Localization of epitopes of herpes simplex virus type 1 glycoprotein D.

0022-538X/85/020634-11$02.00/0

Copyright X) 1985, American Society for Microbiology

Localization of Epitopes of Herpes Simplex Virus Type

1

Glycoprotein

D

ROSELYN J.

EISENBERG,',2*

DEBORAH LONG,2'3 MANUEL PONCE DE LEON,2'3 JAMES T. MATTHEWS,1'2'3t PATRICIA G. SPEAR,4 MARYLOU G. GIBSON,4 LAURENCE A. LASKY,5 PHILLIP BERMAN,5 ELLIS

GOLUB,2'6

ANDGARYH.

COHEN2'3

Department of Pathobiology, School of Veterinary Medicine,1 and Department of Microbiology,3 Department of

Biochemistry,6 andCenterfor Oral Health Research,2 School ofDentalMedicine, University of Pennsylvania,

Philadelphia, Pennsylvania 19104; Department of Microbiology, The University of Chicago, Chicago, Illinois606374; and

Departmentof Vaccine Development, Genentech, Inc., South SanFrancisco, California940805

Received 20August 1984/Accepted 23 October1984

Wepreviously defined eight groupsof monoclonalantibodies which reactwithdistinctepitopes of herpes

simplex virusglycoprotein D (gD). One of these,groupVIIantibody,wasshownto reactwithatype-common

continuous epitope within residues 11 to 19 of the mature glycoprotein (residues 36to 44of the predicted

sequenceofgD). In thecurrentinvestigation,wehavelocalized the sitesofbinding oftwoadditional antibody groups which recognize continuous epitopes of gD. The use of truncated forms of gD aswell ascomputer

predictions of secondarystructureandhydrophilicitywereinstrumental in locatingtheseepitopes andchoosing

syntheticpeptidestomimictheirreactivity. Group II antibodies, whicharetypecommon,reactwithanepitope

within residues 268to287of thematureglycoprotein (residues 293to312 of the predicted sequence). Group

Vantibodies, whicharegD-l specific,reactwithanepitope within residues 340to356 ofthematureprotein

(residues365to381 of the predicted sequence). Fouradditional groupsofmonoclonalantibodiesappear to

reactwithdiscontinuousepitopes of gD-1, since the reactivity of these antibodieswaslost when theglycoprotein

wasdenatured byreduction and alkylation. Truncatedforms of gDwereusedtolocalize these four epitopesto

the first 260 amino acids of the mature protein. Competition experiments were used toassess the relative

positions of binding of various pairs of monoclonal antibodies.Inseveralcases,whenoneantibodywasbound,

therewas nointerference with the binding ofanantibody from anothergroup,indicating that the epitopeswere

distinct. However, in other cases, there was competition, indicating that these epitopes might share some

commonamino acids.

Glycoprotein D (gD) of herpes simplex virus (HSV) is a

structural component ofthe virion envelope which

stimu-lates production of high titers of virus-neutralizing activity (7, 9, 11, 15-17, 34) and islikelytoplayanimportant rolein

the initialstagesof viralinfection. It wasrecently shown that

anti-gD antibodiescanblock the fusion ofinfected cells(39). Inaddition, mice immunized with gDare protectedfroma

lethalHSV challenge(4, 30, 34, 40).Tryptic peptide analysis

(2, 16) and amino-terminal sequencing(14) showed that gD

of HSVtype 2(HSV-2) (gD-2) is structurally similar though

notidenticaltogDof HSV-1(gD-1). Recently,the genesfor gD-1 and gD-2werelocalized and sequenced (29, 32, 50, 51). Although the deduced amino acid sequences for the two

glycoproteins were shown to be 85% homologous, little is

knownaboutthesecondaryortertiarystructureof thesetwo

proteins. Using monoclonal antibodies (MCAb), we previ-ously defined eight epitopes within gD (15), some of which

aretype common and others of which are typespecific. Our

goalis to relate the structureoftheproteinto itsbiological

functions. The high degree of amino acid sequence

homol-ogy between gD-1 and gD-2 probably accounts for the

immunologicalcross-reactivity of polyclonal antibodiesand

MCAb directed againstgD (15, 41). On the otherhand, the

type specificity of other MCAb is undoubtedly related to

differences in the structures of gD-1 and gD-2 and,

conse-quently, inamino acid sequence.

*Correspondingauthor.

tPresentaddress: DepartmentofPathology, HarvardUniversity School ofMedicine, Boston, MA02115.

Recently (7, 12),using synthetic peptides, the type

com-mongroup VIIepitopewaslocalizedtoresidues 11to19of

the mature form of gD (residues 36 to 44 ofthe predicted

sequence [29, 50, 51]). Polyclonal sera to certain of the

synthetic peptidesin the region ofthefirst23amino acids of gD-1 and gD-2alsoexhibited type common virus-neutraliz-ing activity (7), and immunized micewere

protected

from a

lethalviruschallenge(R. J. Eisenberg, G.H. Cohen, andB.

Dietzschold, unpublished data). During these

studies,

we

localized twoadditional epitopes within the first 23 amino acids of gD-2(7, 12). Thetype 2specificity dependedon two

amino acid differences between gD-1 and gD-2 (29, 50, 51).

The purpose ofthecurrent

investigation

was to

continue

todelineatethelocationandcharacteristics of theantigenic

epitopes

of gD. Threedifferentsetsoftermshave beenused

to

distinguish epitopes

which are lost under

denaturing

conditions, such as reduction and alkylation, from those

which are retained:(i) conformationalversussequential (47);

(ii) discontinuousversuscontinuous (1); and

(iii)

assembled

topographic versus segmental (3). We have chosen to use

discontinuous and continuous as operational

definitions,

without any implication that this terminology is preferred

overany other. We havelocalizedthesite ofbindingoftwo

additional MCAb groups. As with group VII, these also recognize continuous epitopes. Groups II and V (15) bind

specifically to residues 268 to 287 ofgD-1 and gD-2 and

residues 340 to 356 of gD-1. In addition, we carried out

competition studies to map the relative positions offour

discontinuousepitopes, correspondingtoMCAb ingroups

I,

III, IV,and VI (15).

634

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MATERIALS AND METHODS

Cells,

virus,

and radioactive

labeling procedures.

Condi-tionsfor the

growth

and maintenance of BHK and KB cells

andforthe

propagation

of virushavebeen described

(8, 11).

For

infection,

an

input

multiplicity

of20 PFUofHSV-1

(HF)

and 10 PFU of HSV-2

(Savage)

per cell was used.

Proce-duresfor

labeling

ofHSV-infected cells with

[35S]methionine

(specific

activity,

600

Ci/mmol) (Amersham

Corp.)

and

[2,3-3H]arginine (specific

activity,

15

Ci/mmol)

(Amersham)

have

been described

previously

(9, 13, 15, 16).

Preparation

of

polyclonal antibody

and MCAb to

gD.

Anti-gD-1

and

anti-gD-2

serawere

prepared

in rabbits

against

immunosorbant-purified

preparations

of

gD-1

and

gD-2 (17).

