regions are involved in Vpu mediated antagonism of Tetherin function.

Although the tyrosine and serine/threonine mutants appear to similarly affect response to

Vpu antagonism and particle release, the STS mutant Tetherin protein demonstrates resistance to Vpu-mediated degradation comparable to that of s-Tetherin (third panel, Figure 2-5). In contrast, the AxA mutant protein levels decrease upon Vpu expression comparably to parental l-

Tetherin (third panel, Figure 2-5). The combined tyrosine and serine/threonine mutant (third panel, lane l-SY) is Vpu resistant like s-Tetherin and the STS mutant. Overall, these findings support an important role for the STS region in Vpu mediated degradation of Tetherin.

42 The l-Tetherin isoform is an inducer of NF-κB

Tetherin has been primarily characterized as a cell intrinsic viral restriction factor.

However, Tetherin has been proposed to have additional activities including a role in induction of the proinflammatory response regulator NF-κB (191). To confirm that Tetherin was able to activate NF-κB, a luciferase reporter assay was employed in 293T cells transiently expressing

Tetherin. Activation of NF-κB by Tetherin displays a bell shaped curve with decreased stimulation at high levels of Tetherin expression (Figure 2-S6), suggesting an optimal range of expression for signaling. Expression of wt Tetherin led to NF-κB activation approximately 20-30 fold over control cells transfected with the expression vector alone (Figure 2-6A). Moreover, Tetherin activation of

NF-κB is specific since there was no effect upon AP1-regulated signaling (Figure 2-6B). TRAF6, an ubiquitin ligase involved in regulating various signal transduction pathways including NF-κB, upregulated both NF-κB and AP1. Supporting the specificity of the observed effect for NF-κB, activation by Tetherin was effectively blocked in a dose dependent manner by expression of a dominant-negative form of the NF-κB activating kinase IKKβ (DN-IKKβ, Figure 2-6C).

We next assessed whether the Tetherin isoforms had differential abilities to activate NF-

κB. Unlike wt Tetherin, the truncated s-Tetherin isoform displayed no NF-κB activation (Figure 2-

6A). Conversely, compared to wild type, the l-Tetherin isoform was, on average, a more potent activator and increased NF-κB activity approximately 40-fold over basal levels. As was seen for wt Tetherin, NF-κB activation by l-Tetherin was sensitive to inhibition by DN-IKKβ (Figure 2-6C).

These findings demonstrate that sequences within the 12 amino acid region absent in s-Tetherin are required for the signaling events downstream of Tetherin that activate NF-κB.

NF-κB activation requires the tyrosine motif and is negatively regulated by s-Tetherin

As described above, Tetherin can assemble into hetero and homodimers of the l- and s- isoforms. Given the observations that s-Tetherin does not activate NF-κB, l-Tetherin is more potent than wt, and wt Tetherin has an intermediate signaling phenotype to that of l- and s- isoforms, we investigated whether the short isoform could modulate signaling activity of the

43 longer species. To address this question, varying ratios of the two isoforms were assessed for their ability to activate NF-κB. As seen in Figure 2-7, l-Tetherin mediated NF-κB activation is strongly diminished by the presence of s-Tetherin. The ability of s-Tetherin to sharply reduce signaling at a 1:1 and 1:3 ratio of l- to s-Tetherin expressing plasmids is supportive of a model in which homo-oligomers of the longer species are needed to signal. Overall, this observation suggests an inhibitory role for s-Tetherin in regulating NF-κB signaling by l-Tetherin.

Within the 12 amino acids unique to l-Tetherin are two highly conserved tyrosine residues at positions 6 and 8 (Figure 2-1). To address whether these residues contributed the ability of the longer isoform to activate NF-κB, alanine substitution mutations were generated in the context of l-Tetherin and tested by transient expression in 293T cells. Compared to the parent l-Tetherin, the ability of the tyrosine mutant (AxA) to activate NF-κB was significantly reduced (Figure 2-6A).

Although impaired, a low but reproducible level of activity was observed for the dual tyrosine mutant. These data suggest that the mutated residues form part of an important signaling motif within the cytoplasmic tail region needed to activate NF-κB that is present only in the longer isoform of Tetherin.

44 DISCUSSION

Here we identify a previously undescribed isoform of human Tetherin generated by translation at an in-frame initiation codon 33 nucleotides downstream of the canonical translation start site, which results in the loss of a 12-amino-acid N-terminal sequence. Alternative translation initiation in eukaryotes can occur by 3 different mechanisms: internal ribosome entry, reinitiation, or leaky ribosome scanning. Our analysis indicates that the tetherin mRNA contains a

“leaky” Kozak sequence around the first AUG codon (M1). Based on the scanning model of translation, this would allow the ribosome to skip over the first start codon a fraction of the time, leading to initiation at the next in frame AUG (M13). Selective disruption of each AUG and introduction of a strong Kozak at the upstream AUG confirmed that alternate sites of translation initiation accounted for each of the observed products. Conservation of an upstream leaky Kozak and a second cytoplasmic methionine in most mammalian Tetherin sequences, along with our analysis of expression from rhesus and murine cDNAs, suggests that multiple isoforms exist in other species and indicate an important biologic role(s) for the two isoforms.

For many messages with alternatively translated isoforms the resultant proteins display distinct functionalities (116, 130, 182, 254). This is clearly the case for Tetherin where we identified unique biologic properties for the two isoforms. Although both isoforms exhibit antiviral activity, the shorter Tetherin isoform, derived by initiation at M13, is highly resistant to the antagonistic effects of HIV-1 Vpu and therefore may play a more important role in restricting virion budding in HIV-1 infected cells. Analysis of mutants in the 12 residues unique to l-Tetherin suggests that the combined loss of two tyrosine and three serine/threonine residues accounts for the Vpu resistance of s-Tetherin. By contrast, l-Tetherin seems to be hypersensitive to Vpu antagonism. However, this longer isoform possesses the ability to induce the immune response regulatory transcription factor NF-κB while s-Tetherin does not have this capability. Expression of the wt Tetherin cDNA, which produces both protein species yielded for Vpu sensitivity and NF-κB signaling that were intermediate between those seen upon expression of

45 the individual isoforms. This likely reflects that fact that when the isoforms are co-expressed both hetero and homodimers form, and that the unique properties observed (e.g. Vpu resistance and

NF-κB signaling) require short-short or long-long homodimers respectively.

In several other systems where alternative translation initiation produces multiple isoforms there are observed differences in the ratios of the isoforms in tissues (130, 182) or under various physiologic conditions (3) suggesting regulation of alternative initiation. For example, the levels of glucocorticoid receptor (GR) isoforms vary significantly in different tissues, indicating that cells can regulate this ratio with important consequences for GR gene regulation (130). Similarly,

LPS regulates differential expression of alternatively translated forms of the CCAAT/enhancer- binding trans-activator proteins C/EBPα and C/EBPβ, producing isoforms with unique properties

(3). In our experiments, the ratios of l- and s-Tetherin differed in transient expression versus endogenous expression. Lysates harvested 48 h post-transient transfection appeared to produce more of the longer species. In contrast, 48 h stimulation with IFN produced roughly equal amounts of both isoforms. Interestingly, tissue specific expression of ovine Tetherin isoforms has been reported (9); however, the mechanism for generation of these isoforms was not investigated. Our analysis of human Tetherin did not query a large number of cell types or conditions, therefore it will be interesting to more fully address whether there are instances in which the ratio of l- and s-Tetherin is regulated. Conversely, the increased sensitivity of l-Tetherin to Vpu compared to s-Tetherin suggests that viral antagonists might alter the isoform ratio.

Because both NF-κB signaling and Vpu sensitivity appear to be affected by the formation of homodimers, this suggests a model whereby subtle changes in isoform stoichiometry, either by differential expression or susceptibility to viral antagonists of one isoform, could rapidly and dramatically affect Tetherin function.

The importance of the shorter Tetherin isoform with increased antiviral activity or resistance to viral antagonists is strongly supported by recent evidence. While this manuscript was in preparation, a polymorphic allele that abrogated expression from the first methionine residue, and thus produced only short isoforms, was described in NZW mice. In these mice,

46 expression of shorter Tetherin isoforms strongly correlated with decreased Friend retrovirus replication and pathogenesis (17). Tetherin sequence variants have also been identified in rhesus macaques, African green monkeys and mice (144, 261). Interestingly, none of the identified macaque polymorphisms account for the single species we observed by Western blot in the macaque FRhK-4 cell line. Further analysis of genomic sequence of tetherin from this rhesus cell line is required to determine the form being expressed.

We find that the ability of s-Tetherin to retain budding HIV-1 virions and to resist Vpu more effectively than l-Tetherin appears to be due to loss of tyrosine and serine threonine motifs found in the cytoplasmic tail of the long isoform. Mutation in either motif rendered Tetherin partially resistant to Vpu. Combining these two sets of mutations recapitulated loss of Vpu sensitivity seen for s-Tetherin suggesting these represent important differences between l- and s-

Tetherin. The tyrosine motif in l-Tetherin is a non-canonical trafficking signal (YXYXXV) that engages the AP-1 and AP-2 clathrin adaptors (140, 181); mutations within this sequence alter cellular trafficking and distribution (78, 92). It has recently been demonstrated that Vpu impairs recycling of Tetherin to the cell surface and that this is a key mechanism for antagonizing

Tetherin function (195). It will be interesting to determine if s-Tetherin effectively recycles in the presence of Vpu or if endocytosis from the cell surface is affected. Also, within the region unique to l-Tetherin is a serine-threonine-serine stretch that has been shown to be important for Vpu mediated Tetherin degradation (229) although more recent studies have questioned this conclusion (72, 229). Our finding that mutations in this motif confer partial resistance to Vpu antagonism and also affect Vpu induced degradation support an import role for this region in interactions with Vpu. Interestingly, particle retention and Tetherin degradation appear to be separable features because the STS mutant appeared to be resistant to Vpu induced degradation while the YxY mutant remained sensitive to Vpu degradation, yet both mutations partially impair

Vpu induced virion release. Our data is consistent with a model in which both decreased protein turnover and altered trafficking account for the ability of s-Tetherin to effectively retain virions in

Vpu-expressing cells.

47 The observation that the long isoform activates NF-κB while the short isoform shows no activity suggests that sequences within the 12 residues unique to l-Tetherin mediate signaling.

Moreover, data demonstrating higher NF-κB induction by expression of the l-Tetherin isoform compared to the wt cDNA, coupled with an inhibitory affect of s-Tetherin on NF-κB activation, support a model in which homodimers of the longer isoform are responsible for signaling. Though

Tetherin does not possess canonical tyrosine-based motifs involved in activating signal transduction pathways, there is evidence that dimerization of CLEC-2 allows approximation of a non-canonical tyrosine-based motif and permits signaling via Syk kinase (87). Furthermore, phosphoproteomic analysis has shown that Tetherin tyrosine residues at positions 6 and 8 can be phosphorylated (211). Moreover, mutation of both these tyrosine residues greatly diminished NF-

κB induction, demonstrating that they are critical for this activity. Because the dual tyrosine residues have been shown to be important in trafficking it will be crucial to assess whether differential localization may also play a role in activating signal transduction. The ability of a dominant negative form of the kinase IKKβ to inhibit Tetherin-induced NF-κB activity indicates that activation likely involves the canonical pathway, however the mechanism by which Tetherin couples to this pathway remains to be elucidated. Similarly to our findings with Tetherin, it has recently been found that the cytoplasmic tail domain of HIV-1 envelope activates NF-κB, but not

AP1 (176). In this case activation occurs via the canonical NF-κB pathway, with HIV-1 envelope directly engaging TGF-β-activated kinase 1 (TAK1). Whether Tetherin utilizes a similar mechanism to specifically activate NF-κB remains to be explored.

It has been demonstrated that Tetherin is a ligand for ILT7 on human dendritic cells and that binding initiates signaling via the ILT7–FcεRIγ complex, inhibiting production of interferon and proinflammatory cytokines by dendritic cells (35). It will be interesting to investigate if ILT7, or an unknown ligand can modulate NF-κB activation by Tetherin. The viral restriction factor TRIM5α has recently been shown to function as a unique pattern recognition receptor recognizing the hexagonal lattice pattern of entering retroviral to activate NF-κB and MAP kinase

48 pathways (172). In a similar vein, we speculate that Tetherin could recognize the “pattern” caused by budding virions to activate NF-κB.

49 MATERIALS AND METHODS

Cell Culture

293T, HT1080 and HeLa cells were maintained in high glucose DMEM + 10% Cosmic Calf

Serum (Hyclone). Primary CD4 T cells (obtained from the University of Pennsylvania Center for

AIDS Research Immunology Core, ND307) were maintained in RPMI + 10% FBS (Invitrogen). All cells were maintained at 37°C with 5% CO2.

Plasmids and Reagents

Human tetherin cDNA in pCMV-SPORT6 was acquired from Open Biosystems. Tetherin mutants were generated using site-directed mutagenesis by PCR. Primers for site-directed mutagenesis were designed using QuikChange Primer Design Program (Agilent Technologies). The HIV-1 gag- encoding construct, psPAX2 was obtained from Addgene (plasmid 12260). HIV-1 vpu and ebolavirus GP cloned into pCAGGS were previously described (100, 204). The NF-κB reporter plasmid, pBIIX-Luciferase was provided by Dr. Michael May (University of Pennsylvania).

The TRAF6 coding region was cloned from a cDNA (Open Biosystems) into pKMyc (Addgene plasmid 19400) to generate an N-terminally FLAG-tagged version using an XbaI restriction site adjacent to the FLAG-tag. The dominant negative FLAG epitope tagged IKKβ (K44M) plasmid was obtained from Addgene (plasmid 11104). The AP1 reporter (Biomyx) and MEKK1-delta plasmids were obtained from Dr. Sunny Shin (University of Pennsylvania).

