Molecula Cha ac e iza ion o he Vi opo in Func ion o Foo -
and-Mou h Disease Vi us Nons uc u al P o ein 2B
D. P. Gladue,
a
E. La go,
b,c
I. de la A ada,
b,c
V. M. Aguilella,
d
A. Alca az,
d
J. L. R. A ondo,
b,c
L. G. Holinka,
a
E. B occhi,
e
E. Rami ez-Medina,
a
E. A. Vuono,
a
K. A. Be gg en,
a
C. Ca illo,
J. L. Nie a,
b,c
M. V. Bo ca
a
a
Plum Island Animal Disease Cen e , ARS, USDA, G eenpo , New Yo k, USA
b
Biofisika Ins i u e (CSIC-UPV/EHU), Uni e si y o he Basque Coun y, Bilbao, Spain
c
Depa men o Biochemis y and Molecula Biology, Uni e si y o he Basque Coun y, Bilbao, Spain
d
Labo a o y o Molecula Biophysics, Depa men o Physics, Uni e si y Jaume I, Cas ellón de la Plana, Spain
e
Is i u o Zoop ofila ico Spe imen ale della Lomba dia e dell’Emilia Romagna, B escia, I aly
Plum Island Animal Disease Cen e , APHIS, USDA, G eenpo , New Yo k, USA
ABSTRACT Nons uc u al p o ein 2B o oo -and-mou h disease (FMD) i us
(FMDV) is comp ised o a small, hyd ophobic, 154-amino-acid p o ein. S uc u e-
unc ion analyses demons a ed ha FMDV 2B is an ion channel- o ming p o ein.
In a ed spec oscopy measu emen s using pa ially o e lapping pep ides ha
spanned egions be ween amino acids 28 and 147 demons a ed he adop ion o
helical con o ma ions in wo pu a i e ansmemb ane egions be ween esidues
60 and 78 and be ween esidues 119 and 147 and a hi d ansmemb ane egion
be ween esidues 79 and 106, adop ing a mainly ex ended s uc u e. Using syn-
he ic pep ides, ion channel ac i i y measu emen s in plana lipid bilaye s and
imaging o single gian unilamella esicles (GUVs) e ealed he exis ence o wo
sequences endowed wi h memb ane-po a ing ac i i y: one spanning FMDV 2B
esidues 55 o 82 and he o he spanning he C- e minal egion o 2B om esi-
dues 99 o 147. Mapping he la e sequence iden ified esidues 119 o 147 as
being esponsible o he ac i i y. Expe imen s o assess he deg ee o inse ion
o he syn he ic pep ides in bilaye s and he inclina ion angle adop ed by each
pep ide ega ding he memb ane plane no mal confi m ha esidues 55 o 82
and 119 o 147 o 2B ac i ely inse as ansmemb ane helices. Using e e se ge-
ne ics, a panel o 13 FMD ecombinan mu an i uses was designed, which ha -
bo ed nonconse a i e as well as alanine subs i u ions in c i ical amino acid esi-
dues in he a ea be ween amino acid esidues 28 and 147. Al e a ions o any o
hese s uc u es in e e ed wi h po e channel ac i i y and he capaci y o he
p o ein o pe meabilize he endoplasmic e iculum (ER) o calcium and we e le-
hal o i us eplica ion. Thus, FMDV 2B eme ges as he fi s membe o he i-
opo in amily con aining wo dis inc po e domains.
IMPORTANCE FMDV nons uc u al p o ein 2B is able o inse i sel in o cellula
memb anes o o m a po e. This po e allows he passage o ions and small mole-
cules h ough he memb ane. In his s udy, we we e able o show ha bo h cu -
en and small molecules a e able o pass hough he po e made by 2B. We also
disco e ed o he fi s ime a i us wi h a po e- o ming p o ein ha con ains
wo independen unc ional po es. By making mu a ions in ou in ec ious clone
o FMDV, we de e mined ha mu a ions in ei he po e esul ed in non iable i-
us. This sugges s ha bo h po e- o ming unc ions a e independen ly equi ed
du ing FMDV in ec ion.
KEYWORDS 2B, FMD, FMDV, i opo in, oo -and-mou h disease
Recei ed 9 Augus 2018 Accep ed 7
Sep embe 2018
Accep ed manusc ip pos ed online 19
Sep embe 2018
Ci a ion Gladue DP, La go E, de la A ada I,
Aguilella VM, Alca az A, A ondo JLR, Holinka
LG, B occhi E, Rami ez-Medina E, Vuono EA,
Be gg en KA, Ca illo C, Nie a JL, Bo ca MV.
2018. Molecula cha ac e iza ion o he
i opo in unc ion o oo -and-mou h disease
i us nons uc u al p o ein 2B. J Vi ol
92:e01360-18. h ps://doi.o g/10.1128/JVI
.01360-18.
Edi o Julie K. P ei e , Uni e si y o Texas
Sou hwes e n Medical Cen e
Copy igh © 2018 Ame ican Socie y o
Mic obiology. All Righ s Rese ed.
Add ess co espondence o M. V. Bo ca,
[email p o ec ed].
D.P.G. and E.L. con ibu ed equally o his
a icle.
