Mutational analysis of the cysteine residues in the hepatitis B virus small envelope protein.

Copyright© 1993, AmericanSocietyforMicrobiology

Mutational Analysis of the Cysteine Residues in the Hepatitis

B

Virus

Small

Envelope Protein

CONSTANCE M. T. MANGOLD* AND ROLF E. STREECK

InstitutfiirMedizinischeMikrobiologie, Johannes Gutenberg-Universitat Mainz,

Augustusplatz, D-55131 Mainz, Germany

Received8February1993/Accepted10May 1993

The smallenvelopeproteinofhepatitisBvirus is the

major

componentof the viralcoatand is alsosecreted from cells as a 20-nm subviral particle, even in the absence of other viral proteins. Such

empty

envelope

particles are composed of

approximately

100 copies of this polypeptide and host-derived lipids and are stabilizedby extensive intermolecular disulfidecross-linking. To studythe contribution of disulfide bondsto

assembly

andsecretion of the viralenvelope, singleand multiplemutantsinvolving all 14 cysteines inHepG2

and COS-7cellswereanalyzed. Of the sixcysteineslocated outside theregion

carrying

the surfaceantigen, Cys-48,Cys-65,andCys-69wereeach foundtobe essential for secretion of 20-nmparticles, whereasCys-76, Cys-90,and Cys-221were dispensable. Byintroduction ofan additional cysteine substituting serine 58, the yield of secretedparticleswasincreased. Of fourmutantsinvolving the eight cysteines located in the antigenic region,

only

the double mutant lacking Cys.121 and Cys-124 was secreted with wild-type efficiency. Secretion-competentenvelopeproteins wereintracellularly retained bysecretion-deficient cysteine mutants. According to

alkylation

studies, both intracellularand secreted envelope proteins contained free sulfhydryl

groups.Disulfide-linked oligomerswerestudiedby gelelectrophoresisunder nonreducingconditions. The human hepatitis B virus (HBV) and the related

hepatotropic viruses discovered in woodchucks (WHV),

ground squirrels (GSHV), ducks (DHBV), and herons

(HHBV)areclassifiedasthefamilyofHepadnaviridae.The virion structures, genome sizes,and modes ofreplicationof

thesevirusesaresimilar. The prototype,HBV, has a diam-eterofapproximately42 nmwith a27-nmcore enclosing a 3.2-kb DNA genome. The lipid-containing envelope carries three closely related protein components. These

polypep-tides are synthesized by initiation of translation at three distinct in-frame AUG codons of the HBV S open reading

frame, givingrise tothe large, middle, and small envelope proteins, respectively. TheC-terminal 226 amino acids cor-respondingtothe small Sprotein containthemajor antigenic determinant,hepatitis Bsurfaceantigen (HBsAg),which is

exposedon the surface of viralparticles. (For reviews, see references 9and24.)

In addition to complete virions, noninfectious HBsAg

lipoprotein particlesin both filamentous andsphericalforms aresecreted from the livers ofpatientswith HBV infections. The spherical 20-nm particles, which are present in large

excess, arepredominantly composed ofapproximately 100

copiesof the small S protein and host-derivedlipids.

Cul-turedcellstransfectedwithvectorsencoding onlythe small

S

protein produce lipoprotein

particles virtually

identicalto the 20-nmparticles found in the blood of HBV carriers.All of the information needed for correct assembly of the

envelopeproteinswithlipids, particle formation, and secre-tionmusttherefore reside in thesmall Sprotein itself.

The Sproteinisinitially synthesized as atransmembrane

protein spanningthe membrane of the endoplasmic reticu-lum (ER) at least twice (6) and is partially glycosylated at

asparagine 146 (22). Secreted 20-nm particles contain both

unglycosylated P24 protein and its glycosylated derivate, GP27. Secreted GP27 carries a complex oligosaccharide

*

Corresponding

author.

chain,whereasonlythehigh-mannose form, whichisabout

1 kDasmaller, isdetectedintracellularly (18).

The Sproteinsin the secretedviraland subviral particles areextensivelycross-linkedby disulfide bridges(10). It has been demonstrated that covalent cross-linking occurs

con-comitantlywith assembly andbudding oflipoprotein

parti-cles(11), but it is unclear whether theformation of disulfide bonds is pivotal for this process. In the present study, we have usedmutant envelope proteinsto approach this ques-tion.

The small, middle, and large HBV envelope proteins contain cysteine residues exclusivelyinthe 226-amino-acid

sequence common to all three polypeptides. Of the 14

cysteines, 8 arelocated in ahydrophilic regioncarrying the surface antigen. Only three of the remainingcysteine resi-duesareconserved among allhepadnaviruses(7, 15, 17, 25,

26).Wehavenowcarried out asystematic analysis of single andmultiplemutants toidentify thecysteinesindispensable

forassemblyand secretion of 20-nm emptyenvelope

parti-cles.

MATERUILSANDMETHODS

Recombinant plasmids and site-directed mutagenesis. For

expressionof the HBV S gene(ayw),plasmid pNI2

carrying

the human metallothionein IIA promoter was used (13). After removal of the uniqueAccI site ofpNI2, the 1.8-kb

XhoI-BglII

fragment of the HBV genome (nucleotides [nt]

129 to 1986 [8]) was inserted into the polylinker of pNI2,

yielding plasmid pW. Plasmid pBAP contains amodified S genewitha36-bp insertionat ntposition303 andencodesa hybrid S protein, HBsPolioAg, carrying an 11-amino-acid insertioncorresponding toapoliovirus type 1 VP1 neutral-ization epitope (5). For epitope tagging, the 552-bp

XhoI-SpeI fragment ofpW (nt 129 to 681) was replaced by the

corresponding

588-bp fragment of pBAP; this construct is

designatedpWP.

For site-directed mutagenesis, we used bacteriophage 4588

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CYSTEINES IN THE HBV ENVELOPE 4589 M13-derivedrecombinants. HBVmpl9 and HBVmp8 carry

the 2.3-kb BglII-BglII fragment (nt 2839 to 1986) and the EcoRI-AccIfragment (nt 1 to 827) of wild-type HBV DNA, respectively. For mutagenesis of cysteines 107, 121, 124, 137, 138, 139, 147, and 149, which are located in the major antigenic determinant, the 734-bp XhoI-AccI fragment of pBAP was cloned into HBVmp8, thereby replacing the 698-bp XhoI-AccI wild-type sequence; the construct is des-ignated HBsPOLmp8. Site-directed mutagenesis was per-formed with the following antisense oligonucleotides:

Cys48Ala, * 5'-ATF11lGGCCTAGAGCCACGGTAGT-3'; Cys48Ser, *

5'-ATTTTGGCCTAGAGACACGGTAGT-3';

Ser58Cys, *

5'-GGTGAGTGATTACATGTTGGGGACTG-3'; His6OAla,

5'-AGGTTGGTGATGCATTGGA-3';

