cells under siege viral glycoprotein interactions at the cell surface

Stuarta,b a Division of Structural Biology, University of Oxford, Wellcome Trust Centre for Human Genetics, Roosevelt Drive, Oxford OX3 7BN, United Kingdom bScience Division, Diamond Lig

Cells under siege: Viral glycoprotein interactions at the cell surface

Thomas A Bowdena,⇑ , E Yvonne Jonesa, David I Stuarta,b

a

Division of Structural Biology, University of Oxford, Wellcome Trust Centre for Human Genetics, Roosevelt Drive, Oxford OX3 7BN, United Kingdom

bScience Division, Diamond Light Source Ltd., Diamond House, Harwell Science and Innovation Campus, Didcot, Oxfordshire 0X11 0DE, United Kingdom

a r t i c l e i n f o

Article history:

Available online 31 March 2011

Keywords:

Glycoprotein structure

Virus entry

Cell signaling

X-ray crystallography

Cell surface receptors

a b s t r a c t

As obligate parasites, viruses are required to enter and replicate within their host, a process which employs many of their proteins to hijack natural cellular processes High resolution X-ray crystallo-graphic analysis has proven to be an ideal method to visualize the mechanisms by which such virus-host interactions occur and has revealed the innovative capacity of viruses to adapt efficiently to their hosts In this review, we draw upon recently elucidated paramyxovirus-, arenavirus-, and poxvirus-host protein complex crystal structures to reveal both the capacity of viruses to appropriate one component of a phys-iological protein–protein binding event (often modifying it to out-compete the host-protein), and the ability to utilize novel binding sites on host cell surface receptors The structures discussed shed light

on a number of biological processes ranging from viral entry to virulence and host antagonism Drawn together they reveal the common strategies which viruses have evolved to interact with their natural host The structures also support molecular level rationales for how viruses can be transmitted to unre-lated organisms and thus pose severe health risks

Ó 2011 Elsevier Inc All rights reserved

1 Introduction

Viruses have tremendous genetic diversity ( Edwards and

Roh-wer, 2005 ), a property largely accounted for by rapid replication,

frequent and unspecific mutations arising from error-prone

poly-merases, and an ability to recombine host genes into their own

genome The resulting capability of a single virion to generate a

genetically diverse complement of progeny provides a simple

mechanism by which virus-host cell interactions can rapidly

be-come specialized for specific host ranges and tissues Combined

with the ability to ‘steal’ host proteins, this provides a powerful

method by which viruses hijack natural host cell functions,

facili-tating processes such as viral attachment and antagonism of the

host’s innate immune response ( Bahar et al., 2011 ).

Crystallographic studies of viral proteins alone and in complex

with their functional ligands have led to a greater appreciation of

how the structurally dissimilar fold architectures resulting from

viral genomic diversity can achieve analogous biological processes.

Structural investigations of viral attachment glycoproteins, for example, have shown that enveloped viruses adopt a wide range

of folds optimized for engagement of their cognate cellular recep-tors These folds vary from the compact and novel a /b fold of Arenaviridae ( Fig 1 A) ( Abraham et al., 2010; Bowden et al., 2009a ), to the trimeric GP1 ‘chalice’ of the Filoviridae ( Fig 1 B) ( Lee et al., 2008 ), the globular six-bladed b-propeller of the Paramyxovirinae ( Fig 1 C) ( Bowden et al., 2010a ), the large trimeric hemagglutinin of the Orthomyxoviridae ( Fig 1 D) ( Weis et al., 1988; Wilson et al., 1981 ), and the highly glycosylated GP120 trimer of Lentiviruses in the Retroviridae ( Wyatt et al., 1998; Zhu et al.,

2003 ) It is noteworthy that the associated fusion glycoproteins from each of the above virus families, in contrast to the attachment glycoproteins, are similar in architecture and have all been grouped into the first of the three known structural classes of fusion proteins ( Eschli et al., 2006; Lamb and Jardetzky, 2007; Lee et al., 2008 ) ( Fig 1 AD) It has been suggested that these proteins are related ( Kadlec et al., 2008 ).