STRUCTURE AND ASSEMBLY
c ossm
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Foo -and-mou h disease (FMD) i us (FMDV), a single-s anded, posi i e RNA i us
wi h a genome o app oxima ely 8,500 bases, belongs o he Aph ho i us genus o
he Pico na i idae amily. S uc u ally, i s genome p esen s a 5=noncoding egion, a
p o ein-coding egion, and a 3=noncoding egion (1). The p o ein-coding egion can be
u he di ided in o he P1 a ea, encoding all ou s uc u al p o eins, and P2 and P3,
encoding nons uc u al p o eins, including he 2B p o ein. 2B p o eins o polio i us,
coxsackie i us, and o he pico na i uses (PVs) ha e been ex ensi ely s udied. These 2B
p o eins con ain wo hyd ophobic egions, and hey can inse hemsel es in o he
memb ane o he endoplasmic e iculum (ER) o he Golgi appa a us o modi y cellula
memb ane pe meabili y once hey a e exp essed in hos cells (2–4). Addi ionally, hese
2B p o eins can dis up he Ca
2⫹
balance in hos cells, a ec ing apop osis (5, 6).
Howe e , ew epo s a e a ailable on he 2B p o ein o FMDV. I has been epo ed ha
he p edic ed s uc u e o FMDV 2B con ains wo hyd ophobic egions and inse s i sel
in o he memb ane o he ER wi h i s N and C e mini o ien ed owa d he cy osol. The
same epo indica ed ha he 2B p o ein inc eases memb ane pe meabili y and can
inc ease he Ca
2⫹
con en in hos cells, he eby inducing au ophagy (7).
He e, we epo a biochemical and unc ional cha ac e iza ion o FMDV 2B using
di e en app oaches, including an in i o model o a ificial memb anes. Resul s
e ealed ha he 2B p o ein possesses a obus ion channel- o ming capaci y and,
uniquely among o he i opo ins, possesses wo di e en s uc u al a eas ha a e able
o independen ly media e po e o ma ion. Consis en wi h hei unc ional ole, hese
sequences olded as ansmemb ane helices ha we e o ien ed almos pe pendicula
o he memb ane plane. In addi ion, we p esen he iden ifica ion o amino acid
esidues loca ed in a eas ha bo ing po e o ma ion ac i i y, which a e c i ical o he
unc ional ac i i y o 2B and he p ocess o i us eplica ion.
RESULTS
FMDV 2B genomic analysis. FMDV nons uc u al p o ein 2B is encoded wi hin a
ac o 462 nucleo ides o he FMDV genome, cha ac e ized as highly conse ed as 2B
has one o he lowes pe cen ages o nucleo ide subs i u ions, wi h only 39% o
nucleo ide posi ions and 24% o amino acids being able o ole a e some change. I s
nucleo ide composi ion has he mos conse a i e ansi ion (Ts)- e sus- ans e sion
(T ) a e o all FMDV genomic egions (Ts/T a e o 6.67), which is e en highe han he
Ts/T a e o he 3D polyme ase (Ts/T a e o 5.53), indica ing he s ong cons ic ion
o admi significan s uc u al modifica ions o he RNA (8). Genomic e idence indica es
ha mos o he nucleo ide changes a e allowed only because hey p oduce a com-
bina ion ha esul s in he same o e y simila amino acids, which is measu ed by he
a io be ween he numbe s o subs i u ions ha code o he same amino acid. The
a io o 2B synonymous (Syn)/nonsynonymous (non-Syn) subs i u ions is 5, while a io
o 1D (encoding he s uc u al p o ein VP1) is 1.03, ha bo ing almos as many Syn as
non-Syn changes. Simila ly, an alignmen o he amino acid sequences o he 2B p o ein
om mo e han 103 isola es (8) ep esen ing all se en se o ypes demons a es ha he
o al numbe o di e ences be ween any wo gi en isola es was ne e mo e han 19
subs i u ions. No ma e he o igins, hos adap a ion his o ies, o an igenic p ofiles o
he pai o i uses compa ed, he e is a ema kably na ow epe oi e o possible amino
acid composi ions o he whole p o ein, measu ed as a pai wise iden i y in 2B o
be ween 81% and 91% (compa ed wi h 48% o 68% in 1D), and when a change occu s,
i is always a e y conse a i e change. The e o e, FMDV 2B is a 154-amino-acid p o ein
con aining 117 in a ian esidues (Fig. 1A), wi h amino acid subs i u ions being limi ed
o only one o wo al e na e esidues pe si e. This simila i y o sequences o he 2B
p o ein ac oss all geno ypes and se o ypes sugges s ha i s s uc u e and base
composi ion ha e a c i ical and specific ole in he p ocess o i us eplica ion. The
hyd ophobici y dis ibu ion along he sequence sugges ed h ee possible hyd ophobic
egions ha a e likely able o o m an
␣
-helix ha could be pa o a ansmemb ane
domain co esponding o amino acids 60 o 78, 84 o 104, and 121 o 141, depic ed as
ed blocks (Fig. 1B). Wi h his in o ma ion, we defined a collec ion o o e lapping
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FIG 1 (Con inued)
Molecula Cha ac e iza ion o FMDV Vi opo in 2B Jou nal o Vi ology
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sequences, which included he 2B hyd ophobic egions, and gene a ed a lib a y o
syn he ic pep ides (Fig. 1C and Table 1).
FMDV 2B p o ein possesses ion channel ac i i y. The FMDV 2B p o ein has been
shown o beha e as a i opo in ega ding i s cy o oxic e ec s upon o e exp ession in
bac e ia and i s capaci y o al e ing a ficking along ea ly sec e o y pa hways in animal
cells, whe e i also seems o induce in acellula calcium elease and au ophagy (7).
Howe e , memb ane pe meabili y measu emen s in es iga ing i s po e o ma ion
mechanisms a he molecula le el had no ye been pe o med. To his end, we ca ied
ou elec ophysiological measu emen s a he le el o single ion channels and mem-
b ane pe meabili y de e mina ions in single esicles, using he pep ide lib a y (Fig. 1C
and Table 1) spanning a eas o FMDV 2B po en ially in ol ed in i opo in unc ion.