His6O Gln,

5'-ACAAGAGGTTGGTGAfJXATTGGA-3';

Cys65 Ala, *

5'-GTTGGAGGAGCAGACGTCGGTGAGTGATT

G-3'; Cys65Ser, *

5'-GTTGGAGGACTAGACGTCGGTGA

GTGATT-3'; Cys69Ala, *

5'-CAGCGATATCCAGGAGCA

GTTGGAG-3'; Cys69Ser, *

5'-CAGCGATATCCAGGACT

AGTTGGAG-3'; Cys65/69Ser, * 5'-CAGCGATATCCAGG

ACTAGTTGGAGGACTAGAGGTTGG-3'; Cys76Ala,

5'-C

GCCGCAGAGCCATCCAGC-3';

Cys76Tyr,5'-AACGCCG

CAGAIACATCCAGCG-3'; Cys9OAla, 5'-AGAAGATGAG

GGCTAGCAGCAGG-3'; Cys9OPhe,

* 5'-CAACAAGAAT

ATTAGGiAATAGCAGCAG-3';

CyslO7Ala, 5'-GAATTAG

AGGAGCAACGGGCAAC-3';

Cysl21/124Ala, 5'-CAGTA

GTCATGCjGGTCCGGGCTGGTCCCGTGC-3';

Cysl37/

138/139Ala, 5'-GTCCGAAGG TTGGTAGCGGCAGG

AGGGATACATAGA3'; Asnl46Gln, * 5'-GGAATACATG

TGCACTGTCCGTCCGAAG-3';

Cysl47/149Ala, 5'-GGAT

GGGAATAGCGGTGGCAl-TICCGTCCG-3';

Cys221Ala,

5'-ACCCAAAGAGCAAAGAAAAT-3'.

For designation of each mutation, the wild-type amino acid is indicated, and then itspositionandthemutantamino acid are listed. In the DNA sequences, the corresponding antisense codons are

underlined; lightface characters refer to thewild-type DNA sequence, and boldface characters refer to mismatches. Asterisks indicate theincorporationorremoval ofdiagnostic restrictionsites. Mutationsoutsidetheunderlinedsequences aregeneticallysilent.

Themutagenic oligonucleotideswere annealed to single-stranded DNAsobtained from the recombinant M13 phages described above. Mutants were isolated by the

phospho-rothioate method (16) asmarketedby Amershamand were screened directly by restriction site analysis or DNA se-quencing. Fragments containing a desired mutation were generated bycleavage withXbaIand AccIorwithXhoIand

SpeI andwereusedtoreplace thecorresponding wild-type

sequenceofplasmid pW;theexchanged regionwasverified

by DNAsequencing. Multiplemutationswereintroduced by combination ofappropriate fragments carrying single muta-tionsorbyrepeatingthemutagenesis withmutanttemplate

DNA.

Cell culture, transfection, and HBsAg detection. Human

hepatoma HepG2(14)orCOS-7cellswere seeded at -5 x

lm

per 6-cm dish in Dulbecco's modified Eagle's medium

plus 10% fetal calf serum 1 day prior to transfection with calciumphosphate precipitatesof DNA(16 ,ug) (29).HepG2 and COS-7 cellswereexposedtotheprecipitatesfor 20to24 h and for 8 to 14 h, respectively, washed twice, and then incubated with fresh Dulbecco'smodifiedEagle'smedium. Three days after transfection, the culture medium was collected. The cells were washed twice with

phosphate-buffered saline(PBS) andlysed byincubationoniceduring

15 minwith 1 ml of0.5% Nonidet P-40 in PBS. Thelysates were collected and vortexed three times for 20 s. Culture

medium and cellular lysates were clarified by centrifugation (10 min at 2,000 xg and 4°C in an Eppendorf Microfuge), andHBsAg reactivity wasdeterminedby anenzyme-linked immunosorbent assay (ELISA; Auszyme II ELISA; Ab-bott).

Density

and sedimentation

velocity analysis.

Mutant and wild-type HBsAgparticles secreted by transfected HepG2

cells were concentrated by centrifugation for 4 h at 45,000 rpm and 4°C in a Beckman SW60 rotor. Thepellets were resuspended, layered onaCsClorsucrosestepgradient (10 to50% [wt/wt]), and centrifuged for 18 h at 35,000 rpm in an SW60rotor. Gradientswerefractionated, and eachfraction was diluted and analyzed for the presence of HBsAg by ELISA; thedensities of CsCl and sucrose were determined byrefractometry.

Metabolic labeling, immunoprecipitation, and gel electro-phoresis. Isotopic labeling was started 36 to 48 h after transfection. Culture medium was removed and samples

weresubjected toELISA asdescribedabove.The cells were washed and incubated for 40 min in 1.5 mlof methionine-free

minimal essential medium with1% dialyzedfetal calfserum. Then, 375 ,uCi of [35S]methionine (>1,000

Ci/mmol;

New

EnglandNuclear)was added. Aftera pulse-labeling period

of 2 to 4 h, a chase was performed byreplacement of the medium with 2 ml of Dulbecco's modified Eagle's medium plus 10% fetalcalfserum,which containsexcess unlabeled methionine.Atthe end of the chaseperiod, the mediumwas collected; the cellswerewashed twice in TN buffer(0.1 M

Tris-HCl [pH

8.0]-0.1

M NaCl, with or without 20 mM

iodoacetamide), lysedwith 1 mlof 0.5%Nonidet P-40 in TN

buffer, and vortexed three times for 20 s. Except for the

experiments shown in Fig. 1 and 3A, B, and D, freshly prepared iodoacetamide (400 mM in water; Sigma) was added (1:20dilution) tothe TN buffer andto the collected culture medium. Forimmune precipitation, we usedrabbit antiserum to HBsAg particles from human serum (Calbio-chem) or ascitic fluid containing the monoclonal antibody C3, which recognizes the inserted poliovirus epitope. Ly-sates and mediawere cleared by centrifugation (10 min at 2,000xgand4°C inaMicrofuge) andwereincubatedonice

for 2 h with antibodies (1:100 dilution) in the presence of detergents(0.5% NonidetP-40,0.05%sodiumdeoxycholate,

and 0.01% sodium dodecyl sulfate [SDS]) and foran addi-tional hour with protein A-Sepharose CL-4B (Pharmacia),

with rotationat4°C. Immune complexeswerewashed three times in RIPA buffer(10 mM Tris-HCl [pH 7.5], 150 mM

NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS) andoncewith 0.1 MTris-HCl, pH6.8. Thiswashing procedure also removed excess iodoacetamide. Samples were prepared for SDS-12% polyacrylamide gel

electro-phoresis(PAGE)by the addition of Laemmli buffer with5% 2-mercaptoethanol and 2% SDS, boiling for 7 min, and incubationonicefor 15to30 min(standardconditions).To obtainnonreducingconditions, 2-mercaptoethanolwas omit-ted from thesamplebuffer. Thegelswerefixed, impregnated with Amplify (Amersham), and dried before exposure to a KodakXARSfilm at -70°C.