MCAb HD-1

(group

I)

and MCAb 170

(group VII)

were

supplied

by

L. Pereira

(15, 41).

MCAb

55S

and 57S

(group

V), 11S

(group

III),

41S

(group IV),

and45S

(group VI)

were

supplied

by

M.

Zweig

(48).

MCAb DL6

(group II)

was

prepared

from mice immunized

intraperitoneally

(34)

with 6 ,ugof

immunosorbant-purified

gD-1

(17).

Three

days

afteran

intravenous boost of1 jig, the

spleen

was removed and the

cellswerefusedto

SP2/0

cells

by

the

procedure

ofMcKearn

(28).

Hybridomas

were cloned in soft agarose

(25),

and

ascites fluids were

prepared

from

Pristane-primed

mice

immunized

intraperitoneally

with cloned cells.

To prepare

immunosorbants,

immunoglobulin

G

(IgG)

was

purified

from ascites fluids of MCAb fromeach group

(15)

andlinkedtocyanogenbromide-activated

Sepharose

4B

(Pharmacia

Fine

Chemicals, Inc.).

The amounts

coupled

ranged

from 5to 12mg of

IgG

per g of

Sepharose.

Purified

immunoglobulins

(50

Rxg)

were iodinated with 1251

(Amer-sham)

by

eitherthe chloramine-T

(19)

orthe

lactoperoxidase

(36)

method. For certain

MCAb,

thechloramine-T method

inactivated the

binding

activity

ofthe

antibody.

Group

IV MCAb were inactivated

by

both

procedures.

Synthetic peptides.

Synthetic peptides

toresidues 1to23of

gD-1

(1-23[1])

and

gD-2

(1-23[2])

were

prepared

asdescribed

previously

(7).

The

peptides

representing

residues340to356

of

gD-1

(340-356[1])

and 268to287of

gD-1 (268-287[1])

were

prepared

by

Peninsula

Laboratories,

Inc.

Cysteine

was

added to the amino terminus of

340-356[1]

and to the

carboxy

terminus of

268-287[1].

The

procedures

for

coupling

of

peptides

to

keyhole

limpet

hemocyanin

(KLH)

were

described

previously

(7,

33).

Briefly,

themaleimidegroupof

the

peptide

was

incorporated

into KLH with

M-malimido-benzol-N-hydroxysuccinimide

esterandthe

M-malimidobe-nzol-N-hydroxysuccinimide

ester-modified

proteins

were

al-lowed to reactwith a 20 Mexcess ofthe

peptide

(22).

The

coupling

ratio of

peptide/carrier

was

previously

determined to be 8

(7).

All

peptides

were dissolved in 0.1 M Tris

(pH

7.8)-0.15 M NaClforassay

by

theimmunoblotmethod

(see

below).

Preparation

of antisera tothe

synthetic peptides.

Afemale

New Zealand White rabbit was immunized with

peptide

340-356[1]

coupled

to KLHatthree

weekly

intervals witha

totalof2.4mgof

coupled peptide.

The animalwas

given

an

intravenous boosterdose of120

p.g

ofthe

coupled peptide

3 to4

days

before each

bleeding.

Atotal of five bleeds were

obtained. Two female New Zealand White rabbits were

immunized with

peptide

268-287[1]

coupled

toKLHatthree

weekly

intervals withatotalof1.4 mgof

coupled peptide

per

rabbit.Theanimalswereboosted

intravenously

oncewith 70 ,ugof

coupled peptide

and then three times with 500 ,ug of free

peptide.

Preparation

of native and denatured

gD.

gD-1

and

gD-2

were each

purified

from

cytoplasmic

extracts of infected

cells by affinity chromatography, using a previously

de-scribed procedure (17). For our purposes, the

proteins

eluted from the immunosorbant column with KSCN and

dialyzed against 0.01 M Tris (pH 7.5)-0.15 M NaCI-0.1%

Nonidet P-40 (TSN buffer) are designatedas "native." For

denaturation, purified gD-1 orgD-2 was suspended in

dis-rupting bufferto yield a final concentration of 3% sodium

dodecyl sulfate (SDS)-100 mM Tris (pH 7.0)-10%

2-mercaptoethanol-0.5% glycerol. The sample was boiled for

5 min. lodoacetamide (0.1 M in 0.1 M Tris, pH 8.0) was

added to give a final concentration of 33 mMiodoacetamide,

and the mixture was incubated for 1 hat roomtemperature.

The samples were dialyzed extensively against TSN buffer.

Preparationof truncated forms of gD. (i) The 38Kfragment

ofgD-1. Preparation of the 38K fragment wasby a

modifi-cation of a previously described procedure (15). Briefly, a

cytoplasmic extract(100 ,ul) of HSV-1-infected cells labeled

with [3H]arginine was added to 50

RI

of

HD-1-IgG-Se-pharose (125 ,ug of IgG). The immunosorbant was washed

extensively with 0.01 M Tris (pH 7.5)-0.1% Nonidet P-40-0.5

M NaCl-0.1 mM phenylmethylsulfonyl fluoride (washing

buffer)and then with V8 enzyme buffer (50 mM Tris,pH8.0)

and incubatedwith 50 p.g ofStaphylococcusaureusprotease

V8 in enzymebufferat37°C for 2 h. The immunosorbantwas

washedextensively withwashing buffer. To test the

prepa-ration for the presence of uncleaved gD, a portion of the

immunosorbant was suspended in SDS-disrupting buffer,

boiled for 3 min, and analyzed by SDS-polyacrylamide

gel

electrophoresis (PAGE). V8 proteolysis resulted in a 38K

fragment (15) and no full-length gD. The 38K fragment

linked to HD-1-IgG-Sepharose was then used to test the

bindingof other MCAb asdescribed in Results.

(ii) TruncatedgD-1, residues 1 to 275.The gene forgD-1

wascloned intoapBR322-simianvirus40 shuttle vector and

included a DNA fragment from a HindlIl site upstream of

thegD gene to a Hinfl site atresidue 300 of the predicted

gD-1 sequence (residue 275 of the mature protein). When

this plasmid is grown in Chinese hamster ovary cells, the

glycoproteinis secreted(30). The glycoproteinwaspurified by affinity chromatography, usingagD-specificMCAb(17).

(iii)

Truncated gD-1, residues 1 to 287. Truncated gD,

residues 1 to 287, was producedand secreted by the HSV

insertion mutant designated 111 (M. G. Gibson and P. G.

Spear, J. Cell Biochem. Suppl. 8B, in press; Gibson and

Spear, 13th Ann. UCLA Symp. 1984, abstr. no. 1337, p.

191).

The virus was constructed by methods previously

described(18)exceptthatatruncated form of thegD-1 gene

(extendingfrom theSaclsite upstream of thegD gene to the

NarI siteatresidue 312of thepredictedsequenceorresidue

287 of thematureprotein)wasinserted into theBglIIsite of

the

thymidine

kinase gene. The expressed proteincontains

48 amino acids at its carboxy terminus that are translated

from the noncodingstrand of HSV-1 DNA atthe 5' end of

thethymidine kinase gene. Theproteinwasaffinity purified

from the medium ofHEp-2cellsinfected with 111 virusbya

previously

described method (17), using a gD-1-specific MCAb

designated

11-436-1 (39).

