Scavenger Receptor Class B Type I and Hepatitis C Virus Infection of Primary Tupaia Hepatocytes

12 Pages·2005·0.79 MB·English

JOURNALOFVIROLOGY,May2005,p.5774–5785 Vol.79,No.9
0022-538X/05/$08.00(cid:1)0 doi:10.1128/JVI.79.9.5774–5785.2005
Copyright©2005,AmericanSocietyforMicrobiology.AllRightsReserved.
Scavenger Receptor Class B Type I and Hepatitis C Virus Infection of
Primary Tupaia Hepatocytes
Heidi Barth,1 Raffaele Cerino,2 Mirko Arcuri,2 Marco Hoffmann,1 Peter Schu¨rmann,1
Mohammed I. Adah,1 Bettina Gissler,1 Xiping Zhao,1 Valeria Ghisetti,3
Bruna Lavezzo,3 Hubert E. Blum,1 Fritz von Weizs¨acker,1
Alessandra Vitelli,2 Elisa Scarselli,2
and Thomas F. Baumert1*
DepartmentofMedicineII,UniversityofFreiburg,Freiburg,Germany,1andIstitutodiRicerchediBiologiaMoleculare
P.Angeletti(IRBM),Pomezia,2andDepartmentofGastroenterologyandMicrobiologyLaboratory,
MolinetteHospital,Turino,3Italy
Received9July2004/Accepted2December2004 D
o
w
Hepatitis C virus (HCV) is a major cause of chronic hepatitis worldwide. The study of early steps during n
HCVinfectionhasbeenhamperedbythelackofsuitableinvitroorinvivomodels.PrimaryTupaiahepatocytes lo
a
(PTH)havebeenshowntobesusceptibletoHCVinfectioninvitroandinvivo.Humanscavengerreceptorclass d
e
BtypeI(SR-BI)representsanHCVreceptorcandidatemediatingthecellularbindingofE2glycoproteinto d
HepG2hepatomacells.However,thefunctionofSR-BIforviralinfectionofhepatocytesisunknown.Inthis f
r
study, we used PTH to assess the functional role of SR-BI as a putative HCV receptor. Sequence analysis of o
m
clonedtupaiaSR-BIrevealedahighhomologybetweentupaiaandhumanSR-BI.TransfectionofCHOcells

h
with human or tupaia SR-BI but not mouse SR-BI cDNA resulted in cellular E2 binding, suggesting that t
t
E2-binding domains between human and tupaia SR-BI are highly conserved. Preincubation of PTH with p
:
anti-SR-BIantibodiesresultedinmarkedinhibitionofE2orHCV-likeparticlebinding.However,anti-SR-BI //
jv
antibodies were not able to block HCV infection of PTH. In conclusion, our results demonstrate that SR-BI i.
representsanimportantcellsurfacemoleculeforthebindingoftheHCVenvelopetohepatocytesandsuggest a
s
thatotheroradditionalcellsurfacemoleculesarerequiredfortheinitiationofHCVinfection.Furthermore, m
the structural and functional similarities between human and tupaia SR-BI indicate that PTH represent a .o
useful model system to characterize the molecular interaction of the HCV envelope and SR-BI on primary rg
hepatocytes. /
o
n

O
c
HepatitisCvirus(HCV)isamajorcauseofposttransfusion mayusethelow-densitylipoproteinreceptorforuptake(2).In to
b
andcommunity-acquiredhepatitisintheworld(8,24,25).The theabsenceofhighlypurifiednativeinfectiousHCVparticles e
majorityofHCV-infectedindividualsdevelopchronichepatitis as a tool for the study of virus-cell interaction, recombinant r
1
that may progress to liver cirrhosis and hepatocellular carci- HCV glycoprotein E2, E1E2 liposomes (22), infectious HCV ,
2
noma (15). Treatment options for chronic HCV infection are pseudotype particles expressing E1 and E2 (HCVpp) (4, 16), 0
1
limited, and a vaccine to prevent HCV infection is not avail- andHCV-likeparticles(HCV-LPs)(3,39,41)havebeenused 7
able(11,15,17). to analyze virus-cell membrane interaction. Based on these b
y
HCVhasbeenclassifiedinaseparategenus(Hepacivirus)of experimental in vitro studies, CD81 (5, 32), dendritic cell- g
theFlaviviridaefamily.Thevirioncontainsapositive-stranded u
specificintercellularadhesionmolecule3grabbingnonintegrin e
RNA genome of approximately 9.6 kb in length (25). The s
genome consists of a highly conserved 5(cid:2) noncoding region (33) and highly sulfated heparan sulfate (3) have been pro- t
posed to play a role in mediating E2 binding and/or HCV
followed by a long open reading frame of 9,030 to 9,099 nu-
internalization.
cleotidesthatistranslatedintoasinglepolyproteinof3,010to
Recently,thescavengerreceptorclassBtypeI(SR-BI)has
3,030 amino acids. Processing of the polyprotein occurs by a
been proposed as a putative HCV receptor candidate (36).
combination of host and viral proteases. The HCV structural
SR-BI, a high-density lipoprotein-binding molecule, plays a
proteins comprise the putative nucleocapsid or core protein
functionalroleinlipidmetabolismandishighlyexpressedin
and the two envelope glycoproteins E1 and E2 (25). The E2
hepatocytes and steroidgenic tissues (34). SR-BI is a 509-
glycoprotein is thought to be responsible for initiating virus
attachmentduetoitsabilitytobindtohumancells(32). residue glycoprotein with a large extracellular loop (LEL)
HCV purified from plasma has been reported to exist in anchored to the plasma membrane at both the N and C
associationwithplasmalipoproteins,suggestingthatthevirus termini by transmembrane domains with short extensions
intothecytoplasm(21).SR-BIhasbeenshowntoplayarole
inmediatingthebindingofrecombinantE2toHepG2hep-
*Corresponding author. Mailing address: Dept. of Medicine II, atoma cells and the entry of recombinant HCVpp into
UniversityofFreiburg,HugstetterStrasse55,D-79106Freiburg,Ger-
Huh-7hepatomacells(5,36).Duetothelackofconvenient
many.Phone:[phone].Fax:[phone].E-mail:Thomas
[email protected]. invitroorinvivomodelsforthestudyofHCVinfection,the
5774
VOL.79,2005 SR-BI AND HCV INFECTION 5775
D
o
w
n
lo
a
d
e
d