Resul s p esen ed in Fig. 2 desc ibe he ion channel ac i i y o he FMDV 2B pep ides
assayed in plana lipid mix u es ha mimicked he composi ion o ER memb anes,
namely, zwi e ionic phospha idylcholine (PC) and phospha idyle hanolamine (PE)
plus anionic phospha idylinosi ol (PI) mixed in a oughly 5:3:2 mola a io (9). Open
channels (conduc ance abo e he backg ound) we e eco ded in he cases o he
FIG 1 (A) Compa a i e mul isequence alignmen o mul iple FMDV 2B isola es and p edic ed s uc u al ea u es. Residues
conse ed among FMDV isola es a e shown as do s. Subs i u ed esidues desc ibed in Table 2 a e shown in ed. (B) Amino acids
o O1 Campos we e used as a ep esen a i e sequence o p edic seconda y s uc u e (H is helical) amphiphilici y (wi h M showing
p edic ed ansmemb ane egions), and a Ky e-Dooli le plo is shown. P edic ions we e done on he ExPASY se e (39, 40). (C)
The pep ides used in his s udy mapped o he amino acid sequence o 2B om O1 Campos. The indica ed esidues in ed a e
changes om he o iginal amino acid sequence.
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pep ides 2B2 and 2B4, spanning esidues 55 o 82 and 99 o 147, espec i ely, wi h
applied ol ages o (⫾)10, 30, 50, and 70 mV, whe eas open channels we e no
de ec ed o 2B1 o 2B3, spanning esidues 28 o 56 o 79 o 106, espec i ely, e en
wi h a s onge po en ial o ⫾100 mV applied (Fig. 2A).
In addi ion, aces showing “opening” and “closing” e en s we e obse ed o plana
bilaye s ea ed wi h 2B2 o 2B4. These cu en - e sus- ime aces we e subsequen ly
analyzed wi h Clampfi 10.1 so wa e so ha a e age cu en alues we e ob ained o
he case o he ac i e sequences unde an applied ol age o 50 mV. His og ams o he
cu en jump ampli udes o he eco ded aces showed ha he mos equen e en s
co esponded o single-channel cu en s o 7 ⫾4 and 5 ⫾3 pA o 2B2(55–82) and
2B4(99–147), espec i ely (Fig. 2B), he eby poin ing o a single-channel conduc ance
(G)equal o 20 ⫾20 pS. This pa e n o changes in conduc ance was una ec ed when
an equimola 2B2-2B4 mix u e was added o he suppo ed bilaye s, sugges ing ha
bo h pep ides wo k independen ly (no shown). In p inciple, channels o med unde
hese condi ions should pe mi he simul aneous anspo o wa e molecules, sol-
a ed ions, and e en small solu es, such as Alexa-based fluo escen compounds (10).
Suppo ing ha assump ion, 2B2 and 2B4 also conduc ed Ca
2⫹
ions e ficien ly ac oss
he ER-mimicking lipid bilaye (Fig. 2C).
Memb ane pe meabiliza ion induced by FMDV 2B p o ein-de i ed syn he ic
pep ides. To gain u he insigh in o he mechanism o pe meabiliza ion by FMDV 2B
TABLE 1 ATR-IR da a o he FMDV 2B syn he ic pep ides
Wa e no. (cm
ⴚ1
) Vib a ion
a
␪
(°)
b
A g dich oic a io ⴞSD A g SⴞSD
c
A g
␥
┴ⴞSD
d
A g
␥
LⴞSD
e
Lipid alone
2,920 as CH
2
s e ching 90 1.94 ⫾0.01 0.04 ⫾0.01 53.19 ⫾0.24
2,850 sCH
2
s e ching 90 1.75 ⫾0.02 0.16 ⫾0.01 48.49 ⫾0.53
2,870 sCH
3
s e ching 0 3.97 ⫾1.00 0.31 ⫾0.10 42.44 ⫾3.95
2B1
2,920 as CH
2
s e ching 90 1.53 ⫾0.07 0.32 ⫾0.05 42.41 ⫾2.04
2,850 sCH
2
s e ching 90 1.40 ⫾0.04 0.42 ⫾0.04 38.24 ⫾1.42
2870 sCH
3
s e ching 0 3.73 ⫾0.33 0.33 ⫾0.04 42.08 ⫾1.63
1656 Amide I–
␣
-helix 30 1.93 ⫾0.04 ⫺0.04 ⫾0.02 56.22 ⫾0.89 58.31 ⫾2.12
2B2
2,920 as CH
2
s e ching 90 1.70 ⫾0.03 0.19 ⫾0.02 47.35 ⫾0.68
2,850 sCH
2
s e ching 90 1.57 ⫾0.02 0.28 ⫾0.01 43.66 ⫾0.53
2,870 sCH
3
s e ching 0 4.19 ⫾0.72 0.36 ⫾0.06 40.55 ⫾2.57
1,656 Amide I–
␣
-helix 30 2.44 ⫾0.02 0.18 ⫾0.01 47.62 ⫾0.28 29.08 ⫾1.99
2B3
2,920 as CH
2
s e ching 90 1.93 ⫾0.08 0.04 ⫾0.05 52.99 ⫾2.02
2,850 sCH
2
s e ching 90 1.73 ⫾0.04 0.17 ⫾0.03 47.98 ⫾1.07
2,870 sCH
3
s e ching 0 7.54 ⫾4.34 0.40 ⫾0.15 38.27 ⫾6.52
1,630 Amide I–
␤
-shee 70 2.50 ⫾0.21 ⫺0.37 ⫾0.13 90.00 ⫾0.00 90.00 ⫾0.00
2B4
2,920 as CH
2
s e ching 90 1.74 ⫾0.03 0.17 ⫾0.02 48.23 ⫾0.72
2,850 sCH
2
s e ching 90 1.66 ⫾0.01 0.22 ⫾0.01 46.17 ⫾0.37
2,870 sCH
3
s e ching 0 4.00 ⫾0.61 0.34 ⫾0.07 41.36 ⫾2.73
1,656 Amide I–
␣
-helix 30 2.05 ⫾0.03 0.02 ⫾0.01 53.84 ⫾0.53 50.71 ⫾2.45
2B4c
2,920 as CH
2
s e ching 90 1.77 ⫾0.03 0.14 ⫾0.02 49.09 ⫾0.88
2,850 sCH
2
s e ching 90 1.66 ⫾0.01 0.22 ⫾0.01 46.10 ⫾0.33
2,870 sCH
3
s e ching 0 4.30 ⫾1.23 0.37 ⫾0.13 40.24 ⫾5.22
1,656 Amide I–
␣
-helix 30 2.41 ⫾0.02 0.17 ⫾0.01 48.01 ⫾0.34 22.42 ⫾3.79
a
Vib a ions a e p esen ed as symme ic (s) o asymme ic (as).
