Methods for lipid droplet biophysical characterization in Flaviviridae infections

micb-09-01951 Augus 18, 2018 Time: 18:53 # 1 REVIEW published: 21 Augus 2018 doi: 10.3389/ micb.2018.01951 Edi ed by: Oc a io Luiz F anco, Uni e sidade Ca ólica de B asília, B azil Re iewed by: Sumana Sanyal, Uni e si y o Hong Kong, Hong Kong Qiyi Tang, Howa d Uni e si y, Uni ed S a es *Co espondence: I o C. Ma ins [email p o ec ed] Nuno C. San os [email p o ec ed] Special y sec ion: This a icle was submi ed o An imic obials, Resis ance and Chemo he apy, a sec ion o he jou nal F on ie s in Mic obiology Recei ed: 20 Ap il 2018 Accep ed: 02 Augus 2018 Published: 21 Augus 2018 Ci a ion: Ma ins AS, Ma ins IC and San os NC (2018) Me hods o Lipid D ople Biophysical Cha ac e iza ion in Fla i i idae In ec ions. F on . Mic obiol. 9:1951. doi: 10.3389/ micb.2018.01951 Me hods o Lipid D ople Biophysical Cha ac e iza ion in Fla i i idae In ec ions Ana S. Ma ins, 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 Lipid d ople s (LDs) a e in acellula o ganelles o neu al lipid s o age, o igina ed om he endoplasmic e iculum. They play an essen ial ole in lipid me abolism and cellula homeos asis. In ac , LDs a e complex o ganelles, in ol ed in many mo e cellula p ocesses han hose ini ially p oposed. They ha e been ex ensi ely s udied in he con ex o LD-associa ed pa hologies. In pa icula , LDs ha e eme ged as c i ical o i us eplica ion and assembly. Vi uses om he Fla i i idae amily, namely dengue i us (DENV), hepa i is C i us (HCV), Wes Nile i us (WNV), and Zika i us (ZIKV), in e ac wi h LDs o usu p he hos lipid me abolism o hei own i al eplica ion and pa hogenesis. In gene al, du ing Fla i i idae in ec ions i is obse ed an inc easing numbe o hos in acellula LDs. Se e al i al p o eins in e ac wi h LDs du ing di e en s eps o he i al li e cycle. The HCV co e p o ein and DENV capsid p o ein, ex ensi ely in e ac wi h LDs o egula e hei eplica ion and assembly. De ailed s udies o LDs in i al in ec ions may con ibu e o he de elopmen o possible inhibi o s o key s eps o i al eplica ion. He e, we e iewed di e en echniques ha can be used o cha ac e ize LDs isola ed om in ec ed o non-in ec ed cells. Mic oscopy s udies ha e been commonly used o obse e LDs accumula ion and localiza ion in in ec ed cell cul u es. Fluo escen dyes, which may a ec LDs di ec ly, a e widely used o p obe LDs bu he e a e also app oaches ha do no equi e he use o luo escence, namely s imula ed Raman sca e ing, elec on and a omic o ce mic oscopy-based app oaches. These h ee a e powe ul echniques o cha ac e ize LDs mo phology. Raman sca e ing mic oscopy allows s udying LDs in a single cell. Elec on and a omic o ce mic oscopies enable a be e cha ac e iza ion o LDs in e ms o s uc u e and in e ac ion wi h o he o ganelles. O he biophysical echniques, such as dynamic ligh sca e ing and ze a po en ial a e also excellen o cha ac e ize LDs in e ms o size in a simple and as way and es possible LDs in e ac ion wi h i al p o eins. These me hodologies a e e iewed in de ail, in he con ex o i al s udies. Keywo ds: lipid d ople , Fla i i idae, i al p o eins, LDs-associa ed p o eins, ligh sca e ing, mic oscopy INTRODUCTION Lipid d ople s (LDs) a e in acellula o ganelles o neu al lipid s o age (Wal he and Fa ese, 2012;Hashemi and Goodman, 2015), o igina ed om he endoplasmic e iculum (ER) whe e some enzymes in ol ed on he gene a ion o neu al lipids a e loca ed (Buhman e al., 2001;Hashemi and Goodman, 2015). Ma u e LDs a e composed o a hyd ophobic co e o neu al lipid, mainly F on ie s in Mic obiology | www. on ie sin.o g 1Augus 2018 | Volume 9 | A icle 1951 micb-09-01951 Augus 18, 2018 Time: 18:53 # 2 Ma ins e al. Lipid D ople Cha ac e iza ion in Fla i i idae In ec ion iacylglyce ols (TAGs) and s e ol es e s (SEs), su ounded by a monolaye o phospholipids and unes e i ied s e ol, wi h a a ie y o in eg al and pe iphe al p o eins (Lin P. e al., 2014;Be suke and Olzmann, 2017). The main LD p o eins a e om he PAT amily: pe ilipin (also known as pe ilipin 1, PLIN1), adipose di e en ia ion- ela ed p o ein (ADRP), also named pe ilipin 2 (PLIN2), and ail-in e ac ing p o ein o 47 kDa (TIP47), also named pe ilipin 3 (PLIN3) (Bickel e al., 2009;K ahme e al., 2009). LDs end o ha e a globula shape (Figu e 1A) wi h a diame e ha a ies om 50 nm o 200 µm, depending on cell ype (K ahme e al., 2009). LDs a e he main cell ese oi o lipids o ene gy p oduc ion (K ahme e al., 2009), as well as o s e ols, a y acids, and phospholipids o ho mone syn hesis and memb ane o ma ion (Thiam e al., 2013), minimizing he olume necessa y o hei s o age. Mo eo e , LDs p o ec cells om he lipo oxic e ec s o unes e i ied lipids (K ahme e al., 2009). Via hei su ace, LDs con ol lipases accessibili y o s o ed TAG, helping o egula e hei enzyma ic b eakdown (Lin P. e al., 2014). Du ing he las decades, se e al s udies ha e been ca ied ou o unde s and LDs biogenesis (Pol e al., 2014;Deslandes e al., 2017;Choudha y e al., 2018) and unc ions (Wal he and Fa ese, 2012;Wel e, 2015). Di e en model sys ems, including bac e ia, yeas , g een algae, Caeno habdi is elegans,D osophila, plan s and se e al ypes o mammalian cells and issues ha e been used. Da a om se e al s udies show ha LDs a e e y complex o ganelles and may be in ol ed in lipid me abolism, memb ane biosyn hesis, memb ane a icking and signal ansduc ion (Ding e al., 2013). LDs we e p oposed o be in ol ed in many o he impo an p ocesses: LDs modula e nuclea unc ions being esponsible o he a ailabili y o p o eins and signaling lipids in he nucleus; LDs may ac as hubs o a y acid a icking o mi ochond ia; LDs a e used by he immune sys em agains pa hogens; howe e , i uses ha e e ol ed s a egies o use LDs as pla o ms o i al assembly (Wel e, 2015;Wang, 2016). Lipid d ople s ha e been ex ensi ely s udied, in pa icula in he con ex o LD-associa ed pa hologies. Gi en he a ailable knowledge abou hese o ganelles as egula o s o lipid and ene gy me abolism, hei in ol emen in human me abolic diseases as well as in i al in ec ions is no su p ising. In ac , he accumula ion o LDs occu s du ing he p og ession o di e en pa hologies (Pol e al., 2014). Se e al impo an in acellula pa hogens om he Fla i i idae amily, such as hepa i is C i us (HCV) (Ba ba e al., 1997) and dengue i us (DENV) (Samsa e al., 2009), inc ease he o ma ion o LDs in he hos cells. I has been shown ha i al RNA eplica ion is egula ed by i al p o eins h ough hei in e ac ion wi h LD su ace p o eins (Vog e al., 2013). Vi uses o he Fla i i idae amily cause se e al se ious human condi ions, such as hemo hagic e e (Rigau-Pé ez, 2006), li e s ea osis (McLauchlan, 2009), and mic ocephaly (Cal e e al., 2016), caused by DENV, HCV, and Zika i us (ZIKV) in ec ion, espec i ely. He e, we e iew di e en app oaches o cha ac e ize LDs in he con ex o Fla i i idae in ec ions, namely DENV and HCV in ec ions. Due o he impo ance o LDs as key componen s o i al eplica ion, de ailed s udies o hese o ganelles and hei in e ac ion wi h i al ac o s a e c ucial. This can lead o no el inhibi o s o key s eps o he i al eplica ion o impo an human pa hogens o he Fla i i idae amily. LDs BIOGENESIS Lipid d ople s biogenesis s a s ollowing he accumula ion o TAG and SE molecules be ween he wo lea le s o he ER memb ane (Joshi e al., 2017;Thiam and Belle , 2017). To main ain s abili y, hese ER lipid bilaye s accommoda e neu al lipids, bu only up o a sa u a ion poin , abo e which he o ma ion o LDs is igge ed (Figu e 1B) (Hamil on, 1989;Hashemi and Goodman, 2015). Al hough LDs can be o med spon aneously om he ER (Deslandes e al., 2017), he ansi ion o a ma u e LD in ol es s uc u al changes (Vanni, 2017). P o eins may play an essen ial ole on LDs o ma ion (Hashemi and Goodman, 2015) and in he s uc u al changes leading o hei ma u a ion (Vanni, 2017). These p o eins include PLIN3, as well as p o eins con aining helical hai pins, such as glyce ol-3-phospha e acyl ans e ase 4 (GPAT4) o diacylglyce ol acyl ans e ase (DGAT) 1 and 2, a s o age- inducing ansmemb ane p o ein 2 (FIT2) and seipin (Pol e al., 2014;Hashemi and Goodman, 2015;Choudha y e al., 2018). Molecula dynamics s udies sugges ha p o eins ha a e ec ui ed o he ER memb ane a e hen expelled p ecisely a he si es o LDs o ma ion, as a consequence o changes in he unde lying memb ane p ope ies (Vanni, 2017). Lipids such as diacylglyce ol (DAG) and phospha idic acid also con ibu e o LDs o ma ion, p omo ing shape change in he same di ec ion ( equi ed o he cu a u e o ma ion) (Skinne e al., 2009; Adeyo e al., 2011;Hashemi and Goodman, 2015;Choudha y e al., 2018). Mo eo e , he ole o DAG on LDs o ma ion may in ol e mo e han i s memb ane-cu a u e p ope ies (Hashemi and Goodman, 2015). Ne e heless, he hypo hesis ha LDs can be spon aneously o med om a symme ical elonga ed lens o he ER memb ane wi hou equi ing any ene gy- consuming machine y, cu a u e-inducing agen o in insic asymme y o he bilaye is s ill a ma e o deba e (Deslandes e al., 2017). Al hough ex ensi e s udies o LDs biogenesis ha e been conduc ed o se e al yea s, some ques ions s ill emain unanswe ed. One o hem is ela ed wi h he loca ion o he o ma ion o LDs a he ER. I is no clea i he e a e speci ic si es o i i is a p ocess ha occu s a andom loca ions (Hashemi and Goodman, 2015;Thiam and Belle , 2017). Ano he ques ion is i he nascen LDs, as hey ansi o ma u e LDs, sepa a e om he ER and mig a e om he cell pe iphe y o he nucleus (Hashemi and Goodman, 2015). Answe ing hese ques ions will con ibu e o ou unde s anding o cell machine y, helping o cla i y he in e ac ions o LDs wi h o he cell o ganelles. LDs IN HEALTH AND DISEASE Lipid d ople s o m s able associa ions no only wi h ER, bu also wi h o he key o ganelles and cellula compa men s, like mi ochond ia, inne