f
r
o
m

h
t
t
p
:
/
/
jv
i.
a
s
m
.
o
r
g
/

o
n

O
c
t
o
b
e
r

1
FIG. 1. Alignmentofaminoacidsequencesoftupaia,mouse,andhumanSR-BI.TupaiaSR-BIcDNAwasclonedandsequencedbyRT-PCR ,
2
oftupaiamRNAwithhumanSR-BI-specificprimersasdescribedinMaterialsandMethods.SR-BIaminoacidsequencesofmouseandhuman 0
SR-BIaredepictedaccordingtoapreviousreport(1).Aminoacidhomologyanddifferencesbetweenspeciesareindicatedbydifferentcolors. 1
7

b
y

g
u
e
functionalroleofSR-BIasa(co)receptorforbindingofthe infectedpatientsresultedintheproductionofinfectiousvirus s
t
viralenvelopetoprimaryhostcellsandinfectionwithnative invitro,indicatingthatPTHprovideamodelforthestudyof
virus is unknown. HCVinfectionandthefunctionalassessmentofHCVreceptor
Thechimpanzee(Pantroglodytes)istheonlynonhumanhost candidates(46).
serving as a model for HCV infection (7, 38). While HCV In this study, we analyzed the functional role of SR-BI for
infection can be successfully studied in chimpanzees, these HCV binding and infection of PTH. We demonstrate that
animalexperimentsareexpensiveandraiseethicalissues.An SR-BI represents an important cell surface molecule mediat-
alternativemodelforHCVinfectionisthetreeshrewTupaia ing binding of the HCV envelope to hepatocytes. Since inhi-
belangeri,asmall,squirrel-likemammalcloselyrelatedtopri- bition of E2–SR-BI interaction did not block viral infection,
mates(29).T.belangerihasbeenshowntobesusceptibletoa ourdatasuggestthatotheroradditionalcellsurfacemolecules
varietyofhumanvirusesincludingherpessimplex,hepatitisB, arerequiredforHCVinfection.
and rotavirus (18, 19, 30, 31, 40, 43). Two studies have dem- (Resultsofthisstudywerepresentedinpartinabstractform
onstrated that T. belangeri can be infected in vivo with HCV at the 10th International Meeting on Hepatitis C Virus and
(20, 42). We have recently shown that primary Tupaia hepa- RelatedVirusesinKyoto,Japan,2to6December2003,andat
tocytes (PTH) can be successfully infected with serum or the 55th Annual Meeting of the American Society for the
plasma derived from chronically HCV-infected humans (46). StudyofLiverDisease[AASLD]inBoston,Mass.,28October
Incubation of PTH with native HCV from chronically HCV- to2November2004.)
5776 BARTH ET AL. J.VIROL.
asdescribedpreviously(6,41).HCV-LPE2concentrationwasdeterminedas
describedpreviously(9,41).Mousemonoclonalanti-E2antibody16A6(41)and
chimpanzeeanti-E2monoclonalantibody49F3weregenerouslyprovidedbyE.
Depla,Innogenetics,N.V.,Ghent,Belgium.Bothmonoclonalanti-E2antibodies
(16A6and49F3)bindtoanE2epitopelocatedbetweenHCVaminoacids516
and530(E.Depla,personalcommunication).Anti-SR-BIpolyclonalserumwas
raisedbygeneticimmunizationofBALB/cmicewithplasmidspVJ-SR-BIhar-
boringthefull-lengthtupaiaandhumanSR-BIcDNAs,respectively.Immuni-
zationprotocolandplasmidvectorhavebeenpreviouslydescribed(47).Preim-
mune control serum was collected from the same mice bled before
immunization.R-phycoerythrin(PE)-conjugatedgoatanti-mouseimmunoglob-
ulinG(IgG)antibody,PE-conjugatedgoatanti-humanIgGantibody,andfluo-
rescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG antibody were
purchased from Jackson ImmunoResearch Laboratories (West Grove, Pa.).
Rabbit anti-SR-BI polyclonal serum (NB 400-104) was obtained from Novus
Biologicals(Littleton,Colo.)andusedaspreviouslydescribed(36).Thisanti-
bodyrecognizesadefinedepitope(CSPAAKGTVLQEAKL,correspondingto
amino acids 496 through 509) in the SR-BI cytoplasmic C-terminal domain.
FIG. 2. Expression of SR-BI in PTH and human hepatoma cells. Horseradishperoxidase-conjugatedanti-rabbitIgGwaspurchasedfromAmer- D
(A) PTH, human hepatoma HepG2 cells, and Sf9 insect cell lysates sham Pharmacia Biotech (Uppsala, Sweden). Penta-His-biotin conjugate and o
weresubjectedtoSDS-PAGE.Followinggeltransfertopolyvinylidene streptavidin-R-PEwerepurchasedfromQIAGEN(Hilden,Germany).Origin w
difluoride membranes, immunoblotting was performed using rabbit and maintenance of HepG2, Sf9 insect and CHO cells have been described n
anti-SR-BIpolyclonalantibody(NB400-104)andhorseradishperox- previously(36,41). loa
idase-conjugatedanti-rabbitIgG.ThepresenceofSR-BIisindicated Isolation,culture,andHCVinfectionofPHT.T.belangeriwasobtainedfrom d
on the left, and molecular weight (MW) is indicated on the right. theGermanPrimateCenter,Go¨ttingen,Germany.Theanimalswerebredand e
d
(B) Analysis of SR-BI expression on freshly isolated PTH by flow maintainedattheanimalfacilitiesofUniversityHospitalFreiburginaccordance
f
cytometryisshown.Followingfixationandpermeabilization,cellswere with institutionally approved protocols and the National Institutes of Health ro
incubatedwithrabbitanti-SR-BIpolyclonalantibody(NB400-104ata guidelinesfortheuseofexperimentalanimals.PTHwereisolatedfromadultT. m
1:500 dilution) and subsequently stained with FITC-conjugated goat belangerishrews(maleandfemale,10to12weeksold,180to200gofbody h
anti-rabbitIgG.Negativecontrol(NC)representsPTHincubatedwith weight)andculturedaspreviouslydescribed(18,19,46).Briefly,freshlyisolated tt
p
controlantibody.xandyaxesshowmeanfluorescenceintensityand PTH were seeded at a density of (cid:3)3 (cid:4) 105 cells/ml of medium (6 (cid:4) 105 :
/
relativenumberofstainedcells,respectively. cells/well)oncollagen-coatedsix-wellplates.Confluenceafterplatingwas80to /jv
90%,withahepatocyteviabilityof(cid:5)90%.Forinfectionofhepatocytes,plasma i.
samplesfrompatientswithchronichepatitisC(genotypes1and4)wereused. a
s
Plasma-derivedHCVusedforinfectionstudieswascharacterizedbygenotyping m
MATERIALSANDMETHODS
andquantitationofviralload.HCVgenotypewasdeterminedusingtheVER .o
Recombinantproteins,antibodies,andcells.Truncatedsolublerecombinant SANTHCVgenotypeassay(LiPA;Bayer,Tarrytown,N.Y.).Quantitationof r
g
HCVglycoproteinE2(aminoacids384through661oftheHCVpolyprotein)of plasmaHCVRNAswasperformedusingtheVERSANTHCVRNA3.0assay /

