71v
v
v
v

h#p://www.nih.go.jp/niid/ja/kansennohanashi/441-hfmd.html

7–9

EV71
手足口病病原体その他

5Echovirus 18

2000

EV71

Coxsackievirus A2
Coxsackievirus A4

件数

1500

Coxsackievirus A5

1975
1978
1997
1998

Coxsackievirus A6

1000

Coxsackievirus A9
500

2016

2015

2014

2013

2012

2011

2010

2009

2008

2007

2006

2005

0

Coxsackievirus A10

44
47
34
78

Coxsackievirus A16

2000, 2001

96

Enterovirus 71

2008

1890

2011

2011
2012

166
63

EV71
A
Sapelovirus

13-15
18

HEV-D

Enterovirus
Teschovirus
Cardiovirus
Senecavirus

Picornaviridae

Erbovirus

HEV-A

0.1%

HRV-A
HRV-C
HRV-B

1960
5000

Aphthovirus
HEV-B
Kobuvirus
Hepatovirus
Tremovirus

HEV-C

EV71

Avihepatovirus
Parechovirus
wikipedia

EV71
2010 6 11

30 nm

Picorna Pico (=) + RNA

ARTICLES
2010
7808

29
2000

Figure 1 Overall structures. (a) Cartoon of themature EV71 virion, looking down an icosahedral
two-fold axis, VP1, VP2, VP3 and VP4 are drawn
in blue, green, red and yellow, respectively.
A single icosahedral protomer is drawn morebrightly. (b) Radius-colored surface representationof the EV71 mature and empty particles. The
surfaces for both are colored from blue to red
according to their distances from the particle
center (blue being closest). (c) Structures of the
EV71 capsid proteins. VP0–VP3 (we treat VP0and VP2 interchangeably) are shown in a similar
orientation to the brighter protomer in a; proteinsfrom the expanded empty particle are colored as
in a, whereas the corresponding chains from themature virion are gray. The proteins have beensuperimposed as rigid bodies. Residues 1–297 of
VP1, 10–254 of VP2, 1–242 of VP3 and 12–69
of VP4 have well-defined electron density in themature virion; residues 73–210 and 219–297 of
VP1, 82–319 of VP0 and 1–238 of VP3 in theempty particle are modeled. Major surface-exposedloops are marked with colored circles: yellow, VP1
BC; orange, VP1 DE; red, VP1 HI; magenta, VP1
GH; light blue, VP2 EF; black, VP3 GH.

3’
VP4

VP0

3’

3B
VP1

2A

2B

2C

P1

P2

P1

P2

VP3

VP1

2A

3A

3C

Poly(A)

3D

P3
P3

2BC

3AB

VP4

3CD
3B

VP2

VP3

VP1

2A

VP2

Mature virion

2B

2C

3A

3C

3D

c

N

C
C

N

C

N
VP1

VOLUME 19

VP2

VP3

are appreciably altered, the interactions that hold the particle t
are strongly reduced (Supplementary Table 1) and the ca
the expanded particle is thinned to 20 Å from 23 Å in thevirus (Fig. 2c). The core structures of the individual polyp
are less affected as CAs superpose with r.m.s. deviations o
0.9 Å and 1.3 Å for VP1, VP0(2) and VP3, respectively (Falthough some important rearrangements occur.

Enteroviruses adopt two fundamental configurations
The ~4% expansion of the EV71 82S empty particle is simila
seen for the poliovirus 135S and 80S uncoating intermediates27,
latter has similar hydrodynamic properties. Cryo-EM analyses rthat the poliovirus 135S and 80S particles are structurally sieach other27 (although the 80S particle has shed the viral genom
are reconfigured compared to the mature virus24,25. We used V
to compare the EV71 expanded particle with the poliovirus p
Unexpectedly, the EV71 particle fit the poliovirus 135S and 80S
densities as well as the poliovirus structure previously modeled
density (Fig. 2d–g); thus, at low resolution the EV71 expanded-a
byproduct is indistinguishable from poliovirus uncoating interm
Furthermore, heat treatment of mature EV71 particles (using a p
similar to that which produces 135S poliovirus particles) conver
to particles that crystallize isomorphously with 82S immature p(data not shown). It therefore seems that the plethora of enteparticles formed during assembly and uncoating possess ofundamental configurations, which are both now defined indetail. The mature virus particle, which is rigid and rendere
stable by cleavage of VP0, is generally converted to the second, ex
configuration after cell attachment. This flexible expanded par
adopt subtly different conformations during the process of un
as it progresses from 135S to 80S form25, but it can also arise fconversion of unstable VP0 containing natural empty particles
for poliovirus10,11 and now EV71.

Mechanics of particle expansion
Particle expansion is accompanied by a 5.4° counterclockwise ro
the protomeric building block (VP1, VP0, VP3), which pivots acorner of VP3 at the icosahedral three-fold axis (Fig. 3a). This sc

NUMBER 4

APRIL 2012

NATURE STRUCTURAL & MOLECULAR BI

EV71
hSCARB2…
SCARB2 and EV71 infection• EV71human
scavenger receptor class b
member(hSCARB2)
is involvedin this 2
pathway.
The involvement of SCARB2 in thispathway should beSCARB2
confirmed experimentally.