Fusion and receptor-binding proteins are often synthesized on the same polypeptide and fold together to form complexes on the virus surface As a result, these protein pairs have not evolved entirely independently Nevertheless, receptor-binding attachment glycoproteins display much greater structural diversity than their fusion glycoprotein counter-parts This is likely to stem from whether there is a functional requirement for a given viral protein

to adapt to and interact with its hosts Viral nucleoproteins and fu-sion glycoproteins (with the exception of the immunosuppressive

1047-8477/$ - see front matter Ó 2011 Elsevier Inc All rights reserved

Abbreviations: GAP, GTPase-activating protein; IPT, Ig-like plexins and

tran-scription factors; HeV, Hendra virus; HeV-G, Hendra virus attachment glycoprotein;

HNV, Henipavirus; HNV-G, Henipavirus attachment glycoprotein; NiV, Nipah virus;

NiV-G, Nipah virus attachment glycoprotein; MACV, Machupo virus; PDB, protein

databank; PSI, plexin-semaphrorin-integrin domain; r.m.s.d., root mean square

deviation; Tf, transferrin; TfR1, transferrin receptor 1; SLAM, Signaling Lymphocytic

Activation Molecule; SPINE, Structural Proteomics In Europe

⇑Corresponding author Fax: +44 (0)1865 287547

E-mail address:tom@strubi.ox.ac.uk(T.A Bowden)

Journal of Structural Biology 175 (2011) 120–126

Contents lists available at ScienceDirect

Journal of Structural Biology

j o u r n a l h o m e p a g e : w w w e l s e v i e r c o m / l o c a t e / y j s b i

segments of some fusion glycoproteins) ( Avota et al., 2010; Cianciolo

et al., 1985; Kleinerman et al., 1987; Volchkov et al., 2001; Yang

et al., 2000 ), interact less-specifically with their host cell and have

a relatively self-contained function (e.g insertion into and merging

of the viral and host envelopes or packaging of genomic material)

which requires minimal adaption Certain non-structural proteins

and attachment glycoproteins, on the other hand, are examples of

viral proteins which more often interact specifically with their host

cell and adapt rapidly to cellular host factors.

Using recently elucidated paramyxovirus-, arenavirus-, and

poxvirus-host complex crystal structures resulting from the Spine

(Structural Proteomics In Europe) 2-Complexes initiative as

exam-ples (detailed in Table 1 ), we draw upon this second, adaptive

group of viral proteins to reveal the varied strategies employed

by viruses when interacting with their hosts Setting these results

in a broader context, through comparison with other structurally

well established viral glycoprotein systems including HIV and Measles virus, we illustrate that viruses not only subvert binding sites that are used in natural physiological signaling processes, but can also exploit novel sites on host proteins previously not used as interaction surfaces.

2 Viral semaphorins and immune antagonism Semaphorins comprise a family of cell surface signaling glyco-proteins which, through binding to the family of plexin glycopro-tein cell surface receptors, activate repulsive guidance pathways which are fundamental to a number of physiological processes including axon guidance, immune regulation and activation, and vascular development ( Kruger et al., 2005; Suzuki et al., 2007 ) There are eight known classes of semaphorins: two found in inver-tebrates, five in verinver-tebrates, and the eighth class in viruses which are known as ‘viral semaphorins’ ( Comeau et al., 1998; Ensser and Fleckenstein, 1995 ) Whilst the ectodomains of cellular sem-aphorins contain C-terminal domain elaborations such as PSI (plexin, semaphorin and integrin) domains, immunoglobulin (Ig)-like domains, thrombospondin domains and PDZ-domain-binding sites which may or may not attach to the cell-surface, the N-terminal portion, comprising a plexin-binding sema-domain, is well conserved The sema-domain is the only component found

in viruses Crystallographic studies, by ourselves and others, have shown that the human Sema3A and mouse Sema4D semadomains consist of a structurally conserved homodimer of seven-bladed b-propellers (1.7 Å root mean square deviation, r.m.s.d., for match-ing C a atoms) ( Antipenko et al., 2003; Janssen et al., 2010; Liu

et al., 2010; Love et al., 2003; Nogi et al., 2010 ).