genotype1a(isolateH77)andgenotype1b(isolateBK)harboringaC-terminal (Bayer).Multiplicityofinfection(MOI)wascalculatedasthenumberofHCV o
n
histidinetagwereproducedin293cellsasdescribedpreviously(28,35).HCV- genomicequivalents(determinedasdescribedabove)presentintheinoculum
O
LPs(derivedfromH77-cDNA,genotype1a)weresynthesizedinSf9insectcells dividedbythenumberofhepatocytesusedforinfection.PTHwereinfectedwith
c
t
o
b
e
r

1
,

2
0
1
7

b
y

g
u
e
s
t
FIG. 3. E2bindingtoCHOcellstransfectedwithmouse,human,andtupaiaSR-BI.CHOcellsweretransfectedwithexpressionconstructs
containingthecDNAformouse(pcDNA3/mSR-BI),human(pcDNA3/hSR-BI),tupaiaSR-BI(pcDNA3/tSR-BI),orcontrolvector(pcDNA3)as
describedpreviously(36).(A)FACSanalysisofanti-SR-BIbinding(usingpolyclonalrabbitanti-SR-BIinteractingwithbothhumanandmouse
SR-BIinpermeabilizedCHOcells[36])intransfectedCHOcellsindicatesthatapproximately30%ofcellstransfectedexpressthereceptoron
thecellsurface.(B)FACSanalysisofE2bindingtotransfectedCHOcellsshowscomparableE2bindingtohumanandtupaiaSR-BIbutnotto
mouseSR-BI.
VOL.79,2005 SR-BI AND HCV INFECTION 5777
D
o
w
n
lo
a
d
e
d

f
r
o
m

h
t
t
p
:
/
/
jv
i.
a
s
m
.
o
r
g
/

o
n

O
c
t
o
b
e
r

1
,

2
0
1
7
FIG. 4. (A) Dose-dependent and saturable binding of E2 to PTH. Hepatocytes were incubated with His-tagged E2 at the concentrations
indicated.CellularE2binding(correspondingtonetmeanfluorescenceintensity[(cid:7)MFI])wasdeterminedbyFACSanalysisusingananti-His- by
biotinylatedmouseantibodyandstreptavidin-R-PEasdescribedinMaterialsandMethods.(B)E2bindinginthepresenceofanti-tupaiaSR-BI g
antibody.PTHwereincubatedwithanti-tupaiaSR-BIantiserum(blackshadowedgraph)orpreimmuneserum(greyshadowedgraph)1hprior u
totheadditionofrecombinantE2(E2concentrationof(cid:3)4(cid:6)g/ml,antiserumdilutionof1:10).Negativecontrol(NC)representingPTHincubated es
withpreimmuneserum(1:10dilution),anti-His-biotinylatedmouseantibody,andstreptavidin-R-PEintheabsenceofE2proteinisshown.Cellular t
E2 binding was analyzed by FACS analysis as described above. (C) Dose-dependent inhibition of E2 binding to PTH by anti-tupaia SR-BI
antiserum.PTHwerepreincubatedwithdifferentdilutionsofanti-tupaiaSR-BI(squares)orpreimmuneserum(circles).AfterwashingwithPBS,
PTHwereincubatedwithrecombinantE2(E2concentrationof(cid:3)1.5(cid:6)g/ml)andcellularE2bindingwasanalyzedbyFACSanalysisasdescribed
forpanelA.DataareshownaspercentbindingcomparedtobindingofE2inthepresenceofPBS(at100%)ofarepresentativeexperiment.
(D)InhibitionofcellularE2bindingbyanti-tupaiaSR-BIandanti-humanSR-BIantibodies.PTHwerepreincubatedwithanti-tupaiaSR-BI,
anti-humanSR-BI,orcontrolantibody(preimmuneserum,alldiluted1:10inPBS)andsubsequentlyanalyzedforE2binding(E2concentration
(cid:8)1.5(cid:6)g/ml)asdescribedforpanelA.Dataareshownaspercentbinding(mean(cid:9)standarddeviationofarepresentativeexperimentperformed
intriplicate)inthepresenceofantibodycomparedtobindingofE2inthepresenceofPBS(at100%).
plasma-derivedHCVonday2afterhepatocyteplatingbyincubationfor6hwith specificRT-PCRusingrTthpolymeraseand5(cid:2)-UTR-specificprimers(23,46).
plasmabyusinganMOIbetween0.05and0.25asdescribedpreviously(46).At ForthesynthesisofcontrolHCVpositive-andnegative-strandRNAs,plasmids
days1and5postincubation,PTHwerecollectedandanalyzedforthepresence pCV-H77C(44)andpGEMT-H77UTRC(46)werelinearizedbydigestionwith
ofpositive-andnegative-strandHCVRNAsbyahighlyspecificstrand-specific XbaIorSpeI,respectively.InvitrotranscriptionandpurificationofHCVRNAs
reversetranscription(RT)-PCR(23,46).Mock-infectedPTHservedasnegative fromlinearizedgel-purifiedcDNAs(10(cid:6)g)wasperformedasdescribedprevi-
controls.TotalRNAfromcellsandmediumwasisolatedtwicebyguanidinium ously(44).TheabsenceofresidualcDNAtemplatewasconfirmedbyPCRusing
isothiocyanate-acid-phenolbasedextraction(RNeasy;QIAGEN).Positive-and HCV-specificprimers.
negative-strandHCVRNAswerethenanalyzedin1/50oftotalRNAsbystrand- FortheanalysisofSR-BI-dependentviralinfection,PTHwerepreincubated
5778 BARTH ET AL. J.VIROL.
D
o
w
n
lo
a
d
e
d

