Effect of specific amino acid substitutions in the putative fusion peptide of structural glycoprotein E2 on Classical Swine Fever Virus replication

E ec o specific amino acid subs i u ions in he pu a i e usion
pep ide o s uc u al glycop o ein E2 on Classical Swine Fe e
Vi us eplica ion
I.J. Fe nández-Sainz
a
, E. La go
b
, D.P. Gladue
a
, P. Fle che
a
,V.O’Donnell
a,c
,
L.G. Holinka
a
, L.B. Ca ey
d
,X.Lu
c
, J.L. Nie a
b
, M.V. Bo ca
a,
n
a
Plum Island Animal Disease Cen e , ARS, USDA, USA
b
Biophysics Uni (CSIC-UPV/EHU), Depa men o Biochemis y and Molecula Biology, Uni e si y o he Basque Coun y (UPV/EHU), P.O. Box 644, 48080
Bilbao, Spain
c
Plum Island Animal Disease Cen e , DHS, G eenpo , NY 11944, USA
d
Depa men o Expe imen al and Heal h Sciences, Uni e si a Pompeu Fab a (UPF), E-08003 Ba celona, Spain
a icle in o
A icle his o y:
Recei ed 6 Feb ua y 2014
Re u ned o au ho o e isions
25 Feb ua y 2014
Accep ed 4 Ma ch 2014
A ailable online 4 Ap il 2014
Keywo ds:
Fusion pep ide
Classical Swine Fe e Vi us
CSFV
Classical swine e e
Pes i i us
abs ac
E2, along wi h E
ns
and E1, is an en elope glycop o ein o Classical Swine Fe e Vi us (CSFV). E2 is
in ol ed in se e al i us unc ions: cell a achmen , hos ange suscep ibili y and i ulence in na u al
hos s. He e we e alua e he ole o a specific E2 egion,
818
CPIGWTGVIEC
828
, con aining a pu a i e usion
pep ide (FP) sequence. Re e se gene ics u ilizing a ull-leng h in ec ious clone o he highly i ulen
CSFV s ain B escia (BIC ) was used o e alua e how indi idual amino acid subs i u ions wi hin his
egion o E2 may a ec eplica ion o BIC . A syn he ic pep ide ep esen ing he comple e E2 FP amino
acid sequence adop ed a
β
- ype ex ended con o ma ion in memb ane mime ics, pene a ed in o model
memb anes, and pe u bed lipid bilaye in eg i y in i o. Simila pep ides ha bo ing amino acid
subs i u ions adop ed compa able con o ma ions bu exhibi ed di e en memb ane ac i i ies. The e o e,
a p elimina y cha ac e iza ion o he pu a i e FP
818
CPIGWTGVIEC
828
indica es a memb ane usion
ac i i y and a c i ical ole in i us eplica ion.
Published by Else ie Inc.
In oduc ion
Classical swine e e (CSF) is a highly con agious disease o
swine caused by CSF i us (CSFV), a small en eloped i us wi h
a posi i e-sense, single-s and RNA genome. CSFV is classified
as a membe o he pes i i us genus wi hin he Fla i i idae
amily along wi h o he i uses o economic impo ance, bo ine
i al dia hea i us (BVDV) and bo de disease i us (BDV)
(Beche e al., 2003). The app oxima ely 12.5-kb CSFV genome
con ains a single open eading ame ha encodes a polyp o-
ein composed o 3898 amino acids ha ul ima ely yields up
o 12 final clea age p oduc s (NH2-Np o-C-E ns-E1-E2-p7-
NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH) h ough co- and pos -
ansla ional p ocessing o he polyp o ein by cellula and i al
p o eases (Rice, 1996).
S uc u al componen s o he i ion include he capsid (C) p o ein
and glycop o eins: E
ns
,E1andE2.E
ns
, a sec e ed p o ein ha
demons a es RNAse ac i i y and is loosely associa ed wi h he i al
en elope (Thiel e al., 1991; Weiland e al., 1990; Weiland e al., 1999),
does no ha e a hyd ophobic ansmemb ane ancho domain. E
ns
does, howe e , possess a C- e minal cha ged amphipa hic segmen
ha can media e ansloca ion o E
ns
ac oss bilaye memb anes
(Langedijk, 2002). E1 and E2 a e ansmemb ane p o eins wi h an N-
e minal ec odomain and a C- e minal hyd ophobic ancho (Thiel e
al., 1991). E2 is conside ed essen ial o CSFV eplica ion, as i us
mu an s con aining pa ial o comple e dele ions o he E2 gene a e
non iable ( an Gennip e al., 2002). E2 has been implica ed, along
wi h E
ns
(Huls and Moo mann, 1997)andE1(Wang e al., 2004), in
i al adso p ion o hos cells (Liange al.,2003; anGennipe al.,
2000). Modifica ions in oduced in o his glycop o ein appea o
ha e an impo an e ec on CSFV i ulence (Risa i e al., 2005, 2006,
2007a, 2007b; Van Gennip e al., 2004).
Using p o eomic compu a ional analysis, E2 has been cha ac-
e ized as a unca ed class II usion p o ein (Ga y and Dash,
2003). Al hough he o e all s uc u es o class I and II usion
p o eins a e dis inc , hey may sha e s uc u al/ unc ional cha -
ac e is ics in he pa s o he molecules ha in e ac wi h and
dis up bilaye memb anes. I is well es ablished ha class I usion
p o eins ha e a usion pep ide a he amino e minus o he
Con en s lis s a ailable a ScienceDi ec
jou nal homepage: www.else ie .com/loca e/y i o
Vi ology
h p://dx.doi.o g/10.1016/j. i ol.2014.03.005
0042-6822/Published by Else ie Inc.
n
Co espondence o: Plum Island Animal Disease Cen e , USDA/ARS/NAA,
P.O. Box 848, G eenpo , NY 11944-0848, USA. Tel.: þ1 631 323 3019;
ax: þ1 631 323 3006.