nuclea en elope, lysosomes/ acuoles, and endosomes (Wel e, 2015;Schuldine and Bohne , 2017). F on ie s in Mic obiology | www. on ie sin.o g 2Augus 2018 | Volume 9 | A icle 1951 micb-09-01951 Augus 18, 2018 Time: 18:53 # 3 Ma ins e al. Lipid D ople Cha ac e iza ion in Fla i i idae In ec ion FIGURE 1 | Majo LDs mo phological ea u es and biogenesis. (A) LDs a e composed by a neu al co e o iacylglyce ols (TAGs) and s e ol es e s (SEs), su ounded by a monolaye o phospholipids and unes e i ied s e ol, wi h se e al p o eins a he su ace and/o pa ially in eg a ed wi hin hei s uc u e, mainly om he PAT amily: pe ilipin 1 (PLIN1), pe ilipin 2 (PLIN2), and pe ilipin 3 (PLIN3). (B) LDs a e de i ed om he endoplasmic e iculum (ER), as a esul o TAG and SE molecules accumula ion be ween he wo lea le s o he ER memb ane. The nascen d ople s g ow in o ma u e LDs, wi h he help o cu a u e-inducing agen s, and may emain a ached o he ER (no shown) o de ach om he ER in o he cy osol. Assuming LDs as he sou ce o lipids o o he o ganelles, hese con ac s may se e o ans e lipids o o he compa men s, enabling memb ane expansion, signaling, and ene gy p oduc ion h ough lipolysis and β-oxida ion o occu . Fo some o hese connec ions, he molecules esponsible o ini ia ing o main aining he con ac we e al eady iden i ied, in pa icula o LDs con ac wi h ER and mi ochond ia (Gao and Goodman, 2015). Seipin, lipin, and FIT2 a e some o he p o eins in ol ed in LDs o ma ion, assembly and TAG ans e o LDs, espec i ely. PLIN5 was iden i ied as a media o o d ople –mi ochond ial in e ac ions, modula ing LD lipases (Wil ling e al., 2013;Gao and Goodman, 2015). Howe e , he ole o LDs a icking wi hin he nucleus, as well as he p ocess o ene gy elease om LDs, is s ill no well unde s ood (Wang, 2016). The neu al lipids s o ed inside o LDs, such as TAG, SE and e inyl es e s, can be used by cells on se e al biological p ocesses. Fo ins ance, LDs a e c i ical o ene gy and memb ane componen s gene a ion (Wel e, 2015). An impai men on LD biogenesis and/o inc eased LD deg ada ion can dis up he no mal lipid me abolism inside he cell, as well as hei ene gy homeos asis (G eenbe g e al., 2011;K ahme e al., 2013). LDs a e impo an o ganelles in adipose issue, li e and in es ine, due o hei in ol emen on ene gy s o age and lipid u no e (Bickel e al., 2009;G oss and Sil e , 2014). Gi en hei ole in lipid s o age, LDs also igu e p ominen ly in se e al pa hologies due o lipid accumula ion, such as obesi y, a y li e , ype 2 diabe es and a he oscle osis (Cohen e al., 2011;G eenbe g e al., 2011; Wal he and Fa ese, 2012;K ahme e al., 2013). The inc easing numbe o LDs in non-adipose issues is a pa hological ea u e o hese me abolic diseases (G eenbe g e al., 2011;K ahme e al., 2013). The associa ion o LDs accumula ion wi h hese diseases is well unde s ood: LDs a e able o seques e oxic lipids, u ning hem in o TAG and s o ing hem, which p e en s he lipo oxici y caused by ee a y acids (Be suke and Olzmann, 2017). Fu he mo e, i was epo ed ha mu a ions in p o eins di ec ly associa ed wi h LDs s uc u e and unc ion may lead o amilial lipodys ophies and neu al lipid s o age diseases (G eenbe g e al., 2011;K ahme e al., 2013). LDs accumula ion also occu s in skele al muscle, mac ophages, mamma y glands, ad enal co ex, o a y, and es is (Wal he and Fa ese, 2012). In he las h ee cases, LDs p o ide he p ecu so o he syn hesis o choles e ol-d i en s e oid ho mones, including glucoco icoids such as co isol, mine aloco icoids such as aldos e one, es os e one and es ogens. LDs-associa ed p o eins such as ADRP (o PLIN2) play a signi ican ole in egula ing he in acellula dis ibu ion o phospholipids and lipids in gene al. The edis ibu ion o LDs occu s p obably due o he educed numbe o he LD-su ace p o ein ADRP, esponsible o main aining he dispe sed in acellula dis ibu ion o hese o ganelles (McIn osh e al., 2010). LDs PROTEOME Now ha he ole o LDs in lipid me abolism is be e unde s ood, a pa o he ocus o he mos ecen esea ch is on o he oles ha LDs play. Mos o hese eme ging oles ha a e s a ing o be s udied a e associa ed wi h pa icula LD p o eins. Thus, unde s anding LDs p o eome and p o ein a ge ing a e some o he main objec i es o ecen s udies (Goodman, 2018). LDs ha e been p oposed o seques e p o eins and, as a esul , ei he modula e hei abili y o in e ac wi h hei binding pa ne s o F on ie s in Mic obiology | www. on ie sin.o g 3Augus 2018 | Volume 9 | A icle 1951 micb-09-01951 Augus 18, 2018 Time: 18:53 # 4 Ma ins e al. Lipid D ople Cha ac e iza ion in Fla i i idae In ec ion simply s o e damaged p o eins be o e deg ada ion. They can also p omo e he assembly o p o ein complexes (Hodges and Wu, 2010). Howe e , i is no well unde s ood ye i hese p o eins a e s ably a ge ed o he LDs and how he elease is con olled. The no ion o LDs as unique o ganelles in he con ol o p o eins cycle inside cells led o he s udy o LDs p o eome in di e en ypes o cells, ep esen a i e o di e en o ganisms o issues (Goodman, 2018). Howe e , a de ailed and accu a e examina ion o he LDs p o eome is challenged by he di icul y o ob aining pu i ied LDs comple ely sepa a ed om o he associa ed o ganelles. One way o ackle his issue is by excluding da a in which p o eins ha a e ma ke s o o he o ganelles a e iden i ied. Howe e , i is no he bes app oach, as i is no possible o asce ain ha such p o ein ma ke s a e ne e ound in LDs (Be suke and Olzmann, 2017). Thus, unde s anding he ole o LD-associa ed p o eins equi es he accu a e de ini ion o LDs p o eome (Be suke e al., 2018). To do so, i is necessa y o isola e pu e LDs. Se e al me hods o isola ing LDs ha e been es ablished, de eloped bo h o p o eomic and unc ional s udies, wi h LDs isola ed om di e en cells. Inc easing LDs pu i y is s ill one o he majo goals, in o de o ensu e a ep oducible amoun o high-quali y LDs (Ding e al., 2013). THE Fla i i idae FAMILY The Fla i i idae amily o i uses is di ided in o ou gene a: Hepaci i us,Fla i i us,Pes i i us and he ecen ly p oposed Pegi i us (Shi e al., 2016). HCV belongs o he i s genus, while o he impo an human pa hogens such as DENV, yellow e e , Wes Nile (WNV), ZIKV and ick-bo ne encephali is i uses all belong o he genus Fla i i us (Mukhopadhyay e al., 2005; Kilpa ick, 2011;Cal e e al., 2016;Shi e al., 2016). The las wo gene a include animal i uses o less di ec ele ance o human heal h. In addi ion, he e a e also he so called “ la i-like” i uses, isola ed om a ange o a h opod species. They a e conside ed dis an ela i es o he known Fla i i idae, which may come o be classi ied in o ha axon (Shi e al., 2016). The be e s udied Fla i i idae a e HCV, DENV, WNV, and, ecen ly, ZIKV, among o he . In common, hese i uses sha e a single s anded posi i e sense RNA [ss(+)RNA], wi h membe s o he Hepaci i us and Fla i i us gene a ha ing genomes be ween 9 and 13 kb (Shi e al., 2016). Besides hei common s uc u e, Fla i i idae eplica ion mechanisms a e e y simila , in ol ing he ansla ion o a single open eading ame in o a polyp o ein. This polyp o ein is clea ed and p ocessed, o la e on o m he ma u e i ion. The li e cycle begins wi h he a achmen o he i us o he cell su ace (Figu e 2). Subsequen ly, i uses a e in e nalized by ecep o -media ed endocy osis and anspo ed o endosomes. Inside he cell, acidi ica ion o he endocy ic esicles wi h i al pa icles igge s con o ma ional ea angemen s in he i ion ha allows he elease o he i al genome in o he cy oplasm. The ss(+)RNA is ansla ed as a single polyp o ein ha i is clea ed by se e al i al and hos p o eases, o igina ing non-s uc u al and s uc u al p o eins (Mukhopadhyay e al., 2005;Hussmann e al., 2014). Genome eplica ion occu s on in acellula memb anes. A e he syn hesis o i al p o eins and o i al RNA, he p ocess o assembly and encapsula ion occu on he ER su ace. These p ocesses in ol e se e al p o eins and memb ane in e ac ions. The imma u e i al pa icles a e anspo ed h ough he ans- Golgi ne wo k, whe e ma u a ion occu s, esul ing in o in ec ious pa icles. The ma u e in ec ious pa icles a e hen eleased in o he ex acellula medium (Mukhopadhyay e al., 2005). A mechanis ic unde s anding o he assembly and encapsula ion p ocesses (as well as o o he s eps o he i al li e cycle) may sugges new a ge s o u u e he apeu ics app oaches (Zhang e al., 2017). LDs ROLE IN Fla i i idae INFECTIONS Posi i e-sense RNA i uses hijack he in acellula memb ane machine y o i al eplica ion (Tang e al., 2014), inc easing he numbe o in acellula LDs and hei diame e . LDs a e used by i uses as an ene gy and lipids ese oi (Samsa e al., 2009; Hea on and Randall, 2010;Hea on e al., 2010;Pe e a e al., 2012). Besides, LDs may also acili a e i al eplica ion, p o iding a pla o m o he assembly and encapsida ion p ocesses (Samsa e al., 2009). LDs also con ibu e o i al genome eplica ion. As his p ocess in ol es an ac i e consump ion o cell ene gy, DENV has been p oposed o use he ene gy s o ed in LDs h ough he p ocess o lipophagy (Hea on and Randall, 2010). B ie ly, DENV induces au ophagy o LDs o elease ee a y acids, esul ing in an inc ease o cellula β-oxida ion and consequen ly in an inc ease o he ATP gene a ed. These p ocesses co ela es wi h he dec ease o he LDs a ea obse ed in DENV in ec ed cells (Hea on and Randall, 2010). As men ioned abo e, Fla i i idae and o he i uses, such as o a i uses, use LDs as pla o ms o i al assembly (Figu e 3) (Roingea d and Melo, 2017;Zhang e al., 2017). This is achie ed h ough he in e ac ion o LDs wi h i al p o eins, namely he equi alen co e and capsid (C) p o eins om HCV and DENV, espec i ely, which play mul iple oles du ing he i al li e cycle (Roingea d and Melo, 2017;Zhang e al., 2017). Recen ly, i was shown ha LDs a e also a ge ed by ZIKV C p o ein (Ma ins e al., 2017;Shang e al., 2018). Mo eo e , ZIKV C–LDs