Yamayoshi et al.

v

It is known that the poliovirus receptor (CD155) and the major[Yamayoshi Nat Med (2009)]
group rhinovirus receptor (intercellular adhesion molecule-1) areable to bind poliovirus and rhinovirus, respectively, and induce•a conformationalhSCARB2change
EV71that leads to the uncoating of the viralgenome.
Chen et al. (2012) reported
that SCARB2,
but not PSGLCoxsackievirus
A7 (CVA7),
CVA14,
1, induced a conformational change from native 160S virions to
135SCVA16
particles. The change was enhanced under an acidic environment (pH 5.6; Chen et al., 2012). It should be further determined[Yamayoshi J Virol (2012)]
whether SCARB2 induces a conformational change to empty
capsids and whether VP4 is released during SCARB2-mediated•conformationalhSCARB2
EV71
change.

vvv
Yamayoshi, S., Fujii, K. &Koike, S. Front Microbiol
3, (2012).

NC

An expanded natural empty particle
The EV71 empty particles are markedly larger than any other picornavirus particle characterized crystallographically (Fig. 1b), with ther.m.s. capsid radius increasing from 132 Å for the mature virion to139 Å for the empty particle. Whereas cooling virus particles to 100 K
often induces modest isotopic shrinkage (~1%), both the 293-K and
100-K empty particles are ~4% larger than the 293-K mature virus.
The 100-K particles are not icosahedral, so refinement imposing
icosahedral symmetry stuck at an R factor of 30%, but it improved
markedly when this constraint was relaxed. Cryo-cooling pushes CAs
in the crystal’s contact region ~1.3 Å toward the particle center, but
the 293-K and 100-K structures are otherwise essentially identical(Supplementary Fig. 5). Such deviations from icosahedral symmetry
have never been observed in mature viruses and reflect the extreme
flexibility of these expanded particles, explaining why it has beendifficult to visualize them at high resolution.
The expansion of the empty particles reflects tectonic movementsin the particle, partially separating the protomeric units, disordering>70,000 protein atoms per particle and forming perforations at the
icosahedral two-fold axes and at the base of the canyon (Fig. 2a,b).
Several external loops, which nestle at the junction of polypeptide
chains, become disordered, including five residues at the C terminus
of VP2 and residues 211–217 of the GH loop of VP1. The VP3 GH
loop also undergoes a major conformational switch, with residues
170–192 converting from loop and helix to almost a B-hairpin upon
expansion, becoming less ordered (for residues 174–190, B factors
exceed 100 Å2) (Supplementary Fig. 5). Inside the particle, the internal festoon comprising VP1’s N-terminal 72 residues and the first81 residues of VP0 is now disordered (Fig. 1). Overall surface properties
426

Empty particle

Wang, X. et al. Nat Struct Mol Biol 19, 424–429 (2012).

EV71

5’
VP3

VP3

EV71

RNA

VP2

b
VP1

(as in other enteroviruses26), and another may interact with VP1 Gln30
and VP3 Gln48. On the outside of the particle, the VP1 BC, DE and HI
loops are flattened away from the five-fold axis (as in BEV1), with residues 96–102 (BC loop) and residues 208–222 (GH loop) being the most
exposed (Fig. 1c). Part of the VP2 EF loop (residues 128–148) is unusually extended and surface dominant, whereas the remainder (residues
151–172) is shorter than usual and less accessible. Exposed residues in
VP3 include 58–69 and 173–190 (Supplementary Fig. 5).

EV71

VPg

a

Neculai, D. et al Nature
504, 172–176 (2013).

ANIMAL MODEL OF EV71 INFECTION[Yamayoshi
J Virol(2013)]using live
To study the neuropathogenicityof EV71,experiments
animals are essential. The most reliable animal model is the monkey model (Hashimoto et al., 1978; Chumakov et al., 1979b;
Hashimoto and Hagiwara, 1982; Hagiwara et al., 1983, 1984;
Nagata et al., 2002, 2004; Arita et al., 2005, 2007; Zhang et al.,2011) because the species barrier caused by receptor differences is
not a critical problem. The localization patterns of EV71-induced
lesions in monkeys after intraspinal and intravenous inoculation
were highly consistent with those observed in humans with severe
EV71 encephalitis at autopsy (Lum et al., 1998; Wang et al., 1999;
Chan et al., 2000; Shieh et al., 2001). Infected monkeys showed
acute flaccid paralysis, which is a sign of involvement of the
pyramidal tract, and tremor and ataxia, which are signs of involvement of the extrapyramidal tract. Histopathological changes wereobserved in the cerebellar and pontine vestibular nuclei, and in the