RESULTS

ThenonconservedcysteinesintheHBVenvelopeproteinare not essential for secretion of 20-nm particles. To

study

the contribution of the cysteine residues in the HBV small

envelope proteintoassembly andsecretion of 20-nm

parti-cles, mutant S genes (subtype

ayw)

were constructed

by

oligonucleotide-directed

mutagenesis.

Mutant genes were VOL.67,1993

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A. sw a b c d e f g h

27 _ ____

24 14151

12 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

B. a w C. a w

43.0-

29.0-18.4

1 2 3 4 1 2 3 4

D.

FIG. 1. Synthesis and secretion ofS proteins lacking

noncon-servedcysteines. COS-7cells transfected with plasmids encoding wild-typeormutant Sproteinswerepulse-labeledwith

[35S]methio-nine for 3 hand thenchasedfor 12(A),6(B),or18(C)h.Aliquots

of cellular lysates (odd-numbered lanes) or cellular supernatants (even-numbered lanes)weresubjectedtoimmunoprecipitationwith

anti-HBsAg antiserum followed by SDS-PAGE under standard

conditions.Arrowheads mark thepositionsofnonglycosylated (24)

andglycosylated (27) Sproteins.Themigrationofmarkerproteins (molecular masses in kilodaltons) is represented on the left (B).

Mutantandwild-type proteinsaredesignated bysmallletters(D).

expressed inCOS-7orHepG2cells fromthe human metal-lothionein IIA promoter by usingthe vectorpNI2 (13).

Initially, cysteine residues thatarenotconserved among

the mammalianhepadnaviruseswerestudied. WhereasHBV

carriesacysteineresidue at eachofthepositions 76, 90,and 221 of theSprotein,both WHVandGSHV havetyrosineat

position 76, leucine at position221, and an additional

cys-teine at position 58; cysteineis also found atposition 90of

the WHVSprotein,butphenylalanineisfound in GSHV(7, 25). To investigate whether the HBV S protein tolerates

similar variation in these positions, HBV Ser-58 was

re-placed by cysteine, Cys-76 was replaced by alanine or

tyrosine, Cys-90was replaced byalanineorphenylalanine,

and Cys-221wasreplaced byalanine.

Theamounts ofSproteinssecreted fromtransfectedcells weredeterminedbyELISA andappearedto beverysimilar

for the wild type, the sixsingle mutants, thedoublemutant

Cys76Ala;Cys9OPhe, and the triple mutant Cys76/221Ala; Cys9OPhe. Subsequently, the transiently expressed S

pro-teins were metabolically labeled, immunoprecipitated, and analyzed bySDS-PAGE(Fig. 1). Asshownbythe compar-ison with wild type (Fig. 1A, lanes 1 and 2), all mutants displayed the same pattern of glycosylated (27-kDa) and nonglycosylated (24-kDa) proteinboth inlysatesandcellular supernatants(Fig. 1A,lanes 3 to 18).TheSer58Cysmutant (lanes a) seemed to have a higher electrophoretic mobility

andto besecretedfasterthan wildtype.This isshownmore

clearlyinFig. 1BandCbyapulse-chase experimentwitha

short(Fig. 1B)and long (Fig. 1C)chase.The other mutants (lanes b to h) were synthesized and secreted like the wild type,consistent with theELISA data.

The finding that all mutants were easily detected by ELISA indicates that the structure of the major antigenic determinantwasnotgreatly disturbed. Moreover,

sucrose-and cesium chloride-gradient analyses (data not shown) revealedthat the secretedproteins formed particleswith the size and buoyant density (1.2 g/ml) characteristic of wild-type 20-nmlipoproteinparticles.

These results demonstrate that cysteines 76, 90, and 221

are dispensable for assembly and secretion of 20-nmHBV lipoprotein particles andsuggestthatan additionalcysteine

atposition58 enhances theefficiencyofsecretion.

Contribution of nonessential cysteines to disulfide

cross-linkingof HBVenvelope proteins.Toinvestigate whether the degreeof intermolecular disulfidecross-linkingwasaffected

by mutation of the nonessential cysteines, wild-type and mutant S proteins were compared by SDS-PAGE under reducingandnonreducingconditions (Fig. 2). Cell lysisand immunoprecipitation were performed in the presence of

iodoacetamidetoprevent the formation of disulfide bondsin vitro. Ser58Cys, the triplemutant Cys76/221Ala;Cys9OPhe, and the followingtwo additional mutantswere includedin this study: a sextuplemutant (i), whichwasobtained from

thetriplemutantbyreplacing cysteines 48, 65,and 69, and the mutantAsnl46Gln(v).Whereasthesextuplemutantwas

deficient for secretion (Fig. 2A, lanes 10 and 11), the nonglycosylated mutantAsnl46Glnwas secreted, although

slightlylessefficientlythanthe wild type(Fig. 2A,lanes 4to 7).

Underreducingconditions, the total amountof immuno-precipitated- S proteins migrates exclusively as monomers

(Fig. 2A). Whenthesameamountofproteinswasanalyzed

undernonreducingconditions, onlydimersandhigher-order oligomerswerefound(Fig. 2B).Three bandsweredetected

in the dimer region (lanes 7 and 9), two of which could tentativelybe ascribed to homodimers ofglycosylated (27/ 27)andnonglycosylated (24/24)Sprotein bycomparingthe nonglycosylatedmutant(Fig. 2B,lane5)with the wild type (Fig. 2B,lane7). Theintermediatebandmaycorrespondto heterodimers (24/27) and/oranalternative form ofthe24/24 homodimer(Fig. 2B, lane5). Inaddition, a diffuse band in

the80- to 90-kDaregion(Fig. 2B, lanes 5, 7, and9) which might represent tetrameric forms was observed.

Higher-orderoligomers apparentlydid not enterthegel. (Notethe relatively strong signalsin theupperpart andontopof the gel,forexamplein lane13 ofFig. 2B.) By comparisonwith cells transfected with the vector pNI2 lacking the S gene

(Fig. 2B,lane2),dimerswerealsodetected in lysatesofcells synthesizingSproteins (even-numbered lanes).

Todemonstrate thatoligomerizationwas duetodisulfide bonding, mutant and wild-type S proteins were analyzed

undermildly reducingconditions (Fig. 2C). Inthepresence

of 1 mM dithiothreitol, both monomers and dimers were detectable(lanes1 to12).Whentheconcentration of dithio-threitolwasgraduallyraisedfrom 0 to30 mM,the

conver-sion ofoligomersinto dimers andofdimers intomonomers wasobserved,as shownfor theSer58Cys mutant(Fig. 2C, lanes 13 to 18).