The domain architecture is conserved amongst the four classes (A–D) of vertebrate plexin type-I membrane glycoproteins and consists of an N-terminal, membrane distal sema-domain which

is anchored to the membrane by PSI domains and IPT (Ig-like, plex-ins and transcription factors) domaplex-ins ( Bork et al., 1999 ) A GTPase-binding domain and a C-terminal segment GAP (GTPase-activating protein) domain constitute the intracellular portion of

Fig 1 Contrasts in fold conservation between attachment and fusion glycoproteins from negative-sense, single-stranded RNA viruses Receptor-binding attachment glycoproteins are shown above and the six-helix bundles from their cognate fusion glycoproteins below from (A) Machupo Arenavirus (PDB identification number 2WFO), (B) Ebola Filovirus (PDB ID 3CSY and 1EBO), (C) Nipah (above) and Parainfluenza type-III (below) Paramyxoviruses (PDB ID 2VSM and 1ZTM, respectively), and (D) Flu Orthomyxovirus (PDB ID 3LZG) Note for panel A, there are currently no known crystal structures of arenaviral fusion glycoproteins, in panel B, Ebola GP1 is shown with its non-covalently associated GP2 subunit (gray cartoon), and in panel D, Flu virus haemagglutinin is shown with the HA1 domain colored as a rainbow and the HA2 domain colored gray

Table 1

Relevant structures solved under the European Spine initiatives

Structure Reference PDB accession code

Semaphorin4DEctoa Love et al (2003) 1OLZ

PlexinA2D1-4b Janssen et al (2010) 3OKT

Semaphorin6AEcto Janssen et al (2010) 3OKW

Semaphorin4DEcto-PlexinB1D1-2 Janssen et al (2010) 3OL2

Semaphorin6AEcto-PlexinA2D1-4 Janssen et al (2010) 3OKY

EphA4LBDc Bowden et al (2009b) 2WO1

EphA4LBD-ephrinB2RBDd Bowden et al (2009b) 2WO2

EphA4LBD-ephrinA2RBD Bowden et al (2009b) 2WO3

EphA2Ecto Seiradake et al (2010) 2X10

EphA2Ecto-ephrinA5RBD Seiradake et al (2010) 2X11

NiV-G Bowden et al (2008b) 2VWD

HeV-G Bowden et al (2010b) 2X9M

NiV-G-ephrinB2 Bowden et al (2008a) 2VSM

HeV-G-ephrinB2 Bowden et al (2008a) 2VSK

MACV-GP1 Bowden et al (2009a) 2WFO

a Ecto, entire ectodomain

b D1–4, domains 1–4

c

LBD, ligand binding domain

d

RBD, receptor binding domain

the glycoprotein and are responsible for activation of Rho family

GTPase signaling pathways within the plexin-expressing cell (

Kru-ger et al., 2005 ).

Immunoregulatory semaphorins including Sema3A, 4A, 4D, and

7A contribute to B cell mediated immunity (Sema4D), T cell

activa-tion and differentiaactiva-tion (Sema4A, Sema3A, and Sema4D), and

inflammation (Sema7A) ( Suzuki et al., 2008 ) Genomic sequencing

of eukaryotic viruses has revealed that Poxviruses (e.g Smallpox

virus and Vaccinia virus) and alcelaphine herpes virus encode viral

semaphorins which have been shown to modulate these processes

( Comeau et al., 1998; Ensser and Fleckenstein, 1995; Suzuki et al.,

2008 ) These viral encoded proteins were presumably ‘stolen’ from

their host during the process of virus-host co-evolution ( Suzuki

et al., 2008 ) The glycoprotein A39R, encoded by Vaccinia virus,

for example, shares highest sequence homology with Sema7A

(30%) and undermines the host immune response by binding to

plexinC1 ( Comeau et al., 1998 ).