ES

ARTICLES

EV71

l structures. (a) Cartoon of theion, looking down an icosahedral
P1, VP2, VP3 and VP4 are drawn
ed and yellow, respectively.
dral protomer is drawn moreius-colored surface representationture and empty particles. The
h are colored from blue to red
ir distances from the particle
ng closest). (c) Structures of theoteins. VP0–VP3 (we treat VP0angeably) are shown in a similare brighter protomer in a; proteins
ed empty particle are colored ase corresponding chains from thee gray. The proteins have beens rigid bodies. Residues 1–297 of
VP2, 1–242 of VP3 and 12–69
l-defined electron density in thesidues 73–210 and 219–297 of
VP0 and 1–238 of VP3 in there modeled. Major surface-exposedd with colored circles: yellow, VP1
DE; red, VP1 HI; magenta, VP1
VP2 EF; black, VP3 GH.

a

VP1

RNA

VP3

VP2
EV71

hSCARB2○○

PSGL-1○×Annexin II

○

?

c

○

?

○

?

Vimenjn○?

C
Nucleolin

○

?

N
VP1

teroviruses26), and another may interact with VP1 Gln30
8. On the outside of the particle, the VP1 BC, DE and HI
ned away from the five-fold axis (as in BEV1), with resiBC loop) and residues 208–222 (GH loop) being the most1c). Part of the VP2 EF loop (residues 128–148) is unusuand surface dominant, whereas the remainder (residues
orter than usual and less accessible. Exposed residues in
8–69 and 173–190 (Supplementary Fig. 5).

natural empty particle
pty particles are markedly larger than any other picornacharacterized crystallographically(Fig. 1b), with theþadius increasing from 132 Å for the mature virion tompty particle. Whereas cooling virus particles to 100 K
modest isotopic shrinkage (~1%), both the 293-K andparticles are ~4% larger than the 293-K mature virus.
articles are not icosahedral, so refinement imposing
ymmetry stuck at anþ
R factor of 30%, but it improvedn this constraint was relaxed. Cryo-cooling pushes CAs
s contact region ~1.3 Å toward the particle center, but
d 100-K structures are otherwise essentially identicalary Fig. 5). Such deviations from icosahedral symmetry
en observed in mature viruses and reflect the extreme
hese expanded particles, explaining why it has beenualize them at high resolution.
ion of the empty particles reflects tectonic movements, partially separating the protomeric units, disordering
in atoms per particle and forming perforations at the
wo-fold axes and at the base of the canyon (Fig. 2a,b).
nal loops, which nestle at the junction of polypeptide
e disordered, including five residues at the C terminus
sidues 211–217 of the GH loop of VP1. The VP3 GH
ergoes a major conformational switch, with residues
erting from loop and helix to almost a B-hairpin uponcoming less ordered (for residues 174–190, B factors) (Supplementary Fig. 5). Inside the particle, the interomprising VP1’s N-terminal 72 residues and the first
VP0 is now disordered (Fig. 1). Overall surface properties
VOLUME 19

Figure 1 Overall structures. (a) Cartoon of themature EV71 virion, looking down an icosahedral
two-fold axis, VP1, VP2, VP3 and VP4 are drawn
in blue, green, red and yellow, respectively.
A single icosahedral protomer is drawn morebrightly. (b) Radius-colored surface representationof the EV71 mature and empty particles. The
surfaces for both are colored from blue to red
Yamayoshi Nat Med (2009)according to their distances from the particle
center (blue being closest). (c) Structures of the
Nishimura Nat Med (2009)
EV71 capsid proteins. VP0–VP3 (we treat VP0and VP2 interchangeably) are shown in a similar
Yang J Virol (2011)
Mature virion
Empty particle
orientation to the brighter protomer in a; proteins
Su BMC Microbiol (2012)
from the expanded empty particle are colored as
in a, whereas the corresponding
Tan J Virol (2013)
N chains from theNmature virion are gray. The proteins
have been
Du J Virol (2014)superimposed as rigid bodies. Residues 1–297 of
Su J Virol (2015)
VP1, 10–254 of VP2, 1–242 of VP3 and 12–69
Cof VP4 have well-defined electron density in theCmature virion; residues 73–210 and 219–297 of
VP1, 82–319 of VP0 and 1–238 of VP3 in theempty particle are modeled. Major surface-exposedN
loops are marked with colored circles: yellow, VP1
C
BC; orange, VP1 DE; red, VP1 HI; magenta, VP1
GH; light blue,
VP2
VP3 VP2 EF; black, VP3 GH.
VP4bhSCARB2

EV71

EV71
a

b

PSGL-1
5
VP1

VP3

VP2
EV71

hSCARB2○○

Yamayoshi Nat Med (2009)

PSGL-1○×Nishimura Nat Med (2009)

Annexin II○?

Yang J Virol (2011)○?

Su BMC Microbiol (2012)

c

○

?

Tan J Virol (2013)

Vimenjn○?

Du J Virol (2014)

C
Nucleolin○?