E-mail add ess: [email p o ec ed] (M.V. Bo ca).
Vi ology 456-457 (2014) 121–130
molecule, o close o i , ha is c i ical o usion (Gallahe , 1987,
1996; Gallahe e al., 1989, 2001). Class II usion p o eins ha e an
in e nal FP ha is loca ed a e seconda y s uc u al olding a
dis al loca ions om he ansmemb ane ancho (Kuhn e al.,
2002; Lesca e al., 2001; Rey e al., 1995). Ga y and Dash
desc ibed a pu a i e FP loca ed be ween amino acid esidues
818–828 o CSFV E2. This pu a i e FP con ains a consensus
sequence wi h a oma ic and hyd ophobic esidues loca ed
be ween wo cys eine esidues. In addi ion, he FP is flanked by
β
shee s in class II usion p o eins. The cys eine esidues as well as
he sequences in be ween a e highly conse ed among pes i-
i uses, as is ue o class I and II FPs om o he en eloped RNA
i uses (Ga y and Dash, 2003). Recen ly he E2 p o ein o BVDV
has been c ys allized, e ealing a h ee
β
-domain s uc u e
(El Oma i e al., 2013). The pu a i e FP appea s o be pa ially
accessible on he su ace o he p o ein. Howe e , i does no o m
a canonical usion loop as hose ound in Class II p o eins, which
sugges s ha o he mechanisms-s uc u es migh ope a e in
pes i i us usion.
He e we e alua e he ole o a specific E2 egion,
818
CPIGWTG-
VIEC
828
, con aining a pu a i e FP sequence. Re e se gene ics
u ilizing a ull-leng h in ec ious clone o he highly i ulen s ain
B escia (BIC ) was used o e alua e he ole o indi idual as well as
combined amino acid subs i u ions in he eplica ion o BIC . Only
double C818S/C828S o iple P819S/I820S/W822S subs i u ions
esul ed in eplica ion-deficien i uses. Fu he mo e, we sough
o es ablish a co ela ion be ween he unc ional e ec s induced
by he amino acid subs i u ions and he capaci y o syn he ic FPs
o inse ing in o memb anes and b each he pe meabili y ba ie .
A he ou se we confi med he e en ion o
β
- ype ex ended
con o ma ions in memb ane mimics o he na i e sequence and
i s a ian s ha bo ing he double C818S/C828S o iple P819S/
I820S/W822S subs i u ions. Ou da a indica e ha amino acid
subs i u ions p ima ily a ec he deg ee o FP pene a ion in o
he lipid bilaye , he iple subs i u ion esul ing in shallowe
inse ion and educed memb ane ac i i y. All in all, ou esul s
sugges ha he pu a i e FP
818
CPIGWTGVIEC
828
is in ol ed in
memb ane usion ac i i y and plays a c i ical ole in i us
eplica ion.
Resul s
Loca ion o he pu a i e usion pep ide in he CSFV E2 p o ein
The pu a i e E2 FP is o med by ele en amino acids si ua ed
be ween posi ions 818 and 828 o he CSFV polypep ide. Compa -
ison o i s amino acid sequence among di e en CSFV isola es
e eals 100% iden i y among isola es (Fig. 1). Compa ed o o he
pes i i uses he amino acid iden i y anges be ween 45 and 72%
simila o BVDV isola es wi h subs i u ions mos ly occu ing a
posi ions 820 and 825–827, 63–72% simila o BDV isola es wi h
amino acid subs i u ions a posi ions 820, 822, 824, 825, and 81%
simila o pes i i us gi a e-1 wi h subs i u ions a 823–825. This
high deg ee o conse a ion wi hin each o he pes i i us sub-
g oups, compa ed o he di e ences be ween he subg oups,
sugges s he pu a i e FP sequence may be in ol ed in an impo -
an i al unc ion such as i us opism.
De elopmen o CSFV in ec ious clones ha bo ing amino acid
subs i u ions in he FP sequence
To e alua e he ole o he pu a i e FP in he in i o and in i o
eplica ion o CSFV as well as in he p oduc ion o disease in swine,
a se ies o ecombinan CSF i uses con aining amino acid sub-
s i u ions in he FP a ea we e designed using he cDNA in ec ious
clone o he B escia s ain (BIC ) as a empla e. A o al o eigh
cDNA cons uc s con aining he amino acid subs i u ions
desc ibed in Table 1 we e cons uc ed.
In ec ious RNA was in i o ansc ibed om each mu a ed ull-
leng h cDNA and used o ans ec SK6 cells. In ec ious i us
con aining indi idual subs i u ions o C818S, C828S, P819S,
I820T, W822S o double subs i u ion V825T/I826T was escued
om ans ec ed cells by day 4 pos - ans ec ion. Pa ial nucleo-
ide sequence o he escued mu an E2 i uses was pe o med o
ensu e he p esence o he p edic ed mu a ions (da a no shown).
Con e sely, in h ee independen ans ec ion e en s, con-
s uc s ha bo ing a double, C818S/C828S, o iple, P819S/I820S/
W822S, subs i u ions we e un a yingly nega i e in e ms o
eco e ing in ec ious pa icles (da a no shown). Immunohis o-
chemis y analysis o ans ec ed cell monolaye s showed ha
cells ans ec ed wi h ei he cons uc C818S/C828S o P819S/
I820S/W822S exp essed E2 (Fig. 2A) al hough a a dec eased le el
when compa ed o hose ound in cells ans ec ed wi h he
cons uc s p oducing iable i us pa icles.