f
r
o
m

h
t
t
p
:
/
/
jv
i.
a
s
m
.
o
r
g
/

o
n

O
c
t
o
b
e
r

1
,

2
0
1
7

b
y

g
u
e
s
t
VOL.79,2005 SR-BI AND HCV INFECTION 5779
withanti-tupaiaSR-BIorpreimmuneserum(1:10dilution)at37°Cfor1h.After anti-SR-BIfor1hatroomtemperaturepriortotheadditionofrecombinantE2
washingwithphosphate-bufferedsaline(PBS),cellswereinfectedbytheaddi- andE2bindingwasdetectedusingbiotinylatedanti-penta-Hismouseantibody
tionofplasmaandprocessedasdescribedabove.ToexcludethatSR-BImole- andstreptavidin-R-PE(1:100dilution).Sincethisassaydoesnotusedye-conju-
cules are accessible for HCV-E2 at the hepatocyte cell surface via de novo gated secondary antibodies for the detection of E2, it can easily distinguish
expressionorintracellularpoolredistributionofSR-BIduringthetimeofviral betweenboundnonbiotinylatedmouseanti-SR-BI(notreactingwithstreptavi-
infection, we performed a second series of experiments in the presence of din-PE)andbiotinylatedanti-His-E2(specificallyinteractingwithstreptavidin-
anti-SR-BIantiserumorpreimmuneserum(1:10dilution)inthetissueculture PE).TostudywhethercellularbindingofHCV-LPswasinhibitedbyanti-SR-BI
mediumbeforeandduringthetimeofinfection. antibodies,cellswerepreincubatedwithdifferentdilutionsofmouseanti-SR-BI
AnalysisoftupaiaSR-BI.TupaiaSR-BIcDNAwasobtainedbyRT-PCR(kit for1hatroomtemperaturepriortotheadditionofrecombinantHCV-LPs,and
Superscript II; Invitrogen) of purified PTH RNA (RNeasy; QIAGEN) with HCV-LPbindingwasdetectedusingchimpanzeemonoclonalanti-E2(49F3)and
humanSR-BI-specificprimers(forward,5(cid:2)ATGGGGCCCCAGGCGCGC R-PE-conjugatedgoatanti-humanIgGantibodies.
AGACATGGGC3(cid:2),andreverse,5(cid:2)AGCGGGGTGTAGGGGCTGGGG Nucleotidesequenceaccessionnumber.ThesequenceofT.belangeriSR-BI
GGCCGG3(cid:2)).ThePCRproductsfromtwoindependentreactionswerecloned was deposited in the GenBank database under GenBank accession number
(pcRZeroBluntkit;Invitrogen)andsequenced.Full-lengthtupaiaSR-BIcDNA AY428553.
wasclonedintoexpressionvectorpcDNA3(Invitrogen)andtransfectedinCHO
cellsbyliposome-mediatedgenetransfer(Lipofectamine;Invitrogen)asprevi-
ouslydescribedforthehumanSR-BI(36).TupaiaSR-BIaminoacidsequence RESULTS
wasalignedwithmouseandhumanSR-BIsequences(1).Fortheanalysisof
SR-BI expression, HepG2 cells, Sf9 cells, and PTH were lysed in a buffer Isolation and characterization of tupaia SR-BI. To assess D
containing50mMTris-HCl,1%NP-40,50mMNaCl,and5mMEDTA,pH7.4. thefunctionofSR-BIforHCVbinding,entry,andinfectionof o
Followingremovalofnucleiandcelldebrisbylow-speedcentrifugation,lysates PTH, we first identified and characterized tupaia SR-BI. To w
were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis n
(SDS-PAGE). Proteins were detected by immunoblotting using rabbit anti- analyzethetupaiaSR-BIprimarystructure,SR-BIcDNAwas lo
SR-BIpolyclonalserum(NB400-104ata1:1,500dilutioninPBScontaining1% cloned from tupaia hepatocyte mRNA with human SR-BI- ad
Tween20)andhorseradishperoxidase-conjugatedanti-rabbitIgG(1:1,000dilu- specificprimers.Sequenceanalysisrevealedamarkedhomol- e
d
tion).CellsurfaceexpressionofSR-BIonfreshlyisolatedPTHandtransfected ogy (88%) between human and tupaia SR-BI. In contrast, f
CHOcellswasanalyzedbyusingafluorescence-activatedcellsorter(FACS) homologyofmouseandhumanSR-BIreachedonly82%(Fig. ro
withmouseanti-tupaiaoranti-humanSR-BIpolyclonalserum(variousdilutions m
inPBS)andR-PE-conjugatedgoatanti-mouseIgGantibody(1:100dilutionin 1). These findings are consistent with the close phylogenetic h
PBS).ForFACSanalysisofSR-BIwithpolyclonalrabbitanti-SR-BI(NB400- relationshipbetweentherespectivespecies.TheLELoftupaia tt
p
104directedagainstanepitopeintheC-terminalintracellulardomainofSR-BI), SR-BI, containing the putative SR-BI HCV E2-binding do- :
/
freshlyisolatedPTHandtransfectedCHOcellswerepermeablizedwith0.1% main, exhibited 44 amino acid changes compared to human /jv
sFaIpToCn-icnopnrjuiogratteodingcouabtaatniotin-rwabitbhitrIagbGbitanantitbi-oSdRy-(B1I:1(010:5d0i0ludtiioluntiionnPiBnSP)B.S)and SR-BI. In contrast, mouse SR-BI LEL differed by 73 amino i.a
AnalysisofcellularE2andHCV-LPbinding.Bindingofrecombinantsoluble acidsfromhumanSR-BILEL. sm
E2toPTHwasperformedasdescribedrecently(35,36).Briefly,freshlyisolated For the analysis of SR-BI expression in PTH, lysates of .
o
PTH(105cellsperassay)wereincubatedwithE2for1hatroomtemperature. culturedPTHweresubjectedtoSDS-PAGEandimmunoblot- r
g
Cellswerewashedtwotimesandincubatedwithabiotinylatedanti-penta-His ting using rabbit anti-SR-BI polyclonal antibody. Tupaia /
mouseantibodydirectedagainsttheHistagofrecombinantE2(1:50dilution) o
SR-BIexhibitedasimilarmolecularmass(approximately85to n
for 1 h atroom temperature. The cells were washed again, and cell-bound
antibodiesweredetectedbytheadditionofstreptavidin-R-PE(1:100dilution) 90 kDa) as human SR-BI expressed in HepG2 cells. By con- O
c
binding to biotin residues of the primary antibody. Flow cytometry was per- trast,polyclonalanti-SR-BIdidnotinteractwithanSR-BI-like t
o
formed using FACSCalibur (Becton Dickinson) and analyzed with CellQuest moleculeinlysatesofSf9insectcells(Fig.2A).Expressionof b
software.ForthemeasurementofHCV-LPbinding,freshlyisolatedPTH(105 e
SR-BI on PTH was confirmed by FACS analysis using rabbit r
cellsperassay)wereincubatedwithHCV-LPsorinsectcellcontrolpreparation 1
(derivedfrominsectcellsinfectedwitharecombinantbaculoviruscontainingthe polyclonalanti-SR-BI(Fig.2B). ,
cDNAfor(cid:10)-glucuronidase)inPBSfor1hat4°C.CellboundHCV-LPswere SR-BI-dependentbindingofHCVenvelopeglycoproteinE2 20
analyzedbyFACSasdescribedpreviouslyusingeithermousemonoclonalan- to PTH. To assess whether tupaia SR-BI was able to bind 1
7
ti-E2(16A6)andR-PE-conjugatedgoatanti-mouseIgGantibodiesorchimpan-
zeemonoclonalanti-E2(49F3)andR-PE-conjugatedgoatanti-humanIgGan- recombinant His-tagged viral envelope glycoprotein E2 (de- b
rivedfromanHCVgenotype1a-cDNA)recentlyusedforthe y
tibodies(41).TostudywhethercellularbindingofrecombinantE2wasinhibited g
by anti-SR-BI antibodies, cells were preincubated with different dilutions of study of SR-BI interaction on human hepatoma cells (36), u
e
s
t
FIG. 5. Specific and dose-dependent binding of anti-tupaia and human SR-BI antibodies to tupaia SR-BI. (A) Specific binding of rabbit
anti-SR-BI (NB 400-104, left panel), mouse anti-tupaia SR-BI (middle panel), and mouse anti-human SR-BI (right panel) to tupaia SR-BI
expressedinCHOcells.CHOcellsweretransfectedwithexpressionconstructscontainingcontrolvector(pcDNA3)orthecDNAfortupaiaSR-BI
(pcDNA3/tSR-BI) as described previously (36). Flow cytometry of transfected cells incubated with anti-SR-BI and R-PE or FITC-conjugated
secondaryantibodiesdemonstratedspecificinteractionofanti-SR-BIantibodieswithtupaiaSR-BI(bottompanels).Incontrast,nointeractionwas
presentinCHOcellstransfectedwithcontrolvectorandincubatedwithanti-SR-BIantibodies(upperpanels).Numbersinsidethepanelrepresent
the percentage of positively stained cells in relationship to the total number of cells. For staining with anti-SR-BI NB 400-104, cells were
permeabilizedpriortoincubationwithantibody.(B)Dose-dependentinteractionofanti-SR-BIwithtupaiaSR-BIexpressedinCHOcells.CHO
cellsweretransfectedwithexpressionconstructscontainingcontrolvector(pcDNA3)orcDNAfortupaiaSR-BI(pcDNA3/tSR-BI)asdescribed
forpanelA.Cellswerethenincubatedwithanti-tupaiaSR-BIinincreasingdilutions,andanti-SR-BIbindingwasdeterminedonnonpermeabilized
cellsasshownforpanelA.ThepercentagesofSR-BI-positivecellsspecificallyrecognizedbyanti-tupaiaSR-BIweredeterminedbysubtracting
the number of positive cells in pcDNA3-transfected cells stained with anti-SR-BI (negative control) from the number of positive cells in
pcDNA3/tSR-BI-transfectedcellsstainedwithanti-SR-BI.Histograms(leftpanel)andpercentagesofcells(rightpanel)recognizedbyanti-tupaia
SR-BIareshowninrelationshiptoantibodydose.(C)DetectionofcellsurfaceSR-BIonPTHbyusingmouseanti-tupaiaandhumanSR-BI
antibodies.NonpermeabilizedPTHwereincubatedwithmouseanti-tupaia(leftpanel)andhumanSR-BI(rightpanel)sera(1:500dilution)or
mouse preimmune serum (1:500 dilution) and subsequently stained with R-PE-conjugated goat anti-mouse IgG. x and y axes show mean
fluorescenceintensityandrelativenumberofstainedcells,respectively.
5780 BARTH ET AL. J.VIROL.
CHO cells were transfected with human, tupaia, or mouse
SR-BI cDNA. Interestingly, transfection of CHO cells with
humanortupaiaSR-BIcDNAresultedinE2binding,whereas
CHOcellstransfectedwithmouseSR-BIcDNAdidnotbind
E2(Fig.3).ThesefindingssuggestedthattupaiaSR-BIbutnot
mouseSR-BIbindsE2similartohumanSR-BI.
ToanalyzewhetherPTHinteractwithrecombinantE2,cel-
lular binding of E2 to PTH was measured by flow cytometry.
Soluble recombinant E2, previously shown to interact with
HepG2cellsinadose-dependentmanner(36),stronglybound
toPTH.E2bindingtoPTHwasdosedependentandsaturable
andreachedaplateauatanE2concentrationofapproximately
4(cid:6)g/ml(Fig.4A).E2derivedfromgenotype1bdemonstrated
asimilarbindingprofile,suggestingthatbindingofE2toPTH
isnotgenotypeorsubtypedependent(datanotshown).
ToinvestigatewhetherHCVenvelopebindingwasmediated D
o
by PTH cell surface SR-BI, we performed competitive assays w
usinganti-SR-BIantibodies.Sincecommerciallyavailablean- n
lo
tibodiesaredirectedagainstepitopesoftheSR-BIintracellu- a
lar cytoplasmic C-terminal domain, these antibodies do not d
e
interact with the SR-BI extracellular loop and are therefore d