Tetherin Protein Expression Analysis

HT1080 cells in a 6 well were transiently transfected with Tetherin expression constructs using

Lipofectamine2000 (Invitrogen) according to manufacturers instructions. For IFN induction experiments, HT1080, 293T, HeLa and primary CD4 T cells were incubated +/- 1000U of recombinant type I IFN (PBL Interferon Source) for 48 h. Two days post-transfection/IFN treatment, cells were washed with PBS and lysed using RIPA buffer (10mM Tris-HCl pH 8.0,

50 5mM EDTA, 140mM NaCl, 1% sodium deoxycholate, 0.1% SDS, 1% NP40). Lysates were passaged through a 25G needle before being cleared by centrifugation at 17,900x g for 15 min at

4°C. Cleared lysates were treated with either treated or not with PNGase (New England BioLabs) for 2 h, then reduced using DTT containing loading buffer. Samples were separated on a 15%

Criterion gel (BioRad) and transferred to PVDF membrane. After blocking in 5% milk TBST (Tris- buffered saline + 0.1% Tween), Western blots were analyzed for Tetherin expression using rabbit anti-BST2 sera (Dr. Klaus Strebel, #11721 National Institutes of Health AIDS Research and

Reference Reagent Program).

VLP Budding Assay

2.5x105 293T cells seeded on a 24 well plate were co-transfected with the indicated tetherin construct (12.5 ng), an HIV-1 Gag-Pol expression vector pSPAX (50 ng) and increasing amounts of (25, 50 or 100 ng) of pCAGSS-vpu or pCAGGS ebolavirus GP using Lipofectamine2000. Cell lysates and supernatants were harvested 48 h post-transfection. Cell lysates were harvested in

Triton X-100 buffer (50mM Tris-HCl pH 8.0, 5mM EDTA, 150mM NaCl, 1% Triton X-100) with

Complete (Roche) protease inhibitor cocktail. Cell lysates were cleared by centrifugation at

17,900x g for 3 min at 4°C. Supernatants containing VLPs were cleared are 1700x g for 2 min at

4°C. VLPs were subsequently purified by pelleting through a 20% sucrose cushion at 40,000 in a

TLA120.1 rotor (Beckman) for 30 min. VLPs were resuspended in PBS on ice for 3 h. Cleared cell lysates and resuspended VLPs were separated on 15% or 4-15% Criterion gels (BioRad) respectively before being transferred to PVDF membrane. Membranes were blocked in 5% milk in TBST for 40 min prior to incubation with primary antibody. HIV-1 Gag p24 was detected in

Western blots for both VLPs and cell lysates using a monoclonal anti-p24 antibody (24-3,

National Institutes of Health AIDS Research and Reference Reagent Program). The GAPDH loading control was analyzed using GAPDH antibody (Calbiochem, CB1001). HIV-1 Vpu was detected using HIV-1 pNL4-3 Vpu antiserum (969, National Institutes of Health AIDS Research

51 and Reference Reagent Program). Ebolavirus GP was detected using rabbit antiserum against the GP1 portion of the protein (125).

Flow Cytometry Analysis of Tetherin Cell Surface Expression

293T cells (2.5x105) seeded on a 24 well plate were transiently co-transfected with 25 ng Tetherin constructs (wt, l-Tetherin, s-Tetherin or l-AxA) and 100 ng HIV-1 vpu or ebolavirus GP. Two days post transfection, cells were washed once with 1xPBS while on the plate. Whole cells were removed using cold 1xPBS and the cells were split into equal aliquots used for flow cytometry and western blot analysis. Cells used for Western blot analysis were pelleted by centrifugation then lysed in Triton X-100 buffer. Lysates were cleared and PNGase treated prior to SDS/PAGE

Western blot analysis as described above. Cells to be used for flow cytometry were resuspended in cold FACS buffer (1xPBS, 1%BSA + 0.05% sodium azide) and pelleted at 2150x g. Cells were resuspended in FACS buffer + PE conjugated anti-BST2 antibody (Biolegend). After 1 h incubation on ice, cells were washed three times with cold FACS buffer and analyzed on a FACS

Calibur (BD Biosciences Immunocytometry Systems). Data analysis performed using FlowJo

9.3.1 (Tree Star).

Luciferase Assay

293T cells (2.5x105) were seeded on a 24 well plate and the following day were transfected with the indicated tetherin constructs (50 ng) and pBIIX-Luciferase reporter (250 ng) using

Lipofectamine2000. In experiments where the long and short isoforms were co-expressed the total amount of tetherin expression plasmid was kept constant at 50 ng. At 30 h post-transfection, cell lysates were harvested in Triton X-100 lysis buffer. Lysates were transferred to a black flat bottom 96-well plate. Luciferase Assay System substrate (Promega E1501) was added to the lysates according to manufacturers directions. Samples were analyzed in a Luminoskan Ascent microplate luminometer (Thermo Scientific). Statistical analysis was performed using PRISM

(GraphPad).

52 Supplementary Methods

Coimmunoprecipitation

293T cells were transfected with l-Tetherin FLAG and/or AU1 s-Tetherin. Cells were lysed in

RIPA buffer and cleared as described above. Lysates incubated with either anti-FLAG rabbit

(Sigma) or anti-AU1 rabbit (Bethyl Lab. Inc.) antibodies bound to Protein A Dynabeads

(Invitrogen) for 4 hours. Lysates washed three times using RIPA buffer. Beads were boiled in

RIPA+6x SDS/PAGE loading buffer for Western blot analysis. FLAG-tagged l-Tetherin was detected using a FLAG-HRP conjugated antibody (Sigma). s-Tetherin was visualized using the anti-AU1 rabbit antibody described above.

53 ACKNOWLEDGEMENTS

We thank Rachel Kaletsky for generating the AU1 s-Tetherin epitope tagged construct, and

Sunny Shin and Michael May for supplying plasmids.

54 FIGURES

Figure 2-1. Alignment of Tetherin sequences and comparison to a consensus Kozak translation initiation sequence. (A) Amino acid alignment comparing the amino-terminal cytoplasmic region of Tetherin from various mammalian species. Accession numbers- Homo sapiens: NP_004326, Pan troglodytes: NP_001177409 XP_552491, Gorilla gorilla: ADI58594, Macaca mulatta: ACV96781, Mus musculus: NP_932763 XP_134266, Rattus norvegicus: NP_937767 XP_579725, Sus scrofa: NP_001155227 (CL Sequencer; 80% limit). Highlighted conserved residues include two cytoplasmic methionine residues, M1 and M13 (arrows), and a previously characterized dual tyrosine motif (*). (B). Comparison of the nucleotide sequences at the M1 and M13 AUGs in human tetherin mRNA to a consensus Kozak translation initiation sequence. Important residues at the −3 and +1 to +4 positions are highlighted in bold black text. Human Tetherin does not conform to the consensus at the −3 position. l-Tetherin and s-Tetherin refer to proteins initiated at M1 or M13 respectively. R = Purine.

55

Figure 2-2. Tetherin exists as two isoforms. (A) Alignment of the Tetherin amino acid (Black text) and cDNA (Grey text) sequences compared to strong Kozak sequence, M1A and M13I point mutations (highlighted in black). (B) HT1080 cells transiently transfected with Tetherin encoding plasmids (WT, Strong Kozak, M1A, M13I or M1A+M13I) were lysed, treated with glycosidase (PNGase) to remove carbohydrate modifications and analyzed by Western blot using a polyclonal Tetherin antibody. l = long isoform, s = short isoform (C) Tetherin isoform expression in cell lysates from IFN stimulated and unstimulated 293T (1st panel), unstimulated HeLa (2nd panel) and primary CD4 T (3rd panel) cells. Deglycosylated tetherin profiles are compared to those from transiently expressing 293T cells (4th panel). l- and s- indicate the long and short Tetherin isoforms. Stable tetherin dimers (d) and an uncharacterized smaller molecular mass species (*) often observed under transient transfected conditions are indicated.

56

Figure 2-3. Tetherin isoforms have different sensitivities to viral antagonists. (A and B) 293T cells were transfected with a constant amount of an HIV-1 Gag-Pol expression vector, a constant amount of the indicated Tetherin expression plasmid and increasing amounts of plasmids expressing the viral antagonists (A) Vpu or (B) ebolavirus GP. VLPs (Top panels) from supernatants were purified and analyzed for HIV-1 Gag p24 release. Cell lysates were probed for cellular levels of HIV Gag and Tetherin. As in Figure 2-2C (*) indicates an undefined species often seen upon transient Tetherin expression in 293T cells. Titration of viral antagonists was assessed using Vpu and GP specific antibodies. GAPDH is used as a lysate loading control.

57

Figure 2-4. Effect of viral antagonists on surface levels of Tetherin. 293T cells were transiently co-transfected with the indicated Tetherin plasmids in the presence viral antagonists Vpu or ebolavirus GP. Half the cells were analyzed by flow cytometry. Graph represents of the mean MFI from multiple experiments (n = 5); error bars = SEM. Surface Tetherin in the absence of viral antagonist was set to 100%. Expression of total cellular Tetherin was analyzed by Western blot of PNGase treated lysates from cells not used for flow cytometry. Reduced monomers, stable dimmers (d) and an unknown species (*) are indicated. Arrow indicates s- Tetherin isoform seen in wt+Vpu expressing cells. GAPDH used as a loading control.

58

Figure 2-5. Resistance to Vpu antagonism requires mutations in the tyrosine and serine/threonine motifs in Tetherin. (A) Amino acid sequences of l-Tetherin mutants that disrupt the tyrosine motif (l-AxA), the serine/threonine residues (l-STS) and the combined mutant (l-SY). (B) 293T cells were transfected with HIV-1 Gag-Pol and Tetherin expression vectors plus increasing amounts of plasmids expressing the viral antagonist Vpu (25 or 100 ng). VLPs (Top panels) from supernatants were purified 48 h post transfection and analyzed for HIV-1 Gag p24 release by Western blot. Cell lysates were probed for cellular levels of HIV-1 Gag and Tetherin. As in Figure 2-2C (*) indicates an unknown species often seen upon transient Tetherin expression in 293T cells. Titration of Vpu was detected using anti-Vpu antibody. GAPDH is used as a lysate loading control.

59

Figure 2-6. Tetherin isoforms differentially activate NF-κB. (A) 293T cells transiently co- transfected with either a wt, l-Tetherin, s-Tetherin or l-AxA encoding plasmid plus an NF-κB responsive firefly luciferase reporter plasmid were lysed and analyzed for luciferase activity 24 h post transfection. Myc-TRAF6 used as a positive control for NF-κB activation. NF-κB signaling experiments (n = 8 in triplicates) were analyzed by one-way ANOVA. (B) Luciferase assay for AP- 1 activation was assessed as in (A) using an AP1 responsive firefly luciferase plasmid (500 ng) in the presence of the same Tetherin encoding plasmids. MEKK1 and myc-TRAF6 were used as positive controls for AP1 activation. AP1 signaling experiments (n = 4 in triplicates) were analyzed by one-way ANOVA. (C) wt and l-Tetherin were co-transfected with increasing amounts of a FLAG epitope tagged dominant negative (DN)-IKKβ and the NF-κB responsive luciferase reporter. Myc-TRAF6 was used as a positive control (n = 3 in triplicates). Representative blot showing expression of co-transfected constructs as well as a GAPDH loading control. On the Western blots, as in Figure 2-2C, (*) indicates unknown species. In the graphs ns = not significant; *** p<0.001.

60

Figure 2-7. s-Tetherin modulates l-Tetherin-mediated NF-κB induction. 293T cells were transiently transfected with an NF-κB luciferase reporter and varying ratios of l- and s-Tetherin expression vectors with the total tetherin expression plasmids amount kept constant. Cells were lysed and analyzed for luciferase activity. In parallel, an l:s titration (black bars) was compared to l-Tetherin titrated with empty plasmid (white bars). wt Tetherin signaling (grey bar) was assessed during each experimental replicate (n = 9 in triplicate). Two-way ANOVA performed to assess statistical significance. A representative Western blot for Tetherin is shown for each set of titrations. *p<0.05; ***p<0.001, **** p<0.0001.

61

Figure 2-S1. Comparison of Kozak translation initiation sequences of mammalian Tetherin messages. Tetherin cDNA sequences from various mammals (Ensembl Gene IDs: CpzBST2 (Chimp)- ENSPRTRT00000019678, GorBST2 (Gorilla)-ENSGGOT00000015329, RhBST2 (Rhesus)- ENSMMUT00000008172, MsBST2 (Mouse)- ENSMUST00000051672; NCBI Ref Sequence: RtBST2 (Rat)- NM_198134.1) restricted to approximately half the cytoplasmic tail and 9 bases upstream of the canonical start codon were aligned and are shown with the deduced amino acid sequences under the corresponding codons. Matches to important residues in the consensus Kozak sequence (−3, and +1 to +4) are denoted in black in each sequence. In all cases, the upstream methionine deviates from the consensus at the −3 position.

62

Figure 2-S2. Resolution of heterogeneous Tetherin expression profile by removal of carbohydrate modification with PNGase. Lysates from HT1080 cells transiently expressing wt Tetherin cDNA were prepared 48 h post transfection. Lysates were analyzed by Western blot using a Tetherin antibody. Left panel (no PNGase), right panel (with PNGase).

63

Figure 2-S3. Endogenous expression of Tetherin isoforms. IFN stimulated or unstimulated HeLa, HT1080 and 293T cells were analyzed at 48 h post exposure. Cellular lysates were used directly or digested with PNGase and analyzed by Western blot. A lighter exposure of PNGase treated profile is shown below the main blot. HT1080 cells transiently expressing wt, l-Tetherin, s- Tetherin, l+s-Tetherin or wt Strong Kozak mutants were harvested, PNGase treated and analyzed adjacent to endogenous Tetherin samples.