Immunoprecipitation

andSDS-PAGE. gDwas

immunopre-cipitated

from HSV-1-orHSV-2-infected cellextracts(cells

infected for 6 h) prepared as previouslydescribed (26, 42),

using

antiseraorMCAb and S. aureusproteinA(IgG Sorb; New

England Enzyme

Center). SDS-PAGEwascarriedout

in slabs of 10%

acrylamide

cross-linked with 0.4%

N,N'-diallyltartardiamide

(13, 49).Forautoradiography, gelswere

dried on filter paper and

placed

in contact with Kodak

XAR-5 film. For

fluorography,

the gels were treated with

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Amplify (Amersham), dried on filterpaper, andexposed to

Kodak XAR-5 film at -70°C.

Immunoblot and neutralization assays. The immunoblot

assay was done as previously described (7, 20), using

antisera or MCAb and iodinated protein A (Amersham).

Virus neutralization assays (50%plaque reduction method),

using HSV-1 (HF) or HSV-2 (Savage), were carried out as

previously described (9, 11).

Competition assays. Twocompetition assays were used.

(i)Immunosorbants. Atotal of 100to 200,ul(representing

150 to 600 ,ug of IgG) of MCAb linked to Sepharose was

incubated for 2 h at37°C with 100,ulofacytoplasmic extract

of unlabeled HSV-1-infected cells (9, 13, 15, 16). The

im-munosorbant was washed 10 times with washing buffer, and

the iodinated second antibody (at least 250,000 cpm) was

added. Thecomplex was washed exhaustively andcounted

in a gammacounter.

(ii)Nitrocellulose. PurifiedgD-1(0.45 to 15 ng) was spotted

onto nitrocellulose strips; the strips were washed as

previ-ously described (7) and incubated with unlabeled first

anti-body and then with iodinated second antibody (ca. 250,000

cpm). The spots were located by autoradiography, cut out,

and countedin a gammacounter. The counts were subjected

to linearregressionanalysis, and the slope of the line (counts

per minute versus concentration of gD) was calculated.

RESULTS

Comparison of the predicted secondary structures of gD-1

and gD-2. Figure 1 shows a computer representation (7) of

thesecondary structure ofgD-1(Fig.1A)and gD-2(Fig.1B),

derivedfromthepredictedamino acidsequences (29,50, 51)

and rules established by Chouand Fasman (5, 6). It should

be noted that these predictive "rules" of secondary

struc-ture result inan accuracyof predictionin a three-state model

(helix-sheet-turn)ofapproximately50versus33%forchance

(24). However, in the absence of any additional structural

information, we have found that these predictions have

heuristicvalue in that they focus attention on certain regions

of the glycoprotein. Furthermore, we have also analyzed

both glycoproteins with a second empirical analysis (Fig. 1)

which assumes that hydrophilic regions of protein structure

have a greater immunological potential (22). For these

calculations, the first 25 amino acids of the predicted

se-quence were excluded from consideration, since direct

N-terminal sequence analysis showed that, for both gD-1 and

gD-2, lysine residue 26 of the deduced sequence was the

aminoterminus of the mature protein (14). In ournumbering

system, this lysineisresidue 1. The criteria used for

predict-ing the probability of

a-turns

(7) were modified to increase

the likelihood of locating possible epitopes. The

modified-turn criteria predict four additional

n-turns

in gD-1 at

resi-dues 200, 225, 255, and 298. None of these additional turns

involves highly hydrophilic regions of the protein.

Interest-ingly, avery hydrophilic region, residues 77 to 95, is not in

a predicted

a-turn.

When the criteria were relaxed even

further, this stretch ofamino acids was still not predicted to

be in a

p-turn.

The working hypothesis is that epitopes are

likely to be located in regions where highly hydrophilic

residues are present in ,-turns (45). If, in addition, the

epitope is continuous, synthetic peptides could be used to

mimic the reactivity of the epitope. The program has also

beenexpanded to indicate the positions of predicted

N-aspar-agine-linkedcarbohydrates (shown as balloons) based on the

sequence Asn-X-Thr or Asn-X-Ser (23). For gD-1 and gD-2,

all three positions are glycosylated (10). Predictions for

hydrophilicity use the same criteria as before (7, 22). The

homologyin aminoacid sequence is reflected in similarities

in both secondary structure andregions ofhydrophilicity in

the two proteins. In at least one case, however,two

differ-ences in amino acid sequence in region 1 to 23 have been

correlated with bothchanges in predicted secondary

struc-ture andantigenicity (7, 12, 29, 50, 51). For bothgD-1 and

gD-2, there are two regions in which ,-turns intersect a

highly hydrophilic region, i.e., residues 11 to 19and 265 to

282. A third region in gD-1, residues 340 to 356, is

hydro-philic and contains a predicted ,-turn overlapping the

hy-drophilic region.Thesea-turnsarepresentevenwhenmore

stringent criteria for

predicting

turns are

applied

(7).In

gD-2,

however, there is no 3-turn in this region, even with the

modified criteria.

Reaction of MCAb in groups I to VII against native and

denaturedgD. To test the structural

predictions,

weused the

immunoblot assay(7, 20) todetermine which of thegroups

of MCAb reacted with discontinuous or continuous

epi-topes. Previous studies indicated that

only

certain MCAb

groups reactedwith denatured gD (32, 37;J. T.

Matthews,

G. H.Cohen,and R. J. Eisenberg, unpublished

data).

Native

gD-1(Fig.2)reacted withpolyclonal

anti-gD

serumand with

MCAbin groups I to VII(rows 1 to8, lanea). Native

gD-2

(lane b) reacted withpolyclonal anti-gD (row 1,lane

b)

and

alsoreactedwithMCAbin groupsI,II, III,

V,

and VII

(rows

2, 3, 4, 6, and8, lane b). The reaction of native

gD-2

with

group V wasunexpected, since group V failedto immuno-precipitategD-2from infectedcell extracts(15,48).

Further-more, denatured gD-2 reacted either

weakly

or not at all

againstgroup VMCAb, whereas native anddenatured

gD-1

reactedequallywellagainstthe sameantibodies. Inaddition

to group V, MCAb in groups II and VII

recognized

the

denaturedformofgD-1andgD-2(lanescand

d).

Thus,

three

groups of MCAb, II, V, and VII,

appeared

to

recognize

continuous

epitopes,

andfourgroupsof

MCAb,

I,

III, IV,

andVI,apparentlyreactedwith discontinuous

epitopes

that

requirethenative conformation of

gD-i

(34).

It should be noted that under the

denaturing

conditions used considerable

secondary

and

tertiary

structure

might

remain ingD. Therefore, thatthe

protein

retained

antigenic

activity for antibodies ingroups

II, V,

and VII is not

proof

per se that these

epitopes

are continuous. In the case of

group VII, the proof was

provided by

the

reactivity

ofa

synthetic

peptide

mimicking

residues8to23 of

gD-1

against

group VII MCAb 170 (7). One ofthe

goals

ofthe present

study was toobtain similar

proof

forthe

epitopes

specified

by MCAb ingroups II and V.

