West Nile virus capsid protein interacts with biologically relevant host lipid systems

ORIGINAL RESEARCH published: 06 Feb ua y 2019 doi: 10.3389/ cimb.2019.00008 F on ie s in Cellula and In ec ion Mic obiology | www. on ie sin.o g 1Feb ua y 2019 | Volume 9 | A icle 8 Edi ed by: Shel on S. B ad ick, The Uni e si y o Texas Medical B anch a Gal es on, Uni ed S a es Re iewed by: Shenngbo Cao, Huazhong Ag icul u al Uni e si y, China Pe e Hin e do e , Johannes Keple Uni e si y o Linz, Aus ia *Co espondence: Nuno C. San os [email p o ec ed] I o C. Ma ins [email p o ec ed] †P esen Add ess: And é F. Faus ino, iBET, Ins i u o de Biologia Expe imen al e Tecnológica, Oei as, Po ugal Special y sec ion: This a icle was submi ed o Vi us and Hos , a sec ion o he jou nal F on ie s in Cellula and In ec ion Mic obiology Recei ed: 17 Augus 2018 Accep ed: 11 Janua y 2019 Published: 06 Feb ua y 2019 Ci a ion: Ma ins AS, Ca alho FA, Faus ino AF, Ma ins IC and San os NC (2019) Wes Nile Vi us Capsid P o ein In e ac s Wi h Biologically Rele an Hos Lipid Sys ems. F on . Cell. In ec . Mic obiol. 9:8. doi: 10.3389/ cimb.2019.00008 Wes Nile Vi us Capsid P o ein In e ac s Wi h Biologically Rele an Hos Lipid Sys ems Ana S. Ma ins, Filomena A. Ca alho, And é F. Faus ino†, I o C. Ma ins*and Nuno C. San os* Ins i u o de Medicina Molecula , Faculdade de Medicina, Uni e sidade de Lisboa, Lisbon, Po ugal Wes Nile and dengue i uses a e closely ela ed la i i uses, o igina ing mosqui o-bo ne i al in ec ions o which he e a e no e ec i e and speci ic ea men s. Thei capsid p o eins sequence and s uc u e a e pa icula ly simila , o ming highly supe imposable α-helical homodime s. Measu ing p o ein-ligand in e ac ions a he single-molecule le el yields de ailed in o ma ion o biological and biomedical ele ance. In his wo k, such an app oach was success ully applied on he cha ac e iza ion o he Wes Nile i us capsid p o ein in e ac ion wi h hos lipid sys ems, namely in acellula lipid d ople s (an essen ial s ep o dengue i us eplica ion) and blood plasma lipop o eins. Dynamic ligh sca e ing measu emen s show ha Wes Nile i us capsid p o ein binds e y low-densi y lipop o eins, bu no low-densi y lipop o eins, and his in e ac ion is dependen o po assium ions. Ze a po en ial expe imen s show ha he in e ac ion wi h lipid d ople s is also dependen o po assium ions as well as su ace p o eins. The o ces in ol ed on he binding o he capsid p o ein wi h lipid d ople s and lipop o eins we e de e mined using a omic o ce mic oscopy-based o ce spec oscopy, p o ing ha hese in e ac ions a e K+-dependen a he han a gene al dependence o ionic s eng h. The capsid p o ein in e ac ion wi h hos lipid sys ems may be a ge ed in u u e he apeu ic s a egies agains di e en la i i uses. The biophysical and nano echnology app oaches employed in his s udy may be applied o cha ac e ize he in e ac ions o o he impo an p o eins om di e en i uses, in o de o unde s and hei li e cycles, as well as o ind new s a egies o inhibi hem. Keywo ds: Wes Nile i us, lipid d ople s, lipop o eins, a omic o ce mic oscopy, o ce spec oscopy, dynamic ligh sca e ing, ze a po en ial INTRODUCTION Wes Nile i us (WNV) is a Fla i i us closely ela ed o Dengue (DENV) and Zika (ZIKV) i uses. I was i s isola ed in Uganda in 1937 (Kilpa ick, 2011), and since hen became endemic ac oss T opical A ica, Sou he n Asia and No he n Aus alia, wi h episodic occu ences in Eu ope (Kilpa ick, 2011). Despi e i s se e i y, WNV in ec ion aised li le conce n un il an ex emely i ulen s ain appea ed in No h Ame ica, a he u n o he millennium (Rei e , 2010; Rossi e al., 2010; Kilpa ick, 2011). The i us is ansmi ed o humans by he bi e o Culex spp. mosqui o ec o s eeding on in ec ed bi ds, wi h mig a o y bi ds cons i u ing he majo ansmission ehicle (Reisen, 2010; Rei e , 2010; Kilpa ick, 2011). In 2012, he e was a esu gence in No h Ame ica Ma ins e al. WNV In e ac s Wi h Lipid Sys ems (5,674 and 428 clinical human cases epo ed in he USA and Canada, espec i ely) and in Eu ope and neighbo coun ies (937 cases) (G ay and Webb, 2014). In 2013, 783 WNV human cases we e epo ed in Eu ope (G ay and Webb, 2014). In 2016, 2,038 cases o WNV disease in human we e epo ed o USA Cen e s o Disease Con ol and P e en ion (CDC), wi h 56% o he cases classi ied as neu oin asi e disease (CDC, h ps://www.cdc.go /wes nile/ s a smaps/p elimina ymapsda a/index.h ml, accessed July 2017). WNV is hus no likely o disappea on i s own acco d and equi es u he esea ch o de elop e ec i e ea men s. To achie e his, i is impo an o unde s and WNV in ec ion, which can ei he lead o mild symp oms, common o o he eb ile diseases o o a mo e se e e clinical o m o neu o-in asi e disease ha includes neck s i ness, s upo , diso ien a ion, meningi is, pa alysis, coma, and dea h (Rossi e al., 2010). Only 1% o he in ec ion cases p og ess o his inal neu o-in asi e s age (Diamond, 2009; Kimu a e al., 2010; Rossi e al., 2010; Lim e al., 2011b; Sej a , 2014). Al hough uncommon, his neu ological s age is li e h ea ening. I is c ucial o a oid WNV in ec ion o e ol e o such condi ion. Fo his, i is necessa y o unde s and WNV in ec ion p og ession. Typically, ollowing a bi e o a WNV in ec ed mosqui o, in he i s s age he i us in ec s ke a inocy es and Lange hans cells, which end up in egional lymph nodes, whe e he i s ound o he ini ial eplica ion occu s (Johns on e al., 2000; Lim e al., 2011a). On a second s age, ano he ound o eplica ion occu s, when WNV