b
␪
, di ec ion o he dipole momen associa ed wi h he ib a ion wi h espec o he di ec ion o he main molecula axis (alipha ic chain o pep ide-seconda y
s uc u e).
c
S, o m ac o .
d
␥
┴, angle be ween he di ec ion o he molecula axis and he pe pendicula o he c ys al plane (simila o he memb ane plane).
e
␥
L, angle be ween he di ec ion o he pep ide-seconda y s uc u e axis and he calcula ed alipha ic chain axis.
Molecula Cha ac e iza ion o FMDV Vi opo in 2B Jou nal o Vi ology
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pep ides, we complemen ed ion channel measu emen s wi h a single- esicle app oach
based on he use o ER-GUVs (Fig. 3A). Con ocal mic og aphs depic ed in Fig. 3A
compa e un ea ed ER-GUVs (nega i e con ol) wi h hose ea ed wi h he po e
domain o classical swine e e i us i opo in p7 (p7C) (posi i e con ol) (11)o he
di e en FMDV 2B pep ides. Nega i e-con ol esicles and hose ea ed wi h he
2B1(28–56) o 2B3(79–106) pep ide we e iewed as da k (emp y) sphe es su ounded
by he o ange-labeled lipid bilaye , agains a g een backg ound con aining he pe -
mean Alexa Fluo 488 dye. In con as , incuba ion wi h he posi i e con ol p7C,
2B2(55–82), o 2B4(99–147) esul ed in g een labeling o he in e nal ER-GUV compa -
men s, indica ing pe meabiliza ion o he lipid bilaye o he dye. O e all, hese esul s
confi m he po e- o ming ac i i y o 2B2(55–82) and 2B4(99–147) sequences, which
e ol es acco ding o a mechanism ha p ese es he in eg i y o he lipid bilaye .
The le el o pe meabiliza ion (pe cen age) o a single ER-GUV could be u he
de e mined by analyzing he balance o fluo escence in ensi ies inside and ou side he
esicle (Fig. 3B). E en hough some o he esicles s ill showed pa ial filling a
equilib ium, ER-GUVs ea ed wi h he 2B2(55–82) o 2B4(99–147) pep ide mos ly
displayed le els o pe meabiliza ion app oaching 100%. Mo eo e , he numbe o
FIG 2 Ion channel ac i i y o 2B pep ides. (A) Cu en eco dings in 150 mM KCl, a di e en po en ials,
a e he addi ion o 2B1, 2B2, 2B3, o 2B4 o ER-like lipid bilaye s. (B) His og ams o he cu en jump
ampli udes eco ded o 2B2 and 2B4 wi h ol age se a 50 mV (le and igh panels, espec i ely). (C)
Cu en eco dings measu ed in 150 mM CaCl
2
a 50 mV.
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FIG 3 Single-ER-GUV pe meabiliza ion induced by 2B pep ides. (A) Mic og aphs depic ing Rho-PE
-labeled ER-GUVs (o ange ci cum e ences) imme sed in a solu ion con aining Alexa Fluo 488 (g een
backg ound). Samples on op co espond o con ol un ea ed ER-GUVs (le ) o ER-GUVs ea ed wi h he
p7C pep ide de i ed om CSFV p7 i opo in (11). Bo om samples co espond o ER-GUVs ea ed wi h
di e en 2B pep ides a he doses displayed on he panels. Ba s co espond o 25
␮
m in all mic og aphs.
(B) Dis ibu ion o ER-GUVs acco ding o hei pe cen age o pe meabiliza ion o Alexa Fluo 488 a e
ea men wi h he di e en 2B pep ides (le ) and mean pe meabiliza ion alues in he samples ( igh ).