in e ac ion can occu in he absence o o he i al p o eins (Shang e al., 2018). In e es ingly, LDs in e ac ion wi h i al p o eins has many mo e nuances, wi h LDs p o eins playing speci ic oles. HCV Co e P o ein–LDs In e ac ion Lipid d ople s play an impo an ole in HCV li e cycle and a e ma ke s in ol ed in s ea osis (Miyana i e al., 2007;McLauchlan, 2009). HCV akes ad an age o hos in acellula lipid sys ems, in pa icula LDs, manipula ing hei p oduc ion and dis ibu ion inside he cells. In ac , HCV induces an inc ease in size and numbe o LDs in hos cells (Mille and K ijnse-Locke , 2008;McLauchlan, 2009). Se e al epo s ha e documen ed he in acellula localiza ion o HCV co e p o ein associa ed o LDs, sugges ing ha his in e ac ion is impo an in HCV li e cycle (Ba ba e al., 1997;McLauchlan and Hope, 2000). The co e p o ein, a s uc u al p o ein a he N- e minal o he polyp o ein encoded by he i al ss(+)RNA, is a dime ic α-helical p o ein F on ie s in Mic obiology | www. on ie sin.o g 4Augus 2018 | Volume 9 | A icle 1951 micb-09-01951 Augus 18, 2018 Time: 18:53 # 5 Ma ins e al. Lipid D ople Cha ac e iza ion in Fla i i idae In ec ion FIGURE 2 | Fla i i us li e cycle. Fla i i us en e in he hos cell by ecep o -media ed endocy osis. Acidi ica ion o he endosomal esicle leads o he usion o he i al and cell memb anes, enabling he elease o he i al genome ss(+)RNA in o he cy osol. The ss(+)RNA is ansla ed in o a polyp o ein ha is p ocessed by i al and hos p o eases, o igina ing se en non-s uc u al and h ee s uc u al p o eins (no shown). Vi us eplica ion and assembly occu nea he ER and LDs. A e i ion ma u a ion by he hos p o ease u in, he ma u e i ion ollows he sec e ion pa hway and is subsequen ly eleased by exocy osis. wi h wo domains (D1 and D2) (McLauchlan, 2000;Boulan e al., 2005, 2006). S udies by se e al labo a o ies p o ided de ailed in o ma ion abou his in e ac ion (Boulan e al., 2007). HCV co e p o ein co-localizes wi h LDs in in ec ed cells in a ime- dependen manne . I has been disco e ed ha he associa ion o he co e o he LDs is media ed by DGAT1, a hos enzyme ha syn hesizes TAG in he ER (He ke e al., 2010). B ie ly, he co e p o ein p og essi ely a aches o he LDs su ace, coa ing i , a e which he LDs coa ed wi h co e p o ein s a o agg ega e a ound he nucleus (Boulan e al., 2008). Mos impo an ly, dis up ing he abili y o co e o coa LDs leads o an inhibi ion o i us p oduc ion, which shows he impo ance o LDs in he i al li e cycle (Boulan e al., 2007). The D2 domain o HCV co e p o ein was iden i ied as c ucial de e minan o i s binding o LDs, an in e ac ion essen ial o i al assembly (Sha inskaya e al., 2007). The in e ac ion o he co e p o ein wi h LDs is also dependen o a C- e minal domain o he co e p o ein, highly conse ed be ween di e en i uses. This in e ac ion o igina es loci, whe e i al RNA and non-s uc u al p o eins in ol ed in genome eplica ion we e de ec ed (McLauchlan, 2009). These e idences sugges ha hese loci may be whe e he assembly and p oduc ion o nascen i ions occu s. In HCV in ec ion, LDs associa e also wi h mic o ubules and agg ega e mainly a ound he mic o ubule-o ganizing cen e . The associa ion o he co e p o ein o LDs may also p omo e hei edis ibu ion and accumula ion a ound he nucleus. Such LDs–co e p o ein associa ion may hen inc ease he p obabili y o in e ac ions be ween he si es o HCV RNA eplica ion and o i ion assembly (Boulan e al., 2008). When associa ed wi h he LDs, he HCV co e p o ein in e e es wi h TAG u no e , s abilizing LDs and consequen ly leading o s ea osis (Ha is e al., 2011). Howe e , LDs p o eins also play an impo an ole in his p ocess. In he absence o PLIN3, HCV co e p o ein-induced s ea osis does no occu (Fe guson e al., 2017). Mo eo e , co e associa ion o LDs leads o an inhibi ion o lipolysis by in e e ing wi h he ac i i y o adipose iacylglyce ol lipase (ATGL), he enzyme esponsible o he i s s ep o deg ada ion o TAG. The co e p o ein al e s he binding o ATGL o i s ac i a o , compa a i e gene iden i ica ion 58, enhancing he associa ion o bo h p o eins wi h LDs (Camus e al., 2014). Ikβkinase-αis ano he c i ical hos ac o o HCV-induced lipogenesis (Li e al., 2013). In ac , HCV in e ac s ex ensi ely wi h hos ac o s o manipula e he lipid me abolism and p omo e i us assembly, which likely con ibu es o i al eplica ion and s ea osis. LDs a e also a ge by an i i al p o eins ha compe e wi h i al p o eins. Vipe in, an in e e on-induced F on ie s in Mic obiology | www. on ie sin.o g 5Augus 2018 | Volume 9 | A icle 1951 micb-09-01951 Augus 18, 2018 Time: 18:53 # 6 Ma ins e al. Lipid D ople Cha ac e iza ion in Fla i i idae In ec ion FIGURE 3 | LDs as pla o m o DENV assembly. LDs (1) a he ER-Golgi in e media e compa men (ERGIC), ARF1 and i s guanine nucleo ide exchange ac o (GEF) GBF1, oge he wi h COPI, deli e adipose iacylglyce ol lipase (ATGL) and ADRP (pe ilipin 2) om ER expo si es (ERES) o he su ace o LDs. DENV sub e s his p ocess o he anspo a ion o he C p o ein o LDs su ace. (2) The accumula ion o DENV C on LDs depends on pe ilipin 3 and in acellula K+concen a ion. (3) Replica ed i al genomes a e eleased h ough he esicle po e and hen engaged in o nucleocapsids ha bud h ough he ER memb ane in close p oximi y. (4) DENV C can be eleased om LDs o he cy osol o o he cellula compa men s o subsequen i al assembly (g ay-dashed ame and enla ged panel). (5) Packed i ions accumula e wi hin he lumen o he esicle packe s-con aining ER ne wo k be o e being anspo ed o he Golgi (adap ed om Zhang e al., 2017). an i i al p o ein, binds o LDs, inhibi ing HCV (Hinson and C esswell, 2009). HCV NS5 P o ein–LDs In e ac ion Hepa i is C i us co e p o ein may be he only p o ein esponsible o in acellula LDs edis ibu ion, bu he e a e o he hos and i al ac o s media ing he in e ac ion o he co e wi h LDs, as well as o he impo an p ocesses du ing i al in ec ion. NS5A, a non-s uc u al p o ein o HCV, is also a key p o ein o HCV pa hogenesis and pe sis ence. I was p e iously desc ibed ha NS5A co-localizes wi h he co e p o ein (Boulan e al., 2007). The C- e minal domain III o NS5A was iden i ied as de e minan o co-localiza ion o he co e p o ein and NS5A a he LDs su ace, which is c ucial o i al assembly (Appel e al., 2008). The co e p o ein–NS5A in e ac ion on LDs su ace is s abilized by apolipop o ein J, also known as clus e in (Lin C.C. e al., 2014). Besides NS5A, o he non-s uc u al p o eins a e in ol ed in HCV assembly. HCV co e p o ein ec ui s hese non-s uc u al p o eins and eplica ion complexes o LD- associa ed memb anes, a c ucial p ocess o p oducing new i us pa icles (Miyana i e al., 2007). HCV NS5 also modula es he unc ion o a K+-speci ic channel (K 2.1) (Mankou i e al., 2009). Mo eo e , HCV uses a i opo in, p7, o p omo e memb ane pe meabili y o po assium and o he ca ions in i s in ec ion p ocess (G i in e al., 2003). DENV Capsid P o ein–LDs In e ac ion Dengue i us o igina es 390 million in ec ions wo ldwide and in he mos se e e cases, he disease p og esses o dengue hemo hagic e e (DHF) (Bha e al., 2013;Pó oa e al., 2014; Guo e al., 2017). The u gen need o an e ec i e accine led o se e al s udies aiming o unde s and he key s eps o he i us li e cycle. The i al assembly and encapsida ion p ocesses, which a e media ed by he C p o ein and in ol e LDs, ha e been s udied by us and o he s in some de ail. I is now clea ha he ma u e DENV C p o ein accumula es on he su ace o LDs ia an in e ac ion ha in ol es speci ic hyd ophobic amino acids. B ie ly, L50 and L54, in he α2 helix o he C p o ein, we e iden i ied as essen ial o DENV C–LDs binding (Samsa e al., 2009;Ma ins e al., 2012). The posi i ely cha ged N- e minal egion o he C p o ein also p omp s his in e ac ion (Ma ins e al., 2012). DENV C in e ac s mainly wi h PLIN3, a he LDs’ su ace, and his in e ac ion is dependen o he high in acellula concen a ion o po assium ions (Ca alho e al., 2012). By inhibi ing he Na+/K+-ATPase in DENV-in ec ed cells, wi hou a ec ing RNA eplica ion, he po assium ions in acellula concen a ion can be lowe ed, which in u n p e en s he C p o ein om in e ac ing wi h LDs and, consequen ly, dec eases he numbe o i al pa icles o med (Ca alho e al., 2012). I was p oposed ha DENV uses a non-canonical unc ion o he COPI sys em o C p o ein accumula ion on LDs (Iglesias e al., 2015). As wi h HCV co e p o ein, DENV C–LDs binding is c ucial o i al eplica ion (Samsa e al., 2009). Dis up ing DENV C associa ion on he LDs su ace dec eases i al RNA ampli ica ion. I also impai s i al pa icle o ma ion. DENV NS4A P o ein–LDs In e ac ion I was ecen ly p oposed ha NS4, a non-s uc u al p o ein o DENV wi h hos immune- esponse modula ion p ope ies, ha e a key ole in he i al li e cycle (Gopala Reddy e al., 2018). NS4A is clea ed om NS3 a i s N- e minal egion and om 2K agmen a i s C- e minal. The clea age o he 2K agmen is essen ial o NS4A o success ully induce hos memb ane al e a ions (Mille e al., 2007). In DENV in ec ion, NS4A associa es wi h a p o ein localized a LDs and ER: he ancien ubiqui ous p o ein 1 (AUP1) (Zhang e al., 2018). This p o ein appea s p edominan ly in he mono-ubiqui yla ed o m in non- in ec ed cells. Howe e , in DENV in ec ion i was epo ed ha AUP1 appea s in he unmodi ied o m and i s exp ession is enhanced (Zhang e al., 2018). In e es ingly, a di e en AUP1 dis ibu ion was obse ed in DENV-in ec ed cells (Zhang e al., 2018). AUP1 associa es o NS4A and elocalizes om LDs o au ophagosomes. NS4A in e ac ion wi h he unmodi ied AUP1, ac i a es i s acyl ans e ase domain o igge lipophagy. This p ocess is also dependen o NS4B. Impo an ly, he F on ie s in Mic obiology | www. on ie sin.o g 6Augus 2018 | Volume 9 | A icle 1951 micb-09-01951 Augus 18, 2018 Time: 18:53 # 7 Ma ins e al. Lipid D ople Cha ac e iza ion in Fla i i idae In ec ion