f
not suitable for the study of inhibition of HCV envelope– r
o
SR-BI interaction on the cell surface. Thus, we generated m

novelantibodiesdirectedagainstepitopesoftheextracellular h
t
loopofhumanandtupaiaSR-BIbythegeneticimmunization tp
:
ofmicebyusingfull-lengthtupaiaandhumanSR-BIcDNAs, //
jv
respectively. To demonstrate that mouse anti-tupaia and hu- i.
a
manSR-BIantibodiesgeneratedbygeneticimmunizationspe- s
m
cificallyinteractwithtupaiaSR-BI,westudiedthebindingof
.
antibodies to tupaia SR-BI expressed on the cell surfaces of o
r
g
transfectedCHOcells.AsshowninFig.5,incubationofCHO /

cells expressing tupaia SR-BI with mouse polyclonal anti- o
n
SR-BI antibodies resulted in a specific interaction of anti- O
tupaia and human SR-BI antibodies with tupaia SR-BI (Fig. c
t
o
5A,bottompanels).Incontrast,nointeractionwaspresentin b
CHO cells transfected with the pcDNA3 control vector and e
r

incubatedwithanti-SR-BI(Fig.5A,upperpanels)orinCHO 1
,
cellstransfectedwithtupaiaSR-BIcDNAandincubatedwith 2
0
preimmuneserum(datanotshown).Interactionofanti-tupaia 1
7
SR-BI(Fig.5B)andanti-humanSR-BI(datanotshown)with
b
tupaia SR-BI expressed in CHO cells was dose dependent. y