Pep ides we e applied a he doses displayed on he panels. (C) S abili y o he memb ane pe meabili-
za ion s a e induced by 2B2 and 2B4. (Top) ER-GUVs pe meabilized o Alexa Fluo 488 a e incuba ion
o 2 h wi h 2B2 o 2B4 (g een) we e supplemen ed ex e nally wi h Alexa Fluo 647 ( ed) and addi ionally
incuba ed o 2 h be o e image p ocessing. The p esence o bo h p obes inside esicles can be obse ed
in he me ged images (bo om igh panels). The “Con ol” panel displays an in ac esicle incuba ed in
he absence o pep ide. (Bo om) Ba s ep esen he deg ee o filling o indi idual ER-GUVs wi h Alexa
Fluo 488 (da k g een) and Alexa Fluo 647 (ligh g een) a e a 4-h incuba ion wi h he 2B pep ides.
Molecula Cha ac e iza ion o FMDV Vi opo in 2B Jou nal o Vi ology
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o ally pe meabilized ER-GUVs inc eased a he highes doses o pep ide es ed, bu no
enhancemen was obse ed o he equimola 2B2-2B4 mix u e (no shown), sugges -
ing again ha hese domains do no unc ion in a conce ed manne . This s a e o
“pe manen ” pe meabiliza ion would be consis en wi h he abo e-desc ibed ion con-
duc ance measu emen s and suppo s ha ER-GUV pe meabiliza ion occu s h ough
po e channels.
To u he es his hypo hesis, we ca ied ou po e s abili y assays in which p obe
di usion ac oss ER-GUV memb anes was measu ed fi s wi h Alexa Fluo 488 and hen
again wi h a second dye, Alexa Fluo 647, which was added a e a 2-h incuba ion o
esicles wi h ei he he 2B2 o 2B4 pep ide (Fig. 3C, op). The pe meabili y o he
second dye clea ly indica es ha esicles ha we e pe meable o he fi s dye also
allowed he en ance o he second one, demons a ing ha mos ER-GUVs emain in
a pe meabilized s a e, a e he fi s pe meabiliza ion e en has occu ed (Fig. 3C,
bo om).
Mapping o po e- o ming ac i i y wi hin he egion spanning C- e minal esi-
dues 99 o 147. The design o he 2B4(99–147) sequence, wi h an app oxima e leng h
o 50 amino acids, was pe o med wi h he conside a ion ha we did no expec o
obse e po e ac i i y in his egion o he p o ein. To s udy in mo e de ail he
unexpec ed ac i i y o his zone, a sublib a y o 3 o e lapping pep ides was c ea ed,
2B4a(96–119), 2B4b(105–131), and 2B4c(119–147) (Table 1 and Fig. 4A), and hei
FIG 4 Mapping o memb ane-po a ing ac i i y wi hin he C- e minal end o FMDV-2B. (A) Sequences and
ange co e ed by he 2B4-de i ed o e lapping pep ides 2B4a, 2B4b, and 2B4c. (B) Ion channel ac i i y o
he pep ides. Shown a e elec ophysiological eco dings a e he addi ion o 2B4a, 2B4b, and 2B4c (le )
and a his og am o cu en jump ampli udes eco ded in lipid bilaye s ea ed wi h 2B4c ( igh ).
Condi ions a e o he wise as desc ibed in he legend o Fig. 2. (C and D) Single-ER-GUV pe meabiliza ion.
Shown a e mic og aphs o ER-GUVs ea ed wi h 2B4-de i ed pep ides (C) and hei dis ibu ions (D)
acco ding o he pe cen age o pe meabiliza ion pe esicle (le ) and calcula ed mean pe meabiliza ion
alues ( igh ). Condi ions a e o he wise as desc ibed in he legend o Fig. 3.
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channel and po e ac i i ies we e subsequen ly compa ed. The da a clea ly demons a e
ha pep ide 2B4c(119–147) possesses channel ac i i y wi h cha ac e is ics simila o
hose o he comple e 2B4(99–147) sequence (Fig. 4B). Also, 2B4c(119–147), bu no
2B4a(96–119) o 2B4b(105–131), was able o pe meabilize ER-GUVs o he Alexa Fluo
488 dye (Fig. 4C). The e o e, i can be concluded ha he C- e minal sequence spanning
amino acids 119 o 147 o he 2B FMDV p o ein displays unc ional cha ac e is ics
compa ible wi h hose o he po e- o ming domains.
S uc u e and o ien a ion o FMDV 2B p o ein-de i ed syn he ic pep ides
in e ac ing wi h memb anes. The s uc u e and o ien a ion adop ed by he di e en
FMDV 2B pep ides in memb anes we e analyzed using pola ized a enua ed o al
eflec ion in a ed (ATR-IR) spec oscopy (Fig. 5). ATR Fou ie ans o m IR (ATR-FTIR)
abso bance spec a we e measu ed o he di e en pep ides using pe pendicula and
pa allel pola ized ligh (Fig. 5A). F om he spec a, he expe imen al a e age dich oic
a ios we e de e mined, and o de pa ame e s Sand il angles we e calcula ed
acco dingly (12, 13)(Table 1).
Figu e 5B compiles he s uc u es and o ien a ions deduced o he di e en 2B
pep ides in he PC-PE-PI lipid bilaye . The spec a o 2B1, 2B2, 2B4, and 2B4c in he
amide I egion showed single peaks a ca. 1,656 cm
⫺1
, compa ible wi h he adop ion o
a helical con o ma ion o he inse ed pep ides. In con as , 2B3 displayed main
abso p ion a 1,630 cm
⫺1
, indica i e o p edominan ex ended con o ma ions, which
seemed unable o inse in o he lipid bilaye . Mo eo e , whe eas he 2B1 helix inse ed
wi h an o ien a ion o app oxima ely 60° ela i e o he memb ane plane no mal,
angles o inse ion deduced o 2B2, 2B4, and 2B4c we e consis en wi h o ien a ions
mo e pe pendicula o he bilaye su ace. Since he calcula ed alues o he il angles
ep esen a e age alues o he angles in he helix popula ion, he diag am also
includes al e na i e models which conside he possibili y o helix sec ions inse ed
wi h di e en angles and sepa a ed by elbows.
