Syncytins and Cell Fusion in the Placenta: Structural Insights Into Lipid Membrane Fusion and Placental Development

Syncytins and Cell Fusion in the Placenta: Structural Insights into Lipid Membrane Fusion and Placental Development

Shira Elion-Jourard

A thesis submitted in conformity with the requirements for the degree of Master of Science Department of Laboratory Medicine and Pathobiology University of Toronto

Syncytins and Cell Fusion in the Placenta: Structural Insights into Lipid Membrane Fusion and Placental Development

Shira Elion-Jourard

Master of Science

Department of Laboratory Medicine and Pathobiology University of Toronto

2018 Abstract

Endogenous retroviruses (ERV) are genetic elements found in eukaryotes of retroviral origin.

Despite making up a substantial portion of vertebrate genomes, very few ERV genes encode functional proteins. Interestingly, many animal species have evolutionarily converged in the adoption of ERV envelopes for facilitating necessary cell fusions during placental development.

These placental ERV envelopes are called syncytins. Structural characterization of syncytins will provide invaluable insight into placental development and evolution as well as contribute to a general understanding of envelope facilitated membrane fusion. Crystallization of and subsequent x-ray crystallographic experiments of human syncytin-2 transmembrane ectodomain in its post-fusion conformation were carried out and the structure was solved at 1.3Å.

Comparative analysis between syncytin-2 and other CX6CC type class I viral fusion proteins reveals, despite high structural conservancy, nuanced structural divergences that may reflect functional differences arising by the distinct contexts in which each protein must facilitate membrane fusion.

Acknowledgments

Joining Dr. Jeffrey Lee’s lab almost three years ago, I embarked on a journey in which I explored the world of science and research and learned more about life and myself than I ever could have imagined. I need to express my sincerest gratitude to Jeff for not only providing me the opportunity to ask scientific questions and the guidance and resources necessary to answer them but also for being an integral part of my scientific and spiritual growth.

My time in the Lee lab would not have been the same without my fellow graduate students, past and present. The senior grad students in our lab: Dr. Halil Aydin, Matthew Taylor, Dr. Jonathan Cook, and Farshad Azimi welcomed me warmly and patiently taught me so much of what I know now. I would especially like to thank Dr. Aydin whose work with syncytin-1 jump started my own project and significantly contributed to this thesis. Wen-Guang He, thank you for joining our lab! You’ve brought both dry Manitoban humour and fresh scientific insight and energy to the lab. I’d also like to thank the past and present senior scientists in our lab whose guidance has been indispensable in the development of my project and my growth in science. I’d like to thank Dr. Karen Siu and Dr. Azmiri Sultana for teaching me the practical basics of working with a crystallography dataset; Dr. Peter Kojo Quashie for his guidance in protein biochemistry, and experiment design and for his mentorship throughout my degree; and Dr. Vitor Serrão, whom, unfortunately, I’ve not had much time to get to know but has clearly revitalized the lab with fresh science, and a proclivity for partying.

I’d like to thank the many scientists outside of our lab who also directly or indirectly contributed to my education and experiences in the program, to name a few: my committee members, Dr. Mario Ostrowski, Dr. Jean-Phillipe Julien; as well as Dr. David Irwin, and Dr. Michael Ohh. I’d also like to thank Dr. Theo Moraes for chairing my defense.

I would like to thank Rama and the other staff from the LMP graduate office for all of their help with scholarship applications and other grad school administrative tasks. I want to give a shoutout to coffee break people and Louella and other admin staff who made sure I was sociable at least one hour per week. Daniel Tarade’s rap serenades and bicycle adventures with Betty Poon will be especially memorable. Thank you for your friendship.

Finally, I need to thank my family and close friends for keeping me grounded and supporting me through the inevitable ups and downs of the science rollercoaster. I would especially like to thank my roommate Jasmin Lantos for her patience with apartment cleanliness during rougher weeks and for taking me swimming that one time I really needed it.

iii

Table of Contents

Acknowledgments...... iii

Table of Contents ...... iv

List of Tables ...... vi

List of Figures ...... vii

List of Abbreviations ...... viii

Chapter 1 ...... 1

Introduction to endogenous retroviruses, syncytins and viral fusion proteins ...... 1

1.1 Endogenous Retroviruses...... 1

1.1.1 Receptor Interference ...... 4

1.1.2 Immunosuppression ...... 4

1.1.3 Somatic Cell Fusion ...... 5

1.2 Placental Development ...... 5

1.2.1 Human and Murine Placentae ...... 9

1.2.2 Syncytins in other Species ...... 10

1.3 Membrane Fusion ...... 15

1.3.1 Fusion Catalysts: The Viral Envelope ...... 17

1.3.2 CX6CC Subtype of Class I Fusion Proteins ...... 20

1.4 Summary and Rationale for Study ...... 21

Chapter 2 ...... 23

Materials and Methods ...... 23

2.1.1 Expression and Purification ...... 23

2.1.2 Crystallization ...... 24

2.1.3 Data Collection and Processing ...... 25

2.1.4 Structure Determination ...... 26

2.1.5 Structure Analysis ...... 26 iv

2.1.6 Phylogenetic Analysis and Clustering ...... 27

Chapter 3 Results and Discussion ...... 28

Results and Discussion ...... 28

3.1 Expression, Purification, and Crystallization...... 28

3.2 Structure Determination and Validation ...... 31

3.3 Overall Structure ...... 35

3.4 Quaternary interfaces ...... 35

3.5 Heptad Stutter and Chloride Coordination ...... 36

3.6 Structural Comparison of Syncytin-1 and Syncytin-2 TM ...... 39

3.7 Structural Comparison of Syncytin-2 with other CX6CC TMs ...... 45

Chapter 4 Future Directions ...... 50

Future Directions ...... 50

4.1 Short-term goals ...... 50

4.2 Conclusions and longer term goals ...... 51

References ...... 53

List of Tables

Table 1 Optimization conditions for crystallization……………………………………………... 25

Table 2 Data collection and refinement statistics……………………………………………...... 34

List of Figures

Figure 1: Entry and proliferation of endogenous retroviruses………………………………...... 3

Figure 2: Placenta morphology………………………………………………………………….. 8

Figure 3: Phylogenetic distribution of syncytins in viviparous animals and each species’ placental characteristics…………………………………………………………………………. 14

Figure 4: Proposed mechanism of membrane fusion…………………………………………… 16

Figure 5 Proposed mechanisms of class I and class II facilitated membrane fusion exemplified by the well characterized proteins Influenza hemagglutinin protein and Dengue E protein as model specimens………………………………………………………………...... 19

Figure 6: The CX CC proteins have a highly conserved layout of features in the TM subunit… 22 6

Figure 7: Purification and histidine tag removal of syncytin-2 TM…………………………………...... 30

Figure 8: Crystallization and diffraction of syncytin-2 TM……………………………………………… 32

Figure 9: Validation of the syncytin-2 TM model using Coot geometry analysis, rotamer outlier analysis, B-factor analysis, and Ramachandran plots……………………………………………………. 33

Figure 10: Structure of syncytin-2 TM………………………………………………………………...... 37

Figure 11 The HR2 interface with the outer groove of the HR1 coiled coil……………………………... 37

Figure 12 TWISTER was used to measure supercoil pitch for trimeric GCN4, fibritin E, syncytin-2 and syncytin-1………………………………………………………………………………………...... 38

Figure 13 Syncytin-1 and syncytin-2 structural homology………………………………………………. 40

Figure 14 Syncytin-1 and syncytin-2 six-helix-bundles are stabilized by salt bridges………………...... 41

Figure 15 The immunosuppressive domain found in the chain reversal region of retroviral and filoviral fusion proteins is a 17 amino acid motif………………………………………...... 43

Figure 16 Syncytin-2 was shown to have immunosuppressive properties and contains a region of high sequence identity to a retroviral immunosuppressive motif……………………………………...... 45

Figure 17 Syncytin-2 alignment with CX6CC proteins…………………………………………………... 48

Figure 18 Phylogenetic clustering of CX6CC fusion proteins…………………………………... 49

vii

List of Abbreviations

ALV Avian leukosis virus AP Alkaline phosphatase ASLV Avian sarcoma leukosis virus BLV Bovine leukemia virus BNC Binucleate cells BSA Bovine serum albumin CASV California Academy of Sciences virus DNA Deoxyribonucleic acid CHO Chinese hamster ovary CV Column volumes ENTV Enzootic nasal tumour virus ERV Endogenous retrovirus FeLV Feline leukemia virus HERV Human endogenous retrovirus HIV Human immunodeficiency virus HR1 Heptad repeat 1 HR2 Heptad repeat 2 HTLV Human T-lymphotropic virus IPTG Isopropyl β-D-1-thiogalactopyranoside ISD Immunosuppressive domain JSRV Jaagsiekte sheep retrovirus enJSRV Endogenous Jaagsiekte sheep retrovirus LB Luria Broth LINE Long Interspersed Element LPS Lipopolysaccharide LTR Long terminal repeat MAPK Mitogen activated protein kinase PEG-MME Polyethylene glycol monomethylether MMLV Moloney murine leukemia virus MPMV Mason pfizer monkey virus mRNA Messenger ribonucleic acid mTOR Mammalian target of rapamycin NCS Non-crystallographic symmetry Ni-NTA Nickel-nitrilotriacetic acid OD Optical density ORF Viral open reading frame SDS-PAGE Sodium dodecyl sulfate-Polyacrylamide gel electrophoresis PBMC Peripheral blood mononuclear cell PBST Phosphate buffered saline Tween 20 PEG Polyethylene glycol PI3 Phosphoinositide 3-kinase viii

PVDF polyvinylidene difluoride RMSD Root mean square deviation SU Surface subunit TLS Translation Libration Screw TM Transmembrane subunit TNC Trinucleate cell Tris-HCl Tris(hydroxymethyl)aminomethane-hydrochloride XMRV Xenotropic murine leukemia virus-related virus

Chapter 1

Introduction to endogenous retroviruses, syncytins and viral fusion proteins 1.1 Endogenous Retroviruses

Endogenous retroviruses (ERV) occupy genomes across the animal kingdom, vertebrates and invertebrates alike. Often ERV elements take up a substantial portion of an organism’s genetic material. ERV elements make up approximately 8% of the human genome and 10% of the mouse genome1,2. To put this in perspective, the coding portion of the human and mouse genomes amounts only to approximately 1%1,2. ERVs occupy a relatively large portion of animal genomes and understanding how they came to be and what impact they may have on an organism’s biology is currently an active area of research.

Endogenous retroviruses are segments of an organism’s genetic material that are homologous to exogenous retroviruses and are classified by their similarity to a retroviral genus. Class I are homologous to gammaretroviruses, class II to betaretroviruses and class III to spumaretroviruses3. Recent and active endogenization of exogenous retroviruses can be observed in sheep with Jaagsiekte sheep retrovirus (JSRV), in cats with Feline leukemia virus (FeLV) and in chickens with Avian leukosis virus (ALV)4–6. Germ cell infection with an exogenous retrovirus and subsequent integration of that virus’s genome into the germ line results in endogenization, such that the retroviral deoxyribonucleic acid (DNA) is transmitted vertically from one generation to the next. Most endogenized retroviruses have numerous copies throughout the organism’s genome and these copies are clustered as an ERV family. The proliferation and resulting tens to thousands of copies of a single ERV family may arise via repeat exogenous germ cell infection, endogenous reinfection of other germ cells, or by retrotransposition mediated by ERV encoded (in cis) or long interspersed element (LINE) encoded (in trans) reverse transcriptase (Figure 1). Reinfection of the germ line by endogenous retroviral particles necessitates a sufficiently intact ERV sequence for functional viral particle generation. Analysis of human endogenous retrovirus (HERV) sequences by Belshaw et al. suggests that reinfection is the primary mechanism by which ERVs proliferate in humans7. The

1 2 eventual mutating destruction of the ERV genetic integrity would prevent continual proliferation and allow for family stabilization in the genome. Retrotransposition in cis or in trans may also contribute to the proliferation of an ERV family. For instance, many HERV-W copies resemble retrosequences rather than pseudogenes in that they are flanked by insertion motifs typical of LINE mediated retrotransposition8,9.

Initially, an endogenous retrovirus retains its proviral structure with gag, pro, pol, and env genes flanked by a 5’ and 3’ long terminal repeat (LTR); however, harbouring infective viruses within a genome would be biologically disruptive and destructive thus silencing by deleterious mutations is highly selected for over evolutionary time3. Over time, ERV proliferation and mutation stabilizes and the majority of ancient ERVs, despite occupying a substantial portion of the genome, are non-functional remnants of their exogenous ancestor. ERV sequence analysis of the human genome reveals that of approximately 7800 HERV regions, only 42 encompass ‘long viral open reading frames (vORF); long vORF are categorised as env or gag genes containing more than 500 codons or pol containing more than 700 codons10. Of these 42 semi-intact viral coding regions, only 18 are outside of the HERV-K family; HERV-K is the youngest HERV family and thought to have been actively infectious as recently as 200,000 years ago11. Interestingly, of the 18 truly ancient HERV genes, 15 are env encoding10. This evolutionary bias for env preservation is likely an indication of the adoption of Env for endogenous purposes. Three potential endogenous purposes a viral envelope may serve are: protection from future infection via receptor interference, contributions to immune modulation via an immunosuppressive domain conserved in retroviral envelope proteins, and adoption of the envelope’s fusogenic capabilities to facilitate cell-cell fusion.

germ cell mRNA exogenous retrovirus genomic DNA

transcription Infection and integration genomic DNA

endogenous retroviruses

exogenous retrovirus

Figure 1 Entry and proliferation of endogenous retroviruses: Endogenization of a retrovirus proceeds first by initial infection of a germ cell by an exogenous retrovirus and subsequent integration of the retroviral genome. Proliferation of the endogenous retrovirus may proceed by i) retrotransposition mediated by ERV or LINE encoded reverse transcriptase; ii) reinfection by germ cell produced ERV particles; iii) multiple exogenous retroviral infections and integrations may also contribute to ERV proliferation. Retroviral genes are represented by red segments in the cartoon, blue represents genomic DNA, infection events are represented by red arrows, and viral particle production is represented by a green arrow.

1.1.1 Receptor Interference

Receptor interference by endogenous retroviral envelope proteins can proceed by competitively inhibiting receptor binding by the exogenous envelope thereby preventing entry. Resistance to exogenous viral infection relayed by ERV receptor interference has been observed in chickens expressing endogenous ALV envelope and sheep expressing endogenous JSRV envelope12,13. Chickens are polymorphic for three endogenous defective ALV proviruses that express the envelope protein, ev3, ev6, and ev9. Expression of these endogenous envelope proteins reduced penetrance of ALV into chicken embryo cells and the amount of endogenous envelope protein present in cells was shown to correlate with resistance to exogenous infection12. The endogenous JSRV envelope shares its receptor with the actively infectious JSRV and Enzootic nasal tumour virus (ENTV): viruses that infect the respiratory tract of sheep and cause neoplasms. Endogenous JSRV is expressed in high amounts in the genital tract and the uterine epithelium in ewes and was shown to confer resistance to JSRV and ENTV infection by inhibition of entry13. ERV receptor interference may have directed adaptation of JSRV and ENTV away from these ERV expressing resistant tissues to the respiratory tract. In both sheep and chicken, relatively young ERVs confer resistance to infection by their exogenous counterparts. Ancient ERV envelopes may continue to play a role in resistance to infection by modern viruses that share the same receptor or have played a role in directing viral evolution away from ERV protected host tissues. For instance, HERV-W env as well as feline endogenous retrovirus RD-114, and baboon endogenous retrovirus shares their receptor, ASCT-2, with a number of gammaretroviruses (reticuloendotheliosis virus A, spleen necrosis virus) and betaretroviruses (simian retroviruses serotypes 1-5). Indeed, HERV-W env has been shown to prevent entry of spleen necrosis virus in vitro14. The evolutionary advantage of resistance to exogenous infection would result in a strong selective pressure to retain an envelope protein capable of interfering with exogenous receptor binding.

