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.). 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