Su J Virol (2015)

NATURE 4STRUCTURAL
26& MOLECULAR BIOLOGY

C

N
N
VP1

v hSCARB2
v Tg
EV71

hSCARB2

VP2Enteroviruses adopt two fundamental configurations
The ~4% expansion of the EV71 82S empty particle is sim
seen for the poliovirus 135S and 80S uncoating intermediate
latter has similar hydrodynamic properties. Cryo-EM analy
that the poliovirus 135S and 80S particles are structurall
each other27 (although the 80S particle has shed the viral ge
are reconfigured compared to the mature virus24,25. We us
to compare the EV71 expanded particle with the polioviru
Unexpectedly, the EV71 particle fit the poliovirus 135S and 8
densities as well as the poliovirus structure previously modedensity (Fig. 2d–g); thus, at low resolution the EV71 expande
byproduct is indistinguishable from poliovirus uncoating int
Furthermore, heat treatment of mature EV71 particles (usinsimilar to that which produces 135S poliovirus particles) coto particles that crystallize isomorphously with 82S immatu(data not shown). It therefore seems that the plethora ofparticles formed during assembly and uncoating posses
fundamental configurations, which are both now defined
detail. The mature virus particle, which is rigid and ren
stable by cleavage of VP0, is generally converted to the second
configuration after cell attachment. This flexible expanded
adopt subtly different conformations during the process o
as it progresses from 135S to 80S form25, but it can also ari
conversion of unstable VP0 containing natural empty parti
for poliovirus10,11 and now EV71.

Mechanics of particle expansion
Particle expansion is accompanied by a 5.4° counterclockwis
the protomeric building block (VP1, VP0, VP3), which pivo
corner of VP3 at the icosahedral three-fold axis (Fig. 3a). Thi

EV71
!!?

VOLUME 19

NUMBER 4

APRIL 2012

%Body Weight

90
80

60

○
×

[Fujii PNAS 2013]

F1
F2
F3
F4
F5
M1
M2
M3
M4
M5

100

70

○
×

NATURE STRUCTURAL & MOLECULA

EV71 106 TCID50

110

○
×

VP3

are appreciably altered, the interactions that hold the parti
are strongly reduced (Supplementary Table 1) and th
the expanded particle is thinned to 20 Å from 23 Å invirus (Fig. 2c). The core structures of the individual po
are less affected as CAs superpose with r.m.s. deviation
0.9 Å and 1.3 Å for VP1, VP0(2) and VP3, respectivel
although some important rearrangements occur.