ubiqui yla ion o NS4A dis up s NS4A-AUP1 in e ac ion and, consequen ly, he lipophagy p ocess essen ial o la i i uses in ec ion. Mo eo e , in he absence o AUP1, cells seem o be esis an o DENV, ZIKV, and WNV p oduc ion (Zhang e al., 2018). Simila i ies Be ween HCV, DENV, and O he Fla i i uses As implied om he abo e, DENV and HCV ha e iden ical in e ac ions be ween i al and cellula p o eins o p omo e physical con ac s wi h LDs. The Rab18 p o ein, a membe o he Rab GTPase amily, is p esen in LDs and ER memb anes, in e ac ing wi h NS5A and NS3, non-s uc u al p o eins o HCV (Salloum e al., 2013) and DENV (Tang e al., 2014), espec i ely. Rab18 co-localizes wi h HCV NS5A a LDs su ace and seems o p omo e he physical associa ion o NS5A and LDs, as well as o he componen s o i al eplica ion (Salloum e al., 2013). In DENV in ec ion, Rab18 seems o coo dina e he localiza ion o a y acid syn hase (FAS), a key enzyme o lipid biosyn hesis, on LDs and ER and i s in e ac ion wi h NS3. Rab18 can be an impo an hos ac o o ensu e ha i us eplica ion occu s a p ecise loca ions wi h su icien lipid supply (Tang e al., 2014). LIPID DROPLET ISOLATION AND PURIFICATION The isola ion and pu i ica ion o hos lipid sys ems can be e y di icul , i no impossible. Ob aining pu i ied in ac ac ions o hos lipid sys ems usually equi es high quan i ies o he sou ce sample. Fo example, he isola ion o human plasma lipop o eins equi es ela i ely high quan i ies o blood. Mo eo e , i should be p o ided om a la ge pool o di e en blood dono s, so ha he composi ion o he lipop o eins is as ep esen a i e and consis en om one isola ion ba ch o he nex as possible. In gene al, he pu i ica ion o hese hos lipid sys ems is done wi h complex and long p o ocols, equen ly equi ing a second pu i ica ion s ep. Howe e , LDs isola ion and pu i ica ion can be ela i ely simple, i compa ed o o he hos lipid sys ems. Typically, LDs isola ion in ol es s imula ion o i s p oduc ion in a pa icula cell line o in e es , a e which he cells a e lysed in a con olled manne and LDs pu i ied. A numbe o cell lines ha e been used o his pu pose, including HeLa cells (Kaczocha e al., 2010;Dejgaa d and P esley, 2014), hepa ocy es (Tu ó e al., 2006), sebaceous gland cells (Dahlho e al., 2015), adipocy es (Ma in and Pa on, 2008), and baby hams e kidney (BHK) cells (Samsa e al., 2009). LDs Isola ion To induce an inc eased p oduc ion o LDs wi hin he cell, cell cul u es a e commonly ea ed o 24–48 h wi h oleic acid (Ca alho e al., 2012), oleic acid complexed o de a ed bo ine se um albumin (Kaczocha e al., 2010) o linoleic acid (Dahlho e al., 2015). These a e a y acids ha s imula e he a y acid ecep o FFAR4 and enhance he numbe and size o LDs (Dahlho e al., 2015). Following, an analysis o lipid accumula ion o e alua e he numbe and size o LDs can be pe o med. A e he induc ion, cells ha e o be washed and esuspended in bu e wi h a p o ease inhibi o cock ail. Inhibi ion o p o eases ac i i y is a key s ep o his p o ocol; o he wise he esul s ob ained in he subsequen s udies can be inaccu a e due o changes in he LDs p o eome. Following, in he p esence o p o ease inhibi o s, cells can be dis up ed by ni ogen ca i a ion using a cell dis up ion essel (Samsa e al., 2009), sonica ion (Ma in and Pa on, 2008), s okes on ice (Rösch e al., 2017) o shea ing wi h small-bo e needles. The me hod chosen o cell dis up ion is di e en in se e al p o ocols desc ibed in he li e a u e and di e s wi h he ype o LDs sou ce (Ding e al., 2013). The e is no e iden co ela ion be ween he me hod used o cell dis up ion and he quan i y and/o pu i y o he LDs ob ained. Lipid d ople s can be pu i ied om a lysa e o cells submi ed o cen i uga ion, since LDs will loa in he aqueous g adien s. Submi ing he cell lysa e o a cen i uga ion a 1,500 ×g o 10 min is su icien o emo e he nuclei and collec he LDs in he supe na an . Wi h an ul acen i uga ion o he supe na an a 250,000 ×g o 70 min, a 4◦C, in a suc ose g adien , i is possible o isola e LDs by collec ing ac ion om he op o he bo om o he g adien (Figu e 4) (Ca alho e al., 2012). Howe e , he ul acen i uga ion condi ions may need o be op imized acco ding o he size o he LDs (Ding e al., 2013). The e a e o he me hods o ex ac LDs, using o ganic sol en s (Ma sumo o e al., 2002), bu hose app oaches a e less equen ly used. Isola ed LDs can be es ed o he absence o cy osolic con amina ion, ac i i y o lac a e dehyd ogenase and he p esence o classical LD p o eins (Ca alho e al., 2012), as well as ia mic oscopy isualiza ion o o he biochemical and biophysical assays (Ma in and Pa on, 2008). LDs Pu i ica ion Issues Seconda y pu i ica ion s eps a e no mally equi ed o ob ain LDs samples wi hou con amina ions. Minimizing con aminan s is o special impo ance when seeking o accu a ely cha ac e ize LDs p o eome, a pa icula di icul s ep gi en LDs’ mul iple con ac s wi h o he in acellula o ganelles and p o eins. LDs-associa ed p o eins a e essen ial o hei biogenesis and indispensable o he unc ions o hese o ganelles. The p o eins associa ed o he su ace o LDs a y be ween cell ypes (Gao and Goodman, 2015). The i s LDs-associa ed p o ein iden i ied was pe ilipin (now e med PLIN1), in 1991 (G eenbe g e al., 1991). Since hen, se e al s udies ha e been done o cha ac e ize he LDs p o eome in di e en cell lines (Dahlho e al., 2015). This was possible wi h he imp o emen o di e en app oaches used o cha ac e ize p o eins, bu also due o he imp o emen o LDs pu i ica ion p o ocols. The ac ions o LDs collec ed om he ul acen i uga ion g adien may be submi ed o an addi ional washing s ep, o educe he con amina ion o p o eins p e enien om o he sou ces (Ding e al., 2013). Se e al washing s eps can be done. Howe e , i is impo an o keep in mind ha e y small LDs may be los wi h sequen ial washings. E en wi h his addi ional s ep, i is almos impossible o elimina e he p o eins om o ganelles o memb ane s uc u es ha a e bound o LDs. To dis up he binding o hese p o eins o LDs, he pH o he washing bu e can be adjus ed o 11.5 (B asaemle F on ie s in Mic obiology | www. on ie sin.o g 7Augus 2018 | Volume 9 | A icle 1951 micb-09-01951 Augus 18, 2018 Time: 18:53 # 8 Ma ins e al. Lipid D ople Cha ac e iza ion in Fla i i idae In ec ion FIGURE 4 | Schema ic ep esen a ion o LDs isola ion and pu i ica ion. e al., 2004). Al hough hese s eps can be e y e icien o emo e LDs’ con amina ing p o eins, LDs’ mo phology and associa ed p o eins may also be a ec ed. To a oid his, one can pe o m a quan i a i e analysis o he p o eins in he wo ac ions sepa a ed ia he densi y g adien ul acen i uga ion o he cell lysa e: he i s one co esponding o he p o eins in he LDs ac ion and he second co esponding o he p o eins in he cell pelle con aining he emaining cellula componen s. LDs-associa ed p o eins can be p ecipi a ed wi h ichlo oace ic acid and ace one (Boulan e al., 2007) o solubilized in Laemmli bu e (Dahlho e al., 2015). P o ein ac ions can hen be sepa a ed by SDS-PAGE and analyzed by mass spec ome y. A quan i a i e compa ison o he p o eins p esen in he LDs and pelle ac ions can p o ide accu a e in o ma ion o LD-associa ed p o eins. The c i e ia de ined by Dahlho e al. (2015) we e ha i he amoun o a p o ein is a leas he double in he LDs ac ion compa ed o he cell pelle ac ion and i his esul is ep oducible in independen measu emen s, he p o ein can be conside ed as pa o he LDs p o eome. Using his a bi a y app oach, i is possible o iden i y he LD-associa ed p o eins o a speci ic ype o cells. Howe e , i is impo an o compa e he esul s ob ained wi h he da a al eady a ailable o he same ype and o he ypes o cells. I a p o ein is iden i ied o he i s ime as LD-associa ed, o he echniques should be used o con i m he esul . Fo ins ance, immuno luo escence s udies may con i m i he p o ein p esen s cy oplasmic localiza ion o i i co-localizes wi h LDs. METHODS FOR LIPID DROPLETS CHARACTERIZATION To mo e o wa d, i is impo an o know he a senal o echniques and app oaches h ough which hese c ucial o ganelles can be s udied and cha ac e ized. LDs ha e been e alua ed conce ning hei physical chemis y p ope ies such as size, su ace cha ge, ze a po en ial and molecula weigh , among o he p ope ies. Addi ionally, de e mining hei composi ion in e ms o su ace p o eins is also ex emely impo an (no desc ibed he e; o mo e in o ma ion see: Be suke e al., 2018; Goodman, 2018;P é os e al., 2018), no only o a undamen al cha ac e iza ion o LD p ope ies, bu also o unde s and he ole o hese o ganelles in pa hologies and i al in ec ion. Di e en echniques and app oaches ha e been used o be e unde s and LD-media ed p ocesses and hei ole in Fla i i idae in ec ion. Recen me hodological imp o emen s p o ided new app oaches o s udy hese o ganelles and o iden i y speci ic i al and hos ac o s in ol ed in key s eps o he i us li e cycle. The in o ma ion ga he ed by hese me hods may be combined F on ie s in Mic obiology | www. on ie sin.o g 8Augus 2018 | Volume 9 | A icle 1951 micb-09-01951 Augus 18, 2018 Time: 18:53 # 9 Ma ins e al. Lipid D ople Cha ac e iza ion in Fla i i idae In ec ion o de elop d ug s a egies agains impo an pa hogens o he Fla i i idae amily. He e, we p esen di e en app oaches o cha ac e ize LDs wi hin he cell en i onmen o isola ed om cell cul u es. In pa icula , we e iew ligh sca e ing, ze a po en ial and mic oscopy echniques, including a omic o ce mic ocopy- based o ce spec oscopy, in he con ex o he use o such echniques o s udy LDs ole in he i al li e cycle. Ligh Sca e ing Dynamic ligh sca e ing (DLS) spec oscopy is a echnique commonly used o de e mine he size dis ibu ion p o ile o pa icles in suspension (Domingues e al., 2008). DLS is used o measu e he 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, in a ime-scale 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, since he di usion a e o pa icles is de e mined by hei size. To calcula e he co ela ion kine ics, which depends on he in ensi y- weigh ed di usion coe icien (D), di e en me hods can be employed, such as CONTIN (P o enche , 1982) o Cumulan s (F isken, 2001). 