1.1.2 Immunosuppression

The immunosuppressive domain conserved amongst retroviral envelopes suppresses cell- mediated immunity by enhancing release of IL-1015. The contribution ERV env proteins make to maternal immune suppression during embryo development has been proposed many times as the human placenta is a hypomethylated tissue, making it more permissive to HERV transcription and expression than other somatic tissues, and placental cytotrophoblasts continually secrete IL-

10 throughout gestation16–20. Mangeney et al. demonstrate the immunosuppressive properties of three placenta specific HERV env proteins: HERV-FRD encoding syncytin-2, ERV-3, and HERV-V env using tumour rejection assays; the immunosuppressive properties of HERV-W encoding syncytin-1 are contested and require further investigation (the details of syncytin immunosuppressive properties are covered in more detail in the discussion section of this thesis)21. The immunosuppressive properties of ERV envelopes are also implicated in disease, particularly in the role they may play in aiding tumour immune evasion. For instance, HERV-K env, and HERV-H env are actively immunosuppressive and expressed in various cancer tissues22–25.

1.1.3 Somatic Cell Fusion

Fully functional ERV envelopes are fusogenic and can mediate cell-cell fusion if the target cell expresses the ERV envelope’s receptor. Two such proteins essential for human placental development were mentioned in passing in the previous section: syncytin-1 and syncytin-2. Indeed, equivalent proteins are found across placentalia, in marsupials and even in a lizard species. These fusogenic ERV envelopes specifically expressed in the placental tissue are the result of entirely independent retroviral integration events and an example of convergence in the evolution of the placenta. Interestingly, despite convergent capture of syncytins for placental development and the similarities in overall function, placental morphology is highly divergent. Syncytins and placental morphology are covered in more detail in the following section. Syncytin-1 mediated fusion has also been implicated in muscle and bone development as well as in cancer progression in humans26–32.

1.2 Placental Development

The placenta is a transient organ made up of fetal trophoblast cells and exists only during gestation. The placenta is the necessary link between fetus and mother, allowing for nutrient, waste and gas exchange to take place. The placenta is the feto-maternal interface, thus it also plays an important role in protecting the fetus from the maternal immune system. Intriguingly, despite carrying out a very similar function in all placental mammals, the placenta is morphologically very divergent. Morphological divergence has been hypothesized to be the

6 evolutionary result of the feto-maternal conflict or a give-and-take scenario between the nutrient demanding fetus and tissue invaded mother33.

Three broad classes of placentas describe their interface type: haemochorial, endotheliochorial and epitheliochorial34. These classes can be further subdivided by overall shape (discoid, zonary, cotylendonary) and the type of feto-maternal interdigitation (villous, labyrinthine, lamellar, folded)34 (Figure 2). The haemochorial placenta is the most invasive; fetal trophoblast cells interface directly with maternal blood, invading the uterine myometrium and disrupting maternal vasculature. The endotheliochorial placenta is of intermediate invasiveness; fetal trophoblast cells interface with maternal vasculature, disrupting only the epithelial tissue of the myometrium. The epitheliochorial placenta is the least invasive; fetal trophoblast cells interface merely with the epithelia of the uterus, without disrupting maternal tissue. The synepitheliochorial placenta results from fusion of trophoblast cells with neighbouring uterine epithelial cells. The ancestral placenta was likely of the haemochorial type35 and evolutionary divergence away from the haemochorial interface may be the maternal self-protective response to fetal demand.

Many placentas across the three classes have syncytial structures (multinucleated protoplasms formed by fused cells) or at least necessitate singular fusion events (i.e. that which occurs in synepitheliochorial placentas between fetal trophoblasts and maternal epithelial cells). As discussed briefly in the previous section, in humans, HERV envelope proteins called syncytin-1 and syncytin-2 are necessary for the formation of the uterine invading trophoblast layer: the syncytiotrophoblast36–39. Over the last 10 years, Thierry Heidmann’s group has identified syncytin-like proteins in species across mammalian clades with highly divergent placental morphologies as well as in a reptilian viviparous genus. By the Heidmann group’s analyses, syncytin-like proteins are ERV envelope proteins that are specifically expressed in the placenta, are functional as fusogens and show purifying selection. Each of these syncytins are the descendants of entirely independent exogenous infection and integration events, thus the purifying selection and adoption of ERV envelopes for placental development is an evolutionary convergent characteristic of the placenta; an interestingly ironic observation given the general divergent nature of placental evolution.

In identifying independently captured ERV envelopes, Heidmann’s group postulates that it is the continual capture of syncytins that has allowed for the independent evolution of the placenta in

7 mammalia and reptillia and a divergent evolutionary response to the feto-maternal conflict40. In the following section, I will outline each of the currently identified syncytins and very briefly discuss the histological context in which they function. See Figure 3 for a summary of species expressing syncytin proteins and the type of placenta in use.

discoid

cotyledonary

zonary

Figure 2 Placenta morphology: Placenta morphology can be categorized by overall shape (A) and by the type of feto-maternal interface (B).

A) Reproduced and edited with permission of the Japanese Society of Toxicologic Pathology from Furukawa, et al. A Comparison of the Histological Structure of the Placenta in Experimental Animals. J Toxicol Pathol 27: 11-18, 2014. B) Reproduced and edited with permission of PNAS from Cornelis, et al. Captured retroviral envelope syncytin gene associated with the unique placental structure of higher ruminants. PNAS. 110(9): E828-E837, 2013

1.2.1 Human and Murine Placentae

The human placenta is haemochorial, of discoid shape and villous interdigitation. Invasion and disruption of maternal vasculature at the decidua basalis allows for placental villi to become bathed in maternal blood41. The core of these villi are composed of mesenchymal cells and fetal vasculature while the outer layers are trophoblastic41. In humans, the inner trophoblast layer are cytotrophoblasts (the undifferentiated stem cell) and the outermost layer of the placental villi is the syncytiotrophoblast, which is composed of fused fully differentiated cytotrophoblasts and is in direct contact with maternal blood41. Although both syncytin-1 and syncytin-2 are expressed in the placental trophoblasts, they appear to be localized differently within the tissue. Consensus on syncytin-1 localization has not been reached. Some groups observe expression restricted to the syncytiotrophoblast (apical only42, basal only43 or unspecified distribution37), and some observe expression in both cytotrophoblasts and the syncytiotrophoblast44,45. ASCT2, the receptor for syncytin-1, is located in the apical cytotrophoblast cell layer bordering the syncytiotrophoblast46. The localization of syncytin-2 expression is fairly undisputed and appears to be restricted to the villous cytotrophoblasts47–49. The expression of the syncytin-2 receptor, MFSD-2, is restricted to the syncytiotrophoblasts; thus, the direction of syncytin-2 mediated infusion of cytotrophoblasts seems to be unidirectional47.

The murine placenta is similar to the human placenta in that it is haemochorial and discoid34,41. The feto-maternal interface is not villous as is in humans, but labyrinthine, and the trophoblast organization at the interface is quite different from than in humans. The labyrinthine interdigitation is essentially a highly complex villous type of digitation in which villous branching is more extensive and interconnected resulting in a labyrinth of fetal villi and intervillous spaces filled with maternal blood, called the maternal lacunae. The trophoblasts in the labyrinthine are organized as three layers, such that the murine placenta is referred to as haemotrichorial. Layer I is the outermost layer and interfaces with maternal blood. This outer layer, unlike human villi, is composed of cytotrophoblasts. Layer II, found underneath layer I, is the first syncytiotrophoblast layer. Importantly, this layer also interfaces with maternal blood as fenestrations traverse the outer layer to contact the maternal blood spaces50. The inner most layer, layer III is also syncytial and interfaces with the mesenchymal and vascular core of the labyrinthine structure. Syncytins in mice, called syncytin-A and syncytin-B, are also restricted in their localization. Syncytin-A is expressed primarily in the layer II syncytium, facilitating the

10 necessary fusion for its formation51. Syncytin-A mice knockouts are embryonic lethal, demonstrating the necessity of syncytin-A for embryogenesis52. Syncytin-B is expressed primarily in the layer III syncytium51 and mice knockouts are viable despite malformation of layer III resulting in unfused cells and enlargement of maternal lacunae. Although viable, these mice experience late-onset growth retardation53.

1.2.2 Syncytins in other Species

As with the murine and the human placenta, syncytins are found in the morphologically diverse placentas of caviomorphs, lagomorphs, marmots, higher ruminants, carnivores, afrotherians, marsupials and Mabuya, a lizard species.

The Caviomorpha (guinea pig) placenta is haemochorial, discoid and labyrinthine as is the murine placenta, but the syncytium of the labyrinthine section is continuous with the uterine invasive junctional zone through which long syncytial columns called syncytial streamers invade the uterine lining and disrupt maternal vasculature54. The caviomorph syncytin-Cav1, identified by Vernochet et al., is localized only to the syncytial streamers and is not found in the labyrinthine zone, unlike the murine syncytins55. The Lagomorpha and Marmotini placenta too are similar to the murine placenta in that they are haemochorial, discoid, and labyrinthine, but consists of only two layers: an inner cytotrophoblast layer and an outer syncytiotrophoblast layer56–58. The lagomorph syncytin-Ory1, identified by Heidmann et al., is found at the interface between the cytotrophoblast and syncytiotrophoblast layer (whether syncytin-Ory1 is restricted to either cytotrophoblasts or the syncytiotrophoblasts was not determined)57. Redelsperger et al. also did not distinguish between cytotrophoblast and syncytiotrophoblast expression of the marmotini syncytin-Mar1, but did localize it to the syncytial front, that is the actively invading and fusing end of the placental labyrinthine58.

The Carnivora placenta is endotheliochorial, zonary and lamellar34. Cytotrophoblast cells migrate to the tips of uterine invading villi to differentiate and fuse, forming the syncytial front. The syncytial front invades the maternal uterine lining by destroying the maternal epithelium but leaving the maternal endothelial cells intact. The Carnivora villi surround the maternal vasculature but are not directly bathed in maternal blood as in the haemochorial type placenta. Syncytin-Car1 expression was localized to the syncytiotrophoblast layer of both dog and cat placentas at the invading syncytial front59.

The Afrotherian tenrec placenta is haemochorial, discoid and labyrinthine but is structurally unique as there are two concentric regions: the peripheral placenta pad where the placenta takes on a labyrinthine character bathed in maternal blood and the central hemophagous region, villous in character and likely important for iron uptake60. The labyrinthine of the peripheral placental pad uniquely does not interface the maternal blood spaces via syncytium but rather with a layer of mononucleated cytotrophoblasts60,61. However, syncytiotrophoblasts are found at the tips of placental villi invading maternal uterine epithelium at the periphery of the central hemophagous region. The Afrotherian syncytin, syncytin-Ten1, is found to have expression restricted only to the syncytiotrophoblasts at the central region villi61.

The placenta of higher ruminants is synepitheliochorial, cotyledonary, and villous. A cotyledonary placenta has specific zones for feto-maternal contact called placentomes. The ruminant placental villi have binucleate cells (BNC) at the invasive front that are formed by acytokinetic mitosis. At placentomes, BNCs fuse with a maternal epithelial cell to form a feto- maternal hybrid trinucleate cell (TNC)62. While in cows, TNCs are transient and degrade rapidly, in sheep, TNCs continue to fuse and form syncytial plaques with 5-25 nuclei that replace regions of the uterine epithelium63,64. Both cows and sheep specifically express a fusogenic ERV env named syncytin-Rum1 in BNCs at placentome villi tips65.

In sheep, syncytin-Rum1 expression is restricted to the BNCs and is not found in the syncytial plaques65. However, the recently endogenized sheep ERV, enJSRV, expresses a functional envelope protein localized to BNCs and syncytial plaques. Inhibition of enJSRV env expression in the placenta impaired trophoblast differentiation and development of the sheep placenta66. The capture of enJSRV may have permitted the ovine development of syncytial plaques, diverging from bovine placentation. Syncytin-Rum1 has not been shown to be absolutely necessary for ovine placentation and the syncytin-Rum1 - enJSRV dichotomy may exemplify a syncytin transition in ovine placentation.

The marsupial opossum placenta is very short lived, lasting only four days. The maternal epithelium is invaded at regular intervals by the fetal syncytiotrophoblast, which envelopes maternal vasculature, making the opossum placenta of mixed epithelio-endothelio-chorial nature67. Syncytin-Opo1, identified by Cornelis et al., is specifically expressed within the syncytiotrophoblast layer of the feto-maternal interface and is likely involved in

12 syncytiotrophoblast formation and opossum placental development68. Another ERV env protein although not specifically expressed in the placenta and is non-fusogenic due to a nonsense mutation immediately preceding the TM domain anchor, is found in the genomes of the opossum as well as two closely related marsupial species, the tammar wallaby and the Tasmanian devil68. Cornelis et al. recognize this non-functional ERV env may be a marsupial syncytin predecessor to the opossum specific Opo1, another potential example of syncytin transition during placental evolution68.

The Mabuya reptilian placenta is, unsurprisingly, morphologically distinct from mammalian placentas. The reptilian placenta is an independently evolved structure as the mammalian and reptilian placentas developed after splitting from their common ancestor. Fetal and maternal epithelial cells fold into each other and interdigitate with microvilli. Fetal cells form giant columnar binucleate cells while the maternal epithelial cells fuse to form a maternal syncytium. These folds are highly vascularized and the microvillous folded interface is sometimes interrupted by small invasive trophoblasts40,69. The syncytin-like protein identified by Cornelis et al., syncytin-Mab1, has widespread expression in many Mabuya tissues, but shows especially high level expression at the feto-maternal interface40. Syncytin-Mab1 expression is highest in the maternal epithelial syncytium40.

A diverse set of independently captured syncytins may play an important role in placental development in mammalian and reptilian species (experimentally, syncytins in humans and mice are shown to be necessary for placental development). Given that mammalian placental evolution precedes the capture of any currently identified syncytins, and hints of placental divergence coincides with new syncytin capture (i.e. sheep divergence from cow placentation and opossum divergence from other marsupial placentation), we may postulate as Heidmann and others have before, a baton-pass progression in syncytin driven placental evolution. That is, as species specific feto-maternal conflict demands placental divergence, new capture of ERV envelopes may enable variant tissue development, and the syncytin-baton may be passed on. The histological and therefore biophysical and biochemical context in which each syncytin facilitates cell-cell fusion is unique. Syncytins are all highly homologous and all classify as CX6-7CC bearing class I viral fusion proteins (except for syncytin-Mab1 that is a CX7C class I fusion protein). The conditions in which they carry out fusion are unique and despite appearing to have high structural similarity based on the presence of motifs in their amino acid sequence, they may

13 have nuanced structural characteristics that lend to enhanced function within the unique conditions for which they have been evolutionarily conserved.