i e becomes high enough o i o sp ead sys emically o isce al o gans, p ima ily in ec ing he kidney and he spleen (Johns on e al., 2000; Samuel and Diamond, 2005; Tesh e al., 2005; Lim e al., 2011a). The disease p og esses o he neu o-in asi e s age only i high i emia is achie ed a his c ucial s age (Samuel and Diamond, 2005; Tesh e al., 2005). The e o e, blocking he in ec ion a he isce al s age is c i ical o p e en ing i s e olu ion o he li e- h ea ening neu ological s age (Diamond, 2009; Kimu a e al., 2010; Rossi e al., 2010; Lim e al., 2011b). Fo his o be possible, i is impo an o examine he simila i ies be ween WNV and closely ela ed la i i uses, especially in he i s s ages o in ec ion. Membe s o Fla i i us genus, Fla i i idae amily, o which WNV belongs, a e s uc u ally simila , wi h homologous p o eins sha ing highly conse ed egions. Fla i i uses such as WNV a e icosahed al en eloped i uses composed o a lipid bilaye su ounding a nucleocapsid con aining a posi i e sense single- s anded genomic RNA complexed wi h mul iple copies o he capsid (C) p o ein (Mukhopadhyay e al., 2005; Bhu anakan ham and Ng, 2013). Vi al assembly, one o he mos impo an p ocesses o he i us li e cycle, is media ed by he C p o ein. The C p o eins ha e oughly 100 amino acid esidues. WNV, DENV, and ZIKV capsid p o eins, o example, ha e 105, 100, and 104 esidues, espec i ely, being highly simila , as p e iously epo ed by us (Ma ins e al., 2012). In solu ion, he C p o eins o WNV and DENV o m a homodime ich in α-helices, wi h each monome composed by ou α-helices (named α1 o α4) connec ed by sho loop egions (Jones e al., 2003; Dokland e al., 2004; Ma e al., 2004), which a e simila in e ms o sequence and s uc u e (Ma ins e al., 2012). WNV and DENV C p o eins ha e an asymme ic cha ge dis ibu ion, being e y posi i ely cha ged p o eins, while also con aining hyd ophobic pocke s. The i s 20 esidues o WNV C, as well o o he Fla i i us C p o eins, namely DENV, a e in insically diso de ed and a e expec ed o acili a e hei unc ions (I anyi-Nagy and Da lix, 2010). Among hem, C p o ein in e ac ion wi h hos lipid s uc u es, essen ial o i al eplica ion, is mos ele an o u u e d ug design (Ma ins e al., 2012; Faus ino e al., 2014, 2015a). Vi uses om he Fla i i idae amily manipula e he hos lipid me abolism o induce he condi ions needed o hei own i al eplica ion (Zhang e al., 2017). Lipid d ople s (LDs) ha e been s udied as impo an in acellula o ganelles o i us pa hogenesis. LDs a e o med in he endoplasmic e iculum (ER) and play a c ucial ole in cell homeos asis. Al hough LDs a e used by he immune sys em agains pa hogens, some i uses ha e e ol ed s a egies o use hese o ganelles as pla o ms o i al assembly and eplica ion. Mo eo e , i uses use LDs as ene gy ese oi s du ing eplica ion o he i al genome, an ene gy- consuming p ocess (Wang, 2016). Fo ins ance, DENV has been p oposed o use LDs h ough he p ocess o lipophagy, o i al eplica ion (Hea on and Randall, 2010). Fu he mo e, LDs ha e been p oposed as a pla o m o i al assembly (Roingea d and Melo, 2017). Impo an ly, LDs a e a ge ed by se e al s uc u al and non-s uc u al i al p o eins du ing he i us li e cycle (Zhang e al., 2017). The associa ion o hese p o eins wi h LDs has been s udied in o de o unde s and hei ole in he key in acellula p ocesses ha occu du ing i us assembly and eplica ion. In some membe s o he Fla i i idae amily, such as DENV and hepa i is C i us (HCV), he in e ac ion o he homologous C p o eins wi h hos lipid sys ems, namely in acellula LDs and blood plasma e y low-densi y lipop o eins (VLDL), was shown o be impo an o hese i uses biological ac i i y (Mukhopadhyay e al., 2005; Samsa e al., 2009; Ca alho e al., 2012; Ma ins e al., 2012; Faus ino e al., 2014). Gi en ha WNV and DENV C a e closely ela ed and highly simila (Mukhopadhyay e al., 2005; Ma ins e al., 2012) and ha he in e ac ion o DENV C wi h hos LDs is essen ial o success ul dengue i al eplica ion (Samsa e al., 2009), such in e ac ion may play a majo ole in WNV and ela ed i uses. This led o s udies on he in e ac ion o hos LDs wi h la i i uses C p o eins. In e es ingly, ZIKV C p o ein was ecen ly epo ed o co-localize wi h LDs as well (Shang e al., 2018). Mo eo e , DENV C-LDs in e ac ion is s ong and speci ic, equi ing LDs su ace p o eins o he binding, as well as he cha ac e is ic high in acellula po assium concen a ions (Ca alho e al., 2012). Impo an ly, he esidues wi hin DENV C hyd ophobic α2–α2′co e in ol ed in LDs in e ac ion a e conse ed among la i i uses, including WNV, bo h in e ms o sequence and s uc u al o ganiza ion (Ma ins e al., 2012). As no speci ic ea men is a ailable o WNV in ec ion, cla i ying he biological ac i i y o WNV C ega ding i s abili y o in e ac wi h hos lipid sys ems may pa e he way o u u e ea men s agains his and ela ed i uses. Wi h his in mind, we i s cha ac e ized ele an hos lipid sys ems, namely LDs isola ed om baby hams e kidney (BHK-21) cells and lipop o eins isola ed om human blood plasma, in he absence o i al p o eins. Following, we es ed WNV C in e ac ion wi h F on ie s in Cellula and In ec ion Mic obiology | www. on ie sin.o g 2Feb ua y 2019 | Volume 9 | A icle 8 Ma ins e al. WNV In e ac s Wi h Lipid Sys ems hese lipid sys ems using ze a po en ial (ζ-po en ial) analysis, dynamic ligh sca e ing (DLS) and a omic o ce mic oscopy (AFM) based o ce spec oscopy. Ou esul s show ha WNV C binds o LDs and VLDL and ha hese in e ac ions a e s ong and speci ic, in