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 (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. I should be no iced ha he exponen ially decaying cu e goes o ze o a ime when he pa icle in mo emen exceeds he wa eleng h o he lase ligh (Uskoko i´ c, 2012). The sca e ing in ensi y dis ibu ion unc ion o DH, [I(DH)], is ob ained, and can be con e ed o n(DH), he pa icle numbe dis ibu ion unc ion o DH h ough he Mie heo y (San os and Cas anho, 1996;Faus ino e al., 2014). The sca e ing in ensi y o a pa icle is p opo ional o he six h powe o i s DH(Rayleigh’s app oxima ion); hus, he con e sion can be done by he ollowing ans o ma ion (San os and Cas anho, 1996;Faus ino e al., 2014): n(DH)≈I(DH) DH6(2) n(DH) exp esses how much a pa icle o a ce ain diame e sca e s ligh . DLS expe imen s can be pe o med in a Mal e n Ze asize Nano ZS equipped wi h a He–Ne lase , λ= 632.8 nm, wi h a backsca e ing de ec ion a 173◦(Faus ino e al., 2014). 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. Dynamic ligh sca e ing can p o ide quan i a i e in o ma ion on pa icle size and shape, wi h ela i ely as measu emen s (Faus ino e al., 2014). Howe e , o he e ogeneous and highly polydispe se sys ems, he esul s can be inaccu a e (Vezoˇ cnik e al., 2015). I he LDs sample in s udy is highly he e ogeneous, which may na u ally occu , he ligh sca e ed om la ge LDs may obscu e he ligh sca e ed om he smalle ones. In his case, he de e mined size dis ibu ion p obably will no co espond o he eal si ua ion o he he e ogenei y in size will no allow a p ope es ima ion o size. Asymme ic- low ield- low ac iona ion echnique (AF4) coupled o a mul i-angle ligh -sca e ing (MALS) enables he sepa a ion o pa icle acco dingly wi h hei size and he de e mina ion o size dis ibu ion, o al numbe , and numbe densi y dis ibu ion o pa icles (Vezoˇ cnik e al., 2015). Recen ly, Si a e al. (2017) in oduced an AF4 o a MALS de ec o wi h an embedded DLS module o s udy he size cha ac e is ics and shape o a i icial LDs. Flow DLS expe imen s, wi h a low a e o 0.2 mL/min, ga e accu a e hyd odynamic adius (RH) alues (Si a e al., 2017). Al hough, wi h inc easing low a es a he DLS de ec o , he accu acy o RHde e mina ion is lowe (Si a e al., 2017). Mo e ecen ly, a new me hod o moni o ing LDs size based on ligh sca e ing was p oposed, nanopa icle acking analysis (NTA) (Mu a o e e al., 2018). The size dis ibu ions o LDs could be measu ed using a Nanosigh LM-10 Nanopa icle T acking Analyze , equipped wi h a 405 nm lase and a high sensi i i y came a. The ligh sca e ed e ealed he empo al posi ions o indi idual LDs, which a e eco ded wi h a came a. To calcula e he RHo LDs, he mo ion o each LD is acked indi idually om he ames o he cap u ed ideos (Mu a o e e al., 2018). The choice o he ype o ligh sca e ing measu emen s should be done based on he sample cha ac e is ics. To de e mine he DHo RHo highly homogeneous LDs samples wi hou high p opensi y o agg ega e, DLS measu emen s assu e accu a e and eliable esul s. O he wise, o he me hods should be chosen. NTA was al eady applied o measu e he size o LDs isola ed om mouse li e . Mo eo e , in he s udy pe o med wi h his echnique, i was possible o analyze he size dis ibu ion o LDs om adul and ge ia ic mice (Mu a o e e al., 2018). This me hod can now be applied o de e mine LDs size isola ed om di e en issues o cells. NTA may be a powe ul echnique o compa e LDs size om non-in ec ed and in ec ed cells. The possible achie emen s may allow unde s anding he e ec o di e en i uses on LDs. Ze a Po en ial Ze a po en ial (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 (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 (Domingues e al., 2008). The ζ-po en ial is calcula ed h ough he elec opho e ic mobili y o he pa icles in solu ion, on an elec ic ield, o he elec ode o opposi e cha ge (Ki by and Hasselb ink, 2004). The iscous o ces oppose he mo emen o he pa icles in suspension un il F on ie s in Mic obiology | www. on ie sin.o g 9Augus 2018 | Volume 9 | A icle 1951 micb-09-01951 Augus 18, 2018 Time: 18:53 # 16 Ma ins e al. Lipid D ople Cha ac e iza ion in Fla i i idae In ec ion he single-molecule le el. The numbe o p o eins bound o he unc ionalized ip is unknown; howe e , his in o ma ion is no equi ed o o ce measu emen s, since i is possible o iden i y single-molecule binding e en s om he shape o he e ac ion cu e. To ha e he da a s a is ically alida ed, he acquisi ion o hund eds o housands o cu es is necessa y, as well as he use o mo e han one AFM ip o each condi ion (Ca alho and San os, 2012;Ca alho e al., 2012). Wi h his in o ma ion, his og ams o dis ibu ion o up u e o ces o dis ances can be gene a ed, and he equency o (un)binding e en s calcula ed om he numbe o cu es wi h (un)binding e en s o e he o al numbe o cu es ob ained. The his og ams dis ibu ion may be analyzed wi h a Gaussian model o ex ac he a e age o ce necessa y o b eak he bond be ween he p o ein a ached o he AFM ip and he LD. I mo e han one p o ein a ached o he AFM ip in e ac s wi h LDs, mul iple peaks o he his og ams dis ibu ion will be i ed wi h he Gaussian model (Ca alho e al., 2010;Ca alho and San os, 2012). Howe e , i he p o ein a ached o he AFM ip does no bind speci ically o he LD, only a peak co esponding o unspeci ic in e ac ions will appea in he his og am, a low o ce alues. Using AFM-based o ce spec oscopy, i is possible o e alua e he ypes o o ce in ol ed in he in e ac ions wi h LDs, pe o ming o ce measu emen s unde di e en bu e condi ions (bu e s wi h di e en ions o di e en concen a ions) (Ca alho e al., 2012) o wi h LDs submi ed o a limi ed p o eolysis (see “Ze a Po en ial” sec ion) (Ma ins e al., 2012). The up u e o ces and he pe cen age o (un)binding e en s o DENV C–LDs in e ac ion in he p esence o di e en ions, as well as in di e en ions concen a ion, ga e de ails abou his in e ac ion (Figu es 11A–D). Only in he p esence o physiological high in acellula po assium concen a ions we e obse ed mul iple peaks on o ce up u e his og ams and he highes pe cen age o binding e en s, demons a ing ha DENV C–LD in e ac ion is speci ic (Ca alho e al., 2012). Al hough his echnique allows he de e mina ion o speci ic in e ac ions as well as hei binding o ce, i is complex and ime-consuming. Howe e , now ha i is well es ablished, i can be pe o med o s udy he in e ac ion o o he i al p o eins wi h LDs. Mo eo e , o ce spec oscopy measu emen s can be pe o med o iden i y speci ic p o eins o he LDs su ace esponsible o he in e ac ion. Fo his, he adhe ed LDs a e p e-incuba ed wi h an ibodies agains speci ic p o eins o he LDs su ace (Ca alho e al., 2012). Using his app oach, AFM-based o ce spec oscopy measu emen s showed ha DENV C mainly in e ac s wi h PLIN3 a he su ace o LDs (Ca alho e al., 2012). The esul s ob ained sugges PLIN3 as he main a ge o DENV C binding. The same expe imen can be pe o med wi h an ibodies agains di e en p o eins a he LDs su ace, and in he p esence o o he i al p o eins. Compa ing he o ce and pe cen age o binding e en s ob ained, i may be possible o iden i y he LD p o eins in ol ed in i us eplica ion. Once hey ha e been iden i ied, i could be impo an o unde s and i hey a e p esen a he su ace o o he lipid sys ems. AFM-based o ce spec oscopy p o ides accu a e da a on he o ce and p obabili y o in e ac ions, a he single-molecule le el. The e o e, i is an excellen echnique o es he e ec i eness o possible inhibi o s o c ucial in e ac ions o i al p o eins wi h LDs. The pep ide pep14-23 was es ed by his app oach, demons a ing i s po en ial applica ion as inhibi o o DENV C–LDs in e ac ion (Figu e 11E) and, he e o e, o DENV eplica ion (Ma ins e al., 2012). As soon as o he i al p o eins in ol ed in speci ic in e ac ions wi h LDs-su ace p o eins ha e been iden i ied, pep14-23 (and o he pep ides) can be es ed as a po en ial b oad- spec um inhibi o . CONCLUSION Lipid d ople s a e essen ial o ganelles, in ol ed on he main aining o he cellula homeos asis and playing an impo an ole in cellula ene gy s o age and lipid me abolism (Be suke and Olzmann, 2017). Vi uses ha e he abili y o hijack he in acellula memb ane machine y o i al eplica ion. Se e al i uses o he Fla i i idae amily ha e been associa ed o he de egula ion o he lipid me abolism. Impo an human pa hogens, such as DENV, HCV, WNV, and ZIKV, a e associa ed, espec i ely, wi h hemo hagic e e (Bha e al., 2013), s ea osis (McLauchlan, 2009), neu ologic illness (Rossi e al., 2010), and mic ocephaly (Cal e e al., 2016). Despi e he knowledge ga he ed by s udies conduc ed in he las yea s, he e a e no e ec i e d ugs a ailable agains hese i uses. The impo an ole o LDs in la i i uses li e cycle makes hem a possible a ge o he de elopmen o new he apeu ics. A be e cha ac e iza ion o LDs mo phology, p o eome and “in e ac ome” may p o ide c ucial in o ma ion o unde s and LDs as undamen al o ganelles in i al eplica ion. Mo eo e , he iden i ica ion and cha ac e iza ion o i al ac o s, as s uc u al and non-s uc u al i al p o eins, as well as hei in e ac ion wi h speci ic LD ac o s may p o ide he in o ma ion needed o de elop e ec i e ea men s. Di e en echniques may be used in u he s udies, depending o he cen al ques ion in analysis. To cha ac e ize LDs size, echniques based on ligh sca e ing such as AF4 and NTA, may p o ide as and accu a e esul s. Howe e , LDs ha e o be isola ed om cell cul u es. Mic oscopy s udies allow he cha ac e iza ion o LDs in cell cul u es o issues, in e ms o size, localiza ion, accumula ion and dynamic. Compa a i e s udies o