Thus, o de e mine i a g oss de ec in glycosyla ion o un-
ca ed exp ession was a he o igin o he le hal pheno ype, he
exp ession o E2 glycop o ein in cells ans ec ed wi h ei he
C818S/C828S o P819S/I820S/W822S was addi ionally analyzed
by Wes e n blo . Cell ex ac s ans ec ed wi h ei he o hese wo
cons uc s demons a ed significan le els o E2 exp ession wi h a
p oduc showing an elec opho e ic mobili y simila o ha
obse ed wi h cell ex ac s ans ec ed wi h an IC encoding o
he pa en al B escia i us (Fig. 2B). These esul s sugges ha he
double and iple subs i u ion cons uc s p oduced a le hal epli-
ca ion i us pheno ype, e en hough he e is e ficien exp ession
o an appa en ly in ac E2 p oduc .
Replica ion o he CSFV FP mu an s in i o
in i o eplica ion cha ac e is ics o he FP E2 mu an i uses
con aining indi idual (C818S, C828S, P819S, I820T, W822S) o
double subs i u ions (V825T/I826T) ela i e o pa en al BIC
we e e alua ed in a single-s ep g ow h cu e. P ima y swine
mac ophage cell cul u es we e in ec ed a a MOI o 0.01 TCID
50
pe cell. Vi uses we e adso bed o 1 h ( ime ze o), and samples
we e collec ed a 72 h pos -in ec ion and i a ed in SK6 cell
cul u es. All mu an i uses exhibi ed g ow h kine ics almos
undis inguishable om ha o he pa en al BIC (Fig. 3A).
Addi ionally, when he FP E2 mu an i uses' plaque sizes we e
compa ed using SK6 cells, all mu an s exhibi ed a plaque size
simila o ha o he pa en al BIC (Fig. 3B). The e o e, indi i-
dual subs i u ions o mos o he esidues o ming he pu a i e
FP do no significan ly a ec he abili y o he i us o eplica e
in cell cul u es.
Vi ulence o CSFV FP mu an s in i o
To examine whe he al e a ions o di e en esidues included
in he pu a i e E2 FP a ec i ulence, di e en g oups o pigs we e
in anasally inocula ed wi h app oxima ely 10
5
TCID
50
o each o
he E2 FP mu an i uses (C818S, C828S, P819S, I820T, W822S,
V825T/I826T) and moni o ed o clinical disease, e alua ed ela i e
o pa en al BIC . All animals in ec ed wi h BIC p esen ed clinical
signs o CSF s a ing 3–4 days pos -in ec ion (DPI), de eloping
classic symp oms o he disease and dying a ound 7–8 DPI
(Table 2). To al whi e blood cells, lymphocy es and pla ele coun s
d opped by 4–6 DPI in animals inocula ed wi h BIC and con-
inued declining un il dea h (da a no shown). All mu an i uses
p esen ed a i ulence pheno ype almos indis inguishable om
ha o he pa en al BIC (Table 2). All animals in ec ed wi h hese
i uses p esen ed clinical signs o CSF s a ing a 3–5 DPI, wi h
clinical p esen a ion and se e i y simila o hose obse ed in
I.J. Fe nández-Sainz e al. / Vi ology 456-457 (2014) 121–130122
animals inocula ed wi h BIC . Whi e blood cells, lymphocy es and
pla ele coun s d opped by 4 DPI and con inued declining un il
dea h (da a no shown) a 6–9 DPI.
Vi emia in animals inocula ed wi h E2 FP mu an s in gene al
accompanied he e olu ion o he clinical disease (da a no
shown), exhibi ing i emia kine ics almos undis inguishable om
ha induced by pa en al BIC i us, p esen ing high i e s ha
emained un il dea h o he animal.
The e o e, wi h he excep ion o he le hal subs i u ions C818S/
C828S o P819S/I820S/W822S, subs i u ions o any o he o he
10 20 30 40 50
....|....|....|....|....|....|....|....|....|....|....|...
B essia YDTSPVVKG-KYNTTLLNGSAFYLVCPIGWTGVIEC-TAVSPTTLRTEVVKTFRREKP
AEA40484.1 F........-..........................-.....................
AEA40482.1 F........-..........................-.....................
AEA40481.1 F........-..........................-.....................
AEA40487.1 F........-..........................-.....................
AEA40478.1 F........-..........................-.....................
AEA40477.1 F........-..........................-..A..................
AEA40480.1 F........-..........................-I....................
AEA40475.1 F........-..........................-.....................
AGI98198.1 F........-..........................-.....................
AFC37768.1 F..T.....-..........................-................K....
AEA40479.1 F........-..........................-..A..................
AGT80624.1 FN.......-..........................-.....................
AGI98192.1 F....I...-..........................-.....................
AGG91155.1 F..T.....-..........................-................K....
AEA40486.1 F........-.F........................-..................D..
AEJ37847.1 F..I.....-..........................-................K....
AFC37766.1 F..T.....-..........................-.....................
BVDV
AAW88355.1 C.A..LIR.-.F.......P..QM.S....P.PVSLYHWANKD.SA.A.GR.YK.PR.
AAW88353.1 CYAR.LLR.-SL...Y..WP..QM..L...P.TVSVYHWAIKV..A.A..R.YK.PR.
CAB45185.1 C.AK.I.R.-.........P..QM........TVS.-MLANRD..D.A..R.Y..SR.
CAB45183.1 C.AK.I.R.-............QM........TVS.-MLANRD..D.A..R.Y..SR.
AAA98608.1 C.SK..I..-.F.AS....P..QM...Q....R...-.L.NQD..D.T..R.Y..TT.