64

Figure 2-S4. Expression profiles of rhesus and murine Tetherin. (A) 293T cells were transfected with murine Tetherin cDNA. Concurrently, murine J774 cells were IFN treated for 48 h. Lysates were PNGase treated and analyzed by Western blot using an anti-mouse CD317 antibody (BioLegend, 127101). Shorter exposure of the transfected 293T cells is shown to the right. (B) Lysates from 293T cells transiently expressing rhesus Tetherin cDNA were analyzed adjacent to lysates of rhesus FRhK-4 cells. The right panel is a lighter exposure of the 293T lane from the same blot. While the J774 cells, the murine and rhesus Tetherin cDNAs produce two isoforms, only a single species that corresponds to the upper (presumably l-Tetherin) isoform is seen in FRhK-4 cells.

65

Figure 2-S5. Isoforms produce homo- and heterodimers. (A) HT1080 cells transiently expressing Tetherin mutants form homodimers. HT1080 transfected with either wt, l-, s- or l+s- Tetherin were lysed in RIPA buffer. Lysates were PNGase treated for 2 h without denaturation. Deglycosylated samples were analyzed under non-reducing conditions and probed for Tetherin using anti-BST2 rabbit sera. l:l=Long homodimers; s:s=short homodimers. (B) Co- immunoprecipitation of epitope tagged isoforms. Cartoon of differentially tagged l- and s-Tetherin expression vectors with tags adjacent to the GPI anchor additions site or at the amino terminus respectively. Epitope tagged Tetherin isoforms were transiently expressed in 293T cells then RIPA lysates were precipitated using the indicated antibodies. Precipitates were analyzed by SDS/PAGE and Western blot. Lane 1, mock transfected; Lane 2, wt-Tetherin FLAG; Lane 3, l- Tetherin FLAG; Lane 4, AU1 s- Tetherin; Lane 5, l-Tetherin FLAG+AU1 s- Tetherin.

66

Figure 2-S6. NF-κB induction varies with Tetherin expression level. 293T cells (2×105) were transiently transfected with a range of amounts of Tetherin expression plasmid (12.5, 25, 50, 100 or 200 ng) and a constant amount NF-κB luciferase reporter. Total DNA transfected was kept constant by including empty vector. The results consistently show a bell shaped response for NF- κB activation by Tetherin. From these results 50 ng was chosen as an optimal amount of plasmid for the NF-κB activation assays. This graph is a representative experiment done in triplicate, bars = SD

CHAPTER 3: CHARACTERIZING ADDITIONAL DETERMINANTS OF

TETHERIN-MEDIATED SIGNALING

Luis J. Cocka and Paul Bates

Department of Microbiology, Perelman School of

Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104

67 ABSTRACT

Tetherin is a potent cellular restriction factor that directly inhibits the egress of a broad range of enveloped viruses. Recent lines of evidence suggest Tetherin can act as a pattern- recognition receptor (PRR) that upon sensing viral particle budding can mediate activation of NF-

κB via a non-canonical tyrosine based motif, correlating with enhanced proinflammatory cytokine expression. Here we report that a previously described Tetherin mutant can activate NF-κB in a tyrosine-independent fashion, via a similar pathway to wt Tetherin. Tyrosine-independent activation requires alteration of a serine-threonine-serine (STS) motif near the amino terminus of the Tetherin cytoplasmic tail. As previously reported, this mutant, SY Tetherin, displays Vpu resistance. We identified minimal sequence in STS that conferred both signaling function and Vpu resistance in the dual tyrosine mutant background, as well as additional determinants of Tetherin- mediated signaling, including single point mutations that affect NF-κB activation. Introduction of acidic residues upstream of the dual tyrosine motif abrogates NF-κB activation. Conversely, increased hydrophobicity via bulk alanine substitution upstream of amino acid position 13 permits signaling. Our panels of mutants provide insight into how the early steps of Tetherin-mediated signaling may be controlled. This data suggests that the first 13 amino acids of Tetherin, unique to the long isoform, may be regulatory, rather than involved in direct recognition of signaling machinery through a proposed hemITAM as has been previously reported.

68 INTRODUCTION

Timely sensing of and response to viral infection is crucial for maintaining host homeostasis and survival. Pattern recognition receptors (PRRs) represent a number of innate and intrinsic immune sensors that recognize broad pathogen associated signatures. Specific localization of PRRs allows recognition of various virus-derived ligands, like viral proteins and nucleic acid, at sites of entry, genome replication and/or egress. Sensing of pathogen-derived ligands can lead to activation of various transcription factors including NF-κB, IRFs and AP1, which regulate expression of various immune stimulatory cytokines like proinflammatory cytokines and interferon (IFN). Cells also express specific intrinsic sensors and can upregulate

IFN-stimulated genes (ISGs) that directly block viral replication. These factors can be broadly acting like viperin and ISG15 (155, 163, 164, 244), disrupting broadly conserved viral replication processes or can be more specific to particular viral families, like TRIM5α, which plays a role specifically recognizing capsid upon HIV-1 entry or APOBEC3G which interfere with retroviral reverse transcription (137, 193, 202, 219)

The mammalian intrinsic cellular restriction factor, Tetherin (BST2, CD317, HM1.24) has been suggested to also act as a pattern-recognition receptor (PRR), mediating-sensing of viral assembly and budding, thus coupling its “tethering” function to its ability to activate the classical

NF-κB pathway (59, 228). Tetherin can enhance expression of NF-κB dependent proinflammatory cytokines, like IL-6 and CXCL10 (59). IFNβ expression is enhanced in the presence of tethered replication competent HIV-1 lacking Vpu (59) presumably from sensing of viral nucleic acids from degraded endocytosed particles (158). Tetherin activation of NF-κB, in part, is mediated by a dual-tyrosine motif (44, 59, 228) previously shown to also regulate Tetherin localization (140,

181). Though the non-canonical tyrosine based motif has been demonstrated to be crucial for signaling, mutational analysis has demonstrated that additional residues in the cytoplasmic tail, as well as residues in the transmembrane domain and ectodomain are important (25, 59, 228) suggesting complexity to how Tetherin may be signaling. The GPI anchor has been identified as

69 important (60), though there are conflicting reports (25, 228), suggesting that merely having a second anchor may be sufficient. Though the cytoplasmic tail is important, it is not sufficient.

Swapping the human Tetherin cytoplasmic tail on other protein scaffolds, like on rhesus Tetherin, which is signaling deficient, does not allow gain-of-function.

Tetherin is a lipid raft resident protein, which places it at the site of budding for various families of enveloped viruses and also proximal to signaling platforms. Like many common signaling receptors, Tetherin clustering, either antibody-mediated or by sensing a “ligand”, activates signaling. When the cell is at rest, Tetherin is believed to interact with the actin cytoskeleton through an actin associating GTPase activator, RICH2 (60). Upon sensing of viral particle budding through the actin cytoskeletal network, a putative hemITAM in the Tetherin cytoplasmic tail is phosphorylated. This event is thought to mediate recruitment of Syk kinase to initiate NF-κB activation. Syk activation subsequently recruits complexes comprised of molecules such as TRAF2, TRAF6, TAK1, TAB1/2 (60).

Here we describe mutations in the cytoplasmic tail of human Tetherin that allows for tyrosine-independent activation of NF-κB. Mutational analysis demonstrates that the STS region, upstream of the putative hemITAM, can affect NF-κB activation in manner that is sensitive to specific substitutions. One mutant, SY, which replaces the STS sequence with alanine residues, had previously been shown to be robustly resistant to HIV-1 Vpu both in VLP and replication competent infection assays (44, 247). Here we show that the SY mutant can robustly activate NF-

κB despite the fact that both tyrosine residues are mutated. Furthermore, we demonstrate that the

SY mutant can activate the classical NF-κB pathway and can signal through similar downstream effectors suggesting normal Tetherin mediated NF-κB activation. The mutational analysis suggests an important role for the amino terminal, in particular the STS region, of Tetherin in regulating NF-κB activation. Together, this work provides evidence to suggest that the dual tyrosine motif may be an important regulatory motif rather than a hemITAM.

70 RESULTS

Identification of a tyrosine-independent, signaling competent Tetherin

We previously mapped residues in the Tetherin cytoplasmic tail that when mutated in the l-Tetherin background, conferred HIV-1 Vpu resistance. Mutations in the dual-tyrosine motif and an upstream serine-threonine-serine (STS) region (mutant SY, Figure 3-1A), were required to produce the Vpu-resistant phenotype observed with s-Tetherin via VLP budding assays and more recently during replication-competent HIV-1 infection in a T cell line (44, 247). Further characterization of the SY mutant, generated in the l-Tetherin background, revealed that compared to wt or long isoform (l-Tetherin) it robustly activated NF-κB independent of both tyrosine residues via transient reporter assay (Figure 3-1A). This result was unexpected, as the dual-tyrosine motif had previously been shown to be important for signaling (44, 59, 228). Thus we sought to further assess the contributions of the individual tyrosine residues for NF-κB activation..

Using site-directed mutagenesis, we generated and analyzed a panel of tyrosine mutants including single alanine substitutions at positions 6 (Y6A) and 8 (Y8A), as well as the more conservative phenylalanine point mutations Y6F and Y8F (Figure 3-1A). In addition we analyzed the dual tyrosine mutations in both alanine (AxA) and phenylalanine (FxF) backgrounds.

Substitution of tyrosine at position 6 with alanine (Y6A) was sufficient to abrogate signaling, while alanine substitution at tyrosine 8 (Y8A) had no effect on NF-κB activation. Unlike the alanine mutant, both individual phenylalanine mutations, Y6F and Y8F are able to active NF-κB. Signaling by FxF is reduced significantly, similar with was previously observed with alanine mutations at both tyrosine residues (Figure 3-1A, (44, 59, 228)). As is the case for the SY mutant signaling function is restored in the FxF background when changes to STS (STS to AAA) are made in combination (mutant SF, Figure 3-1A). Together, these findings suggest an important, novel role of the STS sequence in regulating signaling.

71 The mutant panel above was generated in the (l-Tetherin) background with an isoleucine substitution at position 13 (M13I). To ensure that tyrosine independent-signaling phenotype was not dependent upon this genetic background, we engineered SY in the wt Tetherin context (WT

SY) and analyzed it for NF-κB activation. As seen in Figure 3-1B, transient expression of WT SY activates NF-κB signaling thus confirming the results seen with in the l-Tetherin background.

Additionally, the level of activation by SY in either the wt or l-Tetherin background was constantly higher than that of wt or l-Tetherin. (Figures 3-1A and B). Previous analyses of Tetherin-mediated signaling demonstrated specific activation of NF-κB signaling and not AP-1 (44, 59, 228). In agreement with this observation, the SY mutant did not activate AP-1, nor did it activate a more complex promoter like that of IFNβ (Figure 3-1C), We decided to assess whether SY Tetherin consistently a more robust activator of NF-κB compared to l-Tetherin over a range of transfected plasmid. SY signaling was generally more robust than l-Tetherin (Figure 3-1D). Taken together, we have identified changes in the amino terminal region of Tetherin that specifically activates NF-

κB. in a tyrosine-independent manner. With the data presented above, we raise doubt to the importance of the tyrosine residues and their potential to act as a hemITAM in Tetherin.

SY signaling can be regulated by s-Tetherin

The basic Tetherin functional unit is thought to be a dimer, which multimerizes with other dimeric Tetherin species to enhance signaling function. We previously observed that l-Tetherin signaling could be downregulated by increasing amounts of s-Tetherin. We tested whether SY signaling is dampened similarly by s-Tetherin by transient reporter assay and observed a significant dose dependent decrease in signaling compared to decreasing SY plasmid alone

(Figure 3-2A). In the presence of added l-Tetherin, signaling remains at levels observed with SY alone. Since we previously observed that SY activates much more robustly at lower amounts of plasmid expression (Figure 3-1D), this suggests that SY is the functionally dominant species compared to l-Tetherin. We have observed l-Tetherin to activate NF-κB more robustly than wt

Tetherin over a range of plasmid expression, an observation that can be explained by the

72 expression of both l- and s-Tetherin off the wt plasmid (Figure 3-2B, (44)). Signaling by WT SY, which expresses SY and s-Tetherin off the same plasmid, has reduced signaling compared to SY alone confirming the dominant-negative effect observed when s-Tetherin is independently titrated in (Figure 3-2B).

Identification of additional residues important for Tetherin-mediated signaling

Triple alanine scanning has been performed on the Tetherin cytoplasmic tail, identifying runs of residues important for signaling (59). The observation that the Y6F mutant retains signaling function, we called into question whether the dual tyrosine motif is acting like a bona fide hemITAM. To clarify the requirement within the cytoplasmic tail we sought to identify additional single point mutations that may regulate Tetherin signaling. We performed single alanine scanning on the entire Tetherin cytoplasmic tail using site-directed mutagenesis and identified four residues for which alteration to alanine significantly impair NF-κB activation. In addition to the requirement for Y6, a significant but less dramatic effect was observed with an alanine substitution at the threonine at position 4 (T4) (Figure 3-3A). Additionally, we confirmed the findings that R19 is crucial, and to a lesser extent other membrane proximal basic residues previously observed by triple alanine scanning (59). The loss/reduction in signaling for the Y6A,

R19A, and T4A mutants were confirmed by testing a range of plasmid expression compared to the l-Tetherin parental background (Figure 3-3B). Additionally, we observed a conservative mutation at position 2 (A2V) led to a loss of signal. However, expression of the glycosylated, mature forms of the A2V mutant was poor with the exception potentially explaining the loss of signaling ability (Figure 3-3C, lower panel). In contrast an acidic substitution at this position (A2E) expresses comparably to l-Tetherin, signaling was abrogated. Combined with the finding that alteration of the STS region can restore signaling function to tyrosine mutants, this mutational analysis has identified an important role for N-terminal residues of the cytoplasmic tail in Tetherin- mediated NF-κB activation.