The finding that the secretion-deficient sextuple mutant formeddimers(Fig. 2B,lane10)indicatesthat theremaining eight cysteines located in the major antigenic region are

involved in dimerization. Both dimers and higher-order oligomers were detected for the triple mutant (Fig. 2B,

mutant h). However, the amounts of dimers under

nonre-ducingconditions (Fig. 2B, lanes 6 to 9) and ofmonomers under mildly reducing conditions (Fig. 2C, lanes 4 to 7) appeared to beslightlylargerforthe triplemutantthan for

HBVsmall Sproteinsanalyzed

a Ser58Cys b Cys76Ala

c Cys9OAla d Cys221 Ala e Cys76Tyr

f Cys9OPhe

g Cys76Ala;Cys9OPhe

h Cys76/221 Ala;Cys9OPhe

w wild-type

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[image:3.612.59.298.73.296.2]

CYSTEINES IN THE HBV ENVELOPE 4591 REDUCING

M u v w h a

m. m _- wm m m

B. NONREDUCING

M u v w h a

m rn m m r---m m

200.00u-54!!| | | l l 4-mer[ 68.0-- w ss

43.0- * 27/27

24/27-

24/24-ea.e_

_

_ 0b

_m

w

a,"~-18.4- S

1 2 3 4 5 6 7 8 9 10 11 12 13

w

1 2 3 4 5 6 7 8 9 10 11 1213

1 mM DTT 0 .2 1 5 30 mM DTT

u v w h a a w

s.In

.S.k ". - 0,"_..

4Imer

I

4-er[ i - s 3 3-mer

I

... * *

2-mer[ - 410.|lt tv

.~~

29.0-27_

24_- ..61.

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

FIG. 2. Analysisofoligomerizationof mutantSproteins.COS-7cells weretransfected withplasmidsencodingwild-type (w)ormutant

Sproteins (a, h, i,andv)orwith thevectorpNI2 (u).[35S]methionine-labeledproteins (3-h pulse,12-hchase)wereimmunoprecipitatedand

analyzed bySDS-PAGEinthepresenceof 5%2-mercaptoethanol (A),in the absence ofareducing agent (B),and in thepresenceof various

concentrations ofdithiothreitol (DTT) (C). Molecularmasses(in kilodaltons) of 14C-labeled markerproteins (M) (GIBCO-BRL) and the

positionsofnonglycosylated (24)andglycosylated (27)monomers(arrowheads)and ofputative oligomersofSproteinsareindicatedonthe

left of theautoradiograms. 24/24, 27/27,and24/27markpresumptivehomo- and heterodimers(B). (AandB)Even-numbered lanes,cellular

lysates;odd-numberedlanes,cellularsupernatants;(C)lanes1, 2, 4, 6, 8,and10,lysates;otherlanes,supernatants.In lanes 12 to17,only

one-third of the normalamountof cellularsupernatantwasused forimmunoprecipitation.

the wild type, although virtuallyidentical amounts of these proteins were immunoprecipitated (Fig. 2A, lanes 6 to 9). These results suggestthatcysteines 76, 90, and/or221may

be involved in intermoleculardisulfide bridges.

Asurprisinglysmallamountof dimers andlargeamount of high-molecular-weightmaterial werefound when Ser58Cys

was analyzed (compare lanes 13 of Fig. 2A and B). This indicates that the additionalcysteinepresentin this mutant favors the formation ofhigher-order oligomers. Curiously, both the glycosylated and nonglycosylated forms of Ser58Cyswere splitinto two bands at 30 mM dithiothreitol (Fig. 2C, lane 17). Thiswas also observed with 5% 2-mer-captoethanol (Fig. 2A, lane 13),but not when the proteins

were notalkylated (Fig. 1,mutant a).

Cysteines 48, 65, and 69 and histidine 60 in the HBV S protein are essential for secretion of 20-nm particles. The cysteinesatpositions 48, 65,and 69of theHBVSproteinare

strictlyconservedamong allhepadnaviruses (7, 17, 25, 26). Tostudytheir role in the assemblyand secretion of 20-nm particles, these residues were replaced by alanine and serine. Single and multiple mutants were constructed, in-cludingthetriplemutants(gandq), aquadruplemutant (h) carryinganadditional cysteine76substitution, anda

sextu-ple mutant (i) in which cysteines 90 and 221 were also

replaced (cf. Fig. 3). As shown in Fig. 3A through C, all

mutantswere clearlydetectable in cellular lysatesof

trans-fected COS-7 cells but not in the cell culture media. To

analyzewhether theblock to secretioncould be reversedby A.

29.0

-27

_-24

_-C.

HBVsmall S proteinsanalyzed

a Ser58Cys

h Cys76/221Ala;Cys9OPhe Cys48/65/69/76/221Ala;

Cys9OPhe

u noS proteins

v Asnl46Gln

w wild-type VOL. 67,1993

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4592 MANGOLD AND STREECK

A. M a b c d e f

43.0-

v

*'

._.i

29.0

-27

P-24_ 18.4

-0 ...*...".,

1 2 3 4 5 6 7 8 9 10 11 12 13

C. k w m n o p q

_ 2 3 4x

1 2 3 4 5 6 7 8 9 10 111213141516

B. M g h w

D. u w .

_m '

4 ^,

1 2 3 4 5 6 7 8 91

E. a b c g

.

y--1-0r

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12345 6 7 8 9 10

FIG. 3. Analysisof Sproteins lackingconservedcysteines..COS-7cellstransfected with the vectorpNI2 (u)orwithplasmids encoding wild-type (w) or mutant S proteins (ato t) were pulse-labeled with [35S]methionine for 3 h and then chased for 20 h (A through D). Immunoprecipitated proteinsand markerproteins (M)weresubjectedtoSDS-PAGEunder standard conditions. Molecularmassesof marker

proteins (in kilodaltons) and thepositionsofglycosylated (27) andnonglycosylated (24) S proteins (arrowheads)areindicated. (Aand B) Even-numberedlanes,cellularlysates;odd-numberedlanes,cellular supernatants; (CandD)odd-numberedlanes,lysates;even-numbered

lanes,supernatants; (E)cellularlysatesafter3-h chase(odd-numbered lanes)or20-h chase(even-numbered lanes).

the introduction ofa cysteine residue at another position, Ser-58 was replaced by cysteine in the secretion-deficient single mutants (Fig. 3, mutants r, s, and t). However, no

effect of the Ser58Cyssubstitution onthesecretory pheno-typeof anyof these mutantscould be observed (Fig. 3D). Thisclearlydemonstrates that each of thecysteines 48, 65, and 69 is indispensablefor secretionofSprotein.

For comparison, histidine 60, which is also conserved

among allhepadnaviruses, wasmutatedtoeitherglutamine

oralanine. As shown inFig.3C(lanes1to4),noneofthese mutants was secreted, indicating that His-60 must be in-cluded amongtheessential amino acidsof the Sprotein.