AGV40886.1 C.AK.L.R.-.F.......P..QM...M....TVN.-ALANKD..DITI.R.YM.LE.
AFX69713.1 S.AK.LGR.-.FKS.....A.SKK.W.T.G..TVS.-ALANKD..DVT..R.NT.L..
AAC06278.2 C.AR...R.-.F.A.....P..QM...T....S.S.-.L.NGD..S.T..R.Y..P..
NP_776264.2 C.AK.I.R.-.F.......P..QM........TVS.-.SFNMD..A.T..R.Y..S..
ABW38057.1 C.AK.L.R.-.F.......P..QM..H.....TVS.-ALANKD..ALT..R.YT.H..
BDV
NP_777538.1 C.SR.....-............Q.I..Y..V.RV..-.T..KS..A.....IYKKT..
NP_777537.1 C.SR.....-............Q.I..Y..V.RV..-.T..KS..A.....IYKKT..
AAO26467.1 C.SR.....-............Q....Y..V.RV..-.T..KD..V....RIYKKT..
AAO26466.1 C.SK.....-...S........Q....Y....QV..-....RS..A.....IY..K..
AAO26471.1 C.SK.....-.F.......N..Q....L....QV..-.T..TS..A......Y..SR.
AAO26470.1 C.SK.....-...A........Q....F..V.QV..-.T..TS..A......YK.ST.
AAO26469.1 C.SK.....-...A........Q....F..V.RV..-.T..TS..A......Y.GST.
AAO26468.1 C.SK.....-............QM...F....RV..-.T..TS..A....R.Y..ST.
Gi a e-1
NP_777525.1 C.SR.....-NF....I.H...Q.......V.T...-.L.NTD..A.T...RYT.TT.
NP_620053.1 C.SR.....-NF....I.H...Q.......V.T...-.L.NTD..A.T...RYT.TT.
Fig. 1. Sequence alignmen o show pu a i e FP sequences (highligh ed) o E2 om di e en Pes i i uses (CSF, BVD, BD and Gi a e-1). Pa ial amino acid sequence
su ounding polypep ide esidues 818–828 a e p esen ed.
Table 1
Nucleo ide sequence o p ime s used o he p oduc ion o E2FP ecombinan i uses (only o wa d p ime s a e p esen ed).
Mu an name Fo wa d p ime sequence
C818S 5
0
GGTAGTGCATTCTACCTAGTTTGCCCAATAGGGTGGACGGGTGTT 3
0
C828S 5
0
GGGTGGACGGGTGTTATAGAGTGCACGGCAGTGAGCCCGACAACT 3
0
C818S/C828S
a
P819S 5
0
AGTGCATTCTACCTAGTTTGCTCAATAGGGTGGACGGGTGTTATA 3
0
I820T 5
0
GCATTCTACCTAGTTTGCCCAACAGGGTGGACGGGTGTTATAGAG 3
0
W822S 5
0
TACCTAGTTTGCCCAATAGGGTCGACGGGTGTTATAGAGTGCACG 3
0
P819S/I820S/W822S 5
0
GCATTCTACCTAGTTTGCTCAACAGGGTCGACGGGTGTTATAGAGTGC 3
0
V825T/I826T 5
0
TGCCCAATAGGGTGGACGGGTGATACAGAGTGCACGGCAGTGAGCCCG 3
0
a
P ime s C818S and C828S we e consecu i ely used o c ea e mu an C818S/C828S.
I.J. Fe nández-Sainz e al. / Vi ology 456-457 (2014) 121–130 123
esidues unde s udy wi hin he FP a ea do no significan ly a ec
i us g ow h in cell cul u e o pa hogenesis in swine.
E alua ion in i o o he pu a i e usion pep ide in e ms o s uc u e
and memb ane in e ac ion
To de e mine he e ec o he amino acid subs i u ions on he
adop ed con o ma ions and he in e ac ions wi h memb anes,
h ee FP a ian pep ides we e syn hesized and compa a i ely
analyzed. One o he syn he ic pep ides ep esen s he na i e
sequence o he E2 FP (designa ed as WT) while he o he wo
ha bo a double (C818S/C828S) o a iple subs i u ion (P819S/
I820S/W822S) and we e designa ed as mu -1 and mu -2, espec-
i ely (Fig. 4A). All h ee sequences we e flanked by addi ional
lysine esidues o con e solubili y, and agged a he N- e minus
wi h he NBD fluo escen p obe o assessmen o in e ac ions
wi h lipid bilaye s. The seconda y s uc u es adop ed by hese
syn he ic pep ides a e displayed in Fig. 4B. Ci cula dich oism
measu emen s disclosed compa able amoun s o
β
- ype ex ended
s uc u es ha we e p edominan o he h ee a ian s, and
o e all e ained in he memb ane-mimicking en i onmen s p o-
ided by SDS and DPC micelles. Thus, he amino acid subs i u ions
did no seem o a ec he seconda y s uc u e o he E2 FP, which
o he wise pe o med compa ably in solu ion and he low-pola i y
memb ane en i onmen .
Memb ane in e ac ions o WT, mu -1 and mu -2 a ian s we e
nex assessed in lipid monolaye and unilamella esicle models
(Fig. 5). The h ee sequences we e fi s compa ed in he lipid
monolaye sys em, which allows moni o ing pep ide pene a ion
in o memb anes as an inc ease o he monolaye la e al p essu e
(see La go e al. 2014 o a desc ip ion o he sys em). The sys em
also allows adjus men o he ini ial la e al p essu e (
π
0
). Thus, he
FP capaci y o aising he monolaye la e al p essu e upon injec-
ion a high
π
0
s(i.e., wi h igh ly packed phospholipids) will
co ela e wi h i s capaci y o inse ing in o he i al a ge
memb ane (Nie a and Agi e, 2003; Ra alski e al., 1990). Pene a-
ion le els o he syn he ic FPs depending on
π
0
a e shown in
Fig. 5A. The monolaye exclusion p essu es,
π
ex
, o he maximum
ini ial la e al p essu es a which memb ane-associa ion was
accompanied by pep ide in eg a ion in o he monolaye , we e
Fig. 2. Exp ession o E2 glycop o ein in SK6 cells ans ec ed wi h he indica ed cons uc s (de ec ion was pe o med using an i-E2 mAb WH303) by
(A) immunocy ochemis y, and (B) Wes e n blo .