Recent crystallographic studies of semaphorin-plexin signaling

complexes, in part within the activity of Spine2-Complexes, have

enabled a detailed comparison of the mechanism by which

natural- and viral-semaphorins bind to plexins Structures of

Sema7A-plexinC1 ( Liu et al., 2010 ), Sema6A-plexinA2 ( Janssen

et al., 2010; Nogi et al., 2010 ), and Sema4D-plexinB1( Janssen

et al., 2010 ), have revealed structurally similar signaling

com-plexes, all composed of a Sema-plexin heterotetramer where each

protomer of the semaphorin dimer binds to one plexin

seven-bladed b-propeller sema domain ( Fig 2 A and B) Elucidation of

the crystal structure of vaccinia virus A39R in complex with

plex-inC1 demonstrates that poxviruses take advantage of an almost

identical binding mechanism to that of physiological

semaphorin-plexin signaling, where both the viral and the cell semaphorin

b-propellers bind to their cognate plexin b-propellers in a side-on

orientation ( Fig 2 C) ( Liu et al., 2010 ) Furthermore, although the

binding interface of the viral complex is less extensive to that

observed in analogous physiological complexes

(Sema7A-plex-inC1), the binding strength of the A39R-plexinC1 interaction is

significantly enhanced (Kd of 10 nM versus 300 nM) ( Liu

et al., 2010 ) Whilst Sema-plexin binding affinities are delicately

balanced to contribute to the complex interplay of interactions

required for physiological functions, Vaccinia virus protein A39R

can simply optimize a single interaction These studies are an

example of how genetic variability can give rise to mechanisms

which enhance virus virulence and replication In addition to

incor-porating genes from their host organism, these structures provide a

molecular basis for how viruses can optimize their own proteins to

override normal physiological interactions.

3 Henipavirus entry: The ephrin gateway Nipah virus and Hendra virus compose the genus Henipavirus within the Paramyxoviridae family and are emergent and highly virulent bat-borne pathogens found in Africa, Australia, and South East Asia ( Eaton et al., 2006; Wild, 2009 ) Oligomeric complexes of two glycoproteins extending outward from the viral envelope are required for efficient attachment (G glycoprotein) and fusion (F glycoprotein) into their respective host cells During henipaviral attachment, the F glycoprotein is activated in a pH independent mechanism ( Aguilar et al., 2009 ) to undergo classical class I fusion rearrangements which merge the host and viral membranes ( Lou

et al., 2006 ).

NiV-G and HeV-G (collectively referred to as HNV-G) are type-II transmembrane glycoproteins consisting of an N-terminal cytoplasmic tail, a short transmembrane region, an ectodomain stalk region, and a C-terminal receptor binding six-bladed b-pro-peller domain HNV-G glycoproteins are important in determin-ing the broad species and cellular tropism of these viruses as they have been observed to bind specifically with ephrinB2 and ephrinB3 cell surface receptors at nanomolar affinity ( Bona-parte et al., 2005; Negrete et al., 2005, 2006 ) The sequences of ephrinB2 and ephrinB3 are well conserved amongst many verte-brate species including humans, bats, horses, and pigs (>95% se-quence identity) ( Bossart et al., 2008 ), and they are ubiquitously expressed in most human tissues due to their importance in fun-damental bi-directional cell signaling processes such as osteo-genesis, axon guidance, and vascular development ( Hafner

et al., 2004; Pasquale, 2005 ).

Crystal structures of ephrin ligands alone and in complex with their Eph receptors (determined by others and as part of Spine2-Complexes) have been invaluable for identifying the molecular specificity which underlies normal physiological signaling events These studies reveal that the ephrin ectodomain forms a compact greek-key fold containing a 10 amino acid (GH) binding loop, which is predominantly responsible for Eph receptor binding through its insertion into the receptor binding cleft of the mem-brane-distal Eph receptor b-sandwich domain ( Bowden et al., 2009b; Chrencik et al., 2006a, 2006b; Himanen et al., 2001, 2009,

2010, 2004; Nikolov et al., 2007, 2005; Qin et al., 2010; Seiradake

et al., 2010; Toth et al., 2001 ) ( Fig 3 A).

Site-directed mutagenesis of ephrinB2 and ephrinB3 confirmed that HNV-G subverts natural Eph receptor binding by also utilizing this GH loop during viral attachment ( Negrete et al., 2006 ) How-ever, rather than completely imitating the exact binding mode ob-served in physiological Eph-ephrin interactions, structural studies

Fig 2 Poxviral appropriation of semaphorin-plexin interactions (A) Crystal structure of Sema7a in complex with PlexinC1 (PDB ID 3NVQ) The PlexinC1 seven-bladed b-propeller domains are rendered as gray surfaces and the Sema7A dimer is shown as a cartoon with each protomer colored as a rainbow with the N-terminus in blue and the C-terminus in red Close-up view of protein-protein interactions in the (B) Sema7a-PlexinC1 and (C) poxvirus A39R-PlexinC1 complex (PDB ID 3NVN) interfaces reveal nearly