Although the cysteine and histidine mutants described abovewerenotsecreted, theywerestillglycosylatedtothe

sameextentasthewild-typeprotein (Fig. 3).This indicates

thattranslocation across the membraneof the ERwasnot noticeablyaffected.Accordingtotheelectrophoretic mobil-ities of themutantproteins, theircarbohydratechainswere

not converted to the complex form found in secreted S proteins (see,e.g.,Fig. 3C,lanes 5 and6).Therefore, these mutantsweremostlikelyarrested priortothe medialGolgi compartment. Since allmutantswere also detectable in the cellularlysates byELISA(datanotshown),thestructureof the major antigenic determinant was probably not greatly disturbedbythemutations.

Toinvestigatewhether the differentamountsofSproteins found for some of the mutants should be attributed to differences in stability, pulse-chase experiments were

car-riedout (Fig. 3E).When the amountsofprotein incellular lysateswerecomparedaftera3-h chase(lanes 1, 3, 5, 7,and

9)anda20-h chase(lanes 2, 4, 6, 8,and10),adecreasewas

detectable. Thesinglemutants(a, b,andc)seemedtobe less stablethan the tripleand sextuplemutants.

Disulfide cross-linkingofsecretion-deficient mutant S

pro-teins. To analyze whether substitution of essential amino acids in the HBV S protein noticeablyaffects thedegree of

a Cys48AIa m Cys48Ser

b Cys65AIa n Cys65Ser

c Cys69AIa 0 Cys69Ser

d Cys48/65AIa p Cys65/69Ser

e Cys48/69AIa q Cys48/65/69Ser f Cys65/69AIa r Cys48AIa;Ser58Cys g Cys48/65/69Ala s Cys65AIa;Ser58Cys h Cys48/65/69/76Ala t Cys69AIa;Ser58Cys

Cys48/65/69f76/221Ala;Cys9OPhe

k His6OGln u no S proteins I His6OAla w wild-type

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CYSTEINES IN THE HBV ENVELOPE 4593 NONREDUCING

k w m n o p q u M

s:PX

4 64

bg

a=s I% fi

27 -24~

1 2 3 4 5 6 7 8 9 10 11

FIG. 4. Disulfide cross-linking of secretion-deficient mutant S proteins. [35S]methionine-labeled proteins (3-h pulse, 20-h chase) wereimmunoprecipitated from COS-7 cells transfected with plas-mids encoding wild-type (w) or mutant S proteins (k to q) or with the vectorpNI2(u). Precipitated proteins and marker proteins (M) were subjected to SDS-PAGE under nonreducing conditions. The corre-sponding reducing gel is shown in Fig. 3C, which also specifies the mutations introduced. The positions of S monomers (P24 and GP27) and dimersareindicatedontheleft. Lanes1to 3 and 5 to10, cellular lysates;lane4, cellular supernatant; lane 11, markerproteins.

intermolecular disulfide cross-linking, SDS-PAGE was per-formedunder nonreducingconditions. The resultsobtained forfive mutants in which cysteines 48, 65, and/or 69 were replaced by serine and for thetwo secretion-deficientHis-60

mutantsareshown inFig.4; thecorrespondingreducing gel is shown in Fig. 3C. Small amounts of monomers were

detected for all mutants, and the signals obtained in the

dimer region were equally faint for the wild type and the

single and double mutants (Fig. 4, lanes 1 to 8). This indicates that the majority of polypeptides formed larger

disulfide-linked complexes. In contrast, the triple mutant accumulated in a dimeric form (Fig. 4, lane 9), as was

previously observed for the sextuple mutant(Fig. 2B, lane

10).

Interaction between secretion-deficient and secretion-com-petent S proteins.We next investigated whether the

secre-tion-deficientmutantsassociatewith thewild-typeSprotein

and whether this leads to cosecretion of the mutant or to intracellular retention of the wild type. To be able to

distinguishbetweenthe wildtype and mutants, we initially

chose aswild type thenonglycosylatedmutantAsnl46Gln,

which isefficientlysecreted(Fig.2A,lanes4to7) and forms

20-nm lipoprotein particles (data not shown). When the mutant genes were cotransfected with the phenotypically wild-type gene (v) intoCOS-7 cells at a molar ratio of3:1,

secretion ofAsnl46Glnwas

drastically

reduced(Fig. 5).No

glycosylated S protein could be detected in cellular super-natants, with the exception of

Cys48Ala;Ser58Cys,

which wascosecreted in atinyamount(Fig.5, lane15). Curiously,

the secreted GP27 form of this mutant was split into two bands onelectrophoresis, as wasobserved for the

Ser58Cys

mutant.

Todemonstratemoreclearlyspecificinteraction between

wild-typeand mutant Sproteins, epitopetaggingof the wild

M v v+a v+b v+c v+g v+i v+r u

29.0O- i l l

27

1-

24-18.4-_

-

X

4

a

[image:6.612.331.558.72.177.2]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 FIG. 5. Intracellular retention ofasecretion-competentS protein bysecretion-deficientmutants.COS-7cellsweretransfected witha 1:3mixture of the secretion-competent Asnl46Glnmutant (v) and one of the following secretion-deficient mutants: Cys48Ala (a), Cys65Ala (b), Cys69Ala (c), Cys48/65/69Ala (g), Cys48/65/69/76/ 221Ala;C'ys9OPhe(i), andCys48Ala;Ser58Cys(r). In the control (v), pNI2 DNAwasused instead ofmutant DNA.Cellstransfected with thevectorpNI2 alone (u)wereusedfor lanes 16and 17.

[35S]me-thionine-labeled proteins (4-hpulse, 23-h chase)were immunopre-cipitated and analyzed by SDS-PAGE under standard conditions. Molecularmasses(in kilodaltons) of marker proteins(M) and the positions ofglycosylated (27) and nonglycosylated (24) S proteins (arrowheads)areindicated.Even-numberedlanes, cellularlysates; odd-numbered lanes,cellular supernatants.

type was used. In this approach, cysteine mutants were

coexpressed withamodified Sgeneencoding the

secretion-competent, hybrid protein HBsPolioAg. This polypeptide carriesan11-amino-acidpoliovirus epitope inserted between theduplicated glycine 50 residue ofthe HBVsmall Sprotein (5). Immunoprecipitation was performed by using either HBsAg-specificantiserum(Fig.6A andC)orthe poliovirus-specific monoclonal antibody C3(Fig. 6B andD). Cysteine

mutants, but not the wild-type S protein (w), reduced the

efficiency ofsecretion of the hybrid protein (wp).

Surpris-ingly, inhibition ofsecretion ofHBsPolioAg was less pro-nounced thanthat observed for theAsnl46Gln protein (Fig. 5). Inaddition, smallamountsofsecretion-deficient mutant

proteinsweresecreted under these conditions,as seenmost

clearly for

Cys48Ala;SerS8Cys

(Fig. 6A [wp+r, revealing

twobandsfor P24]). With the antibody C3,

coimmunopre-cipitation of the cysteine mutants with HBsPolioAg was found (see P24 in Fig. 6B and D), indicative of a stable

associationbetween these proteins. Theefficient

coprecipi-tation obtained from thecellularsupernatantssuggests that all of the secreted mutantpolypeptideswereassembled with thehybridprotein into mixed particles (compareP24inFig.