I.J. Fe nández-Sainz e al. / Vi ology 456-457 (2014) 121–130124
compa able o WT and mu -1, and consis en wi h hei pene a-
ion in o memb anes wi h he lipid packing densi y exis ing a he
a ge cell memb ane (
π
c
Z30 mN/m, (Ma sh, 2007). In con as ,
he mu -2 a ian was unable o e ficien ly pene a e in o mono-
laye s comp essed o hose le els.
To de e mine deg ees o associa ion wi h lipid bilaye s, we nex
compa ed pa i ioning o he h ee sequences in o esicles by
moni o ing changes in he emi ed NBD-fluo escence (Fig. 5B).
The pa i ioning coe ficien s, es ima ed upon i a ing he pep ides
wi h inc easing quan i ies o esicles, we e all in he ange o 10
5
,
he e o e deno ing compa able deg ees o associa ion o he h ee
a ian s. Howe e , NBD in ensi y inc eased by a ac o o 10 in he
case o he mu -1 pep ide, while his ac o was educed o 3 o he
mu -2 pep ide. The highe inc ease in emission in ensi y was
consis en wi h deepe pene a ion o he fluo opho e in o he
hyd oca bon co e o he lipid bilaye in he o me case. The WT
NBD emission inc eased by a ac o o 5, consis en wi h an in e -
media e le el o inse ion. In summa y, lipid monolaye and NBD-
fluo escence esul s sugges ha he iple subs i u ion in e e es
wi h FP pene a ion in o he a ge memb ane, while eplacing he CC
esidues esul s in deepe inse ion in o he low-pola i y egion.
Finally, esul s displayed in Fig. 5C e ealed ha WT inse ed
in o memb anes was capable o pe u bing he lipid bilaye
C818S C828S I820T P819S
V826S/I827T W822S BIC
0
1
2
3
4
5
6
7
8
2h 24h 48h 72h
Log10 TCID
50
/ml
Hou s pos -in ec ion
C818S
C828S
P818S
I819S
W822S
V826T/I827T
BIC
Fig. 3. in i o g ow h cha ac e is ics o FP mu an s and pa en al BIC . (A) P ima y swine mac ophage cell cul u es we e in ec ed (MOI¼0.01) wi h each o he FP i us
mu an s o BIC and i us yield i a ed a imes pos -in ec ion in SK6 cells. Da a ep esen means and s anda d de ia ions om wo independen expe imen s. Sensi i i y o
i us de ec ion: Z1.8 TCID50/ml. (B) Plaque o ma ion o he FP i us mu an s and BIC . SK6 monolaye s we e in ec ed, o e laid wi h 0.5% aga ose and incuba ed a 37 1C o
3 days. Pla es we e fixed wi h 50% ( ol/ ol) e hanol-ace one and s ained by immunohis ochemis y wi h mAb WH303.
Table 2
Swine su i al and e e esponse ollowing in ec ion wi h E2FP mu an s and
pa en al BIC .
Vi us No. o
su i o s/
o al no.
Mean ime
o dea h
(days7SD)
Fe e
No. o days
o onse
(days7SD)
Du a ion
(days7SD)
BIC 0/4 7.75 (1.5) 3.75 (0.9) 5 (1.4)
C818S 0/4 7.25 (0.9) 4 (0.0) 4.25 (0.9)
C828S 0/4 6.75 (2.1) 4 (0.0) 3.75 (0.9)
P819S 0/4 7.75 (0.5) 5 (1.1) 4.75 (1.7)
W822S 0/4 9.5 (3) 5.5 (2.3) 4.75 (1.7)
V825T/
I826T
0/4 9.5 (2.3) 3.5 (1) 7 (2.1)
I820T 0/4 8.5 (2.1) 3.5 (0.7) 6 (1.4)
I.J. Fe nández-Sainz e al. / Vi ology 456-457 (2014) 121–130 125

a chi ec u e mo e e ficien ly han he mu an - ep esen ing pep-
ides. In hese assays he in eg i y o he POPG memb ane was
moni o ed using he ANTS/DPX assay (Nie a e al., 1994; Ra alski
e al., 1990). A pep ide- o-lipid a io o 1:700 was equi ed o
induce 50% o esicle pe meabiliza ion by WT, whe eas he a io
inc eased o 1:300 o mu -1 and his le el o pe meabiliza ion
was ba ely a ained wi h he highes dose o mu -2 es ed (1:100).
Thus, o compa able amoun s o pep ide associa ed wi h he
memb ane, he WT sequence was mo e e ficien han he mu an s
a des abilizing he in eg i y o he esicles.
Discussion
This epo a emp ed o cha ac e ize he ole o he pu a i e FP
comp ising he amino acid esidues
818
CPIGWTGVIEC
828
o glycop o-
ein E2 in CSFV. Re e se gene ics and an in i o a ificial memb ane
assays we e used o assess he ole o indi idual esidues wi hin he
E2 FP in he p ocess o i us eplica ion and memb ane inse ion.