73 Tyrosine-independent Tetherin mutant can signal through a similar pathway as wt Tetherin

To determine if the SY mutant might provide insight as to how the amino terminal region of the cytoplasmic tail contributes to regulation of Tetherin-mediated NF-κB activation., we first needed to ascertain if SY behaves like wt Tetherin. As previously observed with wt and l-

Tetherin, NF-κB activation by SY can be blocked with a dominant negative (DN)-IKKβ (Figure 3-

4A). This demonstrates that similar to wt, SY acts through the classical NF-κB pathway Titration of wt IKKβ, which activates NF-κB independent of Tetherin, enhanced signaling in a dose dependent manner for wt, l- and s-Tetherin. Increasing the level of IKKβ did not increase reporter activation with the SY mutant suggesting that robust activation observed might be saturating the available IKKβ in complex present in the cell.

TAK1 has been found to associate with signaling competent Tetherin constructs (59,

228). We performed coimmunoprecipitation (co-IP) to assess whether TAK1 could interact with l- and s-Tetherin isoforms and the SY mutant (Figure 3-4B). Cell lysates containing transiently expressed proteins were immunoprecipitated using an anti-Tetherin Ab. As expected, TAK1 was found to associate with wt Tetherin. Both l-Tetherin and SY pulled down TAK1 while the signaling-deficient s-Tetherin, did not associate with TAK1. The ability of the AxA mutant in the l-

Tetherin background to precipitate TAK1 was highly variable across multiple experiments.

Tetherin has been suggested to mediate signaling via a hemITAM motif that directly recruits Syk kinase (60). We sought to determine whether SY was still Syk dependent. 293T cells transiently transfected with Tetherin mutants and NF-κB reporter were treated for 18h with a Src- family inhibitor, Piceatannol, or a Syk-kinase specific inhibitor, BAY61-3606. Both resulted in a dose dependent decrease in SY-mediated signaling (Figure 3-4C) suggesting Src- and Syk-family kinases are required for signaling. Taken together, SY appears to utilize similar downstream effectors to wt Tetherin for NF-κB signaling function.

74 Ebolavirus glycoprotein antagonizes the SY mutant tethering function

We previously demonstrated that the ebolavirus glycoprotein (EBOV GP) can counteract l- or s-Tetherin equally well to relieve tethering of HIV-1 virus-like particles (VLPs) (44). This agrees with the current model of how EBOV GP may be counteracting Tetherin that suggests a mechanism of action that is cytoplasmic tail independent (126). In contrast to the Vpu resistance observed above, the SY mutant is effectively antagonized with increasing amounts of EBOV GP as determined by efficient VLP release (Figure 3-5, VLP panel). Total Tetherin levels remain unchanged as previously observed with other Tetherin constructs tested. Additionally, all forms of

Tetherin tested are incorporated equally well into VP40 VLPs (Figure 3-5, VLP panel), confirming a recent observation suggesting that mechanistically EBOV GP may counter Tetherin by allowing incorporation into particles (71). Thus SY Tetherin appears to be resistant to Vpu but not EBOV

GP mediated antagonism.

Minimal changes in STS that confer signaling phenotype in tyrosine mutant Tetherin

The SY mutant includes alanine mutations in the STS residues upstream of the dual tyrosine motif. Triple alanine scanning mutational analysis by the Neil group showed that this mutation did not affect signaling in the wt Tetherin background (59), although we observe reduced NF-κB activation in the l-Tetherin background (Figure 3-1A). To identify the requirements within the STS region that confers a signaling phenotype mutational analysis was performed in the AxA background. These studies revealed that the threonine residue at position 4 (T4) is critical to confer NF-κB activation in the AxA background. The level of activation is comparable to the SY mutant when T4 is changed alone or in combination with the two other residues in the motif (S3T4AxA or T4S5AxA) (Figure 3-6A). Alanine mutations in either serine alone, S3 or S5, modestly increases NF-κB activation compared to the AxA mutant (Figure 3-6A) but never to the level observed with the SY mutant.

In addition to the observed effects on NF-κB activation, the changes in SY mutant also make Tetherin highly resistant to HIV-1 Vpu (44). To determine if the individual or combined

75 mutations in the region also affected Vpu-sensitivity the STS region mutant panel was analyzed in a transient budding assay. We observed that rescue of signaling function correlated with resistance to increasing amounts of HIV-1 Vpu (Figure 3-6B). Taken together, though the T4 mutation in the context of l-Tetherin has a modest effect on signaling (Figure 3-3A), it appears to play an important role in allowing signaling function in the AxA background. Furthermore, mutation in T4 synergizes with AxA to enhance resistance to Vpu.

Assessing the plasticity of STS region to mutation

Single alanine substitutions in the STS demonstrated that only residue T4 decreased wt

Tetherin NF-κB activation levels. To more thoroughly assess the role of this region in signaling more disruptive mutations were generated into this region. Substitutions introducing acidic residues at S3 and T4 abrogated signaling, while activation levels were diminished by an acidic mutation at S5 (Figure 3-7A). A triple acidic mutation at STS (DED) also does not signal and cannot compensate for the loss of the dual-tyrosine motif (DEDAxA) (Figure 3-7A). In general, we observed that acidic residues upstream of the dual-tyrosine motif disrupt signaling, which may explain why the A2E mutant does not signal (Figure 3-3C).

The STS region appears to have evolved in primates. Chimp and rhesus sequence from the homologous region, STL and PIL respectively have been recently demonstrated to have differential effects on signaling in the wt Tetherin background (60). We confirmed these findings in the l-Tetherin background. The chimp sequence STL (ChSTL) activated NF-κB to a level that as similar to the SY mutant and greater than the l-Tetherin parental background. This is in agreement with data showing that the ChSTL substitution appeared to induce greater NF-κB activation in the wt background (60). Introduction of rhesus sequence PIL (RhPIL) abrogates signaling. When analyzed in combination with the AxA mutation, the chimp S5L mutation can rescue signaling, while the rhesus sequence PIL does not (Figure 3-7B),

The SY mutant carries five alanine substitutions within the amino-terminal 12 residues of the cytoplasmic tail – the region unique to signaling competent l-Tetherin. To determine if there

76 are additional specific sequence requirements needed to maintain signaling function, the remaining residues in the cytoplasmic tail upstream of position 13 were analyzed by alanine substitution mutagenesis. The additional alanine substitutions were placed in the context of the

SY mutant (Figure 3-7C) and analyzed by transient expression. Sequential alanine substitutions throughout the entirety of the region unique to l-Tetherin did not disrupt signaling function (Figure

3-7C). Indeed a mutant (Ala11) in which residues 2 through 12 are all alanine induces NF-κB activation similarly to SY mutant. Although the region unique to l-Tetherin, in particular the STS region, is an important determinant for regulating Tetherin-mediated NF-κB activation these results suggest a specific sequence dependent interaction with signaling machinery may occur elsewhere on Tetherin which raises questions about the nature of the dual-tyrosine motif and whether it acts as a hemITAM.

77 DISCUSSION

In this work, we utilized a series of mutations to highlight a region in the cytoplasmic tail of Tetherin that appears to be involved in activation of NF-κB signaling. Specifically, mutations identified in a serine-threonine-serine (STS) region near the amino terminus causes Tetherin to signal independently of the dual-tyrosine motif. We previously described this mutant, SY, with changes in both tyrosines and alanine substitutions at all three residues in the STS region as highly resistant to HIV-1 Vpu (44). Here we find that these same changes are associated with high levels of NF-κB activation. Similarly to wt Tetherin, the SY mutant is inhibited by co- expression of the signaling-deficient s-Tetherin. The cellular requirements for SY mutant- mediated signaling appears to be analogous to what has been described for wt Tetherin, in that the classical NF-κB pathway is used and activation is dependent on Syk kinase (44, 59, 60, 228).

However, the fact that signaling occurs in the absence of tyrosine residues is inconsistent with the current hemITAM model for Tetherin-mediated activation ((60), Figure 3-2).

Further analysis revealed that when coupled with a dual tyrosine substitution at positions

6 and 8, the threonine at position 4 (T4) within the STS region is critical for Vpu resistance and

NF-κB activation. The flanking serine residues appeared to modestly contribute additional resistance to Vpu-mediated antagonism in the presence of T4A (Figure 6B) although there is little effect on virion release and NF-κB activation. The STS region was previously identified, including work from our own group, as being important for Vpu-mediated downregulation of Tetherin (44,

229). It has been suggested the STS region can be ubiquitinated in the presence of Vpu, though this observation remains controversial (72, 228). It is unclear to what extent this STS region can be post-transnationally modified in the absence of Vpu, if at all. Modifications like phosphorylation or acetylation could inhibit signaling function as observed with our acidic substitutions.

Furthermore, a modification(s) proximal to the dual tyrosine motif could sterically affect tyrosine phosphorylation in the wt or l-Tetherin backgrounds as well as potentially disrupt functions like such as sorting or protein stability, which could be important for optimal signaling function.

78 The various mutations introduced upstream of the dual-tyrosine motif in the STS region suggest some structural specificity associated with signaling function. The STS region, which appears to have been under selective pressure in primates, is not tolerant of acidic mutations. By contrast, the hemITAM of CLEC-2 is preceded by a similar tri-acidic (DED) sequence that is crucial for hemITAM signaling and triple alanine substitution abrogates its function (88). We observe the opposite effect, where hydrophobic/nonpolar residues appear to be favored in the presence or absence of the dual-tyrosine motif (Figure 3-1A and 3-6). The polyalanine mutations are interesting because these substitutions abrogate any potential motif upstream of residue 13 and suggest that this region serves as a regulatory motif and not a direct protein-docking site.

Additionally, the propensity for this region of the cytoplasmic tail to form a longer hydrophobic, coiled structure also increases. The introduction of a leucine (S5L) from the chimp sequence does not abrogate signaling when the tyrosines are present. In fact, this substitution slightly increases NF-κB activation in the wt (87) or l-Tetherin background and restores signaling to the dual tyrosine mutant. This observation further supports the hypothesis that signaling is favored by hydrophobicity in this region [leucine is one of the most hydrophobic amino acids by hydropathy scoring (110)]. Introducing rhesus Tetherin sequence (PIL) abrogated signaling possibly due to addition of a helix-disrupting proline that might abolish a required structural feature. Supporting the theory that signaling may bot require a tyrosine-based motif is the recent observation that equine Tetherin activates NF-κB when expressed transiently in human cells (259). Both equine

Tetherin proteins have a very short cytoplasmic tail with no tyrosine and no discernable signaling motif. Interestingly, both equine Tetherin proteins contain glycines as the second amino acid suggesting a potential myristoylation site. It is tempting to speculate that a hydrophobic modification of the equine Tetherin amino terminus could behave similarly to hydrophobic mutations we have analyzed in human Tetherin to promote NF-κB activation.

Single-alanine scanning within the l-Tetherin background reveals that residues Y6 and

R19 are also critical for Tetherin activation of NF-κB in agreement with previous results (59). We identified the alanine residue at position 2 (A2) to also be important for NF-κB signaling. Because

79 the second codon is important for translation initiation (+4 position of the Kozak sequence), we designed and generated mutations that would maintain the integrity of the Kozak sequence.

Although reduced expression of the A2V mutant, possibly due to protein stability, may explain why this mutant does not activate NF-κB, the A2E substitution is stable and does not signal. As discussed above for the adjacent STS region, the introduction of an acidic substitution upstream of the dual-tyrosine motif may not be compatible with Tetherin signaling function. Finally we found that the single alanine point mutations at residue 4 (T4A) in the context of the l-Tetherin background constantly reduced the level of NF-κB activation. The importance this residue was likely not uncovered in previously reported triple alanine scanning experiments because, as we have observed, runs of hydrophobic residues upstream of the dual tyrosine motif have no effect on signaling.

The Vpu-resistance phenotype of the SY mutant that we first observed using a VLP budding assay has since been documented in the context of replication competent HIV-1 (247).

However, Vpu from different SIVs and different HIV-1 groups, with the exception of M, have been shown to be more indiscriminant about downregulating s-Tetherin (247), suggesting that SY could be equally sensitive. The dual tyrosine to phenylalanine mutant, SF, which we also show can signal, but not to the degree of SY (Figure 3-1A), has also been tested with live HIV-1, and has been demonstrated to be Vpu-sensitive (247). This is most likely due to impaired trafficking observed in the AxA mutant since bulky phenylalanines can potentially substitute for tyrosines in clathrin-mediated trafficking motifs that interact with AP-2 (249). We hypothesize SY is stable on the cell surface, as has been demonstrated with dual-tyrosine mutants and suggested for s-

Tetherin which lacks the endocytosis motif entirely. Perhaps surface stability allows for some increased degree of clustering, leading to the enhanced signal observed by SY over both wt and l-Tetherin (Figure 3-1D). Surface stability may also partially explain resistance to Vpu, which is thought to interact with Tetherin in the TGN (52).

Tetherin is hypothesized to signal via a non-canonical tyrosine based motif, most likely a hemITAM motif (60). This motif has been identified in a number of proteins that act as immune

80 receptors. Unlike traditional ITAMs, which have interspaced phosphotyrosines, hemITAMs contain of single phosphotyrosine and interact with signaling kinases trough dimerization. For example, CLEC-2 hemITAM dimerization allows binding to Syk via a hemITAM through an unconventional SH2-mediated interaction across single phosphotyrosines residues on adjacent

CLEC-2 molecules (87). Unlike a previously described Y6A mutation, which abrogates signaling, we observed that Tetherin mutants with conservative phenylalanine mutations (Y6F, Y8F) activate NF-κB . This is stark contrast to other described hemITAM containing proteins where phenylalanine substitutions typically abrogate signaling (210, 262). While it is possible that our results can be explained by theorizing that Y6 and Y8 function as redundant, overlapping hemITAM-like signaling motifs, there is no precedent for this arrangement. Moreover, the ability of the SY mutant, which contains no tyrosine residues in the cytoplasmic tail, to interact with TAK1 by co-IP and signal is a Syk-dependent manner supports a model that does not involve a direct hemITAM-mediated interaction on the Tetherin cytoplasmic tail for signaling function.

