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
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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).
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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
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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
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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
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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
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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
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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
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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
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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.
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