Syncytin Name Interface type Shape Interdigitation Syncytin-Ten1 haemochorial discoid labyrinthine

Syncytin-Ory1 haemochorial discoid labyrinthine Syn- A, B, Mar1, Cav1 haemochorial discoid labyrinthine Syncytin- 1, 2 haemochorial discoid villous

Syncytin-Rum1 synepithithelio- cotyledonary villous chorial Syncytin-Car1 endotheliochorial zonary lamellar

Syncytin-Opo1 epithelio/ discoid folded edothelio-chorial

Syncytin-Mab1 epitheliochorial- discoid-like folded like

Figure 3 Phylogenetic distribution of syncytins in viviparous animals (red characters) and each species’ placental characteristics: Syncytin capture temporal estimate is represented by purple arrows on the tree. The large red arrow indicates the probable time of mammalian placenta emergence. The Mabuya placenta likely evolved independently.

Reproduced and edited with permission of PNAS from Cornelis, et al. An endogenous retroviral envelope syncytin and its cognate receptor identified in the viviparous placental Mabuya lizard. PNAS. 114(51): E10991-E11000, 2017 14

1.3 Membrane Fusion

Lipid bilayer fusion is a thermodynamically favourable process but with a large kinetic energy barrier due to the hydration repulsion forces present between lipid bilayer membranes less than 30Å apart70,71. Lipid bilayer membrane fusion likely proceeds via 3 main stages, each involve overcoming an estimated ̴ 30kBT energy barrier in vesicle membrane fusion: i) initial membrane deformation and local increase in membrane curvature allows for target membrane approach and mixing of the membranes’ outer leaflets (i.e. hemifusion stalk formation); ii) hemifusion diaphragm expansion; iii) and fusion pore formation and expansion wherein the inner membrane leaflets of apposing membranes mix and membrane fusion completes (Figure 4)72. Although experiments measuring the activation energies of fusing vesicles corroborate the ̴ 30kBT simulation estimate, a multitude of factors contribute to the activating energy barriers and procession of fusion including: membrane curvature, lipid composition of the membrane, local lateral membrane tension, and pore line tension during pore formation and expansion73–76. For this reason, fusion of biophysically unique membranes (i.e. vesicles, viral envelopes, cellular plasma membranes) may have unique energetic conditions or barriers.

15 16

e-f a-c d

Figure 4 Proposed mechanism of membrane fusion: Membrane fusion proceeds through three activation energy barriers: a)-c) Initial dehydration and membrane deformation to allow outer layer lipid mixing and the hemifusion stalk to form; d) expansion of the hemifusion diaphragm; e)-f) pore formation and expansion. The least energy profile shown above is based on Ryham et al.’s fusion simulation using continuum mechanics.

Top) Reproduced and edited with permissions of Annual Review of Cell and Developmental Biology from Podbilewicz. Virus and cell fusion mechanisms. Annu Rev Cell Dev Bio. 30:111-139, 2014 Bottom) Reproduced and edited with permissions of Biophysical Journal from Ryham, et al. Calculating transition energy barriers and characterizing activation states for steps of fusion. Biophys J. 110(5):1110-1124, 2016

1.3.1 Fusion Catalysts: The Viral Envelope

Importantly, the large kinetic energy barrier to membrane fusion blocks spontaneous fusion between biological membranes permitting the existence of consistent biological compartments necessary for life. Instances in which membrane fusion serves a biological purpose necessitate a protein catalyst. One such instance occurs during enveloped viral infection. Every viral infection begins with entry and for enveloped viruses, entry necessitates fusing its own enveloping membrane with the target cell’s membrane. Enveloped viruses express an envelope protein on their surface to catalyze this energetically costly process. The envelope proteins of enveloped viruses can, for the most part, be classified by structural similarity into three classes77,78.

Class I viral fusion proteins are translated as a single precursor molecule that is post- translationally cleaved into a surface (SU) subunit and a transmembrane (TM) subunit. The TM contains the machinery necessary for fusion but is sequestered by the SU subunit. Each TM monomer has at its N’ terminus a stretch of hydrophobic residues called the fusion peptide or loop that can insert itself into the membrane of the target cell. When triggered, the SU releases the TM and exposes the fusion peptide. Once the fusion peptide has inserted into the target cell’s membrane, each TM monomer is likely in an extended conformation wherein the two heptad repeats, HR1 and HR2, are stacked and separated by a short loop region, thereby bridging the host membrane and the target membrane (Figure 5). Each TM monomer then folds at the HR1/HR2 separating loop region, also called the chain reversal region, to form a helical hairpin. The spontaneous transition from the trimeric prehairpin extended conformation to the trimeric hairpin conformation, also called the six-helix-bundle, likely drives the merger of the two membranes.

Class II viral fusion proteins are found in flaviviruses, alphaviruses, bunyaviruses, pheboviruses and rubiviruses. Class II viral fusion proteins are also translated as a single precursor molecule and through post translational modifications matures as a two subunit protein. Like class I fusion proteins this two subunit protein also contains a fusion subunit, called the E protein in flaviviruses, and a chaperone subunit, called the M protein in flaviviruses. Proteolytic cleavage of the chaperone primes the E protein for fusion and low pH triggers E protein fusion catalysis. The prefusion and postfusion conformations of the class II fusion subunit are similar between all members with available structures. The E protein is arranged as a homodimer in the prefusion

17 18 state and each protomer is composed of three domains. Domain I is a beta sandwich whose topology is well conserved amongst class II viral fusion proteins. Domain II extends away from domain I as a series of long loops or beta strands with a hydrophobic region at its prefusion membrane distal end called the fusion loop. The fusion loop is responsible for the target cell membrane insertion necessary for fusion commencement. Domain II leads into domain III, an Ig- like domain that connects to the transmembrane anchor and is oriented such that stem leading to anchor is most distant from the fusion loop. Like in class I, the post-fusion conformation of the E protein is a trimer of hairpins: domain III is folded over such that the transmembrane anchor and fusion loop are in closer proximity.

Class III viral fusion proteins are found in the viral families Rhabdoviridae, Herpesviridae and Baculoviridae. These proteins are unique in that they are not metastable in their prefusion conformation. Their pre and post-fusion conformations exist in a pH dependent equilibrium. Their postfusion conformation is reminiscent however of both class I and class II viral fusion proteins. The class III protein is a trimer of hairpins that contains at its core a coiled-coil motif like a class I viral fusion protein. Similar to the fusion loop orientation in class II, the fusion loops that insert into the apposing cell membrane are present at the distal turns of an extended beta sheet. Transition from pre to post fusion involves the rotation of the fusion loop and C terminal segment around the coiled coil domain in the protein core.

The common theme amongst all three classes is the procession of membrane fusion via the conformational transition to a trimer of hairpins. The transition may be mediated by the binding of a receptor, by a low pH environment or by a combination of both. The transition to a trimer of hairpins functions to bring the apposing membranes into close apposition and the trimer of hairpins stabilizes the formation and expansion of the fusion pore (Figure 5).

Figure 5 Proposed mechanisms of class I (A) and class II (B) facilitated membrane fusion exemplified by the well characterized proteins Influenza hemagglutinin protein (A) and Dengue E protein as model specimens (B): In both cases, once triggered, the fusion protein transitions into an extended state making contact with the cell membrane, folds back on itself to bring the two membranes into close apposition and then finally forms a trimeric hairpin, stabilizing the fusion pore formation and expansion. In B) after being triggered, E protein proceeds with fusion in a trimeric state; note that the third protomer is not visible in the above diagram.

Reproduced and edited with permissions of Annual Review of Cell and Developmental Biology from Podbilewicz. Virus and cell fusion mechanisms. Annu Rev Cell Dev Bio. 30:111-139, 2014

1.3.2 CX6CC Subtype of Class I Fusion Proteins

Within class I, a subgroup of proteins expressed by filoviruses, retroviruses and two recently identified arenaviruses share certain structural motifs, primarily, a CX6CC motif in the chain 79–84 reversal region, dubbing this group the CX6CC proteins . The first two cysteines of the motif form a chain reversal region stabilizing disulfide bond, while the third links to the SU in its prefusion state and is released following a disulfide isomerization reaction upon fusion activation. Amongst retroviruses, only the lentivirus family expresses a CX7C fusion protein instead; without a third cysteine, these fusion proteins non-covalently bind the SU85,86.

The crystal structures of 8 retroviral, 2 filoviral, and 1 arenaviral CX6CC envelopes are available. In these structures, multiple conserved motifs are present: a heptad stutter near the N’ and C’ termini can guide alignment of a central coiled coil core87, a chloride ion in the center of the coiled coil is coordinated by asparagines, and an immunosuppressive domain (ISD) occupying the C’ terminus of the HR1 and the most membrane distal portion of the chain reversal region (Figure 6). However, despite the high conservation of vital structural motifs, these fusion proteins appear to diverge in the types of stabilization factors present. For instance, the helical hairpins and chain reversal regions of β-, δ-, γ- retroviruses are lined with salt bridges and mutations to residues participating in these salt bridges reduces six-helix-bundle stability and revokes the six-helix-bundle’s capacity to drive fusion79,88. In contrast to this, electrostatic interactions appear to be inconsequential to the stabilization of the Avian Sarcoma Leukosis Virus (ASLV) six-helix-bundle and hydrophobics take the main stage in stabilization80. ASLV mediates fusion in the endosome in a low pH environment; whereas, the electrostatically stabilized β-, δ-, γ- retroviruses mediate fusion at neutral pH, at the plasma membrane. The ASLV six helix bundle has evolved to rely on hydrophobic stabilization that would be resistant to changes in pH. The biochemical environment in which the fusion protein must facilitate the merging of membranes likely drove divergence in the evolution of nuanced fusion protein characteristics.

The ISD is a 17-residue motif encompassing the C terminus of the HR1 and the most distal portion of the chain reversal region. While the details of the underlying mechanism of immunosuppression remain unknown, the ISD appears to activate Ras-MAPK and PI3-mTOR pathways in immune cells resulting in changes in those cells’ cytokine expression profiles89–91.

20 21

An ISD consensus sequence peptide called CKS-17 was shown to decrease expression of the immune stimulating cytokine IL-12 and increase expression of the anti-inflammatory cytokine IL-10 in immune challenged (Peripheral blood mononuclear cells ) PBMCs15. Mutational analysis of the Human Immunodeficiency Virus (HIV) ISD identified necessary residues for immunosuppressive activity92. Alanine substitution to residues in positions 1-4, 9 and 14 of the HIV ISD resulted in abrogation of immunosuppressive function92. In addition, the aforementioned experiments conducted by Mangeney et al. also demonstrated the importance of position 14 in human syncytins, mouse syncytins and MPMV for immunosuppressive activity21. Interestingly, position 14 is highly solvent exposed being oriented at the most membrane distal end of the chain reversal region in CX6CC TMs in their postfusion conformation.

1.4 Summary and Rationale for Study

Being of retroviral origin, all thus far identified syncytin proteins are of the CX6CC class I subtype, with the exception of syncytin-Mab1 and potentially the enJSRV envelope that are the

CX7C subtype (syncytin-Cav1 contains a slightly extended CX7CC motif). There may be several factors driving adoption and evolutionary preservation of syncytin proteins in placental tissue. Receptor interference, for instance, may play a pathogen protective role in the vital reproductive tissue. Perhaps, the protective potential receptor interference offers may have driven initial adoption in reproductive tissues of pre-placenta mammalian ancestors and the immunosuppressive and fusogenic contributions of syncytins may have enabled the development of placental viviparity.

Baton pass adoption of syncytins by trophoblasts or uterine epithelium (as is the case for the Mabuya syncytin) is theorized to enable the divergence evident in placental evolution. Much of the focus for understanding mechanisms of fusion and the evolution of fusion catalysts has been on SNARE mediated intracellular fusion and viral envelope mediated virus-cell fusion. ERV envelope mediated cell-cell fusion offers a unique lens through which fusion can be studied. Heidmann’s group, in identifying species-unique syncytins and localizing their messenger ribonucleic acid (mRNA) expression in host tissue has built a framework on which structural characterization can potentially offer unique insights into the evolution of placental development, and the mechanisms of cell-cell fusion. Human syncytin-1 and -2 are currently the most well characterized syncytins. The syncytin-1 crystal structure was resolved by a previous student in

22 our lab (PDB: 5HA6) and unpublished results from our lab demonstrate the importance of a complex network of salt bridges in stabilizing the syncytin-1 final six-helix-bundle conformation and fusogenic capacity. Syncytin-2 has a more restrictive expression pattern in placental tissue and structural elucidation and further structure-based functional characterization may offer new insights into the nuances of membrane fusion conditions and corresponding mechanisms. A partial syncytin-2 structure is available but is missing some of the HR1 N-terminus and 93 terminates after the CX6CC motif such that it is missing the entire leash region and HR2 (Figure 6). A more complete structure would be informative of hairpin stabilization factors.

We determined the crystal structure of the extracellular region of the postfusion syncytin-2 TM subunit (excluding the MPER and fusion peptide) to 1.3Å resolution. Our structure revealed the presence of the class I conserved heptad stutter near the N-terminus and the stabilization character of the syncytin-2 hairpin. Interestingly, syncytin-2 has only 2 potential salt bridges, a conserved salt bridge in the chain reversal region and one potential salt bridge at the membrane proximal end of the helical hairpin. The HR2 interface with adjacent HR1’s is primarily hydrophobic or polar in nature, thus the stabilization character of the syncytin-2 six-helix-bundle sharply contrasts that of syncytin-1 despite their similar roles in placenta development. Further functional characterization is necessary to confirm the stabilization profile of syncytin-2, but the nuanced structural differences between syncytin-1 and syncytin-2 may reflect their nuanced roles in human placental development and differences in fusion conditions.

Figure 6 The CX CC proteins have a highly 6 conserved layout of features in the TM subunit: Cylinders represent helices, the orange peptide at the N’ terminus represents the fusion peptide, the green segment represents a heptad stutter, the pink segment represents the immunosuppressive domain, the dashed yellow line signifies the location of the hairpin stabilizing disulfide bond of the CX CC motif. The 6 region enclosed in the dashed black lines is the syncytin-2 structure already available (PDB: 1y4m).

Chapter 2

Materials and Methods 2.1.1 Expression and Purification

Human syncytin-2 ectodomain (residues 372-467) (NCBI accession: NM_207582.2) with a point mutation to the third cysteine of the CX6CC motif: C439S, was codon optimized, gene synthesized and subcloned into pET-46 Ek/LIC (henceforth termed pSyncytin-2TM). This construct includes the HR1, the chain reversal region, the HR2 and contains an N-terminal thrombin cleavage site directly after the vector encoded hexahistidine tag. This TM construct does not include hydrophobic regions: the fusion peptide and transmembrane domain. SHuffle T7 E. coli cells were transformed with pSyncytin-2TM by heat shock, plated onto Luria Broth (LB) agar supplemented with 100 μg/mL of ampicillin and incubated overnight at 37°C. 1 L LB culture supplemented with 100 μg/mL ampicillin was inoculated with cells from a single colony in a 20 mL overnight starter culture and grown at 37°C at 180 rpm. The 1 L culture was grown to an optical density at 600nm (OD600) of 0.6 and then induced by addition of isopropyl β -D-1- thiogalactopyranoside (IPTG) to a final concentration of 1 mM. Following an 18 hour incubation at 18°C, cells were harvested by centrifugation at 5000 x g for 20 minutes at 4°C. The cell pellet was stored at -20°C until needed. Dr. Jeffrey Lee kindly assisted with cloning and protein expression.

The frozen pellet was thawed at room temperature and resuspended in nickel-nitrilotriacetic acid (Ni-NTA) Binding Buffer (20 mM Tris(hydroxymethyl)aminomethane-hydrochloride (Tris HCl) pH 8.0, 150 mM NaCl, 20 mM imidazole). Cells were lysed using a hydraulic cell disruption system (Constant Systems) at 30 kpsi. The soluble fraction of the cell lysate was separated by centrifugation at 33,000 x g for 45 minutes at 4°C and then applied directly to a 2 mL Ni-NTA affinity column. The protein bound beads were washed with 5 column volumes (CV) of Ni-NTA Binding Buffer and then progressively eluted in 2 CV fractions of Ni-NTA Binding Buffer supplemented with 50 mM, 100 mM, 200 mM, 300 mM, 400 mM, and 500 mM imidazole. All fractions were assessed by SDS-PAGE (Sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and elutions at 100 mM and 200 mM were combined and selected for further purification.