Whole cell phophoproteomic analysis demonstrated that Tetherin is phosphorylated at Y8 and Y6+8 in combination (211) Interestingly no peptide containing only phoshphorylated Y6 was identified, yet our analysis and that of Galao et al. suggest that Y6 is an important residue for wt

Tetherin signaling, it may be specifically dependent on viral particle sensing. The model for

Tetherin-mediated signaling through a hemITAM motif has been built, in part, around immunoprecipitation of Tetherin with anti-phosphotyrosine antibodies (Abs), which correlated with signaling function. There are some inconsistencies observed with this model. The Y8A mutant, which retains transient signaling function, was shown to IP with a phosphotyrosine Ab only during

MLV infection and not during transient expression (60). Additionally, triple alanine scanning mutagenesis of regions overlapping Y6 only (TSY or SYD mutated to AAA) all retained signaling function (59), paralleling what we have seen with the STS mutation data described above. One hypothesis that fits with our data is that Tetherin phosphorylation specifically, rather than recruitment of Syk directly, induces or stabilizes a conformational change in the cytoplasmic tail important for signaling, and downstream recruitment of Syk kinase by another protein. Tetherin is

81 a lipid raft resident protein and there are numerous examples of proteins that are solely GPI- anchored that propagate signals into the cell, including receptors involved in immunity. However,

Tetherin uniquely contains a cytoplasmic tail in addition to the GPI anchor. Our observation that cytoplasmic tail mutations at the amino terminal end can affect signaling suggests that region of the tail, could act as a regulatory domain.

One aspect of SY mutant-mediated signaling that needs to be explored is whether viral particle budding enhances activation of NF-κB . When the cell is at rest, Tetherin is believed to interact with the actin-binding protein RICH2 via its cytoplasmic tail. The current tyrosine-based signaling model suggests that upon actin-dependent activation by, for example, viral budding, an unknown Src-kinase is thought to phosphorylate the Tetherin cytoplasmic tail, which recruits Syk

(Figure 3-2). The interaction with RICH2 does not appear to depend upon the dual tyrosine motif as this mutant appears to associate with RICH2 comparably to wt Tetherin as measured by co-IP,

(60). We propose that RICH2 interaction remains intact with SY Tetherin as the association does not appear to be tyrosine dependent. If viral particle budding enhances SY signaling, we would hypothesize that src kinase-mediated tyrosine phosphorylation would occur on a currently unknown signaling protein that may associate with Tetherin via another domain.

The ability of src-family and Syk specific inhibitors to impair Tetherin mediated suggests the possibility that the compensatory changes bypass Src-mediated phosphorylation of Tetherin.

SY, and signaling competent variants that do not have tyrosines, could already be primed to signal. In this model Syk recruitment would be through an indirect mechanism and not through a hemITAM on Tetherin, but rather some other phosphorylated protein. We observe SY Tetherin can interact with TAK1. One possibility is that sequence downstream of M13 can bind another effector as long as there is scaffolding or regulatory sequence upstream. As previously stated, a conformational change in the tail could facilitate this interaction. Alternatively, Syk could bind an adaptor that interacts with Tetherin via the GPI anchor. GPI-anchored proteins can propagate signals into the cell though they do not span into the cytosol. This occurs through interactions via adaptor proteins. GPI anchored proteins cross-linked with mAb are frequently observed to be

82 associated with Ca2+ bursts and tyrosine phosphorylation (84). Interestingly, Tetherin mediated signaling has been suggested to involve Ca2+ as it can be dampened with BAPTA treatment

(228). s-Tetherin which has an intact GPI-anchor does not signal; this again raises the possibility that the cytoplasmic tail could primarily play a regulatory function. Interestingly, we, and others, have now shown Tetherin and other signaling competent mutants can interact with TAK1 (Figure,

3-4, (59, 228). The activation of the B cell receptor depends on TAK1 to mediate NF-κB signaling.

As previously mentioned Ca2+ flux though previously implicated in ER stress (228) could be the result of PLCγ activation via Tetherin leading to NF-κB activation. Future experiments will begin to address whether other additional domains mediate interactions that are necessary for Tetherin signaling function.

83 ACKNOWLEDGEMENTS

We thank Rakesh Jambusaria, Nelson Glennie and Katherine St. Denis for generating a subset of the Tetherin point mutants. Ben Dyer for cloning of TAK1 expressing plasmid. Sunny Shin and

Michael May for supplying plasmids.

84 MATERIALS AND METHODS

Cell Culture

293T cells were maintained in DMEM + 10% Fetal Bovine Serum (Hyclone) at 37°C with 5%

CO2.

Plasmids and Reagents

Human tetherin cDNA in pCMV-SPORT6 was acquired from Open Biosystems. All Tetherin mutants tested were generated in the l-Tetherin (M13I) background using Change-IT site-directed mutagenesis by PCR. Primers for site-directed mutagenesis were designed using QuikChange

Primer Design Program (Agilent Technologies). The HIV-1 gag-pol encoding construct, psPAX2 was obtained from Addgene (plasmid 12260). The pCAGGS ebolavirus FLAG-VP40 plasmid used is previously described (100). HIV-1 Vpu and ebolavirus GP cloned into pCAGGS were previously described (100, 204). The NF-κB reporter plasmid, pBIIX-Luciferase was provided by

Dr. Michael May (University of Pennsylvania). The TAK1 constructs were obtained from Open

Biosystems and cloned into pCMV-SPORT6. The wt and dominant negative (K44M) FLAG epitope tagged IKKβ plasmid was obtained from Addgene (plasmid 11104). The AP1 reporter

(Biomyx) and MEKK1-delta plasmids were obtained from Dr. Sunny Shin (University of

Pennsylvania). The Sendai virus stock and IFNβ reporter were provided by Dr. Carolina Lopez .

Transfections were performed using 2.5ul of a 1ug/mL working stock of polyetheylenimine (PEI) per 1ug DNA.

VLP Budding Assay

2.5x105 293T cells seeded on a 24 well plate were transfected with PEI at the described working concentration listed above. Indicated tetherin construct (50 ng) was co-transfected with either:

Gag-Pol expression vector pSPAX (200ng) and increasing amounts of (50 or 200 ng) of pCAGSS-vpu for HIV-1 VLP assays or pCAGGS FLAG-VP40 (100ng) and increasing amounts of

85 (50 or 200 ng) pCAGGS ebolavirus GP for EBOV VLP assay. Cell lysates and supernatants were harvested 48 h post-transfection. Cell lysates were harvested in Triton X-100 buffer (50mM Trs-

HCl pH 8.0, 5mM EDTA, 150mM NaCl, 1% Triton X-100) with Complete (Roche) protease inhibitor cocktail. Cell lysates were cleared by centrifugation at 17,900x g for 3 min at 4°C.

Supernatants containing VLPs were cleared are 1700x g for 2 min at 4°C. VLPs were subsequently purified by pelleting through a 20% sucrose cushion at 40,000 in a TLA120.1 rotor

(Beckman) for 30 min. VLPs were resuspended in PBS on ice for 3 h. Cleared cell lysates and resuspended VLPs were separated on 15% or 4-15% Criterion gels (BioRad) respectively before being transferred to PVDF membrane. Membranes were blocked in 5% milk in TBST for 40 min prior to incubation with primary antibody. HIV-1 Gag p24 was detected in Western blots for both

VLPs and cell lysates using a monoclonal anti-p24 antibody (24-3, National Institutes of Health

AIDS Research and Reference Reagent Program). The loading control used across experiments was αtubulin, which was analyzed using αtubulin mAb (12G10). HIV-1 Vpu was detected using

HIV-1 pNL4-3 Vpu antiserum (969, National Institutes of Health AIDS Research and Reference

Reagent Program). Ebolavirus GP was detected using rabbit antiserum against the GP1 portion of the protein (125). Tetherin expression was assessed using rabbit anti-BST2 sera (Dr. Klaus

Strebel, #11721 National Institutes of Health AIDS Research and Reference Reagent Program).

Luciferase Assay

For single concentration assays: 293T cells (2.5x105) were seeded on a 24 well plate and the following day were transfected with the indicated tetherin constructs (50 ng) and pBIIX-Luciferase reporter (200 ng) using PEI. Total DNA was kept constant using empty pCMV-SPORT6 vector.

At 30 h post-transfection, cell lysates were harvested in Triton X-100 lysis buffer. Lysates were transferred to a black flat bottom 96-well plate. Luciferase Assay System substrate (Promega

E1501) was added to the lysates according to manufacturers directions. Samples were analyzed in a Luminoskan Ascent microplate luminometer (Thermo Scientific). Statistical analysis was performed using PRISM (GraphPad).

86

Co-immunoprecipitation

1.4x106 cells were seeded on 6 well plate. Following day (~20h), PEI transfection performed on cells using 500ng of the indicated Tetherin constructs per well (wt, l-, s-, AxA, SY) + 500ng pCMV-SPORT6 TAK1. Control well with TAK1 and wt Tetherin alone done in parallel, with DNA normalized using empty pCMV-SPORT6. 18h post transfection, cells were lysed on ice for 20min in TritonX-100 buffer in the presence of freshly added Complete (Roche) protease inhibitor cocktail and 1mM sodium orthovanadate (NEB). Lysates were cleared for 10min at 4°C. Aliquot of lysate set aside for input. Remaining lysate incubated with 1.5µg anti-Tetherin Ab (RS39E).

Abs and lysate incubated at 4°C while rocking for 2h after which washed Protein G Dynabeads

(Invitrogen) were added for an additional 1h. After incubation, lysates/bead/Ab complex pulled down over chilled magnet and washed gently 4x using TritonX-100 buffer removing from magnet each time. Protein immunoprecipitated on beads were treated with PNGase via manufacturers instructions then boiled in 1x SDS/PAGE loading buffer for Western blot analysis. Loading control for inputs was determined using αtubulin (12G10) Ab. TAK1 blotted for using TAK1 (D94D7) specific Ab (CST, #5206). Tetherin blotted for using anti-BST2 sera described above.

87 FIGURES

A C

B B κ κ - - 150 1000 F F 800 N N

l l 600 a a

s 400 s 100 a a 200 B B

e 10 e

l- MASTSYDYCRVPIEDGDKRCK v v 8 o o 50 6 b b

STS AAA )

A 4 A

1

d 2 d l B AxA A A l =

ns o 0

o 0 κ - - F l 4 F l- Y6A A l- F SY SY o t

N *

Sendai MEKK1

Y8A A l d

a 3 e SY AAAA A s z i a l

Y6F F B a 2 e m Tetherin r

Y8F F v o o b FxF F F N 1

A s SF AAAF F e αTubulin d l u l

o 0 a F

V l-

( SY SF STS AxA Y6A Y8A Y6F Y8F FxF Tetherin Construct

B D

B κ WT MASTSYDYCRVPMEDGDKRCK - 100

F l- WT AxA A A N T4A

) l 80 1

WT STS AAA a Y6A

s = B

t a WT SY AAAA A κ R19A - 3 60 w

** B F

o N e t

ns l

v 40 a d o s e 2 **** z a b i l B

A 20

a

e m d v l r

o 1 o o 0 b F N

0 50 100 150 200 A

s d e l u

o 0 l Amount Tetherin Plasmid (ng) a F

V wt ( WT SY WT AxA WT STS Tetherin Construct Figure 3-1. Identification of a signaling-competent, tyrosine-independent Tetherin. 293T cells were cotransfected with 50ng of various Tetherin constructs and 200ng of firefly luciferase reporter. Cells were harvested 24h post transfection for luciferase readout (A) Tetherin cartoon with alignment of mutants generated in l-Tetherin background tested in panel assessing the signaling ability of various tyrosine mutants. Tetherin constructs were cotransfected with pBIIX- Luc for NF-κB activation. Values normalized to l-Tetherin background set to 1 (grey dotted line). Graphs represent experiments done in triplicate (n=4); error bars= SEM *p<0.05(B) Alignment of mutants generated in the wt Tetherin background. Tetherin constructs were cotransfected with pBIIX-Luc for NF-κB activation. Graph normalized to wt Tetherin background set to 1 (grey dotted line). Graph represents experiments done in triplicate (n=3); error bars= SEM ** p<0.01, ****p<0.0001 (C) l-Tetherin and SY cotransfected with either AP1 reporter (left) or IFNβ reporter (right). Reporter activation controlled for using MEKK1 for AP1 and incubation with Sendai virus for IFNβ. Graphs are representative of multiple experiments done in triplicate; error bars=SD. Representative lysates ran under denaturing conditions blotted for Tetherin and GAPDH as loading control. (D) Increasing ramp of Tetherin plasmid (12.5ng, 25ng, 50ng, 100ng and 200ng) were cotransfected with pBIIX-Luc for NF-κB activation. Graph is a representative of multiple experiments done in triplicate. 88 A B wt Hyp Ratio from signal

B B κ - κ

- 300 150 ns SY:empty F F N

l N SY:l-

a l s a * SY:s- 100 a s 200 B

a *

e B

v e

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b d l A

o 0 d F l l-

o 0 WT 3:1 1:1 1:3 F 3:1 1:1 1:3 WT SY Hyp RatioRatio from sig l-:s-nal SY only B κ Ratio SY:other Tetherin/empty plasmid - 250 F N

l 200 a

SY:empty SY:l- SY:s- s a 150 B

SY 3:1 1:1 1:3 SY 3:1 1:1 1:3 SY 3:1 1:1 1:3 e 100 v o

Tetherin b 50 A

d l 0 o F aTubulin SY 3:1 1:1 1:3 WT SY Ratio SY:s-

Figure 3-2. SY signaling can be regulated by s-Tetherin. 293T cells were transiently transfected with 50ng of total Tetherin plasmid(s) and 200ng of firefly luciferase reporter. Cells were harvested 24h post transfection for luciferase readout. Graphs from (A) and (B) come from the sets of experiments. (A) 293T cells were transiently transfected with SY Tetherin and titrated with empty vector (black bars), l-Tetherin (grey bars) or s-Tetherin (white bars). Total amount of plasmid transfected was kept constant for titrations. Graph represents multiple experiments done in triplicate (n=5); error bars= SEM. T-test performed between SY:empty and SY:s- conditions *=p<0.01. Lysates from representative experiment, PNGase treated and blotted for Tetherin. Loading controlled for by αtubulin staining. (B) Graphs represent comparison signal of 50ng of either wt Tetherin (top) or wt SY (bottom) compared to different ratios (50ng total)