Both antibodies interacted with SR-BI on the cell surfaces of g
u
PTHwithouttheneedofcellpermeabilization,thusconfirm- e
s
ing the interaction of these antibodies with epitopes of the t
SR-BIextracellularlooponPTH(Fig.5C).SaturationofPTH
FIG. 6. (A)Dose-dependentandsaturablebindingofHCV-LPsto
PTH and human HepG2 cells. Cells were incubated with increasing
concentrationsofHCV-LPs.AfterwashingwithPBS,cellularbinding
ofHCV-LPswasanalyzedbyflowcytometryusingamousemonoclo-
nalanti-E2antibodyandPE-conjugatedanti-mouseIgG.Ontheyaxis, Dataareshownaspercentbinding(mean(cid:9)standarddeviation[SD]
netmeanfluorescenceintensity((cid:7)MFI)valuesforeachHCV-LPE2 ofarepresentativeexperimentperformedintriplicate)inthepresence
concentrationwerecalculatedbysubtractingtheMFIofthenegative ofantibodycomparedtobindingofHCV-LPsinthepresenceofPBS
control (control insect cell preparation) with anti-E2 and PE-conju- (at 100%). (C) SR-BI-dependent binding of HCV-LPs to human
gatedanti-mouseIgGantibodiesfromthatobtainedwiththerespec- HepG2hepatomacells.HepG2cellswereincubatedwithanti-human
tiveHCV-LPE2concentration(xaxis).Squares,bindingofHCV-LPs SR-BIorcontrolantibody(inpreimmuneserumata1:10dilution)1h
to PTH; triangles, binding of HCV-LPs to HepG2 cells. (B) SR-BI- prior to the addition of HCV-LPs. After washing with PBS, HepG2
dependentbindingofHCV-LPstoPTH.PTHwerepreincubatedwith cells were incubated with HCV-LPs in subsaturating concentrations
anti-tupaiaSR-BIorcontrolantibody(inpreimmuneserumata1:10 (HCV-LPE2concentrationof(cid:3)1(cid:6)g/ml)andcellularHCV-LPbind-
dilution).AfterwashingwithPBS,PTHwereincubatedwithHCV-LPs ingwasassessedasdescribedforpanelB.Dataareshownaspercent
in subsaturating concentrations (HCV-LP E2 concentration of (cid:3)1 binding (mean (cid:9) SD of a representative experiment performed in
(cid:6)g/ml)andcellularHCV-LPbindingwasassessedusingachimpanzee triplicate)inthepresenceofantibodycomparedtobindingofE2inthe
monoclonalanti-E2antibodyasdescribedinMaterialsandMethods. presenceofPBS(at100%).
VOL.79,2005 SR-BI AND HCV INFECTION 5781
SR-BIbypolyclonalanti-tupaiaSR-BIantibodywasachieved tientswithchronicHCVinfection.SuccessfulinfectionofPTH
atanantibodydilutionof1:10to1:100(datanotshown). was determined by a time-dependent increase of positive-
Preincubation of PTH with anti-tupaia SR-BI antibody re- strand HCV RNA and the detection of negative-strand HCV
sultedinamarkedandconcentration-dependentinhibitionof RNAinhepatocytesbyahighlystrand-specificRT-PCR(Fig.
E2binding(Fig.4BandC).InhibitionofcellularE2bindingby 7AandB).TostudywhetherHCVinfectioncouldbeinhibited
anti-tupaia SR-BI antibodies was also observed when E2 de- by anti-SR-BI antibodies, PTH were preincubated with anti-
rived from genotype 1b was used as a ligand, suggesting that SR-BIpriortoHCVinfection.AsshowninFig.7C(upperleft
antibody-mediated inhibition of cellular E2 binding is geno- panel),anti-tupaiaSR-BIwasnotabletoblocktheviralinfec-
typeindependent(datanotshown).Inhibitionofcellularbind- tionofPTHatconcentrationsshowntoinhibitE2bindingto
ingreachedaplateauatanantibodydilutionof1:10(Fig.4C) PTH and saturating SR-BI. Similarly, the incubation of PTH
correspondingtotheantibodyconcentrationsaturatingSR-BI withpreimmuneserumdidnotresultinameasurablemodifi-
onPTH.Interestingly,preincubationofPTHwithanti-human cation of HCV infection (Fig. 7C, upper right panel). To ex-
SR-BI also resulted in a marked inhibition of E2 binding, cludethatSR-BImoleculeswereaccessibleforHCV-E2atthe
suggesting that the E2-binding domains of tupaia and human hepatocytecellsurfaceviadenovoexpressionortheintracel-
SR-BI LEL are highly conserved (Fig. 4D). Polyclonal anti- lular pool redistribution of SR-BI during the time of viral
human SR-BI antibody used in the experiment shown in Fig. infection,weperformedasecondseriesofexperimentsinthe D
4DhadbeenpreviouslyshowntoinhibitcellularbindingofE2 presence of anti-SR-BI antiserum or preimmune serum (1:10 o
w
toHepG2hepatomacellsandviralentryofHCVpseudopar- dilution) in the tissue culture medium before and during the n
ticlesintoHuh-7hepatomacellsinaconcentration-dependent time of infection (Fig. 7C, lower panel). Again, no antibody- lo
a
manner(5,36). mediated inhibition of HCV infection was observed (Fig. 7C, d
e
SR-BI and cellular binding of virus-like particles. As an lower panel). Infection experiments were performed at low d