The protein sample was then concentrated to a final volume of 1 mL and isocratically eluted from a BioRad ENrich SEC 70 10 x 300 size exclusion column equilibrated in a Tris Buffered Saline solution (20 mM Tris HCl pH 8.0 and 150 mM NaCl). Fractions containing syncytin-2 TM were combined and incubated with 24 units of restriction-grade thrombin (EMD Millipore) overnight at 4°C. Thrombin cleavage was optimized using immunoblotting. For immunoblotting, samples at various time points of digestion were collected and digestion was stopped by addition of SDS-PAGE loading dye and boiling for 10 min before storing at -20 °C. Samples were separated by SDS-PAGE and then transferred to a polyvinylidene difluoride (PVDF) membrane at 25V for 3 hours at room temperature. Following transfer, the PVDF membrane was incubated in blocking solution for 30 min: 5% Bovine Serum Albumin (BSA) in Phosphate Buffered

Saline-Tween 20 (10mM Na2HPO4, 1.8mM KH2PO4, 137mM NaCl, 2.7mM KCl, 0.1% (v/v) Tween 20; pH 7.4) (PBST). The blocked membrane was then incubated with primary antibody (discontinued Roche mouse anti-His 6(2), cat no. 04905318001) 1:5,000 in blocking solution at 4 °C overnight, rocking. The following day, the membrane was rinsed with PBST and then incubated for 1.5 hours in secondary antibody (Santa Cruz goat anti-mouse IgG-Alkaline Phosphotase (AP) conjugate, cat no. SC-2008), 1:15,000 in blocking solution. Following secondary antibody incubation, the membrane was rinsed with PBST and developed with BCIP/NBT AP substrate (BioShop BCN101.100). The thrombin cleaved sample was concentrated to 10 mg/mL, 17 mg/mL and 24 mg/mL. Concentrations were measured by A280 with a NanoDrop 2000c spectrophotometer.

2.1.2 Crystallization

Prior to crystallization, protein samples were centrifuged at 21,100 x g for 10 min at 4°C to pellet any debris. Three commercially available sparse-matrix screens: JCSG+ (Qiagen), Cryos Suite (Qiagen) and PEG/ION (Hampton Research) were aliquoted into the reservoir wells (70 μL) of 3-well Swissci crystallization plates. The Douglas Instruments Oryx 8 liquid handling system was used to pipette 0.3 μL of protein and 0.3 μL of reservoir solution for each of the three protein concentrations. Plates were incubated at 20°C and imaged using the Formulatrix Rock Imager 1000 system.

Four conditions yielding rod-type crystals were selected for optimization: Condition A (0.1 M Hepes pH 7.0 and 12% (w/v) polyethylene glycol (PEG) 6000), Condition B (0.1 M KSCN and

32% (w/v) polyethylene glycol monomethylether (PEG MME) 2000), Condition C (0.15 M KBr and 32% (w/v) PEG MME 2000), and Condition D (0.2 M NaSCN and 22% (w/v) PEG 3350). Each optimization grid screen was performed in both MRC 48-well sitting drop plates and 24- well VDX hanging drop plates with 1 μL:1 μL and 1 μL:2 μL buffer:protein drop ratios. Large crystals were obtained mainly from three general optimization conditions: (0.1M KSCN, PEG MME 2K; 0.15M KBr, PEG MME 2K; 0.2M NaSCN, PEG3350); the details of crystal-growth conducive conditions are summarized in Table 1. 48 crystals were scooped from optimization plates and initial screens. Perfluoropolyether oil, glucose, sucrose, glycerol, and condition- matching-PEGs were tested as cryoprotectants. Oil was the only cryoprotectant compatible with the crystals. Oil-cryo protected crystals were flash cooled to 100 K and stored under liquid nitrogen in UniPucks until data collection.

Table 1 Optimization conditions for crystallization: conditions yielding crystals determined appropriate for diffraction experiments are shaded blue.

2.1.3 Data Collection and Processing

Crystals were diffracted at the Canadian Light Source on the 08ID-1 beamline. The data were measured to 1.3 Å resolution on a Pilatus3 S 6M detector. A total of 900,  0.2° oscillation frames were collected at a detector to crystal distance of 225.0 mm. Diffraction data were indexed, integrated, and scaled with the program XDS94. The resolution of the data was determined using a cutoff of Rmeas <60% and an I/σ(I) > 2 in the highest resolution shell, while maintaining near 100% completeness of data.

2.1.4 Structure Determination

The syncytin-2 TM structure was determined by molecular replacement using the program phenix.phaser95,96 and the syncytin-1 TM (PDB code: 5ha6) as a search model. In preparation for molecular replacement, the schwarzenbacher algorithm was used in phenix.sculptor95,97 to prune non-homologous residues from syncytin-1 to the last common atom.

The interactive computer graphics program Coot98 was used to rebuild the initial syncytin-2 structure. Several rounds of torsion angle simulated annealing starting at 2500 K using phenix.refine95 were alternated with manual rebuilding of the model aided by |Fo|-|Fc|, 2|Fo|-|Fc| and simulated annealed composite omit 2|Fo|-|Fc| electron density maps in Coot98. The progress of the refinement was monitored by reductions of Rfactor and Rfree. Initially, individual B-factor refinement and application of three-fold non-crystallographic symmetry (NCS) restraints were employed. In later stages of refinement, the NCS restraints were relaxed and Translation, Libration, Screw-motion (TLS) parameters were used. 7 TLS groups for each of the three chains were generated with the TLSMotionDetermination server hosted by the University of Washington99,100. Residues in weak 2|Fo|-|Fc| density at the N- and C-termini were removed and side chains with weak density were trimmed to the last atom for which there was clear density. Water molecules and ions were added manually based on the presence of strong 3σ difference Fourier electron density and the formation of at least one hydrogen bond to a protein or solvent atom. The syncytin-2 TM structure was validated using MolProbity101,102 and Coot98 validation tools. MolProbity reported the percentage of Ramachandran outliers, rotamer outliers, C-beta outliers, and a clashscore for the working model, while Coot98 validation tools allowed for per- residue visualization of Ramachandran and geometric outlier scores, thus aiding in fine-tuned refinement.

2.1.5 Structure Analysis

All structures were visualized and structural figures generated using the program PyMol103. Coot98 was used for measuring all atom-atom distances and bond angles. CLICK104,105 was used for structural alignments and superimpositions. All PDB files inputted for alignment with CLICK were preprocessed using PyMol103 to ensure the presence of a single homotrimeric TM. Some PDB files contained only a single monomer, in which case the symexp PyMol103 command was used to generate symmetry mates. Some PDB files contained extra chains in the

27 crystallographic asymmetric unit; these extra chains were randomly selected, extracted as an object and deleted.

The Phenix module Polygon95,106, which compares the current structure statistics to structures in the PDB of a similar resolution was used to evaluate the refined model statistics. For each statistical measure, Polygon will bin structures with similar values. The deviation of each bin is quantified in Polygon with the ratio of the number of structures in the bin to the average number of structures per bin for that particular statistic. Using Polygon to bin a new model, one can quantify the deviation each of the model’s statistics have from the norm.

Regions of secondary structure were confirmed with the DSSP server107. Quaternary surface analysis of the fully refined structure was carried out using the Protein interfaces, surfaces and assemblies' service PISA at the European Bioinformatics Institute108. Quaternary surface analysis included quantification of inter and intra chain interface area as well as local characterization of interfaces by residue hydrophobicity or polarity. Coiled-coil analysis by the TWISTER server109 was used to reveal supercoil-relaxation effects of the heptad stutter present in the structure.

2.1.6 Phylogenetic Analysis and Clustering

Phylogenetic clustering was conducted based on sequence and structural conservation. Sequence based clustering was carried out using Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets software suite (MEGA7)110. The sequences used were trimmed to within the N and C terminal bounds resolved in their respective crystal structures. Sequences were first aligned using MUSCLE111 and then clustered using the Maximum Likelihood method based on the Le and Gascuel (2008) model112. Initial trees for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms113 to a matrix of pairwise distances estimated using a JTT model114, and then selecting the topology with superior log likelihood value. Clustering was validated by bootstrapping 500 times and the percentage of trees in which the associated sequences clustered together after bootstrapping was reported at each branch. Structural based clustering was distance based. CLICK104,105 alignment reported RMSD’s were used as distances and the PHYLIP server115 was used to apply the Neighbour- Joining Saitou and Nei (1987)116 method for clustering.

Chapter 3 Results and Discussion Results and Discussion 3.1 Expression, Purification, and Crystallization

In the post-fusion conformation, the transmembrane domain of syncytin-2 is expected to form the highly stable six-helix-bundle with its N and C termini anchored in the recently fused membrane. To elucidate the structure of syncytin-2 in its post-fusion conformation, the ectodomain of the transmembrane domain (residues 372-467) was subcloned into pET-46 Ek/LIC and henceforth called syncytin-2 TM. Syncytin-2 TM expressed well, yielding more than 30 mg per L of induced culture. It was isolated successfully with Ni-NTA affinity chromatography and size exclusion chromatography to greater than 95% purity (Figure 7). An elution volume of 10.5 mL from a BioRad ENrich SEC 70 10 x 300 size exclusion column corresponds to a molecular weight of approximately 30kDa indicating syncytin-2 TM is likely trimeric in solution (Figure 7C). Removal of the histidine tag was complete as visualized by anti- His immunoblot (Figure 7E). Concentration of the cleaved, pure sample to greater than 10 mg/mL for crystallization was carried out without difficulty and did not result in precipitation.

Sparse matrix crystallization screening proved to be very fruitful. After 13 days, JCSG+, Cryos Suite, and PEG/ION screens resulted in 20, 11 and 65 hits, respectively (“hits” being anything crystalline or spherulitic). Many of the hits found in PEG precipitant conditions were of rod or needle morphology (Figure 8A). Four conditions yielding higher quality rod shaped crystals were selected for optimization in sitting drop and hanging drop vapour diffusion formats.

Optimization involved decreasing to varying degrees the concentration of PEG precipitant for each chosen condition, as well as setting up larger 1 μL:1 μL and 1 μL:2 μL protein:buffer sized drops in hanging and sitting drop formats. Singular rectangular crystals of approximately 0.1 mm-0.4 mm along at least one axis grew more readily in sitting drop format (Figure 8B). Seemingly, the larger drop size and slightly lower concentration of precipitant promoted growth of singular crystals. Sitting drop Condition D (0.2M NaSCN + PEG 3350) eventually yielded high resolution diffracting crystals. For this condition, low precipitant concentration and 1:2 protein:buffer was necessary for singular crystal growth. Thirty-four crystals were scooped from

28 29 optimization plates and 14 from the initial sparse matrix crystallization screens. Eleven datasets were collected, none of which were of crystals scooped from a sparse matrix crystallization screen. Scooped and dataset yielding crystal optimization conditions are summarized in Table 1.

kDa PL I FT W E1 E2 E3 75

25 20 C 17

D E SEC SEC kDa PL inp peak Time: 0 o/n 24 48 Myoglobin 16.7kDa 75 BSA 66kDa

25kDa > 25 20 11kDa 17 >

11 5

Figure 7 Purification and histidine tag removal of syncytin-2 TM:A) Syncytin-2 construct design: most of the ectodomain of the transmembrane subunit (TM) was cloned into the pET-46 Ek/LIC vector backbone (labelled “Crystallized core” above). The crystallized core contains some of the fusion peptide (FP), the heptad repeat 1 (HR1), the chain reversal region (CR), the leash region (L), and the heptad repeat 2 (HR2). Not included in this construct are the transmembrane domain (TM-grey), the cytoplasmic tail (CT), and the N-terminal surface subunit (SU), which contains a signal peptide (SP) localizing syncytin-2 to the cell membrane and a CXXC domain. B) The Syncytin-2 TM expressed in Shuffle T7 E. coli cells was initially purified via Ni- NTA affinity chromatography. Clarified cell lysate was applied to Ni-NTA beads (I) and was allowed to flow through by gravity (FT). The beads were then washed with 5 CV of binding buffer (W). During this round of purification, syncytin-2 TM was eluted with binding buffer supplemented with 100mM of imidazole (E1), 250mM of imidazole (E2) and 500mM of imidazole (E3). The three elutions were combined and dialyzed for 48 hours with thrombin in Tris-buffered saline (20mM Tris pH 8.0, 150mM NaCl) at 4°C. C) The dialyzed Ni elutions were further purified by size exclusion chromatography on the BioRad SEC70 24mL column. Syncytin-2 TM eluted at the expected volume for a trimeric 30kDa species. Myoglobin and BSA standards are shown above as a grey dashed line. D) The protein injected for SEC purification (SEC inp) and the eluted peak (SEC peak) were visualized by SDS-PAGE. Following SEC purification, the protein sample was >95% pure. E) Immunoblotting with an anti-His antibody and a Coomassie stained band shifted down indicate successful cleavage of the histidine tag. 30

3.2 Structure Determination and Validation

Eleven datasets were collected at the Canadian Light Source (example diffraction image in

Figure 8C), ten of which belong to the space group P21 and ranged in resolution from 3.0 Å -1.7

Å. The eleventh dataset belonged to space group I212121 and diffracted to 1.3 Å resolution. The syncytin-2 TM structure was determined by molecular replacement using the I212121 1.3 Å resolution dataset and syncytin-1 (PDB accession: 5ha6) as the search model. Three syncytin-2 TM chains are found within the asymmetric unit. Clear backbone density was observed for residues 382 to 463, 379 to 465 and 379 to 465 in chains A, B and C, respectively (an example of electron density in Figure 8D). The final overall model was refined to 1.3 Å resolution with a final Rwork/Rfree of 14.9%/17.8%. The refined syncytin-2 TM contains no Ramachandran outliers and has a MolProbity score of 1.48 (Figure 9). A full set of refined statistics can be found in Table 2. The Rwork/Rfree, and Ramachandran statistics, rotamer outliers, RMSD bond lengths and RMSD bond angles all lie within the normal distribution of 1.3 Å resolution structures, as determined in Polygon95,106. The clashscore, Wilson B-factor and mean B-factor are much higher than the normal values for a 1.3 Å structure with Polygon ratios 2.0, 0.25, 0.25, respectively.

A B

D Figure 8 Crystallization and diffraction of syncytin-2 TM: A) One of the selected conditions (0.1M Hepes pH 7.0 and 12% PEG 6000) for optimization and a representative drop of conditions yielding crystals of rod morphology. B) Crystals scooped from optimization screens were mainly of chunky rectangular prism morphology. The above drop is representative of crystals selected for diffraction. The above condition is a 2μL drop (1:1 protein to buffer) with

20mg/mL of protein in 0.15M KBr and 24% PEG MME F440chainA 2000. C) Syncytin-2 TM diffracted to 1.3Å at CLS on the 08ID-1 beamline. Above is a sample image from the L409chainB diffraction experiment. D) Above is a sample of the 2Fo-Fc map at t 1.17σ at the chain reversal region.