Prediction ofthe location of the groupV epitope. Several

linesof evidence enabled ustolocalizethegroup V

epitope.

Previously,

using

V8

proteolysis,

we found that group V

MCAb reacted with a 15K

fragment

of

gD-1

(15).

Tryptic

peptide

analysis

showed that this

fragment

represented

the

carboxyterminus of the

protein

(15;D.

Long,

G. H.

Cohen,

and R. J. Eisenberg,

unpublished

data).

Further evidence

indicated that theepitopewaslocateddownstream fromthe

membrane-anchoring

region

(i.e.,

presumably

after residue 339[51]). First, group V MCAb reacted with

fixed,

but not withunfixed, HSV-1-infected cells (37). This

suggested

that theepitope isnotexposedonthe externalface of the

plasma

membraneof infected cells. Second,when

gD-1

was

synthe-sized and processed in an in vitro system, the

processed

protein was partially protected from

proteolysis by

trypsin

(37). Approximately 3,000 daltons of the

protein

was

re-moved bythis treatment, and the

trypsin-resistant

fragment

could not be

immunoprecipitated by

group V MCAb.

Fur-thermore, when truncatedformsofthe

gD

gene,

lacking

the

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NH2 A

2

gnx

~~~(gD-1)

,w-,q-8-ia v

HOOC~~ ~ ~ ~ ~ ~ ~ H

(gD-2)

*-but4(1

Wit,e=t4;$r~ ,^JA5~ . 4*ea

FIG. 1. Predicted secondary structure and hydrophilicity maps ofgD-1 (A) and gD-2 (B). Secondary structures were predicted by a

computerprogram,usingthe rules of Chou and Fasman fordetermining Pt, Pa,and Pb(5,6). Probabilities for theoccurrenceofa-turnswere evaluatedby usingmodified conditions: Pt>7.5 X 10-'orPt>5 x 1O-5 pt>Paand Pt> Pb.Shaded circles indicatehydrophobic regions;

opencircles indicatehydrophilicareas. Theradiusofacircleoveraresidue isproportionaltothe meanhydrophilicityascalculatedfor that residueplusthenextfive residuesaccordingtothemethod ofHoppand Woods(22). Thevalue is therefore distortedatthe C-terminal end.

Thehexagonalballoons indicatepredictedsites (Asn-X-ThrorSer)of

N-asparagine-Iinked

glycosylation (23).

information for the transmembrane-anchoring region plus thecarboxyterminus, were cloned into Escherichiacoli, the

expressed gD-like protein was not recognized by 57S

anti-body (R. J. Watson, J. H. Weis, J. H. Salstrom, and L. W.

Enquist,J. Invest. Dermatol., in press). These results, taken together, suggested that the group V epitope was located

between residues 340 and 369 of gD-1.

Tolocalize theepitope further, we relied on the computer

predictions (Fig. 1and 3)and differences in the sequences of gD-1 and gD-2 at the carboxy terminus (Fig. 3) (29, 50, 51).

Weargued that the epitope is largely type 1 specific, based

on theimmunoprecipitation data (15), but that certain

simi-larities in sequence might account for the reactivity of gD-2

againstgroupVMCAbin the immunoblot. Region 340 to 356

of gD-1 ishighly hydrophilicandcontainsapredicted j-turn

atresidues 346to349(Fig. 1and 3). The homologous region

of gD-2 is also hydrophilic, but does not contain this

predictedturn. In addition, the sequences of gD-1 and gD-2

in the region 340 to 356 (Fig. 3) show similarities (e.g.,

residues 346to349 areAla-Pro-Lys-Arg and residues 351 to

356 are Arg-Leu-Pro-His-Ileu-Arg in both proteins) and

differences (e.g., residues 343 to 345 are Thr-Arg-Lys in

gD-1 butareAla-Gln-Met ingD-2). These differences appear

tohaveaprofound effectonthe predictedsecondary

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[image:4.612.148.470.74.529.2]

N

ct

J

Xa

0

-0

3

I

I

1

0

.X.. .v.'V.

0

-7

340-356[1] contains the group V epitope; (ii) this

epitope

appears to be immunogenic in the native protein since

several polyclonal seraprepared against gD, including one

against gD-2, reacted with the peptide; and (iii) there is

limitedantigeniccross-reactivity withananalogousregionof

gD-2.

Anti-340-356[1] serum reacted in the immunoblot assay

(Fig. 5A) against nativegD-1 (lane a), gD-2(laneb), andthe

syntheticpeptide 340-356[1](lane h). The specificity ofthis

serumfor thecarboxyterminus of gD isdemonstrated by the

lack ofreactivity against truncated gD-1, residues 1 to 275

(lane e), and the synthetic peptides 1-23[1] and 268-287[1]

(lanes f and g, respectively). The reactions against native gD-1 and gD-2 were stronger than those

against

the

dena-A 3338 340

N H

2

M.H.,9.^

".R.

,F.

3e0 K

[iV' 355 350

365 ,~

[image:5.612.73.287.72.472.2]

~~~~

~

0 0

H

FIG. 2.Immunoblot analysis of monoclonal antibodies directedat

HSVgD. The antibodies used (groupedasdescribed in reference15)

were asfollows: row1,anti-gD-1 (rabbit 1); row2,groupI, HD-1; row3,groupII, DL6;row4,groupIII, 11S;row5,groupIV,41S; row6,groupV,57S;row7,groupVI,45S;row8,groupVII,170. Antigens: lanea,immunosorbant-purified (17) (native) gD-1 (15ng); laneb, native gD-2 (15 ng); lane c,denatured gD-1 (15ng);laned, denaturedgD-2 (15 ng); lanee,truncated gD-1, residues 1to275 (30) (60 ng).

tureof thedownstream residues of thetwoproteins. Based

onthisinformation,residues340to356of gD-1 appeared the

mostlikelyto contain thegroup V epitope.

Recognition of the synthetic peptide 340-356[1] bygroup V

MCAb andby polyclonal anti-gDsera.GroupVMCAb (Fig.

4, row 7) reacted with purified native gD-1 (lane a), gD-2

(lane b),and thesynthetic peptide correspondingtoresidues

340to356 ofgD-1 (lane e).This antibodydidnot reactwith

synthetic peptides corresponding to other portions of gD-1

(lanes c andd). Two polyclonal seraprepared against gD-1

(rows 1and2) reactedwiththis peptide. Inone case(row 1)

thereaction was strong, and in the other case the reaction

was weak(row 2). In addition, the peptide reacted weakly

with one polyclonal serum prepared against purified gD-2

(row 3), but failed to react with a second anti-gD-2 serum

(row 4). These results show that (i) the synthetic peptide

B 340

NH2

V

350

355 360

i: 'i | b R

l...

R

.. .