89 )

A C 1

B =

κ - -

l 2

F ) l-

MASTSYDYCRVPIEDGDKRCK o

t N 1

l A2V d B a = e s A2E κ z - i a - l l 3

B a F 1 o e m t N r v

o l o d b N a

e A s s

z 2 e d i a l l u l

o 0 B a a F

V

( 50 50 e m 200 100 200 100 200 r v o o 1 Amount Tetherin Plasmid (ng) b N

A s

25kDa e d l u

l 20kDa o 0 Tetherin a l- F 15kDa V ( T4A A2V S3A S5A Y6A Y8A D7A C9A M1A V11A P12A E14A R10A D15A G16A D17A K18A R19A C20A K21A M13A Tetherin Construct l- A2V A2E

B

B κ - 150 l- F N

SY l a

s 100 a B

e v

o 50 b A

d l

o 0

F 0 20 40 60 80 100 Amount Tetherin Plasmid (ng)

Figure 3-3. Identification of additional point mutations important for Tetherin-mediated signaling. 293T cells were cotransfected and lysates were harvested 24h post transfection (A) Tetherin cartoon with alignment of alanine mutants generated in l-Tetherin background tested in panel for effects on NF-κB signaling. M13I mutation highlighted in grey. Red letters indicate single point mutations that completely abrogate signaling. Arrows denotes residues that have a modest effect on signaling. Tetherin constructs cotransfected with pBIIX-Luc. Values normalized to l- Tetherin background set to 1 (grey dotted line). Graph represents experiments done in triplicate multiple times (n=4); error bars= SEM (B) Increasing ramp of Tetherin plasmids (12.5ng, 25ng, 50ng, 100ng and 200ng) were cotransfected with pBIIX-Luc for NF-κB activation. Graph is a representative of multiple experiments done in triplicate. (C) Two different A2 mutations tested in ramp with 200ng pBIIX-Luc reporter for NF-κB signaling, compared to 200ng of l-Tetherin background. Denatured lysates from a representative experiment probed for Tetherin expression compared to l-Tetherin background.

90 A wt l- s- SY wt l- s- SY B B B B κ κ κ κ - - - - 10000 10000 10000 10000 F F F F N N N N

l l l l 8000 8000 8000 8000 a a a a s s s s a a a a 6000 6000 6000 6000 B B B B

e e e e

v 4000 v v 4000 v 4000 4000 o o o o b b b b 2000

2000 2000 A

2000 A A A

d d d d l l l l

o 0

o 0

o 0 o 0 F F F F

β β β β β β β β β β β β β β β β β β β β β β β β

No IKK No IKK No IKK No IKK

25ng wt IKK 25ng DN IKK 25ng WT IKK 25ng DN IKK 25ng WT IKK 25ng DN IKK 25ng WT IKK 25ng DN IKK 100ng wt IKK 100ng DN IKK 100ng WT IKK 100ng DN IKK 100ng WT IKK 100ng DN IKK 100ng WT IKK 100ng DN IKK NoIKK NoIKK NoIKK NoIKK Amount IKKβ plasmid (ng) Amount IKKβ plasmid (ng) Amount IKKβ plasmid (ng) Amount IKKβ plasmid (ng)

IKKβ

Tetherin

B C Syk-specific inhibtor treatment B κ

- 400

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l- Amount BAY 61-3606 s- Src-family kinase inhibitor treatment

SY SY

aTubulin WT B AxA κ - 300 l- F WT s- WT only WT N SY SY WT WT l- l TAK1 only TAK1 AxA

a s- s 200 a WT only WT SY B

TAK1 only TAK1 e v

o 100 b A

d l

o 0 F 0 5 0 5 0 5 0 5 10 20 10 20 10 20 10 20 Amount Piceatannol Figure 3-4. Tyrosine-independent Tetherin mutant can signals through a similar pathway as wt Tetherin. 293T cells were transiently cotransfected and lysates were harvested following day (A) wt, l-, s- and SY Tetherin constructs were cotransfected alone, or with increasing amounts (25ng and 100ng) of either wt FLAG-IKKβ or DN FLAG-IKKβ (K44M) plus 200ng of pBIIX-Luc reporter for NF-κB activation. Graphs represent multiple experiments done in triplicate (n=3); error bars= SEM. Lysate from a representative experiment blotted for IKKβ and Tetherin (B) 293T cells transfected with equal amount of Tetherin or TAK1 expressing plasmid. Lysates harvested 20h post transfection. Small input fraction saved for expression control. Remaining lysate coimmunoprecipitated using Tetherin monoclonal antibody. Protein was PNGase treated directly on bead after last wash in protocol. Lysates blotted for Tetherin and TAK1. Inputs included αtubulin. (C) 6h post transfection with Tetherin construct (wt, l-, s- or SY) with pBIIX-Luc reporter for NF-κB activation, media was changed with increasing amounts of either Piceatannol (top) or BAY61-3606 (bottom). Cells treated in drug for 18h before harvest. Graphs represent multiple experiments done in triplicate (Pic, n=3; BAY61, n=4).

91

VLPs - WT l- s- SY VP40only VP40 + GP VP40GP + FLAG-eVP40

Tetherin

Cell FLAG-eVP40 Lysate

Tetherin

eGP

αtubulin

Figure 3-5. Ebolavirus glycoprotein antagonizes the SY mutant tethering function. Ebolavirus (EBOV) VLP budding assay in 293T cells. Cells were transiently transfected with 100ng Tetherin construct (wt, l-, s-, SY), 100ng FLAG-eVP40 and increasing amounts of EBOV GP (eGP) (50ng and 200ng). Supernatants were purified to isolate VLPs and cell lysates generated for total protein assessment 48h post transfection. VLPs probed for FLAG stripped and reprobed for incorporated Tetherin. Cell lysates probed for eGP using a GP specific Ab, FLAG and Tetherin. αtubulin used as protein leading control.

92

- 1 -

used as

tubulin Vpu p24 p55 p41 p24 Tetherin α (A) Cartoon alignment of alignment Cartoon (A)

VLPs Cell Lysates B and cell lysates generated for total protein assessment 48h 1 gag, Tetherin and Vpu using specific Abs. α tubulin

- t T4S5AxA c 1 VLP budding assay in 293T cells. Cells cotransfected with - u

r S3S5AxA t s

polexpression constructpSPAX2 and increasing amountsof HIV n S3T4AxA -

o Tetherin background set to 1 (grey dotted line). Graphs represent S5AAxA - C

n

i T4AAxA r

e 1 gag

SY - S3AAxA h t

e

AxA T

STS l- rified rified to isolate VLPs

4 3 2 1 0

) 1 = - l o t d e z i l a m r o N s e u l a V (

κ B - F N l a s a B e v o b A d l o F EDGDKRCK were were pu

I =3); error bars= SEM. (B) HIV Tetherin background. Panel of Tetherin mutants cotransfected into 293T cells along with pBIIX - in l

. κ B activation. Values normalized to l - A l- MASTSYDYCRVP STS AAA AxA SY AAAA A S3A A T4A A S5A AA A S3T4 AA A S3S5 A AA T4S5 AAA A Minimal changes in STS that confer signaling phenotype in tyrosine mutant Tetherin. mutant tyrosine in phenotype signaling confer that STS in changes Minimal A

6. -

Figure 3 Tetherin mutants generated Luc reporter for NF experiments done in triplicate (n Tetherin mutants (50ng) from (A)with 200ng ofan HIV Vpu (50ng and Supernatants 200ng). post transfection. VLPs probed for p24. Cell lysates probed HIVfor protein leading control

93 A C l- MASTSYDYCRVPIEDGDKRCK l- MASTSYDYCRVPIEDGDKRCK

AxA A A )

1

B S3D D ) STS AAA =

κ 1 -

-

l 3 B

T4D D =

SY AAAA A F

κ o - - l

2 t N

F S5D D Ala7 AAAAAA l o d a t N

e l DED DED s

z 2 d Ala8 AAAAAAA a i a l e s B a z

i DEDAxA DEDA A a

l Ala9 AAAAAAAA e m B a r

1 v

e Ala10 AAAAAAAAA o m o 1 r v b N

o o Ala11 AAAAAAAAAA A s b

N

e d A s l

u l e

o 0 d a l u F l

o 0 V l- a ( SY F STS AxA V l- Ala7 Ala8 Ala9

( Ala10Ala11 S3D T4D S5D DED Tetherin Construct DEDAxA B Tetherin Construct

l- MASTSYDYCRVPIEDGDKRCK STS AAA )

1 ChSTL L B = **

κ - -

l 4

ChSTL AxA LA A F * o t N

RhPIL PIL l ** d

a 3 e s

RhPIL AxA PILA A z i a l B a 2 e m r v o o b N 1

A s

e d l u l

o 0 a F

V l-

( SY STS ChSTL RhPIL

ChSTLAxA RhPILAxA Tetherin Construct

Human MASTSYDYCRVPM-----EDGDKRCK Pan troglodytes MASTLYDYCRVPMDDIWKKDGDKRCK MAPILYDYRKMPMGDIWKEDGDKRCK

Figure 3-7. Assessing the plasticity of STS region to mutation. 293T cells were cotransfected with 50ng of various Tetherin constructs and 200ng of firefly NF-κB luciferase reporter, pBIIX-Luc. Cells were harvested 24h post transfection for luciferase assay. Tetherin cartoon with alignment of mutants generated in l-Tetherin background for each panel tested. Values for all graphs normalized to internal l-Tetherin background, which is set to 1 (grey dotted line). (A) Acidic mutations. Graph represents multiple experiments done in triplicate (n=3); error bars= SEM (B) Hominid sequence swaps with hominid cytoplasmic tail alignment below. Graph represents multiple experiments done in triplicate (n=4); error bars= SEM *p<0.05 , **p<0.01 (C) Polyalanine mutations. Graph represents multiple experiments done in triplicate (n=3); error bars= SEM

94 CHAPTER 4: GENERAL DISCUSSION AND FUTURE PERSPECTIVES

Summary and perspectives on Tetherin isoforms

In this dissertation, we have elucidated novel biology for the cellular restriction factor,

Tetherin. A large body of research has focused on various facets of how Tetherin restricts enveloped virus budding, which includes characterization of structural determinants necessary for tethering function, identification of viruses that are Tetherin-sensitive and whether these viruses encode a Tetherin antagonist, and determining, mechanistically, how virally encoded antagonists counter Tetherin-mediated restriction (See Chapter 1). Tetherin has a short cytoplasmic tail, which is dispensable for tethering viral particles, though it can be targeted directly by virally encoded antagonists for downregulation to allow efficient viral replication. Aside from a tyrosine- based endocytic motif, a second cytoplasmic methionine, downstream of the putative start codon, is highly conserved among Tetherin orthologues.

In Chapter 2 we determined that the nucleotide sequences surrounding the downstream cytoplasmic methionine codon was in an appropriate genetic context to act as a potential alternative start codon. Additionally, we observed that the putative primary start codon was in the context of a suboptimal Kozak sequence (Figure 2-1B) suggesting potential ribosomal leakage.

The pre-initiation ribosomal complex composed of the 40S ribosome and Met-tRNA scan AUG codons in this Kozak context and rather than initiate translation, skip over the potential start site a fraction of the time (108). wt Tetherin cDNA with the endogenous Kozak sequence produces two species: l-Tetherin, which is considered full-length expressing off the upstream open reading frame (uORF), and s-Tetherin, which expresses off an in-frame ORF located 12 codons downstream of the putative start site. Resolution of these species by immunoblotting is enhanced by PNGase treatment of N-linked glycans that produce a diffuse, 30-36kDa banding pattern for

“mature” Tetherin. Interestingly, deglycosylated Tetherin blots had been analyzed prior to our work, including one, which identified the presence of a two species in HeLa cells. However, the

95 bands were misidentified as potential differences in post-translational modifications (6). It is peculiar that the human Tetherin isoforms were not discovered previously. However, it is very likely that prior to our study most labs did not routinely deglycosylate lysates when expressing

Tetherin and/or used Tetherin expression vectors with either strong Kozak sequences at the first start site or codon optimized cDNA. These reasons would account for why this phenomenon was not observed. Strengthening the Kozak upstream of the putative AUG leads to almost exclusive expression of l-Tetherin, suggesting alternative translation initiation off a single polycistronic mRNA as the underlying mechanism. This presents a way by which Tetherin expression, and subsequent function(s), can potentially be modulated raising the question of whether endogenous expression of the isoforms can be regulated by different stimuli. Additionally, the heterogeneity in

Tetherin glycosylation may be a representation of variants that could also have different cellular localizations; changes in Tetherin glycosylation has been shown to impact subcellular localization, but still allow restriction of certain viruses.