FMDV 2B i opo in unc ion is essen ial o i us g ow h. To assess he impo -
ance o he in eg i y o he a ea be ween amino acid esidues 28 and 147 o 2B, using
FIG 5 S uc u es and o ien a ions adop ed by 2B pep ides in ER-like memb anes. (A) Compa ison o ATR-IR spec a o 2B2 and 2B3
pep ides in he amide I egion. Peaks a 1,656 and 1,630 cm
⫺1
a e indica i e o
␣
-helical and ex ended seconda y s uc u es,
espec i ely. The main con ained o ien a ion is in e ed om he a io o peak a eas eco ded wi h inciden ligh pola ized pa allel ()
and pe pendicula (┬) o he memb ane no mal. a.u., a bi a y uni s. (B) Models o he o ien a ion o he memb ane-associa ed
s uc u es adop ed by 2B-de i ed pep ides, which a e based on he alues o he o de pa ame e s and il angles displayed in Table
1. Models conside ing single, con inuous helices a e accompanied by models conside ing kinked helical s uc u es.
Molecula Cha ac e iza ion o FMDV Vi opo in 2B Jou nal o Vi ology
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ypically one unc ional po e is p esen , and could explain he obse a ion ha FMDV
2B p o eins con ain an inse ion and a e la ge han ela ed en e o i us 2B p o eins.
P e ious s udies o o he i uses ha e shown ha in he same i us, wo sepa a e
indi idual p o eins bo h o m po es. Fo example, in se e e acu e espi a o y synd ome
co ona i us (SARS-CoV), bo h p o eins E and 3A ha e po e ac i i y, sugges ing he
possibili y ha in some cases mul iple i opo in unc ions may be necessa y (22, 23).
Howe e , o FMDV 2B, u he wo k elucida ing he indi idual unc ions o ei he po e
will be equi ed o de e mine he possible di e en oles o 2B as a i opo in.
MATERIALS AND METHODS
Gene a ion o FMDV 2B mu an s. The de elopmen o he ull-leng h in ec ious cDNA clone o
FMDV O1 Campos (pO1Ca) was desc ibed p e iously (24). B iefly, he ull-leng h cDNA o O1 Campos was
p eceded by he T7 polyme ase p omo e and wo addi ional G esidues o imp o e ansc ip ion
e ficiency and ollowed by a poly(A) ail o 15 esidues and a unique EcoRV si e. Plasmid O1Ca con aining
he desi ed mu a ions in 2B was cons uc ed by si e-di ec ed mu agenesis. Full-leng h pO1Ca was used
as a empla e in which na i e amino acids we e subs i u ed by si e-di ec ed mu agenesis using he
QuikChange XL si e-di ec ed mu agenesis ki (S a agene, Ceda C eek, TX), pe o med acco ding o he
manu ac u e ’s ins uc ions, whe e he ull-leng h plasmid was amplified by PCR, diges ed wi h DpnI o
lea e only he newly amplified plasmid, ans o med in o XL10-Gold ul acompe en cells, and g own on
Te ific B o h pla es con aining ampicillin (Tekno a, Hollis e , CA). P ime s we e designed by using he
manu ac u e ’s p ime design p og am (Agilen ).
The ob ained ull-leng h in ec ious clones we e comple ely sequenced wi h FMDV-specific p ime s by
he dideoxynucleo ide chain e mina ion me hod (25). Sequencing eac ion mix u es we e p epa ed wi h
he dye e mina o cycle sequencing ki (Applied Biosys ems, Fos e Ci y, CA). Reac ion p oduc s we e
sequenced on a P ism 3730xl au oma ed DNA sequence (Applied Biosys ems). Sequence da a we e
assembled using Sequenche 4.7 so wa e (Gene Codes Co po a ion, Ann A bo , MI). The final DNA
consensus sequence ep esen ed, on a e age, 3- o 4- old edundancy a each base posi ion.
Syn he ic pep ides and lipids. O e lapping 2B sequences o he syn he ic pep ides displayed in
Table 1 we e designed by aking in o accoun sequence conse a ion, hyd ophobici y dis ibu ion, and
he p esence o po en ial u ns a he memb ane in e ace, acco ding o p ocedu es desc ibed in
p e ious wo k (3, 26, 27). Syn hesis was ca ied ou as p e iously desc ibed (26, 27). Phospha idylcholine
(PC), phospha idyle hanolamine (PE), phospha idyle hanolamine-N-(lissamine hodamine B sul onyl)
(Rho-PE), and phospha idylinosi ol (PI) we e pu chased om A an i Pola Lipids (Bi mingham, AL, USA).
Alexa Fluo 488 was ob ained om Molecula P obes (Junc ion Ci y, OR, USA).