345 P

365

HOOC

.Y~' L"

FIG. 3. Comparison of amino acid sequence and predicted

secondary structures ofcarboxy-terminalsequences of gD-1

(resi-dues 338to369)andgD-2(residues 338to368). Probabilities for the

occurrenceofP-turnswereevaluatedasinthelegendtoFig. 1. The single-lettercode designations are asfollows: A, alanine; R,

argi-nine; D, aspartic; N,asparagine; C, cysteine; E, glutamic acid; Q, glutamine; G, glycine; H, histidine; I, isoleucine; L, leucine; K,

lysine; M, methionine; F, phenylalanine; P, proline; S, serine; T, threonine; W, tryptophan; Y, tyrosine; V, valine.

I

2

Adik

qw

A

*

4

4

1

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[image:5.612.318.557.240.648.2]

a

cde

_A

abcdef gh

_{* *}

_*

S

B

S

a

b c d e f

5.5,i

6

ee-7

S

-,Y ._ .._

_

FIG.4. Immunoblot analysis ofsynthetic peptides which mimic portions of gD-1, using polyclonal antibodies and MCAb. The antibodies usedwere asfollows: row1,anti-gD-1 (rabbit 1);row2, anti-gD-1 (rabbit 2); row3, anti-gD-2 (rabbit 3); row4, anti-gD-2 (rabbit 4);row5, group VII,170;row6, groupII,DL6;row7, group V, 57S.Antigens: lane a, native gD-1 (15 ng); lane b, native gD-2 (15 ng); lane c, 8-23[1] (500 ng); lane d, 268-287[1] (1 p.g); lane e,

340-356[1] (100ng).

tured forms oftheseproteins (lanescandd).Anti-340-356[1]

serumimmunoprecipitated both gD-1 (Fig. SB, lane c) and

gD-2 (lane d) from infected cellextracts. Lanesaand b(Fig.

SB) represent negative controls in which the HSV-1 and

HSV-2 extracts were tested with serumobtained from the

animal before immunization with

340-356[1].

As positive controls, the same extracts were immunoprecipitated with

anti-gD-1serum(laneseandf).Itis clear thatanti-340-356[1]

wasmorereactiveagainst

gD-1

than gD-2. Aswithgroup V

MCAb,theanti-340-356[1]serumfailedtoshow any

neutral-izing activity against HSV-1orHSV-2 (data notshown).

Localization of the groupIIepitope. Group II,represented

byDL6MCAb, reacted withthe native anddenaturedforms

of both gD-1 and gD-2 (Fig. 2, row 3), exhibited

type-commonmembrane immunofluorescence (datanotshown), and neutralized HSV-1 at 1:50 dilution and HSV-2at 1:20,

usinga50% endpoint(9, 11). The continuous epitope

recog-nizedbyDL6MCAbwasdistinct fromthoserecognized by

either group VIIorV, sinceDL6didnot reactwith either the

8-23[1]

or the 340-356[1] synthetic peptide (Fig. 4, row 6,

lanes c and e). Preliminary localization of the group II

epitope was accomplished by testing the reactivity oftwo

[image:6.612.64.298.74.406.2]

truncatedforms ofgD-1, representingresidues 1 to275and

FIG. 5. Analysis of anti-340-356[1] serum by immunoblot (A) and SDS-PAGE (B). For immunoblot analysis, the antigens were: a, native gD-1; b, native gD-2; c, denatured gD-1; d, denaturedgD-2; e, truncated gD-1, residues 1 to 275; f, 8-23[1]; g, 268-287[1]; h,

340-356[1].Theconcentrations of theseantigenswerethesameasin

Fig. 2. For SDS-PAGE analysis (B), extracts from HSV-1-infected cells are inlanes a, c, and e and extracts fromHSV-2-infectedcells are in lanes b, d, and f. The sera used were: lanes a and b, preimmunization bleed from rabbit immunized with 340-356[1]; lanes c andd,anti-340-356[1];lanes e and f, anti-gD-1 (17).

1 to 287, against MCAb (Fig. 6). Both forms reacted with

group VII antibody (row 1, lanes c to e), indicating the

presence of residues 8-23 in the truncated proteins. As

expected, neitherform reactedwithgroup V antibody(row

3, lanesc to e). The truncated form, 1-275[1], also failedto

a

bcd e

I

2

*

*S

.0

FIG. 6. Immunoblot analysis of truncated forms of gD-1, using MCAb.Theantibodies usedwereasfollows: row1, group VII,170; row2, groupII,DL6;row3, groupV,57S.Antigens: lane a, native gD-1 (15 ng); lane b, native gD-2 (15 ng); lane c, truncated gD-1, residues1 to275(30),60ng;laned,truncatedgD-1,residues 1 to 287(GibsonandSpear, in press), 100ngofprotein;lanee, residues 1 to287,200ngofprotein.

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react with group II MCAb (row 2, lane c). However, this

antibody didreactwith 1-287[1] (row 2, lanes d and e). These

results suggest that thegroup IIepitope is probably located

between residues 275 and 287, although it could include

several residuesupstream. Fromcomputerpredictions (Fig.

1),theregion from residue 266toapproximately 282

repre-sents a potential epitope in both gD-1 and gD-2 since this

region is highlyhydrophilic and contains a,B-turn (45).This

combination of predictions and data suggest that a peptide

consisting of residues 268to287 wouldencompassthegroup

IIepitope.

Localization of the group II epitope by using synthetic

peptides. Toconfirm thelocation of thegroupIIepitope,we

tested the reactivity of a peptide representing residues

268-287[1]. This peptide reacted withgroupIIMCAb (Fig. 4,

row6,lane d) butnotwithantibodiesingroupV(row7,lane

d) or VII (row 5, lane d). Thus, the group II epitope is

located between residues 268 and 287 of gD-1. In addition,

thepeptide reacted against three of the fourpolyclonalsera

prepared against gD (Fig. 4, rows 1 to3, lane d).

Peptide 268-287[1] was coupled to KLH and used to

immunize tworabbits. After the initial immunization

proto-col andoneintravenous booster dose, serum samples were

assayedfor anti-gD and anti-peptide antibodies by the

im-munoblotassay. Neitherserumreacted with nativeor

dena-tured gD (data not shown). The animals were then given

three intravenous booster doses of the free peptide and

successive serumsampleswere assayed again. Again, none

of theserareacted with native ordenatured gD. One serum

sample reacted with the synthetic peptide 268-287[1] (data

not shown). None of the sera exhibited any neutralizing

activity (data not shown).

Localization of discontinuous epitopes of gD-1.Previously,

we developed a procedure (15) to partially fragment gD,

using S. aureus protease V8, an enzyme which cleaves

specifically at glutamic acid residues. In that procedure,

metabolically labeled gD was immunoprecipitated with a

MCAb plus S. aureus-bearing protein A and the complex

wastreated with the protease (15). Theantibody protected

thatportion of gDtowhich itwasbound, and the bound and

unbound fragments were characterized by SDS-PAGE and

tryptic peptide analysis. We found thatgroupI, IV, and VI

MCAb remained bound to a 38K fragment which, on the

basis ofN-terminal amino acid sequencing (14; D. Long,

G. H. Cohen, R. Hogue-Angeletti, and R. J. Eisenberg,

unpublisheddata), wasfound tocontaintheamino terminus

of pgD (the precursor form ofgD). Analysis of the tryptic

glycopeptides (10) of the 38K fragment showed that

glyco-peptides 1 and 2 were present but that glycopeptide 3 (at

position 262)was missing (data not

$hown).