non-in ec ed and in ec ed cell cul u es may e eal impo an de ails o unde s and he LDs ole in he i al li e cycle. Mo eo e , li e cell imaging and analysis o LDs biogenesis may p o ide c ucial in o ma ion o unde s and hei ole in pa hogenesis. Al hough con ocal mic oscopy is one o he mos used echniques, i equi es he use o luo escen dyes. SRS can also be used o ex ac he same in o ma ion om a single cell and wi hou using dyes. Elec on mic oscopy can be used o cha ac e ize LDs size, o ma ion and in e ac ion wi h o he o ganelles. Mo eo e , his echnique p esen s a highe esolu ion. Fu he mo e, i is known ha i al p o eins play an impo an ole in se e al s eps o he i al li e cycle, namely in i al assembly and encapsida ion. Despi e all he knowledge on i al p o eins in e ac ion wi h LDs, much mo e in o ma ion can be ga he ed F on ie s in Mic obiology | www. on ie sin.o g 16 Augus 2018 | Volume 9 | A icle 1951 micb-09-01951 Augus 18, 2018 Time: 18:53 # 17 Ma ins e al. Lipid D ople Cha ac e iza ion in Fla i i idae In ec ion ia AFM-based o ce spec oscopy. The e o e, aking all o he abo e in o conside a ion, a comp ehensi e unde s anding o LDs ole in i al in ec ion is undamen al o de elop s a egies o inhibi i al eplica ion. AUTHOR CONTRIBUTIONS All au ho s lis ed ha e made a subs an ial, di ec and in ellec ual con ibu ion o he wo k, and app o ed i o publica ion. FUNDING This wo k was suppo ed by Fundação pa a a Ciência e a Tecnologia – Minis é io da Ciência, Tecnologia e Ensino Supe io (FCT-MCTES, Po ugal), including he ellowship PD/BD/113698/2015 and he P og am In es igado FCT ( esea ch con ac IF/00772/2013) o AM and IM, espec i ely. This wo k was also suppo ed by LISBOA01-0145-FEDER- 007391 p ojec , co- unded by FEDER, h ough POR Lisboa 2020 – P og ama Ope acional Regional de Lisboa, Po ugal 2020, and FCT-MCTES. REFERENCES Adeyo, O., Ho n, P. J., Lee, S. K., Binns, D. D., Chand ahas, A., Chapman, K. D., e al. (2011). The yeas lipin o hologue Pah1p is impo an o biogenesis o lipid d ople s. J. Cell Biol. 192, 1043–1055. doi: 10.1083/jcb.201010111 Appel, N., Zayas, M., Mille , S., K ijnse-Locke , J., Schalle , T., F iebe, P., e al. (2008). Essen ial ole o domain III o nons uc u al p o ein 5A o hepa i is C i us in ec ious pa icle assembly. PLoS Pa hog. 4:e1000035. doi: 10.1371/ jou nal.ppa .1000035 Appelq is , H., S anius, K., Bö jesson, K., Nilsson, K. P. R., and Dy age , C. (2017). Speci ic imaging o in acellula lipid d ople s using a benzo hiadiazole de i a i e wi h sol a och omic p ope ies. Bioconjug. Chem. 28, 1363–1370. doi: 10.1021/acs.bioconjchem.7b00048 Assunção-Mi anda, I., Ama al, F. A., Bozza, F. A., Fagundes, C. T., Sousa, L. P., Souza, D. G., e al. (2010). Con ibu ion o mac ophage mig a ion inhibi o y ac o o he pa hogenesis o dengue i us in ec ion. FASEB J. 24, 218–228. doi: 10.1096/ j.09-139469 Ba ba, G., Ha pe , F., Ha ada, T., Koha a, M., Gouline , S., Ma suu a, Y., e al. (1997). Hepa i is C i us co e p o ein shows a cy oplasmic localiza ion and associa es o cellula lipid s o age d ople s. P oc. Na l. Acad. Sci. U.S.A. 94, 1200–1205. doi: 10.1073/pnas.94.4.1200 Be ne, B. J., and Peco a, R. (1990). Dynamic Ligh Sca e ing – Wi h Applica ion o Chemis y, Biology and Physics. Melbou ne, FL: K iege Publishing Company, 4–23. Be suke , K., and Olzmann, J. A. (2017). Es ablishing he lipid d ople p o eome: mechanisms o lipid d ople p o ein a ge ing and deg ada ion. Biochim. Biophys. Ac a 1862, 1166–1177. doi: 10.1016/j.bbalip.2017.06.006 Be suke , K., Pe e son, C. W. H., To, M., Sahl, S. J., Sa ikhin, V., G ossman, E. A., e al. (2018). A p oximi y labeling s a egy p o ides insigh s in o he composi ion and dynamics o lipid d ople p o eomes. De . Cell 44, 97–112.e7. doi: 10.1016/j.de cel.2017.11.020 Bha , S., Ge hing, P. W., B ady, O. J., Messina, J. P., Fa low, A. W., Moyes, C. L., e al. (2013). The global dis ibu ion and bu den o dengue. Na u e 496, 504–507. doi: 10.1038/na u e12060 Bickel, P. E., Tansey, J. T., and Wel e, M. A. (2009). PAT p o eins, an ancien amily o lipid d ople p o eins ha egula e cellula lipid s o es. Biochim. Biophys. Ac a 1791, 419–440. doi: 10.1016/j.bbalip.2009.04.002 Boulan , S., Douglas, M. W., Moody, L., Budkowska, A., Ta ge -Adams, P., and McLauchlan, J. (2008). Hepa i is C i us co e p o ein induces lipid d ople edis ibu ion in a mic o ubule- and dynein-dependen manne . T a ic 9, 1268–1282. doi: 10.1111/j.1600-0854.2008.00767.x Boulan , S., Mon se e , R., Hope, R. G., Ra inie , M., Ta ge -Adams, P., La e gne, J. P., e al. (2006). S uc u al de e minan s ha a ge he hepa i is C i us co e p o ein o lipid d ople s. J. Biol. Chem. 281, 22236–22247. doi: 10.1074/jbc. M601031200 Boulan , S., Ta ge -Adams, P., and McLauchlan, J. (2007). Dis up ing he associa ion o hepa i is C i us co e p o ein wi h lipid d ople s co ela es wi h a loss in p oduc ion o in ec ious i us. J. Gen. Vi ol. 88, 2204–2213. doi: 10.1099/ i .0.82898-0 Boulan , S., Vanbelle, C., Ebel, C., Penin, F., and La e gne, J.-P. (2005). Hepa i is C i us co e p o ein is a dime ic alpha-helical p o ein exhibi ing memb ane p o ein ea u es. J. Vi ol. 79, 11353–11365. doi: 10.1128/JVI.79.17.11353-11365. 2005 B asaemle, D. L., Dolios, G., Shapi o, L., and Wang, R. (2004). P o eomic analysis o p o eins associa ed wi h lipid d ople s o basal and lipoly ically s imula ed 3T3- L1 adipocy es. J. Biol. Chem. 279, 46835–46842. doi: 10.1074/jbc.M409340200 Buhman, K. K., Chen, H. C., and Fa ese, R. V. (2001). The enzymes o neu al lipid syn hesis. J. Biol. Chem. 276, 40369–40372. doi: 10.1074/jbc.R100050200 Cal e , G., Aguia , R. S., Melo, A. S. O., Sampaio, S. A., de Filippis, I., Fab i, A., e al. (2016). De ec ion and sequencing o Zika i us om amnio ic luid o e uses wi h mic ocephaly in B azil: a case s udy. Lance In ec . Dis. 16, 653–660. doi: 10.1016/S1473-3099(16)00095-5 Camus, G., Schweige , M., He ke , E., Ha is, C., Kond a owicz, A. S., Tsou, C. L., e al. (2014). The hepa i is C i us co e p o ein inhibi s adipose iglyce ide lipase (ATGL)-media ed lipid mobiliza ion and enhances he ATGL in e ac ion wi h compa a i e gene iden i ica ion 58 (CGI-58) and lipid d ople s. J. Biol. Chem. 289, 35770–35780. doi: 10.1074/jbc.M114.587816 Cao, C., Zhou, D., Chen, T., S ee s, A. M., and Huang, Y. (2016). Label- ee digi al quan i ica ion o lipid d ople s in single cells by s imula ed Raman mic oscopy on a mic o luidic pla o m. Anal. Chem. 88, 4931–4939. doi: 10. 1021/acs.analchem.6b00862 Ca alho, F. A., Ca nei o, F. A., Ma ins, I. C., Assunção-Mi anda, I., Faus ino, A. F., Pe ei a, R. M., e al. (2012). Dengue i us capsid p o ein binding o hepa ic lipid d ople s (LD) is po assium ion dependen and is media ed by LD su ace p o eins. J. Vi ol. 86, 2096–2108. doi: 10.1128/JVI.06796-11 Ca alho, F. A., Connell, S., Mil enbe ge -Mil enyi, G., Pe ei a, S. V., Ta a es, A., A iëns, R. A. S., e al. (2010). A omic o ce mic oscopy-based molecula ecogni ion o a ib inogen ecep o on human e y h ocy es. ACS Nano 4, 4609–4620. doi: 10.1021/nn1009648 Ca alho, F. A., Ma ins, I. C., and San os, N. C. (2013). A omic o ce mic oscopy and o ce spec oscopy on he assessmen o p o ein olding and unc ionali y. A ch. Biochem. Biophys. 531, 116–127. doi: 10.1016/j.abb.2012.11.007 Ca alho, F. A., and San os, N. C. (2012). A omic o ce mic oscopy-based o ce spec oscopy - biological and biomedical applica ions. IUBMB Li e 64, 465–472. doi: 10.1002/iub.1037 Choudha y, V., Golani, G., Joshi, A. S., Co ie , S., Schnei e , R., P inz, W. A., e al. (2018). A chi ec u e o lipid d ople s in endoplasmic e iculum is de e mined by phospholipid in insic cu a u e. Cu . Biol. 28, 915–926.e9. doi: 10.1016/j. cub.2018.02.020 Cohen, B.-C., Raz, C., Shamay, A., and A go -A gaman, N. (2017). Lipid d ople usion in mamma y epi helial cells is egula ed by phospha idyle hanolamine me abolism. J. Mamma y Gland Biol. Neoplasia 22, 235–249. doi: 10.1007/ s10911-017-9386-7 Cohen, J. C., Ho on, J. D., and Hobbs, H. H. (2011). Human a y li e disease: old ques ions and new insigh s. Science 332, 1519–1523. doi: 10.1126/science. 1204265 Dahlho , M., F öhlich, T., A nold, G. J., Mülle , U., Leonha d , H., Zouboulis, C. C., e al. (2015). Cha ac e iza ion o he sebocy e lipid d ople p o eome e eals no el po en ial egula o s o sebaceous lipogenesis. Exp. Cell Res. 332, 146–155. doi: 10.1016/j.yexc .2014.12.004 Dejgaa d, S. Y., and P esley, J. F. (2014). New au oma ed single-cell echnique o segmen a ion and quan i a ion o lipid d ople s. J. His ochem. Cy ochem. 62, 889–901. doi: 10.1369/0022155414554835 Deslandes, F., Thiam, A. R., and Fo ê , L. (2017). Lipid d ople s can spon aneously bud o om a symme ic bilaye . Biophys. J. 113, 15–18. doi: 10.1016/j.bpj.2017. 05.045 F on ie s in Mic obiology | www. on ie sin.o g 17 Augus 2018 | Volume 9 | A icle 1951 micb-09-01951 Augus 18, 2018 Time: 18:53 # 18 Ma ins e al. Lipid D ople Cha ac e iza ion in Fla i i idae In ec ion DiDona o, D., and B asaemle, D. L. (2003). Fixa ion me hods o he s udy o lipid d ople s by immuno luo escence mic oscopy. J. His ochem. Cy ochem. 51, 773–780. doi: 10.1177/002215540305100608 Ding, Y., Zhang, S., Yang, L., Na, H., Zhang, P., Zhang, H., e al. (2013). Isola ing lipid d ople s om mul iple species. Na . P o oc. 8, 43–51. doi: 10.1038/np o . 2012.142 Domingues, M. M., San iago, P. S., Cas anho, M. A., and San os, N. C. (2008). Wha can ligh sca e ing spec oscopy do o memb ane-ac i e pep ide s udies? J. Pep . Sci. 14, 394–400. doi: 10.1002/psc.1007 Faus ino, A. F., Ca alho, F. A., Ma ins, I. C., Cas anho, M. A., Mohana-Bo ges, R., Almeida, F. C., e al. (2014). Dengue i us capsid p o ein in e ac s speci ically wi h e y low-densi y lipop o eins. Nanomedicine 10, 247–255. doi: 10.1016/j. nano.2013.06.004 Fe guson, D., Zhang, J., Da is, M. A., Helsley, R. N., Vedin, L.