D Figure 9 Validation of the syncytin-2 TM model using Coot geometry analysis, rotamer outlier analysis, B-factor analysis, and Ramachandran plots: A) Deviations of bond angles, planes, and lengths of the working syncytin-2 TM model are plotted in Coot by residue. In the plot above, the height of each bar is the highest Z-score present in that residue’s bonds, thus visually representing the residue’s bond geometry deviation. B) The rotamer likelihood of each residue is visually represented by the height of each bar being inversely proportional to the rotamer probability according to the Molprobity rotamer probability distribution. The tall blue bars simply indicate the residue side chain was trimmed to fit available density. C) B-factors of each residue is represented by the plot above as the height of each bar corresponds to the residue’s total B-factor. Tall red bars marked with an asterisk indicate the high reported B-factor results from side chain atom contributions and the main chain atom B-factors are ≤ 31. D) A Ramachandran plot is used to assess ψ and ϕ angles for the main chain. There are no Ramachandran outliers. 33

1. Statistics for the highest- resolution shell are shown in parentheses. 2. 3% of total reflections are excluded from all stages of refinement

3.3 Overall Structure

Syncytin-2 TM forms a homotrimer of helical hairpins, typical of class I viral fusion proteins

(Figure 10). This six-helix-bundle is 78% α-helical, 3.6% 310 helix and 12% random coil, is about 60 Å long and has an approximate diameter of 29 Å. Extending from the N-terminus along the entire length of the protein, the HR1(residues 383-423) forms the inner coiled-coil. The C- terminus of the HR1 turns to form a 3:10 helix (residues 425-427) almost perpendicular to the HR1. Following the 3:10 helix, a short loop leads into a single turn α-helix (residues 430-434) also perpendicular to the HR1. This region, containing the short 3:10 helix and α-helix, reverses the chain direction and is referred to as the chain reversal region. A long loop referred to as the leash region extends from the chain reversal region parallel to the HR1 and leads up to the HR2 (residues 445-462) at the C terminus. In addition to the peptide backbone linkage, the chain reversal region is also covalently linked to the leash region via a disulfide bond in the CX6CC motif (C431-C438).

3.4 Quaternary interfaces

Syncytin-2 is trimeric; the intermolecular interface between any two chains is approximately 1295 Å2 and is mainly hydrophobic (evaluated with PISA108). Much of this interface is found at the core of syncytin-2’s HR1 coiled-coil. The HR1 residues mostly adhere to a heptad repeat pattern abcdefg, which has positions a and d pointing inward, and except for N391 and N416, all a and d residues are hydrophobic. Despite this near perfect adherence to the heptad repeat typical of coiled coils (one heptad repeat interruption is present and will be discussed in the next section), only three true knobs are present in the syncytin-2 coiled coil, true knobs necessitating complementarity between knob and hole such that the knob residue pairs with a hole on an adjacent chain that also contains that residue, which is in turn a knob for the third chain. The three knobs are V413, M402, and N416.

In addition to stabilization of the six-helix-bundle via classical coiled-coil motifs, the leash region and HR2 are oriented in the groove between the HR1 of the same chain and the HR1 of the counterclockwise chain creating an additional interchain interface. The groove between adjacent HR1’s with which the leash interfaces has mixed hydrophobic and polar character and spans an area of 288 Å2 and 270 Å2 on the same chain HR1 and counterclockwise HR1,

35 36 respectively. In addition to hydrophobic effects and hydrogen bonding, a salt bridge links the leash region to the HR1 of the counterclockwise chain near the chain reversal region; E436 and R418 of the counterclockwise chain are 2.0 Å apart (Figure 14). The groove between adjacent HR1’s with which the HR2 interfaces is almost entirely hydrophobic on the same-chain-HR1- side, spanning 441 Å2, and mainly polar on the counterclockwise-chain-HR1-side spanning 380 2 Å (Figure 11). In addition to the hydrophobic effects and hydrogen bonding enveloping the HR2 in the coiled-coil groove of the adjacent HR1’s, a single salt bridge also stabilizes the HR1-HR2 hairpin; functional groups of D393 and R463 of the same chain are 2.7Å apart (Figure 11 and Figure 14).

3.5 Heptad Stutter and Chloride Coordination

The three-strand coiled-coil heptad repeat of the HR1 is interrupted once by a heptad stutter at M395-A398. The heptad stutter creates a local increase in supercoil pitch resulting in a noticeable relaxation of the supercoil. Trimeric coiled-coils that strictly adhere to the heptad repeat such as, trimeric GCN4 leucine zippers, and the T4 fibritin E protein, have an average supercoil pitch of approximately 130 Å and very little deviation along the length of the coil (Figure 12 A, B). A single stutter serves to increase the supercoil pitch by approximately 100 Å and supercoil relaxation peaks within the stutter region (Figure 12 C, D). Three and half heptad repeats away from the stutter is a putative chloride ion in the center of the six-helix-bundle core, coordinated by N416 of each of the HR1’s.

N’

C’

N416 HR2 Cl N416 N416

~60 Å

HR1 Leash ~29 Å region C438 Cl N416 Cl C438 C431

C431

α-helix

3:10-helix Chain reversal region

Figure 10 Structure of syncytin-2 TM: Ribbon diagrams of the crystallized six-helix-bundle viewed along the length of the bundle (A), from the N’ and C’ terminal end looking down through the center (B) and as a monomeric helical hairpin (C). Monomeric hairpins are coloured blue, red and green. C431 and

C438 form a form the disulfide linkage of the CX6CC motif found in most retroviruses and in filoviruses (the linkage is coloured yellow).N’ A chloride ion (coloured grey) is coordinated by N416 of each monomer. N’ Figure 11 The HR2 interfaces with the outer groove of the HR1 coiled coil: A C’ ribbon diagram of the HR2 with HR1-groove R463 R463 interfacing segments coloured by type (orange: hydrophobic, cyan: polar, red: acidic, blue: basic) demonstrates the biochemical forces contributing to stabilization of the 2.7Å 2.8Å 2.7Å 2.8Å helical hairpin formation. In addition to hydrophobic, and polar forces, a salt bridge (D393-R463) links the HR1 and HR2 of the D393 same chain, further stabilizing the helical D393 hairpin.

HR2A

HR1 A HR1B 37 A

Average supercoil pitch: 129.6 (±8.8)

Figure 12 TWISTER was used to measure supercoil pitch for trimeric GCN4 (PDB access: 4DME) (A), fibritin E (PDB access: 1AA0; residues N421-E454) (B), syncytin-2 (C) and syncytin-1 (PDB access 5ha6) (D). The heptad repeat index (abcdefg) for each residue is listed below it B on the x-axis. “a” and “d” represent residues pointing into the coiled-coil core and are usually hydrophobic. The heptad stutter Average supercoil pitch: 136.3 present in syncytin-1 and syncytin-2 is (±15.4) highlighted in light blue and results in the heptad repeat sequence abcdefgdefg. The local relaxation of supercoil results in a supercoil pitch peak corresponding with the heptad repeat.

Average supercoil pitch: 211.1 (±119.2)

Average supercoil pitch: 251.6 (±220.2)

3.6 Structural Comparison of Syncytin-1 and Syncytin-2 TM

Syncytin-1 TM and syncytin-2 TM are architecturally very similar. Despite only sharing 43% sequence identity, they can be superimposed with a Cα RMSD of 0.85 Å, using the CLICK homology server104,105 (Figure 13B). The central coiled-coil of syncytin-1 also carries a heptad stutter that serves as an acute supercoil relaxation point like the stutter in the syncytin-2 TM

(Figure 12 C, D). Both syncytin-1 TM and syncytin-2 TM contain a CX6CC motif in the chain reversal region, an immunosuppressive domain conserved to 70% sequence identity, as well as the asparagine coordinated chloride ion common to all CX6CC fusion proteins (Figure 13A, B).

Syncytin-1 is notably stabilized by complex salt bridges linking chains and hairpin helices (Figure 14). Two complex salt bridge networks and 3 salt bridge pairs line the length of the 6- helix bundle. At the upper mid-section of the bundle are the two salt bridge networks, one of five residues (E353chain A, D357chain A, R424chain C, R427chain C, R428chain C) and another of three residues (K417chain A, D363chain B, R422chain B). In addition, there are three one-to-one salt bridges at the intermolecular interface (R384chain A-E402chain C, R360chain A-E420chain C and D363chain A-

K417chain C). The large number of residues involved in electrostatically stabilizing the syncytin-1 six-helix-bundle fortifies syncytin-1 against destabilization by single point mutations to salt bridge residues. Alanine point mutations to syncytin-1 salt bridge participating residues results in melting temperature reductions of ≤10°C (Aydin et al., unpublished data). In contrast to this, syncytin-2 contains only the two one-to-one salt bridges mentioned above (Figure 14).

The interchain salt bridge at the chain reversal region (R418chain A- E436chain C) aligns to the syncytin-1 salt bridge R384chain A – E402chain C with an RMSD of 0.48 Å and they are likely equivalent and evolutionarily conserved. Syncytin-2’s second salt bridge, D393chain A – R463chain

A, is an intrachain salt bridge between the HR1 and HR2 near the N and C termini and is unique to syncytin-2.

Figure 13 Syncytin-1 and syncytin-2 structural homology: A) Syncytin-1 and syncytin-2 share 43% sequence identity. Conserved residues are highlighted yellow, and the heptad index for the HR1 is recorded underneath the corresponding residues. The grey chloride ion is placed above it’s coordinating asparagine. B) Using the CLICK algorithm for structural alignment, syncytin-1 and syncytin-2 superimposed to an RMSD of 0.85 Å. Syncytin-1 is coloured cyan and syncytin-2 is coloured green. C) Q427 in syncytin-2 is shown to be necessary for immunosuppressive activity. This residue position is occupied by an arginine (R393) in syncytin-1 and putatively renders syncytin-1 non-immunosuppressive. In the above ribbon diagram, the immunosuppressive domains of superimposed syncytin-1 and syncytin-2 are coloured cyan and green, respectively, and residues Q427syncytin-2 and R393syncytin-1 are displayed as sticks.

Figure 14 Syncytin-1 and syncytin-2 six-helix-bundles are stabilized by salt bridges: A) The syncytin- 2 six-helix bundle is stabilized by one interchain salt bridge at C’ terminal end of the HR1 and the chain reversal region, and another intrachain salt bridge between the HR1 and HR2. B) Syncytin-1 has a similar salt bridge stabilizing the chain reversal region but unique to syncytin-1 is a complex network of salt bridges mediating the interactions of the HR2 with the HR1 groove.

Both syncytin-1 and syncytin-2 serve to fuse placental trophoblast cells, a necessary step for syncytiotrophoblast formation and placental development. Despite serving seemingly redundant roles, the localization patterns of syncytin-1 and syncytin-2 are distinct. While syncytin-1 localization remains ambiguous37,42–45, the localization of syncytin-2 to villous cytotrophoblasts47–49 and the localization of its receptor, MFSD2, to the syncytiotrophoblast47 exclusively near the cyto-syncytio interface, is consistently observed. This polarization of syncytin-2 expression at the cyto-syncytio interface implies a directionality for syncytin-2 mediated infusion of cytotrophoblasts, whereas the directionality of syncytin-1 mediated infusion of cytotrophoblasts remains ambiguous.

The energetic demands of initial membrane contact, lipid mixing, hemifusion stalk formation and nuclear pore formation and expansion are reliant on the physical characteristics of the membranes involved74,117. The syncytiotrophoblast membrane and a cytotrophoblast membrane are biophysically and geometrically dissimilar, thus the directionality of fusion may significantly

41 42 impact the energy costs of fusion. The energy costs of class I viral fusogen mediated fusion are paid by the TM transition from pre-fusion to the highly stable post-fusion six-helix-bundle118.

Despite having nearly identical structural organization and a very low Cα RMSD, syncytin-1 and syncytin-2 six-helix-bundles appear to be subject to remarkably different types of stabilization factors. The syncytin-1 HR1-HR2 interfaces are riddled with charged residues that stabilize the six-helix-bundle fold via numerous paired salt bridges and complex salt bridge networks. In stark contrast to this, syncytin-2 contains only two salt bridges (one of which is found in the chain reversal region and is conserved in syncytin-1) and the six-helix-bundle interfaces are mainly composed of hydrophobic or polar residues. These differences in six-helix bundle stabilization could reflect the variant energetic demands of syncytin-1 and syncytin-2 mediated fusion.

The integration of HERV-W (syncytin-1) and HERV-FRD (syncytin-2) is likely separated by approximately 20 million years, HERV-FRD capture preceding that of HERV-W38,39,119, (although some recent phylogenetic analysis seems to suggest HERV-W may be more ancient that previously assumed120, that HERV-W and HERV-FRD are entirely independent and likely originating from temporally separate integration events still stands). Whether the observed structural differences between syncytin-1 and syncytin-2 do lend themselves to different fusogenic capabilities and whether this in turn contributed to the progressive evolution of the human placenta by syncytin capture is worth exploring.

That both syncytins act, in some capacity, as cell-cell fusogens and are likely necessary for syncytiotrophoblast formation and maintenance is undisputed but, the other proposed function of syncytins in placental health: feto-maternal tolerance via immunosuppression, is less well supported. The small body of research investigating the immunosuppressive activity of syncytin- 1 and syncytin-2 is contradictory and frankly, confusing.

Mangeney et al in 200721 elegantly demonstrated a single residue mutation to the classic retroviral immunosuppressive consensus motif CKS-1715 present in the syncytin-1 immunosuppressive domain (R393) abrogates syncytin-1 immunosuppressive activity. The sequence of this consensus motif as well as the sequences of the syncytin-1, syncytin-2, MPMV, lentivirus, HIV, and filovirus Ebola are aligned in Figure 15. They conferred immunosuppressive activity to syncytin-1 by mutating R393Q to mimic the active immunosuppressive domain of MPMV and syncytin-2. Manganey et al. also successfully inactivated the immunosuppressive

43 domain of syncytin-2 by mimicking the syncytin-1 immunosuppressive domain, mutating Q427R . Immunosuppressive activity was evaluated by tumour rejection assays in mice, wherein mice were injected with tumour cells stably expressing the immunosuppressive protein of interest. Tumour development in the mice indicated active immunosuppression.

Figure 15 The immunosuppressive domain found in the chain reversal region of retroviral and filoviral fusion proteins is a 17 amino acid motif: CKS-17 is an ISD consensus peptide and is bioactive. All CKS-17 conserved residues are highlighted green in the viral ISD sequences. The asparagine in position 3 coordinates a chloride ion in the centre

of CX6CC coiled coil. Position 14 has been shown to be pivotal to the motif’s immunosuppressive activity. According to Mangeney, et al. the glutamine highlighted blue in MPMV (Q471) and syncytin-2 (Q427) retains immunosuppressive activity and the arginine highlighted red in syncytin-1 (R393) abrogates immunosuppressive activity.

Following these seemingly definitive results, experiments monitoring the release of cytokines by PBMC’s after exposure to syncytin-1 or syncytin-2 were conducted and their results generally conflict with Mangeney’s original finding discounting syncytin-1’s immunosuppressive activity. In 2012 Tolosa et al. observed recombinant syncytin-1 mediated suppression of cell mediated immunity via reduction of IFN-γ, CXCL10, and TNF-α release in PBMC’s challenged with lipopolysaccharide (LPS)121. Then, again in 2015, Tolosa et al. observed recombinant syncytin-1 mediated immune suppression in PBMC’s challenged with a strain of H1N1122. In 2015, a third group examined the effect syncytin-1 and syncytin-2 have on maturation of dendritic cells using CHO cells transfected with syncytin-1 or syncytin-2123. They saw both syncytins affecting CD86 expression by dendritic cells but could not significantly implicate syncytin-1 in dendritic maturation inhibition.