Thus the 38K

fragment appearstobe located betweenresidues 1 and 262.

A possible V8 cleavage site is located at glutamic acid

residue 260. The V8 experiments implied that the epitopes

reacting withgroup I, IV, VI, and VII MCAbwerelocated

within this portion of gD-1. Antibodies in groups II and III

couldnotbelocalized by this technique, since nofragments

remained associated with them after V8 proteolysis.

How-ever, we nowknow thatgroup II islocated downstream of

the38K fragment. Thus, it wasalsopossible that thegroup

IIIepitopewaslocated downstream of thecarboxy terminus

of the38K.

Figure 2(lane e) shows thatgroupI, III,IV, VI, and VII

MCAb boundto anothertruncated form of gD-1 consisting

ofresidues 1to275(30). These resultsagreewiththoseofV8

proteolysis (15) with the exception ofgroup III(11S). One

[image:7.612.316.556.89.148.2]

possibility was that thegroup III epitope included residues

TABLE 1. Localization of the 11S epitope on the 38K fragment 1251 bound(cpm)b

Sample" HD-1_251

iiS_125i

57S_125I

Control 9,459 10,205 57,433

gD-1-HD-1 (no V8) 7,868 690,120 350,784 gD-1-HD-1 (+ V8) 10,898 589,136 54,826 a The controlsampleconsistedofHD-1-Sepharose with no gD-1 added.

Foreach assay, 100p.1of acytoplasmic extract ofHSV-1-infected cells was added to 50 ,u1 of HD-1-IgG-Sepharose. The immunosorbant was washed with washing buffer and incubated with 50 ,ug of S. aureus V8 protease in 50 mM Tris (pH 8.0) for 2 h at 37°C. The complex was washed with washing buffer andthenincubated with iodinated MCAb. This complex was washed andcounted in a gamma counter.

between 260 and 275. Anotherpossibility is thatbinding of

group III occurred within the 38K fragment, but that this

binding didnotprotect thefragment

frorn

further

proteolysis

(15). Todetermine directly whether group III MCAb could

bind to the 38K fragment, we carried out the following

experiment. A group I MCAb (HD-1) immunosorbant was

used to bind purified gD-1. This complex was washed

extensively, and a portion was treated with S. aureus

protease V8 and washed again. Then various iodinated

MCAb were added, and the complex was washed

exten-sively and then counted inagamma counter. Table1 shows that, in the HD-1 control

(HD-1

as immunosorbant and

iodinated probe), no significant counts bound above

back-groundineithertheV8-treatedor theuntreated sample. We also probed the V8-treated and untreated samples with

group Vantibody(57S)andfound that theuntreatedsample

containedasignificantnumberofcountsandtheV8-treated sample

contained

no counts above background. This

indi-catedthat the

proteolysis

wascomplete andthat thegroup V

epitopewasnotpresent afterV8 treatment. Withiodinated

11S(groupIIIMCAb)as theprobe, approximately thesame

number ofcounts bound to the V8-treated and untreated

gD-1-HD-1 complexes. These results show that the group IIIepitope ispresent onthe38Kfragmentand,furthermore,

that thisepitope isdistinctfromthe group I epitope.

Topographical relationship

of

epitopes

located in residues 1

to 287 of gD-1. Previous

experiments

indicated that six

epitopes ofgD-1 werelocated withinthefirst 287residuesof

theprotein,twoofwhich werecontinuousand fourofwhich werediscontinuous. Twodifferent experimentswerecarried

out tobegintodefinetherelativepositions oftheseepitopes.

These will be referred to as competition experiments,

al-though we recognize that the term does not accurately describethetypeofanalysis being

performed.

First, representative MCAb from the six groups were

covalently bound to Sepharose. A preliminary

experiment

was carried out todetermine the amount of each

immuno-sorbantrequired to bind agiven amountof gD-1 present in

infected cell extracts. The appropriate amount of each

immunosorbant was used tobind similar amountsof

unlab-eled gD-1. After this, a different and iodinated second

antibodywasadded,and thecomplexeswerewashed

exten-sively and counted in a gamma counter. The underlined

values in Table 2show that each antibody groupcompeted

against itself, sinceonly background levels of counts bound

when the same antibody was used as immunosorbant and

iodinatedprobe.Threetypesofresultswereobtained: (i)no

competition, in which a significant number ofcounts were

bound, e.g., usinggroup III as immunosorbant and group I asprobe, orvice versa; (ii) completecompetition, in which

only a background number of counts were bound, e.g.,

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TABLE 2. Competition analysis usingMCAblinkedtoSepharose 4B'

lodinatedantibodies (cpm of1251bound)

Immuno-sorbant Group I Group II GroupIII Group VI Group VII (HD-1) (DL6) (llS) (45S) (170)

Control 3,166 2,227 710 7,800 1,066

I(HD-1) 2,914 62,135 34,158 15,806 37,909

II(DL6) NDb 3,175 ND ND ND

III(11S) 30,619 80,231 700 2,958 10,972

IV(41S) ND ND 578 32,265 ND

VI(45S) 17,558 85,565 3,304 9,819 30,581 VII(170) 27,618 80,048 30,581 25,435 1,027

aAntibodiesareincluded in groups accordingto originaldefinitions (15). Foreachassay,100 to 200p.lof MCAb linkedtoSepharosewasincubatedfor 2h with100p.lofacytoplasmicextractofHSV-1-infected cells (9,13,15,16).

The immunosorbant was washed and the iodinated second antibody (ca. 250,000 cpm) was added. Thewashed complexeswerecountedin a gamma counter. The underlined values show that eachantibody group competed against itself.

bND, Not done.

comparinggroup IV against group III; (iii) partial competi-tion, in which some counts were bound, e.g., comparing

group VI and groupI. Inthisassay, thereisnoway toknow

what the maximal level of binding should be.

As asecond approach (Table 3), different concentrations of purified gD-1 were spotted onto nitrocellulose strips

which were then incubated with an excess of unlabeled

antibody. The strips were washed and incubated with

iodi-nated second antibody. The maximal level ofbinding (no

competitionor0%in Table 3) wasdetermined fromacontrol inwhichthestrip was incubatedonly with labeledantibody.

The values underlined in Table 3 represent the percent

competition which occurredwhen the same antibody, both

unlabeled and labeled, was used to compete against itself.

Theoretically,these valueshouldapproach 100%. Forgroup

II, this value was 93%; however, forgroups I and III, the

value was

70%.