Ion channel conduc ance measu emen s. Two lipid monolaye s we e made om 5-mg/ml pen ane
solu ions o a lipid mix u e bu e ed wi h 5 mM HEPES wi h KCl (pH 7.4) a bo h sides o Teflon chambe s
pa i ioned by a 15-
␮
m- hick Teflon film wi h 70- o 100-
␮
m-diame e o ifices. Plana lipid bilaye s we e
o med by a monolaye apposi ion on he o ifices p e iously ea ed wi h a 1% solu ion o hexadecane
in pen ane. Pep ides dissol ed in dime hyl sul oxide (DMSO) we e supplemen ed in he lipid solu ions,
p io o monolaye o ma ion in only one o he chambe sides, he cis side. Bilaye o ma ion was di ec ly
de ec ed, and i s hickness can be es ima ed by capaci ance measu emen s. To pe o m channel
conduc ance measu emen s, an elec ic po en ial was applied using Ag/AgCl elec odes in 2 M KCl–1.5%
aga ose b idges assembled wi hin s anda d 250-
␮
l pipe e ips. Po en ial is defined as posi i e when i
is highe a he side o he p o ein addi ion ( he cis side), while he ans side is se o g ound. The cu en
was amplified by an Axopa ch 200B amplifie (Molecula De ices, LLC, Sunny ale, CA, USA) in he ol age
clamp mode (whole-cell
␤
⫽1) wi h a CV-203BU head s age. Da a we e digi ized wi h Digida a 1440A
(Molecula De ices, Sunny ale, CA), while he ou pu signal was fil e ed by an in-line low-pass Bessel fil e
a 10 kHz and di ec ly sa ed in o he compu e memo y wi h a sampling equency o 50 kHz. The
memb ane chambe and he head s age we e isola ed om ex e nal noise sou ces wi h a double me al
sc een (cus om o de ed om Amuneal Manu ac u ing Co p., Philadelphia, PA, USA). Cu en ampli ude
analysis was pe o med using Clampfi 10.6 so wa e (Molecula De ices, LLC, Sunny ale, CA, USA). The
his og ams o he cu en jump ampli ude we e made om a leas 60 eco dings comp ising 10 aces
wi h a du a ion o 20 s, and mo e han 650 e en s we e analyzed o each his og am. The da a we e
no malized o a alue o 1, using he numbe o o al e en s in each case. The his og ams we e fi ed o
a one-peak Gaussian dis ibu ion. Each s anda d de ia ion (SD) alue is he squa e oo a iance o he
co esponding Gaussian dis ibu ion.
Memb ane pe meabili y o single esicles. Fo he single- esicle pe meabili y measu emen s,
gian unilamella esicles (GUVs) made o PC–PE–PI–Rho-PE (50:30:20:0.1 mola a io) we e p epa ed
acco ding o he elec o o ma ion me hod desc ibed in p e ious wo ks (28,29). In b ie , 2
␮
lo he
ER-lipid mix u e (2 mM) was placed on pla inum wi es, and he sol en was e apo a ed. Fo GUV
elec o o ma ion, 2.4 V and 10 Hz we e applied du ing 2 h in a suc ose (300 mM) solu ion. To
p omo e de achmen o he GUVs om he wi es, 2 Hz was finally applied du ing 30 min. Fo
con ocal mic oscopy analyses, 80
␮
l o he solu ion was ans e ed o 320
␮
l o bu e (10 mM
HEPES, 150 mM KCl [pH 7.4]) in a Lab-Tek eigh -chambe ed numbe 1.0 bo osilica e co e glass om
Nalge Nunc In e na ional (Roches e , NY, USA), p e iously blocked wi h 2 mg/ml bo ine se um
albumin (BSA).
Con ocal fluo escence mic oscopy images o indi idual GUVs we e ob ained using a comme cial
Nikon D Eclipse TE2000-U fluo escence mic oscope (Nikon Ins umen s, Tokyo, Japan). Ex en s o
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pe meabiliza ion we e calcula ed o each esicle a e 2ho incuba ion wi h Alexa Fluo 488 and 2B
pep ides. Fo his pu pose, he fluo escence in ensi y alue ou side he esicle is conside ed 100%,
whe eas he in ensi y inside esicles eflec ed he deg ee o dye di usion o he pe cen age o p obe
en y in o he esicle lumen. Fluo escence emission in he di e en egions o he sample was quan i-
a ed a e image p ocessing, and analyses we e ca ied ou wi h ImageJ so wa e ( sb.in o.nih.go /ij/). To
assess he s abili y o he assembled po es, 2 h a e pep ide-GUV incuba ion in he p esence o ee
Alexa Fluo 488, a second ma ke , ee Alexa Fluo 647, was added. The en y o bo h p obes in o esicles
was analyzed in pic u es aken a e an addi ional 2-h incuba ion.
A enua ed o al eflec ion IR spec oscopy. ATR-IR spec a o he 2B pep ides we e measu ed in
a B uke Tenso 27 ins umen a a 2-cm
⫺1
esolu ion. PC-PE-PI (5:3:2 mola a io) lipid mix u es
con aining pep ide (lipid- o-pep ide mola a io, 20:1) we e d ied on he su ace o he ATR Ge c ys al by
flowing d ied ai in o he in a ed spec ome e chambe du ing 5 h. Fo spec um acquisi ion, a pola ized
mi o (Pike Technologies) was adjus ed o 0° and 90°, o gene a e inciden ligh o ien ed pa allel and
pe pendicula o he lipid no mal, espec i ely. A o al o 150 IR spec a we e collec ed unde each
condi ion and a e aged. The dich oic a io o he amide I bond abso p ion was compu ed o pa allel (0°)
e sus pe pendicula (90°) pola ized inciden ligh ela i e o he memb ane no mal and was employed
o calcula e he pep ide o ien a ion as discussed p e iously (12, 30, 31).