The syncytin-2 immunosuppressive domain, region L414-A433, shares 65% sequence identity with CKS-17 (Figure 15). This region encompasses the C terminus of the HR1, including the putative chloride ion, the 3:10 helix and part of the single turn α-helix in the chain reversal region (Figure 16A). Q427, shown by Mangeney, et al. to be necessary for immunosuppressive activity is located at the base of the chain reversal region and the carboxamide side chains of Q427 on chains A and B occupy two conformations rotated at Cγ approximately 90° (Figure

16B). This glutamine is well exposed and near the covalently stabilized CX6CC loop. The equivalent ISD position in syncytin-1, R393, aligns with a Cα RMSD of 0.95Å (Figure 13B). This position’s importance in immune modulation has been previously demonstrated with mutation studies in FeLV and HIV TMs92,124.

How should the contradictory results concerning syncytin-1 immunosuppression activity be considered? The recombinant syncytin-1 construct used in both of the Tolosa lead studies included only residues 350-417. This construct is missing the entire HR2 and has a fully intact

CX6CC motif; the third cysteine in our lab’s crystallizable construct is mutated to a serine to prevent higher order oligomerization by free cysteines. Indeed, in purifying recombinant syncytin-1 (350-417), Tolosa et al. comment on separating the inactive monomer from the active multimeric fraction containing dimers, trimers and other higher order oligomers. The mixture of oligomeric states may have resulted in artifacts during their study. The mechanisms by which the ISD illicit immunosuppression is still unknown and the harbourers of these ISD’s, class I viral fusion proteins, are natively trimeric. Thus, ideally the recombinant protein used to examine immunosuppressive activity should be in its native oligomeric form.

Figure 16 Syncytin-2 was shown to have immunosuppressive properties and contains a region of high sequence identity to a retroviral immunosuppressive motif: This region (coloured pink) encompasses the C’ terminal end of the HR1, including N416 coordinating chloride, as well as most of the chain reversal region (A). Q427, shown to impart immunosuppressive activity to the motif, occupies two conformations on chain A and B (B).

3.7 Structural Comparison of Syncytin-2 with other CX6CC TMs

Syncytin-1 and syncytin-2 both bear the CX6CC motif common amongst all retroviral fusion proteins (except the lentiviruses) as well as filoviruses Ebola virus and Marburg virus fusion proteins, and in the arenavirus California Academy of Sciences Virus (CASV) fusion protein.

Indeed Syncytin-2 aligns with all CX6CC bearing proteins to an RMSD of 1.57 Å or less (Figure 17A). The architecture of the central coiled coil is also highly conserved between all 11 six- helix-bundles in this sub-class of Class I. They all deviate in similar fashions from the classic coiled-coil architecture with the incorporation of a coordinated chloride ion near the chain reversal region (except for the CASV fusion protein which is missing this chloride) and the

heptad stutter closer to the N termini. Interestingly, the CX6CC proteins appear to have consistent spacing between the heptad stutter and the chloride-coordinating-asparagine. The filoviruses, Ebola and Marburg, and the alpharetrovirus, Avian sarcoma leukosis virus (ASLV),

45 46 have 2.5 heptad repeats between stutter and asparagine-chloride, the syncytins and all of the other retroviruses except Bovine leukemia virus (BLV) have 3.5 heptad repeats between stutter and asparagine-chloride. BLV has the greatest distance at 4.5 heptad repeats between stutter and asparagine-chloride (Figure 17B). In all three groups, the asparagine-chloride occupies the d position of the heptad repeat. In 2011, Igonet, et al. identified a common core in the coiled coil of class I viral fusion proteins using the heptad stutter as an anchor. This common core encompasses two heptad repeats after the stutter. In Figure 17B, the top black arrowhead indicates the heptad stutter and the red arrowhead on the Ebola hairpin indicates the C’ terminal end of the minimum common core identified in the Ebola postfusion TM by Igonet, et al87. Thus,

CX6CC TMs can then be further subgrouped based on their extension past the previously identified common core.

Interestingly, these sub-groups are consistent with the subgroupings designated by Henzy and Johnson (2013)125 based on the presence of a fusion peptide (referred to as “gamma-type”) or internal fusion loop (referred to as “avian gamma-type”). Ebola, Marburg and ASLV are avian gamma-type and they have a 2.5 heptad repeat spacing between stutter and chloride-asparagine. Mason Pfizer monkey virus (MPMV), Human T-lymphotropic virus (HTLV), Moloney murine leukemia virus (MMLV), Xenotropic murine leukemia virus-related virus (XMRV), Syncytin-1 and syncytin-2 are gamma-type and they have a spacing of 3.5 heptad repeats between stutter and chloride-asparagine. BLV is also a gamma-type but has a spacing of 4.5 heptad repeats and could potentially be an evolved elongated variant of the 3.5 heptad repeat spacing group.

Using maximum likelihood phylogenetic analysis of CX6CC viral TM amino acid sequences, similar grouping patterns are evident (Figure 18A). The avian gamma-type: ASLV, Ebola, and Marburg, cluster together and the gamma type: MPMV, HTLV, BLV, MMLV, XMRV, syncytin-1 and syncytin-2 cluster together. BLV is hypothesized to be evolutionarily distant from its closest homologue and viral family member, HTLV. To further corroborate CX6CC 104,105 subgrouping, the CLICK alignment server was used to align all available CX6CC postfusion TM structures and a distance based phylogenetic method, Neighbour-joining, was used to visualize structural based evolutionary clustering. Again, ASLV clusters with filoviruses Ebola and Marburg while the other retroviral TM’s cluster together (Figure 18B).

The functional significance of these observations remains unclear. Elongation of the stutter to chloride-asparagine spacing between groups corresponds to overall TM length as well as increases the number of hydrophobic residues stabilizing the coiled-coil. The avian gamma-type TMs are activated in a pH dependent manner and carry out fusion inside the endosome while the gamma-type TMs are pH independent and are activated by receptor binding on the cell surface. The chemical, physical and even geometric conditions for fusion are different between subgroups and the different lengths or enhanced coiled-coil stabilization seen in TM hairpins may reflect fusion constraints.

The only CX6CC prefusion structure available is that of Ebola. The residues corresponding to the postfusion HR1 make up two separate helices. The first residue of the postfusion heptad stutter, T565, generates a 40° kink in the first HR1 prefusion helix. The chloride coordinating asparagine of the postfusion TM is located in the loop connecting the two prefusion HR1 helices and the chloride is not present. Chloride ion binding has previously been postulated to play a role in conformation switching81. The conserved heptad stutter to chloride coordinating asparagine spacing may, alternatively, be more relevant to the early stages of fusion and reflect prefusion mechanisms of fusion activation.

Protein architecture C terminal to the HR1 is more divergent between CX6CC proteins than is the architecture of the HR1. The chain reversal region sometimes contains both a short 3:10 helix and a short α-helix (as in syncytin-2), and sometimes, only the α-helix (as in BLV) and the arrangement and positioning of these helices is not well conserved between proteins (Figure 17B). The length and morphology of the leash region varies as well (Figure 17B). Deltaretroviruses do not appear to have a single helix for the HR2, instead they appear to have two short helices forming the outer arm of helical hairpin. Both helices are fully resolved only in the BLV structure while only the first helix is resolved in the HTLV structure. The first helix lies almost parallel to the HR1 and leads into the second helix with a short loop. The second helix is perpendicular to the first and the HR1 (Figure 17B). In general, the HR1-HR2 relative angles are variant between protein species, markedly so in the Ebola virus hairpin, where the HR2 is almost parallel to the HR1 (Figure 17B).

Figure 17 Syncytin-2 alignment with CX6CC proteins: A) Syncytin-2 aligns to all CX6CC bearing fusion proteins with an RMSD of 1.57 Å or less. The table beside the superimposed six-helix-bundles lists the aligned proteins, their PDB accession codes in brackets, their RMSD and structural overlap (proportion of structure included in RMSD calculation; residues > 4Å away from any partner aligned residue is excluded from RMSD calculation) B) The HR1 is well conserved between CX6CC species but there are divergences in the morphology of the rest of the hairpin. From left to right, Syncytin-2, XMRV, BLV, MPMV, ASLV, Ebola, and CASV hairpins are in an aligned orientation. Black arrowheads indicate heptad stutter location (top) and chloride coordinating asparagine (bottom). Red arrowhead indicates the C’ terminus of the common ancestral core of class I fusion proteins identified by Igonet, et al. in the Ebola postfusion TM

48 49

Figure 18 Phylogenetic clustering of CX6CC fusion proteins: A) The amino acid sequences of 21 CX6CC viral TMs with solved structures were aligned using MUSCLE and the evolutionary history was inferred by using the Maximum Likelihood method based on the Le_Gascuel_2008 22 model . The tree with the highest log likelihood (-1994.33) is shown. The percentage of trees in which the associated taxa clustered together after bootstrapping 500 times is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor- Join and BioNJ algorithms to a matrix of pairwise distances estimated using a JTT model, and then selecting the topology with superior log likelihood value. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 2.3097)). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 11 amino acid sequences. All positions containing gaps and missing data were eliminated. There were a total of 92 positions in the final dataset. Evolutionary analyses were 23 conducted in MEGA7 .

B) CLICK was used to generate 1:1 alignments of all 11 CX6CC TMs and to calculate the RMSD of each aligned pair’s structural overlap. The structural overlap of each aligned pair was at least greater than 80%. PHYLIP Neighbour joining method of Saitou and Nei (1987) and the UPGMA method of clustering was used to generate a distance-based tree based on CLICK reported TM RMSDs

Chapter 4 Future Directions Future Directions 4.1 Short-term goals

Exploring the possibility of syncytin-1 and syncytin-2 functional reciprocity in the placenta may provide insights not only into placental development and placental evolutionary mechanisms but also into fusion mechanisms of class I viral fusion proteins generally. Assessing syncytin-1 and syncytin-2 fusion capabilities in their opposite directions will confirm or deny our hypotheses concerning structural specificity to fusion directionality. Syncytin-1 has already been observed by our lab and many other groups to successfully carry out fusion when expressed on mononucleate cells and the specificity of syncytin-1 expression in the placenta is still unclear. From unpublished results in our lab, we see that disruption of syncytin-1 salt bridges does impact fusogenicity even amongst mononucleate cells. Syncytin-2 however, consistently localizes to cytotrophoblasts, and an experiment evaluating its fusion capabilities when expressed on the syncytium side could reveal a potential link between its structure and function. Primary trophoblasts kept in culture for 72 hours, cease to express syncytin-2 as they form a syncytium48,126. Manipulating expression of syncytin-2 post syncytium formation (potentially by transfecting the primary trophoblasts with syncytin-2 under an inducible promoter) and adding labelled cells expressing MFSD2a (the syncytin-2 receptor) can test for syncytin-2 capacity to fuse a syncytium with mononucleate cells by quantifying in-fusion of the new labelled cells.

In addition to functional studies probing the structural relevance of syncytin-1 and syncytin-2 variance, biophysical characterization of syncytin-2 will provide further insight into its functional mechanism. Circular dichroism spectroscopy melts or differential scanning fluorimetry can be used to determine the melting temperature of syncytin-2 and by extension quantify and compare the stabilization energy of syncytin-2 and syncytin-1. Mutagenesis can be used to disrupt syncytin-2 salt bridges and ascertain their importance to syncytin-2 stability and function.

Our lab consistently purifies syncytin-1 and syncytin-2 as a single trimeric species of greater than 95% purity in the likely native six-helix-bundle conformation. Repeating the experiments of

51 previous groups, exposing PBMC’s to pure trimeric protein instead of the potentially inconsistent multimeric mix used previously, may generate more consistent and informative results. As well, the mechanistic details of immunosuppression by this conserved domain remain elusive. To probe the specific interactions between syncytins and immune cells, cell pulldowns and immunoprecipitation experiments using recombinant syncytin-1 and syncytin-2 may clarify the specific immune cells and/or proteins involved in syncytin mediated immunosuppression.

4.2 Conclusions and longer term goals

Elucidation of the syncytin-2 structure has further enabled a detailed comparative analysis with syncytin-1 and other CX6CC viral TMs and may create opportunities to explore the mechanistic details of fusogen facilitated membrane fusion through in depth structural examination. In comparing syncytin-1 and syncytin-2, it becomes evident that although they are proteins of near identical conformation and similar function, the biochemical factors stabilizing the bundle are dissimilar. A thorough reading of placental synyctin literature also reveals that although these proteins are both placental fusogens, they may play distinct roles in placental development, and may facilitate fusion in distinct environments.

Syncytins generally offer a unique opportunity for studying fusion. They are retroviral proteins captured and then subjected to animalia evolutionary mechanisms. The conditions in which they facilitate fusion are distinct as placental morphology and syncytin localization is unique to each specie. Ten non-human syncytins of unique retroviral origin have thus far been identified and structural elucidation of these postfusion TM’s followed by structure-informed functional characterization may reveal patterns reflecting the unique conditions in which they function. Exploration of the way syncytin structural differences lend themselves to individual functionality may provide invaluable insight into class I fusogen mediated membrane fusion in general. As well, structural characterization of syncytins would likely contribute greatly to our understanding of placental evolution.

Indeed, a more general survey of CX6CC viral TMs reveals structural patterns consistent with sequence based and overall structure based phylogenetic analyses. These structural patterns also appear to be consistent with differences in fusion environments. The HR1 stretch from heptad stutter to chloride coordinating asparagine, appears to be well conserved and consistent with phylogenetic grouping. The first 18 residues of this region correspond to the proposed core

87 remnant of the CX6CC viral TM common ancestor and the observed extensions on the core may be adaptations for specific fusion environments. Here too, lie opportunities for further understanding membrane fusion mechanics by examining fusogen structures in relation to their specific functional demands.

Unfortunately, most of the available structural information for class I mediated membrane fusion pertains to only the postfusion conformation. Only the Ebola prefusion structure is available amongst CX6CC proteins and from that structure, the residues corresponding to most of the chain reversal region, the leash region and the HR2 are unresolved. Vital to a mechanistic understanding of CX6CC mediated membrane fusion is understanding the TM structural transitions during fusion catalysis. To even begin exploring fusogen conformational transitions, knowledge of both before and after states are essential. Additionally, elucidation of syncytin prefusion structures would further our understanding of their roles in placental development and in enabling divergent placental evolution.

References

1. Human Genome Sequencing Consortium I. Finishing the euchromatic sequence of the human genome. Nature. 2004;431(7011):931-945. doi:10.1038/nature03001.

2. Chinwalla AT, Cook LL, Delehaunty KD, et al. Initial sequencing and comparative analysis of the mouse genome. Nature. 2002;420(6915):520-562. doi:10.1038/nature01262.

3. de Parseval N, Heidmann T. Human endogenous retroviruses: from infectious elements to human genes. Cytogenet Genome Res. 2005;110(1-4):318-332. doi:10.1159/000084964.

4. Spencer T, Black S, Arnaud F, Palmarini M. Endogenous retroviruses of sheep: a model system for understanding physiological adaptation to an evolving ruminant genome. Reprod Domest Ruminants. 2010;7(1):95-104. doi:10.5661/RDR-VII-95.

5. Roca AL, Pecon-Slattery J, O’Brien SJ. Genomically Intact Endogenous Feline Leukemia Viruses of Recent Origin. J Virol. 2004;78(8):4370-4375. doi:10.1128/JVI.78.8.4370- 4375.2004.

6. Cosset FL, Ronfort C, Molina RM, et al. Packaging cells for avian leukosis virus-based vectors with various host ranges. J Virol. 1992;66(9):5671-5676.