ol ing K+ions and su ace p o eins om hese hos lipid sys ems. The use o single-molecule me hods o s udy and measu e biologically ele an p o ein-ligand in e ac ions is a p omising a enue o esea ch o nanomedicine, enabling he acquisi ion o de ailed s uc u al knowledge on he sys em unde s udy. He e, we success ully applied such app oaches a he le el o an impo an human pa hogen, by s udying a key i al p o ein, he WNV C p o ein, in he con ex o i s in e ac ion wi h ele an hos lipid sys ems. A s uc u al unde s anding o he majo ac o s modula ing hese key in e ac ions o WNV C is ex emely impo an , since i may lead o new d ug de elopmen app oaches agains WNV and o he la i i uses, such as he closely ela ed Dengue and Zika i uses. MATERIALS AND METHODS Ma e ials Human plasma lipop o eins we e ob ained om Kalen Biomedical LLC (Mon gome y Village, MD, USA). WNV C p o ein, se o ype Kunjin, 105 esidues (11.7 kDa), was chemically syn hesized by VCPBIO (Shenzen, China) wi h >95% pu i y. The C- e minal was amida ed and he N- e minal ace yla ed. The seconda y s uc u e o he p o ein was e alua ed ia ci cula dich oism spec oscopy, showing α-helical and andom coil con en . Expe imen s we e pe o med in wo di e en bu e s: TEE KCl bu e (20 mM T is-HCl, 100 mM KCl, 1 mM EDTA and 1 mM EGTA, pH 7.4) and TEE NaCl bu e (20 mM T is-HCl, 100 mM NaCl, 1 mM EDTA and 1 mM EGTA, pH 7.4). Cell Cul u e and Lipid D ople s Isola ion LDs we e isola ed and pu i ied om baby hams e kidney (BHK-21) cells, by cell ca i a ion ollowed by suc ose-g adien ul acen i uga ion, as p e iously desc ibed (Ca alho e al., 2012; Ma ins e al., 2012). B ie ly, BHK-21 cell line was main ained in high-glucose Dulbecco’s modi ied Eagle’s medium (DMEM) wi h 0.01% sodium py u a e and 4 mM L-glu amine, supplemen ed wi h 10% e al bo ine se um, 100 U/mL penicillin and 100 U/mL s ep omycin in a T75 cul u e lask. Cells we e g own a 37 ◦C, in a humidi ied 5% CO2incuba o . A e 72 h, 24 h be o e LDs isola ion and when app oxima ely 80% con luence was eached, he medium was eplaced o DMEM wi hou e al bo ine se um and an ibio ics, and cells we e ea ed wi h 10 mM oleic acid. A e 24 h o cells incuba ion wi h oleic acid, LDs we e isola ed by washing cells wice and dis up ing hem in TEE bu e wi h 100 mM KCl, cen i uging he cell lysa e and collec ing he supe na an , om which he LDs ac ions we e isola ed ia ul acen i uga ion, ollowing ou p e ious wo k (Ca alho e al., 2012; Ma ins e al., 2012). LD ac ions we e kep a 4◦C, checking be o e use i hey p esen he p e iously epo ed ζ-po en ial alue o , oughly, −20 mV (Ca alho e al., 2012). The same p o ocol was pe o med o isola e LDs in TEE NaCl bu e . DLS Measu emen s o Lipop o eins Dynamic ligh sca e ing (DLS) measu emen s we e ca ied ou on a Mal e n Ze asize Nano ZS (Mal e n, UK) equipped wi h a He-Ne lase (632.8 nm), wi h backsca e ing de ec ion a 173◦. DLS allows o measu e pa icle hyd odynamic diame e and size dis ibu ion o molecules o sup amolecula agg ega es, based on he ligh sca e ing in ensi y luc ua ion on a small olume, on a imescale o mic oseconds, due o he B ownian mo ion o he pa icles (Domingues e al., 2008; S e e eld e al., 2016). The sca e ed ligh is collec ed and measu ed a a gi en angle by a sensi i e de ec o . Size de e mina ions can be pe o med h ough he measu emen o he sca e ing ligh in ensi y luc ua ions as a unc ion o ime. The size o pa icles in suspension can be de e mined in e ms o DH, analyzing he no malized in ensi y au oco ela ion unc ions. Wi h he S okes-Eins ein equa ion, i is possible de e mine he hyd odynamic diame e (DH) om he di usion coe icien (D) alue (Be ne and Peco a, 1990): DH=κT 3πηD(1) whe e ηis he dispe san iscosi y, κ he Bol zmann cons an and T he absolu e empe a u e. Fo hese measu emen s, glass cu e es wi h ound ape u e we e used. VLDL o low- densi y lipop o eins (LDL) we e independen ly dilu ed o a inal concen a ion o 50 µg/mL in TEE KCl bu e . VLDL we e also dilu ed in TEE NaCl bu e a he same inal concen a ion. The DHo bo h lipop o eins was measu ed wi hou WNV C. A e wa ds, successi e olumes o WNV C solu ion we e added, in o de o assess concen a ions om 0.25 o 5 µM, and he DHwas de e mine o each o hem. Samples we e allowed o equilib a e o 15 min a 25 ◦C be o e measu emen s. Fo each sample, 10 measu emen s we e conduc ed, each measu emen being he a e age o 10 uns o 10 s each, wi hou wai ing be ween hem. This p ocedu e was epea ed a leas h ee imes o each condi ion, wi h independen lipop o eins samples. The no malized in ensi y au oco ela ion unc ions we e analyzed wi h he CONTIN me hod (P o enche , 1982). The DH alue o each measu emen was ob ained om he peak o he pa icle numbe dis ibu ion, n(DH), o each o he 10 measu emen s. Lipop o eins size da a was analyzed in a- g oup (o 10 measu emen s) by a e age and s anda d de ia ion, disca ding ou lie s. The a e age wi hou ou lie s was close o he median in all he size da a poin s. Values a e p esen ed as mean ±s anda d e o (SE). Da a se s we e compa ed agains he se o measu emen s wi hou WNV C using he Mann-Whi ney U es . Di e ences we e conside ed s a is ically signi ican when p<0.05. Ze a Po en ial Analysis o LDs Su ace Cha ge Ze a po en ial (ζ-po en ial) expe imen s we e pe o med in he same equipmen used o he DLS measu emen s (Mal e n Ze asize Nano ZS). ζ-po en ial measu emen s a e based on he concep ha cha ged pa icles in suspension a ac o hei su ace ions wi h opposi e cha ge, o which hey can be s ongly