120

h#p://www.redwineandapplesauce.com/

NhSCARB2-Tg

APRIL 2012•••

h#p://www.pz-garden.stardust31.com/

Empty particle

C26), and(as in otherthat
enteroviruses
another may interact with VP1 Gln30
are appreciably altered, the interactions
hold the particle
togetherand VP3 Gln48.
of the particle,
the VP1 BC, DE and HI
are strongly reduced (Supplementary
TableOn1)theandoutside
the capsid
inloopstoare20flattenedthemature
five-fold axis (as in BEV1), with resithe expanded particle is thinned
Å fromaway
23 Åfromin thedues 96–102(BCloop) andpolypeptides
residues 208–222 (GH loop) being the mostvirus (Fig. 2c). The core structuresof theindividual
exposedwith(Fig.r.m.s.
1c). Partof the VP2
are less affected as CAs superpose
deviationsof EF
1.6loopÅ, (residues 128–148) is unusually extended
andrespectively
surface dominant,whereas the remainder (residues
0.9 Å and 1.3 Å for VP1, VP0(2)and VP3,(Fig. 1c),
151–172) is shorter
although some important rearrangements
occur.than usual and less accessible. Exposed residues in
VP3 include 58–69 and 173–190 (Supplementary Fig. 5).
Enteroviruses adopt two fundamental configurations
The ~4% expansion of the EV71
An 82S
expanded
empty particle
natural isemptysimilarparticleto• thatseen for the poliovirus 135S andThe
80SEV71
uncoatingemptyintermediates
particles are 27
markedly, and thelarger than any other picornalatter has similar hydrodynamic
properties.
analysesrevealedvirus
particleCryo-EM
characterized
crystallographically(Fig. 1b), with thethat the poliovirus 135S and 80S
particles
structurally
similarr.m.s.
capsid are
radiusincreasingfrom to132 Å for the mature virion toeach other27 (although the 80S139
Å forhastheshedempty
Whereasparticle
theparticle.
viral genome)
andcooling virus particles to 100 K
24,25. We
28 (~1%), both the 293-K andinducesmodestisotopic
shrinkage
are reconfigured compared tooften
the mature
virus
used VEDA
100-K
empty
are ~4%larger than the 293-K mature EV71
virus.
to compare the EV71 expanded
particle
withparticles
the poliovirus
particles.
The
100-K
particles
so refinement imposing
Unexpectedly, the EV71 particle
fit thepoliovirus
135Sareandnot80Sicosahedral,electron
icosahedral
symmetrymodeled
stuck atinto
an Rthisfactor of 30%, but it improved
densities as well as1the poliovirus
structure previously
this constraint
was relaxed. Cryo-cooling pushes CAs
density (Fig. 2d–g); thus, at lowmarkedly
resolutionwhen
the EV71
expanded-assemblyin thepoliovirus
crystal’s contact
region~1.3 Å toward the particle center, but
byproduct is indistinguishable from
uncoatingintermediates.
293-K
andparticles
100-K structures
are otherwise essentially identical
Furthermore, heat treatment ofthematureEV71(using a protocol
Fig. 5). Suchdeviations
similar to that which produces (Supplementary
135S poliovirus particles)
converts
them from icosahedral symmetry
have never been
observed
in matureviruses and reflect the extreme
to particles that crystallize isomorphouslywith 82S
immature
particles
these
expandedparticles, explaining why it has been(data not shown). It thereforeflexibility
seems thatofthe
plethoraof enterovirus
difficultvisualize themat highparticles formed during assembly
andtouncoating
possess
onlyresolution.
two
The expansionof theemptyinparticles
fundamental configurations, which
are both now
definedatomic reflects tectonic movementsin thewhich
particle,partially
the protomeric units, disordering
detail. The mature virus particle,
is rigidand separating
rendered more
>70,000
proteintoatoms
per particle
and forming perforations at thestable by cleavage of VP0, is generally
converted
the second,
expanded
icosahedral
two-fold
axes andat thecanbase of the canyon (Fig. 2a,b).
configuration after cell attachment.
This flexible
expandedparticle
Several duringexternal
which
nestle at the junction of polypeptide
adopt subtly different conformations
theloops,
processof uncoating
25, but itdisordered,chains,includingas it progresses from 135S to 80S
formbecome
can also arise
from thefive residues at the C terminus
of VP2 andresidues
211–217
ofastheGH loop of VP1. The VP3 GH
conversion of unstable VP0 containing
naturalempty
particles,
seen
loop also undergoes a major conformational switch, with residues
for poliovirus10,11 and now EV71.
170–192 converting from loop and helix to almost a B-hairpin upon
expansion, becoming less ordered (for residues 174–190, B factors
Mechanics of particle expansion2) (Supplementary
exceedParticle expansion is accompanied
by a100
5.4°Åcounterclockwise
rotationFig. 5).
of Inside the particle, the interthe protomeric building block (VP1,
nal festoon
VP0, VP3),
comprisingwhich pivots
VP1’sabout
N-terminal
the72 residues and the firstcorner of VP3 at the icosahedral81three-foldresidues of
axis
VP0
(Fig.
is now
3a). disorderedThis screw-like(Fig. 1). Overall surface properties

NUMBER 4

Mature virion

EV71

hSCARB2-Tg
0

1

2

3

4

5

6

7

8

9

10 11 12 13 14

Days Post Inoculation

1

EV71
EV71

v

EV71

EV71

VP1-145

EV71

v
EV71
v EV71 CVA16hSCARB2hSCARB2-tg

hSCARB2-Tg

EV71

hSCARB2-tg

EV71

EV71

Dr. Mong How Ooi

VP1-145
EV71

EV71

EV71

EV71 genomic RNA
VP2

↓

3A 3B

2B

VP4

VP1-145

VP3

1

VP1

145

2A

2C

3C

3D

297 (a.a.)hSCARB2-Tg
E=

CCT ACC GGG GAA GTT GTC CCG
P T G E V V P
145

E

←

or

EV71

EV71

G=

CCT ACC GGG GGA GTT GTC CCG
P T G G V V P
145

Q=

CCT ACC GGG CAA GTT GTC CCG
P T G Q V V P
145

or

VP1-145

EV71

VP1-145

EV71

ARTICLES
Figure 1 Overall structures. (a) Cartoon of themature EV71 virion, looking down an icosahedral
two-fold axis, VP1, VP2, VP3 and VP4 are drawn
in blue, green, red and yellow, respectively.
A single icosahedral protomer is drawn morebrightly. (b) Radius-colored surface representationof the EV71 mature and empty particles. The
surfaces for both are colored from blue to red
according to their distances from the particle
center (blue being closest). (c) Structures of the
EV71 capsid proteins. VP0–VP3 (we treat VP0and VP2 interchangeably) are shown in a similar
orientation to the brighter protomer in a; proteinsfrom the expanded empty particle are colored as
in a, whereas the corresponding chains from themature virion are gray. The proteins have beensuperimposed as rigid bodies. Residues 1–297 of
VP1, 10–254 of VP2, 1–242 of VP3 and 12–69
of VP4 have well-defined electron density in themature virion; residues 73–210 and 219–297 of
VP1, 82–319 of VP0 and 1–238 of VP3 in theempty particle are modeled. Major surface-exposedloops are marked with colored circles: yellow, VP1
BC; orange, VP1 DE; red, VP1 HI; magenta, VP1
GH; light blue, VP2 EF; black, VP3 GH.