We expanded our observation to rhesus and murine Tetherin, also observing multiple

Tetherin species from cDNA expression further suggesting this is a conserved mechanism by a variety of mammalian species. Mounting evidence suggests the translation of a truncated regulatory Tetherin isoform is common in mammals. During the preparation of the work highlighted in Chapter 2, a naturally occurring polymorphism in the putative Tetherin upstream start codon was observed in a NZW mouse leading to expression of only the shorter isoform lacking the dual-tyrosine motif (17). As observed in human Tetherin, loss of the tyrosine motif stabilizes the protein at the plasma membrane. In a viral challenge experiment with Friend retrovirus, the polymorphism conferred increased protection to viral replication compared to a B6 mouse encoding the wt tetherin sequence (17). The genomes of ruminants have been found to have gene duplications in Tetherin, generating proteins with tails of variable length, including loss of the conserved tyrosine residues. When tested against ovine or bovine specific viruses, the shorter forms were the more potent antiviral product (9, 225). Additionally, feline Tetherins have been described to have full-length and truncated tails although in the context of feline viruses

96 tested, no differences in resistance to viral challenge have been observed (50, 58). Tetherin isoforms identified thus far vary in surface stability, sensitivity to different viral antagonists and to differentially signal. It will be interesting to determine whether there are additional functional differences between the human Tetherin isoforms. The observation that various mammals can express different Tetherin species suggests an underappreciated versatility of the protein. A

Tetherin-like molecule expressed in Chinese hamster cells called GREG (Golgi-resident GPI- anchored protein) was shown to be involved in regulating Golgi integrity (123), a function similar to that of Golgins, which like Tetherin are also integral membrane coiled-coil proteins. This molecule resides almost exclusively in intracellular compartments, but is involved in impacting membrane dynamics, which may parallel what Tetherin does at the cell surface when enveloped viruses perturb membrane integrity.

The dual-tyrosine is important for Tethering trafficking (140, 181). There is evidence of enhanced plasma membrane stability of Tetherin species that lack this motif in vitro, as well as in a naturally occurring mutation in NZW mice (26). These shorter Tetherin variants may be the most important form for restricting viral replication, which may explain why the second cytoplasmic methionine is highly conserved. The lack of the dual tyrosine motif in s-Tetherin makes it highly resistant to HIV-1 Vpu, which is thought to engage Tetherin intracellularly.

Conversely, l-Tetherin is highly sensitive to Vpu-mediated downregulation. Along with the dual tyrosine motif, an upstream STS sequence, when mutated in combination produces s-Tetherin- like, Vpu resistance in the Vpu-sensitive l-Tetherin background. Independently, both these amino terminal regions confer some degree of resistance to HIV-1 Vpu in virus-like particle (VLP)-based systems and subsequently validated with replication competent HIV-1 (247). In the context of viral infection, the dual-tyrosine may serve a regulatory purpose and therefore expression of full-length

Tetherin (l-Tetherin) may be important to properly traffic and relocate the short form after viral particle restriction.

In Chapter 3 we identify the minimal mutations necessary to confer HIV-1 Vpu resistance, highlighting a crucial threonine at position 4 (T4). Based on our data, the flanking

97 serine residues, contribute minimally to the phenotype observe with SY. It is unclear what role these residues play in regulating Tetherin function. Ubiquitination of this motif has been suggested in the presence of Vpu, but that mechanism remains controversial (72, 229). However, this does not rule out the possibility of additional post-translational modifications, as serines and threonines can be phosphorylated and acetylated. We also do not know whether these residues contribute to trafficking. Because of the proximity between STS and the dual tyrosine motif, modifications at either serine or the threonine could sterically hinder machinery engaging the tyrosine residues. Phosphoproteomics has produced evidence of tyrosine phosphorylation of the

Tetherin cytoplasmic tail (211). A similar mass-spectrometry approach could be employed to assess serine/threonine modifications, which may highlight a novel regulatory mechanism for

Tetherin function.

Co-expression of s-Tetherin affects l-Tetherin sensitivity in the presence of Vpu and signaling function (see below), suggesting s-Tetherin has a dominant phenotype, highlighting a potential regulatory role for this novel isoform. Alternative translation initiation of an in-frame polycistronic mRNA generating a truncated isoform with “dominant-negative” properties was recently described with another immune factor, mitochondrial antiviral-signaling protein (MAVS)

(30). The IFN response to viral infection is rapid and crucial for a robust innate immune response.

It is defined by the upregulation of a large array of genes collectively called ISGs. It is estimated that our genome encodes over 300 ISGs with diverse cellular localizations and functions.

Diversification of ISGs through various transcriptional and translational mechanisms enhances the functional repertoire of proteins to counter a myriad of viruses that could infect the host. To date the subset of immune genes identified and validated as being regulated by alternative translation initiation is small including Tetherin, MHC class II invariant chain (220), MAVS (30) and osteopontin (203). The ribosome profiling analysis that identified MAVS isoforms also identified potential internal translation initiation sites (TISs) for several other ISGs (30) suggesting the use of isoforms with differential or regulatory biologic activities may be a common strategy

98 during innate immune responses. Future studies of these putative isoforms should reveal if this is the case.

Expression off polycistronic mRNA is not a new concept, rather an underappreciated mechanism of regulating protein expression and diversity. Advances in sequencing technology have furthered our understanding of how prevalent this mechanism may be. Ribosomal footprinting assays have revealed that between 50-65% of transcripts contain more than one candidate TISs (34, 90, 119). However, few examples of in-frame polycistronic mRNAs have been identified and validated. Ribosome footprinting identified ribosome association with in-frame downstream AUGs predicted to produce an alternative Tetherin isoform in both human and murine cells, supporting our hypothesis (30, 90). Despite the footprinting studies in cultured cells the primary TIS for Tetherin remains unclear. Expression of truncated forms of Tetherin lacking the upstream dual tyrosine motif has been observed in sheep, cats, and mice (9, 17, 26, 37).

Moreover, most upstream sequences tend to be in the context of a suboptimal Kozak. A more in- depth analysis using ribosomal footprinting on a broad range of cell types should be employed to address whether there is preferential ribosomal binding at either start codon..

It is still unclear how, or if, Tetherin isoform expression is regulated. We don’t know whether there is cell-intrinsic machinery that allows one isoform to be preferentially expressed in one cell type versus another or if the isoforms are differentially translated under varying conditions. We observed that interferon alpha (IFNα) stimulation appeared to upregulate both isoforms equally in several cell types/lines tested, including 293T, HT1080, HeLa and primary

CD4 T cells although these studies did not employ stringent quantification. It is unknown whether tetherin mRNA can form secondary structures, like hairpins, around the start sites, which could regulate initiation/scanning of the pre-integration complex and/or processivity of the 80S ribosome leading to differential protein expression.

A common consequence of innate response to viral infection is global repression of translation by the host cell. This mechanism is highly effective against various viral families that replicate in the cytoplasm. During periods of stress eukaryotic translation initiation factor 2 (eIF2)

99 becomes phosphorylated by one of four known kinases, one of which is the IFN induced viral

RNA sensing protein kinase R (PKR) (61). eIF2 phosphorylation leads to a reduction in active ribosomal complexes available for global translation. Among the proteins upregulated by this mechanism are those involved in certain stress responses (15). Ribosome become more selective and may preferentially bind one TIS over another stochastically or depending on sequence context. Neither Tetherin start site is in the context of an optimal Kozak; therefore it is difficult to predict whether one is preferred over the other. However, viral infection assays in the presence or absence of PKR should be done to assess whether there are changes in Tetherin isoform expression. Although none have been seen in human genomic analysis to date, it is unclear whether there exist naturally occurring polymorphisms around either start site or in the untranslated region of the human tetherin mRNA that could lead to changes in isoform expression as is seen in other mammalian species.

Tetherin-Vpu interactions have provided invaluable insight into differential regulation and function of long and short isoforms.in the context of viral infection. There is clear evidence that certain viruses do not discriminate between various isoforms. Host-pathogen co-evolution would suggest that successful viruses could take advantage of Tetherin function to facilitate transmission. Interestingly, challenge with FIV, which utilizes its Env to counter Tetherin function, indiscriminately antagonizes both feline Tetherin isoforms, similar to what we have observed with

EBOV GP. Recently, FIV has been suggested to replicate better in felid Tetherin expressing cells.

The existence of a potent short isoform may be a critical barrier against certain viruses that encode antagonists that do not target the cytoplasmic tail. However, the diversity in mechanisms by which viruses counter Tetherin function may speak more about how viruses may take advantage of a particular feature of Tetherin biology for replication. This has been suggested from observations that Tetherin could facilitate infection (50, 89, 96, 236). Because of differences in surface stability we would hypothesize that Tetherin isoforms could differentially allow for cell-cell spread of certain viruses at virological synapses as observed with HTLV-1 and HIV-1.

100 Various poorly characterized aspects of Tetherin biology (see Chapter 1) could be elucidated by studying the impact of l-Tetherin or s-Tetherin in isolation. Stable cell lines encoding either isoform should be employed to assess potential differential roles of the Tetherin isoforms from various species in cell-cell spread, NK mediated killing and IFN regulation of immunoglobulin-like transcript 7 (ILT7), as all three would require single round infection with virus followed by co-incubation with target cells. We hypothesize that surface stability of s-Tetherin could be a double-edged sword, potentially enhancing cell-cell transmission of tethered viruses as previously described (50, 89, 96), but also aiding in antibody-dependent cell-mediated cytotoxicity (ADCC) (1, 8) by stabilizing antigen (viral particles) on the cell surface. ILT7 on human pDCs negatively regulates pDC-mediated IFN expression upon interaction with Tetherin on the surface of IFN stimulated cells. Recently, tethered viral particles were shown to occlude

Tetherin-ILT7 interactions. By increasing viral tethering on the cell surface, s-Tetherin could potentially interfere with IFNα dampening in pDCs. In this scenario, one would hypothesize that the short isoform might aid in viral clearance by maintaining elevated IFN levels. Prolonged production, however, could have pathological consequences due to prolonged exposure. To date we do not have evidence of individual human Tetherin isoforms being expressed. However, we can take advantage of in vitro systems and murine isoform models to assess various isoform functions and elucidate more of the biology underlying this unique membrane anchored protein.

Summary and perspectives on Tetherin-mediated activation of NF-κB

Our work studying human Tetherin isoforms identified that a previously uncharacterized

NF-κB signaling function is specific to l-Tetherin and that this function is regulated by the signaling-null s-Tetherin isoform (141), Therefore we initially hypothesized that the necessary signaling determinants would localize to first 12 amino acids of the Tetherin cytoplasmic tail. We, and others, discovered that the dual-tyrosine trafficking motif was an important determinant of

NF-κB activation (44, 59, 228). Our preliminary rationale was that the tyrosine-based trafficking motif resembled an ITAM-like sequence therefore potentially serving dual function. A whole cell phosphotyrosine proteomics analysis demonstrated that Tetherin could be phosphorylated (211). 101 In Chapter 3 we continued to build on understanding Tetherin-mediated signaling through mutational analysis of the Tetherin cytoplasmic tail. Work from the Neil and Guatelli groups, published during the time we were conducting the studies highlighted in Chapter 3, described additional structural determinants of Tetherin-mediated signaling. Compilation of these data has led to the current model signaling model where Tetherin can sense viral assembly and budding

(59, 228). However, our work has yielded some results that do not agree with the current model of Tetherin-mediated signaling.

We identified a Tetherin mutant (SY Tetherin) that can activate NF-κB independent of the dual tyrosine motif, which challenges the current model that Tetherin-mediated signaling through

NF-κB occurs via a hemITAM motif. Substitutions at both cytoplasmic tyrosines in combination with changes in the serine-threonine-serine (STS) region at the amino terminus of the Tetherin cytoplasmic tail previously described in Chapter 2 to contribute resistant to HIV-1 Vpu, allow

Tetherin to retain signaling function. In Chapter 3 we identify a single alanine substitution threonine 4, T4A as sufficient to restore signaling function in the dual tyrosine mutant background mirroring the effect of this mutant upon Vpu resistance. To assess the contribution of additional individual residues to NF-κB activation, we engineered cytoplasmic tail point mutants in the l-

Tetherin background to assess contributions of additional individual residues. We observed mutation at tyrosine 6 to alanine, Y6A, abrogated signaling while Y8A had no effect, confirming what had been seen in the wt Tetherin background (60). Interestingly, in their triple alanine substitutions, Galao et al. observed signaling function for mutations at residues 4-6 which includes T4, S5 in combination with the loss of Y6, paralleling what we see with the dual tyrosine motif mutant (59). Conservative phenylalanine substitutions revealed that Y6F, unlike Y6A, still retained the ability to activate NF-κB. Though the dual tyrosine mutant FxF had abrogated signaling, it is unclear whether the overlapping tyrosines are redundant in function. In the case of trafficking, both must be mutated to produce aberrant localization (140, 181).

We assessed whether additional point mutations could abrogate Tetherin signaling. We generated various point mutants in the l-Tetehrin background, highlighting individual residues

102 across the entire cytoplasmic tail to be important via alanine scanning mutagenesis. Y6 and R19 alone had the most drastic effect, and had also been also observed to be important (60, 191). We also identified the importance of position 2. We generated two mutations, a conservative A2V and a charged A2E, ensuring the Kozak context around the putative start codon was not disrupted.

Both mutants failed to signal. A2V, specifically, expressed primarily lower molecular weight products suggesting stability was an issue. In the context of position 2, N-terminal processing of the start methionine is common. N-terminal acetylation has been suggested to play a role in protein stability and localization (10). N-terminal processing predictive software suggests wt

Tetherin and A2E mutant can be potentially be N-acetylated, but not A2V (146). Peptides isolated from the phosphoproteomics screen also identified the wt cytoplasmic tail peptide as being N- terminally acetylated at A2 (211).