alternative to purified HCV particles, truncated forms of gly- MOIs (starting from an MOI of 0.05) corresponding to the fr
o
coprotein E2 are useful to study virus-cell interactions. How- minimal infectious dose to ensure that a lacking inhibitory m
ever, C-truncated recombinant E2 proteins mimic only par- effect of anti-SR-BI was not the result of a high virus input h
t
tially the properties of HCV from infected patients. Since competingwithantibody-SR-BIinteraction.Toevaluateage- tp
HCV-LPs,bycomparison,sharecommonfeatureswithvirions notype-dependenteffectofSR-BIonHCVinfection,weincu- :/
/
in their cellular binding profiles (39, 41), we analyzed the bated PTH with sera and plasma containing different HCV jv
i.
interaction of HCV-LPs with SR-BI on human HepG2 cells isolates, subtypes, or genotypes. Similar to the results pre- a
s
and PTH. As a prerequisite for these studies, we analyzed sented in Fig. 7, no inhibition of infection by anti-SR-BI was m
whether HCV-LPs interact with PTH. Similar to findings ob- observed(datanotshown). .o
r
servedforrecombinantE2,bindingofHCV-LPsdemonstrated g
/
adose-dependentandsaturablebindingtoPTH.Saturationof o
DISCUSSION n
bindingwasreachedatanE2concentrationofapproximately
O
4 (cid:6)g/ml (Fig. 6A). A side-by-side comparison of HCV-LP Thefirststepinviralinfectionistheattachmentandentry c
t
binding to HepG2 hepatoma cells revealed that saturation of of the virion to the host cell requiring the interaction be- o
b
HCV-LP binding is achieved at a similar E2 concentration tween viral and cellular surface proteins, called receptors e
r
(Fig.6A). and coreceptors. Because of the difficulties to propagate 1
To analyze whether cellular HCV-LP binding to PTH is HCVinvitroandthelimitedanimaltropism,thesearchfor ,
2
SR-BIdependent,westudiedcellularHCV-LPbindinginthe HCV receptor candidates has been performed with binding 0
1
presence of anti-SR-BI antibodies. Anti-tupaia SR-BI inhib- studies using truncated versions of recombinant HCV gly- 7

ited binding of HCV-LPs to PTH compared to control anti- coprotein E2 and human hepatoma or lymphoma lines (12, b
y
bodyupto56%(HCV-LPE2concentrationof1(cid:6)g/ml,anti- 13,33).Basedonsuchexperimentalstudies,SR-BIhasbeen g
SR-BIdilutionof1:10).Althoughtheantibodyconcentration recentlysuggestedasanovelHCVreceptorcandidate(36). u
e
requiredforinhibitionofHCV-LPbindingwashigh,theanti- However,thepotentialroleofSR-BIfortheHCVinfection s
t
SR-BI-mediated inhibition of HCV-LP binding was specific, ofnaturaltargetcells,i.e.,primaryhepatocytes,isunknown.
sinceside-by-sideexperimentsusingpreimmuneserumatthe SincePTHcanbesuccessfullyinfectedbyserum-orplasma-
same concentration did not modify cellular HCV-LP binding derived native HCV (46), we used this model to assess the
(Fig.6B).TheseobservationssuggestedthatSR-BIrepresents functional role of SR-BI as a putative HCV receptor on
acellsurfacemoleculemediatingthebindingofHCV-LPsto primary hepatocytes.
PTH. To compare results obtained in PTH with human cell Structuralandfunctionalcharacterizationoftupaiaandhu-
lines,westudiedtheeffectofanti-SR-BIantibodyoncellular man SR-BI validated the tupaia system as a convenient and
HCV-LP binding to the human hepatoma cell line HepG2. useful model for the evaluation of HCV receptor candidates.
PreincubationofHepG2cellswithanti-humanSR-BIantibody Consistentwithourpreviousstudyassessingthestructureand
(previouslyshowntoblockbindingofsolubleE2glycoprotein functionofHCVreceptorcandidateCD81inPTH(46),HCV
and entry of HCVpp in Huh-7 hepatoma cells [5]) partially receptorcandidateSR-BIexhibitedaremarkablyhighdegree
reduced the binding of HCV-LPs to HepG2 hepatoma cells of homology at the amino acid level between tupaias and
(Fig.6C)similartoPTH(Fig.6B). humans. This sequence homology reflects the close genetic
HCV infection of tupaia hepatocytes and SR-BI. To study relationship between tupaias and primates. Interestingly, the
whether SR-BI mediates the HCV infection of hepatocytes, degreeofhomologyofSR-BIattheaminoacidlevelbetween
PTHwereinfectedwithHCVRNA-positiveplasmafrompa- humans, tupaias, and mice (humans versus tupaias, 88%; hu-
5782 BARTH ET AL. J.VIROL.
D
o
w
n
lo
a
d
e
d

f
r
o
m

h
t
t
p
:
/
/
jv
i.
a
s
m
.
o
r
g
/

o
n

O
c
t
o
b
e
r

1
,

2
0
1
7

b
y

g
u
e
s
t
VOL.79,2005 SR-BI AND HCV INFECTION 5783
mansversusmice,82%)correlatedwiththeabilityofSR-BIto betweenproteinsandvirionsplayaroleinthelackingabilityof
bindHCVenvelopeglycoproteinE2(Fig.3).Alignmentofthe anti-SR-BItoblockHCVinfection.
aminoacidsofthedifferentspeciessuggeststhat(i)E2-binding As shown for other viruses, several cellular surface mole-
domains between human and tupaia SR-BI are highly con- culesserveasreceptorsfortheattachmentandentryofviruses
servedand(ii)definedaminoacidchangesintheextracellular (37).Inaddition,viralentrymayrequiretheinteractionofviral
loop of mouse SR-BI abolish E2–SR-BI binding. A detailed proteins with coreceptors or entry receptors that are distinct
comparative and functional analysis of the amino acid differ- from those that mediate initial virus binding (37). For HCV
ences in the three species will finally allow mapping of the CD81 (10, 12, 16, 32, 45), dendritic cell-specific intercellular
E2–SR-BI-binding domain. Furthermore, the marked struc- adhesionmolecule3grabbingnonintegrin(13,26,27,33),the
tural and functional similarities between human and tupaia low-density lipoprotein receptor (2), and heparan sulfate (3)
SR-BI indicate that PTH represent a useful model system to havebeensuggestedtoplayanimportantroleinvirusattach-
characterize the molecular interaction of the HCV envelope mentandentry.Basedonthisandpreviousobservations,HCV
andSR-BIonprimaryhepatocytes. bindingandentryismostlikelyamultifactorialprocesswhich
Concentration-dependent inhibition of E2 or HCV-LPs requiresasetofinitialbindingmoleculesaswellas(co)recep-
binding by anti-tupaia and anti-human SR-BI antibodies torsforviralentry.
clearly demonstrates that SR-BI can mediate binding of the The finding of the present study indicates that SR-BI rep- D
viral envelope to PTH. These observations are in line with a resentsanimportanthepatocytecellsurfacemoleculemediat- o
w
previous study using HCVpp as a surrogate model for HCV ing binding of the viral envelope to the hepatocyte cell mem- n
binding and entry (5). Although recombinant E2, HCV-LPs, brane. SR-BI may act in concert with other E2-binding cell lo
a
and HCVpp represent convenient tools to study envelope- surfacemoleculessuchashighlysulfatedheparansulfate(3)or d
e
glycoprotein-mediated HCV binding and/or entry to or into CD81 (10, 12, 16, 32, 45). The latter hypothesis is underlined d