a

VP1-145G/Q VP1-145Eb
VP1hSCARB2-tg

VP3

VP2

Mature virionc

Empty particle

VP1-145G/Q

N
VP1-145E
N

C
CC

PSGL-1NCN
VP1(as in other enteroviruses26), and another may interact with VP1 Gln30
and VP3 Gln48. On the outside of the particle, the VP1 BC, DE and HI
loops are flattened away from the five-fold axis (as in BEV1), with residues 96–102 (BC loop) and residues 208–222 (GH loop) being the most
exposed (Fig. 1c). Part of the VP2 EF loop (residues 128–148) is unusually extended and surface dominant, whereas the remainder (residues
151–172) is shorter than usual and less accessible. Exposed residues in
VP3 include 58–69 and 173–190 (Supplementary Fig. 5).
An expanded natural empty particle
The EV71 empty particles are markedly larger than any other picornavirus particle characterized crystallographically (Fig. 1b), with ther.m.s. capsid radius increasing from 132 Å for the mature virion to139 Å for the empty particle. Whereas cooling virus particles to 100 K
often induces modest isotopic shrinkage (~1%), both the 293-K and
100-K empty particles are ~4% larger than the 293-K mature virus.
The 100-K particles are not icosahedral, so refinement imposing
icosahedral symmetry stuck at an R factor of 30%, but it improved
markedly when this constraint was relaxed. Cryo-cooling pushes CAs
in the crystal’s contact region ~1.3 Å toward the particle center, but

VP2

VP3

VP4

are appreciably altered, the interactions that hold the particle together
are strongly reduced (Supplementary Table 1) and the capsid in
the expanded
particlethinnedto 20 Åfrom 23 Å in the mature
Nishimuraet.al.isPLoS
Pathog.
2013;9(7):e1003511(modified)virus (Fig. 2c). The core structures of the individual polypeptides
are less affected as CAs superpose with r.m.s. deviations of 1.6 Å,
0.9 Å and 1.3 Å for VP1, VP0(2) and VP3, respectively (Fig. 1c),although some important rearrangements occur.
Enteroviruses adopt two fundamental configurations
The ~4% expansion of the EV71 82S empty particle is similar to thatseen for the poliovirus 135S and 80S uncoating intermediates27, and thelatter has similar hydrodynamic properties. Cryo-EM analyses revealed
that the poliovirus 135S and 80S particles are structurally similar toeach other27 (although the 80S particle has shed the viral genome) andare reconfigured compared to the mature virus24,25. We used VEDA28
to compare the EV71 expanded particle with the poliovirus particles.
Unexpectedly, the EV71 particle fit the poliovirus 135S and 80S electron
densities as well as the poliovirus structure previously modeled into thisdensity (Fig. 2d–g); thus, at low resolution the EV71 expanded-assembly
byproduct is indistinguishable from poliovirus uncoating intermediates.

hSCARB2

VP1-145

EV71
EV71

EV71

VP1-145↓hSCARB2-Tg

EV71

EV71

v
v
v
v

VP1-145G/Q VP1-145E
hSCARB2-tg

REVIEW

Development (2015) 142, 3456-3467 do

C-ter

Endorepellin

v

N-ter

C-ter

N-ter

Agrin

PerlecanCleavage by
metalloproteasesv
Glypicans

N-ter

N-ter

Syndecans

S-S bond

Cleavage byCfurin-like convertase

PSGL-1
Extracellular

hSCARB2

C-ter
er

Cleavage by metalloproteases or ADAMsp

Cleavage by Notum

Intracellular

C1

Poulain, F. E. & Yost, H. J. Development 142,
3456–3467 (2015).

V
C-ter

C2

emerge. Nonetheless, accumulative data clearly indicate that HSPGs
have distinct instructive roles during vertebrate development. For
example, studies have shown that many organs, including thehematopoietic and musculoskeletal systems, and the liver, lungs andkidneys, do not form properly when HSPGs are absent or modified(Tables S1, S2; for reviews, see Thompson et al., 2010; Huegel et al.,2013; Nigam and Bush, 2014). These studies have utilized various
model organisms, including mice, zebrafish and Xenopus, although it
should be noted that some disparities between results have arisen,perhaps owing to the technical challenges of assessing HSPG
functions in vivo (see Box 1). In this Review, we discuss the specificfunctions of HSPGs during vertebrate development, focusing on theearly stages of development and the formation of the cardiovascularand nervous systems. We then evaluate the concept of the sugar code
hypothesis, discussing evidence for and against it, and highlighting
future challenges for the field.
Roles for HSPGs in early development