122 T.A Bowden et al / Journal of Structural Biology 175 (2011) 120–126

of HNV-G alone and in complex with ephrinB2 and ephrinB3 show

that the GH loop is plastic and undergoes a unique rearrangement

that allows it to bind to the top center portion of the HNV-G

b-pro-peller ( Fig 3 B) ( Bowden et al., 2008a; Xu et al., 2008 ) As the GH

loop is well conserved between many vertebrate species including

bats and humans, this observation provides a molecular level

rationale for Henipaviral zoonosis ( Bowden et al., 2008a ) The

conformational changes observed in the ephrins, in addition to

those occurring to the HNV-G b-propeller upon binding ( Bowden

et al., 2010b, 2008a, 2008b; Xu et al., 2008 ), result in a

protein-protein interface which is similarly tight (nanomolar affinity) but

more extensive than physiological Eph-ephrin interactions (an

HNV-G-ephrinB2 interface of approximately 2700 Å2compared to

an average of 2200 Å2buried surface area for an Eph-ephrin

com-plex) Despite differences in the extent of these interactions, the

protein-protein interfaces in both sets of structures are dominated

by hydrophobic contacts between aromatic sidechains of ephrinB2

and ephrinB3 (e.g Phe120ephrinB2and Trp 125ephrinB2) with binding

pockets on the physiological Eph and viral HNV-G glycoproteins.

Such binding surfaces are reminiscent to the hydrophobic contacts

observed in structures of CD4 in complex with MHC class II and

HIV GP120 glycoproteins The interfaces in both CD4 complexes

are dominated by the insertion of Phe43CD4 into hydrophobic

cavities present on the MHC and HIV GP120 glycoproteins ( Kwong

et al., 1998; Wang et al., 2001 ).

The structural properties observed in henipavirus-ephrin

inter-actions, in addition to those observed in the attachment

glycopro-teins from other paramyxoviruses (e.g Measles hemagglutinin in

complex with cell surface SLAM ( Hashiguchi et al., 2011 ) and

CD46 ( Santiago et al., 2010 )), underscore the adaptability of the

viral six-bladed b-propeller scaffold ( Bowden et al., 2010a; Stehle

and Casasnovas, 2009 ), and in a broader context, the innovative

ability of viruses to alter their glycoprotein repertoire to target

new receptors and hosts.

4 Convergent viral attachment through transferrin receptor targeting

The transferrin receptor (TfR1) is a type-2 membrane glycopro-tein which regulates the cellular uptake of iron through binding to its ligand, transferrin (Tf) and is almost ubiquitously expressed in different human tissues ( Ponka and Lok, 1999 ) Upon binding to mono-ferric or di-ferric Tf, the TfR1-Tf complex is internalized through clathrin-dependent endocytosis and later is freed from TfR1 in acidic compartments ( Ponka and Lok, 1999 ) TfR1 exists

as a disulfide-linked dimer which consists of an N-terminal cyto-plasmic domain, a transmembrane region and a 650 amino acid ectodomain A major portion of the TfR1 ectodomain has been crystallized and shown to consist of a protease-like domain, a heli-cal domain and an apiheli-cal domain ( Fig 4 A) ( Bennett et al., 2000; Lawrence et al., 1999 ) Structures of TfR1 in complex with Tf and HFE, a membrane glycoprotein associated with hereditary haemo-chromatosis ( Bomford, 2002; Lebron et al., 1999 ), have been eluci-dated by cryo-electron microscopy ( Cheng et al., 2004 ) and crystallography ( Bennett et al., 2000 ), respectively HFE can com-pete with Tf for binding and both complex structures revealed a 2:2 stoichiometry ( Fig 4 B) In these structures, the Tf and HFE binding sites overlap at the membrane proximal TfR1 helical do-main ( Fig 4 B) and are extensive; the crystal structure of the TfR1–HFE complex revealed the occlusion of approximately

2000 Å2of solvent accessible surface.