bound. These su ace-bound ions o m a laye , he S e n laye F on ie s in Cellula and In ec ion Mic obiology | www. on ie sin.o g 3Feb ua y 2019 | Volume 9 | A icle 8 Ma ins e al. WNV In e ac s Wi h Lipid Sys ems (Uskoko i´ c, 2012). Beyond he S e n laye , ano he laye is o med, whe e ions di use mo e eely. When he pa icle mo es in he solu ion, he ions s ongly a ached o hei su ace mo e wi h i , whe eas he ions in he di use bounda y do no mo e wi h he pa icle. The po en ial ha exis s a his bounda y is de ined as he ζ-po en ial (Ki by and Hasselb ink, 2004; Domingues e al., 2008). The ζ-po en ial o he pa icles can be calcula ed using he Hen y’s ela ion (Domingues e al., 2008): ζ=3ηu 2ε (ka)(2) whe e ζis he ζ-po en ial, u he elec opho e ic mobili y, η he iscosi y o he sol en , εi s dielec ic cons an and (ka) is he Hen y’s unc ion. The physical cons an s used o he calcula ions we e: n0=1.330, η=0.8872 cP, T=298.15 K, λ=632.8 nm and θ=13◦. LDs samples we e equilib a ed o 15 min a 25 ◦C, a he Ze asize , and hen ζ-po en ial was de e mined om he a e age o 15 measu emen s (100 uns each), wi h 90 s o wai ing ime be ween measu emen s. Samples we e analyzed by measu ing independen ly he ζ-po en ial o LDs in a inal olume o 842.5 µL, a e incuba ion o 15 min a oom empe a u e wi h di e en concen a ions o WNV C. Following p e ious app oaches (Ca alho e al., 2012; Ma ins e al., 2012), limi ed p o eolysis o LDs wi h ypsin was pe o med by incuba ing he LDs samples wi h 10 µM ypsin in TEE bu e (wi h KCl o NaCl) o 15 min a oom empe a u e. To s op he eac ion, 1 mM phenylme hylsul onyl luo ide (PMSF) was added o he mix u e o 5 min a oom empe a u e, a e which he ζ- po en ial o ypsinized LDs samples was analyzed by measu ing independen ly LDs in a inal olume o 842.5 µL, a e incuba ion o 15 min a oom empe a u e wi h di e en concen a ions o WNV C. The a ia ion o ze a po en ial (1ζ) o each sample was de e mined by sub ac ing he alue o he ζ-po en ial o LDs in he absence o WNV C om he ζ-po en ial o LDs in he p esence each WNV C concen a ion. Expe imen al da a was i ed using he equa ion: 1ζ =1ζmax[WNV C] C1/2+[WNV C](3) whe e 1ζmax is he i ed maximum ampli ude o a ia ion o he ζ-po en ial induced by he in e ac ion wi h WNV C, and C1/2is he WNV C concen a ion a 1ζmax/2. Ligh sca e ing spec oscopy echniques ha e been used in s udies o di e en ields. He e, we used ζ-po en ial o s udy he in e ac ion o he C p o ein wi h LDs. The same app oach can be used o s udy cha ged pa icles, such as pep ides, e y h ocy es, and bac e ia. Fo ins ance, ζ-po en ial measu emen s we e employed o s udy he e ec o e y h ocy es aging on he in e ac ion wi h ib inogen (Ca alho e al., 2011). The same ype o measu emen s we e also used o e alua e he e ec o an imic obial pep ides on lipid esicles mimicking bac e ia-like memb anes (I azazabal e al., 2019). Fu he mo e, ζ-po en ial has been used o cha ac e ize nanopa icles de eloped o biomedical applica ion, namely o alida e he elec os a ic in e ac ion be ween nanopa icles and hei a ge s and o cha ac e ize he pep ide ancho ing p o ile o nanopa icles (Ca alho e al., 2018). DLS may complemen hese s udies, p o iding quan i a i e in o ma ion on pa icle size dis ibu ion. Besides de e mining he size dis ibu ion o nanopa icles, DLS measu emen s we e used o con i m su ace unc ionaliza ion, cha ac e ize long e m s abili y in di e en condi ions and iden i y he agg ega ion p o ile o nanopa icles (Ca alho e al., 2018). LDs and Lipop o eins P epa a ion o Fo ce Spec oscopy Measu emen s Ten µL o LDs o human plasma lipop o eins (VLDL o LDL) suspensions we e placed on o hin eshly clea ed musco i e mica and allowed o deposi o 30 min a oom empe a u e. Non-adhe en LDs o lipop o eins we e emo ed by 5 sequen ial washing s eps wi h TEE bu e (wi h Na+o K+, depending on he expe imen ). Samples we e loaded in o he AFM appa a us and allowed o equilib a e in he espec i e TEE bu e o 10 min be o e o ce spec oscopy measu emen s. Func ionaliza ion o AFM Tips Wi h WNV C A p o ocol well es ablished in ou lab was used o unc ionalize AFM ips wi h WNV C o o ce spec oscopy measu emen s (Ca alho and San os, 2012; Ca alho e al., 2012; Faus ino e al., 2015b; Guedes e al., 2016). OMCL TR-400- ype silicon ni ide ips (Olympus, Japan) we e cleaned wi h an in ense UV ligh sou ce and silanized in a acuum chambe wi h 3-aminop opyl- ie hoxysilane (APTES, 30 µL) and N,N-di-isop opyle hylamine (10 µL), o 1 h, unde an a gon a mosphe e, o be coa ed wi h a sel -assembled monolaye o amines. Following his, p obes we e insed wi h esh chlo o o m and d ied wi h ni ogen gas. The silaniza ion p ocess esul s in a uni o mly dis ibu ed sel - assembled monolaye o amino- e mina ed APTES molecules on he AFM ips, which we e hen placed in o a 2.5% ( / ) glu a aldehyde solu ion o 20 min and washed 3 imes wi h TEE bu e . Finally, he ips we e placed in o a 187 µM WNV C solu ion du ing 30 min o co alen ly bind he p o ein. P o ein- unc ionalized ips we e immedia ely moun ed on o he AFM ins umen and used o he o ce spec oscopy measu emen s. AFM-Based Fo ce Spec oscopy Measu emen s AFM measu emen s we e pe o med wi h a NanoWiza d II a omic o ce mic oscope (JPK Ins umen s, Be lin, Ge many), moun ed on op o an Axio e 200 in e ed mic oscope (Zeiss, Jena, Ge many), using iangula can ile e s wi h a py amidal ip wi h adius o 15 nm and a esonance equency o 11 kHz in ai (OMCL-TR400, Olympus Eu ope, Ge many). The AFM head is equipped wi h a 15-µm z- ange linea