The first stages of development following egg fertilization(including cell cleavage, gastrulation and patterning) establish thedifferent body axes and overall architecture of the embryo. Studies
of mutants that are defective for genes encoding HSPG biosynthetic
enzymes promote the view that HS sugar chains regulate multiple
steps throughout these early developmental processes (Fig. 4A). HS
chain polymerization has been shown to be crucial for patterningand early developmental function. Homozygous knockout (KO)mutants of Ext1 or Ext2 in mice are embryonic lethal, exhibiting
defects in gastrulation (Lin et al., 2000; Mitchell et al., 2001;
Stickens et al., 2005; Shimokawa et al., 2011). Zebrafish ext2
(dackel) or extl3 (boxer) mutants undergo normal gastrulation
owing to a maternal contribution of both enzymes, but later develop
cartilage and limb defects and display impaired FGF, Wnt and Shh

VP1-145G/Q
VP1-145E

EV71
VP1-145E

VP1-145G/Q

VP1-145G/Q
VP1-145E

VP1-145G/Q

hSCARB2

Fig. 1. Examples of key cell surface
HSPGs. Glypicans (Gpcs) are attach
via a glycosylphosphatidylinositol (GPpossess a large globular domain stabdi-sulfide (S-S) bonds, and HS chain
chains of pink, blue and purple circle
part. They can be released into the efollowing cleavage of their GPI anchor
A furin-like convertase can also cleav
C-terminal end of their globular doma
formation of two subunits that remain aby disulfide bonds. Syndecans (Sdcs
transmembrane proteins with HS cha
N-terminal part. Their intracellular reg
many different partners through two cconstant 1 (C1) and constant 2 (C2), th
more variable region (V). Like Gpcs, S
the extracellular environment after cle
such as matrix metalloproteases andmetalloproteinase (ADAM) disintegrin
are large multidomain proteoglycans
chains and are secreted as different isalternative splicing. The cleavage of tof perlecan by metalloproteases relea
angiogenesis inhibitor. C-ter, C-termi
N-terminus.

SCARB2signaling (Grandel et al., 2000; Norton et al., 20
2008; Holmborn et al., 2012). HS chain modificat
be important, as the absence of both Ndst1 and Nd
enzymes that catalyze HS deacetylation anembryonic lethal in mice (Grobe et al., 2002
disrupted in zebrafish embryos injected with magainst hs2st, which encodes a sulfotransferase t
the C2 position of hexuronic acid residues alo(Cadwalader et al., 2012). Interestingly, the othein HS-modifying enzymes only show later deve
(Table S2), suggesting functional redundancy
gene family members that can contribute to sp
steps in HSPG synthesis. Analyzing combinatio
be required to uncover the early developmental fu
Studies of mutants that are defective for indiv
also provide evidence for early developm
HSPGs. In zebrafish, gpc4 (knypek) mutants
Wnt11-dependent convergent-extension cell mgastrulation, suggesting that the potentiation of W
dependent on HSPG function (Topczewsk
Gastrulation and posterior elongation defects ar
Xenopus embryos lacking or overexpressing gpc4
2003). By contrast, Gpc4 KO mutants in mice dev
only show later defects in neural synapse forma2012). Other reported core proteins mutants
developmental defects (Table S1), including
specific neurodevelopmental defects (discussed
result in perinatal death.
Roles for HSPGs in left-right patterning

Kupffer’s vesicle (KV) is a ciliated organ that(LR) patterning in the zebrafish embryo.

EV71
REVIEW

Development (2015) 142, 3456-3467 doi:10.1242/dev.098178

Ser

Fig. 2. The synthesis of heparan sulfate chains. Heparansulfate (HS) chains are linked by a xylose and a tetrasaccharide
linkage to a specific serine residue of a core protein. This linkage
region is identical in HS and chondroitin (CS) chains. The
addition of the first N-acetylglucosamine is then catalyzed by the
enzyme EXTL3. Elongation of the chains is achieved
through the alternative addition of glucuronic acid andN-acetylglucosamine by the transferases EXT1 and EXT2.
During polymerization, chains undergo several modifications
including epimerization of glucoronic acid and sulfations at
different positions. These modifications occur in different
clusters and generate N-acetylated, N-sulfated and mixed
domains differently involved in interactions with partners. Ser,
serine; NS, N-sulfate group on glucosamine residues; 2S,
2-O-sulfate group on glucuronic or iduronic acid residues; 3S,
3-O-sulfate group on glucosamine residues; 6S, 6-O-sulfate
group on glucosamine residues. Adapted from Esko et al., 2009.

EXTL3
EXT1/EXT2

Ser
GlcNAc N-Deacetylase/N-Sulfotransferases (NDSTs)
Ser
NS

NS

NS

NS

NS

NS

NS

NS

NS

NS 2S NS 2S

GlcA C5 epimerase
Ser
NS

EV71

NS

2-O-Sulfotransferase

SCARB2-Tg

2716-Yamagata-03

Knock out using CRISPR/Cas9 system

EXT1/EXT2

Ser

6 -7

106 TCID50 / i.p.

P0

•
•
•

P1

Ser
NS 2S NS

P2

6-O-Sulfotransferases
6S

6S

6S

6S

6S

Ser
NS 2S NSNSP3

NS 2S NS 2S

3-O-Sulfotransferases
6S

6S

6S

6S

6S

Ser
NS 2S NS
3S

NS

RD, EXT1-KO, EXT2-KO
RD+SCARB2, EXT1-KO+SCARB2, EXT2-KO+SCARB2NS 2S NS 2S
3S

Key

CH2 O SO3–

XyloseO

OSO3–

GalactoseO

Glucuronic acidN-acetylglucosamineO

Iduronic acid

COOH
OH

O

OSO3–

O

NH
SO3–

Poulain, F. E. & Yost, H. J. Development 142, 3456–3467 (2015).