-L., Lee, R. G., e al. (2017). The lipid d ople -associa ed p o ein pe ilipin 3 acili a es hepa i is C i us-d i en hepa ic s ea osis. J. Lipid Res. 58, 420–432. doi: 10.1194/jl . M073734 F isken, B. J. (2001). Re isi ing he me hod o cumulan s o he analysis o dynamic ligh -sca e ing da a. Appl. Op . 40, 4087–4091. doi: 10.1364/AO.40. 004087 Fukumo o, S., and Fujimo o, T. (2002). De o ma ion o lipid d ople s in ixed samples. His ochem. Cell Biol. 118, 423–428. doi: 10.1007/s00418-002-0462-7 Gao, Q., and Goodman, J. M. (2015). The lipid d ople -a well-connec ed o ganelle. F on . Cell De . Biol. 3:49. doi: 10.3389/ cell.2015.00049 Goodman, J. M. (2018). Unde s anding he lipid d ople p o eome and p o ein a ge ing. De . Cell 44, 1–2. doi: 10.1016/j.de cel.2017.12.017 Gopala Reddy, S. B., Chin, W., and Shi ananju, N. S. (2018). Dengue i us NS2 and NS4: mino p o eins, mammo h oles. Biochem. Pha macol. 154, 54–63. doi: 10.1016/j.bcp.2018.04.008 G eenbe g, A. S., Coleman, R. A., K aeme , F. B., McManaman, J. L., Obin, M. S., Pu i, V., e al. (2011). The ole o lipid d ople s in me abolic disease in oden s and humans. J. Clin. In es . 121, 2102–2110. doi: 10.1172/JCI46069 G eenbe g, A. S., Egan, J. J., Wek, S. A., Ga y, N. B., Blanche e-Mackie, E. J., and Londos, C. (1991). Pe ilipin, a majo ho monally egula ed adipocy e-speci ic phosphop o ein associa ed wi h he pe iphe y o lipid s o age d ople s. J. Biol. Chem. 266, 11341–11346. G i in, S. D. C., Beales, L. P., Cla ke, D. S., Wo s old, O., E ans, S. D., Jaege , J., e al. (2003). The p7 p o ein o hepa i is C i us o ms an ion channel ha is blocked by he an i i al d ug aman adine. FEBS Le . 535, 34–38. doi: 10.1016/ S0014-5793(02)03851-6 G oss, D. A., and Sil e , D. L. (2014). Cy osolic lipid d ople s: om mechanisms o a s o age o disease. C i . Re . Biochem. Mol. Biol. 49, 304–326. doi: 10.3109/ 10409238.2014.931337 Guedes, A. F., Ca alho, F. A., Malho, I., Lousada, N., Sa gen o, L., and San os, N. C. (2016). A omic o ce mic oscopy as a ool o e alua e he isk o ca dio ascula diseases in pa ien s. Na . Nano echnol. 11, 687–692. doi: 10.1038/ nnano.2016.52 Guo, C., Zhou, Z., Wen, Z., Liu, Y., Zeng, C., Xiao, D., e al. (2017). Global epidemiology o dengue ou b eaks in 1990-2015: a sys ema ic e iew and me a-analysis. F on . Cell. In ec . Mic obiol. 7:317. doi: 10.3389/ cimb.2017. 00317 Hamil on, J. A. (1989). In e ac ions o iglyce ides wi h phospholipids: inco po a ion in o he bilaye s uc u e and o ma ion o emulsions. Biochemis y 28, 2514–2520. doi: 10.1021/bi00432a025 Ha is, C., He ke , E., Fa ese, R. V., and O , M. (2011). Hepa i is C i us co e p o ein dec eases lipid d ople u no e : a mechanism o co e- induced s ea osis. J. Biol. Chem. 286, 42615–42625. doi: 10.1074/jbc.M111.28 5148 Hashemi, H. F., and Goodman, J. M. (2015). The li e cycle o lipid d ople s. Cu . Opin. Cell Biol. 33, 119–124. doi: 10.1016/j.ceb.2015.02.002 Hea on, N. S., Pe e a, R., Be ge , K. L., Khadka, S., LaCoun , D. J., Kuhn, R. J., e al. (2010). Dengue i us nons uc u al p o ein 3 edis ibu es a y acid syn hase o si es o i al eplica ion and inc eases cellula a y acid syn hesis. P oc. Na l. Acad. Sci. U.S.A. 107, 17345–17350. doi: 10.1073/pnas.101081 1107 Hea on, N. S., and Randall, G. (2010). Dengue i us induced au ophagy egula es lipid me abolism. Cell Hos Mic obe 8, 422–432. doi: 10.1016/j.chom.2010. 10.006 He ke , E., Ha is, C., He nandez, C., Ca pen ie , A., Kaehlcke, K., Rosenbe g, A. R., e al. (2010). E icien hepa i is C i us pa icle o ma ion equi es diacylglyce ol acyl ans e ase-1. Na . Med. 16, 1295–1298. doi: 10.1038/nm. 2238 Hinson, E. R., and C esswell, P. (2009). The an i i al p o ein, ipe in, localizes o lipid d ople s ia i s N- e minal amphipa hic -helix. P oc. Na l. Acad. Sci. U.S.A. 106, 20452–20457. doi: 10.1073/pnas.0911679106 Hodges, B. D. M., and Wu, C. C. (2010). P o eomic insigh s in o an expanded cellula ole o cy oplasmic lipid d ople s. J. Lipid Res. 51, 262–273. doi: 10.1194/jl .R003582 Hussmann, K. L., Vande gaas , R., Zheng, K., Hoo e , L. I., and F ede icksen, B. L. (2014). S uc u al p o eins o Wes Nile i us a e a majo de e minan o in ec ious pa icle p oduc ion and i ness in as ocy es. J. Gen. Vi ol. 95, 1991–2003. doi: 10.1099/ i .0.065474-0 Iglesias, N. G., Mondo e, J. A., Byk, L. A., De Maio, F. A., Samsa, M. M., Al a ez, C., e al. (2015). Dengue i us uses a non-canonical unc ion o he hos GBF1- A -COPI sys em o capsid p o ein accumula ion on lipid d ople s. T a ic 16, 962–977. doi: 10.1111/ a.12305 Joshi, A. S., Zhang, H., and P inz, W. A. (2017). O ganelle biogenesis in he endoplasmic e iculum. Na . Cell Biol. 19, 876–882. doi: 10.1038/ncb3579 Kaczocha, M., Glase , S. T., Chae, J., B own, D. A., and Deu sch, D. G. (2010). Lipid d ople s a e no el si es o N-acyle hanolamine inac i a ion by a y acid amide hyd olase-2. J. Biol. Chem. 285, 2796–2806. doi: 10.1074/jbc.M109.058461 Kilpa ick, A. M. (2011). Globaliza ion, land use, and he in asion o Wes Nile i us. Science 334, 323–327. doi: 10.1126/science.1201010 Ki by, B. J., and Hasselb ink, E. F. (2004). Ze a po en ial o mic o luidic subs a es: 1. Theo y, expe imen al echniques, and e ec s on sepa a ions. Elec opho esis 25, 187–202. doi: 10.1002/elps.200305754 Kozusko, K., Tsang, V. H. M., Bo omley, W., Cho, Y.-H., Gando a, S., Mimmack, M., e al. (2015). Clinical and molecula cha ac e iza ion o a no el PLIN1 ameshi mu a ion iden i ied in pa ien s wi h amilial pa ial lipodys ophy. Diabe es Me ab. Res. Re . 64, 299–310. doi: 10.2337/db14- 0104 K ahme , N., Fa ese, R. V., and Wal he , T. C. (2013). Balancing he a : lipid d ople s and human disease. EMBO Mol. Med. 5, 973–983. doi: 10.1002/emmm. 201100671 K ahme , N., Guo, Y., Fa ese, R. V., and Wal he , T. C. (2009). SnapSho : lipid d ople s. Cell 139, 1024–1024.e1. doi: 10.1016/j.cell.2009.11.023 Li, Q., Pène, V., K ishnamu hy, S., Cha, H., and Liang, T. J. (2013). Hepa i is C i us in ec ion ac i a es an inna e pa hway in ol ing IKK-αin lipogenesis and i al assembly. Na . Med. 19, 722–729. doi: 10.1038/nm.3190 Lin, C. C., Tsai, P., Sun, H. Y., Hsu, M. C., Lee, J. C., Wu, I. C., e al. (2014). Apolipop o ein J, a glucose-up egula ed molecula chape one, s abilizes co e and NS5A o p omo e in ec ious hepa i is C i us i ion p oduc ion. J. Hepa ol. 61, 984–993. doi: 10.1016/j.jhep.2014.06.026 Lin, P., Chen, X., Mok an, H., A ese, E. L., Duan, L., Wang, L., e al. (2014). Memb ane a achmen and s uc u e models o lipid s o age d ople p o ein 1. Biochim. Biophys. Ac a 1838, 874–881. doi: 10.1016/j.bbamem.2013.12.003 Lu, F.-K., Ji, M., Fu, D., Ni, X., F eudige , C. W., Hol om, G., e al. (2012). Mul icolo s imula ed Raman sca e ing (SRS) mic oscopy. Mol. Phys. 110, 1927–1932. doi: 10.1080/00268976.2012.695028 Mankou i, J., Dallas, M. L., Hughes, M. E., G i in, S. D. C., Macdonald, A., Pee s, C., e al. (2009). Supp ession o a p o-apop o ic K+channel as a mechanism o hepa i is C i us pe sis ence. P oc. Na l. Acad. Sci. U.S.A. 106, 15903–15908. doi: 10.1073/pnas.0906798106 Ma in, A., Bodola, F., Sanga , D. V., Goe ge, K., Popo , V., Rijnb and, R., e al. (2003). Ch onic hepa i is associa ed wi h GB i us B pe sis ence in a ama in a e in ahepa ic inocula ion o syn he ic i al RNA. P oc. Na l. Acad. Sci. U.S.A. 100, 9962–9967. doi: 10.1073/pnas.1731505100 Ma in, S., and Pa on, R. G. (2008). Cha ac e iza ion o Rab18, a lipid d ople - associa ed small GTPase. Me hods Enzymol. 438, 109–129. doi: 10.1016/S0076- 6879(07)38008-7 Ma ins, A., Ma ins, I., Faus ino, A., Nascimen o, A., Ca alho, F., and San os, N. (2017). Unde s anding he s uc u e and unc ion o he capsid p o ein o Zika, Wes -Nile and Dengue i uses, namely hei abili y o in e ac wi h hos lipid sys ems. P o ein Sci. 26:150. Ma ins, I. C., Gomes-Ne o, F., Faus ino, A. F., Ca alho, F. A., Ca nei o, F. A., Bozza, P. T., e al. (2012). The diso de ed N- e minal egion o dengue i us F on ie s in Mic obiology | www. on ie sin.o g 18 Augus 2018 | Volume 9 | A icle 1951 micb-09-01951 Augus 18, 2018 Time: 18:53 # 19 Ma ins e al. Lipid D ople Cha ac e iza ion in Fla i i idae In ec ion capsid p o ein con ains a lipid-d ople -binding mo i . Biochem. J. 444, 405–415. doi: 10.1042/BJ20112219 Ma sumo o, S., Fónagy, A., Yamamo o, M., Wang, F., Yokoyama, N., Esumi, Y., e al. (2002). Chemical cha ac e iza ion o cy oplasmic lipid d ople s in he phe omone-p oducing cells o he silkmo h, Bombyx mo i.Insec Biochem. Mol. Biol. 32, 1447–1455. doi: 10.1016/S0965-1748(02)00065-6 McIn osh, A. L., S o ey, S. M., and A sha es, B. P. (2010). In acellula lipid d ople s con ain dynamic pools o sphingomyelin: ADRP binds phospholipids wi h high a ini y. Lipids 45, 465–477. doi: 10.1007/s11745-010-3424-1 McLauchlan, J. (2000). P ope ies o he hepa i is C i us co e p o ein: a s uc u al p o ein ha modula es cellula p ocesses. J. Vi al Hepa . 7, 2–14. doi: 10.1046/j. 1365-2893.2000.00201.x McLauchlan, J. (2009). Lipid d ople s and hepa i is C i us in ec ion. Biochim. Biophys. Ac a 1791, 552–559. doi: 10.1016/j.bbalip.2008.12.012 McLauchlan, J., and Hope, R. G. (2000). Sequence mo i s equi ed o lipid d ople associa ion and p o ein s abili y a e unique o he hepa i is C i us co e p o ein. J. Gen. Vi ol. 81, 1913–1925. doi: 10.1099/0022-1317-81-8-1913 Mille , S., Kas ne , S., K ijnse-Locke , J., Bühle , S., and Ba enschlage , R. (2007). The non-s uc u al p o ein 4A o dengue i us is an in eg al memb ane p o ein inducing memb ane al e a ions in a 2K- egula ed manne . J. Biol. Chem. 282, 8873–8882. doi: 10.1074/jbc.M609919200 Mille , S., and K ijnse-Locke , J. (2008). Modi ica ion o in acellula memb ane s uc u es o i us eplica ion. Na . Re . Mic obiol. 