ized piezoscanne and an in a ed lase . The sp ing cons an o he ips we e calib a ed by he he mal luc ua ion me hod, yielding alues o 22 ±5 mN/m. Fo each con ac o he can ile e wi h LDs o plasma lipop o eins, he AFM ip-sample dis ance was adjus ed in o de o main ain an applied o ce o 200 pN be o e e ac ion. Molecula ecogni ion was sea ched by p essing he ip in e mi en ly on o di e en poin s o LDs o lipop o eins F on ie s in Cellula and In ec ion Mic obiology | www. on ie sin.o g 4Feb ua y 2019 | Volume 9 | A icle 8 Ma ins e al. WNV In e ac s Wi h Lipid Sys ems adso bed o he mica su ace. Da a we e collec ed o each o ce-dis ance cycle a 2 µm/s, leading o a loading a e o 4 nN/s. Expe imen s we e pe o med a oom empe a u e, by main aining he labo a o y be ween 23 and 25 ◦C. Small a ia ions in empe a u e a his ange did no a ec he o ce spec oscopy measu emen s. In he expe imen s wi h LDs, measu emen s we e conduc ed using TEE bu e wi h KCl (10 o 100 mM) o NaCl (100 mM). In he expe imen s wi h lipop o eins, measu emen s we e conduc ed using TEE bu e wi h 100 mM KCl, bo h o VLDL and LDL samples, and using TEE bu e wi h 100 mM NaCl o VLDL. Each expe imen was pe o med a leas h ee imes, each ime on di e en samples and wi h di e en unc ionalized ips. Fo any gi en expe imen , app oxima ely 5,000 o ce-dis ance cu es we e collec ed and analyzed (Ca alho e al., 2010, 2012, 2013; Ma ins e al., 2012; Faus ino e al., 2014). Fo ce cu es we e analyzed using he JPK image p ocessing so wa e . 4.2.61 (JPK Ins umen s, Be lin, Ge many). His og ams o he (un)binding o ces o each s udied p o ein-LD o p o ein-lipop o ein in e ac ion we e cons uc ed choosing he ideal bin size o achie e he bes - i ed Gaussian model peak o ces. The selec ed binning size was 6 pN. Fo ce up u e alues anging be ween 0 and 10 pN we e conside ed o ep esen noise o expe imen al a i ac s, while alues up o 25 pN we e assigned o unspeci ic in e ac ions (Ca alho e al., 2012; Faus ino e al., 2014). F om each his og am, he mos likely single WNV C-hos lipid sys em up u e o ce can be de e mined by i ing he dis ibu ions o he up u e o ces wi h he Gaussian model. Measu emen s wi h ips a di e en s eps o he unc ionaliza ion p ocess (including non- unc ionalized ips) we e conduc ed on mica, and on LDs o lipop o eins samples, which se e as con ols o he AFM ip unc ionaliza ion p ocess. AFM-based o ce spec oscopy was used in his s udy o measu e he in e ac ion o ces be ween molecules. Taking ad an age o i s piconew on sensi i i y, we measu ed he o ce necessa y o b eak he bonds be ween WNV C and LDs o lipop o eins. The same app oach was used o s udy o he molecula in e ac ions (Guedes e al., 2016). AFM is commonly used o cons uc opog aphical images o he su ace o a sample by scanning o apping he sample su ace wi h a ip moun ed unde a lexible can ile e . A lase beam is e lec ed on he back o he can ile e and any small de lec ions a e ampli ied by an op ical le e mechanism, using as de ec o a posi ion-sensi i e pho odiode (Ca alho e al., 2013). These de lec ions a e p ocessed by he elec onic sys em and he sample su ace opog aphy is de e mined (Ca alho e al., 2013). AFM p o ides de ailed in o ma ion o a sample su ace p ope ies. Fo ins ance, AFM has been ex ensi ely used o s udy he e ec o an imic obial pep ides/p o eins on human pa hogens (Domingues e al., 2014; Migliolo e al., 2016; Gonçal es e al., 2017). RESULTS To un a el he de ails o WNV C in e ac ion wi h ele an hos lipid sys ems, a combina ion o biophysics echniques was employed. ζ-po en ial s udies and AFM-based o ce spec oscopy (un)binding analysis we e pe o med o cha ac e ize WNV C in e ac ion wi h LDs, quan i ying he ole o cha ges in he in e ac ions, he binding a ini y, as well as he binding o ces o he in e ac ions a he single-molecule le el. DLS and AFM-based o ce spec oscopy measu emen s we e combined o cha ac e ize he WNV C in e ac ion wi h plasma lipop o eins, namely VLDL and LDL. By combining hese di e en echniques, i was possible o de e mine he single-molecule le el in e ac ion o ces be ween WNV C and VLDL and o obse e he inc emen in VLDL hyd odynamic diame e upon he in e ac ion wi h WNV C. ζ-Po en ial Measu emen s Demons a e a WNV C-LDs Binding In luenced by K+and LDs Su ace P o eins ζ-po en ial measu emen s we e pe o med o de e mine i WNV C is able o bind o LDs. Upon he addi ion o WNV C o LDs in TEE bu e wi h 100 mM KCl, he e was a concen a ion dependen inc ease in he ζ-po en ial (Figu e 1A; da a a e p esen ed as he a ia ion o ζ-po en ial o LDs in he absence o WNV C and LDs in he p esence o WNV C, 1ζ). In he absence o WNV C, LDs in TEE bu e wi h 100 mM KCl p esen a nega i e ζ-po en ial alue (−20.6 ±0.7 mV). The i a ion o LDs suspension wi h WNV C induced a p og essi e inc ease in he sca e ing pa icle cha ge, s abilizing a posi i e alues (+15.8 ± 0.7 mV o a WNV C concen a ion o 5 µM). Wi h an iden ical addi ion o WNV C o ypsinized LDs, he e was a lowe inc ease in 1ζ han o he non- ypsinized LDs (Figu e 1A). T ypsinized LDs ha e an ini ial alue o −18.8 ±1.2 mV ha , upon i a ion, inc eases and s abilizes a +1.5 ±1.7 mV. The inc ease in 1ζo non- ypsinized LDs is much highe han he obse ed o ypsinized LDs a simila concen a ions. The eplacemen o po assium ions on he TEE bu e by he same concen a ion o sodium ions yields simila esul s. Values o ζ-po en ial o LDs in TEE NaCl bu e also inc ease o highe WNV C concen a ions (Figu e 1B). In his sodium bu e , LDs p esen a ζ-po en ial o −16.5 ±0.5 mV in he absence o WNV C. A he highes WNV C concen a ion es ed (5 µM), he ζ-po en ial alue eaches +12.7 ±0.3 mV. The addi ion o WNV C o ypsinized LDs in TEE NaCl bu e lead o a lowe inc ease o LDs ζ-po en ial han he non- ypsinized LDs (Figu e 1B), om −16.6 ±0.6 mV (wi hou WNV C) up o −2.8 ±2.3 mV (a he highes concen a ion o WNV C). O e all, 1ζis consis en ly smalle in sodium bu e han o he po assium bu e o he same WNV C concen a ion. The expe imen al 1ζ-po en ial WNV C-LDs binding cu es we e i ed o an empi ical binding cu e model (Ca alho e al., 2012) (Figu e 1). The alues o he maximum ampli ude o a ia ion o ζ-po en ial induced by he in e ac ion wi h WNV C (1ζmax) and he WNV C concen a ion a 1ζmax/2 ( he hal - maximal e ec , C1/2) a e p esen ed on Table 1. Non- ypsinized LDs in TEE KCl bu e display a C1/2o 112 ±15 nM while o ypsinized LDs a 4.6- old highe C1/2 alue (519 ±168 nM) is obse ed. LDs su ace p o eins a e he e o e impo an o he in e ac ion wi h he i al p o ein. K+ions also seem o play a ole in WNV C-LDs in e ac ion. When TEE KCl bu e is eplaced wi h TEE NaCl bu e , a C1/2o 111 ±21 nM o non- ypsinized F on ie s in Cellula and In ec ion Mic obiology | www. on ie sin.o g 5Feb ua y 2019 | Volume 9 | A icle 8 Ma ins e al. WNV In e ac s Wi h Lipid Sys ems FIGURE 1 | LDs ζ-po en ial analysis a di e en WNV C concen a ions. Va ia ion o ζ-po en ial (1ζ) alues o LDs samples isola ed wi h TEE KCl bu e (K+bu e ) (A) and TEE NaCl bu e (Na+bu e ) (B) in he absence and in he p esence o dis inc WNV C concen a ions. ζ-po en ial alues de e mined o LDs wi hou ypsin p eincuba ion ( illed symbols) and LDs p e-incuba ed wi h ypsin (emp y symbols). WNV C concen a ion axes a e p esen ed in loga i hmic scale. Resul s a e p esen ed as mean ±s anda d e o (SE). Expe imen s we e pe o med in iplica e. Solid lines we e ob ained by i ing he expe imen al da a using Equa ion 3. WNV C binds o LDs inducing a p og essi e inc ease in he su ace cha ge o he LD-WNV C complex. TABLE 1 | ζ-po en ial analysis o LDs i a ion wi h WNV C. Condi ion C1/2(nM) 1ζmax (mV) K+bu e Non- ypsinized 112 ±15 37.6 ±1.2 T ypsinized 519 ±168 26.0 ±2.5 Na+bu e Non- ypsinized 111 ±21 29.8 ±1.5 T ypsinized 1560 ±697 18.8 ±3.1 The alues shown (p esen ed as mean ±SE) a e he maximum ampli ude o a ia ion o he LDs ζ-po en ial (1ζmax) induced by he in e ac ion wi h WNV C, and he WNV C concen a ion a 1ζmax/2 (C1/2). Values o C1/2 and 1ζmax we e ob ained h ough he i ing o ζ-po en ial expe imen al da a using equa ion 3. K+and Na+bu e s a e TEE bu e wi h 100 mM KCl o 100 mM NaCl, espec i ely. LDs is obse ed. Howe e , i he same expe imen is pe o med wi h ypsinized LDs a C1/2o 1560 ±697 nM (14- old highe ) is obse ed. Mo eo e , he di e ence be ween he i ed 1ζmax alues o he se e al condi ions es ed also wo h no e. The 1ζmax alue o non- ypsinized LDs in TEE KCl bu e (+37.6 ±1.2 mV) is highe han he 1ζmax o ypsinized LDs in he same bu e (+26.0 ±2.5 mV), and i is also highe han he 1ζmax o non- ypsinized LDs in TEE NaCl bu e (+29.8 ± 1.5 mV). Jus ocusing on TEE NaCl bu e , he non- ypsinized LDs 1ζmax alue is also highe han he 1ζmax o he ypsinized LDs (+18.8 ±3.1 mV). The e o e po assium ions seems o play a ole in WNV C in e ac ions wi h LDs, in line wi h p e ious indings o DENV C p o ein (Ca alho e al., 2012). AFM WNV C-LDs In e ac ion S udies Co obo a e ζ-Po en ial Da a Single-molecule AFM-based o ce spec oscopy was employed o measu e he speci ic in e ac ions be ween WNV C and LDs. (Un)Binding o ces we e measu ed based on he de lec ion o AFM ips unc ionalized wi h WNV C and allowed o in e ac wi h LDs (Figu es 2D–F). Figu es 2A–C shows he o ce his og ams ob ained o he binding and subsequen unbinding be ween he WNV C- unc ionalized AFM ip and LDs in he p esence o di e en po assium chlo ide concen a ions. The dis ibu ion o he leng h o he up u e adhesion e en s be ween WNV C and LDs was analyzed by i ing he ob ained his og am wi h he Gaussian model desc ibed in ma e ial and me hods. A o ce up u e alue o 30.4 ±0.3 pN was de e mined a 100 mM KCl (Figu e 2A). This alue co esponds o he single-molecule in e ac ion o ce necessa y o b eak he bond be ween one WNV C p o ein dime and one LD. In line wi h p e ious obse a ions, a peak wi h weake in e ac ion o ces is ound (≈17.7 pN), which is a ibu ed o unspeci ic in e ac ions (Ca alho e al., 2012). The wo o he peaks o s onge in e ac ion o ces (52.0 ±2.5 pN and 108.5 ±2.8 pN) likely co espond o he up u e o mul iple bonds due o he in e ac ion o mo e han one p o ein dime a ached o he ip wi h a LD. As such, a clea , s ong and speci ic binding o WNV C o LDs occu s in he p esence o po assium ions. To u he e alua e he ole o K+, i s concen a ion in he bu e was dec eased om 100 o 10 mM (Figu e 2B). In his condi ion, a signi ican dec ease in he (un)binding equency is seen, om 62.7% in TEE bu e wi h 100 mM KCl o 19.4% in TEE bu e wi h 10 mM KCl (Table 2). A he low po assium concen a ion, he o ce his og am also changes signi ican ly: only wo o ce peaks a e obse ed, a 20.1 ± 0.2 pN and 29.6 ±0.9 pN. These peaks a e simila o he i s wo obse ed in TEE bu e wi h 100 mM KCl (≈17.7 pN and 30.4 ±0.3 pN). The weake o ces peak possibly co esponds o unspeci ic in e ac ions and he s onge o he o ce necessa y o b eak he WNV C-LDs binding. Highe in e ac ion o ces, co esponding o he up u e o mul iple bonds, a e no obse ed a low po assium concen a ions. When o ce spec oscopy measu emen s a e pe o med eplacing he