Double (C818S/C828S) o iple (P819S/I820S/W822S) esidue sub-
s i u ions comple ely ab oga e i us eplica ion, addi ionally, he iple
subs i u ion led o a dec eased capaci y o memb ane inse ion.
The cha ac e iza ion o he s uc u e and memb ane in e ac ions
o syn he ic pep ides ep esen ing he double and iple subs i u ions
in he FP indica ed ha he con o ma ions adop ed by he WT and
mu a ed pep ides we e compa ible wi h he ex ended
β
- ype s uc-
u e o he FP. Such s uc u es we e no significan ly al e ed in he
p esence o memb ane mime ics, indica ing he amino acid subs i u-
ions did no a ec he o e all con o ma ion adop ed by he FP in
solu ion o he low-pola i y memb ane-like en i onmen . In con as ,
he h ee pep ides displayed di e en pa e ns o in e ac ions wi h
memb ane models. The abili y o syn he ic pep ides and hei mu an
a ian s o pene a e in o POPG monolaye s was ea lie desc ibed o
he HIV-1 FP (Nie a e al., 1994; Ra alski e al., 1990). Those s udies
demons a ed ha a pola subs i u ion ha inac i a ed he usion
glycop o ein also in e e ed wi h he pene a ion o he FP in o he
POPG monolaye . In ou expe imen s, he iple subs i u ion (P819S/
I820S/W822S) had a compa able blocking e ec on he abili y o he
E2 FP. This mu an associa ed wi h POPG esicles as e ficien ly as WT
and double subs i u ions, bu induced he ein less pe meabiliza ion.
Thus, we assume ha he iple subs i u ion causes a shallowe
associa ion wi h he lipid bilaye , a possibili y suppo ed by he mo e
pola en i onmen sensed by he NBD fluo escen p obe. In his
ega d, p oline and yp ophan esidues sus ain inse ion o he Ebola
i us FP as an in eg al memb ane hai pin, and a e equi ed o he
usogenic ac i i y o he en elope glycop o ein. S uc u e and unc ion
o he comple e in e nal usion loop om Ebola i us glycop o ein 2
(G ego y e al., 2011). By analogy, he lack o FP inse ion induced by
he iple subs i u ion migh in e e e wi h E2 usogenic ac i i y and
p e en CSFV in ec ion. By compa ison he pep ide ha bo ing he
double subs i u ion inse ed e ficien ly in o monolaye s and pene-
a ed deepe in o he lipid bilaye su ounding he esicles (Nie a
and Agi e, 2003). In his case i is possible ha a di-Sulfide b idge
s abilizing a unc ional in eg al haipin would be absen in he FP
mu an , he eby allowing i s deepe inse ion bu hampe ing i s
usogenic ac i i y. None heless, his mu an was s ill less e ficien
han he WT a pe u bing he memb ane. I is pos ula ed ha he
memb ane es uc u ing e ec will be mo e po en o pep ides
inse ed a he le el o he phospholipid glyce ol backbone, han o
pep ides associa ed a he le el o he headg oups o he phospholi-
pids, o han o hose inse ed deepe in o he acyl-chain egion
(Nie a and Agi e, 2003). We in e ha , compa ed o he mu an
de i a i es, he WT pep ide a ains an in e media e dep h o pene a-
ion o exe maximal lipid bilaye des abiliza ion e ec s. This
obse a ion would suppo i s ole as a memb ane-des abilizing
ancho du ing usion and explain i s in ol emen in i al p opaga ion.
Recen ly he E2 p o ein o BVDV has been c ys allized, e ealing a
h ee domain s uc u e. Domains I and II a e simila o Ig-like domains
and domain III is a se ies o h ee small
β
-shee modules; his
s uc u e is belie ed o be simila o CSFV E2 by p edic ion analysis
(Iou in e al., 2013; Li e al., 2013). Using he c ys al s uc u e o
BVDV-1 E2 a ph 8.0 (pdb:2yq2) (El Oma i e al., 2013), he pu a i e FP
appea s o be pa ially accessible, howe e he c i ical esidues P819S/
I820S/W822S appea o be mos ly in e nal si ua ed (Fig. 6). In
summa y, his p elimina y cha ac e iza ion o he unc ionali y o
he pu a i e FP comp ising he amino acid esidues
818
CPIGWTG-
VIEC
828
o glycop o ein E2 in CSFV demons a ed ha al e ing his
a ea al hough do no significan ly al e p o ein exp ession, esul s
c i ical in he p ocess o i us eplica ion and he abili y o he p o ein
o be inse ed in cell memb anes.
Ma e ials and me hods
Vi uses and cells
Swine kidney cells (SK6) (Te ps a 1990), ee o BVDV, we e
cul u ed in Dulbecco's minimal essen ial media (DMEM) (Gibco,
G and Island, NY) wi h 10% e al cal se um (FCS) (A las Biologicals,
Fo Collins, CO). CSFV B escia s ain was p opaga ed in SK6 cells
Fig. 4. Designa ion and s uc u e o E2 FP-de i ed syn he ic pep ides. (A) Designa ion and sequences o he pep ides used in his s udy. Subs i u ed esidues a e indica ed in
bold. (B) Seconda y s uc u es. The s uc u al componen s we e calcula ed o he CD spec a ob ained in bu e (black ba s), o in he p esence o 100 mM DPC (whi e ba s)
o 100 mM SDS (g ay ba s). Means7SD o he ac ion alues es ima ed wi h CONTIN-LL, CDSSTR and SELCON3 p og ams a e plo ed o he pep ides as indica ed in he
panels. H, helix; S, s and; TþU, u nsþuno de ed.