6Awith P24inFig. 6B,e.g., lanes6 or16).

Taken together, these results demonstrate that the

perti-nent cysteine mutations do not prevent association with either of the two phenotypically wild-type proteins (v and

wp). The interaction leads to an almost complete block to

secretion for the Asnl46Gln protein; the hybrid protein HBsPolioAg is retained to a lower extent and enables

inefficient secretion of thecysteinemutants.

Contribution ofcysteines located in the antigenicregionto

oligomerization

and secretion ofHBV S proteins. Of the 14 cysteine residues in the HBV Sprotein, 8 arelocated in a hydrophilic region carryingthemajorantigenic determinant, HBsAg,which is aconformationalepitope(2). Substitution

ofcysteineswas therefore carriedoutin the

hybrid protein

HBsPolioAg, which makes

immunoprecipitation

with the monoclonal antibodyC3 possible, asdescribed above. The

following mutants were constructed:

CyslO7Ala, Cys121/

124Ala, Cysl37/138/139Ala,

and

Cysl47/149Ala.

VOL.67, 1993

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[image:6.612.109.260.74.273.2]

A. wp wp+w wp+a wp+b wp+c wp+g wp+i wp+r u

w

wpw

wp+

wp+

wp+

wp+ wp+

wp+

u 28

25

18.4-B. wp wp+w wp+a wp±bwp+cwp+g wp+qwpr u

2430

C. w

wpw

1wp

wp

wp*

wp+

wp+ u

4.0

-18.4 P;rS;

D. wp wp+w wp+m wp±n wp+o wp+p wp±q u

I--I-- '- '1 1 --- l ----'

43.0

X

,.'WA.sAhX* ,

18.4

[image:7.612.59.296.71.485.2]

-1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

FIG. 6. Interaction betweensecretion-deficientcysteinemutants

andthesecretion-competenthybridprotein HBsPolioAg. Wild-type (w) or secretion-deficient S proteins (a to r) were synthesized

togetherwiththesecretion-competent hybrid protein HBsPolioAg (wp)inCOS-7cellscotransfectedwiththecorresponding plasmids

mixedatamolarratioof3:1. Ascontrol,cellsweretransfectedwith thevectorpNI2alone(u).Thesecretion-deficientsingle (a, b,c, m,

n,ando), double (pandr,triple (gandq),andsextuple (i)mutants

arespecifiedin Fig.3. [3S]methionine-labeledproteins (4-h pulse,

23-h chase) were immunoprecipitated with HBsAg-specific

anti-serum (Aand C)orwith themonoclonal antibody C3 (B and D).

Precipitated proteinswereanalyzed by SDS-PAGE under standard conditions.Themigrationof markerproteins(molecularmasses in

kilodaltons)isrepresentedontheleft. Thepositions ofglycosylated

(28) and nonglycosylated (25) HBsPolioAg (wp) are indicated by

arrows,and thepositions of S proteins (P24 and GP27)areindicated byarrowheads.

Asexpected, none of thesemutants could be detected in supernatantsorlysatesoftransfected COS-7cells by ELISA with a mixture of HBsAg-specific monoclonal antibodies, indicating that the conformation of the antigenic loop was

disturbedby themutations.Asshown in Fig. 7, themutant

S proteins could be immunoprecipitated with rabbit

poly-clonal antiserum to HBsAg particles (Fig. 7B), but more efficiently so with C3 antibodies (Fig. 7E). The double mutant Cysl21/124Ala (bp) was synthesized, glycosylated, andsecreted like the corresponding wild-type protein (wp), which indicates that thesecysteines are not required for the secretion of S proteins. All other mutants were rather poorly secreted (Fig. 7E [ap, cp, and dp]). Interestingly, Cysl47/

149Ala, which had been mutated right next to the carbohy-drateattachment site, Asn-146, was glycosylated to a higher extent than thewild-type protein (Fig. 7 [dp]).

To study the interaction between each of these mutants and the wild-type S protein (w), the corresponding genes were coexpressed in COS-7 cells (Fig. 7C and F). With HBsAg-specific antibodies, partial retention of the wild type (w) was found (Fig. 7C). Moreover, coimmunoprecipitation ofwild-type S proteins was obtained with C3 antibodies (Fig. 7F [P24]), indicative of the formation of mixed protein aggregates between wild-type and mutant S proteins.

Finally, the mutants were analyzed by SDS-PAGE under nonreducing conditions (Fig. 7D). Dimers were found in the cellular lysates for the mutants CyslO7Ala, Cysl21/124Ala, andCysl47/149Ala (ap, bp, and dp) in amounts larger than those for the wild type (wp). Putative multimers were also detected on top of the gel and in secreted Cysl21/124Ala

particles.

Free sulfhydryl groups are present in HBV envelope pro-teins. In the course of this study, we observed that the electrophoretic mobilities of S proteins were reduced through addition of the alkylating agent iodoacetamide dur-ing cell lysis. This agent reacts with free sulfhydryl groups and was used toprevent the formation of disulfide bonds in vitro. Since excess iodoacetamide was removed upon wash-ing of the immune precipitates, the observed shifts in elec-trophoretic mobility upon alkylation should reflect the pres-ence ofcysteines carrying free SH groups at the moment of cell lysis (Fig. 8). Theretardation observed in this analysis was particularly strong for mutants carrying the Ser58Cys

mutation (Fig. 8B, lanes 1 to 4). A minor shift was noticed for thewild-type protein (Fig. 8B, lanes 5 and 6). However, alkylation did not affect migration of the sextuple mutant in which thecysteines 48, 65, 69, 76, 90, and 221 were replaced byeither alanine or phenylalanine (Fig. 8C, lanes 1 and 2). Thisfinding suggests that the remaining eight cysteines in the antigenic region are all disulfide linked. Moreover, it con-firms that the changes in electrophoretic behavior observed were not caused by alkylation of residues other than cys-teines. The corresponding triple mutants, lacking either cysteines 48, 65, and 69 or 76, 90, and 221,respectively, both exhibited a small shift in electrophoretic mobility on alkyla-tion, indicating the presence of free SH groups (Fig. 8C, lanes 5 to 8). Mostnoticeably, alkylation of secreted 20-nm particles also changed the electrophoretic mobility of the wild-type protein (Fig. 8D, lanes 3 and 4). These results strongly suggest that both intracellular and secreted wild-type S proteins contain one or more cysteines that are not involved in disulfide bonding.