Thereasonfor this isnot understood,since,

in each case, the firstantibody was presumably present in

excess. Itmay be a problem of antibody affinity or

presen-tation ofthe antigen on nitrocellulose. Nevertheless, when

heterologous antibody groups were compared, the results

agreedwith the resultsinTable 2. There was nocompetition

between groups I and III or I and VII. Furthermore, there

was partial competitionbetween groups I and IV and I and

VI. Group III showed partial competition with groups IV

and VI. Group II MCAb showed slight

competition

with

group VI and possibly group I. Thus, the two kinds of

experiments leadtothesameconclusions and form thebasis

fortopographical positioning of these epitopesingD-1 (Fig.

7).

DISCUSSION

Inprevious studies, wedefinedeight antigenic epitopes of

gD, based on an analysis with a panel of MCAb (15). We

attempted toassociate the binding ofparticular MCAb with

differentfragments of the protein and found that several of

themwere ina38Kfragment whichencompassesthe amino

terminus. We further defined the position ofonecontinuous

epitope of gD, amino acid residues 11 to 19, which reacts

with group VII MCAb (7, 12). Localization of that epitope

was based on the V8 proteolysis studies (15) as wellas the

use of computer predictions of secondary structure and

hydrophilicity in choosing an appropriate synthetic peptide

to test the predictions. Here, our goal was to localize the

precise locations oftwo other continuous epitopes,

recog-nizedby group II and V MCAb, andto begin to define the

location of discontinuous epitopes of gD-1. Computer

pre-dictions were instrumental in helping to choose synthetic

peptidestodemonstrate thelocation ofcontinuousepitopes.

In each case, the epitope was found to be located within

stretches ofhighlyhydrophilic amino acid residues making

uppredicted ,-turns (45).

The groupVepitope was localized to residues 340to356

ofgD-1 which isdownstreamfromthemembrane-anchoring

region (50, 51). A synthetic peptide consisting of this

se-quence was found to bind specifically to group V MCAb.

The location of this epitope is thus on the portion of gD-1

which faces the inside of the virion or infected cell and

confirms predictions of its location based on other studies

(15, 37; Watsonetal., J. Invest. Dermatol., inpress). Thus,

the failure ofgroupVMCAbtoneutralizevirusortobindto

thesurface of HSV-1-infected cells is due tothe

inaccessi-bility of thisepitope when gDisassociatedwithmembrane.

Recently, Rector et al. (44) used a group V antibody (55S)

(15, 48) to examine whether non-neutralizing antibodies

could be protective in passive immunization studies. Their

data showed that 55S was not protective. Since group V

MCAb wouldnot have reacted with intact virions orintact

infected cells, their result is not surprising.

The results of the presentstudy indicate that thegroupV

epitope is in itself immunogenic in purified gD, since the

synthetic peptide reacted with several polyclonal sera

pre-TABLE 3. Competition analysis usingthe immunoblot assay

Group I (HD-1) Group11(DL6) GroupIII(11S)

Cold antibody Slopeb Competition" Competition Competition

None 173 0 728 0 1,051 0

GroupI(HD-1) 60 70 704 3 1,050 0

Group II(DL6) 139 20 52 93 951 10

Group III (11S) 167 3 695 5 377 64

Group IV(41S) 124 28 ND" 713 32

GroupVI(45S) 137 21 642 20 714 32

GroupVII (170) 167 3 793 0 1,050 0

"Ineach assay, various concentrations of gD-1, ranging from 0.45 to 15 ng, were spotted onto nitrocellulose, incubated with cold antibody for 2h,and then re-actedwith iodinated antibodies for1 h.The spots were cutfrom the nitrocellulose and counted in a gamma counter. The results were plotted as counts bound ver-susconcentration ofgD.

bSlopeisgivenin counts per minute per nanogramofgD.

cPercentcompetitionwasderived by the following equation: 100-(counts per minute pernanogram of gD bound with unlabeledantibody present/counts per minute per nanogram of gD bound with no unlabeled antibody). Values underlined represent the percent competition which occurred when the same antibody,

both,unlabeled andlabeled,was used to compete,against itself.

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[image:8.612.66.559.570.671.2]

NH2' Vil

11-19

. V

V

3340-356

A

OEl~

COOH

[image:9.612.60.303.70.195.2]

268-287

FIG. 7.Topographicmapof HSV-1 gD. Thepositionsofepitopes

bindingtoMCAbingroupsVII, I1,andVareshown. Alsoindicated

(as balloons)are the threeN-asparagine-linked glycosylationsites.

The transmembrane region is depicted as abox. The positions of discontinuous epitopes (ellipses at bottom)were derived fromthe competition experiments. Three of these epitopesappeartoinvolve S-S bonds.

pared against gD, including an antiserum prepared against

gD-2. More recently, we have tested additional polyclonal

sera prepared in rabbits against gD-1 and gD-2 and have

found that six ofeight sera, including two prepared against

gD-2, reacted with this peptide. In addition, antiserum

prepared to the peptide reacted with gD-1 and gD-2 in

immunoblot and immunoprecipitation assays, although the reaction was much stronger against gD-1. A somewhat

puzzling observation was that this antiserum was more

reactive againstthe native than the denaturedforms ofgD-1

and gD-2 (Fig. 5A, lanes a to d). It is possible that the

conformations ofpeptide 340-356[1] on KLH might well be nearertothe conformation of thesamesegmentinthenative

protein thantothe different conformationalensemble of the

denatured protein.

Anotherpuzzling observation was the reactivityofgroup

V MCAb against gD-2detectedbyimmunoblot. Previously,

these antibodies were considered type 1 specific based on

immunoprecipitation and immunofluorescence assays.

Al-though most of the amino acids in the epitope specified by

group V antibodies may be unique to gD-1, it is clear from examination of the sequence in this region that several

amino acids in the epitope must be common to gD-1 and

gD-2. The more sensitive immunoblot assay might better

detect thispartial overlap. Alternatively, gD-2 mightassume

different conformations under the conditions used in dif-ferentassays(27, 38). Thefewtypecommonresidues in this sequence maybearrangedclosetogetherinthe nativegD's, sothataweak typecommonreactivitycanbeseen,although the sequencesotherwise differ substantially. Ifso, it would

explain why group V MCAb appeared to be more reactive

against native than against denatured gD-2 (Fig. 2, row 6,

lanes b and d).Finemappingofthe group Vepitope should take intoaccount the differences in sequence betweengD-1

andgD-2in thisregion.

Localization of the group II epitope was accomplished

first by analyzingthe reactivity oftruncated forms ofgD-1

against DL6 MCAb. The antibody failed to react with a

truncated form ofgD ending at residue 275 of the mature

protein (30)but didreactwithaform ofgD endingatresidue

287(M.G. Gibson and P. G. Spear,inpress). These results

suggested that the epitope was between residues 275 and

287. Thecomputerpredictions (Fig. 1)showedthatgD-1and gD-2 each contained a region with hydrophilic residues within a P-turn. Interestingly, this region is rich in acidic

residues

(both

aspartic

and

glutamic

acids)

and

proline,

but

lacks the basic amino

acids,

such as

lysine,

that are often

associated with

epitopes (31).