I.J. Fe nández-Sainz e al. / Vi ology 456-457 (2014) 121–130126
and was used o he cons uc ion o an in ec ious cDNA clone
(Risa i e al., 2005). G ow h kine ics was assessed using p ima y
swine mac ophage cell cul u es p epa ed as desc ibed by Zsak
e al. (1996). Ti a ion o CSFV om clinical samples was pe -
o med using SK6 cells in 96-well pla es (Cos a , Camb idge, MA).
A e 4 days in cul u e, i al in ec i i y was assessed using an
immunope oxidase assay u ilizing he CSFV monoclonal an ibody
WH303 (mAb WH303) (Edwa ds e al., 1991) and he Vec as ain
ABC ki (Vec o Labo a o ies, Bu lingame, CA). Ti e s we e calcu-
la ed acco ding o he me hod o Reed and Muench (1938) and
exp essed as TCID
50
/ml. As pe o med, es sensi i i y was
Zlog
10
1.8 TCID
50
/ml.
Cons uc ion o CSFV mu an s
A ull-leng h in ec ious clone (IC) o he i ulen B escia s ain
(pBIC) (12) was used as a empla e o ob ain all cDNA IC cons uc s
desc ibed in his epo . Cons uc s con aining mu a ions in he
FP a ea we e ob ained using he QuickChange XL Si e-Di ec ed
Mu agenesis ki (S a agene) pe o med pe manu ac u e 's
ins uc ions using ull-leng h pBIC as empla e and he p ime s
desc ibed in Table 1. The p oduc was hen diges ed wi h Dpn1,
lea ing 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 Aga Pla es
wi h ampicillin (Tekno a). Posi i e colonies we e selec ed o by
sequence analysis o he E2 gene and g own o plasmid pu ifica-
ion using a Maxip ep ki (Qiagen Sciences, MD). Each o he IC
cons uc s we e comple ely sequenced o e i y ha only si e-
di ec ed mu agenesis-induced changes we e p esen .
in i o escue o CSFV B escia and FP mu an s
Full-leng h genomic clones we e linea ized wi h S I and
in i o ansc ibed using he T7 Megasc ip sys em (Ambion,
Aus in, TX) (Risa i e al., 2005). RNA was p ecipi a ed wi h LiCl
and ans ec ed in o SK6 cells by elec opo a ion a 500 V, 720
Ω
,
100 W wi h a BTX 630 elec opo a o (BTX, San Diego, CA). Cells
we e seeded in 12-well pla es and incuba ed o 4 days a 37 1C
Fig. 5. In e ac ions wi h model memb anes o E2 FP-de i ed syn he ic pep ides. (A) Pene a ion in o POPG monolaye s. Maximum inc ease in su ace p essu e induced upon
injec ion o 0.4 μM pep ide in o he subphase was measu ed as a unc ion o he ini ial su ace p essu e o he phospholipid monolaye s. Monolaye exclusion p essu es a e
indica ed in he panels. The do ed line begins a 30 mN/m. (B) Pa i ioning cu es as es ima ed om he ac ional change in NBD-fluo escence in he p esence o inc easing
amoun s o POPG LUV. The solid lines co espond o he bes fi ings o he expe imen al alues o equa ion [1]. The es ima ed K
x
alues a e displayed in he panels. (C) Final
ex en s o ANTS leakage (pe cen age a e 30 min) om POPG LUV as a unc ion o he lipid- o-pep ide mole a io. Lipid concen a ion (100 mM) was fixed. The amoun o
pep ide bound o memb ane was co ec ed acco ding o he pa i ion coe ficien s in panel B. EC
50
alues a e displayed in he panels.
I.J. Fe nández-Sainz e al. / Vi ology 456-457 (2014) 121–130 127
and 5% CO
2
. Vi us was de ec ed by immunope oxidase s aining as
desc ibed abo e, and s ocks o escued i uses we e s o ed a
70 1C.
DNA sequencing and analysis
Full-leng h clones and in i o escued i uses we e comple ely
sequenced wi h CSFV-specific p ime s by he dideoxynucleo ide
chain- e mina ion me hod (Sange e al., 1977). Vi uses eco e ed
om in ec ed animals we e sequenced in he egion o he
genome ha con ained he desi ed mu a ions. Sequencing eac-
ions we e p epa ed wi h he Dye Te mina o Cycle Sequencing Ki
(Applied Biosys ems, Fos e Ci y, CA). Reac ion p oduc s we e
sequenced on a PRISM 3730xl au oma ed DNA sequence (Applied
Biosys ems). Sequence da a was assembled using Sequenche 4.7
™
so wa e (Genes Codes Co po a ion, Ann A bo , MI). The final DNA
consensus sequence ep esen ed, on a e age, a h ee- o ou - old
edundancy a each base posi ion.
Animal in ec ions
Vi ulence o FP mu an i uses ela i e o BIC was ini ially
assessed in 10–12 weeks old, o y-pound comme cial-b eed pigs
inocula ed in anasally (IN) wi h 10
5
TCID
50
o each i us. Pigs
we e andomly alloca ed in o 7 g oups o 4 animals each and we e
inocula ed wi h a FP i us mu an o BIC . Clinical signs (ano exia,
dep ession, pu ple skin discolo a ion, s agge ing gai , dia hea and
cough) and changes in body empe a u e we e eco ded daily
h oughou he expe imen and sco ed as p e iously desc ibed
(Mi elholze e al., 2000). Blood was collec ed a imes pos -
in ec ion om he an e io ena ca a in o EDTA-con aining ubes
(Vacu aine ) o o al and di e en ial whi e blood cell coun s
(pe o med using a Beckman Col e ACT, Beckman, Col e , CA)
and quan ifica ion o i emia by i us i a ion as desc ibed abo e.