DISCUSSION

This paper presents the results of anextensive mutational analysis of the 14 cysteines in the small envelope protein of HBV, subtype ayw. These residues are strictly conserved among all HBV subtypes known (17). To study their contri-bution to the assembly and secretion of 20-nm HBsAg lipoprotein particles, each cysteine was replaced by alanine. Alanine was chosen to minimize secondary effects on the

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CYSTEINES IN THE HBV ENVELOPE 4595

A. - Hybrid proteins analyzed

ap Cysl07AIa

bp Cysl2l/124Ala

cp Cysl37/138/139AIa

dp Cysl47/149AIa

wp wild-type HBsPolioAg

D. M wp ap bp cp dp u

m| rm-mmm----r---i 1l X

4-**a _ ^

B. wp ap bp cp dp

mm

m*

28-. a*

25 W 4

18.4- 440_t - -*

1 2 3 4 5 6 7 8 9 10

C. w+wpw+ap w+bpw+cp w+dp

,, r- r- I

27 _. We z

24_. _ _ _ w.w

18.4

-1 2 3 4 5 6 7 8 9 10

.iw

w

I

il,

U)

w

I'll

I

E

29.0-1 2 3 4 5 6 7 8 9 10 11 1213

E.

43.0-

29.0-wp ap bp cp dp

r---

'

rII

_|fi~~~~~t

Pa....

18.4

-..*.

1 2 3 4

43.0

-18.4

-5 6 7 8 9 10

F. w+wpw+ap w+bp w+cp w+dp

m--- m---i m m m

ce

1 2 3 4

_

-+_

5. 8

5 6 7 8 9 10

FIG. 7. Analysis ofhybridSproteins lacking cysteines in the antigenic region. COS-7 cells were transfected with plasmids encoding the wild-type(wp) or one of the mutant hybrid proteins (ap to dp) (A) or with 3:1 mixtures of plasmids encoding a mutant and the wild-type S protein (w).[35S]methionine-labeledproteins (4-hpulse,20-hchase) wereimmunoprecipitated with rabbit antiserum against HBsAg particles (B and C) or with the monoclonal antibody C3 (D, E, and F). Precipitated proteins were subjected to SDS-PAGE under reducing (B, C, E, andF)ornonreducing conditions (D). The positions of glycosylated (28) and nonglycosylated (25) hybrid proteins are marked by arrows, and thepositions of wild-type S proteins (P24 and GP27) are marked by arrowheads. Migration of marker proteins is represented on the left; molecular masses are in kilodaltons. (B, C, E, and F) Odd-numbered lanes, cellularlysates; even-numbered lanes, cellularsupernatants;(D) even-numbered lanes,lysates;odd-numberedlanes,supernatants;lanes 12 and 13,control cellstransfected with the vector pNI2 (u). The positions of dimersareindicated.

proteinstructure (4), but some mutantsintroducing serine,

phenylalanine,ortyrosinewere alsoconstructed. Sincethe HBV middle and large envelope proteins carry no other

cysteinesbutthose present in the smallprotein,someofthe

resultsobtained for20-nmparticles shouldalso bepertinent

tothe assembly of whole virions.

Contribution ofcysteinestosecretionand

antigenicity

ofthe HBVenvelopeprotein. Each ofthecysteinesatpositions48,

65, and 69 and histidine at position 60 were shown to be

indispensable for secretionof HBVsubviral particles.This is

consistent with the observation that these residues are

strictly conservedamong allhepadnaviruses (7, 17,25, 26), and it also explains why deletion mutants involving this

region of the S protein are secretion defective (23). In contrast,thenonconservedcysteinesatpositions76, 90, and 221 are completely dispensable, since neither single nor

multiple mutations

noticeably

affected the efficiency of

se-cretion of 20-nmlipoprotein particles. The SerS8Cys

muta-tion is perfectly tolerated, but an HBV subtype carrying cysteine 58 has not yet been found (17). It would be

interestingtoanalyzethe effect of theSer58Cys mutationon theassemblyandsecretionof thecompleteHBVvirion and of thehepatitis deltavirus (3).

Theeight cysteines located in the antigenic region were

studiedbyusing the

hybrid

Sprotein HBsPolioAgtoensure immunologic detection after mutagenesis of the HBV sur-face antigen. The additional 12 amino acids of poliovirus capsid protein VP1 inserted at position 50 of the HBV S

protein contain no cysteine residues and do not affect the

efficiency of secretion or the HBs antigenicity (5). The double mutant Cysl21/124Ala was secreted like the wild type,as

previously

reportedfor thesinglemutant

Cysl21Ser

(1). Since the mutants

Cysl07Ala,

Cysl37/138/139Ala,

and

Cysl47/149Alawereratherpoorly secreted, atleast someof

thesixcysteinesinvolved shouldplay a role in the secretion process. Substitution of most of the antigenic region was

previously reportedtobe compatiblewithparticle assembly and secretion. Von Brunn et al. obtained hybrid 20-nm

particleswhen amino acids114 to 156 of the Sproteinwere

replaced byPlasmodium falciparum merozoite

gp190-spe-cificsequences(28). Moreover,the envelopeproteinsof the

avianHBVscompletelylack thesequencecorrespondingto HBV amino acids 105 to 155 (15, 27). This indicates that

assemblyandsecretion ofemptyenvelopeparticlesmay be

achievedwithoutdisulfidebonds involvingcysteinesof the

antigenic determinant. Itshould benoted, however, that the

morphologyof avian viralparticles is atypical(24).

The four mutants carrying cysteine substitutions within theantigenic regionreactedonlymarginallyif at all with the mixture of HBsAg-specific monoclonal antibodies used in theimmunoassay. Similarresults had beenobtainedfor the

singlemutants

Cysl24Ser

and Cysl47Ser (2), whereas the

antigenic reactivity of

Cysl21Ser

waseithergreatly reduced

or enhanced,

depending

onthe monoclonal antibody used (1).The structureof themajorantigenic determinantis most

likely disturbedby theeliminationofdisulfidebonds. How-ever, authentic folding of the antigenic region is not a

prerequisite for secretion of the S protein, since both

Cysl21Ser (1)

and

Cysl21/124Ala

were

efficiently

secreted.

Itisnoteworthythat mutantslackingcysteines48, 65,69, 76,

90, and/or 221 were easily detected by ELISA, which suggests that these residues do not interact withcysteinesin theantigenic region.

Mutationofcysteines 121and 124 shouldpresenta selec-tive advantage to HBV in vivo, helping to evade the host immune system. For in vitro studies, the mutant

Cysl2l/

124Ala could be useful for the selection of monoclonal antibodiesrecognizingother

epitopes

in theS

protein,

e.g., VOL.67,1993

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[image:8.612.78.559.74.281.2]

4596 MANGOLD AND STREECK

A.

B.