6, 363–374. doi: 10.1038/ n mic o1890 Miyana i, Y., A suzawa, K., Usuda, N., Wa ashi, K., Hishiki, T., Zayas, M., e al. (2007). The lipid d ople is an impo an o ganelle o hepa i is C i us p oduc ion. Na . Cell Biol. 9, 1089–1097. doi: 10.1038/ncb1631 Mukhopadhyay, S., Kuhn, R. J., and Rossmann, M. G. (2005). A s uc u al pe spec i e o he la i i us li e cycle. Na . Re . Mic obiol. 3, 13–22. doi: 10.1038/ n mic o1067 Mu a o e, K. A., Naj , C. P., Li ezey, N. M., Ma i, J., Mashek, D. G., and A iaga, E. A. (2018). Sizing lipid d ople s om adul and ge ia ic mouse li e issue ia nanopa icle acking analysis. Anal. Bioanal. Chem. 410, 3629–3638. doi: 10.1007/s00216-018-1016-8 Ne o-Yassa , I., Lo elle, M., Nahmias, Y., Hi schbe g, K., and Sklan, E. H. (2017). Li e cell imaging and analysis o lipid d ople s biogenesis in hepa a is C i us in ec ed cells. Me hods 127, 30–36. doi: 10.1016/j.yme h.2017. 05.010 Ohsaki, Y., Maeda, T., and Fujimo o, T. (2005). Fixa ion and pe meabiliza ion p o ocol is c i ical o he immunolabeling o lipid d ople p o eins. His ochem. Cell Biol. 124, 445–452. doi: 10.1007/s00418-005-0061-5 Pe amuna, A., and Summe s, M. L. (2014). Composi ion and occu ence o lipid d ople s in he cyanobac e ium Nos oc punc i o me.A ch. Mic obiol. 196, 881–890. doi: 10.1007/s00203-014-1027-6 Pe e a, R., Riley, C., Isaac, G., Hop -Jannasch, A. S., Moo e, R. J., Wei z, K. W., e al. (2012). Dengue i us in ec ion pe u bs lipid homeos asis in in ec ed mosqui o cells. PLoS Pa hog. 8:e1002584. doi: 10.1371/jou nal.ppa .1002584 Pol, A., G oss, S. P., and Pa on, R. G. (2014). Biogenesis o he mul i unc ional lipid d ople : lipids, p o eins, and si es. J. Cell Biol. 204, 635–646. doi: 10.1083/ jcb.201311051 Pó oa, T. F., Al es, A. M. B., Oli ei a, C. A. B., Nuo o, G. J., Chagas, V. L. A., and Paes, M. V. (2014). The pa hology o se e e dengue in mul iple o gans o human a al cases: his opa hology, ul as uc u e and i us eplica ion. PLoS One 9:e83386. doi: 10.1371/jou nal.pone.0083386 P é os , C., Sha p, M. E., Ko y, N., Lin, Q., Vo h, G. A., Fa ese, R. V., e al. (2018). Mechanism and de e minan s o amphipa hic helix-con aining p o ein a ge ing o lipid d ople s. De . Cell 44, 73–86.e4. doi: 10.1016/j.de cel.2017. 12.011 P o enche , S. W. (1982). CONTIN: a gene al pu pose cons ained egula iza ion p og am o in e ing noisy linea algeb aic and in eg al equa ions. Compu . Phys. Commun. 27, 229–242. doi: 10.1016/0010-4655(82) 90174-6 Rigau-Pé ez, J. G. (2006). Se e e dengue: he need o new case de ini ions. Lance In ec . Dis. 6, 297–302. doi: 10.1016/S1473-3099(06) 70465-0 Roingea d, P., and Melo, R. C. N. (2017). Lipid d ople hijacking by in acellula pa hogens. Cell. Mic obiol. 19:e12688. doi: 10.1111/cmi. 12688 Rösch, K., Kwia kowski, M., Schlü e , H., and He ke , E. (2017). Lipid d ople isola ion o quan i a i e mass spec ome y analysis. J. Vis. Exp. 122:e55585. doi: 10.3791/55585 Rossi, S. L., Ross, T. M., and E ans, J. D. (2010). Wes Nile i us. Clin. Lab. Med. 30, 47–65. doi: 10.1016/j.cll.2009.10.006 Salloum, S., Wang, H., Fe guson, C., Pa on, R. G., and Tai, A. W. (2013). Rab18 binds o hepa i is C i us NS5A and p omo es in e ac ion be ween si es o i al eplica ion and lipid d ople s. PLoS Pa hog. 9:e1003513. doi: 10.1371/jou nal. ppa .1003513 Samsa, M. M., Mondo e, J. A., Iglesias, N. G., Assunção-Mi anda, I., Ba bosa- Lima, G., Da Poian, A. T., e al. (2009). Dengue i us capsid p o ein usu ps lipid d ople s o i al pa icle o ma ion. PLoS Pa hog. 5:e1000632. doi: 10.1371/jou nal.ppa .1000632 San os, N. C., and Cas anho, M. A. (1996). Teaching ligh sca e ing spec oscopy: he dimension and shape o obacco mosaic i us. Biophys. J. 71, 1641–1650. doi: 10.1016/S0006-3495(96)79369-4 San os, N. C., and Cas anho, M. A. (2004). An o e iew o he biophysical applica ions o a omic o ce mic oscopy. Biophys. Chem. 107, 133–149. doi: 10.1016/j.bpc.2003.09.001 Schuldine , M., and Bohne , M. (2017). A di e en kind o lo e – lipid d ople con ac si es. Biochim. Biophys. Ac a 1862, 1188–1196. doi: 10.1016/j.bbalip. 2017.06.005 Shang, Z., Song, H., Shi, Y., Qi, J., and Gao, G. F. (2018). C ys al s uc u e o he capsid p o ein om Zika i us. J. Mol. Biol. 430, 948–962. doi: 10.1016/j.jmb. 2018.02.006 Sha inskaya, A., Boulan , S., Penin, F., McLauchlan, J., and Ba enschlage , R. (2007). The lipid d ople binding domain o hepa i is C i us co e p o ein is a majo de e minan o e icien i us assembly. J. Biol. Chem. 282, 37158–37169. doi: 10.1074/jbc.M707329200 Shi, M., Lin, X.-D., Vasilakis, N., Tian, J.-H., Li, C.-X., Chen, L.-J., e al. (2016). Di e gen i uses disco e ed in a h opods and e eb a es e ise he e olu iona y his o y o he Fla i i idae and ela ed i uses. J. Vi ol. 90, 659–669. doi: 10.1128/JVI.02036-15 Si a , S., Vezoˇ cnik, V., Maˇ cek, P., Kogej, K., Paho nik, D., and Žaga , E. (2017). Pi alls in size cha ac e iza ion o so pa icles by dynamic ligh sca e ing online coupled o asymme ical low ield- low ac iona ion. Anal. Chem. 89, 11744–11752. doi: 10.1021/acs.analchem.7b03251 Skinne , J. R., Shew, T. M., Schwa z, D. M., Tzeko , A., Lepus, C. M., Abum ad, N. A., e al. (2009). Diacylglyce ol en ichmen o endoplasmic e iculum o lipid d ople s ec ui s pe ilipin 3/TIP47 du ing lipid s o age and mobiliza ion. J. Biol. Chem. 284, 30941–30948. doi: 10.1074/jbc.M109.013995 S e e eld, J., McKenna, S. A., and Pa el, T. R. (2016). Dynamic ligh sca e ing: a p ac ical guide and applica ions in biomedical sciences. Biophys. Re . 8, 409–427. doi: 10.1007/s12551-016-0218-6 Tang, W.-C., Lin, R.-J., Liao, C.-L., and Lin, Y.-L. (2014). Rab18 acili a es dengue i us in ec ion by a ge ing a y acid syn hase o si es o i al eplica ion. J. Vi ol. 88, 6793–6804. doi: 10.1128/JVI.00045-14 Thiam, A. R., and Belle , M. (2017). The why, when and how o lipid d ople di e si y. J. Cell Sci. 130, 315–324. doi: 10.1242/jcs.192021 Thiam, A. R., Fa ese, R. V., and Wal he , T. C. (2013). The biophysics and cell biology o lipid d ople s. Na . Re . Mol. Cell Biol. 14, 775–786. doi: 10.1038/ n m3699 Tu ó, S., Ingelmo-To es, M., Es anyol, J. M., Teba , F., Fe nández, M. A., Albo , C. V., e al. (2006). Iden i ica ion and cha ac e iza ion o associa ed wi h lipid d ople p o ein 1: a no el memb ane-associa ed p o ein ha esides on hepa ic lipid d ople s. T a ic 7, 1254–1269. doi: 10.1111/j.1600-0854.2006. 00465.x Uskoko i´ c, V. (2012). Dynamic ligh sca e ing based mic oelec opho esis: main p ospec s and limi a ions. J. Dispe s. Sci. Technol. 33, 1762–1786. doi: 10.1080/ 01932691.2011.625523 Vahabi, S., Nazemi Salman, B., and Ja anma d, A. (2013). A omic o ce mic oscopy applica ion in biological esea ch: a e iew s udy. I an. J. Med. Sci. 38, 76–83. Vanni, S. (2017). In acellula lipid d ople s: om s uc u e o unc ion. Lipid Insigh s 10, 14–16. doi: 10.1177/1178635317745518 Vezoˇ cnik, V., Rebolj, K., Si a , S., O a, K., Tušek-Žnida iˇ c, M., Š us, J., e al. (2015). Size ac iona ion and size cha ac e iza ion o nanoemulsions o lipid d ople s and la ge unilamella lipid esicles by asymme ic- low ield- low ac iona ion/mul i-angle ligh sca e ing and dynamic ligh F on ie s in Mic obiology | www. on ie sin.o g 19 Augus 2018 | Volume 9 | A icle 1951 micb-09-01951 Augus 18, 2018 Time: 18:53 # 20 Ma ins e al. Lipid D ople Cha ac e iza ion in Fla i i idae In ec ion sca e ing. J. Ch oma og . A 1418, 185–191. doi: 10.1016/j.ch oma.2015. 09.048 Vog , D. A., Camus, G., He ke , E., Webs e , B. R., Tsou, C.-L., G eene, W. C., e al. (2013). Lipid d ople -binding p o ein TIP47 egula es hepa i is C Vi us RNA eplica ion h ough in e ac ion wi h he i al NS5A p o ein. PLoS Pa hog. 9:e1003302. doi: 10.1371/jou nal.ppa .100 3302 Wal he , T. C., and Fa ese, R. V. (2012). Lipid d ople s and cellula lipid me abolism. Annu. Re . Biochem. 81, 687–714. doi: 10.1146/annu e -biochem- 061009-102430 Wang, C. W. (2016). Lipid d ople s, lipophagy, and beyond. Biochim. Biophys. Ac a 1861, 793–805. doi: 10.1016/j.bbalip.2015.12.010 Wel e, M. A. (2015). Expanding oles o lipid d ople s. Cu . Biol. 25, R470–R481. doi: 10.1016/j.cub.2015.04.004 Whi ed, A. M., and Pa k, P. S. H. (2014). A omic o ce mic oscopy: a mul i ace ed ool o s udy memb ane p o eins and hei in e ac ions wi h ligands. Biochim. Biophys. Ac a 1838, 56–68. doi: 10.1016/j.bbamem.2013. 04.011 Wil ling, F., Wang, H., Haas, J. T., K ahme , N., Gould, T. J., Cheng, J., e al. (2013). T iacylglyce ol syn hesis enzymes media e lipid d ople g ow h by Relocalizing om he ER o Lipid D ople s. De . Cell 24, 384–399. doi: 10.1016/j.de cel.2013. 01.013 Willemsen, O. H., Snel, M. M. E., Cambi, A., G e e, J., De G oo h, B. G., and Figdo , C. G. (2000). Biomolecula in e ac ions measu ed by a omic o ce mic oscopy. Biophys. J. 79, 3267–3281. doi: 10.1016/S0006-3495(00)76559-3 Zhang, J., Lan, Y., Li, M. Y., Lame s, M. M., Fusade-Boye , M., Klemm, E., e al. (2018). Fla i i uses exploi he lipid d ople p o ein AUP1 o igge lipophagy and d i e i us p oduc ion. Cell Hos Mic obe 23, 819–831.e5. doi: 10.1016/j. chom.2018.05.005 Zhang, J., Lan, Y., and Sanyal, S. (2017). Modula ion o lipid d ople me abolism- A po en ial a ge o he apeu ic in e en ion in Fla i i idae In ec ions. F on . Mic obiol. 8:2286. doi: 10.3389/ micb.2017.02286 Con lic o In e es S a emen : The au ho s decla e ha he esea ch was conduc ed in he absence o any comme cial o inancial ela ionships ha could be cons ued as a po en ial con lic o in e es . Copy igh © 2018 Ma ins, Ma ins and San os. This is an open-access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License (CC BY). The use, dis ibu ion o ep oduc ion in o he o ums is pe mi ed, p o ided he o iginal au ho (s) and he copy igh owne (s) a e c edi ed and ha he o iginal publica ion in his jou nal is ci ed, in acco dance wi h accep ed academic p ac ice. 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