po assium by 100 mM sodium (Figu e 2C), a d ama ic change occu s, wi h a single up u e o ce peak o 17.9 ±0.1 pN being obse ed, ypical o unspeci ic in e ac ions. In ag eemen wi h F on ie s in Cellula and In ec ion Mic obiology | www. on ie sin.o g 6Feb ua y 2019 | Volume 9 | A icle 8 Ma ins e al. WNV In e ac s Wi h Lipid Sys ems FIGURE 2 | Fo ce up u e his og ams o WNV C-LDs binding, ob ained by AFM-based o ce spec oscopy. Expe imen s we e pe o med in TEE bu e wi h 100 mM KCl (A), 10 mM KCl (B) o 100 mM NaCl (C). The s onge and speci ic in e ac ion o ces be ween WNV C and LD a e only ound a K+concen a ions simila o he in acellula en i onmen . Only weak in e ac ions, cha ac e is ic o unspeci ic binding, a e obse ed o low po assium ion concen a ions o in i s absence. Gaussian i ing lines o each indi idual peak (g een) and cumula i e i lines o he his og am (black) a e p esen ed. Examples o app oach- e ac ion cu es acqui ed du ing he o ce spec oscopy measu emen s o WNV C-LDs adhesion in TEE bu e wi h 100 mM KCl (D), 10 mM KCl (E), o 100 mM NaCl (F). his, a low (un)binding equency o 19.3% is seen o ha condi ion (Table 2). The e o e, he in e ac ion be ween WNV C and LDs is K+-dependen , a he han a gene al dependence o ionic s eng h. DLS Shows Tha WNV C In e ac s Wi h VLDL bu no Wi h LDL in a K+-Dependen Manne Ha ing es ablished ha WNV C binds LDs in a po assium speci ic manne ha equi es LDs su ace p o eins, he WNV C in e ac ion wi h lipop o eins was hen es ed. DLS was employed o measu e he sca e ed ligh in ensi y luc ua ions o lipop o eins size ha occu due o hei B ownian mo ion, and calcula e hei hyd odynamic diame e (DH) using he S okes- Eins ein equa ion (Faus ino e al., 2014). The a e age lipop o eins size in TEE bu e wi h 100 mM KCl was de e mined while hey we e i a ed wi h WNV C. An inc ease in he VLDL size upon i a ion wi h WNV C is clea ly seen (Figu e 3A). DLS da a shows ha in he absence o WNV C, VLDL and LDL p esen a e age hyd odynamic diame e s (DH) o , espec i ely, 38.2 ±0.7 nm and 22.1 ±0.4 nm ( alues a e mean ±SE), in ag eemen wi h li e a u e da a (Cushley and Okon, 2002; Faus ino e al., 2014). Upon i a ion wi h WNV C, a s a ically signi ican inc ease in he a e age size o VLDL occu s, up o 42.8 ±0.5 nm (p<0.005) a he maximum WNV C concen a ion es ed. The VLDL DHin he p esence o WNV C has a 4.5 ±0.6 nm inc ease ela i e o he alue in he absence o he p o ein. By he obse a ion o he in ensi y dis ibu ion, I(DH), o hese measu emen s, i is possible o say ha hese alues do no co espond o VLDL agg ega ion ( ha may occu in a small ac ion o he o al pa icles), bu o an inc ease in he a e age size o VLDL due o he in e ac ion wi h WNV C and o ma ion o a WNV C-VLDL complex. In he case o LDL, upon i a ion wi h WNV C he e was no clea change in DH (Figu e 3A). Po assium ions, ound a highe concen a ions inside cells bu a low concen a ion ou side, we e p e iously shown o be essen ial o DENV C-VLDL in e ac ion (Faus ino e al., 2014). Wi h his in mind, o es he impo ance o K+ o he WNV C- VLDL binding, he size o VLDL and o hei complex wi h WNV C was measu ed eplacing TEE KCl bu e by TEE NaCl bu e (Figu e 3B). VLDL in TEE NaCl bu e ha e a DHini ial alue (40.5 ±0.6 nm) highe han he obse ed in TEE KCl bu e . Upon i a ion wi h WNV C, he e is no signi ican di e ence in DH, sugges ing ha in he absence o po assium ions WNV C canno in e ac wi h VLDL. These esul s indica e ha K+is c ucial o WNV C-VLDL binding. Ha ing de e mined he a e age inc ease in size o VLDL in he p esence o high WNV C p o ein concen a ions, his in o ma ion was used o gene a e a model o WNV C binding o VLDL. Based on DLS da a and on he dimensions o he p o ein molecule ob ained om he s uc u e 1SFK deposi ed a he P o ein Da a Bank (Dokland e al., 2004), we could elabo a e a model o WNV C-VLDL in e ac ion, whe eby WNV C dime s bind o he VLDL su ace, o ming a single i al p o ein laye (Figu e 4). VLDL hyd odynamic adius is ∼19 nm. When WNV C in e ac s wi h i s su ace, he adius inc eases o ∼21.3 nm. This 2.3 nm inc emen in adius co ela es wi h he dimension o he WNV C dime and is in o al ag eemen wi h wha would be F on ie s in Cellula and In ec ion Mic obiology | www. on ie sin.o g 7Feb ua y 2019 | Volume 9 | A icle 8 Ma ins e al. WNV In e ac s Wi h Lipid Sys ems TABLE 2 | Rup u e o ces and pe cen age o (un)binding e en s ob ained by AFM-based o ce spec oscopy o he in e ac ion be ween WNV C and LDs. Expe imen al condi ion % (Un)binding e en s Rup u e Fo ce (pN) 1s peak 2nd peak 3 d peak 4 h peak [KCl] 100 mM 62.7 ≈17.7 30.4 ±0.3 52.0 ±2.5 108.5 ±2.8 10 mM 19.4 20.1 ±0.2 29.6 ±0.9 [NaCl] 100 mM 19.3 17.9 ±0.1 Values a e p esen ed as mean ±SE. FIGURE 3 | DLS analysis o lipop o eins hyd odynamic diame e s upon i a ion wi h WNV C. In TEE bu e wi h 100mM KCl, VLDL (blue iangles) a e age hyd odynamic diame e (DH) inc eases 4.5 ±0.6 nm in he p esence o WNV C, bu no signi ican a ia ion is obse ed o LDL (g een ci cles) (A). In TEE bu e wi h 100 mM NaCl, he DHo VLDL ( ed iangles) does no change signi ican ly upon inc easing WNV C concen a ion (B). K+is he e o e c ucial o WNV C-VLDL in e ac ion. DH alues (p esen ed as mean ±SE) a e he a e age o h ee independen measu emen s o each poin (*p<0.005). Dashed lines co espond o he a e age o each se o esul s. FIGURE 4 | P oposed model o WNV C in e ac ion wi h VLDL. WNV C dimensions i well wi h he obse ed inc ease o ∼2.3 nm on he VLDL hyd odynamic adius. Wi hou WNV C, VLDL p esen a hyd