hepatomacells,itisstillunclearwhetherthesemodelsystems by the finding that preincubation of anti-SR-BI of PTH or fr
o
reflect the early steps of natural infection of hepatocytes by HepG2cells(Fig.6)resultedonlyinanincompleteinhibition m
native virions. Therefore, studies in primary hepatocytes with of HCV-LP binding. It is conceivable that other cell surface h
t
native virions are needed to verify findings obtained with molecules such as highly sulfated heparan sulfate (recently tp
HCVppandothersurrogatesystemsmimickingtheearlysteps showntomediatethebindingofHCV-LPs[3]andE2[14]to :/
/
of viral infection. Using native plasma-derived HCV and pri- HepG2 cells) may contribute to E2-cell surface attachment. jv
i.
mary hepatocytes, we present evidence that SR-BI is not suf- Thepresenceofseveraldifferentcellsurfacemoleculesforthe a
s
ficienttomediatetheHCVinfectionofprimaryhepatocytes.A capture of virions may represent a strategy of the virus to m
polyclonal anti-SR-BI antibody markedly inhibiting the cellu- secureefficientinteractionoftheviruswiththetargetcelland .o
r
larbindingofE2failedtoblockormodulatetheHCVinfec- escapeantibody-mediatedvirusneutralization.Sincetheinhi- g
/
tion of PTH (Fig. 7), although the experimental conditions bitionofE2–SR-BIinteractiondidnotblockormodulateviral o
n
(blocking antibody in concentrations saturating SR-BI and a infection,ourdatasuggestthatSR-BIisnotsufficientforHCV
O
studyofHCVinfectionatMOIscorrespondingtotheminimal entry into hepatocytes. Although mechanisms of viral entry c
t
infectious dose) had been designed to detect a measurable maynotbeidenticalintupaiaandhumanhepatocytesdueto o
b
effectofanti-SRBIonviralinfection.Sincerecombinantpro- species-specificfactorsandSR-BIandCD81havebeenshown e
r
teins(E2andHCV-LPs)andplasma-derivedvirionsusedfor to represent coreceptors for the entry of HCVpp into Huh-7 1
detailed studies of envelope binding (Fig. 3 through 6) and cells (5) and human hepatocytes (4), the findings obtained in ,
2
HCV infection (Fig. 7) consisted of the same genotype (all the HCV-PTH model system suggest that the entry of native 0
1
genotype 1), it is unlikely that genotype-specific differences virus into primary hepatocytes requires additional or other 7

b
y

g
u
e
s
FIG. 7. (A) Sensitivity and specificity of HCV RNA detection from hepatocytes by strand-specific RT-PCR. Different amounts of in vitro- t
synthesizedpositive-ornegative-strandHCVRNAs(synthesizedasdescribedinMaterialsandMethods)wereaddedtothetotalcellularRNAs
of uninfected PTH, respectively, to study the sensitivity and specificity of HCV RNA detection using strand-specific RT-PCR. Positive- and
negative-strandHCVRNAswerethenanalyzedbystrand-specificRT-PCRusingrTthpolymerase5(cid:2)-UTR-specificprimers(23,46).Sensitivity
forbothstrandsrangedfrom0.1to1fgperassayforthecorrectRNAtemplate,whilefortheincorrecttemplate(usesyntheticnegative-strand
HCV RNA as template for the detection of positive-strand HCV RNA or vice versa), at least 0.1 pg of the template was needed to give a
false-positivesignal.(B)InfectionofPTHbyplasma-derivedHCV.PTHwereincubatedwithHCVRNA-positiveplasma(samplesM1,M2,G3,
andG4,allHCVgenotype1)onday2afterplatingasdescribedinMaterialsandMethods.HCVinfectionwasdeterminedbystrand-specific
RT-PCRofcellularpositive-andnegative-strandHCVRNAsondays1and5postincubation(p.i.)inPTHasshownforpanelAanddescribed
inMaterialsandMethods.(C)HCVinfectionofPTHinthepresenceofanti-tupaiaSR-BI.PTHwerepreincubatedwithanti-tupaiaSR-BI(left
panel)orpreimmuneserum(rightpanel)atadilutionof1:101hpriortotheadditionofHCVplasma(samplesM2andG4,bothgenotype1).
Followingthewashingofnonboundanti-SR-BI,plasma-derivedHCVwasaddedtothecellculturemedium.TheMOIs(calculatedasthenumber
ofHCVgenomicequivalentspresentintheinoculumdividedbythenumberofhepatocytes)were0.05(3(cid:4)104genomicequivalents/6(cid:4)105
hepatocytes) and 0.25 (1.5 (cid:4) 105 genomic equivalents/6 (cid:4) 105 hepatocytes) for plasma samples M2 and G4, respectively. HCV infection was
determinedbystrand-specificRT-PCRasdescribedforpanelA.ToexcludethatSR-BImoleculeswereaccessibleforHCV-E2atthehepatocyte
cellsurfaceviathedenovoexpressionorintracellularpoolredistributionofSR-BIduringthetimeofviralinfection,infectionexperimentswere
repeatedinthepresenceofanti-tupaiaSR-BIorpreimmuneserumbeforeandduringthetimeofinfection(lowerpanel).Fortheseexperiments,
PTHwerepreincubatedfor1hwithanti-tupaiaSR-BIorpreimmuneserum(bothserumdilutionsof1:10)andplasma-derivedHCV(samples
andMOIasdescribedforpanelB)wasaddedtothecellculturemediumwithouttheremovalofnon-cell-boundanti-SR-BI.Mock-infectedPTH
servedasnegativecontrols.