3458

therefore important to confirm these observations using
combinations of mutants in the Sdc family.
HS chain modifications catalyzed by 3-O-sulfotransferases(encoded by Hs3st genes) have also been implicated in LR
patterning. The MO-based lineage-specific targeting of hs3st1l2(also known as hs3st5) and hs3st3l (also known as hs3st6) in thezebrafish KV results in normal KV morphogenesis, but causes LR
patterning defects that appear to be due to distinct perturbations in
KV cilia length and cilia motility (Neugebauer et al., 2013). This
study also implicated 3-O-sulfotransferase modifications in theregulation of Fgf8 and other cell signaling pathways. Few othermutants have been reported for the Hs3st family, although it should
be noted that knockouts of Hs3st1 in mice have subtle intrauterine
growth retardation that is background dependent (Shworak et al.,
2002; HajMohammadi et al., 2003).
HSPG functions in cardiovascular development

Roles for core proteins as well as HS modifications in thedevelopment of the cardiovascular system have been identified.
During early organogenesis, the mesoderm lineages that give rise to
the heart and the mesoderm of the gut are present at the lateral edgesof the vertebrate embryo and migrate towards the embryonic
midline, fusing to form the cardiac and gut tube, respectively. The
MO-mediated lineage-specific knockdown of zebrafish sdc2 reveals
a role for this HSPG core protein in the non-cell-autonomous
regulation of ECM formation, which is required for cardiac and gut
mesoderm migration. In particular, the disruption of sdc2 functionin embryonic yolk cells leads to altered ECM formation throughout

EV71

DEVELOPMENT

development, KV cells migrate and undergo a mesoderm-toepithelial transition to form a vesicle. As the vesicle is inflated, each
KV cell grows a specialized apical cilium that is motile and moves
fluid asymmetrically in the KV, thereby establishing LR patterning
throughout the embryo. A number of studies have implicated a rolefor HSPGs during this process of LR patterning (Fig. 4B).
The lineage-specific targeting of sdc2 in zebrafish KV cellswith MOs results in disrupted KV morphogenesis and shortened
cilia, giving rise to altered LR patterning throughout the embryo(Arrington et al., 2013). Whole-embryo sdc2 knockdown also
causes later defects in vasculogenesis (Chen et al., 2004). Roles
for HSPGs in LR patterning have also been assessed in Xenopus
embryos, which offer the ability to identify and target cell lineages
more precisely by microinjection. For example, the lineagespecific expression of dominant-negative mutant Sdc2 isoforms
by targeted injection of mRNAs into individual Xenopus cells
results in LR patterning defects (Kramer and Yost, 2002).
Strikingly, the dominant-negative Sdc2 isoforms, including
phosphomimetic and phosphodeficient forms that have alteredphosphorylation sites in the cytoplasmic domain of the coreprotein, indicate that normal LR patterning requires both nonphosphorylated Sdc2 on the left and protein kinase C gamma(PKCγ)-dependent phosphorylated Sdc2 on the right side of theembryo (Kramer et al., 2002). This reveals a developmental role
for asymmetric Sdc2 phosphorylation in LR patterning. However,
it is possible that both the dominant-negative constructs and theMOs affect more than just Sdc2; dominant-negative Sdc2 might,
for example, disrupt the complex formed by Sdc2 and Sdc4. It is

EV71
EV71

v

RD

EXT1/2-KO

RD+SCARB2

EXT1/2-KO+SCARB2

EV71

v
EV71
v EV71 CVA16hSCARB2hSCARB2-tg

hSCARB2

EV71

VP1-145G/Q
hSCARB2-tgllhSCARB2

EXT1/2-KO+SCARB2

VP1-145G/Q
VP1-145G/Q

Dr. Mong How Ooi

VP1-145G/Q

EV71
VP1-145G/Q VP1-145E

VP1-145G
VP1-145E

hSCARB2-tg

orHS+

VP1-145G/Q

SCARB2+HS+VP1-145G/Q

①

EV71

EV71

VP1-145G
106 TCID50

or± hSCARB2②

EV71

VP1-145E

106 TCID50

EV71
hSCARB2
Tg

Mix

(37

± hSCARB2
non-Tg

VP1-145G

Tg

/ 1h)

or

VP1-145E

hSCARB2-tg

non-TghSCARB2

VP1-145G/Q

EV71

VP1-145G/Q

EV71