HBV small S proteinsanalyzed

a Cys48Ala;Ser58Cys b Ser58Cys

c Cys48/65/69/76/221 Ala;

Cys9OPhe 27

m-d Cys48/65/69AIa

e Cys76/221Ala;Cys9OPhe 24 >, w wild-type

C. c w d e

,, , m m,

Alkylation - + - + - + - +

29

0--a

_k*

a

~~~~~~~~~~~~~~~~~~ip

b

w

.r-* i

,, ;_,. . '

Ai& 'p", a

_ _

1 2 3 4 5 6

D.

w

Al l:..

'..

.-_ f:.

_ 4sos

*_ _

18.4

[image:9.612.61.295.74.413.2]

-1 2 3 4 5 6 7 8 1 2 3 4

FIG. 8. Analysis of free sulfhydrylgroupsin Sproteins.

Dupli-cate cultures ofHepG2 cells

synthesizing

wild-type or mutant S proteins (A)werelabeled with[ S]methionine for2h, chased for 6 h,andsubsequently lysed in thepresence(+)orabsence(-) of the alkylatingagent iodoacetamide.Proteinswereimmunoprecipitated

from the lysates (B and C) and analyzed by SDS-PAGE under standardconditions. (D)Immunoprecipitationofthewild-type (w)

fromlysate (lanes 1 and2) and supernatant(lanes3 and4),half of

which was treated with iodoacetamide (+). Migration of marker proteins is represented on the left (C); molecular masses are in

kilodaltons. Arrowheadsmarkthepositionsof thenonglycosylated (24)andglycosylated (27)S protein.

to improve detection in clinical diagnosis. Finally, these mutantsmightbeusedtofurthercharacterize the role of the various cysteines for the subtype-specific determinants of HBV.

Disulfide cross-linking of envelope proteins. It has been reported that the approximately 100 S protein molecules presentinHBsAg particles areextensively cross-linked by

disulfide bonds (10, 11) and do not contain palmitylated cysteine residues (20) or free sulfhydryl groups (10). In

contrast, the results of alkylation experiments presented heresuggestthepresenceoffree sulfhydrylgroups, evenin

secretedparticles. The disulfide-bonding pattern within the 20-nm particles seems to be heterogeneous, since both oligomersandpolymersweredetectedby gel electrophore-sisunder nonreducing conditions.The existence of hetero-geneityis alsosuggested bytheelectrophoreticbehavior of the SerS8Cys and Cys48Ala;Ser58Cys mutants, showing doublebandsunderreducingconditions.

FIG. 9. Secondary-structure model for the HBV S protein. A secondary-structuremodel(adaptedfromreference27)divides the HBV Sprotein into hydrophobic regions that interact with lipids (shadedarea) andmorehydrophilic regions. The numbers indicate thepositions of the cysteines in the protein. Cysteines in the first hydrophilic loop are initially located in the cytoplasm, whereas cysteines in the second hydrophilic loop carrying the major anti-genicdeterminant, HBsAg,arepositioned in the lumen of theER. After assemblyand secretion of Sproteins, the antigenic loopis exposedonthe surface of(sub)viral lipoprotein particles(outside), while the firstloop isproposedtobe locatedinside theparticles.

Role of cysteines in intracellular transport and

assembly

of S proteins. A secondary-structure model for the S protein

(adaptedfrom reference27) is presented in Fig. 9. According tothismodel, specifichydrophobicregions in the S protein interact with lipids and separate two major hydrophilic

regions. Wheninitially synthesized as transmembrane

pro-tein,the secondhydrophilicloop of the Sproteinis translo-cated into the lumen oftheER, whereas the first hydrophilic loop carrying cysteines 48, 65, 69, and 76 remains in the

cytoplasm(6, 9).Assuggested bythepartialglycosylationof thewild-type protein and all mutants studied, the glycosy-lation attachment site asparagine 146 located in the second hydrophilic loop is only partially accessible. This may partly be duetodisulfide bondsinvolvingcysteines 147 and/or 149, since thedoublemutantCysl47/149Ala was more efficiently glycosylated than the wild type. As demonstrated by the

secretion-competent phenotype of the Asn146Gln mutant and aspreviously shown by tunicamycin experiments (18),

glycosylationofthe S protein is notessential. However, the

nonglycosylated mutant was secreted somewhat less

effi-ciently than the wild type. Glycosylation may speed up secretion by preventing the formation of wrong disulfide bonds.

Recently, evidencehas been obtained thatdisulfide-linked HBVS dimers,whichare formed early in the ER, are first sortedto apost-ER, pre-Golgicompartment andthen con-verted slowly into high-molecular-weight, disulfide-linked oligomers (11). Sincenoneofourcysteinemutants accumu-lated inamonomeric form, proper folding of the monomeric S protein is not a prerequisite for dimerization. Previous studies with other secretoryproteins have shown that aber-rantprotein foldinggenerally causes ER retention (12, 19). Failure of furtheroligomerizationof mutant S proteins may therefore be duetomisfoldingofthedimers.Thefinding that eventhesextuplemutant still formed dimers indicates that disulfidebondinginvolved one or more of the eightcysteines in theantigenic region. Thisconclusion isconsistent with the

secondary-structuremodel(Fig. 9)which postulates that the

antigenic loopistranslocated into the oxidizing environment of the ER lumen. Sincenoneof the mutations involving the

cysteinesintheantigenicregioneliminatedthe capacity for J. VIROL.

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[image:9.612.312.552.74.194.2]

CYSTEINES IN THE HBV ENVELOPE 4597 dimerization, the proteinsforming adimer were mostlikely

cross-linked byseveral disulfide bonds. However,itcannot be excludedthat the elimination ofcysteines causesshuffling of the remaining disulfide bonds.

According to the secondary-structure model, the first hydrophilic loop is initially located in the reducing environ-ment of the cytoplasm. The final disposition of this loop in secreted particles is still disputed (21, 27). It is remarkable that three of the four cysteines present in this loop are indispensable for secretion. If the high-molecular-weight aggregatesformed by the singlemutantsand a double mutant involving these essential residues resemble authentic high-order oligomers, then these residues should be dispensable for the intermolecular disulfide cross-linking within these structures. Cysteines 48, 65, and 69, together with the essential histidine 60, may therefore play a critical role in one of the subsequent steps of the maturation of 20-nm particles, e.g., in the budding from the membrane into the lumen of the intermediate compartment (9, 11).The question of whether cysteines 48, 65, and 69form disulfide bondsor carry free sulfhydryl groups is currently beingaddressedby analyzing the DHBV small S protein which exclusively carries these cysteineresidues.

ACKNOWLEDGMENTS

Wethank G. Lutfalla for the HepG2 cell line, B. Fleischer for the COS-7 cells, F. Delpeyroux forproviding plasmid pBAP and mono-clonal antibody C3, N. Israelfor plasmid pNI2, and B. Mechler for the synthesis of some of the oligonucleotides. We gratefully ac-knowledge thetechnical assistance of B. Zahn and M. Werr.

This work was supported by a grant from the Deutsche For-schungsgemeinschaft (Ma 1212/1-1).

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