Direct phosphorylation of the Tetherin cytoplasmic tail at both tyrosines has been observed in peptides isolated from a phosphoproteomics screen (211). There is a correlation between signaling competent Tetherin and ability to immunoprecipitate with an anti- phosphotyrosine (pY) Ab. However, immunoprecipitation (IP) and co-IP data suggesting that phosphorylation of Tetherin mediates recruitment of Syk via a hemITAM motif does not necessarily show a direct interaction. To test a direct interaction between Syk and Tetherin, synthesized Tetherin cytoplasmic tail peptides with phosphotyrosine modifications could be produced, immobilized and tested by surface plasmon resonance (SPR). Syk specific binding has been assessed for CLEC-2 by SPR (88). Swapping the Tetherin tyrosine motif into another hemITAM containing molecule could also be employed test its ability to act as a bona fide immunotyrosine-based motif. Swapping the entire human Tetherin cytoplasmic tail onto Tetherin proteins from another species does not activate NF-κB (59), though the scaffold used in the study was rhesus Tetherin which has a two amino acid deletion in the TM domain identified as a determinant for signaling (59). It is unclear whether adding the tail to a heterologous protein with similar properties as Tetherin (e.g. type II TM, dimerizes, localizes to lipid-rafts) could be enough to confer function. We would predict that if Tetherin is signaling indirectly through Syk, we could

103 still use pY Abs to IP our signaling competent SY mutant. This finding would suggest the need for an adaptor molecule that could bind Tetherin elsewhere on the tail or through a different domain to activate signaling.

Tetherin was identified as a PRR sensing viral assembly and budding (59, 228).

Determining whether SY can activate signaling by sensing particle release is important to strengthen our working hypothesis that Tetherin is not directly recruiting Syk via a hemITAM. The interaction between Tetherin and RICH2 has been shown to be important for signaling through actin-mediated activation of Src kinases, which are believed to phosphorylate the hemITAM-like sequence. However, RICH2 can bind non-signaling Tetherin variants such as the dual tyrosine mutant equally well (60). The only mutant identified to have very poor binding was a mutant at

R19, which we also observed to be signaling deficient. Based on this observation we would predict that SY would bind RICH2. We have demonstrated that TAK1 can co-IP with the SY mutant as has been observed with wt Tetherin (59, 228). Additionally, both Src-family and Syk kinase specific inhibitors dampened SY-induced signaling in a dose dependent manner suggesting both kinase families are at least indirectly involved in Tetherin mediated NF-κB activation. Additional perturbation of both pathways using siRNA against Syk and inhibitors against TAK1 should be done to confirm our observations and strengthen the model that does not involve a hemITAM.

Additional mutations in the STS region of the cytoplasmic tail of Tetherin suggests

Tetherin phosphorylation could be important for stabilizing a conformational change within

Tetherin rather than directly recruiting Syk. We believe this may be the underlying mechanism regulating Tetherin mediated signaling. Acidic mutations in the l-Tetherin background abrogate or significantly impair signaling, with the exception of the tyrosine proximal serine S5, which is reduced but not to basal levels. This may explain why A2E also decreased signaling. The STS region is thought to be under selective pressure in primates. Interestingly swapping STS with the rhesus sequence PIL abrogates signaling. An S5L substitution, which makes the STS region more like chimp or gorilla Tetherin retains signaling function, and may actually increase activity.

104 These two observations, including an apparent S5L enhancement in signaling, were previously made in the wt background (157). To our surprise, introducing S5L into the dual-tyrosine motif mutant restores signaling. Leucine hydrophobicity may be playing a role. Additionally, a series of polyalanine mutations can be engineered across the entire l-Tetherin specific region of the tail, still allowing l-Tetherin to retain transient signaling function. Taken together, neutrally charged, hydrophobic mutations upstream of the dual-tyrosine motif, but not charged or structure altering in the case of proline from PIL allow Tetherin to signal, suggesting an important and uncharacterized regulatory function for this STS region.

Phosphorylation of the tyrosine residues in the wt Tetherin cytoplasmic tail could lead to a conformational change(s) rather than direct recruitment of Syk kinase (Figure 5-2). The various tyrosine independent, signaling-competent mutants described in Chapter 3 may be primed in a conformation that facilitates NF-κB activation, mimicking what is observed during wt Tetherin signaling. We observe that SY Tetherin constantly induces higher levels of NF-κB activation compared to the l-Tetherin parent. We speculate that a primed Tetherin could access signaling machinery faster without the need of an activation event, like phosphorylation for example.

However, we do know that SY signaling can be regulated and does not have a dominant phenotype, at least in the presence of s-Tetherin, as seen in the titration experiments and with expression of SY in the wt background. The main difference between l- and s-Tetherin is the length of the cytoplasmic tail. Interaction between the juxtaposed tails of Tetherin isoform heterodimers (or tetramers) expressed on the cell surface could be important to signaling. One possible conformational change is enhanced dimerization of the tails, due to a more coiled and hydrophobic amino identity as a result of the substitutions. In the hemITAM model, Syk kinase could be doing the same thing if we are to believe it is functioning as Syk has been suggested to do with CLEC-2 (87). The SH2 domain of Syk or some other SH2 domain containing kinase could bridge phosphotryrosine residues from adjacent tails bringing the two together. Though our drug data suggests Syk is still being recruited by some indirect means, this does not rule out the

105 possibility that another unknown kinase like PI3 kinase, which has been proposed to interact with hemITAM motifs and regulate downstream Syk function, could be involved upstream (135).

Enhanced membrane recruitment of the Tetherin cytoplasmic tail could be a possible mechanism since hydrophobicity has been shown to be important. A recent report described naturally occurring equine Tetherin proteins that activate NF-κB in a tyrosine-independent manner (259). Interestingly, these Tetherins have very short cytoplasmic tails, comparable in length to s-Tetherin. How do these Tetherins activate signaling in the absence of signaling motif?

A simple explanation could be that there is an unidentified motif present. Alternatively, signaling observed by equine Tetherin is occurring through an alternative mechanism compared to human

Tetherin. Horse and donkey Tetherin both have a glycine present at position 2 which may suggest the possibility of these equine Tetherin could be lipidated. Addition of a lipid modification, like myristoylation, onto these short Tetherin species could enhance membrane targeting. The myristoylation signal sequence is not optimal compared to the consensus, though there are examples of myrisoylated proteins that have non-consensus signal sequences.

Tetherin has a membrane proximal basic residue, which could stabilize membrane interactions with the negatively charged heads on plasma membrane lipids localized to the inner leaflet (Figure 5-2B, C). R19 mutants abrogate signaling, and additional single alanine scanning in that region suggests the lysines in the KRCK membrane proximal region could also play a role.

Increased hydrophobicity observed in our tyrosine-independent mutants may target the human

Tetherin cytoplasmic tail to the inner leaflet of the plasma membrane similar a lipid moiety (Figure

5-2C). Acidic residue introduced in this region could repel those interactions explaining why we did not observe activation. Secondary structure analysis using predictive software suggests turn- like structure was identified around Y6 using NetTurnP predictive software (173). Secondary structure predictor suggests coil structures at both ends of the tail (Figure 5-1). (174), The region in between is predicted to be less structured extended strand (63). The coiled nature increases with increasing alanine mutations correlating with signaling function. This leads us to predict that the Tetherin cytoplasmic tail may exist in an inactive state associated with RICH2. Upon

106 phosphorylation Tetherin may undergo a conformational change (and possibly additional modifications), which could mediate membrane targeting. Biophysical assays could provide insight in determining the dynamics of the Tetherin cytoplasmic tail during signaling as well as the extent of membrane interactions. Additionally, assessing membrane insertion of SY and polyalanine Tetherin tails into artificial membranes could suggest membrane interaction in vivo.

As stated earlier, signaling machinery could potentially bind Tetherin on some other part of the protein, including the second half of the cytoplasmic tail, the TM domain or the GPI- anchored end as possible candidates for mediating signaling. GPI-anchored proteins, which don’t access the cytoplasm, have been demonstrated to signal in various immune contexts primarily in lymphocytes (213). The role of the Tetherin GPI-anchored has been debated (25, 60, 228).

Billcliff et al. have discussed Tetherin within the context of the “picket fence” model of membrane organization (25). The “picket fence” model suggests Tetherin can organize these microdomains and upon clustering can coalesce domains, which increases the size and density of the raft domains (Figure 1-3B). A recent publication suggests the outer membrane actually exists in a uniform fluid state and that GPI anchored proteins play a role in limiting lateral diffusion through steric hindrance (199) and thus could maintain the integrity of protein composition in we describe as microdomains. Tetherin, which sits both in and out of raft domains via a GPI anchor and transmembrane domain, respectively, possess this trait as knocking down Tetherin expression has been associated with increased diffusion of proteins and disruption of the underlying actin cytoskeletal network ((25, 180). Rafts are known to be sites of signaling platforms, and Tetherin could be involved in increasing the density to where it and viruses localize when egressing. We propose that SY could potentially bring microdomains into closer proximity while also stabilizing of signaling platforms (Figure 5-2D).

Tetherin-mediated signaling has many implications, not only during viral replication, but also potentially for normal and dysregulated cell biology. Tetherin signaling in cell culture has been a useful tool to elucidate mechanism, however the consequences of Tetherin mediated activation in vivo are not clear. Tetherin can enhance immune responses in mice (122). However,

107 whether this function is dependent on Tetherin-mediated activation of NF-κB is unclear. To assess the role of Tetherin-mediated signaling in vivo, a humanized mouse model would need to be developed. Tetherin knockout mice have been generated without gross developmental effects

(124, 223). However, in cell culture, murine Tetherin does not activate NF-κB, a caveat being that the experiments were performed in human cells. The NF-κB pathway is well conserved which suggests murine Tetherin lacks this function. Identifying whether signaling function is conserved among different Tetherins in their cognate cells would be important to translate cell culture systems into animal models.

Another aspect of Tetherin signaling that is unclear is how similar are the three methods utilized to asses NF-κB activation. Parallel profiles of cytokines have not been assessed.

Transient expression has served as a good tool to streamline various mutants, while crosslinking facilitates kinetic studies. We currently assume particle tethering, crosslinking, and transient overexpression via transfection are initiating NF-κB activation via the same mechanism. Tetherin appears to sense budding particles, but in the absence of viral replication, could this property also be useful during normal cell biology. Tetherin has been suggested to be upregulated in various malignant and invasive cell types, though it is unclear what role Tetherin may play. NF-κB dysregulation has been attributed to various types of cancers and it is tempting to speculate

Tetherin signaling could be somehow be contributing to cell survival, growth and invasion.

Assessing the growth and metastatic nature of cancer cells in the presence and absence of

Tetherin could provide some insight. Taken together, there are still pertinent questions about, mechanistically, how Tetherin is signaling, as well as what are the implications of this function.

We believe much of the machinery that has been implicated plays an important role, but our findings suggest signaling may be more nuanced then previously thought.

108 FIGURES

* * * Tetherin Signal

WT ++ MASTSYDYCRVPMEDGDKRCK GOR4 Ext Coil Strand Coil NetTurnP Turn Turn

MASTSYDYCRVPIEDGDKRCK l- +++ Ext Coil Strand Coil

WTAxA MASTSADACRVPMEDGDKRCK - Coil Coil l-AxA MASTSADACRVPIEDGDKRCK Coil

WTSTS ++ MAAAAYDYCRVPMEDGDKRCK Coil Coil l-STS + MAAAAYDYCRVPIEDGDKRCK

Coil Coil

WT SY ++++ MAAAAADACRVPMEDGDKRCK l-SY MAAAAADACRVPIEDGDKRCK Coil

l-T4AAxA ++++ MASASADACRVPIEDGDKRCK Coil

? ExE MASASADACRVPMEDGDKRCK Prediction only Coil

Figure 5-1. Compilation of secondary structure analysis of Tetherin cytoplasmic tail. Alignment of Tetherin sequences for representative mutants from Chapter 4 mutational analysis. Predictive secondary structure for cytoplasmic tail denoted under sequence. Length of predictive structure highlighted by black bar. GOR4 Secondary Structure Prediction Method was used to identify coiled regions. NetTurnP was used to predict beta-turns. Activation level by each Tetherin qualitatively represented by number of plus symbols: WT signaling = ++. - = signal-deficient. ExE is a phospho-mimetic which has not been tested for signaling (Only predicted structure assessed)

109 C

A B !!!" !!!" !!!"

K C K !!!" R C !!!" !!!" !!!" !!!" !!!" K R D K G D + D G E D M E P M V P R V C R Y C D Y Y D S Y T S S T A S D M A M

!!!" !!!" !!!"

Figure 5-2. Our proposed model for Tetherin-mediated signaling. Tetherin exists as a dimer on the cell surface. (A) The cytoplasmic tails are in a “closed” conformation, preventing signaling machinery from engaging. (B) The open conformation may bind signaling machinery directly, downstream of the dual-tyrosine motif. Tyrosine phosphorylation is likely involved in generating this active state. This interaction may be sufficient to propagate signaling (C) Positive residues in the tail that are membrane proximal could facilitate interactions with the negatively charged inner leaflet of the membrane allowing close proximity with machinery that reside in lipid rafts (D) Alternatively, the Tetherin tail may insert into the membrane allowing Tetherin inhabited microdomains to come in close proximity (arrow), brining signaling machinery together indirectly to initiate signaling

110 APPENDIX- LIST OF ABBREVIATIONS

Ab –antibody APC- antigen presentation cell EBOV – ebolavirus ER – endoplasmic reticulum ESCRT – endosomal sorting complexes required for transport Env – envelope glycoprotein GP - glycoprotein GPI anchor – glycophosphatidylinositol anchor HIV-1 – human Immunodeficiency virus 1 IFN – interferon IP - immunoprecipitation IRF – interferon regulatory factor ISG – IFN stimulated gene ITAM – immunotyrosine-based activation motif JAK/STAT - Janus kinase/signal transducer and activator of transcription l-domain – late budding domain MVBs – multivesicular bodies NF-κB – nuclear factor kappa-light chain-enhancer of activated B cells ORF – open reading frame PM – plasma membrane PRR – pattern recognition receptor RLRs – RIG-I-like receptors SIV – simian immunodeficiency virus TGN – trans-Golgi network TIS – translation initiation site TLR(s) – Toll-like receptor(s) TM – transmembrane VLP(s) – virus-like particle(s) Vpu – viral protein U

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