odynamic adius o ∼19 nm, while wi h 5 µM WNV C i inc eases o ∼21.3 nm. The e o e, a monolaye o WNV C molecules could be o med on he VLDL su ace. expec ed o he o ma ion a laye o WNV C on he su ace o VLDL (Figu e 4). AFM Da a Suppo s WNV C-VLDL Speci ic and K+-Dependen Binding To u he complemen he DLS da a, single-molecule AFM- based o ce spec oscopy was employed o assess WNV C binding o human plasma lipop o eins (VLDL and LDL) (Figu es 5D–F). As epo ed be o e (Faus ino e al., 2014), he peak o o ces a ound 20 pN is a ibu ed o unspeci ic in e ac ions, since i also appea ed in he con ols pe o med wi h non- unc ionalized ips and lipop o eins. Th ough analysis o he his og ams, only o ces abo e 40 pN we e conside ed s ong enough o epo speci ic binding. The dis ibu ion o he o ce o he up u e adhesion e en s be ween WNV C and VLDL o LDL we e analyzed by i ing he ob ained his og ams wi h he Gaussian model. Figu es 5A–C p esen s he his og ams o WNV C in e ac ion wi h VLDL and wi h LDL, bo h in he p esence o TEE bu e wi h 100 mM KCl. Compa ing he his og ams o VLDL and LDL, di e en peaks can be dis inguished, co esponding o di e en up u e o ces. In he VLDL his og am (Figu e 5A), beside he unspeci ic in e ac ions (21.0 ±0.3 pN), s ong and speci ic in e ac ions a e obse ed (82.9 ±0.7 pN). The LDL his og am (Figu e 5B) shows only one peak, wi h weak o ces (24.5 ±0.1 pN), cha ac e is ic o unspeci ic in e ac ions. Mo eo e , as p esen ed in Table 3, he (un)binding equency egis e ed o VLDL (31.1%) is much highe han o LDL (3.1%). As such, i is clea ha WNV C binds speci ically o VLDL and no o LDL, co obo a ing he DLS da a. To de e mine i WNV C-VLDL binding equi es po assium ions, as al eady demons a ed o WNV C-LDs binding, he in e ac ions we e also measu ed in he p esence o TEE bu e wi h 100 mM NaCl (Figu e 5C). In e es ingly, he s ong and speci ic in e ac ions ha occu in TEE bu e wi h 100 mM KCl disappea when he ionic s eng h is main ained bu F on ie s in Cellula and In ec ion Mic obiology | www. on ie sin.o g 8Feb ua y 2019 | Volume 9 | A icle 8 Ma ins e al. WNV In e ac s Wi h Lipid Sys ems FIGURE 5 | Fo ce up u e his og ams o WNV C in e ac ion wi h plasma lipop o eins, ob ained by AFM-based o ce spec oscopy. Expe imen s we e pe o med in TEE bu e wi h 100 mM KCl o VLDL (A) and LDL (B), as well as in TEE bu e wi h 100 mM NaCl o VLDL (C). The single peak o weak up u e o ces obse ed in (B) and (C) co espond o unspeci ic in e ac ions. S ong and speci ic in e ac ions only occu o he in e ac ion o WNV C wi h VLDL in he p esence o po assium ions. Gaussian i ing lines o each indi idual peak a e shown in g een. Examples o app oach- e ac ion cu es acqui ed du ing he o ce spec oscopy expe imen s o WNV C in e ac ion wi h plasma lipop o eins in TEE bu e wi h 100 mM KCl o VLDL (D) and LDL (E), as well as in TEE bu e wi h 100 mM NaCl o VLDL (F). TABLE 3 | Rup u e o ces and pe cen age o (un)binding e en s de e mined wi h AFM-based o ce spec oscopy o he in e ac ion be ween WNV C and VLDL o LDL. Expe imen al condi ion % (Un)binding e en s Rup u e o ces (pN) 1s peak 2nd peak KCl 100 mM VLDL 31.1 21.0 ±0.3 82.9 ±0.7 LDL 3.1 24.5 ±0.1 NaCl 100 mM VLDL 15.1 25.0 ±0.1 Values a e p esen ed as mean ±SE. po assium ions a e eplaced o sodium ions. In he his og am, only a peak o weak o ces is obse ed (25.0 ±0.1 pN), co esponding o unspeci ic in e ac ions. In ag eemen wi h hese esul s, he (un)binding equency also dec eases signi ican ly o 15.1% (Table 3). The e o e, AFM-based o ce spec oscopy also indica es ha po assium ions a e equi ed o he WNV C-VLDL binding o occu . DISCUSSION The esul s ob ained wi h ζ-po en ial measu emen s show ha LDs in 100 mM KCl p esen , as expec ed, a nega i e su ace cha ge, wi h an a e age ζ-po en ial o −20.6 ±0.7 mV, which inc eases in he p esence o WNV C (Figu e 1 and Table 1). Single-molecule AFM-based o ce measu emen s gi e u he suppo o hese indings. WNV C-LD binding is s ong and cha ac e is ic o speci ic binding, wi h a (un)binding o ce o 30.4 ±0.3 pN (Figu e 2 and Table 2). Bo h he AFM and he ζ-po en ial da a a e in excellen ag eemen wi h simila obse a ions ega ding DENV C-LDs binding (Ca alho e al., 2012). Mo eo e , he esul s show ha WNV C-LDs in e ac ion equi es K+concen a ions simila o he in acellula medium and p o eins p esen on LDs su ace. LDs con ain se e al p o eins on hei su ace, majo ly he p o eins o PAT amily, namely, pe ilipin 1 ( o me ly known jus as pe ilipin), pe ilipin 2 (also known as ADRP), pe ilipin 3 (PLIN3; also known as TIP47), as well as o he p o eins in mino quan i ies (Olo sson e al., 2009). Conside ing he p e ious s udies on DENV C-LDs binding (Ca alho e al., 2012), PLIN3 is he mos likely a ge o WNV C in he LDs su ace. Su p isingly, upon incuba ion o LDs wi h inc easing concen a ions o DENV C (Ca alho e al., 2012) o WNV C he alues o C1/2and o 1ζmax a e compa able (LDs binding o WNV C: C1/2=112 ±15 nM, 1ζmax = 37.6 ±1.2 mV and LDs binding o DENV C: C1/2=85.7 ± 17.6 nM, 1ζmax =34.4 ±1.3 mV), sugges ing ha hese wo capsid p o eins, besides being simila , may also bind o he same molecula a ge . Rein o cing hese obse a ions, he speci ic o ce peak a ound 30 pN (WNV C-LDs up u e o ce =30.4 pN and DENV C-LDs up u e o ce =34 pN) and he (un)binding equency de i ed om o ce spec oscopy measu emen s a e also simila be ween DENV C (Ca alho e al., 2012) and WNV F on ie s in Cellula and In ec ion Mic obiology | www. on ie sin.o g 9Feb ua y 2019 | Volume 9 | A icle 8