Cell exp ession and pe meabiliza ion o he ER o calcium. ER localiza ion o he exp essed 2B
p o ein and i s de i ed mu an s was assayed by co ans ec ing 293T cells (2 ⫻10
5
cells) wi h plasmids
encoding GFP usions and mCh-Sec61 be a (1
␮
g each) using calcium phospha e (32). A 36 h pos ans-
ec ion, cells we e fixed wi h 4% o maldehyde in phospha e-bu e ed saline (PBS) and incuba ed wi h
Hoechs dye. Con ocal images we e acqui ed on a Leica TCS SP5 II mic oscope (Leica Mic osys ems
GmbH, We zla , Ge many), using a 63⫻wa e imme sion objec i e.
2B-induced ER pe meabiliza ion was assayed by moni o ing he amoun o hapsiga gin- eleasable
Ca
2⫹
in he cy osol o ans ec ed single cells (33, 34). To his end, changes in he in ensi y o he
indica o p obe hod-2/AM we e eco ded by fluo escence mic oscopy as a unc ion o ime (35, 36).
Thi y-six hou s a e ans ec ion, cells we e loaded wi h hod-2/AM (4
␮
g/ml) a oom empe a u e o
45 min. Dye-loaded cells we e isualized wi h a Nikon Eclipse TE-2000 mic oscope using a 63⫻oil
imme sion objec i e, a e exci a ion/emission a 561 nm/573 o 613 nm. Thapsiga gin was added a a
final concen a ion o 10
␮
M in cul u e. Time-lapse images (512 by 512 pixels) o cells displaying 2B-GFP
exp ession le els compa able o hose o GFP con ols we e collec ed on a Nikon d-Eclipse C1 Si colo
digi al came a ( ame acquisi ion in e al o 1 s).
Vi us p oduc ion. Plasmid pO1C o i s mu an e sions we e linea ized a he EcoRV si e and used
as a empla e o in i o RNA syn hesis using he MEGAsc ip T7 ki (Ambion, Aus in, TX) acco ding
o he manu ac u e ’s p o ocols. A emp s o p oduce gene ically modified i uses we e pe o med
by ans ec ing BHK-21 cells (ATCC CCL-10) wi h in i o- ansc ibed RNA by elec opo a ion (Elec-
ocell Manipula o 600; BTX, San Diego, CA) as p e iously desc ibed (24). B iefly, 0.5 ml o BHK-21
cells a a concen a ion o 1.5 ⫻10
7
cells/ml in PBS was mixed wi h 10
␮
g o RNA in a 4-mm-gap BTX
cu e e. The cells we e hen pulsed once (330 V wi h infini e esis ance and a capaci ance o 1,000
F), dilu ed in cell g ow h medium (Dulbecco’s modified Eagle’s medium [DMEM] con aining 10%
e al cal se um), and allowed o a ach o a T-25 flask. A e 4 h, he medium was emo ed, esh
medium was added, and he cul u es we e incuba ed a 37°C o up o 24 h. The supe na an s om
ans ec ed cells we e blindly passaged a minimum o six imes o un il a cy opa hic e ec (CPE)
appea ed in LFBK-V6 cells (a de i a i e cell line ob ained om po cine kidney LFBK cells exp essing
he bo ine V6 in eg in) (37).
Wes e n blo analysis. Exp ession o FMDV nons uc u al p o ein 2B was analyzed in cell lysa es o
ans ec ed BHK-21 o in ec ed LFBK-V6 cells by Wes e n immunoblo ing. FMDV 2B was de ec ed wi h
a monoclonal an ibody specifically de eloped o his wo k a he Is i u o Zoop ofila ico Spe imen ale
della Lomba dia e dell Emilia-Romagna, B escia, I aly. T ans ec ed BHK-21 cells o in ec ed LFBK-V6 cells
we e ha es ed using adioimmunop ecipi a ion assay (RIPA) bu e and he NuPAGE LDS sample bu e
sys em (In i ogen) and incuba ed a 70°C o 10 min. Samples we e un unde educing condi ions in
p ecas No ex 4% o 12% Bis-T is ac ylamide gels (In i ogen) and ans e ed o poly inylidene difluo-
ide (PVDF) memb anes (In i ogen). Goa an i-mouse IgG conjuga ed o ho se adish pe oxidase (Pie ce,
Rock o d, IL) was used as a seconda y eagen . Wes e n immunoblo s we e isualized using a Supe Signal
Wes Du a ex ended-du a ion subs a e (The mo Scien ific) acco ding o he manu ac u e ’s di ec ions.
Reac i i y was de ec ed wi h an Azu e c300 chemiluminescen Wes e n blo imaging sys em and cSe ies
Cap u e so wa e 2014 (Azu e Biosys ems).
De ec ion o i al RNA. To al RNA was isola ed om in ec ed BHK-21 and LFBK-V6 cells using an
RNeasy miniki (Qiagen, Valencia, CA). Once ex ac ed, RNA was de ec ed by eal- ime e e se
ansc ip ion-PCR ( RT-PCR) on he ABI 7500 sys em (Applied Biosys ems, Aus in, TX) as p e iously
desc ibed, using specific p ime s and p obe co e ing he FMDV genomic a ea encoding nons uc u al
p o ein 3D (38).
ACKNOWLEDGMENTS
This esea ch was pa ially suppo ed by he Basque Go e nmen and a Uni e si y o
he Basque Coun y g an (p ojec IT838-13 o J.L.N.). A.A. and V.M.A. acknowledge
financial suppo om he Spanish Go e nmen (FIS2016-75257-P AEI/FEDER) and
Uni e si a Jaume I (P1.1B2015-28).
Molecula Cha ac e iza ion o FMDV Vi opo in 2B Jou nal o Vi ology
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Also, we especially hank Melanie P a a o edi ing he manusc ip and Elizabe h
Bishop o main aining and p o iding LFBK-V6 cells.
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