7. Belshaw R, Pereira V, Katzourakis A, et al. Long-term reinfection of the human genome by endogenous retroviruses. Proc Natl Acad Sci. 2004;101(14):4894-4899. doi:10.1073/pnas.0307800101.

8. Pavlicek A, Paces J, Elleder D, Hejnar J. Processed Pseudogenes of Human Endogenous Retroviruses Generated by LINEs: Their Integration, Stability, and Distribution. Genome Res. 2002;12(3):391-399. doi:10.1101/gr.216902.

9. Costas J. Characterization of the Intragenomic Spread of the Human Endogenous Retrovirus Family HERV-W. Mol Biol Evol. 2002;19(4):526-533. doi:10.1093/oxfordjournals.molbev.a004108.

10. Villesen P, Aagaard L, Wiuf C, Pedersen FS. Identification of endogenous retroviral reading frames in the human genome. Retrovirology. 2004;1(1):32. doi:10.1186/1742- 4690-1-32.

11. Turner G, Barbulescu M, Su M, Jensen-Seaman MI, Kidd KK, Lenz J. Insertional polymorphisms of full-length endogenous retroviruses in humans. Curr Biol. 2001;11(19):1531-1535. doi:10.1016/S0960-9822(01)00455-9.

12. Robinson HL, Astrin SM, Senior AM, Salazar FH. Host susceptibility to endogenous viruses: defective, glycoprotein-expressing proviruses interfere with infections. J Virol. 1981;40(3):745-751. http://www.ncbi.nlm.nih.gov/pubmed/6275116.

13. Spencer TE, Mura M, Gray CA, Griebel PJ, Palmarini M. Receptor usage and fetal expression of ovine endogenous betaretroviruses: implications for coevolution of

endogenous and exogenous retroviruses. J Virol. 2003;77(1):749-753. doi:10.1128/JVI.77.1.749-753.2003.

14. Ponferrada VG, Mauck BS, Wooley DP. The envelope glycoprotein of human endogenous retrovirus HERV-W induces cellular resistance to spleen necrosis virus. Arch Virol. 2003;148(4):659-675. doi:10.1007/s00705-002-0960-x.

15. Haraguchi S, Good R a, James-Yarish M, Cianciolo GJ, Day NK. Differential modulation of Th1- and Th2-related cytokine mRNA expression by a synthetic peptide homologous to a conserved domain within retroviral envelope protein. Proc Natl Acad Sci. 1995;92(8):3611-3615. doi:10.1073/pnas.92.8.3611.

16. Fuke C, Shimabukuro M, Petronis A, et al. Age related changes in 5-methylcytosine content in human peripheral leukocytes and placentas: an HPLC-based study. Ann Hum Genet. 2004;68(Pt 3):196-204. doi:10.1046/j.1529-8817.2004.00081.x.

17. Gama-Sosa MA, Slagel VA, Trewyn RW, et al. The 5-methylcytosine content of DNA from human tumors. Nucleic Acids Res. 1983;11(19):6883-6894. doi:10.1093/nar/11.19.6883.

18. Hellmann-Blumberg U, Hintz MF, Gatewood JM, Schmid CW. Developmental differences in methylation of human Alu repeats. Mol Cell Biol. 1993;13(8):4523-4530. doi:10.1128/MCB.13.8.4523.

19. Chuong EB, Rumi MAK, Soares MJ, Baker JC. Endogenous retroviruses function as species-specific enhancer elements in the placenta. Nat Genet. 2013;45(3):325-329. doi:10.1038/ng.2553.

20. Roth I, Corry DB, Locksley RM, Abrams JS, Litton MJ, Fisher SJ. Human placental cytotrophoblasts produce the immunosuppressive cytokine interleukin 10. J Exp Med. 1996;184(2):539-548. doi:10.1084/jem.184.2.539.

21. Mangeney M, Renard M, Schlecht-Louf G, et al. Placental syncytins: Genetic disjunction between the fusogenic and immunosuppressive activity of retroviral envelope proteins. Proc Natl Acad Sci U S A. 2007;104(51):20534-20539. doi:10.1073/pnas.0707873105.

22. Yi JM, Kim HM, Kim HS. Human endogenous retrovirus HERV-H family in human tissues and cancer cells: Expression, identification, and phylogeny. Cancer Lett. 2006;231(2):228-239. doi:10.1016/j.canlet.2005.02.001.

23. Rhyu D-W, Kang Y-J, Ock M-S, et al. Expression of human endogenous retrovirus env genes in the blood of breast cancer patients. Int J Mol Sci. 2014;15(6):9173-9183. doi:10.3390/ijms15069173.

24. Morozov VA, Dao Thi VL, Denner J. The transmembrane protein of the human endogenous retrovirus--K (HERV-K) modulates cytokine release and gene expression. PLoS One. 2013;8(8):e70399. doi:10.1371/journal.pone.0070399.

25. Kurth R, Bannert N. Beneficial and detrimental effects of human endogenous retroviruses.

Int J cancer. 2010;126(2):306-314. doi:10.1002/ijc.24902.

26. Bjerregaard B, Talts JF, Larsson L-I. The endogenous envelope protein syncytin is involved in myoblast fusion. In: Larsson L-I, ed. Cell Fusions. Vol Dordrecht: Springer Netherlands; 2011:267-275. doi:10.1007/978-90-481-9772-9_13.

27. Bjerregard B, Ziomkiewicz I, Schulz A, Larsson L-I. Syncytin-1 in differentiating human myoblasts: relationship to caveolin-3 and myogenin. Cell Tissue Res. 2014;357(1):355- 362. doi:10.1007/s00441-014-1930-9.

28. Søe K, Andersen TL, Hobolt-Pedersen A-S, Bjerregaard B, Larsson L-I, Delaissé J-M. Involvement of human endogenous retroviral syncytin-1 in human osteoclast fusion. Bone. 2011;48(4):837-846. doi:10.1016/j.bone.2010.11.011.

29. Møller AMJ, Delaissé J-M, Søe K. Osteoclast fusion: Time-lapse reveals involvement of CD47 and syncytin-1 at different stages of nuclearity. J Cell Physiol. 2017;232(6):1396- 1403. doi:10.1002/jcp.25633.

30. Strick R, Ackermann S, Langbein M, et al. Proliferation and cell-cell fusion of endometrial carcinoma are induced by the human endogenous retroviral Syncytin-1 and regulated by TGF-beta. J Mol Med (Berl). 2007;85(1):23-38. doi:10.1007/s00109-006- 0104-y.

31. Bjerregaard B, Holck S, Christensen IJ, Larsson L-I. Syncytin is involved in breast cancer-endothelial cell fusions. Cell Mol Life Sci. 2006;63(16):1906-1911. doi:10.1007/s00018-006-6201-9.

32. Yan T-L, Wang M, Xu Z, et al. Up-regulation of syncytin-1 contributes to TNF-α- enhanced fusion between OSCC and HUVECs partly via Wnt/β-catenin-dependent pathway. Sci Rep. 2017;7(40983):40983. doi:10.1038/srep40983.

33. Pijnenborg R, Vercruysse L, Hanssens M. Fetal-maternal conflict, trophoblast invasion, preeclampsia, and the red queen. Hypertens pregnancy. 2008;27(2):183-196. doi:10.1080/10641950701826711.

34. Furukawa S, Kuroda Y, Sugiyama A. A comparison of the histological structure of the placenta in experimental animals. J Toxicol Pathol. 2014;27(1):11-18. doi:10.1293/tox.2013-0060.

35. Wildman DE, Chen C, Erez O, Grossman LI, Goodman M, Romero R. Evolution of the mammalian placenta revealed by phylogenetic analysis. Proc Natl Acad Sci U S A. 2006;103(9):3203-3208. doi:10.1073/pnas.0511344103.

36. Blond JL, Lavillette D, Cheynet V, et al. An envelope glycoprotein of the human endogenous retrovirus HERV-W is expressed in the human placenta and fuses cells expressing the type D mammalian retrovirus receptor. J Virol. 2000;74(7):3321-3329. doi:10.1128/JVI.74.7.3321-3329.2000.

37. Mi S, Lee X, Li X, et al. Syncytin is a captive retroviral envelope protein involved in

human placental morphogenesis. Nature. 2000;403(6771):785-789. doi:10.1038/35001608.

38. Blaise S, de Parseval N, Benit L, Heidmann T. Genomewide screening for fusogenic human endogenous retrovirus envelopes identifies syncytin 2, a gene conserved on primate evolution. Proc Natl Acad Sci U S A. 2003;100(22):13013-13018. doi:10.1073/pnas.2132646100.

39. Blaise S, Ruggieri A, Dewannieux M, Cosset F-L, Heidmann T. Identification of an envelope protein from the FRD family of human endogenous retroviruses (HERV-FRD) conferring infectivity and functional conservation among simians. J Virol. 2004;78(2):1050-1054. doi:10.1128/JVI.78.2.1050.

40. Cornelis G, Funk M, Vernochet C, et al. An endogenous retroviral envelope syncytin and its cognate receptor identified in the viviparous placental Mabuya lizard. Proc Natl Acad Sci U S A. 2017;114(51):E10991-E11000. doi:10.1073/pnas.1714590114.

41. Georgiades P, Ferguson-Smith AC, Burton GJ. Comparative developmental anatomy of the murine and human definitive placentae. Placenta. 2002;23(1):3-19. doi:10.1053/plac.2001.0738.

42. Frendo J, Olivier D, Cheynet V, et al. Direct involvement of HERV-W Env glycoprotein in human trophoblast cell fusion and differentiation. Mol Cell Biol. 2003;23(10):3566- 3574. doi:10.1128/MCB.23.10.3566.

43. Lee X, Keith JC, Stumm N, et al. Downregulation of placental syncytin expression and abnormal protein localization in pre-eclampsia. Placenta. 2001;22(10):808-812. doi:10.1053/plac.2001.0722.

44. Holder BS, Tower CL, Abrahams VM, Aplin JD. Syncytin 1 in the human placenta. Placenta. 2012;33(6):460-466. doi:10.1016/j.placenta.2012.02.012.

45. Soygur B, Sati L, Demir R. Altered expression of human endogenous retroviruses syncytin-1, syncytin-2 and their receptors in human normal and gestational diabetic placenta. Histol Histopathol. 2016;31(9):1037-1047. doi:10.14670/HH-11-735.

46. Hayward MD, Potgens AJG, Drewlo S, Kaufmann P, Rasko JEJ. Distribution of human endogenous retrovirus type W receptor in normal human villous placenta. Pathology. 2007;39(4):406-412. doi:10.1080/00313020701444572.

47. Esnault C, Priet S, Ribet D, et al. A placenta-specific receptor for the fusogenic, endogenous retrovirus-derived, human syncytin-2. Proc Natl Acad Sci U S A. 2008;105(45):17532-17537. doi:10.1073/pnas.0807413105.

48. Malassiné A, Blaise S, Handschuh K, et al. Expression of the fusogenic HERV-FRD Env glycoprotein (syncytin 2) in human placenta is restricted to villous cytotrophoblastic cells. Placenta. 2007;28(2-3):185-191. doi:10.1016/j.placenta.2006.03.001.

49. Kudaka W, Oda T, Jinno Y, Yoshimi N, Aoki Y. Cellular localization of placenta-specific

human endogenous retrovirus (HERV) transcripts and their possible implication in pregnancy-induced hypertension. Placenta. 2008;29(3):282-289. doi:10.1016/j.placenta.2007.11.009.

50. Enders AC. A comparative study of the fine structure of the trophoblast in several hemochorial placentas. Am J Anat. 1965;116(1):29-67. doi:10.1002/aja.1001160103.

51. Henke C, Ruebner M, Faschingbauer F, et al. Regulation of murine placentogenesis by the retroviral genes Syncytin-A, Syncytin-B and Peg10. Differentiation. 2013;85(4-5):150- 160. doi:10.1016/j.diff.2013.02.002.

52. Dupressoir A, Vernochet C, Bawa O, et al. Syncytin-A knockout mice demonstrate the critical role in placentation of a fusogenic, endogenous retrovirus-derived, envelope gene. Proc Natl Acad Sci U S A. 2009;106(29):12127-12132. doi:10.1073/pnas.0902925106.

53. Dupressoir A, Vernochet C, Harper F, et al. A pair of co-opted retroviral envelope syncytin genes is required for formation of the two-layered murine placental syncytiotrophoblast. Proc Natl Acad Sci U S A. 2011;108(46):E1164-73. doi:10.1073/pnas.1112304108.

54. Mess A, Zaki N, Kadyrov M, Korr H, Kaufmann P. Caviomorph placentation as a model for trophoblast invasion. Placenta. 2007;28(11-12):1234-1238. doi:10.1016/j.placenta.2007.08.003.

55. Vernochet C, Heidmann O, Dupressoir A, et al. A syncytin-like endogenous retrovirus envelope gene of the guinea pig specifically expressed in the placenta junctional zone and conserved in Caviomorpha. Placenta. 2011;32(11):885-892. doi:10.1016/j.placenta.2011.08.006.

56. Larsen JF. Electron microscopy of the chorioallantoic placenta of the rabbit. J Ultrastruct Res. 1962;7(5-6):535-549. doi:10.1016/S0022-5320(62)90044-8.

57. Heidmann O, Vernochet C, Dupressoir A, Heidmann T. Identification of an endogenous retroviral envelope gene with fusogenic activity and placenta-specific expression in the rabbit: a new “syncytin” in a third order of mammals. Retrovirology. 2009;6(107). doi:10.1186/1742-4690-6-107.

58. Redelsperger F, Cornelis G, Vernochet C, et al. Capture of syncytin-Mar1, a fusogenic endogenous retroviral envelope gene involved in placentation in the Rodentia squirrel- related clade. J Virol. 2014;88(14):7915-7928. doi:10.1128/JVI.00141-14.

59. Cornelis G, Heidmann O, Bernard-Stoecklin S, et al. Ancestral capture of syncytin-Car1, a fusogenic endogenous retroviral envelope gene involved in placentation and conserved in Carnivora. Proc Natl Acad Sci U S A. 2012;109(7):E432-441. doi:10.1073/pnas.1115346109.

60. Carter AM, Blankenship TN, Künzle H, Enders AC. Structure of the definitive placenta of the tenrec, Echinops telfairi. Placenta. 2004;25(2-3):218-232. doi:10.1016/j.placenta.2003.08.009.

61. Cornelis G, Vernochet C, Malicorne S, et al. Retroviral envelope syncytin capture in an ancestrally diverged mammalian clade for placentation in the primitive Afrotherian tenrecs. Proc Natl Acad Sci U S A. 2014;111(41):E4332-E4341. doi:10.1073/pnas.1412268111.

62. Wooding FB. Current topic: The synepitheliochorial placenta of ruminants: Binucleate cell fusions and hormone production. Placenta. 1992;13(2):101-113. doi:10.1016/0143- 4004(92)90025-O.

63. Wathes DC, Wooding FBP. An electron microscopic study of implantation in the cow. Am J Anat. 1980;159(3):285-306. doi:10.1002/aja.1001590305.

64. Wooding FBP. Role of binucleate cells in fetomaternal cell fusion at implantation in the sheep. Am J Anat. 1984;170(2):233-250. doi:10.1002/aja.1001700208.

65. Cornelis G, Heidmann O, Degrelle SA, et al. Captured retroviral envelope syncytin gene associated with the unique placental structure of higher ruminants. Proc Natl Acad Sci U S A. 2013;110(9):E828-E837. doi:10.1073/pnas.1215787110.