In

choosing

a

synthetic

pep-tide for confirmation ofthe

location,

we assumed that the

epitope

could include some residues upstreamof 275.

Pep-tide

268-287[1]

reacted with DL6 MCAb and with three

polyclonal

sera

prepared against gD.

This

peptide

also

reacted with several other

polyclonal anti-gD

rabbitsera, a

total of four to

gD-1

and two to

gD-2.

However,

when

rabbitswereimmunized withthis

peptide coupled

to

KLH,

there was no

antibody

response. After several

injections

with the free

peptide,

one ofthe sera

exhibited

reactivity

against

the

peptide

but not

against gD.

In this case, it is

possible

that none of the conformations of the

peptide

coupled

to KLH

corresponded

to the structure of the

peptide

as it is foundinnative ordenatured

gD.

The

synthetic

peptide approach

wetook inthesestudies is

not yet

likely

to be fruitful in

localizing

discontinuous

epitopes

(1,

45).

This is because these

epitopes depend

ona

certain

tertiary

structure of

gD,

which in part involves

disulfide bonds. The

position

of these bonds is not yet

known,

but such information should aid in localization.

However,

we do know that fourdiscontinuous

epitopes

of

gD-1

are located within the first 260 amino acids of the

protein

(Fig. 7),

since antibodiesingroups

I,

III,

IV,

and VI

reacted with truncated

gD-1 (1

to

275)

as well as with the

38K

fragment

(presumably

residues 1 to

260)

generated

by

V8

proteolysis.

Six ofthe seven

cysteine

residues of

gD-1

arelocated within residues66to202.Itis

quite

possible

that disulfide bonds formed

by

these six

cysteines play

arole in

the structure of discontinuous

epitopes.

Cysteine

atresidue

333ofthemature

protein

is withinthetransmembrane

region

of

gD-1

and is notinvolved in formation of these

epitopes.

Thus,

our data

predict

that this

cysteine

is

probably

not

involved in intramolecular disulfide bonds in

gD-1.

How-ever, it may be involved in intermolecular

disulfides,

per-haps

information ofthe

gD-1

dimer

(17, 18).

Inthis

regard,

it is

interesting

to notethat

gD-2,

which lacks this

cysteine,

does not form dimers

(17,

18).

In

preliminary

experiments,

we have found that the group III

epitope

is

destroyed by

boiling gD-1

in SDS in the absence of

mercaptoethanol,

whereasdestruction ofgroups

I,

IV,

andVI

required

reduc-tion and

alkylation (M.

Ponce de

Leon,

G. H.

Cohen,

and

R. J.

Eisenberg,

unpublished data).

Competition

experiments

were carried out to determine

therelative orientationofthefourdiscontinuous

epitopes.

In

some cases, the

binding

of one

antibody

to

gD-1

had no

effect

(no

competition)

on the

binding

of another. These

results are further evidence that the MCAb

groupings

are

valid and that there are distinct discontinuous

epitopes

on

gD-1.

In other cases, there was

competition.

This indicated

that

(i)

someamino acidsin these

epitopes

are

shared;

or

(ii)

the

epitopes

were soclose that therewassteric hindrance in the

binding

of a second

antibody;

or

(iii)

binding

of one

antibody

altered the conformation of

gD

so that

binding

of

the second

antibody

wasaffected

(35).

Previously,

we

spec-ulated that

binding

ofgroup III MCAb altered the

confor-mation of

gD-1,

making

the molecule more

susceptible

to

protease V8

cleavage (15).

This

explanation

is stillconsistent

with the present results.

On the basis of these

studies,

as well as the studies of

three continuous

epitopes,

we have constructed a two-part

topographic

map for

gD-1

(Fig.

7).

First,

we have

depicted

the

protein essentially

as alinear molecule with the

positions

ofthe three continuous

epitopes

indicated. The

discontinu-ous

epitopes

have been

depicted

in a separate

drawing

as

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http://jvi.asm.org/

ellipses

located downstream fromgroup

VII,

each of which

includesamino acids

prior

toresidue 260. Thediscontinuous

epitopes corresponding

to MCAbwhich exhibited

competi-tion are shown as

overlapping.

Thus,

group III

overlaps

groups IV and VI and group I also

overlaps

IV and VI.

Groups

I and III do not

overlap

at all.

Preliminary

experi-ments indicate that antibodies in all of the MCAb groups

except

possibly

group IV areableto

immunoprecipitate

the

gD-like

protein produced by tunicamycin-treated,

HSV-1-infected cells

(42;

Matthewset

al.,

unpublished data).

Three

of the

epitopes

are

depicted

as

involving

disulfidebonds(S-S

in

Fig.

7),

although

wedonotknowhowmany

cysteines

are

disulfidebonded in

gD

orhow manyare involved in

deter-mining

thestructure ofany one

epitope.

Furtherlocalization of discontinuous

epitopes

will

require

other

approaches, including

amore

complete

understanding

of thecontributionofdisulfide bondstothestructureof

gD.

One

possible

approach

will be to

analyze

the amino acid

changes

associated with mutants which exhibit an altered

pattern

of

reactivity

with MCAb. Such mutants would

include those whichare no

longer

neutralized

by antibody,

suchasthe"mar"mutants

(21).

Another

approach

wouldbe

toexaminethe amino acid

changes

foundin natural isolates

of HSV which exhibit an anomalous

pattern

of

reactivity

with MCAb

(41, 43).

This

approach

was

recently exploited

(43)

to

explain

the

reactivity

of an HSV-1 strain with a

gD-2-specific

MCAbcalled17f3A3

(2).

Analysis

ofthe DNA

sequence of the

gD

gene ofthe isolate revealed a

change

which altered the codon for

asparagine

(residue

72 of the mature

protein)

present

in the prototype HSV-1 strain to

histidine,

normally

present

inthe HSV-2 strain.

Grouping

of

17,A3

hasnot

yet

been

accomplished.

However,

it

might

be

in group VIII

(15).

Our

uncertainty

about the

grouping

of

17,A3

illustrates the need for a common classification of

gD-specific

MCAb. Wearenowinthe process of

grouping

a

numberof additional

gD-specific

MCAb fromseveral

labo-ratoriesto overcome this

difficulty.

ACKNOWLEDGMENTS

This

investigation

was

supported by

Public Health Service grants

DE-02623 from the National Institute of DentalResearch,AI-18289 from the National Institute of

Allergy

andInfectious

Diseases,

and CA-21776 from the NationalCancer Institute.A

portion

of this work

was

supported

by

agranttoG.H.C. and R.J.E. from the American

Cyanamid

Co. M.G.G. is a fellow of the Leukemia Society of

America,and J.T.M.was a

predoctoral

traineesupported byPublic

HealthServicegrantNS-07180 from the National Institute of

Neu-rological

and Communicative Disorders and Stroke.

We thank B.

Hampar,

M.

Zweig,

and L. Pereira for monoclonal

antibodies,

Wesley

Wilcox for

carefully reading

themanuscript,and Madeline Cohen,Valerie Rinaldt, andMichael Nobelfor excellent technicalassistance.

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