In addition to its importance in iron delivery into cells, TfR1 has emerged as an entry receptor for a number of important pathogens including mouse mammary tumor virus ( Ross et al., 2002 ), canine and panleukopenia feline parvoviruses ( Parker et al., 2001 ), and New World hemorrhagic fever arenaviruses ( Radoshitzky et al.,

2007 ) These viruses differ markedly in properties: canine and pan-leukopenia feline parvoviruses are small (26 nm in diameter), icosohedral, single-stranded DNA viruses that do not contain a

Fig 3 Differential utilization of the GHephrinB2loop by Eph receptors and Henipaviruses (A) Crystal structure of EphB2 in complex with ephrinB2 (PDB ID 1KGY) (B) Crystal structure of NiV-G in complex with ephrinB2 (PDB ID 2VSM) For both panels, structures are shown as cartoons with EphB2 and NiV-G colored in gray and ephrinB2 colored as

a rainbow with the N-terminus in blue and the C-terminus in red The primary ephrinB2 interaction loop is highlighted with a thicker radius and the side-chains of residues important for both protein-protein interactions are labeled and shown in a ball and stick representation

lipid bilayer envelope, whilst mouse mammary tumor virus and

New World hemorrhagic fever arenaviruses are large,

plieomor-phic, enveloped viruses (100 and 120 nm in diameter,

respec-tively) which contain single-stranded RNA genomes Structural

and functional studies of these viruses have shown that they attach

to sites on TfR1 which do not overlap with the physiological Tf and

HFE binding sites ( Radoshitzky et al., 2007 ) The structure of the

Machupo virus attachment glycoprotein, GP1, determined as a part

of the Spine2-Complexes project, revealed a novel protein fold

( Bowden et al., 2009a ) A subsequent crystal structure of GP1 in

complex with TfR1 has revealed an extensive binding site

occlud-ing over 1900 Å2 of solvent accessible surface at the tip of the

membrane distal TfR1 apical domain ( Fig 4 C) ( Abraham et al.,

2010 ) Similarly, functional and electron microscopic data suggest

mouse mammary tumor virus and canine and feline parvoviruses

also utilize the TfR1 apical domain for attachment ( Goodman

et al., 2010; Hafenstein et al., 2007; Palermo et al., 2003; Wang

et al., 2006 ).

It has been suggested that this TfR1 ‘viral binding patch’ is used

as it is remote from the known physiological Tf and HFE cellular

binding sites and unlikely to disturb TfR1 endocytosis ( Goodman

et al., 2010 ) Given this hypothesis and the innate ability of viruses

to evolve rapidly, it is not surprising that these diverse viruses have

independently evolved similar molecular mechanisms to rely on

the TfR1 cell surface receptor for virus entry.

5 Concluding remarks

The rapidity of sequence changes in viral genomes is

fundamen-tal for the survival of many viruses It enables co-evolution with

natural host reservoirs as well as opportunities to adapt to and

in-fect new hosts Such genetic variability is thus extremely

problem-atic from a biomedical perspective For example, poxviruses such

as smallpox virus have used these properties to ‘steal’ and optimize

host cell genes such as the plexinC1 interacting semaphorin, A39R,

for antagonism of the host immune system Emergent RNA viruses

such as henipa- and arena-viruses, on the other hand, have relied

on genetic diversity to develop very different attachment

glycopro-tein folds which can be used to bind to a variety of host cell surface

receptors The methodological advances in eukaryotic cell

expres-sion for macromolecular crystallography developed in Spine

( Aricescu et al., 2006; Chang et al., 2007 ) have been invaluable

for understanding the molecular basis of these virus-host cell

interactions.

In this review, recently elucidated virus-host crystal structures, many of which have emerged from the Spine2-Complexes project, have shown how viruses both appropriate existing cellular interac-tions (e.g A39R binding to PlexinC1 and henipavirus attachment to ephrins) as well utilize novel modes for host-interaction (e.g are-naviral attachment to TfR1) These structural insights when drawn together reveal common molecular-level strategies which viruses have evolved to interact with their natural host and result in a dan-ger to human and animal health.

Acknowledgments

We would like to thank Dr Richard Elliott, Dr Max Crispin, and

Dr Xiaoyun Ji for helpful discussions David I Stuart is an Medical Research Council Professor of Structural Biology, E Yvonne Jones is

a Cancer Research UK Principal Research Fellow, Thomas A Bow-den is a Sir Henry Wellcome Postdoctoral Fellow and Junior Re-search Fellow at University College, Oxford This work was funded by the Wellcome Trust [Grant Number 089026/Z/09/Z], Medical Research Council, Cancer Research UK, and Spine2-Complexes [Grant Number FP6-RTD-031220].

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