66. Dunlap KA, Palmarini M, Varela M, et al. Endogenous retroviruses regulate periimplantation placental growth and differentiation. Proc Natl Acad Sci U S A. 2006;103(39):14390-14395. doi:10.1073/pnas.0603836103.

67. Zeller U, Freyer C. Early ontogeny and placentation of the grey short-tailed opossum, Monodelphis domestica (Didelphidae: Marsupialia): contribution to the reconstruction of the marsupial morphotype. J Zool Syst Evol Res. 2001;39(3):137-158. doi:10.1046/j.1439- 0469.2001.00167.x.

68. Cornelis G, Vernochet C, Carradec Q, et al. Retroviral envelope gene captures and syncytin exaptation for placentation in marsupials. Proc Natl Acad Sci U S A. 2015;112(5):E487-496. doi:10.1073/pnas.1417000112.

69. Leal F, Ramírez-Pinilla MP. Morphological variation in the allantoplacenta within the genus Mabuya (Squamata: Scincidae). Anat Rec. 2008;291(9):1124-1139. doi:10.1002/ar.20733.

70. Rand RP, Parsegian VA. Physical force considerations in model and biological membranes. Can J Biochem Cell Biol. 1984;62(8):752-759. doi:10.1139/o84-097.

71. Parsegian V a, Fuller N, Rand RP. Measured work of deformation and repulsion of lecithin bilayers. Proc Natl Acad Sci U S A. 1979;76(6):2750-2754. doi:10.1073/pnas.76.6.2750.

72. Ryham RJ, Klotz TS, Yao L, Cohen FS. Calculating transition energy barriers and characterizing activation states for steps of fusion. Biophys J. 2016;110(5):1110-1124. doi:10.1016/j.bpj.2016.01.013.

73. François-Martin C, Rothman JE, Pincet F. Low energy cost for optimal speed and control of membrane fusion. Proc Natl Acad Sci U S A. 2017;114(6):1238-1241.

doi:10.1073/pnas.1621309114.

74. Kozlov MM, Chernomordik L V. Membrane tension and membrane fusion. Curr Opin Struct Biol. 2015;33:61-67. doi:10.1016/j.sbi.2015.07.010.

75. Salditt T, Aeffner S. X-ray structural investigations of fusion intermediates: Lipid model systems and beyond. Semin Cell Dev Biol. 2016;60:65-77. doi:10.1016/j.semcdb.2016.06.014.

76. Herrmann A, Clague MJ, Puri A, Morris SJ, Blumenthal R, Grimaldi S. Effect of erythrocyte transbilayer phospholipid distribution on fusion with vesicular stomatitis virus. Biochemistry. 1990;29(17):4054-4058. doi:10.1021/bi00469a005.

77. Podbilewicz B. Virus and cell fusion mechanisms. Annu Rev Cell Dev Biol. 2014;30:111- 139. doi:10.1146/annurev-cellbio-101512-122422.

78. Harrison SC. Viral membrane fusion. Virology. 2015;479-480(7):498-507. doi:10.1016/j.virol.2015.03.043.

79. Aydin H, Cook JD, Lee JE. Crystal structures of beta- and gammaretrovirus fusion proteins reveal a role for electrostatic stapling in viral entry. J Virol. 2014;88(1):143-153. doi:10.1128/JVI.02023-13.

80. Aydin H, Smrke BM, Lee JE. Structural characterization of a fusion glycoprotein from a retrovirus that undergoes a hybrid 2-step entry mechanism. FASEB J. 2013;27(12):5059- 5071. doi:10.1096/fj.13-232371.

81. Weissenhorn W, Carfí A, Lee KH, Skehel JJ, Wiley DC. Crystal structure of the Ebola virus membrane fusion subunit, GP2, from the envelope glycoprotein ectodomain. Mol Cell. 1998;2(5):605-616. doi:10.1016/S1097-2765(00)80159-8.

82. Koellhoffer JF, Malashkevich VN, Harrison JS, et al. Crystal structure of the marburg virus GP2 core domain in its postfusion conformation. Biochemistry. 2012;51(39):7665- 7675. doi:10.1021/bi300976m.

83. Stenglein MD, Sanders C, Kistler AL, et al. Identification, characterization, and in vitro culture of highly divergent arenaviruses from boa constrictors and annulated tree boas: candidate etiological agents for snake inclusion body disease. MBio. 2012;3(4):e00180-12. doi:10.1128/mBio.00180-12.

84. Kobe B, Center RJ, Kemp BE, Poumbourios P. Crystal structure of human T cell leukemia virus type 1 gp21 ectodomain crystallized as a maltose-binding protein chimera reveals structural evolution of retroviral transmembrane proteins. Proc Natl Acad Sci U S A. 1999;96(8):4319-4324. http://www.ncbi.nlm.nih.gov/pubmed/10200260.

85. Chan DC, Fass D, Berger JM, Kim PS. Core structure of gp41 from the HIV envelope glycoprotein. Cell. 1997;89(2):263-273. http://www.ncbi.nlm.nih.gov/pubmed/9108481.

86. Pancera M, Majeed S, Ban Y-EA, et al. Structure of HIV-1 gp120 with gp41-interactive

region reveals layered envelope architecture and basis of conformational mobility. Proc Natl Acad Sci U S A. 2010;107(3):1166-1171. doi:10.1073/pnas.0911004107.

87. Igonet S, Vaney M-C, Vonrhein C, et al. X-ray structure of the arenavirus glycoprotein GP2 in its postfusion hairpin conformation. Proc Natl Acad Sci U S A. 2011;108(50):19967-19972. doi:10.1073/pnas.1108910108.

88. Maerz AL, Center RJ, Kemp BE, Kobe B, Poumbourios P. Functional implications of the human T-lymphotropic virus type 1 transmembrane glycoprotein helical hairpin structure. J Virol. 2000;74(14):6614-6621. doi:10.1128/jvi.74.14.6614-6621.2000.

89. Blinov VM, Krasnov GS, Shargunov A V., Shurdov MA, Zverev V V. Immunosuppressive domains of retroviruses: Cell mechanisms of the effect on the human immune system. Mol Biol. 2013;47(5):613-621. doi:10.1134/S0026893313050026.

90. Fan TX, Day NK, Luangwedchakarn V, et al. The phosphorylation of phospholipase C- gamma1, Raf-1, MEK, and ERK1/2 induced by a conserved retroviral peptide. Peptides. 2005;26(11):2165-2174. doi:10.1016/j.peptides.2005.04.009.

91. Luangwedchakarn V, Day NK, Hitchcock R, et al. A retroviral-derived peptide phosphorylates protein kinase D/protein kinase Cμ involving phospholipase C and protein kinase C. Peptides. 2003;24(5):631-637. doi:10.1016/S0196-9781(03)00137-2.

92. Morozov VA, Morozov A V., Semaan M, Denner J. Single mutations in the transmembrane envelope protein abrogate the immunosuppressive property of HIV-1. Retrovirology. 2012;9:67. doi:10.1186/1742-4690-9-67.

93. Renard M, Varela PF, Letzelter C, Duquerroy S, Rey F a., Heidmann T. Crystal structure of a pivotal domain of human syncytin-2, a 40 million years old endogenous retrovirus fusogenic envelope gene captured by primates. J Mol Biol. 2005;352(5):1029-1034. doi:10.1016/j.jmb.2005.07.058.

94. Kabsch W. Xds. Acta Crystallogr Sect D Biol Crystallogr. 2010;66(2):125-132. doi:10.1107/S0907444909047337.

95. Adams PD, Afonine P V., Bunkóczi G, et al. PHENIX: A comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr Sect D Biol Crystallogr. 2010;66(2):213-221. doi:10.1107/S0907444909052925.

96. McCoy AJ, Grosse-Kunstleve RW, Adams PD, Winn MD, Storoni LC, Read RJ. Phaser crystallographic software. J Appl Crystallogr. 2007;40(4):658-674. doi:10.1107/S0021889807021206.

97. Bunkóczi G, Read RJ. Improvement of molecular-replacement models with Sculptor. Acta Crystallogr Sect D Biol Crystallogr. 2011;67(4):303-312. doi:10.1107/S0907444910051218.

98. Emsley P, Lohkamp B, Scott WG, Cowtan K. Features and development of Coot. Acta Crystallogr Sect D Biol Crystallogr. 2010;66(4):486-501.

doi:10.1107/S0907444910007493.

99. Painter J, Merritt EA. TLSMD web server for the generation of multi-group TLS models. J Appl Crystallogr. 2006;39(1):109-111. doi:10.1107/S0021889805038987.

100. Painter J, Merritt EA. Optimal description of a protein structure in terms of multiple groups undergoing TLS motion. Acta Crystallogr Sect D Biol Crystallogr. 2006;62(4):439-450. doi:10.1107/S0907444906005270.

101. Chen VB, Arendall WB, Headd JJ, et al. MolProbity: All-atom structure validation for macromolecular crystallography. Acta Crystallogr Sect D Biol Crystallogr. 2010;66(1):12-21. doi:10.1107/S0907444909042073.

102. Echols N, Grosse-Kunstleve RW, Afonine P V., et al. Graphical tools for macromolecular crystallography in PHENIX. J Appl Crystallogr. 2012;45(3):581-586. doi:10.1107/S0021889812017293.

103. The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC.

104. Nguyen MN, Tan KP, Madhusudhan MS. CLICK--topology-independent comparison of biomolecular 3D structures. Nucleic Acids Res. 2011;39(Web Server issue):W24-8. doi:10.1093/nar/gkr393.

105. Nguyen MN, Madhusudhan MS. Biological insights from topology independent comparison of protein 3D structures. Nucleic Acids Res. 2011;39(14):e94. doi:10.1093/nar/gkr348.

106. Urzhumtseva L, Afonine P V., Adams PD, Urzhumtsev A. Crystallographic model quality at a glance. Acta Crystallogr Sect D Biol Crystallogr. 2009;65(3):297-300. doi:10.1107/S0907444908044296.

107. Touw WG, Baakman C, Black J, et al. A series of PDB-related databanks for everyday needs. Nucleic Acids Res. 2015;43(D1):D364-D368. doi:10.1093/nar/gku1028.

108. Krissinel E, Henrick K. Inference of macromolecular assemblies from crystalline state. J Mol Biol. 2007;372(3):774-797. doi:10.1016/j.jmb.2007.05.022.

109. Strelkov S V., Burkhard P. Analysis of α-helical coiled coils with the program TWISTER reveals a structural mechanism for stutter compensation. J Struct Biol. 2002;137(1-2):54- 64. doi:10.1006/jsbi.2002.4454.

110. Kumar S, Stecher G, Tamura K. MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. Mol Biol Evol. 2016;33(7):1870-1874. doi:10.1093/molbev/msw054.

111. Edgar RC. MUSCLE: Multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 2004;32(5):1792-1797. doi:10.1093/nar/gkh340.

112. Le SQ, Gascuel O. An improved general amino acid replacement matrix. Mol Biol Evol.

2008;25(7):1307-1320. doi:10.1093/molbev/msn067.

113. Gascuel O. BIONJ: an improved version of the NJ algorithm based on a simple model of sequence data. Mol Biol Evol. 1997;14(7):685-695. doi:10.1093/oxfordjournals.molbev.a025808.

114. Jones DT, Taylor WR, Thornton JM. The rapid generation of mutation data matrices from protein sequences. Comput Appl Biosci. 1992;8(3):275-282. http://www.ncbi.nlm.nih.gov/pubmed/1633570.

115. Felsenstein J. PHYLIP (Phylogeny Inference Package). 2005.

116. Saitou N, Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol. 1987;4(4):406-425. doi:10.1093/oxfordjournals.molbev.a040454.

117. Kawamoto S, Klein ML, Shinoda W. Coarse-grained molecular dynamics study of membrane fusion: Curvature effects on free energy barriers along the stalk mechanism. J Chem Phys. 2015;143(24):243112-1-243112-10. doi:10.1063/1.4933087.

118. Markosyan RM, Melikyan GB, Cohen FS. Tension of membranes expressing the hemagglutinin of influenza virus inhibits fusion. Biophys J. 1999;77(2):943-952. doi:10.1016/S0006-3495(99)76945-6.

119. Cáceres M, Thomas JW. The gene of retroviral origin syncytin I is specific to hominoids and is inactive in old world monkeys. J Hered. 2006;97(2):100-106. doi:10.1093/jhered/esj011.

120. Grandi N, Cadeddu M, Blomberg J, Mayer J, Tramontano E. HERV-W group evolutionary history in non-human primates: characterization of ERV-W orthologs in Catarrhini and related ERV groups in Platyrrhini. BMC Evol Biol. 2018;18(6):1-14. doi:10.1186/s12862-018-1125-1.

121. Tolosa JM, Schjenken JE, Clifton VL, et al. The endogenous retroviral envelope protein syncytin-1 inhibits LPS/PHA-stimulated cytokine responses in human blood and is sorted into placental exosomes. Placenta. 2012;33(11):933-941. doi:10.1016/j.placenta.2012.08.004.

122. Tolosa JM, Parsons KS, Hansbro PM, Smith R, Wark PAB. The placental protein syncytin-1 impairs antiviral responses and exaggerates inflammatory responses to influenza. Schmolke M, ed. PLoS One. 2015;10(4):e0118629. doi:10.1371/journal.pone.0118629.

123. Hummel J, Kämmerer U, Müller N, Avota E, Schneider-Schaulies S. Human endogenous retrovirus envelope proteins target dendritic cells to suppress T-cell activation. Eur J Immunol. 2015;45(6):1748-1759. doi:10.1002/eji.201445366.

124. Schlecht-Louf G, Mangeney M, El-Garch H, Lacombe V, Poulet H, Heidmann T. A targeted mutation within the feline leukemia virus (FeLV) envelope protein

immunosuppressive domain to improve a canarypox virus-vectored FeLV vaccine. J Virol. 2014;88(2):992-1001. doi:10.1128/JVI.02234-13.

125. Henzy JE, Johnson WE. Pushing the endogenous envelope. Philos Trans R Soc Lond B Biol Sci. 2013;368(1626):20120506. doi:10.1098/rstb.2012.0506.

126. Malassiné A, Frendo J-L, Evain-Brion D. Trisomy 21- affected placentas highlight prerequisite factors for human trophoblast fusion and differentiation. Int J Dev Biol. 2010;54(2-3):475-482. doi:10.1387/ijdb.082766am.

Figure 2A - Reproduced and edited with permission of the Japanese Society of Toxicologic Pathology from Furukawa, et al. A Comparison of the Histological Structure of the Placenta in Experimental Animals. J Toxicol Pathol 27: 11-18, 2014.

Figure 2B - Reproduced and edited with permission of PNAS from Cornelis, et al. Captured retroviral envelope syncytin gene associated with the unique placental structure of higher ruminants. PNAS. 110(9): E828-E837, 2013

Figure 3 - Reproduced and edited with permission of PNAS from Cornelis, et al. An endogenous retroviral envelope syncytin and its cognate receptor identified in the viviparous placental Mabuya lizard. PNAS. 114(51): E10991-E11000, 2017

Figure 4 Top - Reproduced and edited with permissions of Annual Review of Cell and Developmental Biology from Podbilewicz. Virus and cell fusion mechanisms. Annu Rev Cell Dev Bio. 30:111-139, 2014

Figure 4 Bottom - Reproduced and edited with permissions of Biophysical Journal from Ryham, et al. Calculating transition energy barriers and characterizing activation states for steps of fusion. Biophys J. 110(5):1110-1124, 2016

Figure 5 - Reproduced and edited with permissions of Annual Review of Cell and Developmental Biology from Podbilewicz. Virus and cell fusion mechanisms. Annu Rev Cell Dev Bio. 30:111-139, 2014