Pep ides
Pep ides ep esen ing he CSFV E2 FP (WT) and i s de i ed
mu an s (mu -1 and mu -2, sequences displayed in Fig. 2A) we e
comme cially syn hesized (The mo Scien ific) and agged wi h
he 7-ni obenz-2-oxa-l,3-diazole-4 (NBD) fluo opho e a he
N- e minus. The pu ified pep ides we e dissol ed in dime hyl
sul oxide (DMSO, spec oscopy g ade) and hei concen a ions
de e mined by he bicinchoninic-acid mic oassay (Pie ce, Rock-
o d, IL, USA). Small, dilu ed aliquo s ( ypically 20
μ
L, 1 mg/mL)
we e s o ed ozen and we e hawed only once, upon use.
1-palmi oyl-2-oleoylphospha idylglyce ol (POPG) was pu chased
om A an i Pola Lipids (Bi mingham, AL). The 8-aminonaph ha-
lene-1,3,6- isul onic acid sodium sal (ANTS) and p-xylenebis
(py idinium)b omide (DPX) we e ob ained om Molecula P obes
(Junc ion Ci y, OR). Dodecylphosphocholine (DPC) was om Ana-
ace (Maumee, OH, USA).
Ci cula dich oism s udies
Ci cula dich oism (CD) measu emen s we e ob ained om a
he mally-con olled Jasco J-810 ci cula dich oism spec opola i-
me e calib a ed ou inely wi h (1S)-(þ)-10-campho sul onic acid,
ammonium sal . Samples consis ed o lyophilized pep ides dis-
sol ed a concen a ions o 0.03 mM in 2 mM Hepes (pH 7.4)
bu e . Spec a we e measu ed in a 1 mm pa hleng h qua z cell
ini ially equilib a ed a 25 1C. Da a we e aken wi h a 1 nm
bandwid h a 100 nm/min speed, and he esul s o 20 scans we e
a e aged.
Lipid monolaye pene a ion
Changes in su ace p essu e we e moni o ed as a unc ion o
ime in a fixed-a ea ci cula ough (
μ
T ough S sys em, Kib on,
Fig. 6. Loca ion o pu a i e FP in o schema ic ep esen a ion o a idimensional s uc u e o BVDV E2 (El Oma i e al., 2013). C i ical cys ines a e p esen ed in g een, he PI.W
esidues in blue, and he emainde o he usion pep ide in yellow. The N- e minus is colo ed ed o o ien a ion. (A) A ibbon model is shown wi h he usion pep ide
colo ed and he su ounding β-shee s a e colo ed in o ange (B) The loca ion o he pu a i e FP i is shown a space filling model.
I.J. Fe nández-Sainz e al. / Vi ology 456-457 (2014) 121–130128
Helsinki) measu ing 2 cm in diame e and wi h an aqueous
olume o 1 ml (5 mM Hepes, 100 mM NaCl (pH 7.4)). Lipids,
dissol ed in chlo o o m, we e sp ead o e he su ace and he
desi ed ini ial su ace p essu e (
π
0
) was a ained by changing he
amoun o lipid applied o he ai -wa e in e ace. Pep ides we e
injec ed in o he subphase o a final concen a ion o 0.4
μ
M wi h
a Hamil on mic osy inge.
Lipid esicle assays
La ge unilamella esicles (LUV) made o POPG we e p epa ed
acco ding o he ex usion me hod in 5 mM Hepes, 100 mM NaCl
(pH 7.4) using memb anes wi h a nominal po e-size o 0.1 mm.
Pa i ioning in o memb anes was e alua ed by moni o ing he
change in he emi ed NBD-fluo escence. Co ec ed spec a we e
eco ded using a Fluo oMax-3 (Jobin Y on Ho iba, Longiumeau,
F ance) wi h exci a ion se a 460 nm and 2-nm sli s. Pa i ioning
cu es we e subsequen ly compu ed om he ac ional changes
in emi ed NBD-fluo escence when i a ed wi h inc easing lipid
concen a ions. The appa en mole ac ion pa i ion coe ficien s,
K
x(app)
, we e de e mined by fi ing he expe imen al alues o a
hype bolic unc ion:
F=F
0
¼1þ½ðF
max
=F
0
Þ1½L
Kþ½Lð1Þ
whe e [L] is he lipid concen a ion and Kis he lipid concen a ion
a which he bound pep ide ac ion is 0.5. The e o e, K
x(app)
¼[W]/
Kwhe e [W] is he mola concen a ion o wa e .
lsb-0.009w?>Vesicle pe meabiliza ion was assayed by mon-
i o ing he elease o he medium o encapsula ed fluo escen
ANTS as desc ibed (Nie a e al., 1994). In b ie , LUV con aining
12.5 mM ANTS, 45 mM DPX, 20 mM NaCl and 5 mM Hepes we e
ob ained by sepa a ing he unencapsula ed ma e ial by gel-
fil a ion in a Sephadex G-75 column ha was elu ed wi h 5 mM
Hepes and 100 mM NaCl (pH 7.4). Fluo escence measu emen s
we e pe o med by se ing he ANTS emission a 520 nm and he
exci a ion a 355 nm. A cu o fil e (470 nm) was placed be ween
he sample and he emission monoch oma o . The baseline leak-
age (0%) co esponded o he fluo escence o he esicles a ime 0,
while 100% leakage was he fluo escence alue ob ained a e
addi ion o T i on X-100 (0.5% / ).
Acknowledgmen s
This s udy was in pa suppo ed by Spanish MINECO and
Basque Go e nmen g an s (BIO2011-29792 and IT838-13 o
J.L.N.). We hank he Plum Island Animal Disease Cen e animal
ca e uni s a o excellen echnical assis ance. We specially hank
Melanie P a a o edi ing he manusc ip .
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